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nyse:zts
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Zoetis
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Nov 29th, 2016 12:00AM
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Aug 19th, 2011 12:00AM
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https://www.uspto.gov?id=US09505829-20161129
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Anti-NGF antibodies and their use
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The present disclosure encompasses NGF binding proteins, specifically to antibodies that are chimeric, CDR grafted and canonized antibodies, and methods of making and uses thereof. The antibodies, or antibody portions, of the disclosure are useful for detecting NGF and for inhibiting NGF activity, e.g., in a mammal subject suffering from a disorder in which NGF activity is detrimental.
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9505829
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1. An antibody comprising:
(a) a heavy chain variable region comprising an amino acid sequence having a sequence of SEQ ID NO: 192 and complementary determining regions (CDRs) consisting of SEQ ID NOS: 79, 80, and 81; and
(b) a light chain variable region comprising an amino acid sequence having a sequence of SEQ ID NO: 193 and CDRs consisting of SEQ ID NOS: 82, 83 and 84.
2. The antibody of claim 1, wherein the antibody comprises a heavy chain human immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain.
3. The antibody of claim 1, wherein the antibody comprises a heavy chain canine immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG 1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain.
4. The antibody of claim 1, wherein the antibody comprises a heavy chain feline immunoglobulin constant domain.
5. The antibody of claim 1, wherein the antibody comprises a heavy chain equine immunoglobulin constant domain.
6. The antibody of claim 1, comprising a constant region having an amino acid sequence selected from the group consisting of SEQ ID NO: 52 and SEQ ID NO: 54.
7. The antibody of claim 1, wherein the antibody is selected from the group consisting of: an immunoglobulin molecule, disulfide linked Fv, monoclonal antibody, scFv, chimeric antibody, single domain antibody, CDR-grafted antibody, diabody, humanized antibody, caninized mAb, canine mAb, feline mAb, felinized mAb, equine mAb, equinized mAb, a multispecific antibody, a Fab, a dual specific antibody, a DVD-Ig, a Fab′, a bispecific antibody, an F(ab′)2, and an Fv.
8. The antibody of claim 1, wherein the antibody neutralizes NGF.
9. A pharmaceutical or diagnostic composition comprising an antibody of claim 1, and a pharmaceutically acceptable carrier, diluent or excipient.
10. The pharmaceutical composition of claim 9, comprising a therapeutically effective amount of the antibody.
11. The pharmaceutical composition of claim 9, comprising at least one preservative, wherein the preservative is methylparaben, propylparaben, benzyl alcohol, chlorobutanol or benzalkonium chloride.
12. The pharmaceutical composition of claim 9, wherein the pH of the composition is between 7.0 and 8.0.
13. The pharmaceutical composition of claim 9, wherein the composition has a half-life of from about 8.0 to about 15.0 days when dosed intravenously or subcutaneously.
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13
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CROSS-REFERENCE TO RELATED APPLICATIONS
This is the U.S. national stage entry of International Patent Application No. PCT/US2011/048518, filed on Aug. 19, 2011, which claims priority to U.S. Provisional Patent Application No. 61/375,193, filed on Aug. 19, 2010, the contents of all of which are herein fully incorporated by reference.
TECHNICAL FIELD
The disclosure relates to anti-NGF antibodies and polynucleotides encoding the same, and use of such antibodies and/or polynucleotides in the treatment and/or prevention of pain, including but not limited to post-surgical pain, rheumatoid arthritis pain, cancer pain, and osteoarthritis pain.
BACKGROUND
Nerve growth factor (NGF) is a secreted protein that was discovered over 50 years ago as a molecule that promotes the survival and differentiation of sensory and sympathetic neurons. (See Levi-Montalcini, Science 187: 113 (1975), for a review). The crystal structure of NGF and NGF in complex with the tyrosinekinase A (TrkA) receptor has been determined (McDonald et al., Nature 354: 411 (1991); Wiesmann et al., Nature 401: 184-188 (1999)).
The role of NGF in the development and survival of both peripheral and central neurons has been well characterized. NGF has been shown to be a critical survival and maintenance factor in the development of peripheral sympathetic and embryonic sensory neurons and of basal forebrain cholinergic neurons (see, e.g., Smeyne et al., Nature 368: 246-9 (1994); and Crowley et al., Cell, 76:1001-11 (1994)). It has been shown to inhibit amyloidogenesis that leads to Alzheimer's disease (Calissano et al., Cell Death and Differentiation, 17: 1126-1133 (2010)). NGF up-regulates expression of neuropeptides in sensory neurons (Lindsay et al., Nature, 337:362-364 (1989)) and its activity is mediated through two different membrane-bound receptors, the TrkA receptor and the p75 common neurotrophin receptor (Chao et al., Science, 232:518-521 (1986); Huang et al., Annu. Rev. Neurosci., 24:677-736 (2001); Bibel et al., Genes Dev., 14:2919-2937 (2000)).
NGF is produced by a number of cell types including mast cells (Leon, et al., Proc. Natl. Acad. Sci., 91: 3739-3743 (1994)), B-lymphocytes (Torcia, et al., Cell, 85: 345-356 (1996), keratinocytes (Di Marco, et al., J. Biol. Chem., 268: 22838-22846)), smooth muscle cells (Ueyama, et al., J. Hypertens., 11: 1061-1065 (1993)), fibroblasts (Lindholm, et al., Eur. J. Neurosci., 2: 795-801 (1990)), bronchial epithelial cells (Kassel, et al., Clin, Exp. Allergy, 31:1432-40 (2001)), renal mesangial cells (Steiner, et al., Am. J. Physiol., 261:F792-798 (1991)) and skeletal muscle myotubes (Schwartz, et al., J. Photochem. Photobiol., B66: 195-200 (2002)). In addition, NGF receptors have been found on a variety of cell types outside of the nervous system.
NGF has been implicated in processes outside of the nervous system, e.g., NGF has been shown to enhance vascular permeability (Otten, et al., Eur J. Pharmacol., 106: 199-201 (1984)), enhance T- and B-cell immune responses (Otten, et al., Proc. Natl. Acad. Sci., USA 86: 10059-10063 (1989)), induce lymphocyte differentiation and mast cell proliferation and cause the release of soluble biological signals from mast cells (Matsuda, et al., Proc. Natl. Acad. Sci., 85: 6508-6512 (1988); Pearce, et al., J. Physiol., 372:379-393 (1986); Bischoff, et al., Blood, 79: 2662-2669 (1992); Horigome, et al., J. Biol. Chem., 268: 14881-14887 (1993)).
Both local and systemic administrations of NGF have been shown to elicit hyperalgesia and allodynia (Lewin, G. R. et al., Eur. J. Neurosci. 6: 1903-1912 (1994)). Intravenous infusion of NGF in humans produces a whole body myalgia while local administration evokes injection site hyperalgesia and allodynia in addition to the systemic effects (Apfel, S. C. et al., Neurology, 51: 695-702 (1998)). Furthermore, in certain forms of cancer, excess NGF facilitates the growth and infiltration of nerve fibers with induction of cancer pain (Zhu, Z. et al., J. Clin. Oncol., 17: 241-228 (1999)). Although exogenously added NGF has been shown to be capable of having all of these effects, it is important to note that it has only rarely been shown that endogenous NGF is important in any of these processes in vivo (Torcia, et al., Cell, 85(3): 345-56 (1996)).
An elevated level of NGF has been implicated in certain inflammatory conditions in humans and animals, e.g., systemic lupus erythematosus (Bracci-Laudiero, et al., Neuroreport, 4: 563-565 (1993)), multiple sclerosis (Bracci-Laudiero, et al., Neurosci. Lett., 147:9-12 (1992)), psoriasis (Raychaudhuri, et al., Acta Derm. l'enereol., 78: 84-86 (1998)), arthritis (Falcim, et al., Ann. Rheum. Dis., 55: 745-748 (1996)), interstitial cystitis (Okragly, et al., J. Urology, 161: 438-441 (1999)) and asthma (Braun, et al., Eur. J. Immunol., 28:3240-3251 (1998)). The synovium of patients affected by rheumatoid arthritis expresses high levels of NGF while in non-inflamed synovium NGF has been reported to be undetectable (Aloe, et al., Arch. Rheum., 35:351-355 (1992)). Similar results were seen in rats with experimentally induced rheumatoid arthritis (Aloe, et al., Clin. Exp. Rheumatol., 10: 203-204 (1992)). Elevated levels of NGF have been reported in transgenic arthritic mice along with an increase in the number of mast cells (Aloe, et al., Int. J. Tissue Reactions-Exp. Clin. Aspects, 15: 139-143 (1993)). Additionally, elevated levels of expression of canine NGF has been shown in lame dogs (Isola, M., Ferrari, V., Stabile, F., Bernardini, D., Canner, P., Busetto, R. Nerve growth factor concentrations in the synovial fluid from healthy dogs and dogs with secondary osteoarthritis. Vet. Comp. Orthop. Traumatol. 4: 279 (2011)). PCT Publication No. WO 02/096458 discloses use of anti-NGF antibodies of certain properties in treating various NGF related disorders such as inflammatory condition (e.g., rheumatoid arthritis). It has been reported that a purified anti-NGF antibody injected into arthritic transgenic mice carrying the human tumor necrosis factor (TNF) gene caused reduction in the number of mast cells, as well as a decrease in histamine and substance P levels within the synovium of arthritis mice (Aloe et al., Rheumatol. Int., 14: 249-252 (1995)). It has been shown that exogenous administration of a NGF antibody reduced the enhanced level of TNF occurring in arthritic mice (Manni et al., Rheumatol. Int., 18: 97-102 (1998)).
Increased expression of NGF and high affinity NGF receptor (TrkA) was observed in human osteoarthritis chondrocytes (Iannone et al., Rheumatology, 41: 1413-1418 (2002)). Rodent anti-NGF antagonist antibodies have been reported (Hongo et al., Hybridoma, 19(3):215-227 (2000); Ruberti et al., Cell. Molec. Neurobiol., 13(5): 559-568 (1993)). However, when rodent antibodies are used therapeutically in non-rodent subjects, an anti-murine antibody response develops in significant numbers of treated subjects.
The involvement of NGF in chronic pain has led to considerable interest in therapeutic approaches based on inhibiting the effects of NGF (Saragovi, et al., Trends Pharmacol Sci. 21: 93-98 (2000)). For example, a soluble form of the TrkA receptor was used to block the activity of NGF, which was shown to significantly reduce the formation of neuromas, responsible for neuropathic pain, without damaging the cell bodies of the lesioned neurons (Kryger, et al., J. Hand Surg. (Am.), 26: 635-644 (2001)).
Certain anti-NGF antibodies have been described (PCT Publication Nos. WO 2001/78698, WO 2001/64247, WO 2002/096458, WO 2004/032870, WO 2005/061540, WO 2006/131951, WO 2006/110883; U.S. Publication Nos. US 20050074821, US 20080033157, US 20080182978 and US 20090041717; and U.S. Pat. No. 7,449,616). In animal models of neuropathic pain (e.g., nerve trunk or spinal nerve ligation) systemic injection of neutralizing antibodies to NGF prevents both allodynia and hyperalgesia (Ramer et al., Eur. J. Neurosci., 11: 837-846 (1999); Ro et al., Pain, 79: 265-274 (1999)). Furthermore, treatment with a neutralizing anti-NGF antibody produces significant pain reduction in a murine cancer pain model (Sevcik et al., Pain, 115: 128-141 (2005)). Thus, there is a serious need for anti-NGF antagonist antibodies for humans and animals.
SUMMARY OF THE INVENTION
The present disclosure provides a novel family of binding proteins, CDR grafted antibodies, mammalized (such as bovanized, camelized, caninized, equinized, felinized, humanized etc.) antibodies, and fragments thereof, capable of binding and neutralizing NGF. The disclosure provides a therapeutic means with which to inhibit NGF and provides compositions and methods for treating disease associated with increased levels of NGF, particularly inflammatory disorders.
In one aspect, the present disclosure provides a binding protein, or fragment thereof, comprising hypervariable region sequences wholly or substantially identical to sequences from an antibody from a donor species; and constant region sequences wholly or substantially identical to sequences of antibodies from a target species, wherein the donor and target species are different. The binding protein may for example specifically bind NGF and have a heavy chain having a heavy chain variable region and a light chain having a light chain variable region.
In another aspect, the present disclosure provides a binding protein that specifically binds NGF and which has a heavy chain having a heavy chain variable region and a light chain having a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO:14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 206, SEQ ID NO: 207, or an antigen-binding or an immunologically functional immunoglobulin fragment thereof.
In another aspect, the present disclosure provides a binding protein that specifically binds NGF and which has a heavy chain having a heavy chain variable region and a light chain having a light chain variable region, wherein the light chain variable region comprises an amino acid sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 44, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, or an antigen-binding or an immunologically functional immunoglobulin fragment thereof.
A binding protein of the present disclosure may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 50% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 50% to one of said sequences. Alternatively, the binding protein of the present disclosure may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 70% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 70% to one of said sequences. The binding protein of the present disclosure may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 80% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 80% to one of said sequences. binding protein of the present disclosure may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 90% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 90% to one of said sequences.
A binding protein of the present disclosure may comprise a heavy chain human immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. A binding proteins of the present disclosure may alternatively comprise a heavy chain canine immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. A binding protein of the present disclosure may alternatively comprise a heavy chain feline immunoglobulin constant domain. A binding protein of the present disclosure may alternatively comprise a heavy chain equine immunoglobulin constant domain. A binding protein of the present disclosure may further comprise a constant region having an amino acid sequence selected from the group consisting of SEQ ID NO:52 and SEQ ID NO:54.
Any of the above binding proteins may be selected from the group consisting of; an immunoglobulin molecule, disulfide linked Fv, monoclonal antibody, scFv, chimeric antibody, single domain antibody, CDR-grafted antibody, diabody, humanized antibody, caninized mAb, canine mAb, feline mAb, felinized mAb, equine mAb, equinized mAb, a multispecific antibody, a Fab, a dual specific antibody, a DVD-Ig, a Fab′, a bispecific antibody, a F(ab′)2, and a Fv.
Any of the above binding proteins may be capable of modulating a biological function of NGF, or neutralizing NGF.
Any of the above binding proteins may be capable of neutralizing NGF with a potency (IC50) of at least about 10 nM, at least about 5 nM, at least about 1 nM, at least about 0.5 nM, at least about 0.1 nM, at least about 0.05 nM, at least about 0.01 nM, or at least about 0.001 nM, as measured in the TF-1 cell proliferation assay or the pERK and Pathhunter assays.
Any of the above binding proteins may have an on rate constant (Kon) for NGF of: at least about 102M−1s−1, at least about 103M−1s−1, at least about 104M−1s−1, at least about 105M−1s−1, or at least about 106M−1s−1, or at least about 107M−1s−1, as measured by surface plasmon resonance.
Any of the above binding proteins may have an off rate constant (Koff) for NGF selected from the group consisting of: at most about 10−3s−1, at most about 10−4s−1, at most about 10−5s−1, at most about 10−6s−1, and at most about 10−7s−1, as measured by surface plasmon resonance.
Any of the above binding proteins may have a dissociation constant (KD) for NGF selected from the group consisting of: at most about 10−7 M, at most about 10−8 M, at most about 10−9 M, at most about 10−10 M, at most about 10−11 M at most about 10−12 M, at most about 10−13 M and at most about 10−14M. The dissociation constant (KD) may be, for example, about 1×10−9M, about 1×10−10 M, about 3.14×10−10 M, about 1×10−11 M, about 2.37×10−11 M, about 1×10−12 M, about 1×10−13 M, and about 3.3×10−14 M.
Any of the above binding proteins may further comprise an agent selected from the group consisting of; an immunoadhension molecule, an imaging agent, a therapeutic agent, and a cytotoxic agent. The agent may be, for example, an imaging agent selected from the group consisting of a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin. The imaging agent may be a radiolabel selected from the group consisting of: 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and 153Sm. Alternatively, the agent may be a therapeutic or cytotoxic agent, such as, for example, an anti-metabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an anti-angiogenic agent, an anti-mitotic agent, an anthracycline, toxin, and an apoptotic agent.
Any of the binding proteins may possess a murine, canine, feline, human or equine glycosylation pattern.
Any of the binding proteins may be a crystallized binding protein. The crystallized binding protein may be a carrier-free pharmaceutical controlled release crystallized binding protein.
In another aspect, the present disclosure provides an isolated nucleic acid encoding any of the above binding proteins. The isolated nucleic acid may comprise RNA or DNA.
In another aspect, the present disclosure provides an isolated nucleic acid comprising or complementary to a nucleic acid sequence that encodes a binding protein that specifically binds NGF having a heavy chain having a heavy chain variable region and a light chain having a light chain variable region, wherein the heavy chain variable region is encoded by a nucleotide sequence having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOS: 1, 5, 9, 13, 17, and 21.
In another aspect, the present disclosure provides an isolated nucleic acid comprising or complementary to a nucleic acid sequence that encodes a binding protein that specifically binds NGF having a heavy chain having a heavy chain variable region and a light chain having a light chain variable region, wherein the light chain variable region is encoded by a nucleotide sequence having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOS: 3, 7, 11, 15, 19 and 23.
In another aspect, the present disclosure provides a recombinant vector comprising an isolated nucleic acid encoding a binding protein that specifically binds NGF as described herein. A recombinant vector according to the present disclosure may comprise pcDNA, pTT, pTT3, pEFBOS, pBV, pJV or pBJ. Also provided is a host cell comprising such a recombinant vector. The host cell may be for example a eukaryotic cell, or a prokaryotic cell. The host cell may be a protist cell; an animal cell such as but not limited to a mammalian cell, avian cell; an insect cell, such as but not limited to an insect Sf9 cell; a plant cell; or a fungal cell. The host cell may be for example an E. coli cell. The host cell may be a CHO cell, or a COS cell. Also provided is an isolated cell line that produces a binding protein that specifically binds NGF as described herein.
In another aspect, the present disclosure provides a pharmaceutical or diagnostic composition comprising a binding protein that specifically binds NGF as described herein, and a pharmaceutically acceptable carrier, diluent or excipient. A pharmaceutical composition may comprise a therapeutically effective amount of the NGF binding protein.
In another aspect, the present disclosure provides a composition for the release of a binding protein, the composition comprising: (a) a composition comprising a binding protein that specifically binds NGF as described herein, and a pharmaceutically acceptable carrier, excipient or diluent, and (b) at least one polymeric carrier.
In another aspect, the present disclosure provides a method for reducing NGF activity in a subject (for example, a dog, cat, horse, ferret, etc.) suffering from a disorder in which NGF activity is detrimental, comprising administering to the subject a therapeutically effective amount of a binding protein that specifically binds NGF as described herein.
In another aspect, the present disclosure provides a method for making anti-NGF antibodies comprising: (a) production of murine monoclonal antibodies; (b) screening hybridoma supernatants; (c) grafting of donor CDRs into target frameworks; and (d) introducing backmutations in the framework region of the target antibodies, wherein the anti-NGF antibodies comprise hypervariable region sequences wholly or substantially identical to sequences from an antibody from the donor species and constant region sequences wholly or substantially identical to sequences of an antibody from the target species, wherein the donor and the target species are different. In the method, the donor may be, for example, a mouse and the target a non-murine mammal, such as but not limited to a bovine, canine, equine, or feline mammal, or a camel goat, human or sheep.
In another aspect, the present disclosure provides a method for detecting the presence or amount of NGF in a sample, comprising: providing a reagent comprising any of the above binding proteins that specifically bind NGF; combining the binding protein with the sample for a time and under conditions sufficient for the binding protein to bind to any NGF in the sample; and determining the presence or amount of NGF in the sample based on specific binding of the binding protein to NGF. In the method, the binding protein may be immobilized or may be capable of being immobilized on a solid support. In the method, the binding protein may be coupled to a detectable label, such as, for example, an imaging agent such as but not limited to a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin. The imaging agent may be for example a radiolabel selected from the group consisting of: 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and 153Sm.
In another aspect, the present disclosure provides an immunoassay device for detecting the presence or amount of NGF in a sample, the device comprising any of the above binding proteins that specifically bind NGF, immobilized on a solid support.
In another aspect, the present disclosure provides a kit for detecting the presence or amount of NGF in a sample, the kit comprising: an immunoregamet comprising any of the above binding proteins that specifically bind NGF, and instructions for determining the presence or amount of NGF in the sample based on specific binding of the immunoreagent to NGF. In the kit, the binding protein may be immobilized on a solid support.
In still yet another aspect, the present disclosure relates to an antibody or antigen binding fragment thereof comprising:
a heavy chain variable region comprises an amino acid sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 206, SEQ ID NO: 207, or an antigen-binding or an immunologically functional immunoglobulin fragment thereof; and
a light chain variable region comprises an amino acid sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 44, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, or an antigen-binding or an immunologically functional immunoglobulin fragment thereof.
More specifically, the above-described antibody may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 50% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 50% to one of said sequences. Alternatively, the above-described antibody may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 70% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 70% to one of said sequences. Alternatively, the above-described antibody may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 80% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 80% to one of said sequences. Alternatively, the above-described antibody may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 90% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 90% to one of said sequences.
The above-described antibody may comprise a heavy chain human immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. More specifically, the antibody may comprise a heavy chain canine immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. Alternatively, the antibody comprises a heavy chain feline immunoglobulin constant domain. Still further alternatively, the antibody comprises a heavy chain equine immunoglobulin constant domain. Moreover, the above-described antibody may comprise a constant region having an amino acid sequence selected from the group consisting of SEQ ID NO:52 and SEQ ID NO:54. Still further, the above-described antibody is selected from the group consisting of: an immunoglobulin molecule, disulfide linked Fv, monoclonal antibody, scFv, chimeric antibody, single domain antibody, CDR-grafted antibody, diabody, humanized antibody, caninized mAb, canine mAb, feline mAb, felinized mAb, equine mAb, equinized mAb, a multispecific antibody, a Fab, a dual specific antibody, a DVD-Ig, a Fab′, a bispecific antibody, a F(ab′)2, and a Fv.
In another aspect, the above-identified antibody is capable of modulating a biological function of NGF.
In still yet another aspect, the present disclosure relates to an isolated nucleic acid encoding the above-described antibody.
In another aspect, the present invention relates to an antibody or antigen binding fragment thereof having a heavy chain variable region that comprises an amino acid sequence having at least 90% identity with a sequence of SEQ ID NO:37 and a light chain variable region that comprises an amino acid sequence having at least 90% identity with a sequence of SEQ ID NO:38. The above-described antibody may comprise a heavy chain human immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. More specifically, the antibody may comprise a heavy chain canine immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. Alternatively, the antibody comprises a heavy chain feline immunoglobulin constant domain. Still further alternatively, the antibody comprises a heavy chain equine immunoglobulin constant domain. Moreover, the above-described antibody may comprise a constant region having an amino acid sequence selected from the group consisting of SEQ ID NO:52 and SEQ ID NO:54. Still further, the above-described antibody is selected from the group consisting of: an immunoglobulin molecule, disulfide linked Fv, monoclonal antibody, scFv, chimeric antibody, single domain antibody, CDR-grafted antibody, diabody, humanized antibody, caninized mAb, canine mAb, feline mAb, felinized mAb, equine mAb, equinized mAb, a multispecific antibody, a Fab, a dual specific antibody, a DVD-Ig, a Fab′, a bispecific antibody, a F(ab′)2, and a Fv.
In another aspect, the above-identified antibody is capable of modulating a biological function of NGF.
In still yet another aspect, the present disclosure relates to an isolated nucleic acid encoding the above-described antibody.
In another aspect, the present invention relates to an antibody or antigen binding fragment thereof having a heavy chain variable region comprises an amino acid sequence having at least 90% identity with a sequence of SEQ ID NO: 192 and the light chain variable region comprises an amino acid sequence having at least 90% identity with a sequence of SEQ ID NO: 193. The above-described antibody may comprise a heavy chain human immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. More specifically, the antibody may comprise a heavy chain canine immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. Alternatively, the antibody comprises a heavy chain feline immunoglobulin constant domain. Still further alternatively, the antibody comprises a heavy chain equine immunoglobulin constant domain. Moreover, the above-described antibody may comprise a constant region having an amino acid sequence selected from the group consisting of SEQ ID NO: 52 and SEQ ID NO: 54. Still further, the above-described antibody is selected from the group consisting of: an immunoglobulin molecule, disulfide linked Fv, monoclonal antibody, scFv, chimeric antibody, single domain antibody, CDR-grafted antibody, diabody, humanized antibody, caninized mAb, canine mAb, feline mAb, felinized mAb, equine mAb, equinized mAb, a multispecific antibody, a Fab, a dual specific antibody, a DVD-Ig, a Fab′, a bispecific antibody, a F(ab′)2, and a Fv.
In another aspect, the above-identified antibody is capable of modulating a biological function of NGF.
In still yet another aspect, the present disclosure relates to an isolated nucleic acid encoding the above-described antibody.
In still yet another aspect, the present disclosure relates to a pharmaceutical or diagnostic composition comprising at least one of the above-described antibodies, and a pharmaceutically acceptable carrier, diluent or excipient. More specifically, the pharmaceutical or diagnostic composition may comprise a therapeutically effective amount of at least one of the above-described antibodies. In addition, the pharmaceutical or diagnostic composition may comprise at one preservative. Examples of at least one preservative that may be used is methylparaben, propylparaben, benzyl alcohol, chlorobutanol or benzalkonium chloride.
The pharmaceutical composition can have a pH of greater than about 7.0. Alternatively, the pharmaceutical composition can have a pH of between about 6.8 and about 8.2. Alternatively, the pharmaceutical composition can have a pH of between about 7.2 and about 7.8. Still further alternatively, the pH of the pharmaceutical composition can be between about 7.4 and about 7.6. Still further alternatively, the pH of the pharmaceutical composition can be about 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1 or 8.2.
The pharmaceutical composition of the present disclosure may have a half-life of from about 8.0 days to about 15.0 days when dosed intravenously or subcutaneously. Alternatively, the pharmaceutical composition of the present invention may have a half-life of from about 10.0 days to about 13.0 days. Still further alternatively, the pharmaceutical composition of the present invention may have a half-life of about 8.0 days, about 8.5 days, about 9.0 days, about 9.5 days, about 10.0 days, about 10.5 days, about 11.0 days, about 11.5 days, about 12.0 days, about 12.5 days, about 13.0 days, about 13.5 days, about 14.0 days, about 14.5 days or about 15.0 days.
In another aspect, the present disclosure relates to a method for reducing NGF activity in a subject suffering from a disorder in which NGF activity is detrimental, comprising administering to the subject a therapeutically effective amount of an antibody of antigen binding fragment thereof of at least one of the above-described antibodies or antigen-binding fragments thereof.
BRIEF DESCRIPTION OF FIGURES
FIG. 1 illustrates PR-1254972 VH nucleotide sequence (SEQ ID NO: 1) of mouse anti-NGF antibody.
FIG. 2 illustrates PR-1254972 VH amino acid sequence (SEQ ID NO: 2) of mouse anti-NGF antibody.
FIG. 3 illustrates PR-1254972 VL nucleotide sequence (SEQ ID NO: 3) of mouse anti-NGF antibody.
FIG. 4 illustrates PR-1254972 VL amino acid (SEQ ID NO: 4) of mouse anti-NGF antibody.
FIG. 5 illustrates PR-1254973 VH nucleotide sequence (SEQ ID NO: 5) of mouse anti-NGF antibody.
FIG. 6 illustrates PR-1254973 VH amino acid (SEQ ID NO: 6) of mouse anti-NGF antibody.
FIG. 7 illustrates PR-1254973 VL nucleotide sequence (SEQ ID NO: 7) of mouse anti-NGF antibody.
FIG. 8 illustrates PR-1254973 VL amino acid (SEQ ID NO: 8) of mouse anti-NGF antibody.
FIG. 9 illustrates PR-1254977 VH nucleotide sequence (SEQ ID NO: 9) of mouse anti-NGF antibody.
FIG. 10 illustrates PR-1254977 VH amino acid (SEQ ID NO: 10) of mouse anti-NGF antibody.
FIG. 11 illustrates PR-1254977 VL nucleotide sequence (SEQ ID NO: 11) of mouse anti-NGF antibody.
FIG. 12 illustrates PR-1254977 VL amino acid (SEQ ID NO: 12) of mouse anti-NGF antibody.
FIG. 13 illustrates PR-1254980 VH nucleotide sequence (SEQ ID NO: 13) of mouse anti-NGF antibody.
FIG. 14 illustrates PR-1254980 VH amino acid (SEQ ID NO: 14) of mouse anti-NGF antibody.
FIG. 15 illustrates PR-1254980 VL nucleotide sequence (SEQ ID NO: 15) of mouse anti-NGF antibody.
FIG. 16 illustrates PR-1254980 VL amino acid (SEQ ID NO: 16) of mouse anti-NGF antibody.
FIG. 17 illustrates PR-1254981 VH nucleotide sequence (SEQ ID NO: 17) of mouse anti-NGF antibody.
FIG. 18 illustrates PR-1254981 VH amino acid (SEQ ID NO: 18) of mouse anti-NGF antibody.
FIG. 19 illustrates PR-1254981 VL nucleotide sequence (SEQ ID NO: 19) of mouse anti-NGF antibody.
FIG. 20 illustrates PR-1254981 VL amino acid (SEQ ID NO: 20) of mouse anti-NGF antibody.
FIG. 21 illustrates PR-1254982 VH nucleotide sequence (SEQ ID NO: 21) of mouse anti-NGF antibody.
FIG. 22 illustrates PR-1254982 VH amino acid (SEQ ID NO: 22) of mouse anti-NGF antibody.
FIG. 23 illustrates PR-1254982 VL nucleotide sequence (SEQ ID NO: 23) of mouse anti-NGF antibody.
FIG. 24 illustrates PR-1254982 VL amino acid (SEQ ID NO: 24) of mouse anti-NGF antibody.
FIG. 25 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 25 (72.1 VH amino acid).
FIG. 26 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 26 (72.1 VL amino acid).
FIG. 27 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined) SEQ ID NO: 27 (73.1 VH amino acid).
FIG. 28 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined) SEQ ID NO: 28 (73.1 VL amino acid).
FIG. 29 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 29 (77.1 VH amino acid).
FIG. 30 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 30 (77.1 VL amino acid).
FIG. 31A illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 31 (81.1 VH amino acid).
FIG. 31B illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 177 (81.1B VH amino acid).
FIG. 32 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 32 (81.1 VL amino acid)
FIG. 33 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 33 (82.1 VH amino acid)
FIG. 34 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 34 (82.1 VL amino acid).
FIG. 35 illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 35 (72.2 VH amino acid).
FIG. 36A illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 36 (72.2 VL amino acid).
FIG. 36B illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 179 (72.3 VH amino acid).
FIG. 36C illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 180 (72.4 VH amino acid).
FIG. 36D illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 181 (72.4 VL amino acid).
FIG. 37 illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 37 (73.2 VH amino acid).
FIG. 38A illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 38 (73.2 VL amino acid).
FIG. 38B illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 182 (73.4 VH amino acid).
FIG. 38C illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 183 (73.4 VL amino acid).
FIG. 39 illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 39 (77.2 VH amino acid).
FIG. 40A illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 40 (77.2 VL amino acid).
FIG. 40B illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 184 (77.3 VH amino acid).
FIG. 40C illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 185 (77.4 VH amino acid).
FIG. 40D illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 186 (77.4 VL amino acid).
FIG. 41 illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 41 (81.2 VH amino acid).
FIG. 42A illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 42 (81.2 VL amino acid).
FIG. 42B illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 187 (81.4 VH amino acid).
FIG. 42C illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 188 (81.4 VL amino acid).
FIG. 42D illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 189 (81.2B VH amino acid).
FIG. 42E illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 190 (81.4B VH amino acid).
FIG. 42F illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold, SEQ ID NO:206 (81.5B VH amino acid).
FIG. 42G illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold, SEQ ID NO:207 (81.6B VH amino acid).
FIG. 43 illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 43 (82.2 VH amino acid).
FIG. 44A illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 44 (82.2 VL amino acid).
FIG. 44B illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 191 (82.3 VL amino acid).
FIG. 44C illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 192 (82.4 VH amino acid).
FIG. 44D illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 193 (82.4 VL amino acid).
FIG. 45 illustrates primer sequence to clone canine NGF, SEQ ID NO: 45 (NGF-Dog-S primer).
FIG. 46 illustrates primer sequence to clone canine NGF, SEQ ID NO: 46 (NGF-Dog-AS primer).
FIG. 47 illustrates primer sequence to clone canine NGF, SEQ ID NO: 47 (NGF-d-Ec-S primer).
FIG. 48 illustrates primer sequence to clone canine NGF, SEQ ID NO: 48 (NGF-d-Ec-AS primer).
FIG. 49 illustrates canine NGF C-terminal 6H is (SEQ ID NO: 208) fusion nucleotide sequence, SEQ ID NO: 49.
FIG. 50 illustrates canine NGF C-terminal 6-His (SEQ ID NO: 208) amino acid sequence, SEQ ID NO: 50.
FIG. 51 illustrates canine IgG constant region nucleotide sequence, SEQ ID NO: 51.
FIG. 52 illustrates canine IgG constant region amino acid sequence, SEQ ID NO: 52.
FIG. 53 illustrates canine kappa constant region nucleotide sequence, SEQ ID NO: 53
FIG. 54 illustrates canine kappa constant region amino acid sequence, SEQ ID NO: 54.
FIG. 55 illustrates complementarity determining region, SEQ ID NO: 55 (72.1 VH amino acid; CDR1).
FIG. 56 illustrates complementarity determining region, SEQ ID NO: 56 (72.1 VH amino acid; CDR2).
FIG. 57 illustrates complementarity determining region, SEQ ID NO: 57 (72.1 VH amino acid; CDR3).
FIG. 58 illustrates complementarity determining region, SEQ ID NO: 58 (72.1 VL amino acid; CDR1).
FIG. 59 illustrates complementarity determining region, SEQ ID NO: 59 (72.1 VL amino acid; CDR2).
FIG. 60 illustrates complementarity determining region, SEQ ID NO: 60 (72.1 VL amino acid; CDR3).
FIG. 61 illustrates complementarity determining region, SEQ ID NO: 61 (73.1 VH amino acid; CDR1).
FIG. 62 illustrates complementarity determining region, SEQ ID NO: 62 (73.1 VH amino acid; CDR2).
FIG. 63 illustrates complementarity determining region, SEQ ID NO: 63 (73.1 VH amino acid; CDR3).
FIG. 64 illustrates complementarity determining region, SEQ ID NO: 64 (73.1 VL amino acid; CDR1).
FIG. 65 illustrates complementarity determining region, SEQ ID NO: 65 (73.1 VL amino acid; CDR2).
FIG. 66 illustrates complementarity determining region, SEQ ID NO: 66 (73.1 VL amino acid; CDR3).
FIG. 67 illustrates complementarity determining region, SEQ ID NO: 67 (77.1 VH amino acid; CDR1).
FIG. 68 illustrates complementarity determining region, SEQ ID NO: 68 (77.1 VH amino acid; CDR2).
FIG. 69 illustrates complementarity determining region, SEQ ID NO: 69 (77.1 VH amino acid; CDR3).
FIG. 70 illustrates complementarity determining region, SEQ ID NO: 70 (77.1 VL amino acid; CDR1).
FIG. 71 illustrates complementarity determining region, SEQ ID NO: 71 (77.1 VL amino acid; CDR2).
FIG. 72 illustrates complementarity determining region, SEQ ID NO: 72 (77.1 VL amino acid; CDR3).
FIG. 73 illustrates complementarity determining region, SEQ ID NO: 73 (81.1 VH amino acid; CDR1).
FIG. 74 illustrates complementarity determining region, SEQ ID NO: 74 (81.1 VH amino acid; CDR2).
FIG. 75 illustrates complementarity determining region, SEQ ID NO: 75 (81.1 VH amino acid; CDR3).
FIG. 76 illustrates complementarity determining region, SEQ ID NO: 76 (81.1 VL amino acid; CDR1).
FIG. 77 illustrates complementarity determining region, SEQ ID NO: 77 (81.1 VL amino acid; CDR2).
FIG. 78 illustrates complementarity determining region, SEQ ID NO: 78 (81.1 VL amino acid; CDR3).
FIG. 79 illustrates complementarity determining region, SEQ ID NO: 79 (82.1 VH amino acid; CDR1).
FIG. 80 illustrates complementarity determining region, SEQ ID NO: 80 (82.1 VH amino acid; CDR2).
FIG. 81 illustrates complementarity determining region, SEQ ID NO: 81 (82.1 VH amino acid; CDR3).
FIG. 82 illustrates complementarity determining region, SEQ ID NO: 82 (82.1 VL amino acid; CDR1).
FIG. 83 illustrates complementarity determining region, SEQ ID NO: 83 (82.1 VL amino acid; CDR2).
FIG. 84 illustrates complementarity determining region, SEQ ID NO: 84 (82.1 VL amino acid; CDR3).
FIG. 85 illustrates the sequence of human NGF (SEQ ID NO: 85).
FIG. 86 illustrates the sequences shown in Table 14 illustrating SEQ ID NOs 178, 86-88 from canine heavy chain variable domain sequence derived from canine PBMC.
FIG. 86A illustrates the sequences shown in Table 14 illustrating SEQ ID NOs 89-93 from canine heavy chain variable domain sequences derived from canine PBMC.
FIG. 86B illustrates the sequences shown in Table 14 illustrating SEQ ID NOs 94-98 from canine heavy chain variable domain sequences derived from canine PBMC.
FIG. 86C illustrates the sequences shown in Table 14 illustrating SEQ ID NOs 99-102 from canine heavy chain variable domain sequences derived from canine PBMC.
FIG. 86D illustrates the sequences shown in Table 14 illustrating SEQ ID NOs 103-107 from canine variable domain sequences derived from canine PBMC.
FIG. 86E illustrates the sequences shown in Table 14 illustrating SEQ ID NOs 108-109 from canine heavy variable domain sequences derived from canine PBMC.
FIG. 87 illustrates the sequences shown in Table 15 illustrating SEQ ID NOs 110, 111, 204, 112 from canine lambda light chain variable domain sequences derived from canine PBMC RNA.
FIG. 87A illustrates the sequences shown in Table 15 illustrating SEQ ID NOs 113-117 from canine lambda light chain variable domain sequences derived from canine PBMC RNA.
FIG. 87B illustrates the sequences shown in Table 15 illustrating SEQ ID NOs 118-122 from canine lambda light chain variable domain sequences derived from canine PBMC RNA.
FIG. 87C illustrates the sequences shown in Table 15 illustrating SEQ ID NOs 123-126 from canine lambda light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88 illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 127-131 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88A illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 132-136 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88B illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 137-141 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88C illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 142-146 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88D illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 147-151 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88E illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 152-156 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88F illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 157-161 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88G illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 162-164 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 89 illustrates the sequences shown in Table 17 illustrating SEQ ID NOs 165-168 from mouse anti-NGF CDRs grafted onto Human Ig Frameworks (CDR-grafted Anti-NGF); CDRs underlined.
FIG. 89A illustrates the sequences shown in Table 17 illustrating SEQ ID NOs 169-173 from mouse anti-NGF CDRs grafted onto Human Ig Frameworks (CDR-grafted Anti-NGF); CDRs underlined.
FIG. 89B illustrates the sequences shown in Table 17 illustrating SEQ ID NOs 174-176 from mouse anti-NGF CDRs grafted onto Human Ig Frameworks (CDR-grafted Anti-NGF); CDRs underlined.
FIG. 90 illustrates the sequences shown in Table 18 illustrating SEQ ID NOs 194-196 from Mouse/Canine Chimeric Antibody sequences.
FIG. 90A illustrates the sequences shown in Table 18 illustrating SEQ ID NOs 197-199 from Mouse/Canine Chimeric Antibody sequences.
FIG. 90B illustrates the sequences shown in Table 18 illustrating SEQ ID NOs 200-202 from Mouse/Canine Chimeric Antibody sequences.
FIG. 90C illustrates the sequences shown in Table 18 illustrating SEQ ID NOs 203 from Mouse/Canine Chimeric Antibody sequences.
DETAILED DESCRIPTION OF THE DISCLOSURE
The disclosure describes NGF binding proteins, particularly anti-NGF antibodies, or antigen-binding portions thereof, that bind NGF. Various aspects of the disclosure relate to antibodies and antibody fragments, and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such antibodies and fragments. Methods of using the antibodies of the disclosure to detect human and canine NGF, to inhibit human and canine NGF activity, either in vitro or in vivo; and to regulate gene expression are also encompassed by the disclosure.
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguiy, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.
Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
That the present disclosure may be more readily understood, select terms and phrases as used herein are defined below.
A. DEFINITIONS
The terms “acceptor” and “acceptor antibody” refer to the antibody or nucleic acid sequence providing or encoding at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% of the amino acid sequences of one or more of the framework regions. The term “acceptor” encompasses an antibody amino acid or nucleic acid sequence providing or encoding the constant region(s). The term also encompasses the antibody amino acid or nucleic acid sequence providing or encoding one or more of the framework regions and the constant region(s). For example, the term “acceptor” may refer to a human antibody amino acid or nucleic acid sequence that provides or encodes at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequences of one or more of the framework regions. Such an acceptor may contain at least 1, at least 2, at least 3, least 4, at least 5, or at least 10 amino acid residues that does (do) not occur at one or more specific positions of a human antibody. An acceptor framework region and/or acceptor constant region(s) may be, e.g., derived or obtained from a germline antibody gene, a mature antibody gene, a functional antibody (e.g., antibodies well-known in the art, antibodies in development, or antibodies commercially available).
The term “agonist” refers to a modulator that, when contacted with a molecule of interest, causes an increase in the magnitude of a certain activity or function of the molecule compared to the magnitude of the activity or function observed in the absence of the agonist. Particular agonists of interest may include, but are not limited to, NGF polypeptides or polypeptides, nucleic acids, carbohydrates, or any other molecules that bind to NGF.
The term “antagonist” or “inhibitor” refer to a modulator that, when contacted with a molecule of interest causes a decrease in the magnitude of a certain activity or function of the molecule compared to the magnitude of the activity or function observed in the absence of the antagonist. Particular antagonists of interest include those that block or modulate the biological or immunological activity of NGF. Antagonists and inhibitors of NGF may include, but are not limited to, proteins, nucleic acids, carbohydrates, or any other molecules, which bind to NGF.
The term “antibody” broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art. Nonlimiting examples are discussed herein below.
In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgA1 and IgA2) or subclass.
The term “antibody conjugate” refers to a binding protein, such as an antibody, chemically linked to a second chemical moiety, such as a therapeutic or cytotoxic agent. The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials. In one aspect the therapeutic or cytotoxic agents include, but are not limited to, pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
The term “antibody construct” refers to a polypeptide comprising one or more the antigen binding portions linked to a linker polypeptide or an immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Such linker polypeptides are well known in the art (Holliger, et al., Proc. Natl. Acad. Sci., 90: 6444-6448 (1993); Poljak, et al., Structure 2: 1121-1123 (1994)). An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences are known in the art; canine, equine, and feline are rarer.
The term “antibody fragments” or “antigen-binding moiety” comprises a portion of a full length antibody, generally the antigen binding or variable domain thereof. Examples of antibody fragments include Fab, Fab′, F(ab)2, Fv, sFv fragments, diabodies, linear antibodies, single-chain antibody molecules.
The term “antigen-binding portion” of an antibody (or simply “antibody portion”) refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., NGF). It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. These may also be bispecific, dual specific, or multi-specific formats; specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature, 341: 544-546 (1989); PCT publication WO 90/05144), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv) (Bird et al., Science, 242: 423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci., 85: 5879-5883 (1988)). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (Holliger, et al., Proc. Natl. Acad. Sci., 90: 6444-6448 (1993); Poljak, et al., Structure 2: 1121-1123 (1994)). Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).
Still further, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al., Human Antibodies and Hybridomas, 6: 93-101 (1995)) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, et al., Mol. Immunol., 31: 1047-1058 (1994)). Antibody portions, such as Fab and F(ab′)2 fragments, may be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules may be obtained using standard recombinant DNA techniques, as described herein.
The term “anti-NGF antibody” refers to an antibody which is able to bind to nerve growth factor (NGF) and inhibit NGF biological activity and/or downstream pathway(s) mediated by NGF signaling. An anti-NGF antibody encompasses antibodies that block, antagonize, suppress or reduce (including significantly) NGF biological activity, including downstream pathways mediated by NGF signaling, such as receptor binding and/or elicitation of a cellular response to NGF. Anti-NGF antibodies encompass those that neutralize NGF biological activity, bind NGF and prevent NGF dimerization and/or binding to an NGF receptor (such as p75 and/or trkA), and/or bind NGF and prevent trkA receptor dimerization and/or trkA autophosphorylation. Examples of anti-NGF antibodies are provided herein.
The term “binding protein” refers to a natural or synthetic polypeptide that specifically binds to any portion of a target such as an antigen. The term “binding protein” encompasses antibodies as described herein, including an isolated antibody, antigen-binding portion thereof, or immunologically functional fragment thereof
The term “canine antibody” refers to a naturally-occurring or recombinantly produced immunoglobulin composed of amino acid sequences representative of natural antibodies isolated from canines of various breeds. Canine antibodies are antibodies having variable and constant regions derived from canine germline immunoglobulin sequences. The canine antibodies of the disclosure may include amino acid residues not encoded by canine germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “canine antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto canine framework sequences.
The term “caninization” is defined as a method for transferring non-canine antigen-binding amino acids from a donor antibody to a canine antibody acceptor framework to generate protein therapeutic treatments useful in dogs.
The term “caninized antibody” refers to antibodies which comprise heavy and light chain variable region sequences from a non-canine species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “canine-like”, i.e., more similar to canine germline variable sequences. One type of caninized antibody is a CDR-grafted antibody, in which non-canine CDR sequences are introduced into canine VH and VL sequences to replace the corresponding canine CDR sequences.
Caninized forms of non-canine antibodies provided herein are canine antibodies that contain sequence derived from a non-canine antibody. For the most part, caninized antibodies are canine antibody sequences (“acceptor” or “recipient” antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-canine species (“donor” antibody) such as mouse, rat, rabbit, cat, goat, chicken, bovine, horse, llama, camel, dromedaries, sharks, non-human primates, human, humanized, recombinant sequence, or an engineered sequence having the desired properties. In some instances, framework region (FR) residues of the canine antibody are replaced by corresponding non-canine FR residues. Furthermore, caninized antibodies may include residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. The caninized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc) of a canine antibody. Strategies for caninization of antibodies include, but are not limited to, the strategies disclosed in WO 2003/060080.
The caninized antibody is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a canine antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-canine antibody. A caninized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab′, F(ab′)2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-canine immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a canine immunoglobulin consensus sequence. A caninized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a canine immunoglobulin. A canine or caninized antibody may contain both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. A caninized antibody may only contain a caninized light chain, or may only contain a caninized heavy chain. An exemplary caninized antibody contains a caninized variable domain of a light chain and a caninized variable domain of a heavy chain.
The term “canonical” residue refers to a residue in a CDR or framework that defines a particular canonical CDR structure as defined by Chothia et al. (J. Mol. Biol., 196:901-907 (1987); Chothia et al., J. Mol. Biol., 227:799 (1992). According to Chothia et al., critical portions of the CDRs of many antibodies have nearly identical peptide backbone conformations despite great diversity at the level of amino acid sequence. Each canonical structure specifies primarily a set of peptide backbone torsion angles for a contiguous segment of amino acid residues forming a loop.
The term “CDR” refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. The term “CDR set” refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as L1, L2 and L3 or H1, H2 and H3 where the “L” and the “H” designates the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Still other CDR boundary definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although certain methods described herein use Kabat or Chothia defined CDRs.
The term “CDR-grafted antibody” refers to antibodies which comprise heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of another species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) has been replaced with human CDR sequences.
The term “chimeric antibody” refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human, canine, equine, or feline constant regions. Chimeric antibodies comprise a portion of the heavy and/or light chain that is identical to or homologous with corresponding sequences from antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous with corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, exhibiting the desired biological activity (See e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
The terms “crystal” and “crystallized” refer to an antibody, or antigen binding portion thereof, that exists in the form of a crystal. Crystals are one form of the solid state of matter, which is distinct from other forms such as the amorphous solid state or the liquid crystalline state. Crystals are composed of regular, repeating, three-dimensional arrays of atoms, ions, molecules (e.g., proteins such as antibodies), or molecular assemblies (e.g., antigen/antibody complexes). These three-dimensional arrays are arranged according to specific mathematical relationships that are well-understood in the field. The fundamental unit, or building block, that is repeated in a crystal is called the asymmetric unit. Repetition of the asymmetric unit in an arrangement that conforms to a given, well-defined crystallographic symmetry provides the “unit cell” of the crystal. Repetition of the unit cell by regular translations in all three dimensions provides the crystal. See Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ea., pp. 20 1-16, Oxford University Press, New York, N.Y., (1999).
The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
The terms “donor” and “donor antibody” refer to an antibody providing one or more CDRs. A donor antibody may be an antibody from a species different from the antibody from which the framework regions are obtained or derived. In the context of a humanized antibody, the term “donor antibody” refers to a non-human antibody providing one or more CDRs. In the context of a caninized antibody, the term “donor antibody” refers to a non-canine antibody providing one or more CDRs. In the context of a felinized antibody, the term “donor antibody” refers to a non-feline antibody providing one or more CDRs. In the context of a equinized antibody, the term “donor antibody” refers to a non-equine antibody providing one or more CDRs.
The term “epitope” includes any polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitope determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and may have specific three dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. An antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
The term “equine antibody” refers to a naturally-occurring or recombinantly produced immunoglobulin composed of amino acid sequences representative of natural antibodies isolated from equines of various breeds. Equine antibodies are antibodies having variable and constant regions derived from equine germline immunoglobulin sequences. The equine antibodies of the disclosure may include amino acid residues not encoded by equine germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “equine antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto equine framework sequences.
The term “equinization” is defined as a method for transferring non-equine antigen-binding amino acids from a donor antibody to an equine antibody acceptor framework to generate protein therapeutic treatments useful in horses.
The term “equinized antibody” refers to antibodies which comprise heavy and light chain variable region sequences from a non-equine species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “equine-like”, i.e., more similar to equine germline variable sequences. One type of equinized antibody is a CDR-grafted antibody, in which non-equine CDR sequences are introduced into equine VH and VL sequences to replace the corresponding equine CDR sequences.
Equinized forms of non-equine antibodies provided herein are equine antibodies that contain sequence derived from a non-equine antibody. For the most part, equinized antibodies are equine antibody sequences (“acceptor” or “recipient” antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-equine species (“donor” antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken, bovine, horse, llama, camel, dromedaries, sharks, non-human primates, human, humanized, recombinant sequence, or an engineered sequence having the desired properties. In some instances, framework region (FR) residues of the equine antibody are replaced by corresponding non-equine FR residues. Furthermore, equinized antibodies may include residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. The equinized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc) of an equine antibody.
The equinized antibody is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a equine antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-equine antibody. An equinized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab′, F(ab′)2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-equine immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of an equine immunoglobulin consensus sequence. An equinized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of an equine immunoglobulin. An equine or equinized antibody for example may contain both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. An equinized antibody may only contain an equinized light chain, or an equinized heavy chain. An exemplary equinized antibody contains an equinized variable domain of a light chain an equinized variable domain of a heavy chain. Equine isotypes include, for example, IgGa, IgGb, IgGc, IgG(T), IgM, IgA
The term “Fab” refers to antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to readily crystallize. Pepsin treatment yields a binding cross-linking antigen. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteine(s) from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
The term “feline antibody” refers to a naturally-occurring or recombinantly produced immunoglobulin composed of amino acid sequences representative of natural antibodies isolated from felines of various breeds. Feline antibodies are antibodies having variable and constant regions derived from feline germline immunoglobulin sequences. The feline antibodies of the disclosure may include amino acid residues not encoded by feline germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “feline antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto feline framework sequences.
The term “felinization” is defined as a method for transferring non-feline antigen-binding amino acids from a donor antibody to a feline antibody acceptor framework to generate protein therapeutic treatments useful in cats.
The term “felinized antibody” refers to antibodies which comprise heavy and light chain variable region sequences from a non-feline species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “feline-like”, i.e., more similar to feline germline variable sequences. One type of felinized antibody is a CDR-grafted antibody, in which non-feline CDR sequences are introduced into feline VH and VL sequences to replace the corresponding feline CDR sequences.
Felinized forms of non-feline antibodies provided herein are feline antibodies that contain sequence derived from a non-feline antibody. For the most part, felinized antibodies are feline antibody sequences (“acceptor” or “recipient” antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-feline species (“donor” antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken, bovine, horse, llama, camel, dromedaries, sharks, non-human primates, human, humanized, recombinant sequence, or an engineered sequence having the desired properties. In some instances, framework region (FR) residues of the feline antibody are replaced by corresponding non-feline FR residues. Furthermore, felinized antibodies may include residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. The felinized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc) of a feline antibody.
The felinized antibody is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a feline antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-feline antibody. A felinized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab′, F(ab′)2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-feline immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a feline immunoglobulin consensus sequence. A felinized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a feline immunoglobulin. A feline or felinized antibody may contain both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. A felinized antibody may only contain a felinized light chain or a felinized heavy chain. An exemplary felinized antibody only contains a felinized variable domain of a light chain and a felinized variable domain of a heavy chain.
The term “framework” or “framework sequence” refers to the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence may be determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, -L2, and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy chain) also divide the framework regions on the light chain and the heavy chain into four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying the particular sub-regions as FR1, FR2, FR3 or FR4, a framework region, as referred by others, represents the combined FR's within the variable region of a single, naturally occurring immunoglobulin chain. An FR represents one of the four sub-regions, and FRs represents two or more of the four sub-regions constituting a framework region. Human heavy chain and light chain acceptor sequences are known in the art. Canine heavy chain and light chain acceptor sequences are also known (patent application publication WO03/060080 and U.S. Pat. No. 7,261,890B2).
The term “germline antibody gene” or “gene fragment” refers to an immunoglobulin sequence encoded by non-lymphoid cells that have not undergone the maturation process that leads to genetic rearrangement and mutation for expression of a particular immunoglobulin (Shapiro et al., Crit. Rev. Immunol. 22(3): 183-200 (2002); Marchalonis et al., Adv Exp Med. Biol. 484:13-30 (2001)). One of the advantages provided by the binding proteins of the present disclosure stems from the recognition that germline antibody genes are more likely than mature antibody genes to conserve essential amino acid sequence structures characteristic of individuals in the species, hence less likely to be recognized as from a foreign source when used therapeutically in that species.
The term “Fv” refers to the minimum antibody fragment that contains a complete antigen-recognition and binding site. This region consists of a dimer of one heavy chain and one light chain variable domain.
The term “human antibody” refers to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “human antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
The term “humanized antibody” refers to antibodies which comprise heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “human-like”, i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which non-human CDR sequences are introduced into human VH and VL sequences to replace the corresponding human CDR sequences.
The humanized antibody is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody. A humanized antibody comprises substantially all, or at least one, and typically two, variable domains (Fab, Fab′, F(ab′)2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. A humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. A humanized or caninized antibody may contain both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Alternatively, a humanized antibody may only contain a humanized light chain, or a humanized heavy chain. An exemplary humanized antibody contains a humanized variable domain of a light chain and a humanized variable domain of a heavy chain.
The bovanized, camelized, caninized, equinized, felinized, or humanized antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including without limitation IgG 1, IgG2, IgG3 and IgG4. The bovanized, camelized, caninized, equinized, felinized, or humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions using techniques well-known in the art.
The framework and CDR regions of a bovanized, camelized, caninized, equinized, felinized, or humanized antibody need not correspond precisely to the parental sequences, e.g., the donor antibody CDR or the consensus framework may be mutagenized by substitution, insertion and/or deletion of at least one amino acid residue so that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. Such mutations, however, will not be extensive. Usually, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and at least 95% of the bovanized, camelized, caninized, equinized, felinized, or humanized antibody residues will correspond to those of the parental FR and CDR sequences. The term “consensus framework” refers to the framework region in the consensus immunoglobulin sequence. The term “consensus immunoglobulin sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either may be included in the consensus sequence.
The term “hypervariable region” refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a “complementarity determining region” or “CDR” in the light chain variable domain and in the heavy chain variable domain as defined by Kabat et al., 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and/or as defined by (Chothia and Lesk, Mol. Biol. 196:901-917 (1987) and/or as defined as “AbM loops” by Martin, et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989) and/or as defined by Lefranc et al., Nucleic Acids Res, 27:209-212 (1999) in the international ImMunoGeneTics information systems database. “Framework” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.
The term “identity” refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing their sequences thereof, wherein “identity” refers more specifically to the degree of sequence relatedness between nucleic acid molecules or polypeptides, as determined by the match between strings of two or more nucleotide or two or more amino acid sequences. “Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., “algorithms”) The term “similarity” is used to refer to a related concept with respect to the relationship of two or more nucleic acid molecules or two or more polypeptide molecules. In contrast to “identity,” “similarity” refers to a measure of relatedness, which includes both identical matches and conservative substitution matches. For example, for two polypeptide sequences that have 50/100 identical amino acids, and the remainder are all non-conservative substitutions, then the percent identity and similarity would both be 50%. With respect to the same two sequences, if 25 more positions had conservative substitutions, then the percent identity remains 50%, while percent similarity would be 75% (75/100). Identity and similarity of related nucleic acids and polypeptides may be readily calculated by methods well known and readily available in the art, including but are not limited to, those described in COMPUTATIONAL MOLECULAR BIOLOGY, (Lesk, A. M., ed.), 1988, Oxford University Press, New York; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, (Smith, D. W., ed.), 1993, Academic Press, New York; COMPUTER ANALYSIS OF SEQUENCE DATA, Part 1, (Griffin, A. M., and Griffin, H. G., eds.), 1994, Humana Press, New Jersey; von Heinje, G., SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, 1987, Academic Press; SEQUENCE ANALYSIS PRIMER, (Gribskov, M. and Devereux, J., eds.), 1991, M. Stockton Press, New York; Carillo et al., 1988, SIAM J. Applied Math., 48:1073; and Durbin et al., 1998, BIOLOGICAL SEQUENCE ANALYSIS, Cambridge University Press.
Preferred methods to determine identity are designed to provide the highest match between the compared sequences, and are well described in readily publicly available computer programs. Preferred such computerized methods for determining identity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux et al., 1984, Nucl. Acid. Res., 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., 1990, J. Mol. Biol., 215:403-410). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., 1990, supra). The well-known Smith Waterman algorithm may also be used to determine identity.
The terms “individual,” “patient,” and “subject” are used interchangeably herein, to refer to mammals, including, but not limited to, humans, murines, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian farm and agricultural animals, mammalian sport animals, and mammalian pets. Exemplary subjects companion animals, such as a dog, cat or horse.
An “isolated antibody” refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds NGF is substantially free of antibodies that specifically bind antigens other than NGF). An isolated antibody that specifically binds NGF may, however, have cross-reactivity to other antigens, such as NGF molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.
The terms “isolated polynucleotide” and “isolated nucleic acid” as used interchangeably herein refer to a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a another polynucleotide to which it is not linked in nature, or is not found in nature within a larger sequence.
The term “isolated protein” or “isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation is not associated with naturally associated components that accompany it in its native state; is substantially free of other proteins from the same species; is expressed by a cell from a different species; or does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
The term “Kd” refers to the dissociation constant of a particular antibody-antigen interaction as is known in the art.
The term “Kon” is refers to the on rate constant for association of an antibody to the antigen to form the antibody/antigen complex as is known in the art.
The term “Koff” refers to the off rate constant for dissociation of an antibody from the antibody/antigen complex as is known in the art.
The terms “Kabat numbering”, “Kabat definitions and “Kabat labeling” are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e. hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al., Ann. NY Acad, Sci., 190:382-391 (1971); and Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991)). For the heavy chain variable region, the hypervariable region ranges from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3. For the light chain variable region, the hypervariable region ranges from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.
The term “key residue” refers to certain residues within the variable region that have more impact on the binding specificity and/or affinity of an antibody, in particular a mammalized antibody such as humanized, caninized, equinized or felinized antibody. A key residue includes, but is not limited to, one or more of the following: a residue that is adjacent to a CDR, a potential glycosylation site (may be either N- or O-glycosylation site), a rare residue, a residue capable of interacting with the antigen, a residue capable of interacting with a CDR, a canonical residue, a contact residue between heavy chain variable region and light chain variable region, a residue within the Vernier zone, and a residue in the region that overlaps between the Chothia definition of a variable heavy chain CDR1 and the Kabat definition of the first heavy chain framework.
The term “labeled binding protein” refers to a protein with a label incorporated that provides for the identification of the binding protein. In one aspect, the label is a detectable marker, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that may be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that may be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, 153Sm); fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents, such as gadolinium chelates.
The term “mammalization” refers to a method for transferring donor antigen-binding information to a mammalian antibody acceptor to generate useful therapeutic treatments. More specifically, the invention provides methods for felinization, equinization and caninization of antibodies.
The term “mammalized antibody” refers to antibodies which comprise heavy and light chain variable region sequences from a mammal species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more like “mammal of interest,” see for example, humanized, caninized, equinized or felinized antibodies defined herein. Such mammalized antibodies include, but are not limited to, bovanized, camelized, caninized, equinized, felinized, or humanized antibodies.
The terms “modulate” and “regulate” are used interchangeably and refer to a change or an alteration in the activity of a molecule of interest (e.g., the biological activity of NGF). Modulation may be an increase or a decrease in the magnitude of a certain activity or function of the molecule of interest. Exemplary activities and functions of a molecule include, but are not limited to, binding characteristics, enzymatic activity, cell receptor activation, and signal transduction.
The term “modulator” is a compound capable of changing or altering an activity or function of a molecule of interest (e.g., the biological activity of NGF). For example, a modulator may cause an increase or decrease in the magnitude of a certain activity or function of a molecule compared to the magnitude of the activity or function observed in the absence of the modulator. A modulator may be an inhibitor, which decreases the magnitude of at least one activity or function of a molecule. Exemplary inhibitors include, but are not limited to, proteins, peptides, antibodies, peptibodies, carbohydrates or small organic molecules. Peptibodies are described, e.g., in WO01/83525.
The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone; and not the method by which it is produced and is not limited to antibodies produced through hybridoma technology.
The term “multivalent binding protein” is used in this specification to denote a binding protein comprising two or more antigen binding sites. The multivalent binding protein is engineered to have the three or more antigen binding sites, and is generally not a naturally occurring antibody. The term “multispecific binding protein” refers to a binding protein capable of binding two or more related or unrelated targets. Dual variable domain (DVD) binding proteins are binding proteins that comprise two or more antigen binding sites and are tetravalent or multivalent binding proteins. Such DVDs may be monospecific, i.e capable of binding one antigen or multispecific, i.e. capable of binding two or more antigens. DVD binding proteins comprising two heavy chain DVD polypeptides and two light chain DVD polypeptides are referred to a DVD Ig. Each half of a DVD Ig comprises a heavy chain DVD polypeptide, and a light chain DVD polypeptide, and two antigen binding sites. Each binding site comprises a heavy chain variable domain and a light chain variable domain with a total of 6 CDRs involved in antigen binding per antigen binding site. DVD binding proteins and methods of making DVD binding proteins are disclosed in U.S. patent application Ser. No. 11/507,050 and incorporated herein by reference.
One aspect of the disclosure pertains to a DVD binding protein comprising binding proteins capable of binding NGF. In another aspect, the DVD binding protein is capable of binding NGF and a second target.
The terms “nerve growth factor” and “NGF” refer to nerve growth factor and variants thereof that retain at least part of the biological activity of NGF. NGF includes all mammalian species of native sequence NGF, including murine, rat, human, rabbit, canine, feline, equine, or bovine.
TABLE 1
Sequence of NGF
Protein
Sequence Identifier
Canine NGF C-terminal 6-His
SEQ ID NO: 50
Human NGF
SEQ ID NO: 85
The term “NGF receptor” refers to a polypeptide that is bound by or activated by NGF. NGF receptors include the TrkA receptor and the p75 receptor of any mammalian species, including, but are not limited to, human, canine, feline, equine, primate, or bovine.
The terms “NGF-related disease” and “NGF-related disorder” encompass any disease or disorder in which the activity of NGF in a subject suffering from the disease or disorder has been shown to be or is suspected of being either responsible for the pathophysiology of the disease or disorder, or a factor that contributes to a worsening of the disease or disorder, which may occur as a result of increased levels of NGF or increased sensitivity of the subject to NGF. Accordingly, an NGF-related disease or NGF-related disorder is a disease or disorder in which reduction of NGF activity is expected to alleviate the symptoms and/or progression of the disease or disorder. Such diseases and disorders may be evidenced, for example, by an increase in the concentration of NGF in a biological fluid of a subject suffering from the disorder (e.g., an increase in the concentration of NGF in serum, plasma, synovial fluid, etc. of the subject), which may be detected, for example, using an anti-NGF antibody as described above. Non-limiting examples of diseases and disorders that may be treated with the antibodies of the disclosure include those diseases and disorders discussed in the section below pertaining to pharmaceutical compositions of the antibodies of the disclosure, and encompass acute pain resulting for example from surgery or other trauma, and chronic pain.
The term “neutralizing” refers to neutralization of biological activity of a NGF when a binding protein specifically binds NGF. A neutralizing binding protein is a neutralizing antibody, whose binding to NGF results in inhibition of a biological activity of NGF. The neutralizing binding protein binds NGF and reduces a biologically activity of NGF by at least about 20%, 40%, 60%, 80%, 85% or more Inhibition of a biological activity of NGF by a neutralizing binding protein may be assessed by measuring one or more indicators of NGF biological activity well known in the art, including cell proliferation, cell morphology changes, cell signaling, or any detectable cellular response resulting from binding of NGF to the TrkA receptor.
The term “operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. “Operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. The term “expression control sequence” refers to polynucleotide sequences which are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term “control sequences” is intended to include components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
The term “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody or antibody portion.
The term “polynucleotide” means a polymeric form of two or more nucleotides, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA. The term “isolated polynucleotide” shall mean a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or some combination thereof) that, by virtue of its origin, the “isolated polynucleotide” is not associated with all or a portion of a polynucleotide with which the “isolated polynucleotide” is found in nature; is operably linked to a polynucleotide that it is not linked to in nature; or does not occur in nature as part of a larger sequence.
The term “polypeptide” refers to any polymeric chain of amino acids. The terms “peptide” and “protein” are used interchangeably with the term polypeptide and also refer to a polymeric chain of amino acids. The term “polypeptide” encompasses native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. A polypeptide may be monomeric or polymeric.
The term “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.
The term “recombinant host cell” (or simply “host cell”) is intended to refer to a cell into which exogenous DNA has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but, to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell”. In one aspect, host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life. Eukaryotic cells include protist, fungal, plant and animal cells. In another aspect host cells include, but are not limited to, the prokaryotic cell line E. Coli; mammalian cell lines CHO, HEK 293 and COS; the insect cell line Sf9; and the fungal cell Saccharomyces cerevisiae.
Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.
The term “recombinant antibody” refers to all species of antibodies or immunoglobulins that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library (Hoogenboom, TIB Tech., 15: 62-70 (1997); Azzazy et al., Clin. Biochem., 35: 425-445 (2002); Gavilondo et al., BioTechniques, 29: 128-145 (2002); Hoogenboom et al., Immunology Today, 21: 371-378 (2000)), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann et al., Current Opinion in Biotechnology, 13: 593-597 (2002); Little et al., Immunology Today, 21: 364-370 (2000)) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies have variable and constant regions derived from species-specific germline immunoglobulin sequences. Such recombinant antibodies may be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to species-specific germline VH and VL sequences, may not naturally exist within the antibody germline repertoire in vivo.
The term “recovering” refers to the process of rendering a chemical species such as a polypeptide substantially free of naturally associated components by isolation, e.g., using protein purification techniques well known in the art.
The term “sample” is used in its broadest sense. A “biological sample” includes, but is not limited to, any quantity of a substance from a living thing or formerly living thing. Such living things include, but are not limited to, humans, mice, rats, monkeys, dogs, rabbits and other animals. Such substances include, but are not limited to, blood, serum, urine, synovial fluid, cells, organs, tissues, bone marrow, lymph nodes and spleen.
The term “single-chainFv” or “scFv” refers to antibody fragments comprising the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).
The terms “specific binding” or “specifically binding” in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.
The term “substantially” in the context of a CDR refers to a CDR having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR.
The term “surface plasmon resonance” refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For further descriptions (Jönsson, et al. Ann. Biol. Clin. 51: 19-26 (1993); Jönsson, et al., Biotechniques 11: 620-627 (1991); Johnsson, et al., J. Mol. Recognit. 8:125-131 (1995); and Johnnson, B., et al., Anal. Biochem., 198: 268-277 (1991)).
The term “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount may be the amount and/or duration of a therapy which is sufficient to reduce or ameliorate the severity and/or duration of a disorder or one or more symptoms thereof, prevent the advancement of a disorder, cause regression of a disorder, prevent the recurrence, development, onset or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy (e.g., prophylactic or therapeutic agent). A therapeutically effective amount of the antibody or antibody portion may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody, or antibody portion, are outweighed by the therapeutically beneficial effects.
The term “transformation” refers to any process by which exogenous DNA enters a host cell. Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, lipofection, and particle bombardment. Such “transformed” cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. They also include cells which transiently express the inserted DNA or RNA for limited periods of time.
The term “transgenic organism” refers to an organism having cells that contain a transgene, wherein the transgene introduced into the organism (or an ancestor of the organism) expresses a polypeptide not naturally expressed in the organism. A “transgene” is a DNA construct, which is stably and operably integrated into the genome of a cell from which a transgenic organism develops, directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic organism.
The term “vector” is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The term “Vernier zone” refers to a subset of framework residues that may adjust CDR structure and fine-tune the fit to antigen as described by Foote and Winter (1992, J. Mol. Biol. 224:487-499, which is incorporated herein by reference). Vernier zone residues form a layer underlying the CDRs and may impact on the structure of CDRs and the affinity of the antibody.
B. ANTI NGF BINDING PROTEINS
The present disclosure provides a novel family of binding proteins, murine antibodies, CDR grafted antibodies, mammalized (bovanized, camelized, caninized, equinized, felinized, or humanized) antibodies, and fragments thereof, capable of binding and modulating the biological activity or function of NGF, including the capability of neutralizing NGF. The disclosure thus also provides a therapeutic means with which to inhibit NGF and provides compositions and methods for treating disease associated with increased levels of NGF, particularly a disease, condition or disorder where increased levels of NGF, as compared to NGF levels observed in comparable normal subjects, is detrimental.
Binding proteins of the present disclosure may be made by any of a number of techniques known in the art and as described herein, including culturing a host cell described herein in culture medium under conditions sufficient to produce a binding protein capable of binding NGF.
Monoclonal antibodies may be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies may be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981).
Methods for producing and screening for specific antibodies using hybridoma technology are well known in the art. Such methods include, for example, culturing a hybridoma cell secreting an antibody of the disclosure wherein the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the disclosure with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the disclosure. Briefly, or example, mice may be immunized with an NGF antigen. The NGF antigen may be administered, with or without an adjuvant, to stimulate the immune response. Such adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulating complexes). Such adjuvants may protect the polypeptide from rapid dispersal by sequestering it in a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. If a polypeptide is being administered, the immunization schedule will involve two or more administrations of the polypeptide, spread out over several weeks.
After immunization of an animal with an NGF antigen, antibodies and/or antibody-producing cells may be obtained from the animal. An anti-NGF antibody-containing serum may be obtained from the animal by bleeding or sacrificing the animal. The serum may be used as it is obtained from the animal, an immunoglobulin fraction may be obtained from the serum, or the anti-NGF antibodies may be purified from the serum. Serum or immunoglobulins obtained in this manner are polyclonal, thus having a heterogeneous array of properties.
Once an immune response is detected, e.g., antibodies specific for the antigen NGF are detected in the mouse serum, the mouse spleen may be harvested and splenocytes isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, such as, for example, cells from cell line SP20 available from the ATCC. Hybridomas may be selected and cloned by limited dilution. The hybridoma clones may then be assayed by methods known in the art for cells that secrete antibodies capable of binding NGF. Ascites fluid, which generally contains high levels of antibodies, may be generated by immunizing mice with positive hybridoma clones.
Antibody-producing immortalized hybridomas may be prepared from the immunized animal. After immunization, the animal may be sacrificed and the splenic B cells fused to immortalized myeloma cells as is well known in the art (Harlow et al., supra). Alternatively, the myeloma cells may be from a non-secretory cell line and do not secrete immunoglobulin polypeptides. After fusion and antibiotic selection, the hybridomas may be screened using NGF, or a portion thereof, or a cell expressing NGF. Initial screening may be performed, for example, using an enzyme-linked immunoassay (ELISA) or a radioimmunoassay (RIA). An example of ELISA screening is provided in WO 00/37504.
Anti-NGF antibody-producing hybridomas may be selected, cloned and further screened for desirable characteristics, including robust hybridoma growth, high antibody production and desirable antibody characteristics, as discussed further below. Hybridomas may be cultured and expanded in vivo in syngeneic animals, in animals that lack an immune system, e.g., nude mice, or in cell culture in vitro. Methods of selecting, cloning and expanding hybridomas are well known to those of ordinary skill in the art.
An exemplary animal system for preparing hybridomas is the mouse. Hybridoma production in the mouse is very well established, and immunization protocols and techniques for isolation of immunized splenocytes for fusion are well known. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known. Alternatively, the hybridomas may be produced in a non-human, non-mouse species such as a rat, sheep, pig, goat, cattle or horse. Alternatively, human hybridomas may be produced, in which a human non-secretory myeloma is fused with a human cell expressing an anti-NGF antibody.
Antibody fragments that recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab′)2 fragments of the disclosure may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.
Recombinant antibodies may be generated from single, isolated lymphocytes using a procedure referred to in the art as the selected lymphocyte antibody method (SLAM), as described in U.S. Pat. No. 5,627,052, PCT Publication WO 92/02551 and Babcock et al., Proc. Natl. Acad. Sci., 93: 7843-7848 (1996). In this method, single cells secreting antibodies of interest, e.g., lymphocytes derived from any one of the immunized animals described in Section 1, are screened using an antigen-specific hemolytic plaque assay, wherein the antigen NGF, or a fragment thereof, is coupled to sheep red blood cells using a linker, such as biotin, and used to identify single cells that secrete antibodies with specificity for NGF. Following identification of antibody-secreting cells of interest, heavy- and light-chain variable region cDNAs may be rescued from the cells by reverse transcriptase-PCR and these variable regions may then be expressed, in the context of appropriate immunoglobulin constant regions (e.g., human constant regions), in mammalian host cells, such as COS or CHO cells. The host cells transfected with the amplified immunoglobulin sequences, derived from in vivo selected lymphocytes, may then undergo further analysis and selection in vitro, for example by panning the transfected cells to isolate cells expressing antibodies to NGF. The amplified immunoglobulin sequences further may be manipulated in vitro, such as by in vitro affinity maturation methods such as those described in PCT Publication WO 97/29131 and PCT Publication WO 00/56772.
Antibodies may be produced by immunizing a non-human animal comprising some or all of the human immunoglobulin loci with an NGF antigen. For example, human monoclonal antibodies directed against NGF may be generated using transgenic mice carrying parts of the human immune system rather than the mouse system, referred to in the literature and herein as “HuMab” mice, contain a human immunoglobulin gene minilocus that encodes unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and κ chain loci (Lonberg et al., 1994, Nature 368:856-859). These mice exhibit reduced expression of mouse IgM or κ and in response to immunization, and the introduced human heavy chain and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgG κ monoclonal antibodies. The preparation of HuMab mice is well described in the literature. (See, e.g., Lonberg et al., 1994, Nature 368:856-859; Lonberg, 1994, Handbook of Exp. Pharmacology 113:49-101; Taylor et al., 1994, International Immunology 6:579-591; Lonberg & Huszar, 1995, Intern. Rev. Immunol. 13:65-93; and Harding & Lonberg, 1995, Ann. N.Y. Acad. Sci. 764:536-546). Alternatively, other known mouse strains such as the HCo7, HCo12, and KM transgenic mice strains may be used to generate human anti-NGF antibodies. Another suitable, though non-limiting example of a transgenic mouse is the XENOMOUSE® transgenic mouse, which is an engineered mouse strain that comprises large fragments of the human immunoglobulin loci and is deficient in mouse antibody production. See, e.g., Green et al. Nature Genetics, 7:13-21 (1994); and U.S. Pat. Nos. 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598 and 6,130,364; WO 91/10741, WO 94/02602, WO 96/34096, WO 96/33735, WO 98/16654, WO 98/24893, WO 98/50433, WO 99/45031, WO 99/53049, WO 00 09560, and WO 00/037504. The XENOMOUSE® transgenic mouse produces an adult-like human repertoire of fully human antibodies, and generates antigen-specific human Mabs. The XENOMOUSE® transgenic mouse contains approximately 80% of the human antibody repertoire through introduction of megabase sized, germline configuration YAC fragments of the human heavy chain loci and light chain loci (Mendez et al., Nature Genetics 15:146-156 (1997), Green et al., J. Exp. Med., 188: 483-495 (1998)).
In vitro methods also may be used to make the antibodies of the disclosure, wherein an antibody library is screened to identify an antibody having the desired binding specificity. Methods for such screening of recombinant antibody libraries are well known in the art and include methods described in, for example, U.S. Pat. No. 5,223,409; PCT Publication Nos. WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; Fuchs et al. Bio/Technology, 9:1370-1372 (1991); Hay et al., Hum Antibod Hybridomas, 3: 81-85 (1992); Huse et al., Science, 246: 1275-1281 (1989); McCafferty et al., Nature, 348: 552-554 (1990); Griffiths et al., EMBO J., 12: 725-734 (1993); Hawkins et al., J Mol Biol., 226: 889-896 (1992); Clackson et al., Nature, 352: 624-628 (1991); Gram et al., PNAS, 89: 3576-3580 (1992); Garrad et al., Bio/Technology, 9:1373-1377 (1991); Hoogenboom et al., Nuc Acid Res, 19: 4133-4137 (1991); and Barbas et al., PNAS, 88: 7978-7982 (1991), US patent application publication 20030186374, and PCT Publication No. WO 97/29131.
The recombinant antibody library may be from a subject immunized with NGF, or a portion of NGF. Alternatively, the recombinant antibody library may be from a naïve subject that has not been immunized with NGF, such as a canine antibody library from a canine subject that has not been immunized with canine NGF. Antibodies of the disclosure are selected by screening the recombinant antibody library with the peptide comprising canine NGF to thereby select those antibodies that recognize NGF. Methods for conducting such screening and selection are well known in the art, such as described in the references in the preceding paragraph. To select antibodies of the disclosure having particular binding affinities for hNGF, such as those that dissociate from canine NGF with a particular koff rate constant, the art-known method of surface plasmon resonance may be used to select antibodies having the desired koff rate constant. To select antibodies of the disclosure having a particular neutralizing activity for hNGF, such as those with a particular an IC50, standard methods known in the art for assessing the inhibition of hNGF activity may be used.
For example, the antibodies of the present disclosure may also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular aspect, such phage may be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., canine, human or murine). Phage expressing an antigen binding domain that binds the antigen of interest may be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that may be used to make the antibodies of the present disclosure include those disclosed in Brinkman et al., J. Immunol. Methods, 182: 41-50 (1995); Ames et al., J. Immunol. Methods, 184: 177-186 (1995); Kettleborough et al., Eur. J. Immunol., 24:952-958 (1994); Persic et al., Gene, 187: 9-18 (1997); Burton et al., Advances in Immunology, 57: 191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780, 225; 5,658,727; 5,733,743 and 5,969,108.
As described in the above references, after phage selection, the antibody coding regions from the phage may be isolated and used to generate whole antibodies including human antibodies or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments may also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques, 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science, 240:1041-1043 (1988) (said references incorporated by reference in their entireties). Examples of techniques which may be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258, 498; Huston et al., Methods in Enzymology, 203:46-88 (1991); Shu et al., PNAS, 90:7995-7999 (1993); and Skerra et al., Science, 240:1038-1040 (1988).
Alternatives to screening of recombinant antibody libraries by phage display are known and include other methodologies for screening large combinatorial libraries which may be applied to the identification of dual specificity antibodies of the disclosure. One type of alternative expression system is one in which the recombinant antibody library is expressed as RNA-protein fusions, as described in PCT Publication No. WO 98/31700, and in Roberts et al., Proc. Natl. Acad. Sci., 94:12297-12302 (1997). In this system, a covalent fusion is created between a mRNA and the peptide or protein that it encodes by in vitro translation of synthetic mRNAs that carry puromycin, a peptidyl acceptor antibiotic, at their 3′ end. Thus, a specific mRNA may be enriched from a complex mixture of mRNAs (e.g., a combinatorial library) based on the properties of the encoded peptide or protein, e.g., antibody, or portion thereof, such as binding of the antibody, or portion thereof, to the dual specificity antigen. Nucleic acid sequences encoding antibodies, or portions thereof, recovered from screening of such libraries may be expressed by recombinant means as described above (e.g., in mammalian host cells) and, moreover, may be subjected to further affinity maturation by either additional rounds of screening of mRNA-peptide fusions in which mutations have been introduced into the originally selected sequence(s), or by other methods for affinity maturation in vitro of recombinant antibodies, as described above.
In another approach the antibodies of the present disclosure may also be generated or affinity matured using yeast display methods known in the art. In yeast display methods, genetic methods are used to tether antibody domains to the yeast cell wall and display them on the surface of yeast. In particular, such yeast may be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Examples of yeast display methods that may be used to make the antibodies of the present disclosure include those disclosed Wittrup, et al. U.S. Pat. No. 6,699,658 incorporated herein by reference.
The antibodies or antigen binding fragments described herein may also be produced by genetic engineering. For example, the technology for expression of both heavy and light chain genes in E. coli is the subject of the PCT patent applications: publication number WO 901443, WO901443, and WO 9014424 and in Huse et al., 1989 Science 246:1275-81. The present disclosure thus also encompasses the isolated nucleic acids encoding any of the binding proteins described herein, as well as a recombinant vector comprising such a nucleic acid molecule, and a host cell comprising such a recombinant vector.
A vector is a nucleic acid molecule, which may be a construct, capable of transporting another nucleic acid to which it has been linked. A vector may include any preferred or required operational elements. Preferred vectors are those for which the restriction sites have been described and which contain the operational elements needed for transcription of the nucleic acid sequence. Such operational elements include for example at least one suitable promoter, at least one operator, at least one leader sequence, at least one terminator codon, and any other DNA sequences necessary or preferred for appropriate transcription and subsequent translation of the nucleic acid sequence. Such vectors contain at least one origin of replication recognized by the host organism along with at least one selectable marker and at least one promoter sequence capable of initiating transcription of the nucleic acid sequence. A vector may be a plasmid into which additional DNA segments may be ligated. A vector may be a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as a plasmid is the most commonly used form of vector. However, the present disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. By way of example and not limitation, suitable vectors include pcDNA, pTT (Durocher et al., Nucleic Acids Research, Vol 30, No. 2 (2002)); pTT3 (pTT with additional multiple cloning site, pEFBOS (Mizushima et al., Nucleic acids Research, Vol 18, No. 17 (1990)), pBV, pJV, pBJ, or pHybE (patent publication no.: US 2009/0239259 A1).
Sequences that are operably linked are in a relationship permitting them to function in their intended manner. A control sequence operably linked to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. Operably linked sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences are polynucleotide sequences that are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, such control sequences generally include promoters and transcription termination sequence. Control sequences may include components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
A host cell may be transformed with a vector that introduces exogenous DNA into a host cell. Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, lipofection, calcium-phosphate precipitation, DEAE-dextran transfection, particle bombardment and the like. Transformed cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, and cells which transiently express the inserted DNA or RNA for limited periods of time.
Host organisms such as host cells are cultured under conditions appropriate for amplification of the vector and expression of the protein, as well known in the art. Expressed recombinant proteins may be detected by any of a number of methods also well known in the art.
Suitable host organisms include for example a prokaryotic or eukaryotic cell system. A eukaryotic cell may be a protist cell, animal cell, plant cell or fungal cell. A eukaryotic cell is for example an animal cell which may be a mammalian cell, avian cell, or an insect cell such as an insect Sf9 cell. Cells from established and readily available may be used, such as but not limited to HeLa, MRC-5 or CV-1. The host cell may be an E. coli cell, or a yeast cell such as but not limited to Saccharomyces cerevisiae. Mammalian host cells for expressing the recombinant antibodies of the disclosure also include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub et al., Proc. Natl. Acad. Sci., 77: 4216-4220 (1980), used with a DHFR selectable marker, e.g., as described in Kaufman et al., Mol. Biol., 159: 601-621 (1982)), NS0 myeloma cells, COS cells and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells, or by secretion of the antibody into the culture medium in which the host cells are grown. Antibodies may be recovered from the culture medium using standard protein purification methods.
Host cells may also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It will be understood that variations on the above procedure are within the scope of the present disclosure. For example, it may be desirable to transfect a host cell with DNA encoding functional fragments of either the light chain and/or the heavy chain of an antibody of this disclosure. Recombinant DNA technology may also be used to remove some, or all, of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigens of interest. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the disclosure. In addition, bifunctional antibodies may be produced in which one heavy and one light chain are an antibody of the disclosure and the other heavy and light chain are specific for an antigen other than the antigens of interest by crosslinking an antibody of the disclosure to a second antibody by standard chemical crosslinking methods.
In a system for recombinant expression of an antibody, or antigen-binding portion thereof, of the disclosure, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked to CMV enhancer/AdMLP promoter regulatory elements to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody from the culture medium. Still further the disclosure provides a method of synthesizing a recombinant antibody of the disclosure by culturing a host cell of the disclosure in a suitable culture medium until a recombinant antibody of the disclosure is synthesized. The method may further comprise isolating the recombinant antibody from the culture medium.
The present disclosure thus provides anti NGF binding proteins that are specific for and substantially neutralize NGF polypeptides, including active human NGF. Also provided are antibody heavy and light chain amino acid sequences which are substantially specific for and substantially neutralize NGF polypeptides when they are bound to them. This specificity enables the anti-human NGF human antibodies, and human monoclonal antibodies with like specificity, to be effective immunotherapy for NGF associated diseases.
The present disclosure encompasses anti NGF binding proteins comprising at least one of the amino acid sequences selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 206, SEQ ID NO: 207 and SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 44, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:198, SEQ ID NO: 200, SEQ ID NO: 202, and which binds an NGF polypeptide epitope with substantially high affinity as described herein and has the capacity to substantially modulate, including substantially reduce, NGF polypeptide activity.
Examples of such binding proteins include binding proteins comprising a variable heavy chain polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 206 and SEQ ID NO: 207; and a variable light chain polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 44, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200 and SEQ ID NO: 202.
Exemplary pairings of a variable heavy chain polypeptide and a variable light chain polypeptide are represented by the following pairings: SEQ ID NO: 2 and SEQ ID NO: 4; SEQ ID NO: 6 and SEQ ID NO: 8; SEQ ID NO: 10 and SEQ ID NO: 12; SEQ ID NO: 14 and SEQ ID NO: 16; SEQ ID NO: 18 and SEQ ID NO: 20; SEQ ID NO: 22 and SEQ ID NO: 24; SEQ ID NO: 25 and SEQ ID NO: 26; SEQ ID NO: 27 and SEQ ID NO: 28; SEQ ID NO: 29 and SEQ ID NO: 30; SEQ ID NO: 31 and SEQ ID NO: 32; SEQ ID NO: 177 and SEQ ID NO: 32; SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO:36; SEQ ID NO: 37 and SEQ ID NO: 38; SEQ ID NO: 39 and SEQ ID NO: 40; SEQ ID NO: 41 and SEQ ID NO: 42; SEQ ID NO: 43 and SEQ ID NO: 44; SEQ ID NO: 180 and SEQ ID NO: 181, SEQ ID NO: 182 and SEQ ID NO: 183; SEQ ID NO: 185 and SEQ ID NO: 186; SEQ ID NO: 187 and SEQ ID NO: 188; and SEQ ID NO: 192 and SEQ ID NO: 193.
Also encompassed in the disclosure are binding proteins that specifically bind NGF as described herein and comprise a heavy chain variable region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO:27, SEQ ID NO: 29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 206, SEQ ID NO: 207. Also encompassed are binding proteins that specifically bind NGF as described herein and comprise a light chain variable region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 44, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202.
Exemplary binding proteins that specifically bind NGF as described herein preferably comprise a heavy chain variable region and a light chain variable region as follows:
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 2, or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and the light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 4 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 6 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 8 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 10 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 12 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 14 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 16 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 18 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 20 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 22 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 24 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 25 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 26 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 27 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 28 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 29 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 30 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 31 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 32 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 177 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 32 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 33 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 34 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 35 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 36 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 37 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 38 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 39 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 40 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 41 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 42 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 43 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 44 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 180 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 181 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 182 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 183 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 185 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 186 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 187 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 188 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 189 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 42 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 190 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 188 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 206 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 42 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 207 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 188 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof; and
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 192 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 193 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof.
Exemplary binding proteins as disclosed herein may include at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NOS: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81; or modified CDR amino acid sequences having a sequence identity of at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% to one of said sequences; b) light chain CDRs consisting of SEQ ID NOS: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84; or modified CDR amino acid sequences having a sequence identity of at least 50% %, at least 60%, at least 70%, at least 80%, or at least 90% to one of said sequences.
It should be understood that variations are contemplated in any of the nucleic acid and amino acid sequences described herein. Such variations include those that will result in a nucleic acid sequence that is capable of directing production of analogs of the corresponding NGF binding proteins. It will be understood that due to the degeneracy of the genetic code, many substitutions of nucleotides may be made that will lead to a DNA sequence that remains capable of directing production of the corresponding protein or its analogs. All such variant DNA sequences that are functionally equivalent to any of the sequences described herein, are encompassed by the present disclosure.
A variant of any of the binding proteins described herein means a protein (or polypeptide) that differs from a given protein (e.g., an anti-NGF antibody) in amino acid sequence by the addition (e.g., insertion), deletion, or conservative substitution of amino acids, but that retains the biological activity of the given protein. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity and degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes may be identified, in part, by considering the hydropathic index of amino acids, as understood in the art (see, e.g., Kyte et al., J. Mol. Biol. 157: 105-132 (1982)). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes may be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids also may be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity (see, e.g., U.S. Pat. No. 4,554,101, which is incorporated herein by reference). Substitution of amino acids having similar hydrophilicity values may result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. In one aspect, substitutions are performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties. “Variant” also may be used to describe a polypeptide or fragment thereof that has been differentially processed, such as by proteolysis, phosphorylation, or other post-translational modification, yet retains its biological activity or antigen reactivity, e.g., the ability to bind to NGF. Use of “variant” herein is intended to encompass fragments of a variant unless otherwise contradicted by context.
The binding proteins described herein encompass an immunoglobulin molecule, disulfide linked Fv, scFv, monoclonal antibody, murine antibody, chimeric antibody, single domain antibody, CDR-grafted antibody, diabody, mammalized (bovanized, camelized, caninized, equinized, felinized, or humanized) antibody, a canine antibody, feline antibody, equine antibody, murine antibody, multispecific antibody, Fab, dual specific antibody, DVD, Fab′, bispecific antibody, F(ab′)2, or Fv including a single chain Fv fragment.
A binding protein may comprise a particular heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. An exemplary binding protein includes an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. Furthermore, the antibody may comprise a light chain constant region, such as a kappa light chain constant region or a lambda light chain constant region. An exemplary binding protein comprises a kappa light chain constant region.
Replacements of amino acid residues in the Fc portion to alter antibody effector function are known in the art (Winter, et al. U.S. Pat. Nos. 5,648,260; 5,624,821). The Fc portion of an antibody mediates several important effector functions e.g. cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC) and half-life/clearance rate of antibody and antigen-antibody complexes. In some cases these effector functions are desirable for therapeutic antibody but in other cases might be unnecessary or even deleterious, depending on the therapeutic objectives. Certain human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcγRs and complement C1q, respectively. Neonatal Fc receptors (FcRn) are the critical components determining the circulating half-life of antibodies. At least one amino acid residue may be replaced in the constant region of the antibody, for example the Fc region of the antibody, such that effector functions of the antibody are altered.
Binding proteins according to the present disclosure may comprise a heavy chain immunoglobulin constant domain such as, for example, a human or canine or equine or feline IgM constant domain, a human or canine or equine or feline IgG4 constant domain, a human or canine or equine or feline IgG1 constant domain, a human or canine or equine or feline IgE constant domain, a human or canine or equine or feline IgG2 constant domain, a human or canine or equine or feline IgG3 constant domain, and a human or canine or equine or feline IgA constant domain. A binding protein as described herein may comprise a light chain immunoglobulin constant domain such as but not limited to any of human, canine, equine or feline, kappa or lambda constant domains, or any of canine, equine or feline kappa or lambda equivalent constant domains. An exemplary such binding protein has a constant region having an amino acid sequence of SEQ ID NO: 52 or SEQ ID NO: 54.
Binding proteins as described herein may also encompass an NGF anti-idiotype antibody relative to at least one NGF binding protein of the present disclosure. The anti-idiotype antibody includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule such as, but not limited to, at least one complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or; any portion thereof, which may be incorporated into a binding protein of the present disclosure.
The binding proteins of the disclosure are capable of binding to human and canine NGF with high specificity, and additionally are capable of modulating the biological activity or function of NGF in an organism or a subject, including substantially neutralizing human and canine NGF. Also encompassed by the present disclosure are isolated murine monoclonal antibodies, or antigen-binding portions thereof, that bind to NGF with a substantially high affinity, have a slow off rate and/or have a substantially high neutralizing capacity. An exemplary binding protein as disclosed herein is capable of neutralizing NGF with a potency (IC50) of at least about 10 nM, at least about 5 nM, at least about 1 nM, at least about 0.5 nM, at least about 0.1 nM, at least about 0.05 nM, at least about 0.01 nM, or at least about 0.001 nM, as measured in the TF-1 cell proliferation assay or the pERK and Pathhunter assays. Binding proteins as described herein may have an on rate constant (Kon) to NGF of at least about 102M−1s−1; at least about 103M−1s1; at least about 104M−1s−1; at least about 105M−1s−1; at least about 106M−1s−1; or at least about 107M−1s−1′ as measured by surface plasmon resonance. Binding proteins as described herein may have an off rate constant (Koff) to NGF of at most about 10−3s−1; at most about 10−4s−1; at most about 10−5s−1; at most about 10−6s−1 or at most about 10−7s−1, as measured by surface plasmon resonance. Binding proteins as described herein may have a dissociation constant (KD) to NGF of at most about 10−7 M; at most about 10−8 M; at most about 10−9 M; at most about 10−10 M; at most about 10−11 M; at most about 10−12 M; at most about 10−13 M, or at most about 10−14 M. For example, a binding protein as described herein may have a dissociation constant (KD) of about 1×10−9M, about 1×10−10 M, about 3.14×10−10 M, about 1×10−11 M, about 2.37×10−11 M, about 1×10−12 M, about 1×10−13 M, or about 3.3×10−14 M.
Binding proteins as described herein including an isolated antibody, or antigen-binding portion thereof, or immunologically functional fragment thereof, may bind NGF and dissociate from NGF with a koff rate constant of about 0.1s−1 or less, as determined by surface plasmon resonance, or may inhibit NGF activity with an IC50 of about 1×10−6M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from NGF with a koff rate constant of about 1×10−2s−1 or less, as determined by surface plasmon resonance, or may inhibit NGF activity with an IC50 of about 1×10−7M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from NGF with a koff rate constant of about 1×10−3s−1 or less, as determined by surface plasmon resonance, or may inhibit NGF with an IC50 of about 1×10−8M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from NGF with a koff rate constant of about 1×10−4s−1 or less, as determined by surface plasmon resonance, or may inhibit NGF activity with an IC50 of about 1×10−9M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from NGF with a koff rate constant of about 1×10−5s−1 or less, as determined by surface plasmon resonance, or may inhibit NGF activity with an IC50 of about 1×10−10 M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from NGF with a koff rate constant of about 1×10−5s−1 or less, as determined by surface plasmon resonance, or may inhibit NGF activity with an IC50 of about 1×10−11 M or less.
A binding protein as described herein may bind canine NGF, wherein the antibody, or antigen-binding portion thereof, may dissociate from canine NGF with a koff rate constant of about 0.1s−1 or less, as determined by surface plasmon resonance, or may inhibit canine NGF activity with an IC50 of about 1×10−6M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from canine NGF with a koff rate constant of about 1×10−2s−1 or less, as determined by surface plasmon resonance, or may inhibit canine NGF activity with an IC50 of about 1×10−7M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from canine NGF with a koff rate constant of about 1×10−3s−1 or less, as determined by surface plasmon resonance, or may inhibit canine NGF with an IC50 of about 1×10−8M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from canine NGF with a koff rate constant of about 1×10−4s−1 or less, as determined by surface plasmon resonance, or may inhibit canine NGF activity with an IC50 of about 1×10−9M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from canine NGF with a koff rate constant of about 1×10−5s−1 or less, as determined by surface plasmon resonance, or may inhibit canine NGF activity with an IC50 of about 1×10−10M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from canine NGF with a koff rate constant of about 1×10−5s−1 or less, as determined by surface plasmon resonance, or may inhibit canine NGF activity with an IC50 of about 1×10−11 M or less.
The binding proteins of the disclosure further encompass binding proteins coupled to an immunoadhesion molecule, imaging agent, therapeutic agent, or cytotoxic agent. Non-limiting examples of suitable imaging agents include an enzyme, fluorescent label, luminescent label, bioluminescent label, magnetic label, biotin or a radiolabel including, but not limited to, 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and 153Sm. The therapeutic or cytotoxic agent may be an anti-metabolite, alkylating agent, antibiotic, growth factor, cytokine, anti-angiogenic agent, anti-mitotic agent, anthracycline, toxin, or apoptotic agent. Also provided herein is a labeled binding protein wherein an antibody or antibody portion of the disclosure is derivatized or linked to another functional molecule (e.g., another peptide or protein). For example, a labeled binding protein of the disclosure may be derived by functionally linking an antibody or antibody portion of the disclosed binding protein (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that may mediate associate of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
Useful detectable agents with which an antibody or antibody portion of the disclosure may be derivatized, may include fluorescent compounds. Exemplary fluorescent detectable agents include, for example, fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and the like. An antibody may also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. When an antibody is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, when the detectable agent horseradish peroxidase is present, addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is detectable. An antibody may also be derivatized with biotin, and detected through indirect measurement of avidin or streptavidin binding.
The binding proteins described herein may be in crystallized form. Crystallized binding proteins according to the present disclosure may be produced according to methods known in the art, as disclosed for example in WO 02072636. Preferably the crystallized binding protein retains biological activity after crystallization. The binding proteins may thus be provided as crystals of whole anti-NGF antibodies or portions or fragments thereof as disclosed herein. Such crystals may be used to prepare formulations and compositions incorporating anti NGF binding proteins, including diagnostic and therapeutic compositions. An exemplary such crystallized binding protein is a carrier-free, controlled release crystallized binding protein. An exemplary crystallized binding protein demonstrates a greater half-life in vivo than the soluble counterpart of the binding protein.
Anti NGF binding proteins as described herein may be glycosylated. The glycosylation may demonstrate, for example, a bovine, camel, canine, murine, equine, feline, or human glycosylation pattern. Glycosylated binding proteins as described herein include the antibody or antigen-binding portion coupled to one or more carbohydrate residues. Nascent in vivo protein production may undergo further processing, known as post-translational modification. Sugar (glycosyl) residues may be added enzymatically, a process known as glycosylation. The resulting proteins bearing covalently linked oligosaccharide side chains are known as glycosylated proteins or glycoproteins. Protein glycosylation depends on the amino acid sequence of the protein of interest, as well as the host cell in which the protein is expressed. Different organisms may produce different glycosylation enzymes (eg., glycosyltransferases and glycosidases), and have different substrates (nucleotide sugars) available. Due to such factors, protein glycosylation pattern, and composition of glycosyl residues, may differ depending on the host system in which the particular protein is expressed. Glycosyl residues useful in the disclosure may include, but are not limited to, glucose, galactose, mannose, fucose, n-acetylglucosamine and sialic acid. The glycosylated binding protein comprises glycosyl residues such that the glycosylation pattern is human, murine, canine, feline, bovine or equine.
It is known to those skilled in the art that differing protein glycosylation may result in differing protein characteristics. For instance, the efficacy of a therapeutic protein produced in a microorganism host, such as yeast, and glycosylated utilizing the host endogenous pathway may be reduced compared to that of the same protein expressed in a mammalian cell, such as a CHO cell line. Such glycoproteins may also be immunogenic in humans and show reduced half-life in vivo after administration. Specific receptors in humans and other animals may recognize specific glycosyl residues and promote the rapid clearance of the protein from the bloodstream. Other adverse effects may include changes in protein folding, solubility, susceptibility to proteases, trafficking, transport, compartmentalization, secretion, recognition by other proteins or factors, antigenicity, or allergenicity. Accordingly, a practitioner may prefer a therapeutic protein with a specific composition and pattern of glycosylation, such as a glycosylation composition and pattern identical, or at least similar, to that produced in human cells or in the species-specific cells of the intended subject animal.
Expressing glycosylated proteins different from that of a host cell may be achieved by genetically modifying the host cell to express heterologous glycosylation enzymes. Using techniques known in the art, a practitioner may generate antibodies or antigen-binding portions thereof exhibiting human protein glycosylation. For example, yeast strains have been genetically modified to express non-naturally occurring glycosylation enzymes such that glycosylated proteins (glycoproteins) produced in these yeast strains exhibit protein glycosylation identical to that of animal cells, especially human cells (U.S. patent applications 20040018590 and 20020137134).
Further, it will be appreciated by those skilled in the art that a protein of interest may be expressed using a library of host cells genetically engineered to express various glycosylation enzymes such that member host cells of the library produce the protein of interest with variant glycosylation patterns. A practitioner may then select and isolate the protein of interest with particular novel glycosylation patterns. The protein having a particularly selected novel glycosylation pattern exhibits improved or altered biological properties.
Anti NGF Chimeric Antibodies
A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a non-murine immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art, see e.g., Morrison, Science, 229: 1202 (1985); Oi et al., BioTechniques, 4: 214 (1986); Gillies et al., J. Immunol. Methods, 125: 191-202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397. In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81: 851-855 (1984); Neuberger et al., Nature, 312:604-608 (1984); Takeda et al., Nature, 314: 452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity may be used.
Anti NGF CDR Grafted Antibodies
CDR-grafted antibodies of the disclosure may comprise heavy and light chain variable region sequences from a non-murine antibody wherein one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of the murine antibodies of the disclosure. A framework sequence from any non-murine antibody may serve as the template for CDR grafting. However, straight chain replacement onto such a framework often leads to some loss of binding affinity to the antigen. The more homologous a non-murine antibody is to the original murine antibody, the less likely the possibility that combining the murine CDRs with the non-murine framework will introduce distortions in the CDRs that could reduce affinity.
A non-murine variable framework that is chosen to replace the murine variable framework apart from the CDRs may have at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity with the murine antibody variable region framework. The non-murine variable framework, apart from the CDRs, that is chosen to replace the murine variable framework, apart from the CDRs, may be a bovine, camel, canine, equine, feline or human variable framework. For example, the non-murine variable framework that is chosen to replace the murine variable framework, apart from the CDRs, is a canine variable framework and has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the murine antibody variable region framework.
Methods for producing CDR-grafted antibodies are known in the art (see EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), and include veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5): 489-498 (1991); Studnicka et al., Protein Engineering, 7(6):805-814 (1994); Roguska et al., PNAS, 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,352).
Anti NGF Humanized Antibodies
The process of modifying a monoclonal antibody from an animal to render it less immunogenic for therapeutic administration to humans (humanization) has been aggressively pursued and has been described in a number of publicatons (Antibody Engineering: A practical Guide. Carl A. K. Borrebaeck ed. W.H. Freeman and Company, 1992; and references cited above). Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Known human Ig sequences are disclosed in a variety of websites which are available on the Internet (such as the NCBI website, Antibody Resource, and known to those skilled in the art as well as in Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Dept. Health (1983), which is incorporated herein by reference. Additional sequences are shown in Table 1A below. Such imported sequences may be used to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic of the antibody, as known in the art.
TABLE 1A
Mouse Anti-NGF mAb CDRs Grafted onto Human Ig
Frameworks (CDR-Grafted Anti-NGF Abs (This Table
1A is identical to Table 17 in the Examples)
Name
Sequence (CDRs are underlined)
HU72 VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYM
(CDR GRAFT VH3-
FWVRQATGKGLEWVSTISDGGSYTYYTDNVKGRF
13/JH5)
TISRENAKNSLYLQMNSLRAGDTAVYYCARDWSD
SEGFAYWGQGTLVTVSS (SEQ ID NO: 165)
Hu73 VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWM
(CDR GRAFT VH1-
HWVRQAPGQGLEWMGRIDPYGGGTKHNEKFKRRV
18/JH6)
TMTTDTSTSTAYMELRSLRSDDTAVYYCARSGYD
YYFDVWGQGTTVTVSS (SEQ ID NO: 166)
HU77 VH
QVQLVQSGAEVKKPGSSVKVSCKASGFNIKDTYI
(CDR GRAFT VH1-
YWVRQAPGQGLEWMGRIDPANGNTIYASKFQGRV
69/JH6)
TITADKSTSTAYMELSSLRSEDTAVYYCARYGYY
AYWGQGTTVTVSS (SEQ ID NO: 167)
HU80 VH
QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYI
(CDR GRAFT VH1-
YWVRQAPGQGLEWMGRIDPANGNTIYASKFQGRV
18/JH6)
TMTTDTSTSTAYMELRSLRSDDTAVYYCARYGYY
AYWGQGTTVTVSS (SEQ ID NO: 168)
HU81 VH
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNHYM
(CDR GRAFT VH3-
YWVRQAPGKGLEWVGSISDGGAYTFYPDTVKGRF
15/JH1)
TISRDDSKNTLYLQMNSLKTEDTAVYYCTTEESA
NNGFAFWGQGTLVTVSS (SEQ ID NO: 169)
HU82 VH
QVTLKESGPVLVKPTETLTLTCTVSGFSLTGYNI
(CDR GRAFT VH2-
NWIRQPPGKALEWLAMIWGYGDTDYNSALKSRLT
26/JH6)
ISKDTSKSQVVLTMTNMDPVDTATYYCARDHYGG
NDWYFDVWGQGTTVTVSS (SEQ ID NO: 170)
HU72 VL
DIVMTQTPLSLPVTPGEPASISCRSSQSIVQSNG
(CDR GRAFT
NTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFS
01/JK2)
GSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPFT
FGQGTKLEIKR (SEQ ID NO: 171)
HU73 VL
DIQMIQSPSFLSASVGDRVSIICRASENIYSFLA
(CDR GRAFT
WYLQKPGKSPKLFLYNANTLAEGVSSRFSGRGSG
L22/JK2)
TDFTLTIISLKPEDFAAYYCQHHFGTPFTFGQGT
KLEIKR (SEQ ID NO: 172)
HU77 VL
DIVMTQTPLSLPVTPGEPASISCKSTKSLLNGDG
(CDR GRAFT
FTYLDWYLQKPGQSPQLLIYLVSNRFSGVPDRFS
01/JK2)
GSGSGTDFTLKISRVEAEDVGVYYCFESNYLFTF
GQGTKLEIKR (SEQ ID NO: 173)
HU80 VL
DIVMTQTPLSLPVTPGEPASISCKSTKSLLNGDG
(CDR GRAFT
FTYLDWYLQKPGQSPQLLIYLVSNRFSGVPDRFS
01/JK2)
GSGSGTDFTLKISRVEAEDVGVYYCFESNYLFTF
GQGTKLEIKR (SEQ ID NO: 174)
HU81 VL
DIVMTQTPLSLPVTPGEPASISCRSSQSILHSNG
(CDR GRAFT
NTYLEWYLQKPGQSPQLLIYRVSNRFSGVPDRFS
01/JK2)
GSGSGTDFTLKISRVEAEDVGVYYCFQGAHVPFT
FGQGTKLEIKR (SEQ ID NO: 175)
HU82 VL
DIQMTQSPSSLSASVGDRVTITCRASQDITNYLN
(CDR GRAFT
WYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSG
08/JK2)
TDFTFTISSLQPEDIATYYCQQGKTLPRTFGQGT
KLEIKR (SEQ ID NO: 176)
Framework residues in the human framework regions may be substituted with the corresponding residue from the CDR donor antibody to alter or improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues may be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding. Antibodies may be humanized using a variety of techniques known in the art, such as but not limited to those described in Jones et al., Nature 321:522 (1986); Verhoeyen et al., Science 239:1534 (1988)), Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994); PCT publication WO 91/09967, PCT/US98/16280, US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596, EP 239,400, U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539; and 4,816,567.
Anti NGF Caninized Antibodies
The process of modifying a monoclonal antibody from an animal to render it less immunogenic for therapeutic administration to canines (caninization) has been described in U.S. Pat. No. 7,261,890 B2 2007). The amino acid sequence of canine IgG1 is provided in GenBank (AF354264). Determination of the amino acid sequence of the variable regions of both a canine IgM and a canine IgA heavy chain (Wasserman et al., Biochem., 16, 3160 (1977), determination of the amino acid sequence of the κ light chain from a canine IgA (Wasserman et al., Immunochem., 15, 303 (1978)), complete amino-acid sequence of a canine μ chain was disclosed (McCumber et al., Mol. Immunol., 16, 565 (1979)), a single canine IgG-Aγ chain cDNA and four canine IgG-Aγ chain protein sequences were disclosed (Tang et al., Vet. Immunology Immunopathology, 80, 259 (2001)). It describes PCR amplification of a canine spleen cDNA library with a degenerate oligonucleotide primer designed from the conserved regions of human, mouse, pig, and bovine IgGs. Canine immunoglobulin variable domains, caninized antibodies, and methods for making and using them are disclosed in US Patent Application No. 2004/0181039 and U.S. Pat. Nos. 7,261,890; 6,504,013; 5,852,183; 5,5225,539.
Table 2 below is a list of amino acid sequences of VH and VL regions of selected caninized anti-NGF antibodies of the disclosure.
TABLE 2
SEQ ID NO:
Region
25
72.1 VH
26
72.1 VL
27
73.1 VH
28
73.1 VL
29
77.1 VH
30
77.1 VL
31
81.1 VH
32
81.1 VL
33
82.1 VH
34
82.1 VL
35
72.2 VH
36
72.2 VL
37
73.2 VH
38
73.2 VL
39
77.2 VH
40
77.2 VL
41
81.2 VH
42
81.2 VL
43
82.2 VH
44
82.2 VL
177
81.1B VH
179
72.3 VH
180
72.4 VH
181
72.4 VL
182
73.4 VH
183
73.4 VL
184
77.3 VH
185
77.4 VH
186
77.4 VL
187
81.4 VH
188
81.4 VL
189
81.2B VH
190
81.4B VH
191
82.3 VL
192
82.4 VH
193
82.4 VL
206
81.5B VH
207
81.6B VH
C. USES OF ANTI-NGF ANTIBODIES
Binding proteins as described herein may be used in a method for detecting the presence of NGF in a sample in vivo or in vitro (e.g., in a biological sample, such as serum, plasma, tissue, biopsy). The in vitro method may be used for example to diagnose a disease or disorder, e.g., an NGF-associated disorder. The method includes (i) contacting the sample or a control sample with the anti-NGF antibody or fragment thereof as described herein; and (ii) detecting formation of a complex between the anti-NGF antibody or fragment thereof, and the sample or the control sample, wherein a statistically significant change in the formation of the complex in the sample relative to the control sample is indicative of the presence of the NGF in the sample.
Binding proteins as described herein may be used in a method for detecting the presence of NGF in vivo (e.g., in vivo imaging in a subject). The method may be used to diagnose a disorder, e.g., an NGF-associated disorder. The method includes: (i) administering the anti-NGF antibody or fragment thereof as described herein to a subject or a control subject under conditions that allow binding of the antibody or fragment to NGF; and (ii) detecting formation of a complex between the antibody or fragment and NGF, wherein a statistically significant change in the formation of the complex in the subject relative to the control subject is indicative of the presence of NGF.
Given the ability to bind to NGF, the anti-NGF antibodies, or portions thereof, or combinations thereof, as described herein may be used as immunoreagent(s) to detect NGF (e.g., in a biological sample, such as serum or plasma), in a conventional immunoassay, such as an enzyme linked immunosorbent assays (ELISA), a radioimmunoassay (RIA) or tissue immunohistochemistry. A method for detecting NGF in a biological sample involves contacting a biological sample with an antibody, or antibody portion, of the disclosure and detecting either the antibody (or antibody portion) bound to NGF or unbound antibody (or antibody portion), to thereby detect NGF in the biological sample. The binding protein may be directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, or 153Sm.
NGF may alternatively be assayed in biological fluids by a competition immunoassay utilizing recombinant NGF standards labeled with a detectable substance and an unlabeled anti-NGF antibody. In this assay, the biological sample, the labeled recombinant NGF standards and the anti-NGF antibody are combined and the amount of labeled rNGF standard bound to the unlabeled antibody is determined. The amount of NGF in the biological sample is inversely proportional to the amount of labeled rNGF standard bound to the anti-NGF antibody. Similarly, NGF may also be assayed in biological fluids by a competition immunoassay utilizing rNGF standards labeled with a detectable substance and an unlabeled anti-NGF antibody.
The disclosure thus also contemplates immunoassay reagents, devices and kits including one or more of the presently disclosed binding proteins for detecting the presence or amount of NGF in a sample. It is contemplated for example that an immunoreagent comprising one or more of the presently disclosed binding proteins may be provided in the form of a kit with one or more containers such as vials or bottles, with each container containing a separate reagent such as an anti-NGF binding protein, or a cocktail of anti-NGF binding proteins, detection reagents and washing reagents employed in the assay. The immunoreagent(s) may be advantageously provided in a device in which the immunoreagents(s) is immobilized on a solid support, such as but not limited to a cuvette, tube, microtiter plates or wells, strips, chips or beads. The kit may comprise at least one container for conducting the assay, and/or a buffer, such as an assay buffer or a wash buffer, either one of which may be provided as a concentrated solution, a substrate solution for the detectable label (e.g., an enzymatic label), or a stop solution. Preferably, the kit comprises all components, i.e., reagents, standards, buffers, diluents, etc., which are necessary to perform the assay. The kit may contain instructions for determining the presence or amount of NGF in the sample based on specific binding of the immunoreagent to NGF, in paper form or computer-readable form, such as a disk, CD, DVD, or the like, and/or may be made available online.
The binding proteins in the kit may be labeled with a detectable label such as those described above including a fluorophore, a radioactive moiety, an enzyme, a biotin/avidin label, a chromophore, a chemiluminescent label, or the like; or the kit may include reagents for carrying out detectable labeling. The antibodies, calibrators and/or controls can be provided in separate containers or pre-dispensed into an appropriate assay format, for example, into microtiter plates.
Optionally, the kit includes quality control components (for example, sensitivity panels, calibrators, and positive controls). Preparation of quality control reagents is well-known in the art and is described on insert sheets for a variety of immunodiagnostic products. Sensitivity panel members optionally are used to establish assay performance characteristics, and further optionally are useful indicators of the integrity of the immunoassay kit reagents, and the standardization of assays.
The kit can also optionally include other reagents required to conduct a diagnostic assay or facilitate quality control evaluations, such as buffers, salts, enzymes, enzyme co-factors, enzyme substrates, detection reagents, and the like. Other components, such as buffers and solutions for the isolation and/or treatment of a test sample (e.g., pretreatment reagents), also can be included in the kit. The kit can additionally include one or more other controls. One or more of the components of the kit can be lyophilized, in which case the kit can further comprise reagents suitable for the reconstitution of the lyophilized components.
The various components of the kit optionally are provided in suitable containers as necessary, e.g., a microtiter plate. The kit can further include containers for holding or storing a sample (e.g., a container or cartridge for a urine sample). Where appropriate, the kit optionally also can contain reaction vessels, mixing vessels, and other components that facilitate the preparation of reagents or the test sample. The kit can also include one or more instruments for assisting with obtaining a test sample, such as a syringe, pipette, forceps, measured spoon, or the like. Instructions:
It will be appreciated that the antibodies and antibody portions of the disclosure are capable of substantially neutralizing NGF activity both in vitro and in vivo. Accordingly, such antibodies and antibody portions of the disclosure can also be used to substantially inhibit NGF activity, e.g., in a cell culture containing NGF, in mammalian subjects having NGF with which an antibody of the disclosure cross-reacts. The disclosure thus provides a method for inhibiting NGF activity comprising contacting NGF with an antibody or antibody portion of the disclosure such that NGF activity is substantially inhibited. For example, in a cell culture containing, or suspected of containing NGF, an antibody or antibody portion of the disclosure can be added to the culture medium to inhibit NGF activity in the culture.
Accordingly, the disclosure also provides a method for inhibiting NGF activity comprising contacting NGF with a binding protein such that NGF activity is substantially inhibited. In another aspect, the disclosure provides a method for inhibiting NGF activity in a subject suffering from a disorder in which NGF activity is detrimental, comprising administering to the subject a binding protein disclosed above such that NGF activity in the subject is substantially inhibited and treatment is achieved.
The disclosure also provides a method for reducing NGF activity in a subject, such as a subject suffering from a disease or disorder in which NGF activity is detrimental. The disclosure provides methods for reducing NGF activity in a subject suffering from such a disease or disorder, which method comprises administering to the subject an antibody or antibody portion of the disclosure such that NGF activity in the subject is reduced. The subject can be a mammal expressing an NGF to which an antibody of the disclosure is capable of binding. Still further the subject can be a mammal into which NGF has been introduced (e.g., by administration of NGF or by expression of an NGF transgene). An antibody of the disclosure can be administered to a subject in need thereof for therapeutic purposes.
An antibody of the disclosure can be administered for veterinary purposes to a non-human mammal expressing an NGF with which the antibody is capable of binding. For example, an antibody of the disclosure can be administered for veterinary purposes to a non-human mammal such as a dog, horse, cat, or livestock (beef and dairy cattle, swine, sheep, goats, poultry, etc.) expressing an NGF with which the antibody is capable of binding.
In another aspect, the disclosure provides a method of treating (e.g., curing, suppressing, ameliorating, delaying or preventing or decreasing the risk of the onset, recurrence or relapse of) or preventing an NGF associated disorder, in a subject. The method includes: administering to the subject a disclosed NGF binding protein (particularly an antagonist), e.g., an anti-NGF antibody or fragment thereof as described herein, in an amount sufficient to treat or prevent the NGF associated disorder. The NGF antagonist, e.g., the anti-NGF antibody or fragment thereof, can be administered to the subject, alone or in combination with other therapeutic modalities as described herein.
An antibody of the disclosure can be administered to a non-human mammal expressing an NGF with which the antibody is capable of binding as an animal model of human disease. Such animal models may be useful for evaluating the therapeutic efficacy of antibodies of the disclosure (e.g., testing of dosages and time courses of administration).
In another aspect, the antibodies and binding proteins of the disclosure are useful for treating NGF-related diseases and disorders including or involving acute or chronic pain. Non-limiting examples of NGF-related diseases and disorders include general inflammation, surgical and post-surgical pain including pain from amputation, dental pain, pain from trauma, fracture pain, pain from abscess, neuropathic pain, hyperalgesia and allodynia, neuropathic pain, post-herpetic neuralgia, diabetes including, but not limited to, diabetic neuropathy pain, stroke, thalamic pain syndrome, gout joint pain, osteoarthritis or rheumatoid arthritis pain, rheumatic diseases, lupus, psoriasis, sciatica, pain associated with musculoskeletal diseases including, but not limited to, chronic low back pain, fibromyalgia, sprains, pains associated with sickle cell crises, general headache, migraine, cluster headache, tension headache, trigeminal neuralgia, dysmenorrhea, endometriosis, ovarian cysts, visceral pain, prostatitis, cystitis, interstitial cystitis, erythromelalgia or pain caused by pancreatitis or kidney stones, general gastrointestinal disorders including, but not limited to, colitis, gastric ulceration and duodenal ulcers, gastroesophageal reflux, dyspepsia, inflammatory bowel disorders, irritable bowel syndrome, inflammatory bladder disorders, incisional pain, pain from burns and/or wounds, ankylosing spondilitis, periarticular pathologies, cancer pain including, but not limited to, pain from bone metastases and pain from cancer treatment, and pain from HIV or AIDS. Other examples of NGF-related diseases and conditions include malignant melanoma, Sjogren's syndrome, rhinitis, bronchial disorders, and asthma, such as uncontrolled asthma with severe airway hyper-responsiveness, intractable cough; and pain from skin diseases or disorders with an inflammatory component such as, but not limited to, sunburn, allergic skin reactions, dermatitis, pruritis, and vitiligo.
The disclosure also provides a method of treating a subject suffering from a disorder in which NGF is detrimental comprising administering a binding protein before, concurrent, or after the administration of a second agent. In another aspect, the additional therapeutic agent that can be coadministered and/or coformulated with one or more NGF antagonists, (e.g., anti-NGF antibodies or fragments thereof,) include, but are not limited to, TNF antagonists; a soluble fragment of a TNF receptor; ENBREL®; TNF enzyme antagonists; TNF converting enzyme (TACE) inhibitors; muscarinic receptor antagonists; TGF-beta antagonists; interferon gamma; perfenidone; chemotherapeutic agents, methotrexate; leflunomide; sirolimus (rapamycin) or an analog thereof, CCI-779; COX2 or cPLA2 inhibitors; NSAIDs; immunomodulators; p38 inhibitors; TPL-2, MK-2 and NFκB inhibitors; budenoside; epidermal growth factor; corticosteroids; cyclosporine; sulfasalazine; aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide; antioxidants; thromboxane inhibitors; anti-IL-6 antibodies; growth factors; elastase inhibitors; pyridinyl-imidazole compounds; antibodies or agonists of TNF, CGRP, substance P, bradykinin, MMP-2, MMP-9, MMP-13, LT, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, EMAP-II, GM-CSF, FGF, or PDGF; antibodies of CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands; FK506; rapamycin; mycophenolate mofetil; ibuprofen; prednisolone; phosphodiesterase inhibitors; adensosine agonists; antithrombotic agents; complement inhibitors; adrenergic agents; IRAK, NIK, IKK, p38, or MAP kinase inhibitors; IL-1P converting enzyme inhibitors; converting enzyme inhibitors; T-cell signaling inhibitors; metalloproteinase inhibitors; 6-mercaptopurines; angiotensin converting enzyme inhibitors; soluble cytokine receptors; soluble p55 TNF receptor; soluble p75 TNF receptor; sIL-1RI; sIL-1RII; sIL-6R; anti-inflammatory cytokines; IL-4; IL-10; IL-11; and TGFβ.
D. PHARMACEUTICAL COMPOSITIONS
The antibodies and antibody-portions of the disclosure can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises at least one antibody or antibody portion of the disclosure and a pharmaceutically acceptable carrier. Such compositions can be used for example in a method for treating a mammal for a disease or disorder involving increased levels of NGF by administering to the mammal an effective amount of the composition. A pharmaceutical composition may include a therapeutically effective amount of the antibody or antibody portion. The pharmaceutical compositions as described herein may be used for diagnosing, detecting, or monitoring a disorder or one or more symptoms thereof; preventing, treating, managing, or ameliorating a disorder or one or more symptoms thereof; and/or research. As used herein, the phrase “increased levels of NGF” refers to a level of NGF in a subject, such as a mammal, that is greater or higher than an established or predetermined baseline level of NGF such as, for example, a level previously established for said subject or averaged from a group of subjects.
A pharmaceutical composition may comprise, for example, a binding protein and a pharmaceutically acceptable carrier, excipient or diluent. For example, pharmaceutical compositions may comprise a therapeutically effective amount of one or more of the binding proteins as disclosed herein, together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant. The pharmaceutical composition may contain one or more various formulation materials for modifying, maintaining or preserving the composition or properties of the composition, for example, the color, consistency, isotonicity, odor, osmolarity, pH, sterility, stability, viscosity and other properties of the composition. Such formulation materials are generally well known and many suitable formulation materials are described for example in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed. (A. R. Gennaro, ed.) 1990, Mack Publishing Company. Non-limiting examples of suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. In addition, the pharmaceutical composition can also contain one or more preservatives. Examples of suitable preservatives that can be used include, but are not limited to, methylparaben, propylparaben, benzyl alcohol, chlorobutanol, and benzalkonium chloride. Optimal pharmaceutical formulations can be readily determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage.
The pharmaceutical composition may comprise at least one additional therapeutic agent for treating a disorder in which NGF activity is detrimental. The additional agent can be, for example, a therapeutic agent, imaging agent, cytotoxic agent, angiogenesis inhibitors, kinase inhibitors, co-stimulation molecule blockers, adhesion molecule blockers, anti-cytokine antibody or functional fragment thereof, methotrexate, cyclosporine, rapamycin, FK506, detectable label or reporter, TNF antagonist, anti-rheumatic, muscle relaxant, narcotic, non-steroid anti-inflammatory drug (NSAID), analgesic, anesthetic, sedative, local anesthetic, neuromuscular blocker, antimicrobial, antipsoriatic, corticosteriod, anabolic steroid, erythropoietin, immunoglobulin, immunosuppressive, growth hormone, hormone replacement drug, radiopharmaceutical, antidepressant, antipsychotic, stimulant, asthma medication, beta agonist, inhaled steroid, oral steroid, epinephrine or analog, cytokine, or a cytokine antagonist.
The pharmaceutical composition of the present disclosure may have a pH greater than about 7.0 or between about 7.0 and about 8.0. Alternatively, the pharmaceutical composition may have a pH of between about 7.2 to about 7.8. Still further alternatively, the pH of the pharmaceutical composition may be between about 7.4 to about 7.6. Still further alternatively, the pH of the pharmaceutical composition may be about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 7.7, 7.8, 7.9 or 8.0. With respect to the pharmaceutical compositions of the present disclosure, there is an increase in degradation, an increase in fragmentation or an increase in degradation and an increase in fragmentation at a pH of 6.0 or less. This finding was surprising as many pharmaceutical compositions comprising humanized antibodies exhibit an increase in degradation, an increase fragmentation or an increase in degradation and an increase in fragmentation at a pH lower than 5.0 and again at a pH higher than about 6.0. Accordingly, most pharmaceutical compositions containing humanized antibodies are stable at a pH between about 5.0 to about 6.0.
A composition for the release of a binding protein may comprise, for example, a formulation including an amount of a crystallized binding protein, crystallized antibody construct or crystallized antibody conjugate as disclosed above. The composition may further comprise an additional ingredient, such as carrier, excipient or diluent, and at least one polymeric carrier. The polymeric carrier can comprise one or more polymers selected from the following: poly(acrylic acid), poly(cyanoacrylates), poly(amino acids), poly(anhydrides), poly(depsipeptide), poly(esters), poly(lactic acid), poly(lactic-co-glycolic acid) or PLGA, poly(b-hydroxybutryate), poly(caprolactone), poly(dioxanone); poly(ethylene glycol), poly((hydroxypropyl) methacrylamide, poly[(organo)phosphazene], poly(ortho esters), poly(vinyl alcohol), poly(vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends and copolymers thereof. The additional ingredient may be, for example, albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-β-cyclodextrin, methoxypolyethylene glycol and polyethylene glycol.
The polymeric carrier may be capable of affecting the release of the binding protein from the composition as described further herein below. Polymeric materials can be used in the formulation of pharmaceutical compositions comprising the disclosed binding proteins to achieve controlled or sustained release of the disclosed binding proteins (Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger et al., J. Macromol. Sci. Rev. Macromol. Chem., 23:61 (1983); Levy et al., Science, 228: 190 (1985); During et al., Ann. Neurol., 25: 351 (1989); Howard et al., J. Neurosurg., 7 1:105 (1989); U.S. Pat. Nos. 5,679,377; 5, 916,597; 5,912,015; 5,989,463; 5,128,326; PCT Publication Nos. WO 99/15154; and WO 99/20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. The polymer used in a sustained release formulation may be inert, free of leachable impurities, stable on storage, sterile, and biodegradable. A controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
Controlled release systems are discussed in the review by Langer (Science, 249:1527-1533 (1990)). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more therapeutic agents of the disclosure (U.S. Pat. No. 4,526,938; PCT publication Nos. WO 91/05548 and WO 96/20698; Ning et al., Radiotherapy & Oncology, 39: 179-189 (1996), Song et al., PDA Journal of Pharmaceutical Science &Technology, 50: 372-397 (1995); Cleek et al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater., 24: 853-854 (1997); and Lam et al., Proc. Int'l. Symp. Control Rel. Bioact. Mater., 24: 759-760 (1997)).
The binding proteins of the present disclosure can be administered by a variety of methods known in the art. For example, the binding proteins of the present disclosure may be administered by subcutaneous injection, intravenous injection or infusion. Administration can be systemic or local. As will be appreciated by the skilled artisan, the route and/or mode of administration may vary depending upon the desired results. The active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
For example, such pharmaceutical compositions may be administered to a subject by parenteral, intradermal, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal. Methods of administering a prophylactic or therapeutic agent of the disclosure also include, but are not limited to, epidural administration, intratumoral administration, and mucosal administration (e.g., intranasal and oral routes). In addition, pulmonary administration can be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent (U.S. Pat. Nos. 6,019,968; 5,985,320; 5,985,309; 5,934, 272; 5,874,064; 5,855,913; 5,290, 540; and 4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903). The antibodies and antibody portions described herein can be administered for example using Alkermes AIR® pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.). The prophylactic or therapeutic agents may be administered by any convenient route, and may be administered together with other biologically active agents.
Various delivery systems are known and can be used to administer one or more disclosed binding proteinsor the combination of one or more disclosed binding proteins and a prophylactic agent or therapeutic agent useful for preventing, managing, treating, or ameliorating a disorder or one or more symptoms thereof, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or antibody fragment, receptor-mediated endocytosis (see, e.g., Wu et al., J. Biol. Chem., 262: 4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. It may be desirable to administer the disclosed binding proteins locally to the area in need of treatment, which may be achieved by, for example, and not by way of limitation, local infusion, by injection, or by means of an implant, said implant being of a porous or non-porous material, including membranes and matrices, such as sialastic membranes, polymers, fibrous matrices (e.g., Tissuel®), or collagen matrices. An effective amount of one or more disclosed binding proteins can be administered locally to the affected area to a subject to prevent, treat, manage, and/or ameliorate a disorder or a symptom thereof. Alternatively, an effective amount of one or more of the disclosed binding proteins is administered locally to the affected area in combination with an effective amount of one or more therapies (e.g., one or more prophylactic or therapeutic agents) other than disclosed binding proteins of a subject to prevent, treat, manage, and/or ameliorate a disorder or one or more symptoms thereof.
The disclosed binding proteins can be delivered in a controlled release or sustained release system such as, for example, an infusion pump device operable to achieve controlled or sustained release of the disclosed binding proteins (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:20 (1987); Buchwald et al., Surgery, 88: 507 (1980); Saudek et al., N. Engl. J. Med., 321: 574 (1989)).
When a composition as described herein comprises a nucleic acid encoding a binding protein as described herein as a prophylactic or therapeutic agent, the nucleic acid can be administered in vivo to promote expression of its encoded prophylactic or therapeutic agent, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (Joliot et al., Proc. Natl. Acad. Sci., 88: 1864-1868 (1991)). Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression by homologous recombination.
A pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. For example, a composition may be formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to human beings and companion animals. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocamne to ease pain at the site of the injection.
If the compositions of the disclosure are to be administered topically, the compositions can be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art (Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995)). For non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity greater than water are typically employed. Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which are, if desired, sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, such as, for example, osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as Freon) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well-known in the art.
The pharmaceutical composition of the present disclosure can have a half-life of from about 8 days to about 15 days when dosed intravenously or subcutaneously. Alternatively, the pharmaceutical composition of the present invention can have a half-life of from about 10 days to about 13 days. Still further alternatively, the pharmaceutical composition of the present invention can have a half-life of about 8 days, such as about 8.5 days, about 9 days, such as about 9.5 days, about 10 days, such as about 10.5 days, about 11 days, such as about 11.5 days, about 12 days, about 12.5 days, about 13 days, such as about 13.5 days, about 14 days, such as about 14.5 days, or about 15 days.
If the method of the disclosure comprises intranasal administration of a composition, the composition can be formulated in an aerosol form, spray, mist or in the form of drops. In particular, prophylactic or therapeutic agents for use according to the present disclosure can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (composed of, e.g., gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
If the method of the disclosure comprises oral administration, compositions can be formulated orally in the form of tablets, capsules, cachets, gelcaps, solutions, suspensions, and the like. Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well-known in the art. Liquid preparations for oral administration may take the form of, but not limited to, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of a prophylactic or therapeutic agent(s).
The method of the disclosure may comprise pulmonary administration, e.g., by use of an inhaler or nebulizer, of a composition formulated with an aerosolizing agent (U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903). For example, an antibody of the disclosure, combination therapy, and/or composition of the disclosure may be administered using Alkermes AIR® pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).
The method of the disclosure may comprise administration of a composition formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.
The methods of the disclosure may additionally comprise administration of compositions formulated as depot preparations. Such long acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).
The methods of the disclosure encompass administration of compositions formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions, such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
The ingredients of the disclosed compositions may be supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or a substantially water-free concentrate in a hermetically sealed container such as an ampoule or sachette which may indicate the quantity of active agent. Where the mode of administration is infusion, the disclosed compositions can be dispensed with an infusion solution containing sterile pharmaceutical grade solution such as water or saline. Where the mode of administration is by injection, an ampoule of sterile solution such as water or saline can be provided so that the ingredients may be mixed prior to administration.
In particular, the disclosure also provides that one or more of disclosed binding proteins or pharmaceutical compositions thereof is packaged in a hermetically sealed container such as an ampoule or sachette which may indicate the quantity of the agent. One or more of the disclosed binding proteins or pharmaceutical compositions thereof may be supplied as a dry sterilized lyophilized powder or substantially water-free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. One or more of the disclosed binding proteins or pharmaceutical compositions thereof may be supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least about 0.5 mg, 1 mg, 2 mg, 4 mg, 5 mg, 10 mg, 15 mg, 25 mg, 35 mg, 45 mg, 50 mg, 75 mg, or 100 mg. The lyophilized disclosed binding proteins or pharmaceutical compositions thereof may be stored at any suitable temperature, such as, for example, between about 2° C. and about 8° C. and may be stored in its original container. The disclosed binding proteins or pharmaceutical compositions thereof may be administered within about 1 week, within about 5 days, within about 72 hours, within about 48 hours, within about 24 hours, within about 12 hours, within about 6 hours, within about 5 hours, within about 3 hours, or within about 1 hour after being reconstituted. Alternatively, one or more of the disclosed binding proteins or pharmaceutical compositions thereof may be supplied in liquid form in a hermetically sealed container which may indicate the quantity and concentration of the agent. The liquid form of the administered composition may be supplied in a hermetically sealed container at concentrations of at least about 0.01 mg/mL, at least about 0.05 mg/mL, at least about 0.1 mg/mL, at least about 0.2 mg/mL, at least about 0.25 mg/ml, at least about 0.5 mg/ml, at least about 1 mg/ml, at least about 2.5 mg/ml, at least about 5 mg/ml, at least about 8 mg/ml, at least about 10 mg/ml, at least about 15 mg/kg, at least about 25 mg/ml, at least about 50 mg/ml, at least about 75 mg/ml, or at least about 100 mg/ml. The liquid form may be stored at any suitable temperature such as between about 2° C. and about 8° C. and may be stored in its original container.
The binding proteins of the disclosure can be incorporated into a pharmaceutical composition suitable for parenteral administration. In one aspect, binding proteins are prepared as an injectable solution containing between about 0.1 and about 250 mg/ml antibody. The injectable solution can be composed of either a liquid or lyophilized dosage form in a flint or amber vial, ampoule or pre-filled syringe. The buffer can be any suitable buffer such as L-histidine or a phosphate buffer saline at a concentration of about 1-50 mM, or about 5-10 mM Other suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate and potassium phosphate. Buffers may be used to modify the toxicity of the pharmaceutical composition. For example, sodium chloride can be used to modify the toxicity of the binding protein solution at a concentration of from about 0.1 and about 300 mM, such as about 150 mM saline to modify the toxicity of a liquid dosage form. Cryoprotectants, such as sucrose, can be included in a lyophilized dosage form at a concentration of about 0.1 to about 10% or from about 0.5 to about 1.0% may be used. Other suitable cryoprotectants include, but are not limited to, trehalose and lactose. Bulking agents, such as mannitol, can be included in a lyophilized dosage form at a concentration of about 1 to about 10%, or from about 2 to about 4%. Stabilizers, such as L-Methionine, can be used in both liquid and lyophilized dosage forms at a concentration of about 1 to about 50 mM, or about 5 to about 10 mM). Other suitable bulking agents include, but are not limited to, glycine and arginine. Surfactants, such as polysorbate-80, can be included in both liquid and lyophilized dosage forms at a concentration of about 0.001 to about 0.05% or about 0.005 to about 0.01%. Additional surfactants include, but are not limited to, polysorbate 20 and BRIJ surfactants.
An exemplary pharmaceutical formulation or composition of the present disclosure may be a liquid pharmaceutical composition having a pH between about 7.4 to about 8.0. The liquid pharmaceutical composition comprises about 5 mg/ml to about 50 mg/ml of an antibody comprising a heavy chain variable region comprising an amino acid sequence having a sequence of SEQ ID NO: 37 and a light chain variable region comprising an amino acid sequence comprising a sequence of SEQ ID NO: 38. The liquid pharmaceutical composition further comprises at least one buffer (such as, phosphate buffer saline, tris or histidine). The molarity of buffer that can be used can be from about 1 mM to about 60 mM. Optionally, said pharmaceutical composition or formulation can also contain at least one preservative, such as, methylparaben, propylparaben, benzyl alcohol, chlorobutanol or benzalkonium chloride. The amount of preservative that can be used can be from about 0.01 percent by volume to about 5.0% by volume depending on the preservative used.
Another exemplary pharmaceutical formulation or composition of the present disclosure may be a liquid pharmaceutical composition comprising a pH between about 7.4 to about 8.0. The liquid pharmaceutical composition comprises about 5 mg/ml to about 50 mg/ml of an antibody comprising a heavy chain variable region comprising an amino acid sequence having a sequence of SEQ ID NO: 192 and a light chain variable region comprising an amino acid sequence comprising a sequence of SEQ ID NO: 193. The liquid pharmaceutical composition further comprises at least one buffer (such as, phosphate buffer saline, tris or histidine). The molarity of buffer that can be used can be from about 1 mM to about 60 mM. Optionally, said pharmaceutical composition or formulation can also contain at least one preservative, such as, methylparaben, propylparaben, benzyl alcohol, chlorobutanol or benzalkonium chloride. The amount of preservative that can be used can be from about 0.01 percent by volume to about 5.0% by volume depending on the preservative used.
The compositions of this disclosure may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The form of the disclosed composition may depend on the intended mode of administration and therapeutic application. The disclosed compositions may be in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies. The mode of administration may be parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). The disclosed binding proteins may be administered by intravenous infusion or injection, or by intramuscular or subcutaneous injection.
Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other suitable ordered structure such as those suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., antibody or antibody portion) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Dispersions may be prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile, lyophilized powders for the preparation of sterile injectable solutions, the methods of preparation include, but are not limited to, vacuum drying and spray-drying that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption such as, for example, monostearate salts and gelatin.
An antibody or antibody portion of the disclosure may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the disclosure by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.
The disclosed binding proteins may be co-administered with other active compounds which may also be incorporated into the disclosed compositions. An antibody or antibody portion of the disclosure may be coformulated with and/or coadministered with one or more additional therapeutic agents that are useful for treating disorders in which NGF activity is detrimental. For example, an anti-NGF antibody or antibody portion of the disclosure may be coformulated and/or coadministered with one or more additional antibodies that bind other targets (e.g., antibodies that bind other cytokines or that bind cell surface molecules). Furthermore, one or more disclosed binding proteins may be used in combination with two or more of the foregoing therapeutic agents. Such combination therapies may, for example, enable the use of lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.
An antibody to NGF or fragment thereof may be formulated with a vehicle that extends the half-life of the binding protein. Suitable vehicles known in the art include, but are not limited to, the Fc domain, polyethylene glycol, and dextran. Such vehicles are described, e.g., in U.S. application Ser. No. 09/428,082 and published PCT Application No. WO 99/25044.
Isolated nucleic acid sequences comprising nucleotide sequences encoding disclosed binding proteins or another prophylactic or therapeutic agent of the disclosure may be administered to treat, prevent, manage, or ameliorate a disorder or one or more symptoms thereof by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid, wherein the nucleic acids produce their encoded antibody or prophylactic or therapeutic agent of the disclosure that mediates a prophylactic or therapeutic effect.
Any of the methods for gene therapy available in the art can be used according to the present disclosure. For general reviews of the methods of gene therapy, see Goldspiel et al., Clinical Pharmacy, 12: 488-505 (1993); Wu et al., Biotherapy, 3: 87-95 (1991); Tolstoshev, Ann. Rev. Phamacol. Toxicol., 32: 573-596 (1993); Mulligan, Science, 260: 926-932 (1993); and Morgan et al., Ann. Rev. Biochem., 62: 191-217 (1993); TIBTECH, 11(5):155-215 (1993). Methods commonly known in the art of recombinant DNA technology which can be used are described in, for example, Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990). Detailed descriptions of various methods of gene therapy are disclosed in US20050042664A1.
Antibodies of the disclosure, or antigen binding portions thereof, can be used alone or in combination to treat NGF related diseases. It should be understood that the antibodies of the disclosure or antigen binding portion thereof can be used alone or in combination with an additional agent, e.g., a therapeutic agent, said additional agent being selected by the skilled artisan for its intended purpose. For example, the additional agent can be a therapeutic agent art-recognized as being useful to treat the disease or condition being treated by the antibody of the present disclosure. The additional agent also can be an agent that imparts a beneficial attribute to the therapeutic composition e.g., an agent which affects the viscosity of the composition.
It should further be understood that the combinations which are to be included within this disclosure are those combinations useful for their intended purpose. The agents set forth below are illustrative for purposes and not intended to be limited. The combinations, which are part of this disclosure, can be the antibodies of the present disclosure and at least one additional agent selected from the lists below. The combination can also include more than one additional agent, e.g., two or three additional agents if the combination is such that the formed composition can perform its intended function.
Combinations include non-steroidal anti-inflammatory drug(s) also referred to as NSAIDS which include drugs like ibuprofen. Other combinations are corticosteroids including prednisolone; the well known side-effects of steroid use can be reduced or even eliminated by tapering the steroid dose required when treating patients in combination with the anti-NGF antibodies of this disclosure. Non-limiting examples of therapeutic agents for rheumatoid arthritis or pain with which an antibody, or antibody portion, of the disclosure can be combined include the following: cytokine suppressive anti-inflammatory drug(s) (CSAIDs); antibodies to or antagonists of cytokines or growth factors, for example, TNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, IL-21, interferons, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of the disclosure, or antigen binding portions thereof, can be combined with antibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, CTLA or their ligands including CD154 (gp39 or CD40L).
Combinations of therapeutic agents may interfere at different points in the autoimmune and subsequent inflammatory cascade; examples include TNF antagonists like chimeric, humanized or human TNF antibodies, D2E7, (PCT Publication No. WO 97/29131), CA2 (Remicade™), CDP 571, and soluble p55 or p75 TNF receptors, derivatives, thereof, (p75TNFR1gG (Enbrel™) or p55TNFR1gG (Lenercept), and also TNFα converting enzyme (TACE) inhibitors; similarly other IL-1 inhibitors (Interleukin-1-converting enzyme inhibitors, IL-1RA etc.) may be effective for the same reason. Other combinations include Interleukin 11.
The antibodies of the disclosure, or antigen binding portions thereof, may also be combined with agents, such as methotrexate, 6-MP, azathioprine sulphasalazine, mesalazine, olsalazine chloroquinine/hydroxychloroquine, pencillamine, aurothiomalate (intramuscular and oral), azathioprine, cochicine, corticosteroids (oral, inhaled and local injection), beta-2 adrenoreceptor agonists (salbutamol, terbutaline, salmeteral), xanthines (theophylline, aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium and oxitropium, cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adensosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, agents which interfere with signalling by proinflammatory cytokines such as or IL-1 (e.g. IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1β converting enzyme inhibitors, converting enzyme (TACE) inhibitors, T-cell signalling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors and derivatives thereof (e.g. soluble p55 or p75 TNF receptors and the derivatives p75TNFRIgG (Enbrel™ and p55TNFRIgG (Lenercept)), sIL-1RI, sIL-1RII, sIL-6R), antiinflammatory cytokines (e.g. IL-4, IL-10, IL-11, IL-13 and TGFβ), celecoxib, folic acid, hydroxychloroquine sulfate, rofecoxib, etanercept, infliximab, naproxen, valdecoxib, sulfasalazine, methylprednisolone, meloxicam, methylprednisolone acetate, gold sodium thiomalate, aspirin, triamcinolone acetonide, propoxyphene napsylate/apap, folate, nabumetone, diclofenac, piroxicam, etodolac, diclofenac sodium, oxaprozin, oxycodone hcl, hydrocodone bitartrate/apap, diclofenac sodium/misoprostol, fentanyl, anakinra, human recombinant, tramadol hcl, salsalate, sulindac, cyanocobalamin/fa/pyridoxine, acetaminophen, alendronate sodium, prednisolone, morphine sulfate, lidocaine hydrochloride, indomethacin, glucosamine sulf/chondroitin, amitriptyline hcl, sulfadiazine, oxycodone hcl/acetaminophen, olopatadine hcl, misoprostol, naproxen sodium, omeprazole, cyclophosphamide, rituximab, IL-1 TRAP, MRA, CTLA4-IG, IL-18 BP, anti-IL-18, Anti-IL15, BIRB-796, SCIO-469, VX-702, AMG-548, VX-740, Roflumilast, IC-485, CDC-801, and Mesopram. Other combinations include methotrexate or leflunomide and in moderate or severe rheumatoid arthritis cases, cyclosporine. The antibodies of the disclosure, or antigen binding portions thereof, may also be combined with agents, such as cancer chemotherapeutics, antimicrobials, anti-inflammatories, and anthelmintics used in animals.
The NSAID may be any non-steroidal anti-inflammatory compound. NSAIDs are categorized by virtue of their ability to inhibit cyclooxygenase. Cyclooxygenase 1 and cyclooxygenase 2 are two major isoforms of cyclooxygenase and most standard NSAIDs are mixed inhibitors of the two isoforms. Most standard NSAIDs fall within one of the following five structural categories: (1) propionic acid derivatives, such as ibuprofen, naproxen, naprosyn, diclofenac, and ketoprofen; (2) acetic acid derivatives, such as tolmetin and slindac; (3) fenamic acid derivatives, such as mefenamic acid and meclofenamic acid; (4) biphenylcarboxylic acid derivatives, such as diflunisal and flufenisal; and (5) oxicams, such as piroxim, sudoxicam, and isoxicam. Another class of NSAID has been described which selectively inhibit cyclooxygenase 2. Cox-2 inhibitors have been described (U.S. Pat. Nos. 5,616,601; 5,604,260; 5,593,994; 5,550,142; 5,536,752; 5,521,213; 5,475,995; 5,639,780; 5,604,253; 5,552,422; 5,510,368; 5,436,265; 5,409,944; and 5,130,311). Certain exemplary COX-2 inhibitors include celecoxib (SC-58635), rofecoxib, DUP-697, flosulide (CGP-28238), meloxicam, 6-methoxy-2 naphthylacetic acid (6-MNA), MK-966, nabumetone (prodrug for 6-MNA), nimesulide, NS-398, SC-5766, SC-58215, T-614; or combinations thereof.
The NGF antagonist and/or an additional therapeutic agent, such as NSAID, can be administered to a subject via any suitable route. For example, they can be administered together or separately, and/or simultaneously and/or sequentially, orally, intravenously, sublingually, subcutaneously, intraarterially, intramuscularly, rectally, intraspinally, intrathoracically, intraperitoneally, intraventricularly, sublingually, transdermally or by inhalation. Administration can be systemic, e.g., intravenous, or localized. The nerve growth factor antagonist and the additional therapeutic agent may be present together with one or more pharmaceutically acceptable carriers or excipients, or they may be present in separate compositions. In another aspect, the invention provides a synergistic composition of an NGF antagonist and an NSAID.
The pharmaceutical compositions of the disclosure may include a “therapeutically effective amount” or a “prophylactically effective amount” of an antibody or antibody portion of the disclosure. Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antibody portion of the disclosure is about 0.001 to about 20 mg/kg or about 0.001 to about 10 mg/kg. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the disclosure described herein are obvious and may be made using suitable equivalents without departing from the scope of the disclosure or the embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting of the disclosure.
E. EXAMPLES
The following examples are provided for exemplary guidance to make and use the disclosed binding proteins and pharmaceutical compositions thereof according to the inventive subject matter. However, it should be recognized that numerous modifications may be made without departing from the inventive concept presented herein.
Example 1
Immunization of Mice with NGF
To generate mouse anti-NGF mAbs, female A/J mice were immunized subcutaneously with 25 μg of human β NGF (R&D Systems catalog #256-GF/CF) in CFA. Animals were boosted every three weeks with 25 μg human β NGF in IFA. Four days prior to fusion, the mice were boosted with 10 μg of human β NGF in sterile saline intravenously. Spleen cells from the immunized mouse were fused with SP2/0-Ag14 myeloma cells at a 5:1 ratio spleen to SP2/0 cells, using standard techniques. Seven to ten days post fusion, when macroscopic colonies were observed, supernatants were tested in a capture ELISA format for binding to biotinylated human or rat β NGF. ELISA-positive wells were expanded to 24 well plates and tested for binding to biotinylated rat β NGF. Supernatants from hybridoma cell lines testing positive for both human and rat NGF were evaluated in a bioassay format. Cell lines of interest were cloned by limiting dilution to isolate an NGF-specific mouse monoclonal antibody.
Example 2
Screening Hybridoma Supernatants to Identify Secreted Anti-NGF MAbs
A. Indirect Binding ELISA
To determine if anti-NGF mAbs were present in hybridoma supernatants, ELISA plates were coated with goat anti-murine IgG Fc (Jackson ImmunoResearch, cat #115-005-164) and incubated overnight at 4° C. The plates were washed three times with wash buffer. The plates were blocked with 200 μl of 2% milk and incubated for 1 hour at room temperature. The plates were washed as above. Hybridoma supernatants were diluted 5-fold, 25-fold, 125-fold and 1625-fold with PBS and then added to the plate wells and incubated for 1 hour at room temperature. The positive control was crude sera (diluted 1:500 with PBS) isolated from a β NGF immunized mouse and the negative control was hybridoma supernatant derived from a mouse immunized with an antigen other than NGF. The plates were washed and then 50 μl of biotinylated human or rat β NGF at 50 ng/ml was added and incubated for 1 hour at room temperature. The plates were washed. Streptavidin-HRP (Thermo, cat #21126) conjugate was diluted at 10,000 and added to the plates. The plates were incubated for 30 minutes at room temperature. The plates were washed and then TMB substrate (Invitrogen, catalog #00-2023) was added. The reaction was stopped using 2N H2SO4 (VWR, catalog #BDH3500-1). The absorbance at 450 nm was read on a Spectromax 2E plate reader (Molecular Devices); these absorbance readings are shown in Tables 1 and 2. The numerical value indicates binding of mouse anti-NGF antibodies to biotinylated human or rat β NGF. This data indicates that several hybridoma supernatants contained anti-NGF antibodies.
TABLE 3
Biotinylated Human NGF Indirect Binding ELISA data
Supernatant
dilution (fold)
30F11
23F1
22E1
3C3
16B9
17G6
23H2
25E5
29E6
7H1
19C1
30A1
5
1.066
1.143
1.288
1.137
0.821
1.122
0.913
1.299
1.196
1.155
0.936
1.090
25
1.005
1.171
1.255
1.108
0.644
1.127
0.529
1.254
1.127
1.159
0.555
0.926
125
0.873
0.979
0.772
0.948
0.340
1.017
0.191
0.988
0.889
1.002
0.234
0.507
625
0.436
0.696
0.296
0.571
0.107
0.713
0.085
0.512
0.426
0.673
0.100
0.223
Supernatant
dilution (fold)
29A7
27A5
26D5
26H12
23D7
22A9
22G3
21D4
3E9
3F9
2G11
1D6
5
1.198
1.116
0.954
0.943
1.087
0.707
0.662
1.154
1.167
0.974
1.038
0.545
25
1.092
0.887
0.903
0.794
1.060
0.549
0.498
1.042
0.996
0.694
0.992
0.457
125
0.762
0.395
0.823
0.381
0.857
0.348
0.240
0.899
0.655
0.323
0.819
0.164
625
0.293
0.174
0.542
0.135
0.489
0.168
0.126
0.543
0.298
0.145
0.486
0.066
Supernatant
dilution (fold)
4B6
8E4
9E2
9H2
20B10
14G6
12H12
11D1
5
1.252
1.294
1.126
1.167
1.098
1.274
1.222
0.642
25
1.131
1.076
1.085
0.915
0.997
1.206
1.083
0.497
125
0.768
0.595
0.938
0.395
0.576
0.956
0.741
0.275
625
0.341
0.250
0.605
0.171
0.143
0.598
0.363
0.117
Supernatant
Positive
dilution (fold)
control
4E2
12D6
1D10
2D8
3F7
4F11
4H2
5D8
5G9
6B2
6F10
3
1.018
1.078
0.985
1.105
1.046
1.282
1.192
1.013
0.790
1.052
1.231
1.096
15
0.981
0.991
0.844
0.963
0.868
1.166
1.016
0.800
0.654
0.919
0.939
1.045
75
1.020
0.705
0.501
0.655
0.436
1.049
0.702
0.447
0.420
0.534
0.505
0.999
Supernatant
Nega-
dilution
tive
Negative
(fold)
6H2
7C10
7G1
8G9
10A12
10B6
11A9
12A5
12F6
13E3
14A9
control
3
1.322
0.745
0.233
0.849
0.192
1.135
0.056
0.725
1.003
1.003
1.107
0.054
15
1.221
0.378
0.106
0.548
0.089
1.088
0.051
0.401
0.944
0.881
1.082
0.053
75
0.791
0.151
0.066
0.220
0.060
0.872
0.050
0.183
0.681
0.463
0.951
0.051
TABLE 4
Biotinylated Rat NGF Indirect Binding ELISA data
Supernatant
dilution
(fold)
30F11
23F1
22E1
3C3
16B9
17G6
23H2
25E5
29E6
7H1
19C1
30A1
5
0.694
0.764
1.054
0.698
0.443
0.749
0.670
1.091
0.677
0.733
0.660
0.690
25
0.734
0.767
0.936
0.729
0.350
0.758
0.412
1.099
0.655
0.664
0.462
0.681
125
0.603
0.737
0.557
0.628
0.218
0.751
0.176
0.803
0.523
0.603
0.197
0.445
625
0.361
0.528
0.229
0.520
0.094
0.567
0.083
0.396
0.261
0.401
0.088
0.180
Supernatant
dilution
(fold)
29A7
27A5
26D5
26H12
23D7
22A9
22G3
21D4
3E9
3F9
2G11
1D6
5
0.967
0.610
0.611
0.538
0.684
0.508
0.521
0.787
1.098
0.633
0.705
0.327
25
0.907
0.514
0.571
0.368
0.775
0.417
0.384
0.760
0.945
0.502
0.669
0.278
125
0.441
0.236
0.516
0.169
0.654
0.240
0.209
0.671
0.530
0.264
0.588
0.132
625
0.224
0.113
0.413
0.082
0.396
0.117
0.107
0.453
0.219
0.117
0.353
0.063
Supernatant
dilution
(fold)
4B6
8E4
9E2
9H2
20B10
14G6
12H12
11D1
5
0.607
0.685
0.632
0.453
0.472
0.755
0.676
0.122
25
0.508
0.518
0.559
0.310
0.431
0.739
0.571
0.095
125
0.438
0.317
0.529
0.157
0.261
0.665
0.357
0.076
625
0.234
0.150
0.382
0.085
0.108
0.424
0.173
0.060
Supernatant
dilution
Positive
(fold)
control
4E2
12D6
1D10
2D8
3F7
4F11
4H2
5D8
5G9
6B2
6F10
3
0.773
0.777
0.459
1.023
0.590
1.097
0.952
0.945
0.565
0.952
1.122
0.937
15
0.736
0.651
0.379
0.877
0.599
1.125
0.690
0.684
0.467
0.767
0.876
1.005
75
0.760
0.471
0.210
0.548
0.323
1.044
0.576
0.348
0.294
0.406
0.453
0.849
Supernatant
dilution
Negative
(fold)
6H2
7C10
7G1
8G9
10A12
10B6
11A9
12A5
12F6
13E3
14A9
control
3
1.108
0.541
0.197
0.681
0.145
0.440
0.058
0.521
0.904
0.786
0.845
0.055
15
0.860
0.275
0.093
0.396
0.077
0.784
0.052
0.334
0.810
0.737
0.777
0.053
75
0.603
0.115
0.061
0.155
0.060
0.727
0.051
0.153
0.565
0.413
0.582
0.052
B. TrkA Binding ELISA
To determine if anti-NGF mAbs in hybridoma supernatants blocked NGF from binding to the TrkA receptor, ELISA plates were coated with goat anti-human IgG Fc (Jackson ImmunoResearch, cat #109-005-008) at 2 μg/ml in PBS and incubated over night at 4° C. The plates were washed three times with PBS/Tween. The plates were blocked with 200 μl/well of 2% milk in PBS for 1 hour at room temperature. The plates were washed three times as above. Rat TrkA/Fc chimera (R&D Systems, catalog #1056-TK) was added at 1 μg/ml (50 μl/well) in PBS/0.1% BSA and then incubated for 1 hour at room temperature. Biotinylated human NGF was titered and pre-incubated with anti-NGF antibody supernatants diluted 1-fold, 5-fold, and 25-fold, or purified anti-NGF mAbs diluted to 0.08, 0.4, 2, or 10 μg/ml for 1 hour at room temperature on a plate shaker. The negative control was unrelated conditioned supernatant. The positive control was sera from a mouse immunized with NGF. The plates were washed and then 50 μl of each biotinylated NGF/Ab mix was added to the appropriate wells. The plates were incubated for 1 hour at room temperature. The plates were washed. 50 μl of streptavidin-HRP (Thermo, cat #21126) was added at 10,000 dilution. The plates were incubated for 30 min at room temperature. The plates were washed. 50 μl of TMB (Invitrogen, cat #00-2023) was added and the reaction was stopped using 2N H2SO4 (VWR, cat #BDH3500-1). The absorbance at 450 nm was read on a Spectromax 2E plate reader (Molecular Devices), and the absorbance readings are shown in Table 5. The numerical value indicates binding of biotinylated human β NGF to rat TrkA/Fc chimera. This data indicates that several hybridoma supernatants contained anti-NGF receptor-blocking antibodies.
TABLE 5
Rat TrkA Inhibition Binding ELISA Data for Anti-NGF Hybridoma Supernatants
Supernatant
dilution
Negative
Positive
(fold)
control
control
30F11
23F1
22E1
3C3
16B9
17G6
23H2
25E5
29E6
7H1
1
0.465
0.050
0.158
0.108
0.357
0.146
0.142
0.091
0.379
0.304
0.291
0.217
5
0.456
0.055
0.210
0.140
0.429
0.195
0.249
0.123
0.622
0.354
0.600
0.419
25
0.462
0.102
0.331
0.276
0.558
0.345
0.409
0.210
0.418
0.505
0.881
0.758
Supernatant
dilution
(fold)
19C1
30A1
29A7
27A5
26D5
26H12
23D7
22A9
22G3
21D4
3E9
3F9
1
0.285
0.148
0.427
0.444
0.063
0.344
0.131
0.322
0.150
0.133
0.328
0.186
5
0.567
0.281
0.462
0.800
0.076
0.621
0.212
0.362
0.211
0.242
0.416
0.295
25
0.686
0.464
0.502
0.680
0.101
0.665
0.393
0.453
0.337
0.404
0.682
0.498
Supernatant
dilution
(fold)
Neg
Pos
2G11
1D6
4B6
8E4
9E2
9H2
1
0.372
0.052
0.138
0.226
0.169
0.273
0.103
0.380
5
0.336
0.073
0.205
0.281
0.287
0.669
0.125
0.604
25
0.318
0.228
0.328
0.343
0.424
0.693
0.151
0.521
Supernatant
dilution
(fold)
20B10
14G6
12H12
11D1
19A12
2B12
PBS
PBS
1
0.166
0.113
0.060
0.101
0.100
0.065
0.295
0.315
5
0.200
0.192
0.099
0.152
0.170
0.070
0.334
0.297
25
0.289
0.334
0.190
0.264
0.295
0.095
0.306
0.289
Supernatant
dilution
−ve
+ve
+ve
+ve
(fold)
contrl
contrl
contrl
contrl
13E3
14A9
4E2
12D6
1D10
2D8
3F7
4F11
1
0.386
0.112
0.145
0.104
0.400
0.121
0.283
0.145
0.248
0.359
0.056
0.286
5
0.388
0.164
0.234
0.140
0.383
0.208
0.290
0.211
0.312
0.588
0.083
0.356
25
0.386
0.308
0.488
0.216
0.497
0.376
0.334
0.364
0.447
0.497
0.149
0.541
Supernatant
dilution
(fold)
4H2
5D8
5G9
6B2
6F10
6H2
7C10
8G9
10B6
12A5
12F6
PBS
1
0.396
0.363
0.344
0.096
0.206
0.400
0.230
0.409
0.329
0.306
0.172
0.436
5
0.457
0.398
0.387
0.215
0.212
0.523
0.489
0.473
0.364
0.328
0.227
0.351
25
0.606
0.504
0.473
0.451
0.242
0.738
0.487
0.413
0.399
0.406
0.324
0.338
C. SureFire Cellular Phospho-ERK (pERK) Assay
To determine if anti-NGF mAbs in hybridoma supernatants blocked downstream signaling as a result of blocking NGF from binding to TrkA, Neuroscreen-1 cells (Thermo Fisher Scientific) were grown on collagen I-coated flasks in RPMI medium supplemented with 10% horse serum, 5% FBS, 100 units/ml penicillin/streptomycin, 2 mM L-glutamine, and 10 mM HEPES at 37° C. in a humidified atmosphere at 95% air and 5% CO2. For the ERK phosphorylation assay, 5×104 cells were seeded in each well of a 96-well plate coated with collagen I (Becton Dickinson). Cells were then serum starved for 24 hours before stimulation. 130 pM human β NGF (R&D Systems catalog #256-GF/CF) was mixed into diluted hybridoma supernatants (to achieve a final supernatant dilution (fold) of 10-fold, 100-fold, 500-fold or 1,000-fold) and mixtures were pre-incubated for 15 min at 37° C. before being added to the cells. Each diluted hybridoma supernatant was tested in quadruplicate. After 5 min of stimulation, the medium was removed and replaced with SureFire™ AlphaScreen cell lysis (PerkinElmer). Cell lysates were then processed according to the manufacturer's instructions and fluorescence signals quantified using an EnVision plate reader (PerkinElmer); the fluorescence data is summarized in Table 6. The numerical value indicates ERK phosphorylation due to TrkA signaling in the presence of human β NGF and is expressed as the percentage of signal vs. maximum signal. The maximum signal is defined as 100% response from cells showing ERK phosphorylation in the presence of only β NGF (no hybridoma supernatant). This data indicates that several hybridoma supernatants contained neutralizing anti-NGF antibodies.
TABLE 6
SureFire pERK Assay Data Generated with Anti-NGF mAb Hybridoma Supernatants
Supernatant
dilution
(fold)
23F1
17G6
30F11
3C3
100
5
2
4
2
2
0
1
1
5
2
2
0
5
2
5
3
1000
5
3
4
3
2
2
2
1
4
2
4
3
6
3
5
4
5000
8
7
8
8
13
7
15
8
30
26
28
25
24
25
22
23
10000
35
33
32
32
44
25
43
23
65
45
56
42
57
52
68
62
Supernatant
dilution
(fold)
2B12
21D4
4B6
22G3
100
0
0
−1
0
4
3
5
2
5
4
1
0
13
7
7
2
1000
0
0
0
0
5
3
5
3
1
0
2
1
3
2
4
2
5000
18
16
21
17
11
7
12
8
8
8
7
8
23
18
25
20
10000
51
43
49
41
38
23
37
23
30
34
30
35
51
45
47
43
Supernatant
dilution
(fold)
2G11
14G6
16B9
19A12
100
5
2
6
3
4
3
0
−1
3
3
3
2
2
1
2
1
1000
6
3
6
3
−1
0
0
0
65
57
70
60
3
2
3
2
5000
14
8
14
8
7
7
8
7
72
63
73
62
47
36
46
32
10000
44
30
74
48
38
36
36
36
76
62
77
62
69
55
81
65
Supernatant
dilution
(fold)
30A1
26D5
23D7
23H2
100
0
0
1
0
0
0
0
0
2
2
−1
0
47
41
44
44
1000
1
2
2
2
1
1
1
1
1
0
1
1
86
80
79
82
5000
40
41
42
40
37
30
35
30
59
48
56
54
85
80
85
83
10000
62
67
63
64
64
52
80
71
74
63
70
60
85
84
82
84
Supernatant
dilution
(fold)
9E2
20B10
12H12
11D1
100
3
3
3
4
2
1
1
1
3
3
3
2
69
75
70
78
1000
5
7
5
8
1
1
1
0
30
30
30
32
71
84
72
85
5000
37
57
36
55
20
28
19
29
62
64
60
61
76
78
80
76
10000
55
69
56
67
55
71
73
84
69
78
76
77
89
95
102
101
D. PathHunter Assay
To determine if anti-NGF mAbs in hybridoma supernatants blocked downstream signaling as a result of blocking NGF from binding to TrkA, the PathHunter U2OS stable cell line stably expressing the NGF receptor TrkA and the co-activator protein SHC1 fused to complementing fragments of β-galactosidase was purchased from DiscoveRx. Cells were grown in MEM media supplemented with 10% FBS, 100 units/ml penicillin/streptomycin, 2 mM L-glutamine, 500 μg/ml Geneticin G418, and 250 μg/ml Hygromycin at 37° C. in a humidified atmosphere at 95% air and 5% CO2. Sixteen hours before the assay, 2×104 cells were seeded in each well of a 96-well half-volume black plate in 40 μl of MEM media supplemented with 0.5% horse serum. 440 pM human β NGF (R and D Systems catalog #256-GF/CF) was mixed into diluted hybridoma supernatants (to achieve a final supernatant dilution of 10-fold, 100-fold, 500-fold or 1,000-fold) and mixtures were pre-incubated for 15 min at 37° C. before being added to the cells. Cell plates were incubated for 5 min at room temperature before stimulation with 10 μl per well of NGF/antibody mixture. After 3 hours of cell induction at room temperature, 25 μl of PathHunter detection reagent was added to each well according to the manufacturer's instructions. The chemiluminescent signal was detected 1 hour later using a TopCount plate reader (PerkinElmer); the chemiluminescence signal data is shown in Table 7. The numerical value indicates β-galactosidase generation due to TrkA signaling in the presence of human β NGF and is expressed as the percentage of signal vs. maximum signal. The maximum signal is defined as 100% response from cells showing in the presence of β-galactosidase generation in the presence of only β NGF (no hybridoma supernatant). This data indicates that several hybridoma supernatants contained neutralizing anti-NGF antibodies.
TABLE 7
PathHunter Data Generated with Hybridoma Supernatants
Supernatant
dilution
(fold)
30F11
23F1
3C3
16B9
17G6
19A12
100
21
22
5
5
16
29
19
24
10
14
25
28
1000
42
37
23
13
40
46
117
114
23
24
21
30
5000
97
99
69
70
71
81
120
127
100
115
93
92
10000
94
93
92
91
78
84
114
129
115
120
89
98
Supernatant
dilution
(fold)
2B12
30A1
26D5
23D7
23H2
22G3
100
89
90
21
24
83
84
31
28
142
176
16
16
1000
64
70
50
57
49
51
54
53
88
134
20
23
5000
128
127
126
139
92
96
112
131
117
120
89
99
10000
128
133
133
129
95
84
124
148
111
136
86
101
Supernatant
dilution
(fold)
21D4
2G11
4B6
9E2
20B10
100
9
10
22
22
24
31
102
104
6
5
1000
16
17
29
27
46
49
77
91
0
4
5000
107
100
66
72
88
94
137
152
25
31
10000
108
112
66
72
102
109
137
143
52
58
Supernatant
dilution
Unrelated
(fold)
14G6
12H12
11D1
26H12
hybridoma
100
18
17
26
23
117
101
106
119
156
185
1000
27
27
46
56
127
118
124
115
136
144
5000
105
83
145
137
109
99
137
166
126
132
10000
113
109
122
145
91
98
151
167
138
137
Example 3
Hybridoma Sub-Cloning
Hybridoma cell lines were subcloned using standard limiting dilution techniques. Cells were diluted to a concentration of 50, 5, or 0.5 cells/mL. 200 uL of the diluted cell suspensions were plated into 96 well tissue culture plates. The plates were incubated at 37° C. with 5% CO2 and ˜90% relative humidity. The growth was visually checked at day 7 for macroscopic colonies. Supernatants from wells were screened for antibody production when colony growth was visible. Table 8 shows the subclone identification nomenclature and monikers. This data indicates that several anti-NGF antibodies could be isolated from a clonal population of cells.
TABLE 8
Hybridoma Subclone Identification and Monikers
Hybridoma
Subcloned
Supernatant Name
Hybridoma Name
Moniker
Lot #
14G6
ML129-14G6.3H3
PR-1254970
1734671
2G11
ML129-2G11.3B1
PR-1254971
1734673
20B10
ML129-20B10.3F4
PR-1254972
1734675
2B12
ML129-2B12.5G9
PR-1254973
1734676
17G6
ML129-17G6.3E7
PR-1254974
1734677
21D4
ML129-21D4.4A11
PR-1254977
1734678
4B6
ML129-4B6.4H3
PR-1254978
1734679
22G3
ML129-22G3.3F3
PR-1254979
1734680
23F1
ML129-23F1.4G3
PR-1254980
1734681
14A9
ML130-14A9.5B12
PR-1254981
1734682
3F7
ML130-3F7.4A8
PR-1254982
1734683
Example 4
Scale Up and Purification of Monoclonal Antibodies
Subcloned hybridoma cell lines were expanded into Hybridoma SFM (Invitrogen catalog #12045) with 5% Low IgG Fetal bovine serum (Invitrogen catalog #16250-078). Supernatants were harvested, centrifuged and filtered to remove cellular debris, and concentrated. Antibodies were mixed with Pierce binding buffer A (Thermo, catalog #21001) in a 1 ratio. The antibodies were loaded onto a recombinant Protein A sepharose (GE Healthcare, catalog #17-1279-04) chromatography column, eluted using Pierce elution buffer (Thermo, catalog #21004), neutralized using 2M Tris pH 7.5, and then dialyzed into PBS. This work allowed the isolation of anti-NGF mAbs for characterization studies.
Example 5
Cloning of Canine NGF
The coding region of canine NGF was amplified from canine universal cDNA (Biochain Institute, catalog #4734565) using primers of SEQ ID NO: 45 and SEQ ID NO: 46 or primers of SEQ ID NO: 47 and SEQ ID NO: 48 and cloned into a mammalian or bacterial expression vector, respectively. The PCR reactions were set up as recommended by the manufacturer (Novagen, KOD Hot Start Master Mix, catalog #71842-3). The mammalian clone was made as a C-terminal 6-His fusion protein (SEQ ID NO: 208) by ligating the PCR product with pTT6 vector (Abbott) at the KpnI/XbaI restriction sites. The bacterial clone was made with the pro-NGF sequence using the mammalian clone as a template and ligated with pET15B (Novagen) at the NdeI/XhoI restriction sites. The DNA sequence and amino acid sequence of the canine NGF isolated are listed as SEQ ID NO: 49 and SEQ ID NO: 50, respectively. This work allowed expression of canine NGF protein for purification.
Example 6
Expression of Canine NGF
The canine NGF clone in the bacterial expression vector was grown at 37° C. in overnight express auto inducing Terrific Broth (Novagen) in Rosetta2 (DE3) E. coli host (EMD Biosciences) in 2 L non-baffled flasks. The cells were centrifuged down and the cell paste was resuspended in 100 mL of lysis buffer (25 mM Tris, 300 mM NaCl, 10% glycerol, 0.1% Triton X 100 pH 8.0) with lysonase and sonicated for 2 min on ice. The sample was centrifuged at 15000 RPM and the pellet was solubilized in 50 mL of 25 mM Tris, 6 M GdHCl pH 8.0. The sample was centrifuged at 15000 rpm for 30 min and the supernatant was loaded on to a 10 ml IMAC resin.
A. IMAC Chromatography
A 10 ml GE-Ni FF column was prepared. Buffer A: 25 mM Tris, 6 M GdHCl pH 8.0, Buffer B: A+500 mM Imidazole. The resin was equilibrated and loaded with recirculation to allow for complete binding (−10 passes) overnight at 4° C. The column was washed with Buffer A. Batch elution was carried out with 40 ml Buffer A, followed by 30 ml Buffer A. 5 ml fractions were collected and pooled.
B. Refolding by Rapid Dilution
The pooled fraction was reduced by adding 50 mM DTT, and EDTA was added to 10 mM, and incubated for 1 h at RT. The sample was acidified by adding 6M HCl to pH 4.0 and dialyzed into 6M GdHCl pH 5.0 to remove excess DTT. Refolding was performed by diluting the reduced/acidified sample in 1 L of 100 mM Tris, 1 M Arginine, 5 mM EDTA, 5 mM GSH, 1 mM GSSG (SEQ ID NO: 209) pH 9.5 for 4 h at 4° C. The refolded protein was dialyzed against 25 mM Tris, 200 mM NaCl, 10% Glycerol pH 8.0. Precipitation was cleared by filtration. The clarified sample was concentrated and diafiltered into 25 mM Tris, 200 mM NaCl, 10% Glycerol pH 8.0 using a 10K membrane.
C. Ni-IMAC
Refolded pro-NGF was loaded on a 5 ml Ni-IMAC. Buffer A: 25 mM Tris, 300 mM NaCl, 10% Glycerol pH 8.0. Buffer B: A+500 mM Imidazole. The column was washed with Buffer A. 8 ml fractions were collected. Elution was performed with a linear gradient 0-100% Buffer B. 5 ml fractions were collected. Samples of each fraction were mixed with non-reducing NuPage SLB (Invitrogen) and separated on a 4-12% NuPAGE Novex Bis-Tris Midi gel for analysis. Fractions containing protein were pooled and dialyzed against 20 mM Na Phosphate, 50 mM NaCl, 10% glycerol pH 7.4.
D. Trypsin Digestion
Pro-β NGF was mixed with trypsin in resuspension buffer and incubated on ice for 30 min. Immobilized inhibitor was added and incubated for 15 min and then filtered.
E. Sepharose Cation Exchange Chromatography
The sample was loaded on a 5 ml SP Sepharose high performance chromatography column (GE Healthcare). Buffer A: 20 mM Na Phosphate, 50 mM NaCl, 10% Glycerol pH 7.4, Buffer B: A+1 M NaCl. The column was washed with Buffer A. Elution was performed with linear gradient 0-100% Buffer B. 5 ml fractions were collected. The fractions were separated on a 4-12% Criterion XT Bis-Tris Midi gel for analysis. Fractions containing protein were pooled, dialyzed in PBS pH 7.4, and concentrated.
This work resulted in the production of several milligrams of purified canine NGF for characterization studies and for studies of anti-NGF canine antibodies.
Example 7
Characterization of Sublconed and Purified Hybridoma Antibodies
A. Canine NGF Direct Binding ELISA
To determine if purified mouse anti-NGF mAbs bind to canine β NGF, ELISA plates were coated with 50 μl/well of canine NGF (Abbott Laboratories) at 1 μg/ml in PBS and incubated over night at 4° C. The plates were washed three times with PBS+Tween buffer. The plates were blocked with 200 μl/well of 2% milk in PBS for 1 hour at room temperature. The plates were washed three times as above. Purified antibodies were diluted to 0.4, 2, or 10 μg/ml. 50 μl of each concentration of purified antibody was added to the plates. The plates were incubated for 1 hour at room temperature. The plates were washed. 50 μl of a 5000-fold diluted goat anti-mouse IgG Fc-HRP (Thermo, catalog #31439) was added. The plates were incubated for 1 hour at room temperature. 50 μl of TMB (Invitrogen, catalog #00-2023) was added and the reaction was stopped using 2N H2SO4 (VWR, catalog #BDH3500-1). The absorbance at 450 nm was read on a Spectromax 2E plate reader (Molecular Devices). The results are shown in Table 9, and the numerical value indicates binding of mouse anti-NGF antibodies to canine β NGF.
TABLE 9
Canine NGF Direct Binding ELISA Data Using Purified Anti-NGF mAbs
μg/ml
PR-
PR-
PR-
PR-
PR-
PR-
PR-
PR-
PR-
PR-
PR-
Mab
1254970
1254971
1254972
1254973
1254974
1254977
1254978
1254979
1254980
1254981
1254982
10
0.530
0.497
0.154
0.905
0.552
0.552
0.579
0.683
0.491
0.610
0.208
2
0.342
0.324
0.091
0.836
0.383
0.414
0.458
0.566
0.334
0.458
0.142
0.4
0.176
0.165
0.071
0.769
0.209
0.223
0.253
0.313
0.168
0.229
0.095
B. TF-1 Cell Proliferation Potency Assay
TF-1 is a human erythroleukaemic cell line that expresses human TrkA and proliferates in response to recombinant β NGF. To determine if purified anti-NGF mAbs blocked NGF-induced proliferation, TF-1 cells (ATCC# CRL-2003) were maintained at 37° C. and 5% CO2 in RPMI (Gibco, cat #11875-093) media containing recombinant human GM-CSF at 2 ng/mL (R&D Systems, cat #215-GM) and fetal bovine serum (FBS, Hyclone, cat #SH 30070.03). GM-CSF and FBS was removed 24 hours before the assay. On day one of the assay each anti-NGF mAb was titrated (concentrations ranging from 33.3 nM to 1.7 fM) and added to a fixed concentration of recombinant canine NGF (70 pM) and TF-1 cells (2.5×104 cells/well) in RPMI+4% FBS for 72 hours. Cell proliferation was measured using Cell Titer-glo (Promega, cat #G7571). The IC50 values of each anti-NGF mAb on canine NGF-induced TF-1 cell proliferation is shown in Table 10, and the data shows that in the presence of 70 pM canine NGF, most of the anti-NGF antibodies display sub-nM potencies, and some display potencies of less than 50 pM.
TABLE 10
Potency of Mouse Anti-NGF Antibodies on Canine NGF-Induced
TF-1 Cell Proliferation
Moniker
Lot
IC50 (nM)
PR-1254970
1734671
0.662
PR-1254971
1734673
1.088
PR-1254972
1734675
0.303
PR-1254973
1734676
0.039
PR-1254974
1734677
0.230
PR-1254977
1734678
0.217
PR-1254978
1734679
0.978
PR-1254979
1734680
0.288
PR-1254980
1734681
0.343
PR-1254981
1734682
0.046
PR-1254982
1734683
0.025
C. SureFire Cellular pERK and PathHunter Assays
To determine if purified mouse anti-NGF mAbs blocked canine NGF-induced cellular responses, purified antibodies were characterized by titration in the SureFire cellular pERK (using 128 pM canine β NGF in each test well) and PathHunter assays (using 441 pM canine NGF in each test well) as described in Example 2 Sections C and D. The IC50 of each anti-NGF mAb on canine β NGF-induced cellular responses is summarized in Table 11, and the data shows that in the presence of 128 pM canine NGF all the anti-NGF antibodies display sub-nM potencies, and some display potencies of less than 50 pM (pERK assay). Also, in the presence of 441 pM canine NGF, all the anti-NGF antibodies display sub-nM potencies, and some display potencies of less than 150 pM (PathHunter assay).
TABLE 11
Summary of pERK and Path Hunter Assay Data for Purified
Anti-NGF mAbs
SureFire pERK
PathHunter
Antibody
IC50 (nM)
IC50 (nM)
PR-1254970
0.02711
0.3346
PR-1254971
0.04750
0.4986
PR-1254972
0.2282
0.3133
PR-1254973
0.01876
0.1428
PR-1254974
0.01561
0.2464
PR-1254977
0.01759
0.1810
PR-1254978
0.02466
0.3559
PR-1254979
0.01627
0.2414
PR-1254980
0.01371
0.3812
PR-1254981
0.02135
0.2794
PR-1254982
0.005804
0.1505
Example 8
Characterization of Purified Anti-NGF Antibodies Following Hybridoma Subcloning
A. Mass Spectrophotometry (MS) and Size Exclusion Chromatography (SEC) Analysis on Anti-NGF Antibodies
The mouse anti-NGF mAbs were reduced using 1M DTT and analyzed using HPLC/MS on a 6224 TOF mass spectrometer and a 1200 HPLC (Agilent technologies) using a Vydac C4, 1 MM×150 mm column (CN#214TP5115, the Nest Group) at a flow rate of 50 μl/min Buffer A: 99.9% HPLC water+0.1% FA+0.01% TFA and buffer B: 99.9% ACN+0.1% FA+0.01% TFA. The LC equilibrium and sample desalting was performed using 5% buffer B for 7 min. The separation gradient was performed using 30% to 50% Buffer B for 10 min and a washing step was performed at 95% buffer B for 10 mins. The TOF acquisition parameters were: gas temperature at 350 C and OCT/RF at 750V. The mass range was from 600-3200 m/z and the rate specified was 1.03 spectra/s. Qualitative analysis software (Agilent) was used to deconvolute antibody molecular weights.
The antibodies were analyzed on Shimadzu LC-10AVP system (Shimadzu Scientific). The SEC column used was a Superdex-200 10/300L (GE Healthcare). The flow rate was 0.75 ml/min and UV280 was used to monitor peaks. The buffer used was Na2SO4+92 mM NaPO4+5 mM NaZ3, pH 7.0. The reagent antibody was injected in 10 μL (10 μg). The gel protein markers on SEC were from Bio-Rad (CN#151-1901). The MS and SEC results are summarized in Table 12. This data determined the hybridoma-derived antibodies were highly monomeric following purification. In addition, the molecular weights of the heavy and light chains comprising the hybridoma-derived antibodies were determined.
B. Antibody Isotype Determination
The isotype of the anti-NGF mAbs was determined using the Zymed Mouse MonoAb-ID Kit (Invitrogen catalog#90-6550 lot#1407589). The isotyping results are summarized in Table 12. This data indicates that murine IgG1/k, IgG2a/k, and IgG2b/k mouse antibodies are capable of binding and neutralizing NGF.
TABLE 12
Isotyping, Size Exclusion Chromatography, and Mass
Spectrometry Analysis of Anti-NGF Antibodies
Molecular
Molecular
%
weight (Dal)
weight (Dal)
Hybridoma Name
Moniker
Lot
Isotype
Monomer
Light Chain
Heavy Chain
ML129-14G6.3H3
PR-1254970
1734671
IgG1 Kappa
96.9
24221.43
49479.67
ML129-2G11.3B1
PR-1254971
1734673
IgG1 Kappa
96.8
24156.26
49491.69
ML129-20B10.3F4
PR-1254972
1734675
IgG2b Kappa
99.0
24159.34
50329.24
ML129-2B12.5G9
PR-1254973
1734676
IgG2b Kappa
99.4
23539.38
51102.21
ML129-17G6.3E7
PR-1254974
1734677
IgG1 Kappa
98.8
24221.43
49479.45
ML129-21D4.4A11
PR-1254977
1734678
IgG1 Kappa
98.4
24221.46
49479.70
ML129-4B6.4H3
PR-1254978
1734679
IgG1 Kappa
96.7
24170.40
49533.92
ML129-22G3.3F3
PR-1254979
1734680
IgG2a Kappa
99.0
24221.42
50123.17
ML129-23F1.4G3
PR-1254980
1734681
IgG1 Kappa
99.5
24221.42
49493.95
ML130-14A9.5B12
PR-1254981
1734682
IgG1 Kappa
99.1
24180.28
50241.85
ML130-3F7.4A8
PR-1254982
1734683
IgG1 Kappa
99.4
23708.54
50289.13
Example 9
Binding Kinetics of Anti-NGF Antibodies
A biomolecular protein interaction analysis was used to evaluate the binding kinetics of the interaction between the purified anti-NGF hybridoma antibodies and recombinant canine β NGF. The antibodies were captured using a goat anti-mouse IgG FC (10000 RU) surface which was directly immobilized to a CM5 chip using an amine coupling procedure according to the manufacturer's instructions (Biacore). A sample size of 5 μl of antibody at a concentration of 1 μg/ml was captured at 10 μL/minute. Recombinant canine NGF was used as the antigen. Canine NGF was injected at 75 μl/min (concentration range: 5-0.039 nM) for mouse antibodies. The association rate was monitored for 3.3 minutes and the dissociation rate was monitored for 10 minutes. Aliquots of canine NGF were also simultaneously injected over a reference reaction CM surface to record any nonspecific binding background. The instrument sensitivity for on-rate is 1×107, such that any on-rate that is faster than 1×107 may not be accurately measured; the instrument sensitivity for off-rate is 1×10−6, such that any off-rate that is slower than 1×10−6 may not be accurately measured. Therefore, an on-rate that is faster than 1×107 is recorded as >1×107 and an off-rate that is slower than 1×10−6 is recorded as <1×10−6. The biomolecular protein interaction analysis results are summarized in Table 13. This data indicates that the isolated murine anti-NGF mAbs have fast on-rates (from greater than 7×106) and slow off-rates (from less than 1×10−3). The overall KDs of the murine anti-NGF mAbs range from about 300 pM to 0.1 pM demonstrating efficient binding of the purified anti-NGF hybridoma antibodies to recombinant canine β NGF.
TABLE 13
Binding Kinetics of Anti-NGF mAbs to Canine NGF
On rate
Off rate
Overall affinity
Antibody
(1/Ms)
(1/s)
(M)
PR-1254972
Expt 1
>1 × 107
3.14 × 10−3
<3.14 × 10−10
lot: 1734675
Expt 2
>1 × 107
3.21 × 10−3
<3.21 × 10−10
Average
>1 × 107
3.18 × 10−3
<3.18 × 10−10
PR-1254973
Expt 1
>1 × 107
1.21 × 10−4
<1.21 × 10−11
lot: 1734676
Expt 2
>1 × 107
1.38 × 10−4
<1.38 × 10−11
Average
>1 × 107
1.30 × 10−4
<1.30 × 10−11
PR-1254977
Expt 1
>1 × 107
1.39 × 10−4
<1.39 × 10−11
lot: 1734678
Expt 2
>1 × 107
1.60 × 10−4
<1.6 × 10−11
Average
>1 × 107
1.50 × 10−4
<1.5 × 10−11
PR-1254980
Expt 1
>1 × 107
2.37 × 10−4
<2.37 × 10−11
lot: 1734681
Expt 2
>1 × 107
2.25 × 10−4
<2.25 × 10−11
Average
>1 × 107
2.31 × 10−4
<2.31 × 10−11
PR-1254981
Expt 1
8.67 × 106
1.27 × 10−4
1.47 × 10−11
lot: 1734682
Expt 2
7.48 × 106
1.40 × 10−4
1.87 × 10−11
Average
8.08 × 106
1.34 × 10−4
1.67 × 10−11
PR-1254982
Expt 1
>1 × 107
<1 × 10−6
<1 × 10−13
lot: 1734683
Expt 2
>1 × 107
<1 × 10−6
<1 × 10−13
Average
>1 × 107
<1e × 10−6
<1 × 10−13
Example 10
Method for Identifying Anti-NGF Antibody Sequences from Hybridomas by Cloning and Sequencing
To identify the nucleotide and amino acid sequence of the six subcloned hybridoma mAbs shown in Table 13, the RNA from individual hybridoma cultures was extracted with Qiagen RNeasy kit (Qiagen, cat #74104). RNA was reverse-transcribed and cDNA antibody sequences amplified using the Qiagen One-Step RT-PCR kit (Qiagen, catalog #210212). Forward primers were degenerate and designed to anneal to the variable regions (heavy chain primers: 1HA, 1HB, 1HC, 1HD, 1HE, 1HF; and light chain primers: 1LA, 1LB, 1LC, 1LD, 1LE, 1LF, 1LG) (EMD4 Biosciences catalog #69896). Reverse primers were also degenerate and made to constant regions of gamma (heavy chains) and kappa (light chains). PCR products of approximately 400-450 base pairs were gel isolated and purified with Qiagen Gel Extraction kit (Qiagen, cat #28706).
Purified PCR products were cloned into TOPO TA cloning vectors (Invitrogen, cat #K4500-01SC). Each topoisomerase reaction mixture was used to transform TOP 10 chemically competent bacteria and plated on LB plates with 75 μg/ml Ampicillin and 60 μl 2% Bluo-Gal (Invitrogen, cat #15519-028). Isolated colonies were picked from the LB plate to inoculate 20 μl LB broth/100 μg/ml carbenicillin. One μl of this mini-culture was used in a PCR reaction with M13 forward and reverse primers to amplify the insert in the TOPO vector. PCR products were separated on 2% agarose gels; samples indicating an appropriately-sized insert in the vector were sequenced using an Applied Biosystems model 3730S DNA sequencer. DNA sequences derived from the identification of all murine mAb heavy and light chain variable domains were translated into protein sequence and are shown in FIG. 1 to FIG. 24.
Example 11
Homology Modeling of Murine Anti-NGF Antibodies
The sequences of the heavy and light chain variable regions of each anti-NGF antibody were imported into InsightII (Accelrys, San Diego, Calif.). Each sequence was used as a template for BLAST to find the Xray crystal structures from the Protein Data Bank (www.rcsb.org) which were closest in identity. One structure for each of the heavy and light chains was selected based both on percent identity and on matching the exact length of all CDR loops. The sequences of each template and each query sequence were aligned and standard homology modeling techniques used to construct homology models of each chain. The complex of both modeled chains was then minimized for 50 cycles of restrained (500 Kcal/Angstrom for all heavy atoms) conjugate gradient minimization using the CVFF forcefield in the DISCOVER program (Accelrys, San Diego, Calif.).
The likelihood that a given framework residue would impact the binding properties of the antibody depends on its proximity to the CDR residues. Therefore, using the model structures, residues that fell within 5 Å of any CDR atom were identified as most important and were recommended to be candidates for retention of the murine residue in the caninized antibody sequences. A change in nucleotide(s) in a mutant gene that restores the original sequence and hence the original phenotype is often referred to as a back mutation. Therefore, we refer to residues that are candidates for retention of the murine residue in the caninized antibody sequences as backmutations.
Example 12
Identification of Canine Heavy and Light Chain Antibody Sequences from Canine PBMCs
To identify canine Ig heavy and lamda light chain antibody variable domain amino acid sequences, RNA was isolated from mongrel canine peripheral blood mononuclear cells (PBMCs) using an RNEasy kit (Qiagen #74104). Canine PBMC mRNA was reverse transcribed (RT) with SuperScript III reverse transcriptase (Invitrogen catalog #18080-093) and cDNAs were amplified using the 5′ RACE System (Rapid Amplification of cDNA Ends) (Invitrogen #18374-058). RT and PCR primers (RK323, RK324, RK122, LG010, LG011, LG012) are described in patent publication number: U.S. Pat. No. 7,261,890 B2 entitled Methods for Using Canine Immunoglobulin Variable Domains and Caninized Antibodies). Primers RK323 and RK324 were used for canine IgG reverse transcription followed by nested PCR with RK326 and the Abridged Anchor Primer (AAP) (Invitrogen). LG011 was used for canine lambda light chain RT PCR, followed by nested PCR with LG010 and LG012 and AAP.
The resulting PCR products were separated by agarose gel electrophoresis. The 600 base pair (canine lambda and kappa light chains) and 800 base pair (canine Ig heavy chain) PCR products were purified from the agarose using a Gel Extraction kit (Qiagen #28706) and cloned into the TA site of the pCR2.1 TOPO vectors using the TOPO-TA Cloning system (Invitrogen #K4500-01SC). Transformed TOP10 bacteria were selected and plasmid DNA was isolated using Qiaprep Spin Mini-Prep Kit (Qiagen #27104). Plasmid DNA from 25 heavy chain, 38 kappa light chain and 23 lambda light chain colonies was sequenced to identify the nucleotide and corresponding amino acid sequences. Complete variable domain sequence data were obtained from 25 heavy chain, 38 kappa light chain and 19 lambda light chain clones. Variable domain sequence data including the leader peptide (when identified) are shown in Tables 14, 15 and 16. All derived heavy chain and light chain sequence are unique compared to those disclosed in patent publication number: U.S. Pat. No. 7,261,890 B2.
TABLE 14
Canine Heavy Chain Variable Domain Sequences
Derived from Canine PBMC RNA
Name
Sequence
Ca-1005
EVQLEESGGDLVKPGGSLRLSCVASGFSIGSYGMSWVRQSPGK
GLQWVAWIKYDGSRTFYADAVKGRFTISRDNAKNTLFLQMNSL
RAEDTAVYFCVKGPNSSWLPSTYFASWGQGTLVTVSS
(SEQ ID NO: 178)
Ca-2301
EMQLVESGGDLVRPGGSLRLSCVASGFTFSTYGMTWVRQSPGK
GLQWVATIGPGGRNTYYADAVKGRFTISRDDAENTLFLQMNSL
RAEDTAVYYCAQAFDATYYTSFDCWGRGSLVAVSS
(SEQ ID NO: 86)
Ca-2302
MESVLSWVFLVALLQGIQGEIRLVESGGDLVKPGGSLRLSCVA
SGFIFGNYDMSWVRQAPGKGLQWVAAVRYDGSSTYYSDAVKGR
ITISRDDPGNTVYLQLDSLRAEDTATYYCVRGGYYSSSFYIGG
AFGHWGPGTLITVSS (SEQ ID NO: 87)
Ca-2303
MECVLGWVFLVAILRGVQGEVQLVESGGDLVKPGGSLRLSCVA
SGFTFSDYYMSWIRQAPGKGLQWVADISDGGDGTGYAGAVKGR
FTVSRENVKNTLYLQMNDLRAEDTAIYYCTKAREMYGYRDFDS
WGPGTLVTVSS (SEQ ID NO: 88)
Ca-2304
MESVLGLVALLTILKGVQGEVQLVESGGDLVKPGGSLRLSCVA
SGFTFSNYYMTWVRQAPGKGLEWVGYIHNGGTYTYYADAVKGR
FTISRDDAKNTLYLEMNSLRAEDTAVYYCGKMIFDYWGQGTLV
TVSS (SEQ ID NO: 89)
Ca-2305
MESALSWVFLVTILKGVQGEVLLVESGGDLVKPGGSLRLSCLT
SGFTFNTYDWGWVRQAPGKGLQWIAYIKKGGSDVRYADAVKGR
FTISRDDAKNTLYLQMNSLRAEDTAVYYCARSAWDSFDYWGQG
TLVTVSS (SEQ ID NO: 90)
Ca-2306
MESVFCWVFLVAILKGVRGVQGEVQLVESGGDLVKPAGSLRLS
CVASGFTFTDYSMNWVRQAPGKGLQWVATISNDGTSTDYTDAV
KGRFTVSRDSARNTVYLQMTSLRADDTATYYCVSRHSYSLLAD
YWGQGTLVTVSS (SEQ ID NO: 91)
Ca-2307
MQMPWSLLCLLAAPLGVLSEVTLQESGPGLVKPSQTLSLTCAV
SGGSVIRNYYWHWIRQRPGRGLEWMGCWSETTYYSPAFRGRIS
ITIDAATDQFSLHLNSMTTDDTAVYYCARALYPTSSWYDGMDY
WGHGASVVVSS (SEQ ID NO: 92)
Ca-2308
EVQLVESGGDLVKPGGSLRLSCESSGFIFSQYAMNWVRQAPGK
GLQWVAYIGGAGFITYHADDVKGRFTISRDNAKNTLYLQMNSL
TINDTAVYYCVRSNSRIPDYWGQGTLVAVSS
(SEQ ID NO: 93)
Ca-2309
MESVFCWVFLVAILKGVQGEVQLVESGGDLVKPGGSLRLSCVA
SGFTFSSVYMSWVRQAPGKGLQWVARITTDGTDTFYADAVKGR
FTISRDNVKNMLYLEMNSLRAEDTAIYYCGDPWQPAYPDLWGQ
GTMVTVSS (SEQ ID NO: 94)
Ca-2310
MESVLCWVFLVAILKGVQGEVHLVESGGDLVKPGGTLRLSCVA
SGFTFSQYDMSWVRQSPGKGLQWVALSRYHGGGTYYADAVKGR
FTISRDNAKNMLYLQMNSLRAEDTAVYYCVKEGSRWDLRGDYD
YWGQGTLVTVSS (SEQ ID NO: 95)
Ca-2311
MQMPWSLLCLLAAPLGVLSELTLQESGPGLVKPSQTLSLTCVV
SGGSVTSSHYWNWIRQRPGRGLEWMGYWTGNVNYNPAFQGRIS
IIGDAAKNQFSLHLSSMTTDDTAVYYCARCGIVAPGFLPIGDF
DFWGQGTLVTVSS (SEQ ID NO: 96)
Ca-2312
MESVFCWVFLVAILKGVQGEVQLVESGGDLVKPGGSLRLSCVA
SGFSFSNYFMFWGRQAPGKGLQWVARIRSDGGSTYYADAVKGR
FTISRDNARNTLYLQMNSLRAEDTATYYCAKADIIKLPEYRGQ
GTLVTVSS (SEQ ID NO: 97)
Ca-2401
ESVLGWIFLATILKGVQGEVQLVESGGDLVKPGGSLRLSCVGS
GFTFSSSWMNWVRQAPGKGLQWIAEISGTGSSTNYADAVKGRF
TISRDNDKNTLYLQMNSLRAEDTAMYYCARAAYYGNYRNDLDY
WGQGTLVTVSS (SEQ ID NO: 98)
Ca-2402
KPAGSLRLSCVASGFTFSSHSVTWVRQAPGKGLQFVAGITSGG
NNRYYTDAVRGRFTLSRDNAKNTVYLQMNSLRAEDTAMYFCAL
GSYEWLSGEFDYWGQGTLVTVSS (SEQ ID NO: 99)
Ca-2403
MESVFCWVFLVAILKGVQGEVQLVESGGDLVKPGGSLRLSCVA
SGFTLNNYFMYWVRQAPGKGLQWVARLNSNGDSTFYADAVKGR
FTISRDNAKNTLYLQMNSLRAEDTSMYYCAKDLIYGYTLWGQG
TLVTVSS (SEQ ID NO: 100)
Ca-2404
MASVLSWVFLVAIVKGVQGEVQLVESGGDLVKPGGSLRLSCVA
SGFIFNKYEVYWVRQAPGKGLEWVARILESGNPTYYAEAVEGR
FTISRDNAKNMAYLQMNSLRADDTAVYYCATPSVSSTVAIDYW
GQGALVTVSS (SEQ ID NO: 101)
Ca-2405
MQMPWSLLCLLATPLGVLSELTLQESGPGLVKPSQTLSLTCVV
SRGSVTSDYYWNWIRQRPGRGLEWMGHWIGSTAYNPAFQGRIS
ITADTAKNQLSLQLRSMTTEDTAVYFCARGSSWTPSGDSWGQG
TLVTVSS (SEQ ID NO: 102)
Ca-2406
MASVLKLGFSCRYCKKVSRVRCNXVESGGDLVKPGGSLRLSCV
ASGFIFNKYEVYWVRQAPGKGLEWVARILESGNPTYYAEAVEG
RFTISRDNAKNMAYLQMNSLRADDTAVYYCATPSVSSTVAIDY
WGQGALVTVSS (SEQ ID NO: 103)
Ca-2407
MDCSWRIFFLLALATGVHSEVQLVQSAAEVKKPGASVKVSCKT
SGYTLTDYYIHWVQQAPGTGLHWMGWIDPEXGTTDYAQKFQGX
VTLTADTSTNTAYMELSGLRAEDTAVYYCARFPRSLDYGSFPF
DYWGQGTLVTVSS (SEQ ID NO: 104)
Ca-2408
MESVLCWVFLVAILKGVQGEVRLVESGGDLVKPGGSLRLSCVA
SGFTFRNYGMSWVRQRPGKGLQWVAAIRSDGVTYYADDLKVRF
TVSRDDARNTLYLQLNSLGAEDTAVYYCAKAPWGLYDAWGQGT
LVTVSS (SEQ ID NO: 105)
Ca-2409
MESVLSWVFLVAILQGVQGEVQVVESGGDLVKPAGSLRLSCVA
SGYSISTYTMTWVRQVPGKGLQLVAGINGDGSSTYYTDAVKGR
FTISRDNARNTVYLQMNSLRAEDTAMYYCLGEYSWFYYWGQGT
LVTVSS (SEQ ID NO: 106)
Ca-2410
MQMPWSLLCLLAAPLGVLSELTLQESGPRLVKPSQTLSLTCAV
SGGSVTTTSYWSWIRQRPGRGLEWVGYWTGTTNYSPAFQGRIS
ISADTAKNQFSLHLSSVTTEDTALYFCASKSASTSWYFSLFES
WGQGTLVTVSS (SEQ ID NO: 107)
Ca-2411
MESVLGLVFLLTILKGVQGEVQLVESGGDLVKPGGSLRLSCVA
SGFTFSSYSMSWVRQAPGKGLQWVGYIDNGGTSTYYADAVKGR
FTISRDNAKNTLYLQMNSLRAEDTAVYYCGRGSYGMEYWGHGT
SLFVSS (SEQ ID NO: 108)
Ca-2412
MESVLGLLFLVAILKGVQGEIQLVESGGDLLKPGGSLRLSCVA
SGFTFSGSDMNWIRQAPGKGLQWVAHITHEGIGTSYVGSVKGR
FTISRDNAKNTLYLQMNDLRAEDTAMYYCAYSPWNYYSFDSWG
QGTLVTVSS (SEQ ID NO: 109)
TABLE 15
Canine Lambda Light Chain Variable Domain
Sequences Derived from Canine PBMC RNA
Name
Sequence
Ca-1001
MTSTMAWSPLLLTLLTHCTVSWAQTVLTQSPSVSAVLGRRVT
ISCTGSDTNIGSHRDVQWYQLVPGKSPKTLIYGTDNRPSGIP
VRFSGSKSGNSGTLTITGIQAEDEADYYCQSYDDDLSMNVFG
GGTHLTVLG (SEQ ID NO: 110)
Ca-1002
MDWVPFYILPFIFSTGFCALPVLTQPTNASASLEESVKLTCT
LSSEHSNYIVRWYQQQPGKAPRYLMYVRSDGSYKRGDGIPSR
FSGSSSGADRYLTISNIKSEDEDDYYYCGADYTISGQYGSVF
GGGTHLTVLG (SEQ ID NO: 111)
Ca-1003
LWISGGSALGTPTMAWTHLLLPVLTLCTGSVASSVLTQPPSV
SVSLGQTATISCSGESLSKYYAQWFQQKAGQVPVLVIYKDTE
RPSGIPDRFSGSSSGNTHTLTISRARAEDEADYYCESEVSTG
TYCVRRRHPSNRPRSAQGLPLGHTLPALL
(SEQ ID NO: 204)
Ca-1006
MTSTMAWSPLLLTLLTHCTGSWAQSVLTQPASLSGSLGQRVT
ISCTGSSSNIGGYSVNWLQQLPGTGPRTIIYNNSNRPSGVPD
RFSGSRSGTTATLTISGLQAEDEADYYCSTWDSNLRTIVFGG
GTHLTVLG (SEQ ID NO: 112)
Ca-1007
MTSTMDWSPLLLTLLAHCTGSWAQSVLTQPASVSGSLGQRVT
ISCTGSTSNLGTYNVGWLQQVPGTGPRTVIYTNIYRPSGVPD
RFSGSESGSTATLTISDLQAEDEAEYYCTAWDSSLNAYVFGS
GTQLTVLG (SEQ ID NO: 113)
Ca-1008
MTSNMAWCPFLLTLLAYCTGSWAQSVLTQPTSVSGSLGQRVT
ISCSGSTNNIGIVGASWYQQLPGKAPKLLVYSDGDRPSGVPD
RFSGSNSGNSDTLTITGLQAEDEADYYCQSFDTTLDAAVFGG
GTHLTVLG (SEQ ID NO: 114)
Ca-1009
MTSTMAWSPLLLTLLAHCTVSWAQAVLTQPPSVSAALGQRVT
ISCTGSDTNIGSGYEVHWYRQVPGKSPAIIIYGNSNRPSGVP
VRFSGSKSGSTATLTITGIEAEDEADYHCQSYDGNLDGGVFG
GGTHLTVLG (SEQ ID NO: 115)
Ca-1010
MTSTMGWFPLILTLLAHCAGSWAQSVLTQPASVSGSLGQRVT
ISCTGSSPNVGYGDFVAWYQQVPGTSPRTLIYNTRSRPSGVP
DRFSASRSGNTATLTISGLQAEDEADYYCSSYDNTLIGIVFG
GGTHLTVLG (SEQ ID NO: 116)
Ca-1011
MTSTMGWSPLLLTLLAHCTGSWAQSVLTQPASVSGSLGQRVT
ITCTGSSSNIGRANVAWFQQVPGTGPRTVIYTSVKRPSGVPD
RFSGSKSGSTATLTISGLQAEDEADYYCSSWDNSLDAGVFGG
GTHLTVLG (SEQ ID NO: 117)
Ca-1012
MTSTMGWFPLLLTLLAHSTGSWAQSVLTQPASVSGSLGQRVT
ITCTGGTSNIGRGFVSWFQQVPGIGPKILIFDAYRRPSGVPD
RFSGSRSGNTATLTISGLQAEDEADYYCAVYDSRLDVGVFGS
GSQLTVLS (SEQ ID NO: 118)
Ca-1202
MTSNMAWCPFLLTLLTYCTGSWARSVLTQPASVSGSPGQKVT
IYCSGTMSDIGVLGANWYQQLPGKAPKLLVDNDGDRPSGVPD
RFSASKSGHSDTLTITGLQPEDEGDYYCQSFDSSLDAAIFGE
GTHLTVLG (SEQ ID NO: 119)
Ca-1203
SVASYVLTQSPSQNVTLRQAAHITCEGHNIGTKSVHWYQQKQ
GQAPVLIIYDDKSRPSGIPERFSGANSGNTATLTISGALAED
EADYYCLVWDSSAIWVFGEGTHLTVLG
(SEQ ID NO: 120)
Ca-1204
MTSTMAWSPLLLTLLAHFTGSWAQSVLTQPTSVSGSLGQRVT
ISCTASSSNIDRDYVAWYQQLPGTRPRALIYANSNRPSGVPD
RFSGSKSGSTATLTISGLQAEDEADYYCSTWDNSLTYVFGSG
TQLTVLG (SEQ ID NO: 121)
Ca-1205
SVASYVLTQVPSVSVNLGKTATITCEGDNVGEKYTHWYQQEY
GQAPVLIIYEDSRRPSGIPERFSGSNSGNTATLTISGARAED
ETDYYCQVWDDSGNVFGGGTHLTVLG (SEQ ID NO: 122)
Ca-1206
MTSTMGWFPLILTLLAHCAGSWAQSVLTQPASVSGSLGQRVT
ISCTGSDSNVGYGDSIAYGDSVAWYQQVPGTSPRTLIYDVTS
RPSGVPDRFSGSRSGTTATLTISGLQAEDEADYYCSSFDKTL
NGLIVGGGTHLTVLG (SEQ ID NO: 123)
Ca-1207
MTSNMAWSPLLLTLLAYCTGSWAQSALTQPTSVSGSLGQRVS
ISCSGGIHNIGSVGATWYQQLPGKAPKLLVSSDGDRPSGIPD
RFSGSRSGNSVTLTITGLQAEDEAEYYCQSFDSTLGVHVVFG
GGTHLTVLG (SEQ ID NO: 124)
Ca-1208
LCSAVGPPKTESVMTSTMGWSPLLLTLLAHCTGSWAQSVLTQ
PASVSGSLGQRVTIPCTGSSSNIDRYNVAWFQQLPGTGPKPS
SIVLLTDPQGSLIDSLAPSQAA (SEQ ID NO: 205)
Ca-1209
MTSTMAWFPLLLTLLAHYTGSWARSDLTQPASVSGSLGQRIT
ISCTGSSSNIGRNYVGWYQQLPGRGPRTVVYGINSRPSGVPD
RFSGSKSGSTVTLTISGLQAEDEADYYCSTWDDSLSVVVFGG
GTHLTVLG (SEQ ID NO: 125)
Ca-1210
MTSTMGWSPLLLTLTHWTGSWAQSVLSQPASMSGSLGLRITI
CCTGKNSNINNSYVDWNQPLAGTGPRTVIHDDGDRPSGVPDQ
FSGSKSGNTATLTISRLQAEDEADYNGASFETSFNAVFGGGT
HVTVLG (SEQ ID NO: 126)
TABLE 16
Canine Kappa Light Chain Variable Domain
Sequences Derived from Canine PBMC RNA
Ca Ka016-A1
LSWLRQKPGHSPQRLIHQVSSRDPGVPDRFSGSGSGT
DFTLTISRVEADDGGVYYCGQGSQSIPTFG QGTKVE
IKR (SEQ. ID NO. 127)
Ca Ka016-A2
MRFPSQLLGLLMLWIPGSAGDIVMTQTPLSLSVSPGE
PASISCKASQSLLHSNGNTYLYWFRQKPGQSPQRLIY
KVSNRDPGVPDRFSGSGSGTDFTLRISRVETDDAGVY
YCGQVIQDPWTFGVGTKLELKR
(SEQ. ID NO. 128)
Ca Ka016-A3
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSVSPGE
TASISCRASQTLLYSNGKNYLFWYRQKPGQSPQRLID
LASNRDPGVPDRFSGSGSGTDFTLRISRVEADDAGVY
YCGQGMEIPWTFGAGTKVELKR
(SEQ. ID NO. 129)
Ca Ka016-A4
MKFPSLLLGLLMLWIPGSTGEAVMTQTPLSLAVTPGE
VATISCRASQSLLHSDGKSYLNWYLQKPGQTPRPLIY
EASKRFSGVSDRFSGSGSGTDFTLKINRVEAEDVGVY
YCQQSLHFPPTFGPGTKVELKR
(SEQ. ID NO. 130)
Ca Ka016-A5
PDRFSGSGSGTDFTLTISRVEADDAGIYYCGQATQTP
PTFGAGTKLDLKR (SEQ. ID NO. 131)
Ca Ka016-A6
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSVRPGE
SASISCKASQSLLHSGGGTYLNWFRQRPGQSPQRLIY
EVSKRDTGVPDRFSGSGSGTDFTLRITRVEADDTGIY
YCGQNTQLPLTFGQGTKVEIKR
(SEQ. ID NO. 132)
Ca Ka016-A7
MRFPSQLLGLLMLWIPGSTGDIVMTQTPLSLSVSPGE
PASISCKASQSLLHSNGNTYLFWLRQKPGQSPQRLIY
RVSNRDPGVPDRFSGSGSGTDFTLRISRVEADDAGVY
YCGQRVRSPWTFGAGTKVEVKR
(SEQ. ID NO. 133)
Ca Ka016-A8
MRFPSQLLGLLMLWIPGSAGDIVMTQTPLSLSVSPGE
PASISCKASQSLLHSNGNTYLYWFRQKPGQSPQRLIY
KVSNRDPGVPDRFSGSGSGTDFTLRISRVETDDAGVY
YCGQVIQDPWTFGVGTKLELKR
(SEQ. ID NO. 134)
Ca Ka016-A9
MRFPSQLLGLLMLWIPGSSGDVVMAQTPLSLSVSPGE
TASISCRASQSLLHSNGNTFLFWFRQKPGQSPQRLIN
FLSNRDPGVPDRFSGSGSGTDFTLRINRVEADDAGLY
YCGQGLQAPLTFGQGTKLEIKR
(SEQ. ID NO. 135)
Ca Ka016-A10
MRFPSQLLGLLMLWIPGSNGDDVLTQTPLSLSVRPGE
TVSILCKASESLLHSDGNTYLSWVRQKAGQSPQRLMY
RVSDRDTGVPDRFSGSGSGTDFTLTISGVEADDAGIY
YCGQATHYPLEFGQGTRVEIKR
(SEQ. ID NO. 136)
Ca Ka016-A11
LMLWIPGSTGEIVLTQTPLSLSVSPGEPASISCKASQ
SLLHPNGVTYLYWFRQKPGQSPQRLIYKVSNRDPGVP
DRFSGSGSEIDFTLIISRVEADDGGIYYCGQGIQNPF
TFGQGTKLEIKR (SEQ. ID NO. 137)
Ca Ka016-A12
MRFPSQLLGLLMLWIPGSIGDIVMTQTPLSLSVSPGE
SASISCKASQSLLHSNGNTYLYWFRQKPGHSPQRLIH
QVSSRDPGVPDRFSGSGSGTDFTLRISRVEADDAGLY
YCGQGTQFPFTFGQGTKVEIKR
(SEQ. ID NO. 138)
Ca Ka016-B1
MRFPSQLLGLLMLWIPGSIGDIVMTQTPLSLSVSPGE
SASISCKASQSLLHSNGNTYLYWFRQKPGH SPQRLI
HQVSSRDPGVPDRFSGSGSGTDFTLRISRVEADDAGL
YYCGQGTQFPFTFGQGTKVEIKR
(SEQ. ID NO. 139)
Ca Ka016-B2
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSVSPGE
TASISCRASQSLLHSNGNTYSFWFRQKPGQSPQRLIN
LVSSRGPGVPDRFSGSGSGTDFTLIISRVEADDAGVY
YCGHGKEAPYTFSQGTKLEIKR
(SEQ. ID NO. 140)
Ca Ka016-B3
MRFPSQLLGLLMLWIPGSVGDIVMTQSPMSLSVGPGE
SASMSCKANQSLLYSDGITYLSWFLQRPGQSPQRLIY
EVSKRDTGVPGRFIGSGAGTDFTLRISRVEADDAGVY
YCGQALQFPLTFSQGAKLEIER
(SEQ. ID NO. 141)
Ca Ka016-B4
MRFPSQLLGLLMLWIPGSSGDVVMTQTPLSLSVRPGE
TASISCRASQSLLIISSGITKLFWYRQKPGQSPQRLV
YWVSNRDPGVPDRFTGSGSGTDFTLRISRLEADDAGI
YYCGHAIGFPLTFGQGTKVEIKR
(SEQ. ID NO. 142)
Ca Ka016-B5
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSVRPGE
SASISCKASQSLLHSGGGTYLNWFRQRPGQSPQRLIY
EVSKRDTGVPDRFSGSGSGTDFTLRITRVEADDTGIY
YCGQNTQFPLTFGQGTKVEIKR
(SEQ. ID NO. 143)
Ca Ka016-B6
MRFPSQLLGLLMLWIPGSSGGIVMTQTPLSLSVRPGE
TASISCRASQSLLYSDGNTYLFWFRQKPGQSPQRLMY
RVSDRDTGVPDRFSGSGSGTDFTLTISGVEADDAGIY
YCGQATHYPLEFGQGTXVEIKR
(SEQ. ID NO. 144)
Ca Ka016-B7
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSVRPGE
SASISCKASQSLLHSGGGTYLNVVFRQRPGQSPQRLI
YEVSKRDTGVPDRFIGSGAGTDFTLRISRVEADDAGV
YYCGQGVQGPWTIGAGTKLELQR
(SEQ. ID NO. 145)
Ca Ka016-B8
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSVSVSPGE
TASISCKASQSLLSHDGNTYLHWFRQKPGQSPQRLIY
KVSNRDTGVPDRFSGSGSGTDFTLKISRVEADDTGVY
YCGQITQDPFTFGQGTKLEIKR
(SEQ. ID NO. 146)
Ca Ka016-B9
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSVSPGE
TASISCRASQSLLHSNGNTYLFWFRQKPGQSPQRLIN
WVSNRDPGVPDRFGGSGSGTDFTLRISRVEADDAGIY
YCGQGIQGPYTFSQGTKLEIKR
(SEQ. ID NO. 147)
Ca Ka016-B10
MRFPSQFLGLLMLWIPGSSGDIAMTQTPLSLSVGPGE
TASITCKASQSLLHSNGNTYLFWFRQKPGQSPQRLIY
LVSNRDPGVPDRFSGSGSGTDFTLTISRVEADDAGIY
YCGQATQTPPTFGAGTKLDLKR
(SEQ. ID NO. 148)
Ca Ka016-B11
MRFPSQLLGLLMLWIPGSSGDIVMAQTPLSLSVSPGE
PASISCKASQSLLHSDGRTCLSWFRQKSGQSPQRLIY
EVSNRDTGVPDRFSGSGSGTDFTLRISRVEADDTGIY
YCGQTVQFPLTFGQGTKLEIKR
(SEQ. ID NO. 149)
Ca Ka016-B12
GQSPQRLIYKVSNRDPGVPDRFSGSGSGTDFTLRISR
VEPEDVGVYYCGQGTLNPWTFGAGTKVELK R
(SEQ. ID NO. 150)
Ca Ka017-1
MRFPSQLLGLLMLWIPGSSGDVVMTQTPLSLSVSPGE
TASISCRASQSLLHSNGNTFLFWFRQ*PGQSPQRLIN
FVSNRDPGVPDRFSGSGSGTDFTLRISRVEADDAGIY
YCGQGLLAPPTFGQGTKVEIRR (SEQ. ID NOS
151 and 210, respectively)
NOTE: * INDICATES A STOP CODON
Ca Ka017-2
MRFPSQLLGLLMLWIPGSGGDIVMTQTPPSLSVSPRE
PASISCKASQSLLRSNGNTYLYWFRQKPGQSPEGLIY
RVSNRFTGVSDRFSGSGSGTDFTLRISTVEADDAGVY
YCGQATQFPSTFSQGTKLEIKR
(SEQ. ID NO. 152)
Ca Ka017-3
MRFPSQLLGLLMLWIPGSXGDIVLTQTPLSLSVSPGE
PASISCKASQSLLHSNGITYLNWYRQRPGQSPQXLIY
KVSNRDTGVPDRFSGSGSGTDFTLRXSKVEADDTGIY
YCGQDTQFPLTLGXGTHXEIKR
(SEQ. ID NO. 153)
Ca Ka017-5
MRFPSQLLGLLMLWIPGSTGDIVMTQTPLSLSVSPGE
PASIYCKASQSLLHSNGKTFLYWFRQKPGQS PQRLI
YRVSNRDPGVPDRFSGSGSGTDFTLRISRVEADDAGI
YYCGQGIQDPTFGQGTKVEIKR
(SEQ. ID NO. 154)
Ca Ka017-6
MRFPSQLLGLLMLWIPGSGGDIVMTQTPPSLSVSPRE
AASISCKASQSLLKSNGNTYFYWFRQKPG QVSEGLI
YKVSSRFTGVSDRFSGSGSGTDFTLRISRVEADDAGV
YFCGQALQFPYTFSQGTKLDIKR
(SEQ. ID NO. 155)
Ca Ka017-10
MRFPSQLLGLLMLWIPESGGDVVLTQTPPSLSLSPGE
TASISCKASRSLLNSDGSTYLDWYLQKPGQSPRLLIY
LVSNRFSGVSDRFSGSGSGTDFTLTISRVEADDAGVY
YCGQGSRVPLTFGQGTKVEIKR
(SEQ. ID NO. 156)
Ca Ka017-11
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSVSPGE
TASISCRASQSLLHRNGITYLSWFRQRPGQSP QRLI
NLVSNRDPGVPDRFSGSGSGTDFTLRISRVEADDVGV
YYCGHGLQTPYTFGQGTSLEIER
(SEQ. ID NO. 157)
Ca Ka017-12
MRFPSQLLGLLVLWIPGSSGDIVMTQTPLSLSVSPGE
TVSISCRASQSLLYSDGNIYLFWFRRKPGQSP QHLI
NLVSNRDPGVPDRFSGSGSGTDFTLRISRVEADDAGV
YYCGQGTQPPYTFSQGTKVEIKR
(SEQ. ID NO. 158)
Ca Ka017-13
MRFPSQLLGLLMLWIPESGGDVVLTQTPPSLSLSPGE
TASISCKASRSLLNSDGSTYLDWYLQKPGQS PRLLI
YLVSNRFSGVSDRFSGSGSGTDFTLTISRVEADDAGV
YYCGQGSRVPLTFGQGTKVEIKR
(SEQ. ID NO. 159)
Ca Ka017-14
MRFPSQLLGLLMLWIPGSSGDIVMAQTPLSLSVSPGE
TASISCRASQSLLHSNGITYLFWYRQKPGQS PQRLI
SMVFNRDPGVPDRFGGSGSGTDFTLRISRVEADDAGL
YFCGHGTQIPYSFSQGTKLEIKR
(SEQ. ID NO. 160)
Ca Ka017-16
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSISPGE
PASISCKASQSLLHSGGDTYLNWFRQRPGQS PQLLI
NRVSSRKKGVPDRFSGSGSGTEFTLRISRVEADDAGI
YFCGQGTQFPYTFSQGTKLEIKR
(SEQ. ID NO. 161)
Ca Ka017-20
MRFPSQLLGLLMLWIPGSGGDIVMTQTPPSLSVSPGE
PASMSCKASQSLLHSNGNTYLYWFRQKP GQSPEALI
YKVSNRFTGVSDRFSGSGSGTDFTLRINRVEADDVGV
YYCGQGIQIPYTFSQGTKLEIKR
(SEQ. ID NO. 162)
Ca Ka017-23
MRFPSQLLGLLMLWIPGSTGEIVLTQTPLSLSVSPGE
SASISCKASQSLLYSNGNTYLYWFRQKAGQSP QRVI
YRVSNRDPGVPDRFSGSGSGTDFTLRISSVENDDAGV
YYCGQGSEDPPTFGAGTKVELKR
(SEQ. ID NO. 163)
Ca Ka017-24
MRFPSQLLGLLTLWIPGSTGDIVMTQTPLSLSVSPGE
PASISCKASQSLLHSNGNTYLYWFRQKPGQS PQRLI
YKVSNRDPGVPXRFSGSGSGTDFTLRVSXVEADDAGV
YYCGQGVQDPFTFGQGTKLEIKR
(SEQ. ID NO. 164)
Example 13
CDR-Grafting to Create Caninized Monoclonal Antibodies
To generate caninized antibody sequences from mouse anti-NGF antibodies, each murine variable heavy chain antibody gene sequence was separately aligned against 36 canine Ig germline variable heavy chain sequences using Vector NTI software. Eleven canine Ig germline variable heavy chain sequences were derived from U.S. Pat. No. 7,261,890 B2, (Methods for Using Canine Immunoglobulin Variable Domains and Caninized Antibodies), the contents of which are herein incorporated by reference, and 25 canine Ig germline variable heavy chain sequences were derived from Table 14 (Canine Heavy Chain Variable Domain Sequences Derived from Canine PBMC RNA). Each murine variable light chain gene sequence was separately aligned against 68 germline variable light chain sequences (derived from U.S. Pat. No. 7,261,890 B2) using Vector NTI software. Canine variable domain sequences having the highest overall homology to the original murine sequences were selected for each heavy chain and light chain sequence to provide the framework sequence. In silico construction of complete CDR grafted antibodies was accomplished by substitution of canine variable domain CDR sequences with murine CDR sequences (derived from the subcloned anti-NGF antibody hybridoma mAbs). To identify residues in each sequence, the first amino acid in each listed sequence was defined as 1, and all remaining residues numbered consecutively thereafter using Kabat numbering system.
The heavy chain CDR sequences from PR-1254972 were grafted in silico onto canine 894 as follows: (1) One N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Six back-mutations (Q3H, V37I, Q46E, D73N, T77N, R83K) were introduced to make the 72.2 VH sequence. (3) One, two, three, four, five, or six of the back-mutations disclosed above could be introduced into 72.2 VH to maintain antibody affinity to NGF after caninization of mAb 72.2. (4) One, two, three, four, five, or six of these back-mutations may be substituted during subsequent affinity maturation of 72.2 VH. 72.3 VH was generated by introducing the back-mutations in 72.2 VH with the addition of H39Q back-mutation. 72.4 VH was generated by introducing back-mutations Q3H, H39Q, Q46E, D73N. The light chain CDR sequences from PR-1254972 were grafted in silico onto canine 1001 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Four back-mutations (I2V, V3L, Q45K, S59P) were introduced to make the 72.2 VL sequence. (3) One, two, three, or four of these back-mutations could be introduced into 72.2 VL to maintain antibody affinity to NGF after caninization of mAb 72.2. (4) One, two, three, or four of these back-mutations may be substituted during subsequent affinity maturation of 72.2 VL. 72.4 VL was generated by introducing back-mutations Q45K, and S59P.
The heavy chain CDR sequences from PR-1254973 were grafted in silico onto canine 894 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Eight back-mutations (T24A, M48I, V67A, L69V, T73K, N76S, V78A, A93T) were introduced to make the 73.2 VH sequence. (3) One, two, three, four, five, six, seven, or eight of these back-mutations could be introduced into 73.2 VH to maintain antibody affinity to NGF after caninization of mAb 73.2. (4) One, two, three, four, five, six, seven, or eight of these eight back-mutations may be substituted during subsequent affinity maturation of 73.2 VH. 73.4 VH was generated by introducing back-mutations T24A, T73K, A93T. The light chain CDR sequences from PR-1254973 were grafted in silico onto canine 1034 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Eight back-mutations (I1D, V3Q, S22T, F36H, R46L, 148V, D60S, D70Q) were introduced to make the 73.2 VL sequence. (3) One, two, three, four, five, six, seven, or eight of these back-mutations could be introduced into 73.2 VL to maintain antibody affinity to NGF after caninization of mAb 73.2. (4) One, two, three, four, five, six, seven, or eight of these eight back-mutations may be substituted during subsequent affinity maturation of 73.2 VL. 73.4 VL was generated by introducing back-mutations I1D, V3Q, F36H, R46L, D60S, D70Q.
The heavy chain CDR sequences from PR-1254977 were grafted in silico onto canine 894 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Eight back-mutations (T24A, Q38K, M48I, R66K, V67A, T68S, L69I, V78A) were introduced to make the 77.2 VH sequence. (3) One, two, three, four, five, six, seven, or eight of these back-mutations could be introduced into 77.2 VH to maintain antibody affinity to NGF after caninization of mAb 77.2. (4) One, two, three, four, five, six, seven, or eight of these back-mutations may be substituted during subsequent affinity maturation of 77.2 VH. 77.3 VH was generated by introducing the back-mutations in 77.2 VH with the addition of R94G back-mutation. 77.4 VH was generated by introducing back-mutations T24A, Q38K, and R94G. The light chain CDR sequences from PR-1254977 were grafted in silico onto canine 997 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Four back-mutations (L2V, F36Y, R46L, S98G) were introduced to make the 77.2 VL sequence. (3) One, two, three, or four of these back-mutations could be introduced into 77.2 VL to maintain antibody affinity to NGF after caninization of mAb 77.2. (4) One, two, three, or four of these back-mutations may be substituted during subsequent affinity maturation of 77.2 VL. 77.4 VL was generated by introducing back-mutations F36Y and R46L.
The heavy chain CDR sequences from PR-1254981 were grafted in silico onto canine 876 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Six back-mutations (Q46E, G49A, T77N, R83K, L91Y, E93T) were introduced to make the 81.2 VH sequence. (3) One, two, three, four, five, or six of these back-mutations could be introduced into 81.2 VH to maintain antibody affinity to NGF after caninization of mAb 81.2. (4) One, two, three, four, five, or six of these six back-mutations may be substituted during subsequent affinity maturation of 81.2 VH. 81.4 VH was generated by introducing back-mutations Q46E, G49A, L91Y, and E93T.
The light chain CDR sequences from PR-1254981 were grafted in silico onto canine 1011 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Four back-mutations (V3L, A7T, F36Y, R46L) were introduced to make the 81.2 VL sequence. (3) One, two, three, or four of these back-mutations could be introduced into 81.2 VL to maintain antibody affinity to NGF after caninization of mAb 81.2. (4) One, two, three, or four of these back-mutations may be substituted during subsequent affinity maturation of 81.2 VL. 81.4 VL was generated by introducing back-mutations A7T, F36Y, and R46L.
Alternatively, the heavy chain CDR sequences from PR-1254981 were grafted in silico onto canine 1005 VH as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Seven back-mutations (Q46E, T77N, F79Y, R83K, F91Y, V93T, K94R) were introduced to make the 81.5B VH sequence. (3) One, two, three, four, five, six, or seven of these back-mutations could be introduced into 81.5B VH to maintain antibody affinity to NGF after caninization of mAb 81.5B. (4) One, two, three, four, five, six, or seven of these seven back-mutations may be substituted during subsequent affinity maturation of 81.5B VH. 81.6B was generated by introducing back-mutations Q46E, F79Y, F91Y, and V93T. Variants 81.2B and 81.4B were generated by introducing A84K mutation to 81.5B and 81.6B, respectively.
The heavy chain CDR sequences from PR-1254982 were grafted in silico onto canine 892 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Twelve back-mutations (I3Q, 137V, M48L, 167L, T705, A71K, G73N, N76S, H77Q, L78V, S79F, T93A) were introduced to make the 82.2 VH sequence. (3) One, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve of these back-mutations could be introduced into 82.2 VH to maintain antibody affinity to NGF after caninization of mAb 82.2. (4) One, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve of these back-mutations may be substituted during subsequent affinity maturation of 82.2 VH. 82.4 VH was generated by introducing back-mutations I3Q, A71K, H77Q, S79F, and T93A. The light chain CDR sequences from PR-1254982 were grafted in silico onto canine 1034 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Ten back-mutations (I1D, V3Q, S22T, F36Y, Q45K, R46L, D60S, F71Y, T72S, Y87F) were introduced to make the 82.2 VL sequence. (3) One, two, three, four, five, six, seven, eight, nine, or ten of these back-mutations could be introduced into 82.2 VL to maintain antibody affinity to NGF after caninization of mAb 82.2. (4) One, two, three, four, five, six, seven, eight, nine, or ten of these back-mutations may be substituted during subsequent affinity maturation of 82.2 VL. 82.3 VH was generated by introducing the back-mutations in 82.2 VH with the addition of P44V back-mutation. 82.4 VL was generated by introducing back-mutations HD, V3Q, F36Y, Q45K, R46L, D60S, F71Y, and Y87F.
Example 14
Isoelectric Point of Canine Framework Amino Acids
The heavy chain framework amino acids (i.e. non-CDR amino acids) of the caninized IgG1 kappa antibodies yield a calculated isoelectric point of less than 8.0. The light chain framework amino acids, when the light chain is kappa, yield a calculated isoelectric point of less than 6.5. The isoelectric point of the caninized antibodies as a whole, i.e. heavy and light chain combined, due to the framework amino acids, and when the light chain is kappa, is less than 8.0. In comparison, the framework amino acids of human IgG1 heavy chains typically yield isoelectric points of greater than 8.0. The framework amino acids of human kappa light chains typically yield isoelectric points of greater than 6.5. The framework amino acids of whole human IgG1/k antibodies typically yield isoelectric points of greater than 8.0.
Example 15
CDR-Grafting to Create Humanized Monoclonal Antibodies
Each murine variable heavy and variable light chain antibody gene sequence (as set forth in Table 16) was separately aligned against 44 human immunoglobulin germline variable heavy chain or 46 germline variable light chain sequences (derived from NCBI Ig Blast website which is well known to those skilled in the art) using Vector NTI software. Human variable domain sequences having the highest overall homology to the original murine sequences were selected for each heavy chain and light chain antibody sequence to provide the framework (FW) 1, 2 and 3 sequences for CDR-grafting purposes. Identification of a suitable human variable heavy and light chain FW4 region (also known as the “joining” region) was accomplished by separately aligning each murine heavy chain and light chain FW4 region with 6 human immunoglobulin germline joining heavy chain and 5 germline joining light chain sequences in the NCBI database. In silico construction of complete CDR grafted variable domains was accomplished by substitution of human variable domain CDR sequences (derived from the NCBI website) with murine CDR sequences (derived from the murine antibodies) with addition of a FW4 region (derived from the NCBI website) to each 3′ end. Further humanization may be accomplished by identification of back-mutations. Full length human Igs may be produced by expressing the variable domains of each CDR-grafted mAb with an in-frame human IgG constant domain. Mouse Anti-NGF mAb CDRs grafted onto human Ig frameworks (CDR-grafted Anti-NGF Abs) produced are those listed in Table 17.
TABLE 17
Mouse Anti-NGF mAb CDRs Humanized by CDR
Grafting onto Human Ig Frameworks
Name
Sequence (CDRs are underlined)
HU72 VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYM
(CDR-GRAFT VH3-
FWVRQATGKGLEWVSTISDGGSYTYYTDNVKGRF
13/JH5)
TISRENAKNSLYLQMNSLRAGDTAVYYCARDWSD
SEGFAYWGQGTLVTVSS (SEQ ID NO: 165)
Hu73 VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWM
(CDR-GRAFT VH1-
HWVRQAPGQGLEWMGRIDPYGGGTKHNEKFKRRV
18/JH6)
TMTTDTSTSTAYMELRSLRSDDTAVYYCARSGYD
YYFDVWGQGTTVTVSS (SEQ ID NO: 166)
HU77 VH
QVQLVQSGAEVKKPGSSVKVSCKASGFNIKDTYI
(CDR-GRAFT VH1-
YWVRQAPGQGLEWMGRIDPANGNTIYASKFQGRV
69/JH6)
TITADKSTSTAYMELSSLRSEDTAVYYCARYGYY
AYWGQGTTVTVSS (SEQ ID NO: 167)
HU80 VH
QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYI
(CDR-GRAFT VH1-
YWVRQAPGQGLEWMGRIDPANGNTIYASKFQGRV
18/JH6)
TMTTDTSTSTAYMELRSLRSDDTAVYYCARYGYY
AYWGQGTTVTVSS (SEQ ID NO: 168)
HU81 VH
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNHYM
(CDR-GRAFT VH3-
YWVRQAPGKGLEWVGSISDGGAYTFYPDTVKGRF
15/JH1)
TISRDDSKNTLYLQMNSLKTEDTAVYYCTTEESA
NNGFAFWGQGTLVTVSS (SEQ ID NO: 169)
HU82 VH
QVTLKESGPVLVKPTETLTLTCTVSGFSLTGYNI
(CDR-GRAFT VH2-
NWIRQPPGKALEWLAMIWGYGDTDYNSALKSRLT
26/JH6)
ISKDTSKSQVVLTMTNMDPVDTATYYCARDHYGG
NDWYFDVWGQGTTVTVSS (SEQ ID NO: 170)
HU72 VL
DIVMTQTPLSLPVTPGEPASISCRSSQSIVQSNG
(CDR-GRAFT
NTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFS
01/JK2)
GSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPFT
FGQGTKLEIKR (SEQ ID NO: 171)
HU73 VL
DIQMIQSPSFLSASVGDRVSIICRASENIYSFLA
(CDR-GRAFT
WYLQKPGKSPKLFLYNANTLAEGVSSRFSGRGSG
L22/JK2)
TDFTLTIISLKPEDFAAYYCQHHFGTPFTFGQGT
KLEIKR (SEQ ID NO: 172)
HU77 VL
DIVMTQTPLSLPVTPGEPASISCKSTKSLLNGDG
(CDR-GRAFT
FTYLDWYLQKPGQSPQLLIYLVSNRFSGVPDRFS
01/JK2)
GSGSGTDFTLKISRVEAEDVGVYYCFESNYLFTF
GQGTKLEIKR (SEQ ID NO: 173)
HU80 VL
DIVMTQTPLSLPVTPGEPASISCKSTKSLLNGDG
(CDR-GRAFT
FTYLDWYLQKPGQSPQLLIYLVSNRFSGVPDRFS
01/JK2)
GSGSGTDFTLKISRVEAEDVGVYYCFESNYLFTF
GQGTKLEIKR (SEQ ID NO: 174)
HU81 VL
DIVMTQTPLSLPVTPGEPASISCRSSQSILHSNG
(CDR-GRAFT
NTYLEWYLQKPGQSPQLLIYRVSNRFSGVPDRFS
01/JK2)
GSGSGTDFTLKISRVEAEDVGVYYCFQGAHVPFT
FGQGTKLEIKR (SEQ ID NO: 175)
HU82 VL
DIQMTQSPSSLSASVGDRVTITCRASQDITNYLN
(CDR-GRAFT
WYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSG
08/JK2)
TDFTFTISSLQPEDIATYYCQQGKTLPRTFGQGT
KLEIKR (SEQ ID NO: 176)
Example 16
Method for Constructing Full-Length Mouse/Canine Chimeric and Caninized Antibodies
Using conventional molecular biology techniques, a cDNA fragment encoding the canine IgG1 constant region (which was obtained from the IMGT®, the International ImMunoGeneTics information system, which is the global reference in immunogenetics and immunoinformatics, created in by Marie-Paule Lefranc (Université Montepellier 2 and CNRS)) was synthesized and ligated to the 3′ end of each of the heavy chain variable domains derived from murine anti-NGF monoclonal antibodies PR-1254972, PR-1254973, PR-1254977, PR-1254981, PR-1254982. For these same anti-NGF mAbs, a cDNA fragment encoding the canine kappa constant region obtained from U.S. Pat. No. 5,852,183A, (Sequence ID No. 54) was synthesized and ligated to the 3′ end of each of the light chain variable domains. The complete canine IgG heavy chain constant domain nucleotide sequence and amino acid sequence is shown as SEQ ID NO: 51 and SEQ ID NO: 52, respectively. The complete canine kappa light chain constant domain nucleotide sequence and amino acid sequence is shown as SEQ ID NO: 53 and SEQ ID NO: 54, respectively. Complete heavy chain and light chain chimeric cDNAs were ligated into the pHybE expression plasmid; the sequences of these chimeric mAbs are in Table 18 below.
TABLE 18
Mouse/Canine Chimeric Antibody Sequences
Name
Sequence (CDRs are underlined)
PR-1290646
DVLMTQTPLSLPVSLGDQASISCRSSQSIVQSNGNTYL
light chain
EWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDF
amino acid
TLKISREAEDLGVYYCFQGSHVPFTFGSGTKLEIKRND
sequence
AQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKW
KVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYL
SHELYSCEITHKSLPSTLIKSFQRSECQRVD
(SEQ ID NO: 194)
PR-1290646
EVHLVESGGGLVKPGGFLILSCAASGFTFSDYYMFWIR
heavy chain
QTPGKRLEWVATISDGGSYTYYTDNVKGRFTISRDNVK
amino acid
NNLYLQMSHLKSADTAMYYCARDWSDSEGFAYWGQGTL
sequence
VTVSAASTTAPSVFPLAPSCGSTSGSTVALACLVSGYF
PEPVTVSWNSGSLTSGVHTFPSVLQSSGLHSLSSMVTV
PSSRWPSETFTCNVVHPASNTKVDKPVFNECRCTDTPP
CPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDL
GREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVS
VLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGR
AHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDV
EWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKS
RWQQGDPFTCAVMHETLQNHYTDLSLSHSPGK
(SEQ ID NO: 195)
PR-1290654
DIQMTQSPASLSASVGETVTVTCRASENIYSFLAWHQQ
light chain
KQGKSPQLLVYNANTLAEGVPSRFSGSGSGTQFSLKIN
amino acid
SLQPEDFGSYYCQHHFGTPFTFGSGTKLEIKRNDAQPA
sequence
VYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDG
VIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHEL
YSCEITHKSLPSTLIKSFQR SECQRVD
(SEQ ID NO: 196)
PR-1290654
QVQLQQPGAELVKPGASVKLSCKASGYTFTNYWMHWVK
heavy chain
QRPGQGLEWIGRIDPYGGGTKHNEKFKRKATVTADKSS
amino acid
STAYILLSSLTSEDSAVYYCTRSGYDYYFDVWGTGTTV
sequence
TVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFP
EPVTVSWNSGSLTSGVHTFPSVLQSSGLHSLSSMVTVP
SSRWPSETFTCNVVHPASNTKVDKPVFNECRCTDTPPC
PVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLG
REDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSV
LPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRA
HKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVE
WQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSR
WQQGDPFTCAVMHETLQNHYTDLSLSHSPGV
(SEQ ID NO: 197)
PR-1290656
DVVLTQTPLSLPVNIGDQASISCKSTKSLLNGDGFTYL
light chain
DWYLQKPGQSPQLLIYLVSNRFSGVPDRFSGSGSGTDF
amino acid
TLKISRVEAEDLGVYYCFESNYLFTFGSGTKLEMKRND
sequence
AQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKW
KVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYL
SHELYSCEITHKSLPSTLIKSFQRSECQRVD
(SEQ ID NO: 198)
PR-1290656
EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYIYWVK
heavy chain
QRPEQGLEWIGRIDPANGNTIYASKFQGKASITADTSS
amino acid
NTAYMQLSSLTSGDTAVYYCAGYGYYAYWGQGTTLTVS
sequence
SASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPV
TVSWNSGSLTSGVHTFPSVLQSSGLHSLSSMVTVPSSR
WPSETFTCNVVHPASNTKVDKPVFNECRCTDTPPCPVP
EPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGRED
PEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPI
EHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKP
SVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQS
NGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQ
GDPFTCAVMHETLQNHYTDLSLSHSPGK
(SEQ ID NO: 199)
PR-1290657
DVLMTQTPLSLPVSLGDQASISCRSSQSILHSNGNTYL
light chain
EWYLQKPGQSPNLLIYRVSNRFSGVPDRFSGSGSGTDF
amino acid
TLKISRVEAEDLGVYYCFQGAHVPFTFGSGTKLEIKRN
sequence
DAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVK
WKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEY
LSHELYSCEITHKSLPSTLI KSFQRSECQRVD
(SEQ ID NO: 200)
PR-1290657
EVQLVESGGGAVKPGGSLTLSCAASGFTFSNHYMYWVR
heavy chain
QTPEKRLEWVASISDGGAYTFYPDTVKGRFTISRDNVN
amino acid
NNLYLQMRHLKSEDTAMYYCTREESANNGFAFWGQGTL
sequence
VTVSAASTTAPSVFPLAPSCGSTSGSTVALACLVSGYF
PEPVTVSWNSGSLTSGVHTFPSVLQSSGLHSLSSMVTV
PSSRWPSETFTCNVVHPASNTKVDKPVFNECRCTDTPP
CPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDL
GREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVS
VLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGR
AHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDV
EWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKS
RWQQGDPFTCAVMHETLQNHYTDLSLSHSPGV
(SEQ ID NO: 201)
The canine IgG1 constant region nucleotide sequence described above was also ligated to the 3′ end of each of the cDNAs encoding heavy chain variable domains derived from caninized anti-NGF monoclonal antibodies 72.2 VH, 72.3 VH, 72.4 VH, 73.2 VH, 73.4 VH, 77.2 VH, 77.3 VH, 77.4 VH, 81.2 VH, 81.4 VH, 81.2B, 81.4B, 81.5B, 81.6B, 82.2 VH, 82.4 VH. The canine kappa light chain constant domain nucleotide sequence described above was also ligated to the 3′ end of each of the cDNAs encoding light chain variable domains derived from caninized anti-NGF monoclonal antibodies 72.2 VL, 72.4, 73.2 VL, 73.4 VL, 77.2 VL, 77.4 VL, 81.2 VL, 81.4 VL, 82.2 VL.
Full-length chimeric or caninized antibodies were transiently expressed in 293-6E cells by co-transfection of combinations of heavy and light chain pHybE plasmids. Table 20 highlights all possible combination of caninized heavy and light chains that may be combined to produce a caninized antibody per the name in the table (Table 20). In Table 20, the heavy chain plasmids encoding caninized versions of murine heavy chains are listed on the top line and proceed rightward. The light chain plasmids encoding caninized versions of murine light chains are listed on the left-hand column and proceed downward. At each point where these boxes intersect, a name has been indicated to describe a potential resulting caninized antibody.
Example 17
Caninized Monoclonal Antibody Expression and Purification
Selected heavy chain and light chain mouse/canine chimeric and caninized antibody plasmids were co-transfected into 293-6e cells in suspension and allowed to grow for 7-8 days. Cell supernatants were harvested, centrifuged, and filtered. For each expressed antibody, supernatant was mixed with an equal volume of Pierce binding buffer to perform Protein A Sepharose affinity chromatography according to manufacturers instructions (GE Healthcare #17-1279-04). Although according to several sources canine IgGs bind directly to Protein A moderately well (GE Healthcare Antibody Purification Handbook package insert; Scott, M. A., et. al., Vet Immunol-Immunopatho, 59:205, 1997; Warr, G. W and Hart, I. R., Am J Vet Res, 40:922, 1979; Thermo Scientific Pierce Antibody Production and Purification Technical Handbook) the monoclonal canine mAbs did not quantitatively bind to Protein A and therefore could not be purified from supernatants without modification to the Protein A purification methodology.
To allow quantitative binding of canine IgGs to Protein A, supernatants were concentrated and mixed with an equal volume of Pierce binding buffer (Thermo #21007). To the concentrated and diluted supernatants, NaCl was added to a final concentration of 2.5 M. NaCl-adjusted supernatant was loaded onto Protein A Sepharose by continuous over-night loading, washed with Pierce binding buffer, and eluted using Pierce elution buffer (Thermo #21004). The eluates were neutralized by dropwise addition of 1M Tris pH 8.0; following this the neutralized antibodies were dialyzed into PBS and amounts of antibody were quantified spectrophotometrically by OD280. The amount purified was mathematically divided by the total volume of cell supernatant purified to determine the overall estimated expression levels in ug/mL. The isolation and purification of theses canine IgG1/k mAbs allowed analytical characterization studies of the mAbs to be completed.
For purification of large-scale cell supernatants (10-15 L), cell supernatants were concentrated, then mixed with Pierce binding buffer A (Thermo, catalog #21001) in a 1 to 1 ratio. To this mixture, 5 M NaCl was added to 1.3 M final concentration. The pH of the mixture was adjusted to 8.5 with 10 N NaOH. The pH-adjusted cell supes were loaded onto a Protein A MabSelect SuRe (GE Healthcare, catalog #17-5438-03) chromatography column and eluted using two steps. The first step of the elution was performed using 20 mM Tris, 25 mM NaCl, pH 8.0, 7.4 mS/cm. Fractions containing antibodies were identified by OD280 and size exclusion chromatography. To quantitatively isolate the remaining antibody bound to the Protein A column, the second step elution was performed using Pierce elution buffer (Thermo, catalog #21004), pH 2.7, 3.7 mS/cm, and fractions containing antibodies were identified by OD280 and size exclusion chromatography. All fractions containing antibodies were neutralized using 2M Tris pH 8.5, and then dialyzed into PBS. The method employed to purify large volumes of cell supernatant containing canine monoclonal antibodies (ex. 10-15 L) differs from the method typically employed to purify human antibodies from large volumes. For human antibodies, Protein A purification is typically accomplished with cell supernatant binding conditions of pH 7.0 to 8.3 and 15 to 20 mS/cm, washing with similar conditions (1×PBS) and a 1 step elution of human antibodies with 0.1 M acetic acid, 0.15 M sodium chloride, pH 2.7 at 15 to 20 mS/cm or Thermo IgG elution buffer, pH 2.7, at 15 mS/cm.
Purified canine antibodies were analyzed by mass spectroscopy (MS) to confirm the expressed antibody protein molecular weight matched the expected weight based on amino acid sequence. In addition, canine antibodies were analyzed by size exclusion chromatography (SEC) to determine the percent monomer. This data indicated that mouse/canine chimierc IgG1/k mAbs may be expressed transiently in 293-6e cells and are 81% or greater monomeric following purification. This data also indicated that caninized IgG1/k mAbs may be expressed transiently in 293-6e cells and in most cases are 80% or greater monomeric following purification. In some cases, expression of protein may not be detected and in some cases purified caninized mAb is between 24 and 34% monomeric. The data is summarized in Tables 19 and 20.
TABLE 19
Mouse/Canine Chimeric Monoclonal Antibody Characterization Data
Estimated
Name of
Moniker of
Expression
Mouse/
Mouse/
Level
Canine
Canine
in Cell
%
Hybridoma
Chimeric
Chimeric
Supernatants
Monomeric
Moniker
Version
Version
(ug/mL)
mAb
PR-1254972
Mu72 Canine
PR-1290646
3.2
97
IgG1/k
Chimera
PR-1254973
Mu73 Canine
PR-1290654
7
88.3
IgG1/k
Chimera
PR-1254977
Mu77 Canine
PR-1290656
0.3
82.4
IgG1/k
Chimera
PR-1254981
Mu81 Canine
PR-1290657
0.9
81
IgG1/k
Chimera
PR-1254982
Mu82 Canine
PR-1290658
11.9
92.3
IgG1/k
Chimera
TABLE 20
Production of Caninized Antibodies by Combinations of Caninized
Heavy and Light Chains
Heavy
Light chain
chain
72.2 VH
72.3 VH
72.4 VH
73.2 VH
73.4 VH
77.2 VH
77.3 VH
72.2 VL
72VHv2/72VLv2
72.3 CaIgG1/k
72VHv4/72VLv2
73.5 CaIgG1/k
73VHv4/72VLv2
77VHv2/72VLv2
77.5 CaIgG1/k
72.4 VL
72VHv2/72VLv4
72VHv3/72VLv4
72.4 CaIgG1/k
73VHv2/72VLv4
73VHv4/72VLv4
77VHv2/72VLv4
77VHv3/72VLv4
73.2 VL
72VHv2/73VLv2
72.5 CaIgG/k
72VHv4/73VLv2
73.2 CaIgG1/k
73VHv4/73VLv2
77VHv2/73VLv2
77.6 CaIgG1/k
73.4 VL
72VHv2/73VLv4
72VHv3/73VLv4
72VHv4/73VLv4
73VHv2/73VLv4
73.4 CaIgG/k
77VHv2/73VLv4
77VHv3/73VLv4
77.2 VL
72VHv2/77VLv2
72.6 CaIgG/k
72VHv4/77VLv2
73.6 CaIgG1/k
73VHv4/77VLv2
77VHv2/77VLv2
77.3 CaIgG1/k
77.4 VL
72VHv2/77VLv4
72VHv3/77VLv4
72VHv4/77VLv4
73VHv2/77VLv4
73VHv4/77VLv4
77VHv2/77VLv4
77VHv3/77VLv4
81.2 VL
72VHv2/81VLv2
72.7 CaIgG/k
72VHv4/81VLv2
73.7 CaIgG1/k
73VHv4/81VLv2
77VHv2/81VLv2
77.7 CaIgG1/k
81.4 VL
72VHv2/81VLv4
72VHv3/81VLv4
72VHv4/81VLv4
73VHv2/81VLv4
73VHv4/81VLv4
77VHv2/81VLv4
77VHv3/81VLv4
82.2 VL
72VHv2/82VLv2
72VHv3/82VLv2
72VHv4/82VLv2
73VHv2/82VLv2
73VHv4/82VLv2
77VHv2/82VLv2
77VHv3/82VLv2
82.3 VL
72VHv2/82VLv3
72.8 CaIgG/k
72VHv4/82VLv3
73.8 CaIgG1/k
73VHv4/82VLv3
77VHv2/82VLv3
77.8 CaIgG1/k
82.4 VL
72VHv2/82VLv4
72VHv3/82VLv4
72VHv4/82VLv4
73VHv2/82VLv4
73VHv4/82VLv4
77VHv2/82VLv4
77VHv3/82VLv4
Light chain
Heavy chain
77.4 VH
81.2 VH
81.4 VH
82.2 VH
82.4 VH
72.2 VL
77VHv4/72VLv2
81.5 CaIgG1/k
81VHv4/72VLv2
82.5 CaIgG1/k
82VHv4/72VLv2
72.4 VL
77VHv4/72VLv4
81VHv2/72VLv4
81VHv4/72VLv4
82VHv2/72VLv4
82VHv4/72VLv4
73.2 VL
77VHv4/73VLv2
81.6 CaIgG1/k
81VHv4/73VLv2
82.6 CaIgG1/k
82VHv4/73VLv2
73.4 VL
77VHv4/73VLv4
81VHv2/73VLv4
81VHv4/73VLv4
82VHv2/73VLv4
82VHv4/73VLv4
77.2 VL
77VHv4/77VLv2
81.7 CaIgG1/k
81VHv4/77VLv2
82.7 CaIgG1/k
82VHv4/77VLv2
77.4 VL
77.4 CaIgG1/k
81VHv2/77VLv4
81VHv4/77VLv4
82VHv2/77VLv4
82VHv4/77VLv4
81.2 VL
77VHv4/81VLv2
8.12 CaIgG1/k
81VHv4/81VLv2
82.8 CaIgG1/k
82VHv4/81VLv2
81.4 VL
77VHv4/81VLv4
81VHv2/81VLv4
81.4 CaIgG1/k
82VHv2/81VLv4
82VHv4/81VLv4
82.2 VL
77VHv4/82VLv2
81VHv2/82VLv2
81VHv4/82VLv2
82VHv2/82VLv2
82VHv4/82VLv2
82.3 VL
77VHv4/82VLv3
81.8 CaIgG1/k
81VHv4/82VLv3
82.3 CaIgG1/k
82VHv4/82VLv3
82.4 VL
77VHv4/82VLv4
81VHv2/82VLv4
81VHv4/82VLv4
82VHv2/82VLv4
82.4 CaIgG1/k
TABLE 21
Caninized Monoclonal Antibody Characterization Data
Estimated
Expression
Level in
%
Cell
Mono-
Supernatants
meric
Name
Moniker
Lot
(ug/mL)
mAb
72.3 Canine IgG1/k
PR-1313524
1804091
2.63
88.3
72.4 Canine IgG1/k
PR-1314949
1805928
1.6
81.5
73.2 Canine IgG1/k
PR-1313520
1810546
13.4
96.5
73.4 Canine IgG1/k
PR-1314950
1805932
1.8
90
77.3 Canine IgG1/k
N/A
N/A
0.7
24.8
77.4 Canine IgG1/k
N/A
N/A
1
34.6
81.2 Canine IgG1/k
N/A
No mAb
No mAb
N/A
detected
detected
81.4 Canine IgG1/k
N/A
No mAb
No mAb
N/A
detected
detected
82.3 Canine IgG1/k
PR-1313519
1810585
4.4
80.7
82.4 Canine IgG1/k
PR-1313521
1816320
9.8
94.2
Example 18
Affinity Analysis of Canine Antibodies
Purified mouse/canine chimeric antibodies and caninized antibodies were analyzed for affinity to canine NGF using a Biacore T100 instrument. Goat anti Canine IgG (Southern Biotech) was immobilized at 5000-10000 RU on a CM5 chip using an amine coupling procedure according to the manufacturer's instructions (Biacore). Canine NGF was injected at 50 uL/min at a concentration range of 50-0.156 nM for the mouse/canine chimeric antibodies or 10-0.156 nM for the caninized antibodies. The association rate was monitored for 5 min and the dissociation rate was monitored for 10-20 min. The chip surface was regenerated using 50-75 uL 10 mM glycine pH 1.5 at a flow rate of 50-100 uL/min. Data was analyzed using Biaevaluation T100 software version 2.0.2, software, GE Healthcare Life Sciences (Piscataway, N.J.). Overall affinity parameters established for mouse/canine chimeric antibodies is summarized in Table 22 and for caninized antibodies in Table 23. This data indicates that the isolated mouse/canine chimeric anti-NGF mAbs have fast on-rates (from greater than 2×106) and slow off-rates (from less than 3×10−3). The overall KDs of the mouse/canine anti-NGF mAbs range from about 1300 pM to 1.6 pM. This data also indicates that the isolated caninized chimeric anti-NGF mAbs have fast on-rates (from greater than 6×106) and slow off-rates (from less than 2×10−4). The overall KDs of the caninized anti-NGF mAbs range from about 42 pM to 1.2 pM.
TABLE 22
Affinity Parameters of Mouse/Canine Chimeric Monoclonal
Antibodies to Canine NGF
On-rate
Off-rate
Overall
Name
Moniker
(1/M·S)
(1/S)
Affinity (M)
Mu72 Canine
PR-1290646
2.9 × 106
3.8 × 10−3
1.3 × 10−9
IgG1/k Chimera
Mu73 Canine
PR-1290654
6.3 × 106
9 × 10−5
1.4 × 10−11
IgG1/k Chimera
Mu77 Canine
PR-1290656
9.1 × 106
1.9 × 10−4
2.1 × 10−11
IgG1/k Chimera
Mu81 Canine
PR-1290657
4.2 × 106
3.5 × 10−4
8.2 × 10−11
IgG1/k Chimera
Mu82 Canine
PR-1290658
8.7 × 106
1.4 × 10−5
1.6 × 10−12
IgG1/k Chimera
TABLE 23
Affinity Parameters of Caninized Monoclonal Antibodies
to Canine NGF
On-rate
Off-rate
Overall affinity
Name
(1/M · s)
(1/s)
(M)
73.2 canine IgG1/k
Expt 1
6.3 × 106
2.8 × 10−4
4.4 × 10−11
PR-13113520
Expt 2
6.9 × 106
2.9 × 10−4
4.2 × 10−11
Average
6.6 × 106
2.9 × 10−4
4.3 × 10−11
82.3 canine IgG1/k
Expt 1
8.2 × 106
2 × 10−5
2.4 × 10−12
PR-13113519
Expt 2
8.5 × 106
1.3 × 10−5
1.6 × 10−12
Average
8.4 × 106
1.7 × 10−5
2 × 10−12
82.4 canine IgG1/k
Expt 1
8.6 × 106
1.1 × 10−5
1.2 × 10−12
PR-13113521
Expt 2
7.7 × 106
1.2 × 10−5
1.5 × 10−12
Average
8.2 × 106
1.2 × 10−5
1.4 × 10−12
Example 19
Characterization of Canine Antibodies by the TF-1 Cell Proliferation Potency Assay
Purified mouse/canine chimeric antibodies and caninized antibodies were characterized using the TF-1 Cell Proliferation Potency Assay (described previously) using 70 pM canine NGF in the assay. The summarized potency data is in Tables 20 and 21. The data shows that in the presence of 70 pM canine NGF, all of the mouse/canine chimeric anti-NGF antibodies display sub-nM potencies, and all display potencies of less than 50 pM. The data shows that in the presence of 70 pM canine NGF, some of the caninized anti-NGF antibodies have no neutralization potency on 70 pM canine NGF. Some caninized mAbs have sub-nM potencies, and some have potencies of less than 20 pM.
TABLE 24
Potency of Mouse/Canine Chimeric NGF Monoclonal Antibodies on
Canine NGF-Induced TF-1 Cell Proliferation
Name
Moniker
Lot
IC50 (nM)
Mu72 Canine IgG1/k Chimera
PR-1290646
1785614
0.041
Mu73 Canine IgG1/k Chimera
PR-1290654
1785658
0.008
Mu77 Canine IgG1/k Chimera
PR-1290656
1785699
0.028
Mu81 Canine IgG1/k Chimera
PR-1290657
1778832
0.012
Mu82 Canine IgG1/k Chimera
PR-1290658
1785732
0.007
TABLE 25
Potency of Caninized NGF Monoclonal Antibodies on Canine
NGF-Induced TF-1 Cell Proliferation (N/A = not applicable)
Name
Moniker
Lot
IC50 (nM)
72.3 Canine IgG1/k
PR-1313524
1804091
0
72.4 Canine IgG1/k
PR-1314949
1805928
0
73.2 Canine IgG1/k
PR-1313520
1810546
0.422
73.4 Canine IgG1/k
PR-1314950
1805932
0
77.3 Canine IgG1/k
N/A
N/A
0.625
77.4 Canine IgG1/k
N/A
N/A
0
82.3 Canine IgG1/k
PR-1313519
1810585
0.017
82.4 Canine IgG1/k
PR-1313521
1816320
0.016
Example 20
Characterization of Solubility and Stability of Caninized Anti-NGF Antibodies
Stock solutions of two caninized anti-NGF antibodies (73.2 canine IgG1/k and 82.4 canine IgG1/k) were obtained. [Are these the canonized Abs produced in Ex. 13?] The antibodies were formulated in phosphate buffer saline (PBS) at concentrations below 5 mg/ml (PBS contains, but is not limited to, the following ingredients: 15 mM phosphate buffer and 150 mM sodium chloride at pH 7.4).
Solubility:
The solubility of the caninized antibodies at high concentrations in PBS were evaluated by concentrating the antibodies with Amicon 30K molecular weight cutoff centrifuge spin filters. The final concentrations were determined by UV absorbance.
At room temperature, 73.2 canine IgG1/k was soluble to at least 54 mg/ml and 82.4 canine IgG1/k was soluble to at least 83 mg/ml. When stored at 5° C. for 5 hours at those concentrations, 73.2 canine IgG1/k formed a gel layer at the bottom of the container while 82.4 canine IgG1/k remained as a uniform solution. When re-equilibrated to room temperature, 73.2 canine IgG1/k became a uniform solution. When 73.2 canine IgG1/k was diluted to 27 mg/ml, it remained as a uniform solution at 5° C.
In comparison, adalimumab, a human antibody, demonstrated a solubility of at least 150 mg/ml at 5° C. and at room temperature. This was observed in a formulation with a pH of 7 and with a sodium chloride concentration of 150 mM. The observations are described in Table 26.
TABLE 26
Solubility of 73.2 canine IgG1/k, 82.4 canine IgG1/k, and human
antibody adalimumab in PBS.
Room temperature
Observations when
Antibody
solubility (mg/ml)
placed at 5° C.
73.2 canine IgG1/k
≧54
Gel layer formed at
container bottom*
82.4 canine IgG1/k
≧83
Remained as solution
adalimumab
≧150
Remained as solution
*returned to uniform solution when brought back to room temperature; when diluted to 27 mg/ml, remained as uniform solution at 5° C.
The solubility of 73.2 canine IgG1/k 82.4 canine IgG1/k was also evaluated in 15 mM histidine buffer pH 6.0. This is a buffer typically used to formulate human therapeutic antibodies. The PBS buffer comprising the stock solutions of 73.2 canine IgG1/k and 82.4 canine IgG1/k were exchanged with the histidine buffer using Amicon 30K molecular weight cutoff centrifuge spin filters. Following buffer exchange, the antibodies exhibited white precipitation and solubilities of less than 2 mg/ml at room temperature, as determined by UV absorbance. In comparison, the human antibody adalimumab was observed to reach a concentration of at least 150 mg/ml in 15 mM histidine buffer pH 6.0 at room temperature. These observations are summarized in Table 27.
TABLE 27
Solubility of anti-NGF caninized antibodies 73.2 canine IgG1/k,
82.4 canine IgG1/k and human antibody adalimumab in 15 mM
histidine buffer pH 6.0.
Room temperature
Antibody
solubility (mg/ml)
Observations
73.2 canine IgG1/k
<2
White precipitate observed
82.4 canine IgG1/k
<2
White precipitate observed
adalimumab
≧150
Remained as solution
Freeze-Thaw Stability
An assessment of the freeze-thaw (FT) stability of 73.2 canine IgG1/k and 82.4 canine IgG1/k in PBS, and after dilution with PBS to 1 mg/ml, was performed. Both antibodies were frozen at −80° C. for at least 4 hours. They were then thawed in a 30° C. water bath (this constitutes one freeze-thaw cycle). Stability was assessed for four freeze-thaw cycles by size exclusion HPLC (SEC). The freeze-thaw analysis is summarized in Table 28.
TABLE 28
Freeze-thaw stability of 73.2 canine IgG1/k and 82.4 canine
IgG1/k at 1 mg/ml in PBS.
Percentage Species
Post
Post
Post
Antibody
Species
Pre-FT
FT#1
FT#2
FT#4
73.2 canine
Monomer
97.4
97.3
97.3
97.2
IgG1/k
Aggregate
1.7
1.8
1.8
1.8
Fragment
0.9
0.9
0.9
1
82.4 canine
Monomer
96.6
96.6
96.6
96.1
IgG1/k
Aggregate
2.9
2.9
2.9
3.2
Fragment
0.5
0.5
0.5
0.7
Storage Stability and Accelerated Stability:
The stability of 73.2 canine IgG1/k and 82.4 canine IgG1/k when formulated at a concentration of 10 mg/mL and within a pH range of 5 to 8 and at low (˜7.5 mM) and high (˜>150 mM) ionic strengths was assessed. Stability at these conditions was assessed by monitoring the stability of the antibodies in the following buffers and salt concentrations: (A) 15 mM acetate pH 5; (B) 15 mM acetate pH 5+150 mM NaCl; (C) 15 mM histidine pH 6+150 mM NaCl; (D) 15 mM phosphate pH 7.4; (E) PBS pH 7.4; (F) 15 mM Tris pH 7.5; (G) 15 mM Tris pH 8.0. Sodium azide (0.02%) was added to all buffers as an anti-microbial agent.
Stock solutions of 73.2 canine IgG1/k and 82.3 canine IgG1/k in PBS were concentrated up to 15 mg/ml using 30K molecular weight cutoff centrifuge spin filters. They were then dialyzed against the buffers listed above for 18 hours using mini-dialysis 1 kDa molecular weight cut-off dialysis tubes (GE Healthcare). Following dialysis, samples were diluted with the respective buffers to a final concentration of 10 mg/ml. 150 μl of each sample was aliquoted to cryovials which were then stored at 40° C. or 5° C. Samples were analysed at time=0 hours (T0), at 7 days (T7d), and at 21 days (T21d) and stability was assessed by SEC.
After 21 days at 40° C., accelerated stability testing showed that 73.2 canine IgG1/k and 82.3 canine IgG1/k have much greater fragmentation at pH values below 7.4 than at pH values above 7.4. In comparison, the human antibody adalimumab, exhibited less fragmentation within the pH range 4 to 8 over 21 days at 40° C. In particular, the fragmentation of adalimumab at pH 6 was much less than the fragmentation of 73.2 canine IgG1/k or 82.4 canine IgG1/k at pH 6. Also, adalimumab at the higher stress condition of pH 4 showed equal or less fragmentation compared to 73.2 canine IgG1/k or 82.4 canine IgG1/k at the lower stress condition of pH 5. The results of the stability analyses and fragmentation profiles are shown, respectively, in Tables 25 and 26. These data suggest that canine IgG1/k monoclonal antibodies have a different degradation profile compared to that of human IgG1/k monoclonal antibodies. Specifically, the fragmentation appears to be more extensive for canine IgG1/k antibodies than for human antibodies at pH 6 and below.
TABLE 29
Stability data from SEC for 73.2 canine IgG1/k, 82.4 canine IgG1/k and
human antibody adalimumab in different formulations at 7 and 21 days at
5° C. and at 40° C.
Percentage
Percentage
Percentage
Monomer
Aggregate
Fragment
Buffer
T0
T7d
T21d
T0
T7d
T21d
T0
T7d
T21d
73.2 canine IgG1/k at 5° C.
A (pH 5)
94.6
91.4
90.3
2.9
3.1
3.4
2.6
5.5
6.2
B (pH 5)
95.2
98.2
98.2
3.4
0.4
0.5
1.4
1.4
1.3
C (pH 6)
93.9
98.0
97.8
4.4
0.5
0.7
1.8
1.5
1.5
D (pH 7.4)
94.3
97.9
97.7
4.7
0.6
0.8
1.0
1.5
1.5
E (pH 7.4)
94.5
97.9
97.8
4.5
0.5
0.8
1.0
1.6
1.5
F (pH 7.5)
94.5
98.0
97.8
4.0
0.5
0.8
1.6
1.5
1.5
G (pH 8.0)
93.7
97.8
97.6
4.6
0.7
0.9
1.7
1.5
1.5
73.2 canine IgG1/k at at 40° C.
A (pH 5)
94.6
81.3
79.0
2.9
4.5
5.1
2.6
14.2
15.9
B (pH 5)
95.2
92.1
90.9
3.4
0.6
1.2
1.4
7.4
7.8
C (pH 6)
93.9
94.4
91.6
4.4
0.6
1.1
1.8
5.0
7.3
D (pH 7.4)
94.3
97.5
96.6
4.7
0.9
1.6
1.0
1.5
1.8
E (pH 7.4)
94.5
98.0
97.3
4.5
0.6
1.1
1.0
1.4
1.6
F (pH 7.5)
94.5
97.5
96.3
4.0
0.9
1.8
1.6
1.6
1.9
G (pH 8.0)
93.7
97.0
95.2
4.6
1.2
2.6
1.7
1.8
2.2
82.4 canine IgG1/k at 5° C.
A (pH 5)
96.7
98.1
98.7
2.3
1.3
.8
1.0
0.5
0.5
B (pH 5)
96.3
97.2
97.3
2.4
2.3
2.3
1.3
0.5
0.4
C (pH 6)
97.0
97.1
97.1
2.6
2.3
2.3
0.4
0.6
0.6
D (pH 7.4)
96.4
97.0
97.1
2.5
2.4
2.5
1.0
0.5
0.4
E (pH 7.4)
96.7
96.8
96.8
2.8
2.5
2.6
0.5
0.7
0.6
F (pH 7.5)
96.8
97.1
96.9
2.9
2.5
2.5
0.2
0.5
0.6
G (pH 8.0)
96.6
96.9
96.9
2.5
2.5
2.5
0.9
0.6
0.6
82.4 canine IgG1/k at 40° C.
A (pH 5)
96.7
93.1
87.8
2.3
2.8
4.2
1.0
4.1
8.0
B (pH 5)
96.3
93.3
91.3
2.4
2.5
3.1
1.3
4.3
5.6
C (pH 6)
97.0
94.1
92.5
2.6
2.3
2.7
0.4
3.5
4.8
D (pH 7.4)
96.4
93.5
93.8
2.5
3.4
3.3
1.0
3.1
2.9
E (pH 7.4)
96.7
95.1
93.6
2.8
2.4
2.6
0.5
2.5
3.8
F (pH 7.5)
96.8
93.4
923.6
2.9
2.6
3.0
0.2
4.0
0.5
G (pH 8.0)
96.6
94.8
92.7
2.5
2.8
3.5
0.9
2.4
0.4
adalimumab at 40° C.
Percentage
Percentage
Percentage
Monomer
Aggregate
Fragment
PH
T0
T7d
T21d
T0
T7d
T21d
T0
T7d
T21d
4
99
98
95
<1
<2
0.5
<1
<2
4.5
6
99
99
99
<1
<1
0.3
<1
<1
0.7
8
99
98
98
<1
<2
1.2
<1
<2
0.8
TABLE 30
Fragmentation profile from SEC for 73.2 canine IgG1/k,
82.4 canine IgG1/k and human antibody adalimumab in
different formulations at 21 days and at 40° C.
Increase in
Percent
Fragmentation
over 21 days at
Antibody
Buffer
40° C.
73.2 canine IgG1/k
A (pH 5)
13.3
B (pH 5)
6.4
C (pH 6)
5.5
D (pH 7.4)
0.8
E (pH 7.4)
0.6
F (pH 7.5)
0.3
G (pH 8.0)
0.5
82.4 canine IgG1/k
A (pH 5)
7.0
B (pH 5)
4.3
C (pH 6)
4.4
D (pH 7.4)
1.9
E (pH 7.4)
3.3
F (pH 7.5)
0.3
G (pH 8.0)
−0.5
adalimumab
pH 4
<4.5
pH 6
<0.7
pH 8
<0.8
Example 21
Canine Single Dose PK Study and Antigen Bridging Assay for PK Serum Sample Analysis
The serum levels of 73.2 canine IgG1/k and 82.4 canine IgG1/k were analyzed following a single dose of 4.5 mg/kg (intravenous or subcutaneous) in mongrel dogs. Following the injection, 13 samples of venous blood were collected over 672 hours. Blood samples were allowed to clot and the serum removed for antibody quantitation.
An NGF bridging assay was developed to quantitate canine anti-NGF mAbs in serum. Streptavidin-coated 96-well plates (MSD #L11SA-1) were blocked with Blocker A (MSD #R93BA-4). Canine anti-NGF antibody present in serum (or in PBS) was mixed with equi-molar ratios of biotin-tagged NGF and Sulfo-tagged NGF (Sulfo Reagent MSD #R91AN-1) and incubated to form an NGF+antibody complex. The final concentration of the biotin-tagged NGF and sulfo-tagged NGF in the assay was between 1-2 nM. NGF-antibody complexes were added to the streptavidin-coated plate and allowed to bind for 60 minutes. Following incubation, plates were washed with PBS plus 0.05% Tween-20, and bound NGF-antibody complexes were detected in Read Buffer T (MSD #R92TC-1) on an MSD SECTOR Imager 6000. Data was quantitated to estimate the total amount of antibody in ug/mL of a sample liquid and is provided below in Tables 27-30.
TABLE 31
Serum Concentrations of 82.4 canine IgG1/k Following a
Single Subcutaneous Dose
Dog #
Hours post
1073305
1072602
1072104
1072306
1072105
injection
ug/mL
0
0.0
0.0
0
0
0.1
0.25
2.6
0.4
0.0
2.24
0.1
1
9.0
2.2
0.9
9.07
0.4
8
31.5
17.7
13.8
32.55
12.8
12
41.1
20.5
18.7
33.31
24.5
24
42.2
25.6
23.2
35.73
23.8
48
52.3
37.2
36.3
41.35
35.3
72
51.2
41.1
34.4
38.91
36.8
144
47.1
42.6
35.2
33.46
36.7
240
39.0
32.4
29.0
26.03
31.7
336
30.9
28.2
24.1
19.99
26.8
504
20.1
18.3
16.0
11.03
18.6
672
16.3
12.8
10.4
5.81
5.4
TABLE 32
Serum Concentrations of 82.4 canine IgG1/k Following a
Single Intravenous Dose
Dog #
Hours post
1072705
1073804
1073303
1073903
1074502
injection
ug/mL
0
0
0
0
0
0
0.25
102.6
83.1
84.0
80.1
81.7
1
106.7
70.1
87.4
74.1
81.9
8
88.4
65.7
68.1
67.4
68.9
12
91.5
61.6
62.6
62.1
59.3
24
87.4
60.0
57.2
53.9
53.0
48
76.6
49.9
52.6
50.9
50.2
72
60.3
49.1
46.0
40.8
44.6
144
45.7
43.1
36.1
35.7
36.0
240
37.2
34.4
32.6
24.3
32.6
336
31.7
32.7
24.8
20.6
20.1
504
23.5
17.4
18.5
12.2
12.8
672
15.2
10.7
12.5
7.3
8.3
TABLE 33
Serum Concentrations of 73.2 canine IgG1/k Following a
Single Subcutaneous Dose
Dog #
1072607
1074307
1072606
1074503
Hours post injection
ug/mL
0
0
0
0
0
0.25
0
0
2.1
0
1
1.0
5.9
4.7
0.0
8
18.6
31.3
18.7
7.4
12
22.7
32.5
21.3
8.7
24
26.8
33.2
24.2
12.0
48
33.7
35.9
28.4
16.2
72
35.0
37.6
30.7
19.4
144
34.9
37.4
30.2
21.8
240
31.6
31.8
26.8
22.0
336
24.4
24.7
22.6
16.5
504
15.3
14.3
13.8
10.6
672
6.8
9.2
5.1
5.4
TABLE 34
Serum Concentrations of 73.2 canine IgG1/k Following a
Single Intravenous Dose
Dog #
Hours post
1072804
1073304
1072604
injection
ug/mL
0
0
0
0
0.25
93.6
33.2
108.5
1
86.3
30.8
103.1
8
76.9
22.1
85.4
12
72.1
21.4
80.5
24
63.0
17.1
68.0
48
54.6
14.6
56.8
72
49.4
13.5
50.8
144
41.4
10.2
41.4
240
35.4
8.9
30.8
336
30.5
6.3
20.9
504
22.2
4.0
3.4
672
14.8
3.4
0.0
Example 22
Pharmacokinetic Analysis of Serum Concentration Data
Pharmacokinetic parameters for both intravenous (IV) and subcutaneous (SC) dosing routes were calculated for each animal using WinNonlin software (Pharsight Corporation, Mountain View, Calif.) by noncompartmental analysis. Other calculations, e.g. mean, standard deviation (SD), and percent subcutaneous bioavailability (F: %) were carried out using Microsoft Excel software (Microsoft Corporation Redmond, Wash.). The data is shown in Table 35 and 36.
TABLE 35
Pharmacokinetic Analysis of 73.2 canine IgG1/k
Following a Single Intravenous Dose
IV
SC
T½
Vss
CI
T½
Cmax
Tmax
(day)
(mL/kg)
(mL/h/kg)
(day)
(ug/mL)
(day)
% F
14.8*
71
0.15
8.0*
31.3
4.8
51
*Harmonic Mean
TABLE 36
Pharmacokinetic Analysis of 82.4 canine IgG1/k Following a
Single Intravenous Dose
IV
SC
T½
Vss
CI
T½
Cmax
Tmax
(day)
(mL/kg)
(mL/h/kg)
(day)
(ug/mL)
(day)
% F
10.9*
73
0.19
11.6*
41.9
3.0
94
*Harmonic Mean
The data indicates that canine mAbs 73.2 and 82.4 have a half-life of about 8 to about 15 days when dosed IV or SC, suggesting that these molecules exhibit mammalian antibody-like half-lives and overall PK parameters.
Example 23
ELISA for Titering Canine Antibodies
To quantitate canine antibodies in cell supernatants (or other liquids), high-binding EIA plates (Costar #9018) were coated with polyclonal goat anti-dog IgG antibodies (Rockland #604-1102) at 4 ug/ml in PBS. After blocking with 2% non-fat milk in PBS, canine monoclonal antibodies were added to the plates and the plates were washed with PBS plus 0.05% Tween-20. Bound canine mAbs were detected with HRP-tagged goat anti-dog IgG antibodies (Rockland #604-1302) at 0.1 ug/ml. Plates were washed with PBS plus 0.05% Tween-20. Canine mAbs were detected by addition of TMB substrate (Neogen #308177), and the reaction was stopped with 1N HCl. Bound canine antibodies were quantitated by absorption at 450 nM to estimate the total amount of antibody in ug/mL of a sample liquid.
The present disclosure incorporates by reference in their entirety techniques well known in the field of molecular biology. These techniques include, but are not limited to, techniques described in the following publications:
Ausubel, F. M. et al. eds., Short Protocols In Molecular Biology (4th Ed. 1999) John Wiley & Sons, NY. (ISBN 0-471-32938-X).
Lu and Weiner eds., Cloning and Expression Vectors for Gene Function Analysis (2001) BioTechniques Press. Westborough, Mass. 298 pp. (ISBN 1-881299-21-X).
Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).
Old, R. W. & S. B. Primrose, Principles of Gene Manipulation: An Introduction To Genetic Engineering (3d Ed. 1985) Blackwell Scientific Publications, Boston. Studies in Microbiology; V.2:409 pp. (ISBN 0-632-01318-4).
Sambrook, J. et al. eds., Molecular Cloning: A Laboratory Manual (2d Ed. 1989) Cold Spring Harbor Laboratory Press, NY. Vols. 1-3. (ISBN 0-87969-309-6).
Winnacker, E. L. From Genes To Clones: Introduction To Gene Technology (1987) VCH Publishers, NY (translated by Horst Ibelgaufts). 634 pp. (ISBN 0-89573-614-4).
All references, publications, and patent applications disclosed herein are hereby incorporated by reference in their entirety.
Although a number of embodiments, aspects and features have been described above, it will be understood by those skilled in the art that modifications and variations of the described embodiments and features may be made without departing from the present disclosure or the disclosure as defined in the appended claims.
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13817721
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zoetis belgium s.a.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Mar 31st, 2022 03:01PM
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Mar 31st, 2022 03:01PM
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Zoetis
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:zts
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Zoetis
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Nov 13th, 2018 12:00AM
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Jul 13th, 2015 12:00AM
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https://www.uspto.gov?id=US10125192-20181113
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Caninized anti-NGF antibodies and their use
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The present disclosure encompasses NGF binding proteins, specifically to antibodies that are chimeric, CDR grafted and canonized antibodies, and methods of making and uses thereof. The antibodies, or antibody portions, of the disclosure are useful for detecting NGF and for inhibiting NGF activity, e.g., in a mammal subject suffering from a disorder in which NGF activity is detrimental.
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10125192
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1. A method of inhibiting the biological function of Nerve Growth Factor (NGF) in a canine comprising administration of a therapeutically effective amount of a composition comprising a caninized antibody that specifically binds to NGF and inhibits NGF from binding to TrkA receptors that comprises:
a) a variable heavy chain (VH) region comprising:
i) CDR1 comprising SEQ ID NO. 61,
ii) CDR2 comprising SEQ ID NO. 62; and
iii) CDR3 comprising SEQ ID NO. 63; and
b) a variable light chain (VL) region comprising:
i) CDR1 comprising SEQ ID NO. 64,
ii) CDR2 comprising SEQ ID NO. 65; and
iii) CDR3 comprising SEQ ID NO. 66; and,
wherein the inhibition of NGF reduces pain in a canine.
2. The method according to claim 1 wherein the caninized antigen binding protein comprises a monoclonal antibody.
3. A method of treating a canine suffering from pain comprising the administration of a therapeutically effective amount of a caninized antibody that specifically binds to Nerve Growth Factor (NGF) and inhibits the binding of NGF to TrkA that comprises:
a) a variable heavy chain (VH) region comprising:
i) CDR1 comprising SEQ ID NO. 61,
ii) CDR2 comprising SEQ ID NO. 62; and
iii) CDR3 comprising SEQ ID NO. 63; and
b) a variable light chain (VL) region comprising:
i) CDR1 comprising SEQ ID NO. 64,
ii) CDR2 comprising SEQ ID NO. 65; and
iii) CDR3 comprising SEQ ID NO. 66.
4. The method according to claim 3 wherein the caninized antigen binding protein comprises a monoclonal antibody.
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4
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CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 13/817,721, now granted U.S. Pat. No. 9,505,829, which is a national stage 371 application of the international application PCT/US2011/048518 filed on Aug. 19, 2011 which claims priority to the U.S. Provisional Application No. 61/375,193, filed Aug. 19, 2010, all contents of which are incorporated by reference in their entirety
TECHNICAL FIELD
The disclosure relates to anti-NGF antibodies and polynucleotides encoding the same, and use of such antibodies and/or polynucleotides in the treatment and/or prevention of pain, including but not limited to post-surgical pain, rheumatoid arthritis pain, cancer pain, and osteoarthritis pain.
BACKGROUND
Nerve growth factor (NGF) is a secreted protein that was discovered over 50 years ago as a molecule that promotes the survival and differentiation of sensory and sympathetic neurons. (See Levi-Montalcini, Science 187: 113 (1975), for a review). The crystal structure of NGF and NGF in complex with the tyrosine kinase A (TrkA) receptor has been determined (McDonald et al., Nature 354: 411 (1991); Wiesmann et al., Nature 401: 184-188 (1999)).
The role of NGF in the development and survival of both peripheral and central neurons has been well characterized. NGF has been shown to be a critical survival and maintenance factor in the development of peripheral sympathetic and embryonic sensory neurons and of basal forebrain cholinergic neurons (see, e.g., Smeyne et al., Nature 368: 246-9 (1994); and Crowley et al., Cell, 76: 1001-11 (1994)). It has been shown to inhibit amyloidogenesis that leads to Alzheimer's disease (Calissano et al., Cell Death and Differentiation, 17: 1126-1133 (2010)). NGF up-regulates expression of neuropeptides in sensory neurons (Lindsay et al., Nature, 337:362-364 (1989)) and its activity is mediated through two different membrane-bound receptors, the TrkA receptor and the p75 common neurotrophin receptor (Chao et al., Science, 232:518-521 (1986); Huang et al., Annu. Rev. Neurosci., 24:677-736 (2001); Bibel et al., Genes Dev., 14:2919-2937 (2000)).
NGF is produced by a number of cell types including mast cells (Leon, et al., Proc. Natl. Acad. Set, 91: 3739-3743 (1994)), B-lymphocytes (Torcia, et al., Cell, 85: 345-356 (1996), keratinocytes (Di Marco, et al., J. Biol. Chem., 268: 22838-22846)), smooth muscle cells (Ueyama, et al., J. Hypertens., 11: 1061-1065 (1993)), fibroblasts (Lindholm, et al., Eur. J. Neurosci., 2: 795-801 (1990)), bronchial epithelial cells (Kassel, et al., Clin, Exp. Allergy, 31: 1432-40 (2001)), renal mesangial cells (Steiner, et al., Am. J. Physiol., 261:F792-798 (1991)) and skeletal muscle myotubes (Schwartz, et al., J Photochem. Photobiol., B66: 195-200 (2002)). In addition, NGF receptors have been found on a variety of cell types outside of the nervous system.
NGF has been implicated in processes outside of the nervous system, e.g., NGF has been shown to enhance vascular permeability (Otten, et al., Eur J Pharmacol., 106: 199-201 (1984)), enhance T- and B-cell immune responses (Otten, et al., Proc. Natl. Acad. Sci., USA 86: 10059-10063 (1989)), induce lymphocyte differentiation and mast cell proliferation and cause the release of soluble biological signals from mast cells (Matsuda, et al., Proc. Natl. Acad. Sci., 85: 6508-6512 (1988); Pearce, et al., J. Physiol, 372:379-393 (1986); Bischoff, et al., Blood, 79: 2662-2669 (1992); Horigome, et al., J. Biol. Chem., 268: 14881-14887 (1993)).
Both local and systemic administrations of NGF have been shown to elicit hyperalgesia and allodynia (Lewin, G. R. et al., Eur. J. Neurosci. 6: 1903-1912 (1994)). Intravenous infusion of NGF in humans produces a whole body myalgia while local administration evokes injection site hyperalgesia and allodynia in addition to the systemic effects (Apfel, S. C. et al., Neurology, 51: 695-702 (1998)). Furthermore, in certain forms of cancer, excess NGF facilitates the growth and infiltration of nerve fibers with induction of cancer pain (Zhu, Z. et al., J Clin. Oncol., 17: 241-228 (1999). Although exogenously added NGF has been shown to be capable of having all of these effects, it is important to note that it has only rarely been shown that endogenous NGF is important in any of these processes in vivo (Torcia, et al., Cell, 85(3): 345-56 (1996)).
An elevated level of NGF has been implicated in certain inflammatory conditions in humans and animals, e.g., systemic lupus erythematosus (Bracci-Laudiero, et al., Neuroreport, 4: 563-565 (1993)), multiple sclerosis (Bracci-Laudiero, et al., Neurosci. Lett., 147:9-12 (1992)), psoriasis (Raychaudhuri, et al., Acta Derm. Venereol, 78: 84-86 (1998)), arthritis (Falcim, et al., Ann. Rheum. Dis., 55: 745-748 (1996)), interstitial cystitis (Okragly, et al., J. Urology 6: 438-441 (1999)) and asthma (Braun, et al., Eur. J Immunol., 28:3240-3251 (1998)). The synovium of patients affected by rheumatoid arthritis expresses high levels of NGF while in non-inflamed synovium NGF has been reported to be undetectable (Aloe, et al., Arch. Rheum., 35:351-355 (1992)). Similar results were seen in rats with experimentally induced rheumatoid arthritis (Aloe, et al., Clin. Exp. Rheumatol., 10: 203-204 (1992)). Elevated levels of NGF have been reported in transgenic arthritic mice along with an increase in the number of mast cells (Aloe, et al., Int. J. Tissue Reactions-Exp. Clin. Aspects, 15: 139-143 (1993)). Additionally, elevated levels of expression of canine NGF has been shown in lame dogs (Isola, M., Ferrari, V., Stabile, F., Bernardini, D., Carnier, P., Busetto, R. Nerve growth factor concentrations in the synovial fluid from healthy dogs and dogs with secondary osteoarthritis. Vet. Comp. Orthop. Traumatol. 4: 279 (2011)). PCT Publication No. WO 02/096458 discloses use of anti-NGF antibodies of certain properties in treating various NGF related disorders such as inflammatory condition (e.g., rheumatoid arthritis). It has been reported that a purified anti-NGF antibody injected into arthritic transgenic mice carrying the human tumor necrosis factor (TNF) gene caused reduction in the number of mast cells, as well as a decrease in histamine and substance P levels within the synovium of arthritis mice (Aloe et al., Rheumatol. Int., 14: 249-252 (1995)). It has been shown that exogenous administration of a NGF antibody reduced the enhanced level of TNF occurring in arthritic mice (Manni et al., Rheumatol. Int., 18: 97-102 (1998)).
Increased expression of NGF and high affinity NGF receptor (TrkA) was observed in human osteoarthritis chondrocytes (Iannone et al., Rheumatology, 41: 1413-1418 (2002)). Rodent anti-NGF antagonist antibodies have been reported (Hongo et al., Hybridoma, 19(3):215-227 (2000); Ruberti et al., Cell. Molec. Neurobiol., 13(5): 559-568 (1993)). However, when rodent antibodies are used therapeutically in non-rodent subjects, an anti-murine antibody response develops in significant numbers of treated subjects.
The involvement of NGF in chronic pain has led to considerable interest in therapeutic approaches based on inhibiting the effects of NGF (Saragovi, et al., Trends Pharmacol Sci. 21: 93-98 (2000)). For example, a soluble form of the TrkA receptor was used to block the activity of NGF, which was shown to significantly reduce the formation of neuromas, responsible for neuropathic pain, without damaging the cell bodies of the lesioned neurons (Kryger, et al., J. Hand Surg. (Am.), 26: 635-644 (2001)).
Certain anti-NGF antibodies have been described (PCT Publication Nos. WO 2001/78698, WO 2001/64247, WO 2002/096458, WO 2004/032870, WO 2005/061540, WO 2006/131951, WO 2006/110883; U.S. Publication Nos. US 20050074821, US 20080033157, US 20080182978 and US 20090041717; and U.S. Pat. No. 7,449,616). In animal models of neuropathic pain (e.g., nerve trunk or spinal nerve ligation) systemic injection of neutralizing antibodies to NGF prevents both allodynia and hyperalgesia (Ramer et al., Eur. J. Neurosci., 11: 837-846 (1999); Ro et al., Pain, 79: 265-274 (1999)). Furthermore, treatment with a neutralizing anti-NGF antibody produces significant pain reduction in a murine cancer pain model (Sevcik et al., Pain, 115: 128-141 (2005)). Thus, there is a serious need for anti-NGF antagonist antibodies for humans and animals.
SUMMARY OF THE INVENTION
The present disclosure provides a novel family of binding proteins, CDR grafted antibodies, mammalized (such as bovanized, camelized, caninized, equinized, felinized, humanized etc.) antibodies, and fragments thereof, capable of binding and neutralizing NGF. The disclosure provides a therapeutic means with which to inhibit NGF and provides compositions and methods for treating disease associated with increased levels of NGF, particularly inflammatory disorders.
In one aspect, the present disclosure provides a binding protein, or fragment thereof, comprising hypervariable region sequences wholly or substantially identical to sequences from an antibody from a donor species; and constant region sequences wholly or substantially identical to sequences of antibodies from a target species, wherein the donor and target species are different. The binding protein may for example specifically bind NGF and have a heavy chain having a heavy chain variable region and a light chain having a light chain variable region.
In another aspect, the present disclosure provides a binding protein that specifically binds NGF and which has a heavy chain having a heavy chain variable region and a light chain having a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 206, SEQ ID NO: 207, or an antigen-binding or an immunologically functional immunoglobulin fragment thereof.
In another aspect, the present disclosure provides a binding protein that specifically binds NGF and which has a heavy chain having a heavy chain variable region and a light chain having a light chain variable region, wherein the light chain variable region comprises an amino acid sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 44, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, or an antigen-binding or an immunologically functional immunoglobulin fragment thereof.
A binding protein of the present disclosure may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 50% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 50% to one of said sequences. Alternatively, the binding protein of the present disclosure may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 70% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 70% to one of said sequences. The binding protein of the present disclosure may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 80% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 80% to one of said sequences, binding protein of the present disclosure may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 90% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 90% to one of said sequences.
A binding protein of the present disclosure may comprise a heavy chain human immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. A binding proteins of the present disclosure may alternatively comprise a heavy chain canine immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. A binding protein of the present disclosure may alternatively comprise a heavy chain feline immunoglobulin constant domain. A binding protein of the present disclosure may alternatively comprise a heavy chain equine immunoglobulin constant domain. A binding protein of the present disclosure may further comprise a constant region having an amino acid sequence selected from the group consisting of SEQ ID NO: 52 and SEQ ID NO: 54.
Any of the above binding proteins may be selected from the group consisting of; an immunoglobulin molecule, disulfide linked Fv, monoclonal antibody, scFv, chimeric antibody, single domain antibody, CDR-grafted antibody, diabody, humanized antibody, caninized mAb, canine mAb, feline mAb, felinized mAb, equine mAb, equinized mAb, a multispecific antibody, a Fab, a dual specific antibody, a DVD-Ig, a Fab′, a bispecific antibody, a F(ab′)2, and a Fv.
Any of the above binding proteins may be capable of modulating a biological function of NGF, or neutralizing NGF.
Any of the above binding proteins may be capable of neutralizing NGF with a potency (IC50) of at least about 10 nM, at least about 5 nM, at least about 1 nM, at least about 0.5 nM, at least about 0.1 nM, at least about 0.05 nM, at least about 0.01 nM, or at least about 0.001 nM, as measured in the TF-1 cell proliferation assay or the pERK and Pathhunter assays.
Any of the above binding proteins may have an on rate constant (Kon) for NGF of: at least about 102 M−1s−1 at least about 103 M−1s−1, at least about M−1s−1, at least about 105 M−1s−1, or at least about 106 M−1s−1, or at least about 107 M−1s−1, as measured by surface plasmon resonance.
Any of the above binding proteins may have an off rate constant (Koff) for NGF selected from the group consisting of: at most about 10−3s−1, at most about 10−4 s−1, at most about 10−5 s−1, at most about 10−6 s−1, and at most about 10−7s−1, as measured by surface plasmon resonance.
Any of the above binding proteins may have a dissociation constant (KD) for NGF selected from the group consisting of: at most about 10−7 M, at most about 10−8 M, at most about 10−9 M, at most about 10−10 M, at most about 10−11 M at most about 10−12 M, at most about 10−13 M and at most about 10−14 M. The dissociation constant (KD) may be, for example, about 1×10−9 M, about 1×10−10M, about 3.14×10−10M, about 1×10−11 M, about 2.37×10−11 M, about 1×10−12 M, about 1×10−13 M, and about 3.3×10−14 M.
Any of the above binding proteins may further comprise an agent selected from the group consisting of; an immunoadhension molecule, an imaging agent, a therapeutic agent, and a cytotoxic agent. The agent may be, for example, an imaging agent selected from the group consisting of a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin. The imaging agent may be a radiolabel selected from the group consisting of: 3H, 14C, 35S, 90Y, 99Tc, 111In, 1251, 1311, 177Lu, 166Ho, and 153Sm. Alternatively, the agent may be a therapeutic or cytotoxic agent, such as, for example, an anti-metabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an anti-angiogenic agent, an anti-mitotic agent, an anthracycline, toxin, and an apoptotic agent.
Any of the binding proteins may possess a murine, canine, feline, human or equine glycosylation pattern.
Any of the binding proteins may be a crystallized binding protein. The crystallized binding protein may be a carrier-free pharmaceutical controlled release crystallized binding protein.
In another aspect, the present disclosure provides an isolated nucleic acid encoding any of the above binding proteins. The isolated nucleic acid may comprise RNA or DNA.
In another aspect, the present disclosure provides an isolated nucleic acid comprising or complementary to a nucleic acid sequence that encodes a binding protein that specifically binds NGF having a heavy chain having a heavy chain variable region and a light chain having a light chain variable region, wherein the heavy chain variable region is encoded by a nucleotide sequence having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, and 21.
In another aspect, the present disclosure provides an isolated nucleic acid comprising or complementary to a nucleic acid sequence that encodes a binding protein that specifically binds NGF having a heavy chain having a heavy chain variable region and a light chain having a light chain variable region, wherein the light chain variable region is encoded by a nucleotide sequence having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOS: 3, 7, 11, 15, 19 and 23.
In another aspect, the present disclosure provides a recombinant vector comprising an isolated nucleic acid encoding a binding protein that specifically binds NGF as described herein. A recombinant vector according to the present disclosure may comprise pcDNA, pTT, pTT3, pEFBOS, pBV, pJV or pBJ. Also provided is a host cell comprising such a recombinant vector. The host cell may be for example a eukaryotic cell, or a prokaryotic cell. The host cell may be a protist cell; an animal cell such as but not limited to a mammalian cell, avian cell; an insect cell, such as but not limited to an insect Sf9 cell; a plant cell; or a fungal cell. The host cell may be for example an E. coli cell. The host cell may be a CHO cell, or a COS cell. Also provided is an isolated cell line that produces a binding protein that specifically binds NGF as described herein.
In another aspect, the present disclosure provides a pharmaceutical or diagnostic composition comprising a binding protein that specifically binds NGF as described herein, and a pharmaceutically acceptable carrier, diluent or excipient. A pharmaceutical composition may comprise a therapeutically effective amount of the NGF binding protein.
In another aspect, the present disclosure provides a composition for the release of a binding protein, the composition comprising: (a) a composition comprising a binding protein that specifically binds NGF as described herein, and a pharmaceutically acceptable carrier, excipient or diluent, and (b) at least one polymeric carrier.
In another aspect, the present disclosure provides a method for reducing NGF activity in a subject (for example, a dog, cat, horse, ferret, etc.) suffering from a disorder in which NGF activity is detrimental, comprising administering to the subject a therapeutically effective amount of a binding protein that specifically binds NGF as described herein.
In another aspect, the present disclosure provides a method for making anti-NGF antibodies comprising: (a) production of murine monoclonal antibodies; (b) screening hybridoma supernatants; (c) grafting of donor CDRs into target frameworks; and (d) introducing backmutations in the framework region of the target antibodies, wherein the anti-NGF antibodies comprise hypervariable region sequences wholly or substantially identical to sequences from an antibody from the donor species and constant region sequences wholly or substantially identical to sequences of an antibody from the target species, wherein the donor and the target species are different. In the method, the donor may be, for example, a mouse and the target a non-murine mammal, such as but not limited to a bovine, canine, equine, or feline mammal, or a camel goat, human or sheep.
In another aspect, the present disclosure provides a method for detecting the presence or amount of NGF in a sample, comprising: providing a reagent comprising any of the above binding proteins that specifically bind NGF; combining the binding protein with the sample for a time and under conditions sufficient for the binding protein to bind to any NGF in the sample; and determining the presence or amount of NGF in the sample based on specific binding of the binding protein to NGF. In the method, the binding protein may be immobilized or may be capable of being immobilized on a solid support. In the method, the binding protein may be coupled to a detectable label, such as, for example, an imaging agent such as but not limited to a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin. The imaging agent may be for example a radiolabel selected from the group consisting of: 3H, 14C, 35S, 90Y, 99Tc, 111 In, 125I, 131I, 177Lu, 166Ho, and 153Sm.
In another aspect, the present disclosure provides an immunoassay device for detecting the presence or amount of NGF in a sample, the device comprising any of the above binding proteins that specifically bind NGF, immobilized on a solid support.
In another aspect, the present disclosure provides a kit for detecting the presence or amount of NGF in a sample, the kit comprising: an immunoreagent comprising any of the above binding proteins that specifically bind NGF and instructions for determining the presence or amount of NGF in the sample based on specific binding of the immunoreagent to NGF. In the kit, the binding protein may be immobilized on a solid support.
In still yet another aspect, the present disclosure relates to an antibody or antigen binding fragment thereof comprising:
a heavy chain variable region comprises an amino acid sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO:168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 206, SEQ ID NO: 207, or an antigen-binding or an immunologically functional immunoglobulin fragment thereof; and
a light chain variable region comprises an amino acid sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 44, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, or an antigen-binding or an immunologically functional immunoglobulin fragment thereof.
More specifically, the above-described antibody may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 50% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 50% to one of said sequences. Alternatively, the above-described antibody may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 70% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 70% to one of said sequences. Alternatively, the above-described antibody may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 80% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 80% to one of said sequences. Alternatively, the above-described antibody may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 90% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 90% to one of said sequences.
The above-described antibody may comprise a heavy chain human immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. More specifically, the antibody may comprise a heavy chain canine immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. Alternatively, the antibody comprises a heavy chain feline immunoglobulin constant domain. Still further alternatively, the antibody comprises a heavy chain equine immunoglobulin constant domain. Moreover, the above-described antibody may comprise a constant region having an amino acid sequence selected from the group consisting of SEQ ID NO: 52 and SEQ ID NO: 54. Still further, the above-described antibody is selected from the group consisting of: an immunoglobulin molecule, disulfide linked Fv, monoclonal antibody, scFv, chimeric antibody, single domain antibody, CDR-grafted antibody, diabody, humanized antibody, caninized mAb, canine mAb, feline mAb, felinized mAb, equine mAb, equinized mAb, a multispecific antibody, a Fab, a dual specific antibody, a DVD-Ig, a Fab′, a bispecific antibody, a F(ab′)2, and a Fv.
In another aspect, the above-identified antibody is capable of modulating a biological function of NGF.
In still yet another aspect, the present disclosure relates to an isolated nucleic acid encoding the above-described antibody.
In another aspect, the present invention relates to an antibody or antigen binding fragment thereof having a heavy chain variable region that comprises an amino acid sequence having at least 90% identity with a sequence of SEQ ID NO:37 and a light chain variable region that comprises an amino acid sequence having at least 90% identity with a sequence of SEQ ID NO:38. The above-described antibody may comprise a heavy chain human immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. More specifically, the antibody may comprise a heavy chain canine immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. Alternatively, the antibody comprises a heavy chain feline immunoglobulin constant domain. Still further alternatively, the antibody comprises a heavy chain equine immunoglobulin constant domain. Moreover, the above-described antibody may comprise a constant region having an amino acid sequence selected from the group consisting of SEQ ID NO: 52 and SEQ ID NO: 54. Still further, the above-described antibody is selected from the group consisting of: an immunoglobulin molecule, disulfide linked Fv, monoclonal antibody, scFv, chimeric antibody, single domain antibody, CDR-grafted antibody, diabody, humanized antibody, caninized mAb, canine mAb, feline mAb, felinized mAb, equine mAb, equinized mAb, a multispecific antibody, a Fab, a dual specific antibody, a DVD-Ig, a Fab′, a bispecific antibody, a F(ab′)2, and a Fv.
In another aspect, the above-identified antibody is capable of modulating a biological function of NGF.
In still yet another aspect, the present disclosure relates to an isolated nucleic acid encoding the above-described antibody.
In another aspect, the present invention relates to an antibody or antigen binding fragment thereof having a heavy chain variable region comprises an amino acid sequence having at least 90% identity with a sequence of SEQ ID NO: 192 and the light chain variable region comprises an amino acid sequence having at least 90% identity with a sequence of SEQ ID NO: 193. The above-described antibody may comprise a heavy chain human immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. More specifically, the antibody may comprise a heavy chain canine immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. Alternatively, the antibody comprises a heavy chain feline immunoglobulin constant domain. Still further alternatively, the antibody comprises a heavy chain equine immunoglobulin constant domain. Moreover, the above-described antibody may comprise a constant region having an amino acid sequence selected from the group consisting of SEQ ID NO: 52 and SEQ ID NO: 54. Still further, the above-described antibody is selected from the group consisting of: an immunoglobulin molecule, disulfide linked Fv, monoclonal antibody, scFv, chimeric antibody, single domain antibody, CDR-grafted antibody, diabody, humanized antibody, caninized mAb, canine mAb, feline mAb, felinized mAb, equine mAb, equinized mAb, a multispecific antibody, a Fab, a dual specific antibody, a DVD-Ig, a Fab′, a bispecific antibody, a F(ab′)2, and a Fv.
In another aspect, the above-identified antibody is capable of modulating a biological function of NGF.
In still yet another aspect, the present disclosure relates to an isolated nucleic acid encoding the above-described antibody.
In still yet another aspect, the present disclosure relates to a pharmaceutical or diagnostic composition comprising at least one of the above-described antibodies, and a pharmaceutically acceptable carrier, diluent or excipient. More specifically, the pharmaceutical or diagnostic composition may comprise a therapeutically effective amount of at least one of the above-described antibodies. In addition, the pharmaceutical or diagnostic composition may comprise at one preservative. An example of at least one preservative that may be used is methylparaben, propylparaben, benzyl alcohol, chlorobutanol or benzalkonium chloride.
The pharmaceutical composition can have a pH of greater than about 7.0. Alternatively, the pharmaceutical composition can have a pH of between about 6.8 and about 8.2. Alternatively; the pharmaceutical composition can have a pH of between about 7.2 and about 7.8. Still further alternatively, the pH of the pharmaceutical composition can be between about 7.4 and about 7.6. Still further alternatively, the pH of the pharmaceutical composition can be about 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1 or 8.2.
The pharmaceutical composition of the present disclosure may have a half-life of from about 8.0 days to about 15.0 days when dosed intravenously or subcutaneously. Alternatively, the pharmaceutical composition of the present invention may have a half-life of from about 10.0 days to about 13.0 days. Still further alternatively, the pharmaceutical composition of the present invention may have a half-life of about 8.0 days, about 8.5 days, about 9.0 days, about 9.5 days, about 10.0 days, about 10.5 days, about 11.0 days, about 11.5 days, about 12.0 days, about 12.5 days, about 13.0 days, about 13.5 days, about 14.0 days, about 14.5 days or about 15.0 days.
In another aspect, the present disclosure relates to a method for reducing NGF activity in a subject suffering from a disorder in which NGF activity is detrimental, comprising administering to the subject a therapeutically effective amount of an antibody of antigen binding fragment thereof of at least one of the above-described antibodies or antigen-binding fragments thereof.
BRIEF DESCRIPTION OF FIGURES
FIG. 1 illustrates PR-1254972 VH nucleotide sequence (SEQ ID NO: 1) of mouse anti-NGF antibody.
FIG. 2 illustrates PR-1254972 VH amino acid sequence (SEQ ID NO: 2) of mouse anti-NGF antibody.
FIG. 3 illustrates PR-1254972 VL nucleotide sequence (SEQ ID NO: 3) of mouse anti-NGF antibody.
FIG. 4 illustrates PR-1254972 VL amino acid (SEQ ID NO: 4) of mouse anti-NGF antibody.
FIG. 5 illustrates PR-1254973 VH nucleotide sequence (SEQ ID NO: 5) of mouse anti-NGF antibody.
FIG. 6 illustrates PR-1254973 VH amino acid (SEQ ID NO: 6) of mouse anti-NGF antibody.
FIG. 7 illustrates PR-1254973 VL nucleotide sequence (SEQ ID NO: 7) of mouse anti-NGF antibody.
FIG. 8 illustrates PR-1254973 VL amino acid (SEQ ID NO: 8) of mouse anti-NGF antibody.
FIG. 9 illustrates PR-1254977 VH nucleotide sequence (SEQ ID NO: 9) of mouse anti-NGF antibody.
FIG. 10 illustrates PR-1254977 VH amino acid (SEQ ID NO: 10) of mouse anti-NGF antibody.
FIG. 11 illustrates PR-1254977 VL nucleotide sequence (SEQ ID NO: 11) of mouse anti-NGF antibody.
FIG. 12 illustrates PR-1254977 VL amino acid (SEQ ID NO: 12) of mouse anti-NGF antibody.
FIG. 13 illustrates PR-1254980 VH nucleotide sequence (SEQ ID NO: 13) of mouse anti-NGF antibody.
FIG. 14 illustrates PR-1254980 VH amino acid (SEQ ID NO: 14) of mouse anti-NGF antibody.
FIG. 15 illustrates PR-1254980 VL nucleotide sequence (SEQ ID NO: 15) of mouse anti-NGF antibody.
FIG. 16 illustrates PR-1254980 VL amino acid (SEQ ID NO: 16) of mouse anti-NGF antibody.
FIG. 17 illustrates PR-1254981 VH nucleotide sequence (SEQ ID NO: 17) of mouse anti-NGF antibody.
FIG. 18 illustrates PR-1254981 VH amino acid (SEQ ID NO: 18) of mouse anti-NGF antibody.
FIG. 19 illustrates PR-1254981 VL nucleotide sequence (SEQ ID NO: 19) of mouse anti-NGF antibody.
FIG. 20 illustrates PR-1254981 VL amino acid (SEQ ID NO: 20) of mouse anti-NGF antibody.
FIG. 21 illustrates PR-1254982 VH nucleotide sequence (SEQ ID NO: 21) of mouse anti-NGF antibody.
FIG. 22 illustrates PR-1254982 VH amino acid (SEQ ID NO: 22) of mouse anti-NGF antibody.
FIG. 23 illustrates PR-1254982 VL nucleotide sequence (SEQ ID NO: 23) of mouse anti-NGF antibody.
FIG. 24 illustrates PR-1254982 VL amino acid (SEQ ID NO: 24) of mouse anti-NGF antibody.
FIG. 25 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 25 (72.1 VH amino acid).
FIG. 26 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 26 (72.1 VL amino acid).
FIG. 27 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined) SEQ ID NO: 27 (73.1 VH amino acid).
FIG. 28 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined) SEQ ID NO: 28 (73.1 VL amino acid).
FIG. 29 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 29 (77.1 VH amino acid).
FIG. 30 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 30 (77.1 VL amino acid).
FIG. 31 A illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 31 (81.1 VH amino acid).
FIG. 31 B illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 177 (81.1B VH amino acid).
FIG. 32 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 32 (81.1 VL amino acid)
FIG. 33 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 33 (82.1 VH amino acid)
FIG. 34 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 34 (82.1 VL amino acid).
FIG. 35 illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 35 (72.2 VH amino acid).
FIG. 36A illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 36 (72.2 VL amino acid).
FIG. 36B illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 179 (72.3 VH amino acid).
FIG. 36C illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 180 (72.4 VH amino acid).
FIG. 36D illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 181 (72.4 VL amino acid).
FIG. 37 illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 37 (73.2 VH amino acid).
FIG. 38A illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 38 (73.2 VL amino acid).
FIG. 38B illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 182 (73.4 VH amino acid).
FIG. 38C illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 183 (73.4 VL amino acid).
FIG. 39 illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 39 (77.2 VH amino acid).
FIG. 40A illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 40 (77.2 VL amino acid).
FIG. 40B illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 184 (77.3 VH amino acid).
FIG. 40C illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 185 (77.4 VH amino acid).
FIG. 40D illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 186 (77.4 VL amino acid).
FIG. 41 illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 41 (81.2 VH amino acid).
FIG. 42A illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 42 (81.2 VL amino acid).
FIG. 42B illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 187 (81.4 VH amino acid).
FIG. 42C illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 188 (81.4 VL amino acid).
FIG. 42D illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 189 (81.2B VH amino acid).
FIG. 42E illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 190 (81.4B VH amino acid).
FIG. 42F illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold, SEQ ID NO: 206 (81.5B VH amino acid).
FIG. 42G illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold, SEQ ID NO: 207 (81.6B VH amino acid).
FIG. 43 illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 43 (82.2 VH amino acid).
FIG. 44A illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 44 (82.2 VL amino acid).
FIG. 44B illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 191 (82.3 VL amino acid).
FIG. 44C illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 192 (82.4 VH amino acid).
FIG. 44D illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 193 (82.4 VL amino acid).
FIG. 45 illustrates primer sequence to clone canine NGF, SEQ ID NO: 45 (NGF-Dog-S primer).
FIG. 46 illustrates primer sequence to clone canine NGF, SEQ ID NO: 46 (NGF-Dog-AS primer).
FIG. 47 illustrates primer sequence to clone canine NGF, SEQ ID NO: 47 (NGF-d-Ec-S primer).
FIG. 48 illustrates primer sequence to clone canine NGF, SEQ ID NO: 48 (NGF-d-Ec-AS primer).
FIG. 49 illustrates canine NGF C-terminal 6His fusion nucleotide sequence, SEQ ID NO: 49.
FIG. 50 illustrates canine NGF C-terminal 6-His amino acid sequence, SEQ ID NO: 50.
FIG. 51 illustrates canine IgG constant region nucleotide sequence, SEQ ID NO: 51.
FIG. 52 illustrates canine IgG constant region amino acid sequence, SEQ ID NO: 52.
FIG. 53 illustrates canine kappa constant region nucleotide sequence, SEQ ID NO: 53
FIG. 54 illustrates canine kappa constant region amino acid sequence, SEQ ID NO: 54.
FIG. 55 illustrates complementarity determining region, SEQ ID NO: 55 (72.1 VH amino acid; CDR1).
FIG. 56 illustrates complementarity determining region, SEQ ID NO: 56 (72.1 VH amino acid; CDR2).
FIG. 57 illustrates complementarity determining region, SEQ ID NO: 57 (72.1 VH amino acid; CDR3).
FIG. 58 illustrates complementarity determining region, SEQ ID NO: 58 (72.1 VL amino acid; CDR1).
FIG. 59 illustrates complementarity determining region, SEQ ID NO: 59 (72.1 VL amino acid; CDR2).
FIG. 60 illustrates complementarity determining region, SEQ ID NO: 60 (72.1 VL amino acid; CDR3).
FIG. 61 illustrates complementarity determining region, SEQ ID NO: 61 (73.1 VH amino acid; CDR1).
FIG. 62 illustrates complementarity determining region, SEQ ID NO: 62 (73.1 VH amino acid; CDR2).
FIG. 63 illustrates complementarity determining region, SEQ ID NO: 63 (73.1 VH amino acid; CDR3).
FIG. 64 illustrates complementarity determining region, SEQ ID NO: 64 (73.1 VL amino acid; CDR1).
FIG. 65 illustrates complementarity determining region, SEQ ID NO: 65 (73.1 VL amino acid; CDR2).
FIG. 66 illustrates complementarity determining region, SEQ ID NO: 66 (73.1 VL amino acid; CDR3).
FIG. 67 illustrates complementarity determining region, SEQ ID NO: 67 (77.1 VH amino acid; CDR1).
FIG. 68 illustrates complementarity determining region, SEQ ID NO: 68 (77.1 VH amino acid; CDR2).
FIG. 69 illustrates complementarity determining region, SEQ ID NO: 69 (77.1 VH amino acid; CDR3).
FIG. 70 illustrates complementarity determining region, SEQ ID NO: 70 (77.1 VL amino acid; CDR1).
FIG. 71 illustrates complementarity determining region, SEQ ID NO: 71 (77.1 VL amino acid; CDR2).
FIG. 72 illustrates complementarity determining region, SEQ ID NO: 72 (77.1 VL amino acid; CDR3).
FIG. 73 illustrates complementarity determining region, SEQ ID NO: 73 (81.1 VH amino acid; CDR1).
FIG. 74 illustrates complementarity determining region, SEQ ID NO: 74 (81.1 VH amino acid; CDR2).
FIG. 75 illustrates complementarity determining region, SEQ ID NO: 75 (81.1 VH amino acid; CDR3).
FIG. 76 illustrates complementarity determining region, SEQ ID NO: 76 (81.1 VL amino acid; CDR1).
FIG. 77 illustrates complementarity determining region, SEQ ID NO: 77 (81.1 VL amino acid; CDR2).
FIG. 78 illustrates complementarity determining region, SEQ ID NO: 78 (81.1 VL amino acid; CDR3).
FIG. 79 illustrates complementarity determining region, SEQ ID NO: 79 (82.1 VH amino acid; CDR1).
FIG. 80 illustrates complementarity determining region, SEQ ID NO: 80 (82.1 VH amino acid; CDR2).
FIG. 81 illustrates complementarity determining region, SEQ ID NO: 81 (82.1 VH amino acid; CDR3).
FIG. 82 illustrates complementarity determining region, SEQ ID NO: 82 (82.1 VL amino acid; CDR1).
FIG. 83 illustrates complementarity determining region, SEQ ID NO: 83 (82.1 VL amino acid; CDR2).
FIG. 84 illustrates complementarity determining region, SEQ ID NO: 84 (82.1 VL amino acid; CDR3).
FIG. 85 illustrates the sequence of human βNGF (SEQ ID NO: 85).
FIG. 86 illustrates the sequences shown in Table 14 illustrating SEQ ID NOs 178, 86-88 from canine heavy chain variable domain sequences derived from canine PBMC.
FIG. 86A illustrates the sequences shown in Table 14 illustrating SEQ ID NOs 89-93 from canine heavy chain variable domain sequences derived from canine PBMC.
FIG. 86B illustrates the sequences shown in Table 14 illustrating SEQ ID NOs 94-98 from canine heavy chain variable domain sequences derived from canine PBMC.
FIG. 86C illustrates the sequences shown in Table 14 illustrating SEQ ID NOs 99-102 from canine heavy chain variable domain sequences derived from canine PBMC.
FIG. 86D illustrates the sequences shown in Table 14 illustrating SEQ ID NOs 103-107 from canine heavy chain variable domain sequences derived from canine PBMC.
FIG. 86E illustrates the sequences shown in Table 14 illustrating SEQ ID NOs 108-109 from canine heavy chain variable domain sequences derived from canine PBMC.
FIG. 87 illustrates the sequences shown in Table 15 illustrating SEQ ID NOs 110,111,204,112 from canine Iambda light chain variable domain sequences derived from canine PBMC RNA.
FIG. 87A illustrates the sequences shown in Table 15 illustrating SEQ ID NOs 113-117 from canine lambda light chain variable domain sequences derived from canine PBMC RNA.
FIG. 87B illustrates the sequences shown in Table 15 illustrating SEQ ID NOs 118-122 from canine lambda light chain variable domain sequences derived from canine PBMC RNA.
FIG. 87C illustrates the sequences shown in Table 15 illustrating SEQ ID NOs 123-126 from canine lambda light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88 illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 127-131 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88A illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 132-136 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88B illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 137-141 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88C illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 142-146 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88D illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 147-151 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88E illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 152-156 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88F illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 157-161 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88G illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 162-164 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 89 illustrates the sequences shown in Table 17 illustrating SEQ ID NOs 165-168 from mouse anti-NGF CDRs grafted onto Human Ig Frameworks gCDR-grafted Anti-NGF); CDRs underlined.
FIG. 89A illustrates the sequences shown in Table 17 illustrating SEQ ID NOs 169-173 from mouse anti-NGF CDRs grafted onto Human Ig Frameworks (CDR-grafted Anti-NGF); CDRs underlined.
FIG. 89B illustrates the sequences shown in Table 17 illustrating SEQ ID NOs 174-176 from mouse anti-NGF CDRs grafted onto Human Ig Frameworks (CDR-grafted Anti-NGF); CDRs underlined.
FIG. 90 illustrates the sequences shown in Table 18 illustrating SEQ ID NOs 194-196 from Mouse/Canine Chimeric Antibody sequences.
FIG. 90A illustrates the sequences shown in Table 18 illustrating SEQ ID NOs 197-199 from Mouse/Canine Chimeric Antibody sequences.
FIG. 90B illustrates the sequences shown in Table 18 illustrating SEQ ID NOs 200-202 from Mouse/Canine Chimeric Antibody sequences.
FIG. 90C illustrates the sequences shown in Table 18 illustrating SEQ ID NOs 203 from Mouse/Canine Chimeric Antibody sequences.
DETAILED DESCRIPTION OF THE DISCLOSURE
The disclosure describes NGF binding proteins, particularly anti-NGF antibodies, or antigen-binding portions thereof, that bind NGF. Various aspects of the disclosure relate to antibodies and antibody fragments, and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such antibodies and fragments. Methods of using the antibodies of the disclosure to detect human and canine NGF, to inhibit human and canine NGF activity, either in vitro or in vivo; and to regulate gene expression are also encompassed by the disclosure.
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.
Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
That the present disclosure may be more readily understood, select terms and phrases as used herein are defined below.
Definitions
The terms “acceptor” and “acceptor antibody” refer to the antibody or nucleic acid sequence providing or encoding at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% of the amino acid sequences of one or more of the framework regions. The term “acceptor” encompasses an antibody amino acid or nucleic acid sequence providing or encoding the constant region(s). The term also encompasses the antibody amino acid or nucleic acid sequence providing or encoding one or more of the framework regions and the constant region(s). For example, the term “acceptor” may refer to a human antibody amino acid or nucleic acid sequence that provides or encodes at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequences of one or more of the framework regions. Such an acceptor may contain at least 1, at least 2, at least 3, least 4, at least 5, or at least 10 amino acid residues that does (do) not occur at one or more specific positions of a human antibody. An acceptor framework region and/or acceptor constant region(s) may be, e.g., derived or obtained from a germline antibody gene, a mature antibody gene, a functional antibody (e.g., antibodies well-known in the art, antibodies in development, or antibodies commercially available).
The term “agonist” refers to a modulator that, when contacted with a molecule of interest, causes an increase in the magnitude of a certain activity or function of the molecule compared to the magnitude of the activity or function observed in the absence of the agonist. Particular agonists of interest may include, but are not limited to, NGF polypeptides or polypeptides, nucleic acids, carbohydrates, or any other molecules that bind to NGF.
The term “antagonist” or “inhibitor” refer to a modulator that, when contacted with a molecule of interest causes a decrease in the magnitude of a certain activity or function of the molecule compared to the magnitude of the activity or function observed in the absence of the antagonist. Particular antagonists of interest include those that block or modulate the biological or immunological activity of NGF. Antagonists and inhibitors of NGF may include, but are not limited to, proteins, nucleic acids, carbohydrates, or any other molecules, which bind to NGF.
The term “antibody” broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art. Non-limiting examples are discussed herein below.
In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgAI and IgA2) or subclass.
The term “antibody conjugate” refers to a binding protein, such as an antibody, chemically linked to a second chemical moiety, such as a therapeutic or cytotoxic agent. The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials. In one aspect the therapeutic or cytotoxic agents include, but are not limited to, pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
The term “antibody construct” refers to a polypeptide comprising one or more the antigen binding portions linked to a linker polypeptide or an immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Such linker polypeptides are well known in the art (Holliger, et al., Proc. Natl. Acad. Set, 90: 6444-6448 (1993); Poljak, et al., Structure 2: 1121-1123 (1994)). An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences are known in the art; canine, equine, and feline are rarer.
The term “antibody fragments” or “antigen-binding moiety” comprises a portion of a full length antibody, generally the antigen binding or variable domain thereof. Examples of antibody fragments include Fab, Fab′, F(ab)2, Fv, scFv fragments, diabodies, linear antibodies, single-chain antibody molecules.
The term “antigen-binding portion” of an antibody (or simply “antibody portion”) refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., NGF). It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. These may also be bispecific, dual specific, or multi-specific formats; specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature, 341: 544-546 (1989); PCT publication WO 90/05144), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv) (Bird et al., Science, 242: 423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci., 85: 5879-5883 (1988)). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed.
Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (Holliger, et al., Proc. Natl. Acad. Sci., 90: 6444-6448 (1993); Poljak, et al., Structure 2: 1121-1123 (1994)). Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).
Still further, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al., Human Antibodies and Hybridomas, 6: 93-101 (1995)) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, et al., Mol. Immunol., 31: 1047-1058 (1994)). Antibody portions, such as Fab and F(ab′)2 fragments, may be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules may be obtained using standard recombinant DNA techniques, as described herein.
The term “anti-NGF antibody” refers to an antibody which is able to bind to nerve growth factor (NGF) and inhibit NGF biological activity and/or downstream pathway(s) mediated by NGF signaling. An anti-NGF antibody encompasses antibodies that block, antagonize, suppress or reduce (including significantly) NGF biological activity, including downstream pathways mediated by NGF signaling, such as receptor binding and/or elicitation of a cellular response to NGF. Anti-NGF antibodies encompass those that neutralize NGF biological activity, bind NGF and prevent NGF dimerization and/or binding to an NGF receptor (such as p75 and/or trkA), and/or bind NGF and prevent trkA receptor dimerization and/or trkA autophosphorylation. Examples of anti-NGF antibodies are provided herein.
The term “binding protein” refers to a natural or synthetic polypeptide that specifically binds to any portion of a target such as an antigen. The term “binding protein” encompasses antibodies as described herein, including an isolated antibody, antigen-binding portion thereof, or immunologically functional fragment thereof
The term “canine antibody” refers to a naturally-occurring or recombinantly produced immunoglobulin composed of amino acid sequences representative of natural antibodies isolated from canines of various breeds. Canine antibodies are antibodies having variable and constant regions derived from canine germline immunoglobulin sequences. The canine antibodies of the disclosure may include amino acid residues not encoded by canine germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “canine antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto canine framework sequences.
The term “caninization” is defined as a method for transferring non-canine antigen-binding amino acids from a donor antibody to a canine antibody acceptor framework to generate protein therapeutic treatments useful in dogs.
The term “caninized antibody” refers to antibodies which comprise heavy and light chain variable region sequences from a non-canine species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “canine-like”, i.e., more similar to canine germline variable sequences. One type of caninized antibody is a CDR-grafted antibody, in which non-canine CDR sequences are introduced into canine VH and VL sequences to replace the corresponding canine CDR sequences.
Caninized forms of non-canine antibodies provided herein are canine antibodies that contain sequence derived from a non-canine antibody. For the most part, caninized antibodies are canine antibody sequences (“acceptor” or “recipient” antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-canine species (“donor” antibody) such as mouse, rat, rabbit, cat, goat, chicken, bovine, horse, llama, camel, dromedaries, sharks, non-human primates, human, humanized, recombinant sequence, or an engineered sequence having the desired properties. In some instances, framework region (FR) residues of the canine antibody are replaced by corresponding non-canine FR residues. Furthermore, caninized antibodies may include residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. The caninized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc) of a canine antibody. Strategies for caninization of antibodies include, but are not limited to, the strategies disclosed in WO 2003/060080.
The caninized antibody is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a canine antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-canine antibody. A caninized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab′, F(ab′) 2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-canine immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a canine immunoglobulin consensus sequence. A caninized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a canine immunoglobulin. A canine or caninized antibody may contain both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. A caninized antibody may only contain a caninized light chain, or may only contain a caninized heavy chain. An exemplary caninized antibody contains a caninized variable domain of a light chain and a caninized variable domain of a heavy chain.
The term “canonical” residue refers to a residue in a CDR or framework that defines a particular canonical CDR structure as defined by Chothia et al. (J. Mol. Biol., 196:901-907 (1987); Chothia et al., J. Mol. Biol., 227:799 (1992). According to Chothia et al., critical portions of the CDRs of many antibodies have nearly identical peptide backbone conformations despite great diversity at the level of amino acid sequence. Each canonical structure specifies primarily a set of peptide backbone torsion angles for a contiguous segment of amino acid residues forming a loop.
The term “CDR” refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. The term “CDR set” refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as L1I, L2 and L3 or H1, H2 and H3 where the “L” and the “H” designates the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan (FASEB J. 9: 133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Still other CDR boundary definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although certain methods described herein use Kabat or Chothia defined CDRs.
The term “CDR-grafted antibody” refers to antibodies which comprise heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of another species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) has been replaced with human CDR sequences.
The term “chimeric antibody” refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human, canine, equine, or feline constant regions. Chimeric antibodies comprise a portion of the heavy and/or light chain that is identical to or homologous with corresponding sequences from antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous with corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, exhibiting the desired biological activity (See e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
The terms “crystal” and “crystallized” refer to an antibody, or antigen binding portion thereof, that exists in the form of a crystal. Crystals are one form of the solid state of matter, which is distinct from other forms such as the amorphous solid state or the liquid crystalline state. Crystals are composed of regular, repeating, three-dimensional arrays of atoms, ions, molecules (e.g., proteins such as antibodies), or molecular assemblies (e.g., antigen/antibody complexes). These three-dimensional arrays are arranged according to specific mathematical relationships that are well-understood in the field. The fundamental unit, or building block, that is repeated in a crystal is called the asymmetric unit. Repetition of the asymmetric unit in an arrangement that conforms to a given, well-defined crystallographic symmetry provides the “unit cell” of the crystal. Repetition of the unit cell by regular translations in all three dimensions provides the crystal. See Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ea., pp. 20 1-16, Oxford University Press, New York, N.Y., (1999).
The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
The terms “donor” and “donor antibody” refer to an antibody providing one or more CDRs. A donor antibody may be an antibody from a species different from the antibody from which the framework regions are obtained or derived. In the context of a humanized antibody, the term “donor antibody” refers to a non-human antibody providing one or more CDRs. In the context of a caninized antibody, the term “donor antibody” refers to a non-canine antibody providing one or more CDRs. In the context of a felinized antibody, the term “donor antibody” refers to a non-feline antibody providing one or more CDRs. In the context of an equinized antibody, the term “donor antibody” refers to a non-equine antibody providing one or more CDRs.
The term “epitope” includes any polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitope determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and may have specific three dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. An antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
The term “equine antibody” refers to a naturally-occurring or recombinantly produced immunoglobulin composed of amino acid sequences representative of natural antibodies isolated from equines of various breeds. Equine antibodies are antibodies having variable and constant regions derived from equine germline immunoglobulin sequences. The equine antibodies of the disclosure may include amino acid residues not encoded by equine germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “equine antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto equine framework sequences.
The term “equalization” is defined as a method for transferring non-equine antigen-binding amino acids from a donor antibody to an equine antibody acceptor framework to generate protein therapeutic treatments useful in horses.
The term “equinized antibody” refers to antibodies which comprise heavy and light chain variable region sequences from a non-equine species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “equine-like”, i.e., more similar to equine germline variable sequences. One type of equinized antibody is a CDR-grafted antibody, in which non-equine CDR sequences are introduced into equine VH and VL sequences to replace the corresponding equine CDR sequences.
Equinized forms of non-equine antibodies provided herein are equine antibodies that contain sequence derived from a non-equine antibody. For the most part, equinized antibodies are equine antibody sequences (“acceptor” or “recipient” antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-equine species (“donor” antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken, bovine, horse, llama, camel, dromedaries, sharks, non-human primates, human, humanized, recombinant sequence, or an engineered sequence having the desired properties. In some instances, framework region (FR) residues of the equine antibody are replaced by corresponding non-equine FR residues. Furthermore, equinized antibodies may include residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. The equinized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc) of an equine antibody.
The equinized antibody is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of an equine antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-equine antibody. An equinized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab′, F(ab′)2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-equine immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of an equine immunoglobulin consensus sequence. An equinized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of an equine immunoglobulin. An equine or equinized antibody for example may contain both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. An equinized antibody may only contain an equinized light chain, or an equinized heavy chain. An exemplary equinized antibody contains an equinized variable domain of a light chain an equinized variable domain of a heavy chain. Equine isotypes include, for example, IgGa, IgGb, IgGc, IgG (T), IgM, and IgA
The term “Fab” refers to antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to readily crystallize. Pepsin treatment yields a binding cross-linking antigen. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteine(s) from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
The term “feline antibody” refers to a naturally-occurring or recombinantly produced immunoglobulin composed of amino acid sequences representative of natural antibodies isolated from felines of various breeds. Feline antibodies are antibodies having variable and constant regions derived from feline germline immunoglobulin sequences. The feline antibodies of the disclosure may include amino acid residues not encoded by feline germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “feline antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto feline framework sequences.
The term “felinization” is defined as a method for transferring non-feline antigen-binding amino acids from a donor antibody to a feline antibody acceptor framework to generate protein therapeutic treatments useful in cats.
The term “felinized antibody” refers to antibodies which comprise heavy and light chain variable region sequences from a non-feline species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “feline-like”, i.e., more similar to feline germline variable sequences. One type of felinized antibody is a CDR-grafted antibody, in which non-feline CDR sequences are introduced into feline VH and VL sequences to replace the corresponding feline CDR sequences.
Felinized forms of non-feline antibodies provided herein are feline antibodies that contain sequence derived from a non-feline antibody. For the most part, felinized antibodies are feline antibody sequences (“acceptor” or “recipient” antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-feline species (“donor” antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken, bovine, horse, llama, camel, dromedaries, sharks, non-human primates, human, humanized, recombinant sequence, or an engineered sequence having the desired properties. In some instances, framework region (FR) residues of the feline antibody are replaced by corresponding non-feline FR residues. Furthermore, felinized antibodies may include residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. The felinized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc) of a feline antibody.
The felinized antibody is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a feline antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-feline antibody. A felinized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab′, F(ab′) 2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-feline immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a feline immunoglobulin consensus sequence. A felinized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a feline immunoglobulin. A feline or felinized antibody may contain both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. A felinized antibody may only contain a felinized light chain or a felinized heavy chain. An exemplary felinized antibody only contains a felinized variable domain of a light chain and a felinized variable domain of a heavy chain.
The term “framework” or “framework sequence” refers to the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence may be determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, -L2, and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy chain) also divide the framework regions on the light chain and the heavy chain into four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying the particular sub-regions as FR1, FR2, FR3 or FR4, a framework region, as referred by others, represents the combined FR's within the variable region of a single, naturally occurring immunoglobulin chain. An FR represents one of the four sub-regions, and FRs represents two or more of the four sub-regions constituting a framework region. Human heavy chain and light chain acceptor sequences are known in the art. Canine heavy chain and light chain acceptor sequences are also known (patent application publication WO03/060080 and U.S. Pat. No. 7,261,890B2).
The term “germline antibody gene” or “gene fragment” refers to an immunoglobulin sequence encoded by non-lymphoid cells that have not undergone the maturation process that leads to genetic rearrangement and mutation for expression of a particular immunoglobulin (Shapiro et al., Crit. Rev. Immunol. 22(3): 183-200 (2002); Marchalonis et al, Adv Exp MedBiol. 484: 13-30 (2001)). One of the advantages provided by the binding proteins of the present disclosure stems from the recognition that germline antibody genes are more likely than mature antibody genes to conserve essential amino acid sequence structures characteristic of individuals in the species, hence less likely to be recognized as from a foreign source when used therapeutically in that species.
The term “Fv” refers to the minimum antibody fragment that contains a complete antigen-recognition and binding site. This region consists of a dimer of one heavy chain and one light chain variable domain.
The term “human antibody” refers to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “human antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
The term “humanized antibody” refers to antibodies which comprise heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “human-like”, i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which non-human CDR sequences are introduced into human VH and VL sequences to replace the corresponding human CDR sequences.
The humanized antibody is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody. A humanized antibody comprises substantially all, or at least one, and typically two, variable domains (Fab, Fab′, F(ab′) 2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. A humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. A humanized or caninized antibody may contain both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Alternatively, a humanized antibody may only contain a humanized light chain, or a humanized heavy chain. An exemplary humanized antibody contains a humanized variable domain of a light chain and a humanized variable domain of a heavy chain.
The bovanized, camelized, caninized, equinized, felinized, or humanized antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including without limitation IgG1, IgG2, IgG3 and IgG4. The bovanized, camelized, caninized, equinized, felinized, or humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions using techniques well-known in the art.
The framework and CDR regions of a bovanized, camelized, caninized, equinized, felinized, or humanized antibody need not correspond precisely to the parental sequences, e.g., the donor antibody CDR or the consensus framework may be mutagenized by substitution, insertion and/or deletion of at least one amino acid residue so that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. Such mutations, however, will not be extensive. Usually, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and at least 95% of the bovanized, camelized, caninized, equinized, felinized, or humanized antibody residues will correspond to those of the parental FR and CDR sequences. The term “consensus framework” refers to the framework region in the consensus immunoglobulin sequence. The term “consensus immunoglobulin sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either may be included in the consensus sequence.
The term “hypervariable region” refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a “complementarity determining region” or “CDR” in the light chain variable domain and in the heavy chain variable domain as defined by Kabat et al., 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and/or as defined by (Chothia and Lesk, Mol. Biol. 196:901-917 (1987) and/or as defined as “AbM loops” by Martin, et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989) and/or as defined by Lefranc et al., Nucleic Acids Res, 27:209-212 (1999) in the international ImMunoGeneTics information systems database. “Framework” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.
The term “identity” refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing their sequences thereof, wherein “identity” refers more specifically to the degree of sequence relatedness between nucleic acid molecules or polypeptides, as determined by the match between strings of two or more nucleotide or two or more amino acid sequences. “Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., “algorithms”). The term “similarity” is used to refer to a related concept with respect to the relationship of two or more nucleic acid molecules or two or more polypeptide molecules. In contrast to “identity,” “similarity” refers to a measure of relatedness, which includes both identical matches and conservative substitution matches. For example, for two polypeptide sequences that have 50/100 identical amino acids, and the remainder are all non-conservative substitutions, then the percent identity and similarity would both be 50%. With respect to the same two sequences, if 25 more positions had conservative substitutions, then the percent identity remains 50%, while percent similarity would be 75% (75/100). Identity and similarity of related nucleic acids and polypeptides may be readily calculated by methods well known and readily available in the art, including but are not limited to, those described in COMPUTATIONAL MOLECULAR BIOLOGY, (Lesk, A. M., ed.), 1988, Oxford University Press, New York; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, (Smith, D. W., ed.), 1993, Academic Press, New York; COMPUTER ANALYSIS OF SEQUENCE DATA, Part 1, (Griffin, A. M., and Griffin, H. G., eds.), 1994, Humana Press, New Jersey; von Heinje, G., SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, 1987, Academic Press; SEQUENCE ANALYSIS PRIMER, (Gribskov, M. and Devereux, J., eds.), 1991, M. Stockton Press, New York; Carillo et al., 1988, SIAM J. Applied Math., 48:1073; and Durbin et al., 1998, BIOLOGICAL SEQUENCE ANALYSIS, Cambridge University Press.
Preferred methods to determine identity are designed to provide the highest match between the compared sequences, and are well described in readily publicly available computer programs. Preferred such computerized methods for determining identity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux et al., 1984, Nucl Acid. Res., 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., 1990, J. Mol Biol., 215:403-410). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., 1990, supra). The well-known Smith Waterman algorithm may also be used to determine identity.
The terms “individual,” “patient,” and “subject” are used interchangeably herein, to refer to mammals, including, but not limited to, humans, murines, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian farm and agricultural animals, mammalian sport animals, and mammalian pets. Exemplary subjects companion animals, such as a dog, cat or horse.
An “isolated antibody” refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds NGF is substantially free of antibodies that specifically bind antigens other than NGF). An isolated antibody that specifically binds NGF may, however, have cross-reactivity to other antigens, such as NGF molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals. The terms “isolated polynucleotide” and “isolated nucleic acid” as used interchangeably herein refer to a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a another polynucleotide to which it is not linked in nature, or is not found in nature within a larger sequence. The term “isolated protein” or “isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation is not associated with naturally associated components that accompany it in its native state; is substantially free of other proteins from the same species; is expressed by a cell from a different species; or does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
The term “Kd” refers to the dissociation constant of a particular antibody-antigen interaction as is known in the art.
The term “Kon” is refers to the on rate constant for association of an antibody to the antigen to form the antibody/antigen complex as is known in the art.
The term “Koff” refers to the off rate constant for dissociation of an antibody from the antibody/antigen complex as is known in the art.
The terms “Kabat numbering”, “Kabat definitions” and “Kabat labeling” are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e. hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al., Ann. NY Acad, Sci., 190:382-391 (1971); and Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991)). For the heavy chain variable region, the hypervariable region ranges from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3. For the light chain variable region, the hypervariable region ranges from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.
The term “key residue” refers to certain residues within the variable region that have more impact on the binding specificity and/or affinity of an antibody, in particular a mammalized antibody such as humanized, caninized, equinized or felinized antibody. A key residue includes, but is not limited to, one or more of the following: a residue that is adjacent to a CDR, a potential glycosylation site (may be either N- or O-glycosylation site), a rare residue, a residue capable of interacting with the antigen, a residue capable of interacting with a CDR, a canonical residue, a contact residue between heavy chain variable region and light chain variable region, a residue within the Vernier zone, and a residue in the region that overlaps between the Chothia definition of a variable heavy chain CDR1 and the Kabat definition of the first heavy chain framework.
The term “labeled binding protein” refers to a protein with a label incorporated that provides for the identification of the binding protein. In one aspect, the label is a detectable marker, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that may be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that may be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g. 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 165Ho, 153Sm); fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents, such as gadolinium chelates.
The term “mammalization” refers to a method for transferring donor antigen-binding information to a mammalian antibody acceptor to generate useful therapeutic treatments. More specifically, the invention provides methods for felinization, equinization and caninization of antibodies.
The term “mammalized antibody” refers to antibodies which comprise heavy and light chain variable region sequences from a mammal species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more like “mammal of interest,” see for example, humanized, caninized, equinized or felinized antibodies defined herein. Such mammalized antibodies include, but are not limited to, bovanized, camelized, caninized, equinized, felinized, or humanized antibodies.
The terms “modulate” and “regulate” are used interchangeably and refer to a change or an alteration in the activity of a molecule of interest (e.g., the biological activity of NGF). Modulation may be an increase or a decrease in the magnitude of a certain activity or function of the molecule of interest. Exemplary activities and functions of a molecule include, but are not limited to, binding characteristics, enzymatic activity, cell receptor activation, and signal transduction.
The term “modulator” is a compound capable of changing or altering an activity or function of a molecule of interest (e.g., the biological activity of NGF). For example, a modulator may cause an increase or decrease in the magnitude of a certain activity or function of a molecule compared to the magnitude of the activity or function observed in the absence of the modulator. A modulator may be an inhibitor, which decreases the magnitude of at least one activity or function of a molecule. Exemplary inhibitors include, but are not limited to, proteins, peptides, antibodies, peptibodies, carbohydrates or small organic molecules. Peptibodies are described, e.g., in WO01/83525.
The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone; and not the method by which it is produced and is not limited to antibodies produced through hybridoma technology.
The term “multivalent binding protein” is used in this specification to denote a binding protein comprising two or more antigen binding sites. The multivalent binding protein is engineered to have the three or more antigen binding sites, and is generally not a naturally occurring antibody. The term “multispecific binding protein” refers to a binding protein capable of binding two or more related or unrelated targets. Dual variable domain (DVD) binding proteins are binding proteins that comprise two or more antigen binding sites and are tetravalent or multivalent binding proteins. Such DVDs may be monospecific, i.e. capable of binding one antigen or multispecific, i.e. capable of binding two or more antigens. DVD binding proteins comprising two heavy chain DVD polypeptides and two light chain DVD polypeptides are referred to a DVD Ig. Each half of a DVD Ig comprises a heavy chain DVD polypeptide, and a light chain DVD polypeptide, and two antigen binding sites. Each binding site comprises a heavy chain variable domain and a light chain variable domain with a total of 6 CDRs involved in antigen binding per antigen binding site. DVD binding proteins and methods of making DVD binding proteins are disclosed in U.S. patent application Ser. No. 11/507,050 and incorporated herein by reference.
One aspect of the disclosure pertains to a DVD binding protein comprising binding proteins capable of binding NGF. In another aspect, the DVD binding protein is capable of binding NGF and a second target.
The terms “nerve growth factor” and “NGF” refer to nerve growth factor and variants thereof that retain at least part of the biological activity of NGF. NGF includes all mammalian species of native sequence NGF, including murine, rat, human, rabbit, canine, feline, equine, or bovine.
TABLE 1
Sequence of NGF
Protein
Sequence Identifier
Canine NGF C-terminal 6-His
SEQ ID NO: 50
Human NGF
SEQ ID NO: 85
The term “NGF receptor” refers to a polypeptide that is bound by or activated by NGF. NGF receptors include the TrkA receptor and the p75 receptor of any mammalian species, including, but are not limited to, human, canine, feline, equine, primate, or bovine.
The terms “NGF-related disease” and “NGF-related disorder” encompass any disease or disorder in which the activity of NGF in a subject suffering from the disease or disorder has been shown to be or is suspected of being either responsible for the pathophysiology of the disease or disorder, or a factor that contributes to a worsening of the disease or disorder, which may occur as a result of increased levels of NGF or increased sensitivity of the subject to NGF. Accordingly, an NGF-related disease or NGF-related disorder is a disease or disorder in which reduction of NGF activity is expected to alleviate the symptoms and/or progression of the disease or disorder. Such diseases and disorders may be evidenced, for example, by an increase in the concentration of NGF in a biological fluid of a subject suffering from the disorder (e.g., an increase in the concentration of NGF in serum, plasma, synovial fluid, etc. of the subject), which may be detected, for example, using an anti-NGF antibody as described above. Non-limiting examples of diseases and disorders that may be treated with the antibodies of the disclosure include those diseases and disorders discussed in the section below pertaining to pharmaceutical compositions of the antibodies of the disclosure, and encompass acute pain resulting for example from surgery or other trauma, and chronic pain.
The term “neutralizing” refers to neutralization of biological activity of a NGF when a binding protein specifically binds NGF. A neutralizing binding protein is a neutralizing antibody, who's binding to NGF results in inhibition of a biological activity of NGF. The neutralizing binding protein binds NGF and reduces a biologically activity of NGF by at least about 20%, 40%, 60%, 80%, 85% or more. Inhibition of a biological activity of NGF by a neutralizing binding protein may be assessed by measuring one or more indicators of NGF biological activity well known in the art, including cell proliferation, cell morphology changes, cell signaling, or any detectable cellular response resulting from binding of NGF to the TrkA receptor.
The term “operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. “Operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. The term “expression control sequence” refers to polynucleotide sequences which are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term “control sequences” is intended to include components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
The term “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody or antibody portion.
The term “polynucleotide” means a polymeric form of two or more nucleotides, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA. The term “isolated polynucleotide” shall mean a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or some combination thereof) that, by virtue of its origin, the “isolated polynucleotide” is not associated with all or a portion of a polynucleotide with which the “isolated polynucleotide” is found in nature; is operably linked to a polynucleotide that it is not linked to in nature; or does not occur in nature as part of a larger sequence.
The term “polypeptide” refers to any polymeric chain of amino acids. The terms “peptide” and “protein” are used interchangeably with the term polypeptide and also refer to a polymeric chain of amino acids. The term “polypeptide” encompasses native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. A polypeptide may be monomelic or polymeric.
The term “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.
The term “recombinant host cell” (or simply “host cell”) is intended to refer to a cell into which exogenous DNA has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but, to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell”. In one aspect, host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life. Eukaryotic cells include protist, fungal, plant and animal cells. In another aspect host cells include, but are not limited to, the prokaryotic cell line E. coli; mammalian cell lines CHO, HEK 293 and COS; the insect cell line Sf9; and the fungal cell Saccharomyces cerevisiae.
Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.
The term “recombinant antibody” refers to all species of antibodies or immunoglobulins that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library (Hoogenboom, TIB Tech., 15: 62-70 (1997); Azzazy et al., Clin. Biochem., 35: 425-445 (2002); Gavilondo et al., BioTechniques, 29: 128-145 (2002); Hoogenboom et al., Immunology Today, 21: 371-378 (2000)), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann et al., Current Opinion in Biotechnology, 13: 593-597 (2002); Little et al., Immunology Today, 21: 364-370 (2000)) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies have variable and constant regions derived from species-specific germline immunoglobulin sequences. Such recombinant antibodies may be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to species-specific germline VH and VL sequences, may not naturally exist within the antibody germline repertoire in vivo.
The term “recovering” refers to the process of rendering a chemical species such as a polypeptide substantially free of naturally associated components by isolation, e.g., using protein purification techniques well known in the art.
The term “sample” is used in its broadest sense. A “biological sample” includes, but is not limited to, any quantity of a substance from a living thing or formerly living thing. Such living things include, but are not limited to, humans, mice, rats, monkeys, dogs, rabbits and other animals. Such substances include, but are not limited to, blood, serum, urine, synovial fluid, cells, organs, tissues, bone marrow, lymph nodes and spleen.
The term “single-chainFv” or “scFv” refers to antibody fragments comprising the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).
The terms “specific binding” or “specifically binding” in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.
The term “substantially” in the context of a CDR refers to a CDR having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR.
The term “surface plasmon resonance” refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For further descriptions (Jonsson, et al. Ann. Biol. Clin. 51: 19-26 (1993); Jonsson, et al., Biotechniques 11: 620-627 (1991); Johnsson, et al., J. Mol. Recognit. 8: 125-131 (1995); and Johnnson, B., et al, Anal. Biochem., 198: 268-277 (1991)).
The term “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount may be the amount and/or duration of a therapy which is sufficient to reduce or ameliorate the severity and/or duration of a disorder or one or more symptoms thereof, prevent the advancement of a disorder, cause regression of a disorder, prevent the recurrence, development, onset or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy (e.g., prophylactic or therapeutic agent). A therapeutically effective amount of the antibody or antibody portion may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody, or antibody portion, are outweighed by the therapeutically beneficial effects.
The term “transformation” refers to any process by which exogenous DNA enters a host cell. Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, lipofection, and particle bombardment. Such “transformed” cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. They also include cells which transiently express the inserted DNA or RNA for limited periods of time.
The term “transgenic organism” refers to an organism having cells that contain a transgene, wherein the transgene introduced into the organism (or an ancestor of the organism) expresses a polypeptide not naturally expressed in the organism. A “transgene” is a DNA construct, which is stably and operably integrated into the genome of a cell from which a transgenic organism develops, directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic organism.
The term “vector” is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The term “Vernier zone” refers to a subset of framework residues that may adjust CDR structure and fine-tune the fit to antigen as described by Foote and Winter (1992, J. Mol. Biol. 224:487-499, which is incorporated herein by reference). Vernier zone residues form a layer underlying the CDRs and may impact on the structure of CDRs and the affinity of the antibody.
Anti NGF Binding Proteins
The present disclosure provides a novel family of binding proteins, murine antibodies, CDR grafted antibodies, mammalized (bovanized, camelized, caninized, equinized, felinized, or humanized) antibodies, and fragments thereof, capable of binding and modulating the biological activity or function of NGF, including the capability of neutralizing NGF. The disclosure thus also provides a therapeutic means with which to inhibit NGF and provides compositions and methods for treating disease associated with increased levels of NGF, particularly a disease, condition or disorder where increased levels of NGF, as compared to NGF levels observed in comparable normal subjects, is detrimental.
Binding proteins of the present disclosure may be made by any of a number of techniques known in the art and as described herein, including culturing a host cell described herein in culture medium under conditions sufficient to produce a binding protein capable of binding NGF.
Monoclonal antibodies may be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies may be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981).
Methods for producing and screening for specific antibodies using hybridoma technology are well known in the art. Such methods include, for example, culturing a hybridoma cell secreting an antibody of the disclosure wherein the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the disclosure with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the disclosure. Briefly, for example, mice may be immunized with an NGF antigen. The NGF antigen may be administered, with or without an adjuvant, to stimulate the immune response. Such adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulating complexes). Such adjuvants may protect the polypeptide from rapid dispersal by sequestering it in a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. If a polypeptide is being administered, the immunization schedule will involve two or more administrations of the polypeptide, spread out over several weeks.
After immunization of an animal with an NGF antigen, antibodies and/or antibody-producing cells may be obtained from the animal. An anti-NGF antibody-containing serum may be obtained from the animal by bleeding or sacrificing the animal. The serum may be used as it is obtained from the animal, an immunoglobulin fraction may be obtained from the serum, or the anti-NGF antibodies may be purified from the serum. Serum or immunoglobulins obtained in this manner are polyclonal, thus having a heterogeneous array of properties.
Once an immune response is detected, e.g., antibodies specific for the antigen NGF are detected in the mouse serum, the mouse spleen may be harvested and splenocytes isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, such as, for example, cells from cell line SP20 available from the ATCC. Hybridomas may be selected and cloned by limited dilution. The hybridoma clones may then be assayed by methods known in the art for cells that secrete antibodies capable of binding NGF. Ascites fluid, which generally contains high levels of antibodies, may be generated by immunizing mice with positive hybridoma clones.
Antibody-producing immortalized hybridomas may be prepared from the immunized animal. After immunization, the animal may be sacrificed and the splenic B cells fused to immortalized myeloma cells as is well known in the art (Harlow et al., supra). Alternatively, the myeloma cells may be from a non-secretory cell line and do not secrete immunoglobulin polypeptides. After fusion and antibiotic selection, the hybridomas may be screened using NGF, or a portion thereof, or a cell expressing NGF. Initial screening may be performed, for example, using an enzyme-linked immunoassay (ELISA) or a radioimmunoassay (PJA). An example of ELISA screening is provided in WO 00/37504.
Anti-NGF antibody-producing hybridomas may be selected, cloned and further screened for desirable characteristics, including robust hybridoma growth, high antibody production and desirable antibody characteristics, as discussed further below. Hybridomas may be cultured and expanded in vivo in syngeneic animals, in animals that lack an immune system, e.g., nude mice, or in cell culture in vitro. Methods of selecting, cloning and expanding hybridomas are well known to those of ordinary skill in the art.
An exemplary animal system for preparing hybridomas is the mouse. Hybridoma production in the mouse is very well established, and immunization protocols and techniques for isolation of immunized splenocytes for fusion are well known. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known. Alternatively, the hybridomas may be produced in a non-human, non-mouse species such as a rat, sheep, pig, goat, cattle or horse. Alternatively, human hybridomas may be produced, in which a human non-secretory myeloma is fused with a human cell expressing an anti-NGF antibody.
Antibody fragments that recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab′)2 fragments of the disclosure may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.
Recombinant antibodies may be generated from single, isolated lymphocytes using a procedure referred to in the art as the selected lymphocyte antibody method (SLAM), as described in U.S. Pat. No. 5,627,052, PCT Publication WO 92/02551 and Babcock et al., Proc. Natl. Acad. Sci, 93: 7843-7848 (1996). In this method, single cells secreting antibodies of interest, e.g., lymphocytes derived from any one of the immunized animals described in Section 1, are screened using an antigen-specific hemolytic plaque assay, wherein the antigen NGF, or a fragment thereof, is coupled to sheep red blood cells using a linker, such as biotin, and used to identify single cells that secrete antibodies with specificity for NGF. Following identification of antibody-secreting cells of interest, heavy- and light-chain variable region cDNAs may be rescued from the cells by reverse transcriptase-PCR and these variable regions may then be expressed, in the context of appropriate immunoglobulin constant regions (e.g., human constant regions), in mammalian host cells, such as COS or CHO cells. The host cells transfected with the amplified immunoglobulin sequences, derived from in vivo selected lymphocytes, may then undergo further analysis and selection in vitro, for example by panning the transfected cells to isolate cells expressing antibodies to NGF. The amplified immunoglobulin sequences further may be manipulated in vitro, such as by in vitro affinity maturation methods such as those described in PCT Publication WO 97/29131 and PCT Publication WO 00/56772.
Antibodies may be produced by immunizing a non-human animal comprising some or all of the human immunoglobulin loci with an NGF antigen. For example, human monoclonal antibodies directed against NGF may be generated using transgenic mice carrying parts of the human immune system rather than the mouse system, referred to in the literature and herein as “HuMab” mice, contain a human immunoglobulin gene minilocus that encodes unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and κ chain loci (Lonberg et al., 1994, Nature 368:856-859). These mice exhibit reduced expression of mouse IgM or κ and in response to immunization, and the introduced human heavy chain and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgG κ monoclonal antibodies. The preparation of HuMab mice is well described in the literature. (See, e.g., Lonberg et al., 1994, Nature 368:856-859; Lonberg, 1994, Handbook of Exp. Pharmacology 113:49-101; Taylor et al., 1994, International Immunology 6:579-591; Lonberg & Huszar, 1995, Intern. Rev. Immunol. 13:65-93; and Harding & Lonberg, 1995, Ann. N.Y. Acad. Sci 764:536-546). Alternatively, other known mouse strains such as the HCo7, HCol2, and KM transgenic mice strains may be used to generate human anti-NGF antibodies.
Another suitable, though non-limiting example of a transgenic mouse is the XENOMOUSE® transgenic mouse, which is an engineered mouse strain that comprises large fragments of the human immunoglobulin loci and is deficient in mouse antibody production. See, e.g., Green et al. Nature Genetics, 7: 13-21 (1994); and U.S. Pat. Nos. 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598 and 6,130,364; WO 91/10741, WO 94/02602, WO 96/34096, WO 96/33735, WO 98/16654, WO 98/24893, WO 98/50433, WO 99/45031, WO 99/53049, WO 00 09560, and WO 00/037504. The XENOMOUSE® transgenic mouse produces an adult-like human repertoire of fully human antibodies, and generates antigen-specific human mAbs. The XENOMOUSE® transgenic mouse contains approximately 80% of the human antibody repertoire through introduction of megabase sized, germline configuration YAC fragments of the human heavy chain loci and light chain loci (Mendez et al., Nature Genetics 15: 146-156 (1997), Green et al., J. Exp. Med., 188: 483-495 (1998)).
In vitro methods also may be used to make the antibodies of the disclosure, wherein an antibody library is screened to identify an antibody having the desired binding specificity. Methods for such screening of recombinant antibody libraries are well known in the art and include methods described in, for example, U.S. Pat. No. 5,223,409; PCT Publication Nos. WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; Fuchs et al. Bio/Technology, 9: 1370-1372 (1991); Hay et al., Hum Antibody Hybridomas, 3: 81-85 (1992); Huse et al., Science, 246: 1275-1281 (1989); McCafferty et al., Nature, 348: 552-554 (1990); Griffiths et al., EMBO J., 12: 725-734 (1993); Hawkins et al., J o/Biol, 226: 889-896 (1992); Clackson et al., Nature, 352: 624-628 (1991); Gram et al., PNAS, 89: 3576-3580 (1992); Garrad et al., Bio/Technology, 9: 1373-1377 (1991); Hoogenboom et al., Nuc Acid Res, 19: 4133-4137 (1991); and Barbas et al., PNAS, 88: 7978-7982 (1991), US patent application publication 20030186374, and PCT Publication No. WO 97/29131.
The recombinant antibody library may be from a subject immunized with NGF, or a portion of NGF. Alternatively, the recombinant antibody library may be from a naive subject that has not been immunized with NGF, such as a canine antibody library from a canine subject that has not been immunized with canine NGF. Antibodies of the disclosure are selected by screening the recombinant antibody library with the peptide comprising canine NGF to thereby select those antibodies that recognize NGF. Methods for conducting such screening and selection are well known in the art, such as described in the references in the preceding paragraph. To select antibodies of the disclosure having particular binding affinities for hNGF, such as those that dissociate from canine NGF with a particular koff rate constant, the art-known method of surface plasmon resonance may be used to select antibodies having the desired koff rate constant. To select antibodies of the disclosure having a particular neutralizing activity for hNGF, such as those with a particular an IC5o, standard methods known in the art for assessing the inhibition of hNGF activity may be used.
For example, the antibodies of the present disclosure may also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular aspect, such phage may be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e. g., canine, human or murine). Phage expressing an antigen binding domain that binds the antigen of interest may be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and MI 3 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that may be used to make the antibodies of the present disclosure include those disclosed in Brinkman et al., J. Immunol. Methods, 182: 41-50 (1995); Ames et al., J. Immunol. Methods, 184: 177-186 (1995); Kettleborough et al., Eur. J. Immunol, 24:952-958 (1994); Persic et al., Gene, 187: 9-18 (1997); Burton et al., Advances in Immunology, 57: 191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780, 225; 5,658,727; 5,733,743 and 5,969,108.
As described in the above references, after phage selection, the antibody coding regions from the phage may be isolated and used to generate whole antibodies including human antibodies or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments may also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques, 12(6):864-869 (1992); and Sawai et al., AJPJ 34:26-34 (1995); and Better et al., Science, 240: 1041-1043 (1988) (said references incorporated by reference in their entireties).
Examples of techniques which may be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203:46-88 (1991); Shu et al., PNAS, 90:7995-7999 (1993); and Skerra et al., Science, 240: 1038-1040 (1988).
Alternatives to screening of recombinant antibody libraries by phage display are known and include other methodologies for screening large combinatorial libraries which may be applied to the identification of dual specificity antibodies of the disclosure. One type of alternative expression system is one in which the recombinant antibody library is expressed as RNA-protein fusions, as described in PCT Publication No. WO 98/31700, and in Roberts et al., Proc. Natl. Acad. Sci., 94: 12297-12302 (1997). In this system, a covalent fusion is created between a mRNA and the peptide or protein that it encodes by in vitro translation of synthetic mRNAs that carry puromycin, a peptidyl acceptor antibiotic, at their 3′ end. Thus, a specific mRNA may be enriched from a complex mixture of mRNAs (e.g., a combinatorial library) based on the properties of the encoded peptide or protein, e.g., antibody, or portion thereof, such as binding of the antibody, or portion thereof, to the dual specificity antigen. Nucleic acid sequences encoding antibodies, or portions thereof, recovered from screening of such libraries may be expressed by recombinant means as described above (e.g., in mammalian host cells) and, moreover, may be subjected to further affinity maturation by either additional rounds of screening of mRNA-peptide fusions in which mutations have been introduced into the originally selected sequence(s), or by other methods for affinity maturation in vitro of recombinant antibodies, as described above.
In another approach the antibodies of the present disclosure may also be generated or affinity matured using yeast display methods known in the art. In yeast display methods, genetic methods are used to tether antibody domains to the yeast cell wall and display them on the surface of yeast. In particular, such yeast may be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e. g., human or murine). Examples of yeast display methods that may be used to make the antibodies of the present disclosure include those disclosed Wittrup, et al. U.S. Pat. No. 6,699,658 incorporated herein by reference.
The antibodies or antigen binding fragments described herein may also be produced by genetic engineering. For example, the technology for expression of both heavy and light chain genes in E. coli is the subject of the PCT patent applications: publication number WO 901443, WO901443, and WO 9014424 and in Huse et al., 1989 Science 246: 1275-81. The present disclosure thus also encompasses the isolated nucleic acids encoding any of the binding proteins described herein, as well as a recombinant vector comprising such a nucleic acid molecule, and a host cell comprising such a recombinant vector.
A vector is a nucleic acid molecule, which may be a construct, capable of transporting another nucleic acid to which it has been linked. A vector may include any preferred or required operational elements. Preferred vectors are those for which the restriction sites have been described and which contain the operational elements needed for transcription of the nucleic acid sequence. Such operational elements include for example at least one suitable promoter, at least one operator, at least one leader sequence, at least one terminator codon, and any other DNA sequences necessary or preferred for appropriate transcription and subsequent translation of the nucleic acid sequence. Such vectors contain at least one origin of replication recognized by the host organism along with at least one selectable marker and at least one promoter sequence capable of initiating transcription of the nucleic acid sequence. A vector may be a plasmid into which additional DNA segments may be ligated. A vector may be a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as a plasmid is the most commonly used form of vector. However, the present disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. By way of example and not limitation, suitable vectors include pcDNA, pTT (Durocher et al., Nucleic Acids Research, Vol 30, No. 2 (2002)); pTT3 (pTT with additional multiple cloning site, pEFBOS (Mizushima et al., Nucleic acids Research, Vol 18, No. 17 (1990)), pBV, pJV, pBJ, or pHybE (patent publication no.: US 2009/0239259 AI).
Sequences that are operably linked are in a relationship permitting them to function in their intended manner. A control sequence operably linked to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. Operably linked sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences are polynucleotide sequences that are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, such control sequences generally include promoters and transcription termination sequence. Control sequences may include components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
A host cell may be transformed with a vector that introduces exogenous DNA into a host cell. Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, lipofection, calcium-phosphate precipitation, DEAE-dextran transfection, particle bombardment and the like. Transformed cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, and cells which transiently express the inserted DNA or RNA for limited periods of time.
Host organisms such as host cells are cultured under conditions appropriate for amplification of the vector and expression of the protein, as well known in the art. Expressed recombinant proteins may be detected by any of a number of methods also well known in the art.
Suitable host organisms include for example a prokaryotic or eukaryotic cell system. A eukaryotic cell may be a protist cell, animal cell, plant cell or fungal cell. A eukaryotic cell is for example an animal cell which may be a mammalian cell, avian cell, or an insect cell such as an insect Sf9 cell. Cells from established and readily available may be used, such as but not limited to HeLa, MRC-5 or CV-1. The host cell may be an E. coli cell or a yeast cell such as but not limited to Saccharomyces cerevisiae. Mammalian host cells for expressing the recombinant antibodies of the disclosure also include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub et al., Proc. Natl. Acad. Sci., 77: 4216-4220 (1980), used with a DHFR selectable marker, e.g., as described in Kaufman et al., Mol. Biol, 159: 601-621 (1982)), NS0 myeloma cells, COS cells and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells, or by secretion of the antibody into the culture medium in which the host cells are grown. Antibodies may be recovered from the culture medium using standard protein purification methods.
Host cells may also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It will be understood that variations on the above procedure are within the scope of the present disclosure. For example, it may be desirable to transfect a host cell with DNA encoding functional fragments of either the light chain and/or the heavy chain of an antibody of this disclosure. Recombinant DNA technology may also be used to remove some, or all, of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigens of interest. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the disclosure. In addition, bifunctional antibodies may be produced in which one heavy and one light chain are an antibody of the disclosure and the other heavy and light chain are specific for an antigen other than the antigens of interest by crosslinking an antibody of the disclosure to a second antibody by standard chemical crosslinking methods.
In a system for recombinant expression of an antibody, or antigen-binding portion thereof, of the disclosure, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked to CMV enhancer/AdMLP promoter regulatory elements to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium.
Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody from the culture medium. Still further the disclosure provides a method of synthesizing a recombinant antibody of the disclosure by culturing a host cell of the disclosure in a suitable culture medium until a recombinant antibody of the disclosure is synthesized. The method may further comprise isolating the recombinant antibody from the culture medium.
The present disclosure thus provides anti NGF binding proteins that are specific for and substantially neutralize NGF polypeptides, including active human NGF. Also provided are antibody heavy and light chain amino acid sequences which are substantially specific for and substantially neutralize NGF polypeptides when they are bound to them. This specificity enables the anti-human NGF human antibodies and human monoclonal antibodies with like specificity, to be effective immunotherapy for NGF associated diseases.
The present disclosure encompasses anti NGF binding proteins comprising at least one of the amino acid sequences selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 206, SEQ ID NO: 207 and SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 44, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, and which binds an NGF polypeptide epitope with substantially high affinity as described herein and has the capacity to substantially modulate, including substantially reduce, NGF polypeptide activity.
Examples of such binding proteins include binding proteins comprising a variable heavy chain polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 206 and SEQ ID NO: 207; and a variable light chain polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 44, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200 and SEQ ID NO: 202.
Exemplary pairings of a variable heavy chain polypeptide and a variable light chain polypeptide are represented by the following pairings: SEQ ID NO: 2 and SEQ ID NO: 4; SEQ ID NO: 6 and SEQ ID NO: 8; SEQ ID NO: 10 and SEQ ID NO: 12; SEQ ID NO: 14 and SEQ ID NO: 16; SEQ ID NO: 18 and SEQ ID NO: 20; SEQ ID NO: 22 and SEQ ID NO: 24; SEQ ID NO: 25 and SEQ ID NO: 26; SEQ ID NO: 27 and SEQ ID NO: 28; SEQ ID NO: 29 and SEQ ID NO: 30; SEQ ID NO: 31 and SEQ ID NO: 32; SEQ ID NO: 177 and SEQ ID NO: 32; SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO:36; SEQ ID NO: 37 and SEQ ID NO: 38; SEQ ID NO: 39 and SEQ ID NO: 40; SEQ ID NO: 41 and SEQ ID NO: 42; SEQ ID NO: 43 and SEQ ID NO: 44; SEQ ID NO: 180 and SEQ ID NO: 181, SEQ ID NO: 182 and SEQ ID NO: 183; SEQ ID NO: 185 and SEQ ID NO: 186; SEQ ID NO: 187 and SEQ ID NO: 188; and SEQ ID NO: 192 and SEQ ID NO: 193.
Also encompassed in the disclosure are binding proteins that specifically bind NGF as described herein and comprise a heavy chain variable region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO:27, SEQ ID NO: 29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 206, SEQ ID NO: 207. Also encompassed are binding proteins that specifically bind NGF as described herein and comprise a light chain variable region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 44, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202.
Exemplary binding proteins that specifically bind NGF as described herein preferably comprise a heavy chain variable region and a light chain variable region as follows:
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 2, or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and the light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 4 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof; a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 6 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 8 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 10 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 12 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 14 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 16 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof; a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 18 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 20 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 22 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 24 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 25 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 26 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 27 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 28 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 29 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 30 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 31 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 32 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 177 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 32 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 33 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 34 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 35 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 36 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 37 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 38 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 39 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 40 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 41 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 42 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 43 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 44 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 180 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 181 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 182 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 183 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 185 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 186 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 187 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 188 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 189 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 42 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 190 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 188 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 206 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 42 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 207 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 188 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof; and
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 192 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 193 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof.
Exemplary binding proteins as disclosed herein may include at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NOS: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81; or modified CDR amino acid sequences having a sequence identity of at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% to one of said sequences; b) light chain CDRs consisting of SEQ ID NOS: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84; or modified CDR amino acid sequences having a sequence identity of at least 50% %, at least 60%, at least 70%, at least 80%, or at least 90% to one of said sequences.
It should be understood that variations are contemplated in any of the nucleic acid and amino acid sequences described herein. Such variations include those that will result in a nucleic acid sequence that is capable of directing production of analogs of the corresponding NGF binding proteins. It will be understood that due to the degeneracy of the genetic code, many substitutions of nucleotides may be made that will lead to a DNA sequence that remains capable of directing production of the corresponding protein or its analogs. All such variant DNA sequences that are functionally equivalent to any of the sequences described herein are encompassed by the present disclosure.
A variant of any of the binding proteins described herein means a protein (or polypeptide) that differs from a given protein (e.g., an anti-NGF antibody) in amino acid sequence by the addition (e.g., insertion), deletion, or conservative substitution of amino acids, but that retains the biological activity of the given protein. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity and degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes may be identified, in part, by considering the hydropathic index of amino acids, as understood in the art (see, e.g., Kyte et al., J. Mol. Biol. 157: 105-132 (1982)). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes may be substituted and still retains protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids also may be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity (see, e.g., U.S. Pat. No. 4,554,101, which is incorporated herein by reference). Substitution of amino acids having similar hydrophilicity values may result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. In one aspect, substitutions are performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties. “Variant” also may be used to describe a polypeptide or fragment thereof that has been differentially processed, such as by proteolysis, phosphorylation, or other post-translational modification, yet retains its biological activity or antigen reactivity, e.g., the ability to bind to NGF. Use of “variant” herein is intended to encompass fragments of a variant unless otherwise contradicted by context.
The binding proteins described herein encompass an immunoglobulin molecule, disulfide linked Fv, scFv, monoclonal antibody, murine antibody, chimeric antibody, single domain antibody, CDR-grafted antibody, diabody, mammalized (bovanized, camelized, caninized, equinized, felinized, or humanized) antibody, a canine antibody, feline antibody, equine antibody, murine antibody, multispecific antibody, Fab, dual specific antibody, DVD, Fab′, bispecific antibody, F(ab′)2, or Fv including a single chain Fv fragment.
A binding protein may comprise a particular heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. An exemplary binding protein includes an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. Furthermore, the antibody may comprise a light chain constant region, such as a kappa light chain constant region or a lambda light chain constant region. An exemplary binding protein comprises a kappa light chain constant region.
Replacements of amino acid residues in the Fc portion to alter antibody effector function are known in the art (Winter, et al. U.S. Pat. Nos. 5,648,260; 5,624,821). The Fc portion of an antibody mediates several important effector functions e.g. cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC) and half-life/clearance rate of antibody and antigen-antibody complexes. In some cases these effector functions are desirable for therapeutic antibody but in other cases might be unnecessary or even deleterious, depending on the therapeutic objectives. Certain human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcyRs and complement C1q, respectively. Neonatal Fc receptors (FcRn) are the critical components determining the circulating half-life of antibodies. At least one amino acid residue may be replaced in the constant region of the antibody, for example the Fc region of the antibody, such that effector functions of the antibody are altered.
Binding proteins according to the present disclosure may comprise a heavy chain immunoglobulin constant domain such as, for example, a human or canine or equine or feline IgM constant domain, a human or canine or equine or feline IgG4 constant domain, a human or canine or equine or feline IgG1 constant domain, a human or canine or equine or feline IgE constant domain, a human or canine or equine or feline IgG2 constant domain, a human or canine or equine or feline IgG3 constant domain, and a human or canine or equine or feline IgA constant domain. A binding protein as described herein may comprise a light chain immunoglobulin constant domain such as but not limited to any of human, canine, equine or feline, kappa or lambda constant domains, or any of canine, equine or feline kappa or lambda equivalent constant domains. An exemplary such binding protein has a constant region having an amino acid sequence of SEQ ID NO: 52 or SEQ ID NO: 54.
Binding proteins as described herein may also encompass an NGF anti-idiotype antibody relative to at least one NGF binding protein of the present disclosure. The anti-idiotype antibody includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule such as, but not limited to, at least one complimentarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or; any portion thereof, which may be incorporated into a binding protein of the present disclosure.
The binding proteins of the disclosure are capable of binding to human and canine NGF with high specificity, and additionally are capable of modulating the biological activity or function of NGF in an organism or a subject, including substantially neutralizing human and canine NGF. Also encompassed by the present disclosure are isolated murine monoclonal antibodies, or antigen-binding portions thereof, that bind to NGF with a substantially high affinity, have a slow off rate and/or have a substantially high neutralizing capacity. An exemplary binding protein as disclosed herein is capable of neutralizing NGF with a potency (IC50) of at least about 10 nM, at least about 5 nM, at least about 1 nM, at least about 0.5 nM, at least about 0.1 nM, at least about 0.05 nM, at least about 0.01 nM, or at least about 0.001 nM, as measured in the TF-1 cell proliferation assay or the pERK and Pathhunter assays. Binding proteins as described herein may have an on rate constant (Kon) to NGF of at least about 102M−1s−1 at least about 103 M−1s−1; at least about 104M−1s−1; at least about 105M−1s−1; at least about 106M−1s−1; or at least about 107M−1s−1 as measured by surface plasmon resonance. Binding proteins as described herein may have an off rate constant (Koff) to NGF of at most about 10−3s−1; at most about 10−4s−1; at most about 10−5s−1; at most about 10−6s−1 or at most about 10−7s−1, as measured by surface plasmon resonance. Binding proteins as described herein may have a dissociation constant (KD) to NGF of at most about 10−7 M; at most about 10−8 M; at most about 10−9 M; at most about 10−10M; at most about 10−11M; at most about 10−12M; at most about 10−13M, or at most about 10−14M. For example, a binding protein as described herein may have a dissociation constant (KD) of about 1×10−9M, about 1×10−10M, about 3.14×10−10M, about 1×10−11M, about 2.37×10−11M, about 1×10−12M about 1×10−13M or about 3.3×10−14M.
Binding proteins as described herein including an isolated antibody, or antigen-binding portion thereof, or immunologically functional fragment thereof, may bind NGF and dissociate from NGF with a koff rate constant of about 0.1 s−1 or less, as determined by surface plasmon resonance, or may inhibit NGF activity with an IC50 of about 1×10−6M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from NGF with a koff rate constant of about 1×10−2 s−1 less, as determined by surface plasmon resonance, or may inhibit NGF activity with an IC50 of about 1×10−7 M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from NGF with a koff rate constant of about 1×10−3 s−1 or less, as determined by surface plasmon resonance, or may inhibit NGF with an IC50 of about 1×10−8M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from NGF with a koff rate constant of about 1×10−4 s−1 or less, as determined by surface plasmon resonance, or may inhibit NGF activity with an IC50 of about 1×10−9M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from NGF with a koff rate constant of about 1×10−5 s−1 or less, as determined by surface plasmon resonance, or may inhibit NGF activity with an IC50 of about 1×10−10M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from NGF with a koff rate constant of about 1×10−5 s−1 less, as determined by surface plasmon resonance, or may inhibit NGF activity with an IC50 of about 1×10″11M or less.
A binding protein as described herein may bind canine NGF, wherein the antibody, or antigen-binding portion thereof, may dissociate from canine NGF with a koff rate constant of about 0.1 s−1 or less, as determined by surface plasmon resonance, or may inhibit canine NGF activity with an IC50 of about 1×10−6M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from canine NGF with a koff rate constant of about 1×10−2 s−1 or less, as determined by surface plasmon resonance, or may inhibit canine NGF activity with an IC50 of about 1×10−7M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from canine NGF with a k0ff rate constant of about 1×10−3 s−1 or less, as determined by surface plasmon resonance, or may inhibit canine NGF with an IC5o of about 1×10−8M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from canine NGF with a koff rate constant of about 1×10−4 s−1 or less, as determined by surface plasmon resonance, or may inhibit canine NGF activity with an IC5o of about 1×10−9M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from canine NGF with a koff rate constant of about 1×10−5 s−1 or less, as determined by surface plasmon resonance, or may inhibit canine NGF activity with an IC50 of about 1×10−10M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from canine NGF with a koff rate constant of about 1×10−5 s−1 or less, as determined by surface plasmon resonance, or may inhibit canine NGF activity with an IC50 of about 1×10−11M or less.
The binding proteins of the disclosure further encompass binding proteins coupled to an immunoadhesion molecule, imaging agent, therapeutic agent, or cytotoxic agent. Non-limiting examples of suitable imaging agents include an enzyme, fluorescent label, luminescent label, bioluminescent label, magnetic label, biotin or a radiolabel including, but not limited to, 3H, 14C, 35S, 90Y, 99Tc, 111 In, 125 I, 1311, 177Lu, I66Ho, and 153Sm. The therapeutic or cytotoxic agent may be an anti-metabolite, alkylating agent, antibiotic, growth factor, cytokine, anti-angiogenic agent, anti-mitotic agent, anthracycline, toxin, or apoptotic agent. Also provided herein is a labeled binding protein wherein an antibody or antibody portion of the disclosure is derivatized or linked to another functional molecule (e.g., another peptide or protein). For example, a labeled binding protein of the disclosure may be derived by functionally linking an antibody or antibody portion of the disclosed binding protein (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that may mediate associate of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
Useful detectable agents with which an antibody or antibody portion of the disclosure may be derivatized, may include fluorescent compounds. Exemplary fluorescent detectable agents include, for example, fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-I-napthalenesulfonyl chloride, phycoerythrin and the like. An antibody may also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. When an antibody is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, when the detectable agent horseradish peroxidase is present, addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is detectable. An antibody may also be derivatized with biotin, and detected through indirect measurement of avidin or streptavidin binding.
The binding proteins described herein may be in crystallized form. Crystallized binding proteins according to the present disclosure may be produced according to methods known in the art, as disclosed for example in WO 02072636. Preferably the crystallized binding protein retains biological activity after crystallization. The binding proteins may thus be provided as crystals of whole anti-NGF antibodies or portions or fragments thereof as disclosed herein. Such crystals may be used to prepare formulations and compositions incorporating anti NGF binding proteins, including diagnostic and therapeutic compositions. An exemplary such crystallized binding protein is a carrier-free, controlled release crystallized binding protein. An exemplary crystallized binding protein demonstrates a greater half-life in vivo than the soluble counterpart of the binding protein.
Anti NGF binding proteins as described herein may be glycosylated. The glycosylation may demonstrate, for example, a bovine, camel, canine, murine, equine, feline, or human glycosylation pattern. Glycosylated binding proteins as described herein include the antibody or antigen-binding portion coupled to one or more carbohydrate residues. Nascent in vivo protein production may undergo further processing, known as post-translational modification. Sugar (glycosyl) residues may be added enzymatically, a process known as glycosylation. The resulting proteins bearing covalently linked oligosaccharide side chains are known as glycosylated proteins or glycoproteins. Protein glycosylation depends on the amino acid sequence of the protein of interest, as well as the host cell in which the protein is expressed. Different organisms may produce different glycosylation enzymes (ex., glycosyltransferases and glycosidases), and have different substrates (nucleotide sugars) available. Due to such factors, protein glycosylation pattern, and composition of glycosyl residues, may differ depending on the host system in which the particular protein is expressed. Glycosyl residues useful in the disclosure may include, but are not limited to, glucose, galactose, mannose, fucose, n-acetylglucosamine and sialic acid. The glycosylated binding protein comprises glycosyl residues such that the glycosylation pattern is human, murine, canine, feline, bovine or equine.
It is known to those skilled in the art that differing protein glycosylation may result in differing protein characteristics. For instance, the efficacy of a therapeutic protein produced in a microorganism host, such as yeast, and glycosylated utilizing the host endogenous pathway may be reduced compared to that of the same protein expressed in a mammalian cell, such as a CHO cell line. Such glycoproteins may also be immunogenic in humans and show reduced half-life in vivo after administration. Specific receptors in humans and other animals may recognize specific glycosyl residues and promote the rapid clearance of the protein from the bloodstream. Other adverse effects may include changes in protein folding, solubility, susceptibility to proteases, trafficking, transport, compartmentalization, secretion, recognition by other proteins or factors, antigenicity, or allergenicity. Accordingly, a practitioner may prefer a therapeutic protein with a specific composition and pattern of glycosylation, such as a glycosylation composition and pattern identical, or at least similar, to that produced in human cells or in the species-specific cells of the intended subject animal.
Expressing glycosylated proteins different from that of a host cell may be achieved by genetically modifying the host cell to express heterologous glycosylation enzymes. Using techniques known in the art, a practitioner may generate antibodies or antigen-binding portions thereof exhibiting human protein glycosylation. For example, yeast strains have been genetically modified to express non-naturally occurring glycosylation enzymes such that glycosylated proteins (glycoproteins) produced in these yeast strains exhibit protein glycosylation identical to that of animal cells, especially human cells (U.S. patent applications 20040018590 and 20020137134).
Further, it will be appreciated by those skilled in the art that a protein of interest may be expressed using a library of host cells genetically engineered to express various glycosylation enzymes such that member host cells of the library produce the protein of interest with variant glycosylation patterns. A practitioner may then select and isolate the protein of interest with particular novel glycosylation patterns. The protein having a particularly selected novel glycosylation pattern exhibits improved or altered biological properties.
Anti NGF Chimeric Antibodies
A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a non-murine immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art, see e.g., Morrison, Science, 229: 1202 (1985); Oi et al., BioTechniques, 4: 214 (1986); Gillies et al., J. Immunol. Methods, 125: 191-202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397. In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81: 851-855 (1984); Neuberger et al., Nature, 312:604-608 (1984); Takeda et al., Nature, 314: 452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity may be used.
Anti NGF CDR Grafted Antibodies
CDR-grafted antibodies of the disclosure may comprise heavy and light chain variable region sequences from a non-murine antibody wherein one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of the murine antibodies of the disclosure. A framework sequence from any non-murine antibody may serve as the template for CDR grafting. However, straight chain replacement onto such a framework often leads to some loss of binding affinity to the antigen. The more homologous a non-murine antibody is to the original murine antibody, the less likely the possibility that combining the murine CDRs with the non-murine framework will introduce distortions in the CDRs that could reduce affinity.
A non-murine variable framework that is chosen to replace the murine variable framework apart from the CDRs may have at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity with the murine antibody variable region framework. The non-murine variable framework, apart from the CDRs, that is chosen to replace the murine variable framework, apart from the CDRs, may be a bovine, camel, canine, equine, feline or human variable framework. For example, the non-murine variable framework that is chosen to replace the murine variable framework, apart from the CDRs, is a canine variable framework and has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the murine antibody variable region framework.
Methods for producing CDR-grafted antibodies are known in the art (see EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), and include veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5): 489-498 (1991); Studnicka et al., Protein Engineering, 7(6):805-814 (1994); Roguska et al., PNAS, 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,352).
Anti NGF Humanized Antibodies
The process of modifying a monoclonal antibody from an animal to render it less immunogenic for therapeutic administration to humans (humanization) has been aggressively pursued and has been described in a number of publications (Antibody Engineering: A practical Guide. Carl A. K. Borrebaeck ed. W.H. Freeman and Company, 1992; and references cited above). Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Known human Ig sequences are disclosed in a variety of websites which are available on the Internet (such as the NCBI website, Antibody Resource, and known to those skilled in the art as well as in Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Dept. Health (1983), which is incorporated herein by reference. Additional sequences are shown in Table 1 A below. Such imported sequences may be used to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic of the antibody, as known in the art.
TABLE 1A
Mouse Anti-NGF mAb CDRs Grafted onto Human Ig Frameworks CDR-Grafted Anti-
NGFAbs(This Table 1A is identical to Table 15 in the Examples)
HU72 VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMFWVRQATGKGLEWVSTISDGGSYT
(CDR GRAFTVH3-
YYTDNVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCARDWSDSEGFAYWGQGTLVT
13/JH5)
VSS
(SEQ ID NO: 165)
Hu73 VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGRIDPYGGGT
(CDR GRAFT VH1-
KHNEKFKRRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSGYDYYFDVWGQGTTVTV
18/JH6)
SS
(SEQ ID NO: 166)
HU77 VH
QVQLVQSGAEVKKPGSSVKVSCKASGFNIKDTYIYWVRQAPGQGLEWMGRIDPANGNT
(CDR GRAFT VH1-
IYASKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARYGYYAYWGQGTTVTVSS
69/JH6)
(SEQ ID NO: 167)
HU80 VH
QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIYWVRQAPGQGLEWMGRIDPANGNT
(CDR GRAFT VH1-
IYASKFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARYGYYAYWGQGTTVTVSS
18/JH6)
(SEQ ID NO: 168)
HU81 VH
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNHYMYWVRQAPGKGLEWVGSISDGGAYT
(CDR GRAFT VH3-
FYPDTVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTEESANNGFAFWGQGTLVT
15/JH1)
VSS
(SEQ ID NO: 169)
HU82 VH
QVTLKESGPVLVKPTETLTLTCTVSGFSLTGYNINWIRQPPGKALEWLAMIWGYGDTD
(CDR GRAFT VH2-
YNSALKSRLTISKDTSKSQWLTMTNMDPVDTATYYCARDHYGGNDWYFDVWGQGTTVT
26/JH6)
VSS
(SEQ ID NO: 170)
HU72 VL
DIVMTQTPLSLPVTPGEPASISCRSSQSIVQSNGNTYLEWYLQKPGQSPQLLIYKVSN
(CDR GRAFT
RFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPFTFGQGTKLEIKR
01/JK2)
(SEQ ID NO: 171)
HU73 VL
DIQMIQSPSFLSASVGDRVSIICRASENIYSFLAWYLQKPGKSPKLFLYNANTLAEGV
(CDR GRAFT
SSRFSGRGSGTDFTLTIISLKPEDFAAYYCQHHFGTPFTFGQGTKLEIKR
L22/JK2)
(SEQ ID NO: 172)
HU77 VL
DIVMTQTPLSLPVTPGEPASISCKSTKSLLNGDGFTYLDWYLQKPGQSPQLLIYLVSN
(CDR GRAFT
RFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFESNYLFTFGQGTKLEIKR
01/JK2)
(SEQ ID NO: 173)
HU80 VL
DIVMTQTPLSLPVTPGEPASISCKSTKSLLNGDGFTYLDWYLQKPGQSPQLLIYLVSN
(CDR GRAFT
RFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFESNYLFTFGQGTKLEIKR
01/JK2)
(SEQ ID NO: 174)
HU81 VL
DIVMTQTPLSLPVTPGEPASISCRSSQSILHSNGNTYLEWYLQKPGQSPQLLIYRVSN
(CDR GRAFT
RFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGAHVPFTFGQGTKLEIKR
01/JK2)
(SEQ ID NO: 175)
HU82 VL
DIQMTQSPSSLSASVGDRVTITCRASQDITNYLNWYQQKPGKAPKLLIYYTSRLHSGV
(CDR GRAFT
PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGKTLPRTFGQGTKLEIKR
08/JK2)
(SEQ ID NO: 176)
Framework residues in the human framework regions may be substituted with the corresponding residue from the CDR donor antibody to alter or improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues may be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding. Antibodies may be humanized using a variety of techniques known in the art, such as but not limited to those described in Jones et al., Nature 321:522 (1986); Verhoeyen et al., Science 239: 1534 (1988)), Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994); PCT publication WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596, EP 239,400, U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539; and 4,816,567.
Anti NGF Caninized Antibodies
The process of modifying a monoclonal antibody from an animal to render it less immunogenic for therapeutic administration to canines (caninization) has been described in U.S. Pat. No. 7,261,890 B2 2007). The amino acid sequence of canine IgG1 is provided in GenBank (AF354264). Determination of the amino acid sequence of the variable regions of both a canine IgM and a canine IgA heavy chain (Wasserman et al., Biochem., 16, 3160 (1977), determination of the amino acid sequence of the κ light chain from a canine IgA (Wasserman et al., Immunochem., 15, 303 (1978)), complete amino-acid sequence of a canine μ chain was disclosed (McCumber et al., Mol. Immunol, 16, 565 (1979)), a single canine IgG-Aγ chain cDNA and four canine IgG-Aγ chain protein sequences were disclosed (Tang et al., Vet. Immunology Immunopathology, 80, 259 (2001)). It describes PCR amplification of a canine spleen cDNA library with a degenerate oligonucleotide primer designed from the conserved regions of human, mouse, pig, and bovine IgGs. Canine immunoglobulin variable domains, caninized antibodies, and methods for making and using them are disclosed in US Patent Application No. 2004/0181039 and U.S. Pat. Nos. 7,261,890; 6,504,013; 5,852,183; 5,5225,539.
Table 2 below is a list of amino acid sequences of VH and VL regions of selected caninized anti-NGF antibodies of the disclosure.
TABLE 2
SEQ ID NO:
Region
25
72.1
VH
26
72.1
VL
27
73.1
VH
28
73.1
VL
29
77.1
VH
30
77.1
VL
31
81.1
VH
32
81.1
VL
33
82.1
VH
34
82.1
VL
35
72.2
VH
36
72.2
VL
37
73.2
VH
38
73.2
VL
39
77.2
VH
40
77.2
VL
41
81.2
VH
42
81.2
VL
43
82.2
VH
44
82.2
VL
177
81.1B
VH
179
72.3
VH
180
72.4
VH
181
72.4
VL
182
73.4
VH
183
73.4
VL
184
77.3
VH
185
77.4
VH
186
77.4
VL
187
81.4
VH
188
81.4
VL
189
81.2B
VH
190
81.4B
VH
191
82.3
VL
192
82.4
VH
193
82.4
VL
206
81.5B
VH
207
81.6B
VH
Uses of Anti-NGF Antibodies
Binding proteins as described herein may be used in a method for detecting the presence of NGF in a sample in vivo or in vitro (e.g., in a biological sample, such as serum, plasma, tissue, biopsy). The in vitro method may be used for example to diagnose a disease or disorder, e.g., an NGF-associated disorder. The method includes (i) contacting the sample or a control sample with the anti-NGF antibody or fragment thereof as described herein; and (ii) detecting formation of a complex between the anti-NGF antibody or fragment thereof, and the sample or the control sample, wherein a statistically significant change in the formation of the complex in the sample relative to the control sample is indicative of the presence of the NGF in the sample.
Binding proteins as described herein may be used in a method for detecting the presence of NGF in vivo (e.g., in vivo imaging in a subject). The method may be used to diagnose a disorder, e.g., an NGF-associated disorder. The method includes: (i) administering the anti-NGF antibody or fragment thereof as described herein to a subject or a control subject under conditions that allow binding of the antibody or fragment to NGF; and (ii) detecting formation of a complex between the antibody or fragment and NGF, wherein a statistically significant change in the formation of the complex in the subject relative to the control subject is indicative of the presence of NGF.
Given the ability to bind to NGF, the anti-NGF antibodies, or portions thereof, or combinations thereof, as described herein may be used as immunoreagent(s) to detect NGF (e.g., in a biological sample, such as serum or plasma), in a conventional immunoassay, such as an enzyme linked immunosorbent assays (ELISA), a radioimmunoassay (RIA) or tissue immunohistochemistry. A method for detecting NGF in a biological sample involves contacting a biological sample with an antibody, or antibody portion, of the disclosure and detecting either the antibody (or antibody portion) bound to NGF or unbound antibody (or antibody portion), to thereby detect NGF in the biological sample. The binding protein may be directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include H 14 C 5S, 90Y, 99Tc, 111In, 1251, 131I, 177Lu, 166Ho, or 15 Sm.
NGF may alternatively be assayed in biological fluids by a competition immunoassay utilizing recombinant NGF standards labeled with a detectable substance and an unlabeled anti-NGF antibody. In this assay, the biological sample, the labeled recombinant NGF standards and the anti-NGF antibody are combined and the amount of labeled rNGF standard bound to the unlabeled antibody is determined. The amount of NGF in the biological sample is inversely proportional to the amount of labeled rNGF standard bound to the anti-NGF antibody. Similarly, NGF may also be assayed in biological fluids by a competition immunoassay utilizing rNGF standards labeled with a detectable substance and an unlabeled anti-NGF antibody.
The disclosure thus also contemplates immunoassay reagents, devices and kits including one or more of the presently disclosed binding proteins for detecting the presence or amount of NGF in a sample. It is contemplated for example that an immunoreagent comprising one or more of the presently disclosed binding proteins may be provided in the form of a kit with one or more containers such as vials or bottles, with each container containing a separate reagent such as an anti-NGF binding protein, or a cocktail of anti-NGF binding proteins, detection reagents and washing reagents employed in the assay. The immunoreagent(s) may be advantageously provided in a device in which the immunoreagents(s) is immobilized on a solid support, such as but not limited to a cuvette, tube, microtiter plates or wells, strips, chips or beads. The kit may comprise at least one container for conducting the assay, and/or a buffer, such as an assay buffer or a wash buffer, either one of which may be provided as a concentrated solution, a substrate solution for the detectable label (e.g., an enzymatic label), or a stop solution. Preferably, the kit comprises all components, i.e., reagents, standards, buffers, diluents, etc., which are necessary to perform the assay. The kit may contain instructions for determining the presence or amount of NGF in the sample based on specific binding of the immunoreagent to NGF, in paper form or computer-readable form, such as a disk, CD, DVD, or the like, and/or may be made available online.
The binding proteins in the kit may be labeled with a detectable label such as those described above including a fluorophore, a radioactive moiety, an enzyme, a biotin/avidin label, a chromophore, a chemiluminescent label, or the like; or the kit may include reagents for carrying out detectable labeling. The antibodies, calibrators and/or controls can be provided in separate containers or pre-dispensed into an appropriate assay format, for example, into microtiter plates.
Optionally, the kit includes quality control components (for example, sensitivity panels, calibrators, and positive controls). Preparation of quality control reagents is well-known in the art and is described on insert sheets for a variety of immunodiagnostic products. Sensitivity panel members optionally are used to establish assay performance characteristics, and further optionally are useful indicators of the integrity of the immunoassay kit reagents, and the standardization of assays.
The kit can also optionally include other reagents required to conduct a diagnostic assay or facilitate quality control evaluations, such as buffers, salts, enzymes, enzyme co-factors, enzyme substrates, detection reagents, and the like. Other components, such as buffers and solutions for the isolation and/or treatment of a test sample (e.g., pretreatment reagents), also can be included in the kit. The kit can additionally include one or more other controls. One or more of the components of the kit can be lyophilized, in which case the kit can further comprise reagents suitable for the reconstitution of the lyophilized components.
The various components of the kit optionally are provided in suitable containers as necessary, e.g., a microtiter plate. The kit can further include containers for holding or storing a sample (e.g., a container or cartridge for a urine sample). Where appropriate, the kit optionally also can contain reaction vessels, mixing vessels, and other components that facilitate the preparation of reagents or the test sample. The kit can also include one or more instruments for assisting with obtaining a test sample, such as a syringe, pipette, forceps, measured spoon, or the like. Instructions:
It will be appreciated that the antibodies and antibody portions of the disclosure are capable of substantially neutralizing NGF activity both in vitro and in vivo. Accordingly, such antibodies and antibody portions of the disclosure can also be used to substantially inhibit NGF activity, e.g., in a cell culture containing NGF, in mammalian subjects having NGF with which an antibody of the disclosure cross-reacts. The disclosure thus provides a method for inhibiting NGF activity comprising contacting NGF with an antibody or antibody portion of the disclosure such that NGF activity is substantially inhibited. For example, in a cell culture containing, or suspected of containing NGF, an antibody or antibody portion of the disclosure can be added to the culture medium to inhibit NGF activity in the culture.
Accordingly, the disclosure also provides a method for inhibiting NGF activity comprising contacting NGF with a binding protein such that NGF activity is substantially inhibited. In another aspect, the disclosure provides a method for inhibiting NGF activity in a subject suffering from a disorder in which NGF activity is detrimental, comprising administering to the subject a binding protein disclosed above such that NGF activity in the subject is substantially inhibited and treatment is achieved.
The disclosure also provides a method for reducing NGF activity in a subject, such as a subject suffering from a disease or disorder in which NGF activity is detrimental. The disclosure provides methods for reducing NGF activity in a subject suffering from such a disease or disorder, which method comprises administering to the subject an antibody or antibody portion of the disclosure such that NGF activity in the subject is reduced. The subject can be a mammal expressing an NGF to which an antibody of the disclosure is capable of binding. Still further the subject can be a mammal into which NGF has been introduced (e.g., by administration of NGF or by expression of an NGF transgene). An antibody of the disclosure can be administered to a subject in need thereof for therapeutic purposes.
An antibody of the disclosure can be administered for veterinary purposes to a non-human mammal expressing an NGF with which the antibody is capable of binding. For example, an antibody of the disclosure can be administered for veterinary purposes to a non-human mammal such as a dog, horse, cat, or livestock (beef and dairy cattle, swine, sheep, goats, poultry, etc.) expressing an NGF with which the antibody is capable of binding.
In another aspect, the disclosure provides a method of treating (e.g., curing, suppressing, ameliorating, delaying or preventing or decreasing the risk of the onset, recurrence or relapse of) or preventing an NGF associated disorder, in a subject. The method includes: administering to the subject a disclosed NGF binding protein (particularly an antagonist), e.g., an anti-NGF antibody or fragment thereof as described herein, in an amount sufficient to treat or prevent the NGF associated disorder. The NGF antagonist, e.g., the anti-NGF antibody or fragment thereof, can be administered to the subject, alone or in combination with other therapeutic modalities as described herein.
An antibody of the disclosure can be administered to a non-human mammal expressing an NGF with which the antibody is capable of binding as an animal model of human disease. Such animal models may be useful for evaluating the therapeutic efficacy of antibodies of the disclosure (e.g., testing of dosages and time courses of administration).
In another aspect, the antibodies and binding proteins of the disclosure are useful for treating NGF-related diseases and disorders including or involving acute or chronic pain. Non-limiting examples of NGF-related diseases and disorders include general inflammation, surgical and postsurgical pain including pain from amputation, dental pain, pain from trauma, fracture pain, pain from abscess, neuropathic pain, hyperalgesia and allodynia, neuropathic pain, post-herpetic neuralgia, diabetes including, but not limited to, diabetic neuropathy pain, stroke, thalamic pain syndrome, gout joint pain, osteoarthritis or rheumatoid arthritis pain, rheumatic diseases, lupus, psoriasis, sciatica, pain associated with musculoskeletal diseases including, but not limited to, chronic low back pain, fibromyalgia, sprains, pains associated with sickle cell crises, general headache, migraine, cluster headache, tension headache, trigeminal neuralgia, dysmenorrhea, endometriosis, ovarian cysts, visceral pain, prostatitis, cystitis, interstitial cystitis, erythromelalgia or pain caused by pancreatitis or kidney stones, general gastrointestinal disorders including, but not limited to, colitis, gastric ulceration and duodenal ulcers, gastroesophageal reflux, dyspepsia, inflammatory bowel disorders, irritable bowel syndrome, inflammatory bladder disorders, incisional pain, pain from burns and/or wounds, ankylosing spondilitis, periarticular pathologies, cancer pain including, but not limited to, pain from bone metastases and pain from cancer treatment, and pain from HIV or AIDS. Other examples of NGF-related diseases and conditions include malignant melanoma, Sjogren's syndrome, rhinitis, bronchial disorders, and asthma, such as uncontrolled asthma with severe airway hyper-responsiveness, intractable cough; and pain from skin diseases or disorders with an inflammatory component such as, but not limited to, sunburn, allergic skin reactions, dermatitis, pruritis, and vitiligo.
The disclosure also provides a method of treating a subject suffering from a disorder in which NGF is detrimental comprising administering a binding protein before, concurrent, or after the administration of a second agent. In another aspect, the additional therapeutic agent that can be co-administered and/or co-formulated with one or more NGF antagonists, (e.g., anti-NGF antibodies or fragments thereof) include, but are not limited to, TNF antagonists; a soluble fragment of a TNF receptor; ENBREL®; TNF enzyme antagonists; TNF converting enzyme (TACE) inhibitors; muscarinic receptor antagonists; TGF-beta antagonists; interferon gamma; perfenidone; chemotherapeutic agents, methotrexate; leflunomide; sirolimus (rapamycin) or an analog thereof, CCI-779; COX2 or cPLA2 inhibitors; NSAIDs; immunomodulators; p38 inhibitors; TPL-2, MK-2 and NFKB inhibitors; budenoside; epidermal growth factor; corticosteroids; cyclosporine; sulfasalazine; am inosalicylates; 6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide; antioxidants; thromboxane inhibitors; anti-IL-6 antibodies; growth factors; elastase inhibitors; pyridinyl-imidazole compounds; antibodies or agonists of TNF, CGRP, substance P, bradykinin, MMP-2, MMP-9, MMP-13, LT, IL-1a, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, EMAP-II, GM-CSF, FGF, or PDGF; antibodies of CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands; FK506; rapamycin; mycophenolate mofetil; ibuprofen; prednisolone; phosphodiesterase inhibitors; adenosine agonists; antithrombotic agents; complement inhibitors; adrenergic agents; IRAK, NIK, IKK, p38, or MAP kinase inhibitors; IL-1β converting enzyme inhibitors; TNF converting enzyme inhibitors; T-cell signaling inhibitors; metalloproteinase inhibitors; 6-mercaptopurines; angiotensin converting enzyme inhibitors; soluble cytokine receptors; soluble p55 TNF receptor; soluble p75 TNF receptor; sIL-IRI; sIL-IRII; sIL-6R; anti-inflammatory cytokines; IL-4; IL-10; IL-11; and TGFβ.
Pharmaceutical Compositions
The antibodies and antibody-portions of the disclosure can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises at least one antibody or antibody portion of the disclosure and a pharmaceutically acceptable carrier. Such compositions can be used for example in a method for treating a mammal for a disease or disorder involving increased levels of NGF by administering to the mammal an effective amount of the composition. A pharmaceutical composition may include a therapeutically effective amount of the antibody or antibody portion. The pharmaceutical compositions as described herein may be used for diagnosing, detecting, or monitoring a disorder or one or more symptoms thereof; preventing, treating, managing, or ameliorating a disorder or one or more symptoms thereof; and/or research. As used herein, the phrase “increased levels of NGF” refers to a level of NGF in a subject, such as a mammal, that is greater or higher than an established or predetermined baseline level of NGF such as, for example, a level previously established for said subject or averaged from a group of subjects.
A pharmaceutical composition may comprise, for example, a binding protein and a pharmaceutically acceptable carrier, excipient or diluent. For example, pharmaceutical compositions may comprise a therapeutically effective amount of one or more of the binding proteins as disclosed herein, together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant. The pharmaceutical composition may contain one or more various formulation materials for modifying, maintaining or preserving the composition or properties of the composition, for example, the color, consistency, isotonicity, odor, osmolality, pH, sterility, stability, viscosity and other properties of the composition. Such formulation materials are generally well known and many suitable formulation materials are described for example in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed. (A. R. Gennaro, ed.) 1990, Mack Publishing Company. Non-limiting examples of suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. In addition, the pharmaceutical composition can also contain one or more preservatives. Examples of suitable preservatives that can be used include, but are not limited to, methylparaben, propylparaben, benzyl alcohol, chlorobutanol, and benzalkonium chloride. Optimal pharmaceutical formulations can be readily determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage.
The pharmaceutical composition may comprise at least one additional therapeutic agent for treating a disorder in which NGF activity is detrimental. The additional agent can be, for example, a therapeutic agent, imaging agent, cytotoxic agent, angiogenesis inhibitors, kinase inhibitors, co-stimulation molecule blockers, adhesion molecule blockers, anti-cytokine antibody or functional fragment thereof, methotrexate, cyclosporine, rapamycin, FK506, detectable label or reporter, TNF antagonist, anti-rheumatic, muscle relaxant, narcotic, non-steroid anti-inflammatory drug (NSAID), analgesic, anesthetic, sedative, local anesthetic, neuromuscular blocker, antimicrobial, antipsoriatic, corticosteroid, anabolic steroid, erythropoietin, immunoglobulin, immunosuppressive, growth hormone, hormone replacement drug, radiopharmaceutical, antidepressant, antipsychotic, stimulant, asthma medication, beta agonist, inhaled steroid, oral steroid, epinephrine or analog, cytokine, or a cytokine antagonist.
The pharmaceutical composition of the present disclosure may have a pH greater than about 7.0 or between about 7.0 and about 8.0. Alternatively, the pharmaceutical composition may have a pH of between about 7.2 to about 7.8. Still further alternatively, the pH of the pharmaceutical composition may be between about 7.4 to about 7.6. Still further alternatively, the pH of the pharmaceutical composition may be about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 7.7, 7.8, 7.9 or 8.0. With respect to the pharmaceutical compositions of the present disclosure, there is an increase in degradation, an increase in fragmentation or an increase in degradation and an increase in fragmentation at a pH of 6.0 or less. This finding was surprising as many pharmaceutical compositions comprising humanized antibodies exhibit an increase in degradation, an increase fragmentation or an increase in degradation and an increase in fragmentation at a pH lower than 5.0 and again at a pH higher than about 6.0. Accordingly, most pharmaceutical compositions containing humanized antibodies are stable at a pH between about 5.0 to about 6.0.
A composition for the release of a binding protein may comprise, for example, a formulation including an amount of a crystallized binding protein, crystallized antibody construct or crystallized antibody conjugate as disclosed above. The composition may further comprise an additional ingredient, such as carrier, excipient or diluent, and at least one polymeric carrier. The polymeric carrier can comprise one or more polymers selected from the following: poly (acrylic acid), poly(cyanoacrylates), poly (amino acids), poly (anhydrides), poly (depsipeptide), poly (esters), poly (lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutryate), poly (caprolactone), poly (dioxanone); poly (ethylene glycol), poly ((hydroxypropyl) methacrylamide, poly [(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends and copolymers thereof. The additional ingredient may be, for example, albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-P-cyclodextrin, methoxypolyethylene glycol and polyethylene glycol.
The polymeric carrier may be capable of affecting the release of the binding protein from the composition as described further herein below. Polymeric materials can be used in the formulation of pharmaceutical compositions comprising the disclosed binding proteins to achieve controlled or sustained release of the disclosed binding proteins (Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger et al., J. Macromol. Sci. Rev. Macromol. Chem., 23:61 (1983); Levy et al., Science, 228: 190 (1985); During Qt al, Ann. Neurol., 25: 351 (1989); Howard et al., J. Neurosurg., 7 1: 105 (1989); U.S. Pat. Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; 5,128,326; PCT Publication Nos. WO 99/15154; and WO 99/20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. The polymer used in a sustained release formulation may be inert, free of leachable impurities, stable on storage, sterile, and biodegradable. A controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 1 15-138 (1984)).
Controlled release systems are discussed in the review by Langer (Science, 249: 1527-1533 (1990)). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more therapeutic agents of the disclosure (U.S. Pat. No. 4,526,938; PCT publication Nos. WO 91/05548 and WO 96/20698; Ning et al., Radiotherapy & Oncology, 39: 179-189 (1996), Song et al., PDA Journal of Pharmaceutical Science & Technology, 50: 372-397 (1995); Cleek et al., Pro. Intl Symp. Control. Rel. Bioact. Mater., 24: 853-854 (1997); and Lam et al., Proc. Intl Symp. Control Rel. Bioact. Mater., 24: 759-760 (1997)).
The binding proteins of the present disclosure can be administered by a variety of methods known in the art. For example, the binding proteins of the present disclosure may be administered by subcutaneous injection, intravenous injection or infusion. Administration can be systemic or local. As will be appreciated by the skilled artisan, the route and/or mode of administration may vary depending upon the desired results. The active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
For example, such pharmaceutical compositions may be administered to a subject by parenteral, intradermal, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal. Methods of administering a prophylactic or therapeutic agent of the disclosure also include, but are not limited to, epidural administration, intratumoral administration, and mucosal administration (e.g., intranasal and oral routes). In addition, pulmonary administration can be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent (U.S. Pat. Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903). The antibodies and antibody portions described herein can be administered for example using Alkermes AIR® pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.). The prophylactic or therapeutic agents may be administered by any convenient route, and may be administered together with other biologically active agents.
Various delivery systems are known and can be used to administer one or more disclosed binding proteins or the combination of one or more disclosed binding proteins and a prophylactic agent or therapeutic agent useful for preventing, managing, treating, or ameliorating a disorder or one or more symptoms thereof, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or antibody fragment, receptor-mediated endocytosis (see, e. g., Wu et al., J. Biol. Chem., 262: 4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. It may be desirable to administer the disclosed binding proteins locally to the area in need of treatment, which may be achieved by, for example, and not by way of limitation, local infusion, by injection, or by means of an implant, said implant being of a porous or non-porous material, including membranes and matrices, such as sialastic membranes, polymers, fibrous matrices (e.g., Tissuel®), or collagen matrices. An effective amount of one or more disclosed binding proteins can be administered locally to the affected area to a subject to prevent, treat, manage, and/or ameliorate a disorder or a symptom thereof. Alternatively, an effective amount of one or more of the disclosed binding proteins is administered locally to the affected area in combination with an effective amount of one or more therapies (e. g., one or more prophylactic or therapeutic agents) other than disclosed binding proteins of a subject to prevent, treat, manage, and/or ameliorate a disorder or one or more symptoms thereof.
The disclosed binding proteins can be delivered in a controlled release or sustained release system such as, for example, an infusion pump device operable to achieve controlled or sustained release of the disclosed binding proteins (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:20 (1987); Buchwald et al., Surgery, 88: 507 (1980); Saudek et al., N. Engl. J. Med., 321: 574 (1989)).
When a composition as described herein comprises a nucleic acid encoding a binding protein as described herein as a prophylactic or therapeutic agent, the nucleic acid can be administered in vivo to promote expression of its encoded prophylactic or therapeutic agent, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (Joliot et al., Proc. Natl. Acad. Sci., 88: 1864-1868 (1991)). Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression by homologous recombination.
A pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. For example, a composition may be formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to human beings and companion animals. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection.
If the compositions of the disclosure are to be administered topically, the compositions can be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art (Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995)). For non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity greater than water are typically employed. Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which are, if desired, sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, such as, for example, osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as Freon) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well-known in the art.
The pharmaceutical composition of the present disclosure can have a half-life of from about 8 days to about 15 days when dosed intravenously or subcutaneously. Alternatively, the pharmaceutical composition of the present invention can have a half-life of from about 10 days to about 13 days. Still further alternatively, the pharmaceutical composition of the present invention can have a half-life of about 8 days, such as about 8.5 days, about 9 days, such as about 9.5 days, about 10 days, such as about 10.5 days, about 11 days, such as about 11.5 days, about 12 days, about 12.5 days, about 13 days, such as about 13.5 days, about 14 days, such as about 14.5 days, or about 15 days.
If the method of the disclosure comprises intranasal administration of a composition, the composition can be formulated in an aerosol form, spray, mist or in the form of drops. In particular, prophylactic or therapeutic agents for use according to the present disclosure can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (composed of, e.g., gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
If the method of the disclosure comprises oral administration, compositions can be formulated orally in the form of tablets, capsules, cachets, gelcaps, solutions, suspensions, and the like. Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well-known in the art. Liquid preparations for oral administration may take the form of, but not limited to, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of a prophylactic or therapeutic agent(s).
The method of the disclosure may comprise pulmonary administration, e.g., by use of an inhaler or nebulizer, of a composition formulated with an aerosolizing agent (U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903). For example, an antibody of the disclosure, combination therapy, and/or composition of the disclosure may be administered using Alkermes AIR® pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).
The method of the disclosure may comprise administration of a composition formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.
The methods of the disclosure may additionally comprise administration of compositions formulated as depot preparations. Such long acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).
The methods of the disclosure encompass administration of compositions formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions, such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
The ingredients of the disclosed compositions may be supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or a substantially water-free concentrate in a hermetically sealed container such as an ampoule or sachette which may indicate the quantity of active agent. Where the mode of administration is infusion, the disclosed compositions can be dispensed with an infusion solution containing sterile pharmaceutical grade solution such as water or saline. Where the mode of administration is by injection, an ampoule of sterile solution such as water or saline can be provided so that the ingredients may be mixed prior to administration.
In particular, the disclosure also provides that one or more of disclosed binding proteins or pharmaceutical compositions thereof is packaged in a hermetically sealed container such as an ampoule or sachette which may indicate the quantity of the agent. One or more of the disclosed binding proteins or pharmaceutical compositions thereof may be supplied as a dry sterilized lyophilized powder or substantially water-free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. One or more of the disclosed binding proteins or pharmaceutical compositions thereof may be supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least about 0.5 mg, 1 mg, 2 mg, 4 mg, 5 mg, 10 mg, 15 mg, 25 mg, 35 mg, 45 mg, 50 mg, 75 mg, or 100 mg. The lyophilized disclosed binding proteins or pharmaceutical compositions thereof may be stored at any suitable temperature, such as, for example, between about 2° C. and about 8° C. and may be stored in its original container. The disclosed binding proteins or pharmaceutical compositions thereof may be administered within about 1 week, within about 5 days, within about 72 hours, within about 48 hours, within about 24 hours, within about 12 hours, within about 6 hours, within about 5 hours, within about 3 hours, or within about 1 hour after being reconstituted. Alternatively, one or more of the disclosed binding proteins or pharmaceutical compositions thereof may be supplied in liquid form in a hermetically sealed container which may indicate the quantity and concentration of the agent. The liquid form of the administered composition may be supplied in a hermetically sealed container at concentrations of at least about 0.01 mg/mL, at least about 0.05 mg/mL, at least about 0.1 mg/mL, at least about 0.2 mg/mL, at least about 0.25 mg/ml, at least about 0.5 mg/ml, at least about 1 mg/ml, at least about 2.5 mg/ml, at least about 5 mg/ml, at least about 8 mg/ml, at least about 10 mg/ml, at least about 15 mg/kg, at least about 25 mg/ml, at least about 50 mg/ml, at least about 75 mg/ml, or at least about 100 mg/ml. The liquid form may be stored at any suitable temperature such as between about 2° C. and about 8° C. and may be stored in its original container.
The binding proteins of the disclosure can be incorporated into a pharmaceutical composition suitable for parenteral administration. In one aspect, binding proteins are prepared as an injectable solution containing between about 0.1 and about 250 mg/ml antibody. The injectable solution can be composed of either a liquid or lyophilized dosage form in a flint or amber vial, ampoule or pre-filled syringe. The buffer can be any suitable buffer such as L-histidine or a phosphate buffer saline at a concentration of about 1-50 mM, or about 5-10 mM. Other suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate and potassium phosphate. Buffers may be used to modify the toxicity of the pharmaceutical composition. For example, sodium chloride can be used to modify the toxicity of the binding protein solution at a concentration of from about 0.1 and about 300 mM, such as about 150 mM saline to modify the toxicity of a liquid dosage form.
Cryoprotectants, such as sucrose, can be included in a lyophilized dosage form at a concentration of about 0.1 to about 10% or from about 0.5 to about 1.0% may be used. Other suitable cryoprotectants include, but are not limited to, trehalose and lactose. Bulking agents, such as mannitol, can be included in a lyophilized dosage form at a concentration of about 1 to about 10%, or from about 2 to about 4%. Stabilizers, such as L-Methionine, can be used in both liquid and lyophilized dosage forms at a concentration of about 1 to about 50 mM, or about 5 to about 10 mM). Other suitable bulking agents include, but are not limited to, glycine and arginine. Surfactants, such as polysorbate-80, can be included in both liquid and lyophilized dosage forms at a concentration of about 0.001 to about 0.05% or about 0.005 to about 0.01%. Additional surfactants include, but are not limited to, polysorbate 20 and BRIJ surfactants.
An exemplary pharmaceutical formulation or composition of the present disclosure may be a liquid pharmaceutical composition having a pH between about 7.4 to about 8.0. The liquid pharmaceutical composition comprises about 5 mg/ml to about 50 mg/ml of an antibody comprising a heavy chain variable region comprising an amino acid sequence having a sequence of SEQ ID NO: 37 and a light chain variable region comprising an amino acid sequence comprising a sequence of SEQ ID NO: 38. The liquid pharmaceutical composition further comprises at least one buffer (such as, phosphate buffer saline, tris or histidine). The molarity of buffer that can be used can be from about 1 mM to about 60 mM. Optionally, said pharmaceutical composition or formulation can also contain at least one preservative, such as, methylparaben, propylparaben, benzyl alcohol, chlorobutanol or benzalkonium chloride. The amount of preservative that can be used can be from about 0.01 percent by volume to about 5.0% by volume depending on the preservative used.
Another exemplary pharmaceutical formulation or composition of the present disclosure may be a liquid pharmaceutical composition comprising a pH between about 7.4 to about 8.0. The liquid pharmaceutical composition comprises about 5 mg/ml to about 50 mg/ml of an antibody comprising a heavy chain variable region comprising an amino acid sequence having a sequence of SEQ ID NO: 192 and a light chain variable region comprising an amino acid sequence comprising a sequence of SEQ ID NO: 193. The liquid pharmaceutical composition further comprises at least one buffer (such as, phosphate buffer saline, tris or histidine). The molarity of buffer that can be used can be from about 1 mM to about 60 mM. Optionally, said pharmaceutical composition or formulation can also contain at least one preservative, such as, methylparaben, propylparaben, benzyl alcohol, chlorobutanol or benzalkonium chloride. The amount of preservative that can be used can be from about 0.01 percent by volume to about 5.0% by volume depending on the preservative used.
The compositions of this disclosure may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The form of the disclosed composition may depend on the intended mode of administration and therapeutic application. The disclosed compositions may be in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies. The mode of administration may be parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). The disclosed binding proteins may be administered by intravenous infusion or injection, or by intramuscular or subcutaneous injection.
Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other suitable ordered structure such as those suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., antibody or antibody portion) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Dispersions may be prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile, lyophilized powders for the preparation of sterile injectable solutions, the methods of preparation include, but are not limited to, vacuum drying and spray-drying that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption such as, for example, monostearate salts and gelatin.
An antibody or antibody portion of the disclosure may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the disclosure by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.
The disclosed binding proteins may be co-administered with other active compounds which may also be incorporated into the disclosed compositions. An antibody or antibody portion of the disclosure may be co-formulated with and/or co-administered with one or more additional therapeutic agents that are useful for treating disorders in which NGF activity is detrimental. For example, an anti-NGF antibody or antibody portion of the disclosure may be co-formulated and/or co-administered with one or more additional antibodies that bind other targets (e.g., antibodies that bind other cytokines or that bind cell surface molecules). Furthermore, one or more disclosed binding proteins may be used in combination with two or more of the foregoing therapeutic agents. Such combination therapies may, for example, enable the use of lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.
An antibody to NGF or fragment thereof may be formulated with a vehicle that extends the half-life of the binding protein. Suitable vehicles known in the art include, but are not limited to, the Fc domain, polyethylene glycol, and dextran. Such vehicles are described, e.g., in U.S. application Ser. No. 09/428,082 and published PCT Application No. WO 99/25044.
Isolated nucleic acid sequences comprising nucleotide sequences encoding disclosed binding proteins or another prophylactic or therapeutic agent of the disclosure may be administered to treat, prevent, manage, or ameliorate a disorder or one or more symptoms thereof by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid, wherein the nucleic acids produce their encoded antibody or prophylactic or therapeutic agent of the disclosure that mediates a prophylactic or therapeutic effect.
Any of the methods for gene therapy available in the art can be used according to the present disclosure. For general reviews of the methods of gene therapy, see Goldspiel et al., Clinical Pharmacy, 12: 488-505 (1993); Wu et al., Biotherapy, 3: 87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol., 32: 573-596 (1993); Mulligan, Science, 260: 926-932 (1993); and Morgan et al., Ann. Rev. Biochem., 62: 191-217 (1993); TIBTECH, 11(5): 155-215 (1993). Methods commonly known in the art of recombinant DNA technology which can be used are described in, for example, Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, N Y (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990). Detailed descriptions of various methods of gene therapy are disclosed in US20050042664A1.
Antibodies of the disclosure, or antigen binding portions thereof, can be used alone or in combination to treat NGF related diseases. It should be understood that the antibodies of the disclosure or antigen binding portion thereof can be used alone or in combination with an additional agent, e.g., a therapeutic agent, said additional agent being selected by the skilled artisan for its intended purpose. For example, the additional agent can be a therapeutic agent art-recognized as being useful to treat the disease or condition being treated by the antibody of the present disclosure. The additional agent also can be an agent that imparts a beneficial attribute to the therapeutic composition e.g., an agent which affects the viscosity of the composition.
It should further be understood that the combinations which are to be included within this disclosure are those combinations useful for their intended purpose. The agents set forth below are illustrative for purposes and not intended to be limited. The combinations, which are part of this disclosure, can be the antibodies of the present disclosure and at least one additional agent selected from the lists below. The combination can also include more than one additional agent, e.g., two or three additional agents if the combination is such that the formed composition can perform its intended function.
Combinations include non-steroidal anti-inflammatory drug(s) also referred to as NSAIDS which include drugs like ibuprofen. Other combinations are corticosteroids including prednisolone; the well-known side-effects of steroid use can be reduced or even eliminated by tapering the steroid dose required when treating patients in combination with the anti-NGF antibodies of this disclosure. Non-limiting examples of therapeutic agents for rheumatoid arthritis or pain with which an antibody, or antibody portion, of the disclosure can be combined include the following: cytokine suppressive anti-inflammatory drug(s) (CSAIDs); antibodies to or antagonists of cytokines or growth factors, for example, TNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, IL-21, interferons, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of the disclosure, or antigen binding portions thereof, can be combined with antibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, CTLA or their ligands including CD154 (gp39 or CD40L).
Combinations of therapeutic agents may interfere at different points in the autoimmune and subsequent inflammatory cascade; examples include TNF antagonists like chimeric, humanized or human TNF antibodies, D2E7, (PCT Publication No. WO 97/29131), CA2 (Remicade™), CDP 571, and soluble p55 or p75 TNF receptors, derivatives, thereof, (p75TNFR1gG (Enbrel™) or p55TNFR1gG (Lenercept), and also TNFa converting enzyme (TACE) inhibitors; similarly other IL-1 inhibitors (Interleukin-1-converting enzyme inhibitors, (L-IRA etc.) may be effective for the same reason. Other combinations include Interleukin 11.
The antibodies of the disclosure, or antigen binding portions thereof, may also be combined with agents, such as methotrexate, 6-MP, azathioprine sulphasalazine, mesalazine, olsalazine chloroquinine/hydroxychloroquine, pencillamine, aurothiomalate (intramuscular and oral), azathioprine, cochicine, corticosteroids (oral, inhaled and local injection), beta-2 adrenoreceptor agonists (salbutamol, terbutaline, salmeteral), xanthines (theophylline, aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium and oxitropium, cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adensosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, agents which interfere with signaling by proinflammatory cytokines such as TNF or IL-1 (e.g. IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1β converting enzyme inhibitors, TNFa converting enzyme (TACE) inhibitors, T-cell signaling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors and derivatives thereof (e.g. soluble p55 or p75 TNF receptors and the derivatives p75TNFRIgG (Enbrel™ and p55TNFRIgG (Lenercept)), sIL-IRI, sIL-IRII, sIL-6R), anti-inflammatory cytokines (e.g. IL-4, IL-10, IL-11, IL-13 and TGFβ), celecoxib, folic acid, hydroxychloroquine sulfate, rofecoxib, etanercept, infliximab, naproxen, valdecoxib, sulfasalazine, methylprednisolone, meloxicam, methylprednisolone acetate, gold sodium thiomalate, aspirin, triamcinolone acetonide, propoxyphene napsylate/apap, folate, nabumetone, diclofenac, piroxicam, etodolac, diclofenac sodium, oxaprozin, oxycodone hcl, hydrocodone bitartrate/apap, diclofenac sodium/misoprostol, fentanyl, anakinra, human recombinant, tramadol hcl, salsalate, sulindac, cyanocobalamin/fa/pyridoxine, acetaminophen, alendronate sodium, prednisolone, morphine sulfate, lidocaine hydrochloride, indomethacin, glucosamine sulf/chondroitin, amitriptyline hcl, sulfadiazine, oxycodone hcl/acetaminophen, olopatadine hcl, misoprostol, naproxen sodium, omeprazole, cyclophosphamide, rituximab, IL-1 TRAP, MRA, CTLA4-IG, IL-18 BP, anti-IL-18, Anti-IL15, BIRB-796, SCIO-469, VX-702, AMG-548, VX-740, Roflumilast, IC-485, CDC-801, and Mesopram. Other combinations include methotrexate or leflunomide and in moderate or severe rheumatoid arthritis cases, cyclosporine. The antibodies of the disclosure, or antigen binding portions thereof, may also be combined with agents, such as cancer chemotherapeutics, antimicrobials, anti-inflammatories, and anthelmintics used in animals.
The NSAID may be any non-steroidal anti-inflammatory compound. NSAIDs are categorized by virtue of their ability to inhibit cyclooxygenase. Cyclooxygenase 1 and cyclooxygenase 2 are two major isoforms of cyclooxygenase and most standard NSAIDs are mixed inhibitors of the two isoforms. Most standard NSAIDs fall within one of the following five structural categories: (1) propionic acid derivatives, such as ibuprofen, naproxen, naprosyn, diclofenac, and ketoprofen; (2) acetic acid derivatives, such as tolmetin and slindac; (3) fenamic acid derivatives, such as mefenamic acid and meclofenamic acid; (4) biphenylcarboxylic acid derivatives, such as diflunisal and flufenisal; and (5) oxicams, such as piroxim, sudoxicam, and isoxicam. Another class of NSAID has been described which selectively inhibit cyclooxygenase 2. Cox-2 inhibitors have been described (U.S. Pat. Nos. 5,616,601; 5,604,260; 5,593,994; 5,550,142; 5,536,752; 5,521,213; 5,475,995; 5,639,780; 5,604,253; 5,552,422; 5,510,368; 5,436,265; 5,409,944; and 5,130,311). Certain exemplary COX-2 inhibitors include celecoxib (SC-58635), rofecoxib, DUP-697, flosulide (CGP-28238), meloxicam, 6-methoxy-2 naphthylacetic acid (6-MNA), MK-966, nabumetone (prodrug for 6-MNA), nimesulide, NS-398, SC-5766, SC-58215, T-614; or combinations thereof.
The NGF antagonist and/or an additional therapeutic agent, such as NSAID, can be administered to a subject via any suitable route. For example, they can be administered together or separately, and/or simultaneously and/or sequentially, orally, intravenously, sublingually, subcutaneously, intraarterially, intramuscularly, rectally, intraspinally, intrathoracically, intraperitoneally, intraventricularly, sublingually, transdermally or by inhalation. Administration can be systemic, e.g., intravenous, or localized. The nerve growth factor antagonist and the additional therapeutic agent may be present together with one or more pharmaceutically acceptable carriers or excipients, or they may be present in separate compositions. In another aspect, the invention provides a synergistic composition of an NGF antagonist and an NSAID.
The pharmaceutical compositions of the disclosure may include a “therapeutically effective amount” or a “prophylactically effective amount” of an antibody or antibody portion of the disclosure. Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antibody portion of the disclosure is about 0.001 to about 20 mg/kg or about 0.001 to about 10 mg/kg. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the disclosure described herein are obvious and may be made using suitable equivalents without departing from the scope of the disclosure or the embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting of the disclosure.
EXAMPLES
The following examples are provided for exemplary guidance to make and use the disclosed binding proteins and pharmaceutical compositions thereof according to the inventive subject matter. However, it should be recognized that numerous modifications may be made without departing from the inventive concept presented herein.
Example 1: Immunization of Mice with NGF
To generate mouse anti-NGF mAbs, female A/J mice were immunized subcutaneously with 25 μg of human β NGF (R&D Systems catalog #256-GF/CF) in CFA. Animals were boosted every three weeks with 25 μg human β NGF in IFA. Four days prior to fusion, the mice were boosted with 10 μg of human β NGF in sterile saline intravenously. Spleen cells from the immunized mouse were fused with SP2/0-Agl4 myeloma cells at a 5:1 ratio spleen to SP2/0 cells, using standard techniques. Seven to ten days post fusion, when macroscopic colonies were observed; supernatants were tested in a capture ELISA format for binding to biotinylated human or rat β NGF. ELISA-positive wells were expanded to 24 well plates and tested for binding to biotinylated rat β NGF. Supernatants from hybridoma cell lines testing positive for both human and rat NGF were evaluated in a bioassay format. Cell lines of interest were cloned by limiting dilution to isolate an NGF-specific mouse monoclonal antibody.
Example 2: Screening Hybridoma Supernatants to Identify Secreted Anti-NGF MAbs
A. Indirect Binding ELISA
To determine if anti-NGF mAbs were present in hybridoma supernatants, ELISA plates were coated with goat anti-murine IgG Fc (Jackson ImmunoResearch, cat #115-005-164) and incubated overnight at 4° C. The plates were washed three times with wash buffer. The plates were blocked with 200 μl of 2% milk and incubated for 1 hour at room temperature. The plates were washed as above. Hybridoma supernatants were diluted 5-fold, 25-fold, 125-fold and 1625-fold with PBS and then added to the plate wells and incubated for 1 hour at room temperature. The positive control was crude sera (diluted 1:500 with PBS) isolated from a β NGF immunized mouse and the negative control was hybridoma supernatant derived from a mouse immunized with an antigen other than NGF. The plates were washed and then 50 μl of biotinylated human or rat β NGF at 50 ng/ml was added and incubated for 1 hour at room temperature. The plates were washed. Streptavidin-HRP (Thermo, cat #21 126) conjugate was diluted at 10,000 and added to the plates. The plates were incubated for 30 minutes at room temperature. The plates were washed and then TMB substrate (Invitrogen, catalog #00-2023) was added. The reaction was stopped using 2N H2SO4 (VWR, catalog # BDH3500-1). The absorbance at 450 nm was read on a Spectromax 2E plate reader (Molecular Devices); these absorbance readings are shown in Tables 1 and 2. The numerical value indicates binding of mouse anti-NGF antibodies to biotinylated human or rat β NGF. This data indicates that several hybridoma supernatants contained anti-NGF antibodies.
TABLE 3
Biotinylated Human NGF Indirect Binding ELISA data
Supernatant
dilution
(fold)
30F11
23F1
22E1
3C3
16B9
17G6
23H2
25E5
29E6
7H1
19C1
30A1
5
1.066
1.143
1.288
1.137
0.821
1.122
0.913
1.299
1.196
1.155
0.936
1.09
25
1.005
1.171
1.255
1.108
0.644
1.127
0.529
1.254
1.127
1.159
0.555
0.926
125
0.873
0.979
0.772
0.948
0.34
1.017
0.191
0.988
0.889
1.002
0.234
0.507
625
0.436
0.696
0.296
0.571
0.107
0.713
0.085
0.512
0.426
0.673
0.1
0.223
Supernatant
dilution
(fold)
29A7
27A5
26D5
26H12
23D7
22A9
22G3
21D4
3E9
3F9
2G11
1D6
5
1.198
1.116
0.954
0.943
1.087
0.707
0.662
1.154
1.167
0.974
1.038
0.545
25
1.092
0.887
0.903
0.794
1.06
0.549
0.498
1.042
0.996
0.694
0.992
0.457
125
0.762
0.395
0.823
0.381
0.857
0.348
0.24
0.899
0.655
0.323
0.819
0.164
625
0.293
0.174
0.542
0.135
0.489
0.168
0.126
0.543
0.298
0.145
0.486
0.066
Supernatant
dilution
(fold)
4B6
8E4
9E2
9H2
20B10
14G6
12H12
11D1
5
1.252
1.294
1.126
1.167
1.098
1.274
1.222
0.642
25
1.131
1.076
1.085
0.915
0.997
1.206
1.083
0.497
125
0.768
0.595
0.938
0.395
0.576
0.956
0.741
0.275
625
0.341
0.25
0.605
0.171
0.143
0.598
0.363
0.117
Supernatant
dilution
Positive
(fold)
control
4E2
12D6
1D10
2D8
3F7
4F11
4H2
5D8
5G9
6B2
6F10
3
1.018
1.078
0.985
1.105
1.046
1.282
1.192
1.013
0.79
1.052
1.231
1.096
15
0.981
0.991
0.844
0.963
0.868
1.166
1.016
0.8
0.654
0.919
0.939
1.045
75
1.02
0.705
0.501
0.655
0.436
1.049
0.702
0.447
0.42
0.534
0.505
0.999
Supernatant
dilution
Negative
Negative
(fold)
6H2
7C10
7G1
8G9
10A12
10B6
11A9
12A5
12F6
13E3
14A9
control
3
1.322
0.745
0.233
0.849
0.192
1.135
0.056
0.725
1.003
1.003
1.107
0.054
15
1.221
0.378
0.106
0.548
0.089
1.088
0.051
0.401
0.944
0.881
1.082
0.053
75
0.791
0.151
0.06
0.220
0.060
0.872
0.050
0.183
0.681
0.463
0.951
0.051
TABLE 4
Biotinylated Rat NGF Indirect Binding ELISA data
Supernatant
dilution
(fold)
30F11
23F1
22E1
3C3
16B9
17G6
23H2
25E5
29E6
7H1
19C1
30A1
5
0.694
0.764
1.054
0.698
0.443
0.749
0.670
1.091
0.677
0.733
0.660
0.690
25
0.734
0.767
0.936
0.729
0.350
0.758
0.412
1.099
0.655
0.664
0.462
0.681
125
0.603
0.737
0.557
0.628
0.218
0.751
0.176
0.803
0.523
0.603
0.197
0.445
625
0.361
0.528
0.229
0.520
0.094
0.567
0.083
0.396
0.261
0.401
0.088
0.180
Supernatant
dilution
(fold)
29A7
27A5
26D5
26H12
23D7
22A9
22G3
21D4
3E9
3F9
2G11
1D6
5
0.967
0.610
0.611
0.538
0.684
0.508
0.521
0.787
1.098
0.633
0.705
0.327
25
0.907
0.514
0.571
0.368
0.775
0.417
0.384
0.760
0.945
0.502
0.669
0.278
125
0.441
0.236
0.516
0.169
0.654
0.240
0.209
0.671
0.530
0.264
0.588
0.132
625
0.224
0.113
0.413
0.082
0.396
0.117
0.107
0.453
0.219
0.117
0.353
0.063
Supernatant
dilution
(fold)
4B6
8E4
9E2
9H2
20B10
14G6
12H12
11D1
5
0.607
0.685
0.632
0.453
0.472
0.755
0.676
0.122
25
0.508
0.518
0.559
0.310
0.431
0.739
0.571
0.095
125
0.438
0.317
0.529
0.157
0.261
0.665
0.357
0.076
625
0.234
0.150
0.382
0.085
0.108
0.424
0.173
0.060
Supernatant
dilution
Positive
(fold)
control
4E2
12D6
1D10
2D8
3F7
4F11
4H2
5D8
5G9
6B2
6F10
3
0.773
0.777
0.459
1.023
0.590
1.097
0.952
0.945
0.565
0.952
1.122
0.937
15
0.736
0.651
0.379
0.877
0.599
1.125
0.690
0.684
0.467
0.767
0.876
1.005
75
0.760
0.471
0.210
0.548
0.323
1.044
0.576
0.348
0.294
0.406
0.453
0.849
Supernatant
dilution
Negative
(fold)
6H2
7C10
7G1
8G9
10A12
10B6
11A9
12A5
12F6
13E3
14A9
control
3
1.108
0.541
0.197
0.681
0.145
0.440
0.058
0.521
0.904
0.786
0.845
0.055
15
0.860
0.275
0.093
0.396
0.077
0.784
0.052
0.334
0.810
0.737
0.777
0.053
75
0.603
0.115
0.061
0.155
0.060
0.727
0.051
0.153
0.565
0.413
0.582
0.052
B. TrkA Binding ELISA
To determine if anti-NGF mAbs in hybridoma supernatants blocked NGF from binding to the TrkA receptor, ELISA plates were coated with goat anti-human IgG Fc (Jackson ImmunoResearch, cat #109-005-008) at 2 μg/ml in PBS and incubated over night at 4° C. The plates were washed three times with PBS/Tween. The plates were blocked with 200 μl/well of 2% milk in PBS for 1 hour at room temperature. The plates were washed three times as above. Rat TrkA/Fc chimera (R&D Systems, catalog #1056-TK) was added at 1 μg/ml (50 μl/well) in PBS/0.1% BSA and then incubated for 1 hour at room temperature. Biotinylated human NGF was titered and pre-incubated with anti-NGF antibody supernatants diluted 1-fold, 5-fold, and 25-fold, or purified anti-NGF mAbs diluted to 0.08, 0.4, 2, or 10 μg/ml for 1 hour at room temperature on a plate shaker. The negative control was unrelated conditioned supernatant. The positive control was sera from a mouse immunized with NGF. The plates were washed and then 50 μl of each biotinylated NGF/Ab mix was added to the appropriate wells. The plates were incubated for 1 hour at room temperature. The plates were washed. 50 μl of streptavidin-HRP (Thermo, cat #21 126) was added at 10,000 dilution. The plates were incubated for 30 min at room temperature. The plates were washed. 50 μl of TMB (Invitrogen, cat #00-2023) was added and the reaction was stopped using 2N H2SO4 (VWR, cat # BDH3500-1). The absorbance at 450 nm was read on a Spectromax 2E plate reader (Molecular Devices), and the absorbance readings are shown in Table 5. The numerical value indicates binding of biotinylated human β NGF to rat TrkA/Fc chimera. This data indicates that several hybridoma supernatants contained anti-NGF receptor-blocking antibodies.
TABLE 5
Rat TrkA Inhibition Binding ELISA Data for Anti-NGF Hybridoma Supernatants
Supernatant
dilution
Negative
Positive
(fold)
control
control
30F11
23F1
22E1
3C3
16B9
17G6
23H2
25E5
29E6
7H1
1
0.465
0.050
0.158
0.108
0.357
0.146
0.142
0.091
0.379
0.304
0.291
0.217
5
0.456
0.055
0.210
0.140
0.429
0.195
0.249
0.123
0.622
0.354
0.600
0.419
25
0.462
0.102
0.331
0.276
0.558
0.345
0.409
0.210
0.418
0.505
0.881
0.758
Supernatant
dilution
(fold)
19C1
30A1
29A7
27A5
26D5
26H12
23D7
22A9
22G3
21D4
3E9
3F9
1
0.285
0.148
0.427
0.444
0.063
0.344
0.131
0.322
0.150
0.133
0.328
0.186
5
0.567
0.281
0.462
0.800
0.076
0.621
0.212
0.362
0.211
0.242
0.416
0.295
25
0.686
0.464
0.502
0.680
0.101
0.665
0.393
0.453
0.337
0.404
0.682
0.498
Supernatant
Dilution
(fold)
Neg
Pos
2G11
1D6
4B6
8E4
9E2
9H2
1
0.372
0.052
0.138
0.226
0.169
0.273
0.103
0.380
5
0.336
0.073
0.205
0.281
0.287
0.669
0.125
0.604
25
0.318
0.228
0.328
0.343
0.424
0.693
0.151
0.521
Supernatant
dilution
(fold)
20B10
14G6
12H12
11D1
19A12
2B12
PBS
PBS
1
0.166
0.113
0.060
0.101
0.100
0.065
0.295
0.315
5
0.200
0.192
0.099
0.152
0.170
0.070
0.334
0.297
25
0.289
0.334
0.190
0.264
0.295
0.095
0.306
0.289
Supernatant
dilution
−ve
+ve
+ve
+ve
(fold)
contrl
contrl
contrl
contrl
13E3
14A9
4E2
12D6
1D10
2D8
3F7
4F11
1
0.386
0.112
0.145
0.104
0.400
0.121
0.283
0.145
0.248
0.359
0.056
0.286
5
0.388
0.164
0.234
0.140
0.383
0.208
0.290
0.211
0.312
0.588
0.083
0.356
25
0.386
0.308
0.488
0.216
0.497
0.376
0.334
0.364
0.447
0.497
0.149
0.541
Supernatant
dilution
(fold)
4H2
5D8
5G9
6B2
6F10
6H2
7C10
8G9
10B6
12A5
12F6
PBS
1
0.396
0.363
0.344
0.096
0.206
0.400
0.230
0.409
0.329
0.306
0.172
0.436
5
0.457
0.398
0.387
0.215
0.212
0.523
0.489
0.473
0.364
0.328
0.227
0.351
25
0.606
0.504
0.473
0.451
0.242
0.738
0.487
0.413
0.399
0.406
0.324
0.338
C. SureFire Cellular Phospho-ERK (pERK) Assay
To determine if anti-NGF mAbs in hybridoma supernatants blocked downstream signaling as a result of blocking NGF from binding to TrkA, Neuroscreen-1 cells (Thermo Fisher Scientific) were grown on collagen I-coated flasks in RPMI medium supplemented with 10% horse serum, 5% FBS, 100 units/ml penicillin/streptomycin, 2 mM L-glutamine, and 10 mM HEPES at 37° C. in a humidified atmosphere at 95% air and 5% CO2. For the ERK phosphorylation assay, 5×104 cells were seeded in each well of a 96-well plate coated with collagen I (Becton Dickinson). Cells were then serum starved for 24 hours before stimulation. 130 pM human β NGF (R&D Systems catalog #256-GF/CF) was mixed into diluted hybridoma supernatants (to achieve a final supernatant dilution (fold) of 10-fold, 100-fold, 500-fold or 1,000-fold) and mixtures were pre-incubated for 15 min at 37° C. before being added to the cells. Each diluted hybridoma supernatant was tested in quadruplicate. After 5 min of stimulation, the medium was removed and replaced with SureFire™ AlphaScreen cell lysis (PerkinElmer). Cell lysates were then processed according to the manufacturer's instructions and fluorescence signals quantified using an En Vision plate reader (PerkinElmer); the fluorescence data is summarized in Table 6. The numerical value indicates ERK phosphorylation due to TrkA signaling in the presence of human β NGF and is expressed as the percentage of signal vs. maximum signal. The maximum signal is defined as 100% response from cells showing ERK phosphorylation in the presence of only β NGF (no hybridoma supernatant). This data indicates that several hybridoma supernatants contained neutralizing anti-NGF antibodies.
TABLE 6
SureFire pERK Assay Data Generated with Anti-NGF mAb Hybridoma Supernatants
Supernatant
dilution
(fold)
23F1
17G6
30F11
3C3
100
5
2
4
2
2
0
1
1
5
2
2
0
5
2
5
3
1000
5
3
4
3
2
2
2
1
4
2
4
3
6
3
5
4
5000
8
7
8
8
13
7
15
8
30
26
28
25
24
25
22
23
10000
35
33
32
32
44
25
43
23
65
45
56
42
57
52
68
62
Supernatant
dilution
(fold)
2B12
21D4
4B6
22G3
100
0
0
−1
0
4
3
5
2
5
4
1
0
13
7
7
2
1000
0
0
0
0
5
3
5
3
1
0
2
1
3
2
4
2
5000
18
16
21
17
11
7
12
8
8
8
7
8
23
18
25
20
10000
51
43
49
41
38
23
37
23
30
34
30
35
51
45
47
43
Supernatant
dilution
(fold)
2G11
14G6
16B9
19A12
100
5
2
6
3
4
3
0
−1
3
3
3
2
2
1
2
1
1000
6
3
6
3
−1
0
0
0
65
57
70
60
3
2
3
2
5000
14
8
14
8
7
7
8
7
72
63
73
62
47
36
46
32
10000
44
30
74
48
38
36
36
36
76
62
77
62
69
55
81
65
Supernatant
dilution
(fold)
30A1
26D5
23D7
23H2
100
0
0
1
0
0
0
0
0
2
2
−1
0
47
41
44
44
1000
1
2
2
2
1
1
1
1
1
0
1
1
86
80
79
82
5000
40
41
42
40
37
30
35
30
59
48
56
54
85
80
85
83
10000
62
67
63
64
64
52
80
71
74
63
70
60
85
84
82
84
Supernatant
dilution
(fold)
9E2
20B10
12H12
11D1
100
3
3
3
4
2
1
1
1
3
3
3
2
69
75
70
78
1000
5
7
5
8
1
1
1
0
30
30
30
32
71
84
72
85
5000
37
57
36
55
20
28
19
29
62
64
60
61
76
78
80
76
10000
55
69
56
67
55
71
73
84
69
78
76
77
89
95
102
101
D. PathHunter Assay
To determine if anti-NGF mAbs in hybridoma supernatants blocked downstream signaling as a result of blocking NGF from binding to TrkA, the PathHunter U20S stable cell line stably expressing the NGF receptor TrkA and the co-activator protein SHC1 fused to complementing fragments of β-galactosidase was purchased from DiscoveRx. Cells were grown in MEM media supplemented with 10% FBS, 100 units/ml penicillin/streptomycin, 2 mM L-glutamine, 500 μg/ml Geneticin G418, and 250 μg/ml Hygromycin at 37° C. in a humidified atmosphere at 95% air and 5% CO2. Sixteen hours before the assay, 2×104 cells were seeded in each well of a 96-well half-volume black plate in 40 μl of MEM media supplemented with 0.5% horse serum. 440 pM human β NGF (R and D Systems catalog #256-GF/CF) was mixed into diluted hybridoma supernatants (to achieve a final supernatant dilution of 10-fold, 100-fold, 500-fold or 1,000-fold) and mixtures were pre-incubated for 15 min at 37° C. before being added to the cells. Cell plates were incubated for 5 min at room temperature before stimulation with 10 μl per well of NGF/antibody mixture. After 3 hours of cell induction at room temperature, 25 μl of PathHunter detection reagent was added to each well according to the manufacturer's instructions. The chemiluminescent signal was detected 1 hour later using a TopCount plate reader (PerkinElmer); the chemiluminescence signal data is shown in Table 7. The numerical value indicates β-galactosidase generation due to TrkA signaling in the presence of human NGF and is expressed as the percentage of signal vs. maximum signal. The maximum signal is defined as 100% response from cells showing in the presence of β-galactosidase generation in the presence of only β NGF (no hybridoma supernatant). This data indicates that several hybridoma supernatants contained neutralizing anti-NGF antibodies.
TABLE 7
PathHunter Data Generated with Hybridoma Supernatants
Supernatant
dilution
(fold)
30F11
23F1
3C3
16B9
17G6
19A12
100
21
22
5
5
16
29
19
24
10
14
25
28
1000
42
37
23
13
40
46
117
114
23
24
21
30
5000
97
99
69
70
71
81
120
127
100
115
93
92
10000
94
93
92
91
78
84
114
129
115
120
89
98
Supernatant
dilution
(fold)
2B12
30A1
26D5
23D7
23H2
22G3
100
89
90
21
24
83
84
31
28
142
176
16
16
1000
64
70
50
57
49
51
54
53
88
134
20
23
5000
128
127
126
139
92
96
112
131
117
120
89
99
10000
128
133
133
129
95
84
124
148
111
136
86
101
Supernatant
dilution
(fold)
21D4
2G11
4B6
9E2
20B10
100
9
10
22
22
24
31
102
104
6
5
1000
16
17
29
27
46
49
77
91
0
4
5000
107
100
66
72
88
94
137
152
25
31
10000
108
112
66
72
102
109
137
143
52
58
Supernatant
dilution
Unrelated
(fold)
14G6
12H12
11D1
26H12
hybridoma
100
18
17
26
23
117
101
106
119
156
185
1000
27
27
46
56
127
118
124
115
136
144
5000
105
83
145
137
109
99
137
166
126
132
10000
113
109
122
145
91
98
151
167
138
137
Example 3: Hybridoma Sub-Cloning
Hybridoma cell lines were subcloned using standard limiting dilution techniques. Cells were diluted to a concentration of 50, 5, or 0.5 cells/mL. 200 ul of the diluted cell suspensions were plated into 96 well tissue culture plates. The plates were incubated at 37° C. with 5% CO2 and −90% relative humidity. The growth was visually checked at day 7 for macroscopic colonies. Supernatants from wells were screened for antibody production when colony growth was visible. Table 8 shows the subclone identification nomenclature and monikers. This data indicates that several anti-NGF antibodies could be isolated from a clonal population of cells.
TABLE 8
Hybridoma Subclone Identification and Monikers
Hybridoma Super-
Subcloned
natant Name
Hybridoma Name
Moniker
Lot #
14G6
ML129-14G6.3H3
PR-1254970
1734671
2G11
ML129-2G11.3B1
PR-1254971
1734673
20B10
ML129-20B10.3F4
PR-1254972
1734675
2B12
ML129-2B12.5G9
PR-1254973
1734676
17G6
ML129-17G6.3E7
PR-1254974
1734677
21D4
ML129-21D4.4A11
PR-1254977
1734678
4B6
ML129-4B6.4H3
PR-1254978
1734679
22G3
ML129-22G3.3F3
PR-1254979
1734680
23F1
ML129-23F1.4G3
PR-1254980
1734681
14A9
ML130-14A9.5B12
PR-1254981
1734682
3F7
ML130-3F7.4A8
PR-1254982
1734683
Example 4: Scale Up and Purification of Monoclonal Antibodies
Subcloned hybridoma cell lines were expanded into Hybridoma SFM (Invitrogen catalog #12045) with 5% Low IgG Fetal bovine serum (Invitrogen catalog #16250-078). Supernatants were harvested, centrifuged and filtered to remove cellular debris, and concentrated. Antibodies were mixed with Pierce binding buffer A (Thermo, catalog #21001) in a 1 ratio. The antibodies were loaded onto a recombinant Protein A sepharose (GE Healthcare, catalog #17-1279-04) chromatography column, eluted using Pierce elution buffer (Thermo, catalog #21004), neutralized using 2M Tris pH 7.5, and then dialyzed into PBS. This work allowed the isolation of anti-NGF mAbs for characterization studies.
Example 5: Cloning of Canine NGF
The coding region of canine NGF was amplified from canine universal cDNA (Biochain Institute, catalog #4734565) using primers of SEQ ID NO: 45 and SEQ ID NO: 46 or primers of SEQ ID NO: 47 and SEQ ID NO: 48 and cloned into a mammalian or bacterial expression vector, respectively. The PCR reactions were set up as recommended by the manufacturer (Novagen, KOD Hot Start Master Mix, catalog #71842-3). The mammalian clone was made as a C-terminal 6-His fusion protein by ligating the PCR product with pTT6 vector (Abbott) at the Kpnl/Xbal restriction sites. The bacterial clone was made with the pro-NGF sequence using the mammalian clone as a template and ligated with pET15B (Novagen) at the Ndel/Xhol restriction sites. The DNA sequence and amino acid sequence of the canine NGF isolated are listed as SEQ ID NO: 49 and SEQ ID NO: 50, respectively. This work allowed expression of canine NGF protein for purification.
Example 6: Expression of Canine NGF
The canine NGF clone in the bacterial expression vector was grown at 37° C. in overnight express auto inducing Terrific Broth (Novagen) in Rosetta2 (DE3) E. coli host (EMD Biosciences) in 2 L non-baffled flasks. The cells were centrifuged down and the cell paste was resuspended in 100 mL of lysis buffer (25 mM Tris, 300 mM NaCl, 10% glycerol, 0.1% Triton X 100 pH 8.0) with lysonase and sonicated for 2 min on ice. The sample was centrifuged at 15000 RPM and the pellet was solubilized in 50 mL of 25 mM Tris, 6 M GdHCl pH 8.0. The sample was centrifuged at 15000 rpm for 30 min and the supernatant was loaded on to a 10 ml IMAC resin.
A. IMAC Chromatography
A 10 ml GE-Ni FF column was prepared. Buffer A: 25 mM Tris, 6 M GdHCl pH 8.0, Buffer B: A+500 mM Imidazole. The resin was equilibrated and loaded with recirculation to allow for complete binding (˜10 passes) overnight at 4° C. The column was washed with Buffer A. Batch elution was carried out with 40 ml Buffer A, followed by 30 ml Buffer A. 5 ml fractions were collected and pooled.
B. Refolding by Rapid Dilution
The pooled fraction was reduced by adding 50 mM DTT, and EDTA was added to 10 mM, and incubated for 1 h at RT. The sample was acidified by adding 6M HC1 to pH 4.0 and dialyzed into 6M GdHCl pH 5.0 to remove excess DTT. Refolding was performed by diluting the reduced/acidified sample in 1 L of 100 mM Tris, 1 M Arginine, 5 mM EDTA, 5 mM GSH, and 1 mM GSSG pH 9.5 for 4 h at 4° C. The refolded protein was dialyzed against 25 mM Tris, 200 mM NaCl, 10% Glycerol pH 8.0. Precipitation was cleared by filtration. The clarified sample was concentrated and diafiltered into 25 mM Tris, 200 mM NaCl, 10% Glycerol pH 8.0 using a 10K membrane.
C. Ni-IMAC
Refolded pro-NGF was loaded on a 5 ml Ni-IMAC. Buffer A: 25 mM Tris, 300 mM NaCl, 10% Glycerol pH 8.0. Buffer B: A+500 mM Imidazole. The column was washed with Buffer A. 8 ml fractions were collected. Elution was performed with a linear gradient 0-100% Buffer B. 5 ml fractions were collected. Samples of each fraction were mixed with non-reducing NuPage SLB (Invitrogen) and separated on a 4-12% NuPAGE Novex Bis-Tris Midi gel for analysis. Fractions containing protein were pooled and dialyzed against 20 mM Na Phosphate, 50 mM NaCl, 10% glycerol pH 7.4.
D. Trypsin Digestion
Pro-β NGF was mixed with trypsin in resuspension buffer and incubated on ice for 30 min. Immobilized inhibitor was added and incubated for 15 min and then filtered.
E. Sepharose Cation Exchange Chromatography
The sample was loaded on a 5 ml SP Sepharose high performance chromatography column (GE Healthcare). Buffer A: 20 mM Na Phosphate, 50 mM NaCl, 10% Glycerol pH 7.4, Buffer B: A+1 M NaCl. The column was washed with Buffer A. Elution was performed with linear gradient 0-100% Buffer B. 5 ml fractions were collected. The fractions were separated on a 4-12% Criterion XT Bis-Tris Midi gel for analysis. Fractions containing protein were pooled, dialyzed in PBS pH 7.4, and concentrated.
This work resulted in the production of several milligrams of purified canine NGF for characterization studies and for studies of anti-NGF canine antibodies.
Example 7: Characterization of Subcloned and Purified Hybridoma Antibodies
A. Canine NGF Direct Binding ELISA
To determine if purified mouse anti-NGF mAbs bind to canine β NGF, ELISA plates were coated with 50 μl/well of canine NGF (Abbott Laboratories) at 1 μg/ml in PBS and incubated over night at 4° C. The plates were washed three times with PBS+Tween buffer. The plates were blocked with 200 μl/well of 2% milk in PBS for 1 hour at room temperature. The plates were washed three times as above. Purified antibodies were diluted to 0.4, 2, or 10 μg/ml. 50 μl of each concentration of purified antibody was added to the plates. The plates were incubated for 1 hour at room temperature. The plates were washed. 50 μl of a 5000-fold diluted goat anti-mouse IgG Fc-HRP (Thermo, catalog #31439) was added. The plates were incubated for 1 hour at room temperature. 50 μl of TMB (Invitrogen, catalog #00-2023) was added and the reaction was stopped using 2N H2SO4 (VWR, catalog # BDH3500-1). The absorbance at 450 nm was read on a Spectromax 2E plate reader (Molecular Devices). The results are shown in Table 9, and the numerical value indicates binding of mouse anti-NGF antibodies to canine NGF.
TABLE 9
Canine NGF Direct Binding ELISA Data Using Purified Anti-NGF mAbs
μg/ml
PR-
PR-
PR-
PR-
PR-
PR-
PR-
PR-
PR-
PR-
PR-
Mab
1254970
1254971
1254972
1254973
1254974
1254977
1254978
1254979
1254980
1254981
1254982
10
0.530
0.497
0.154
0.905
0.552
0.552
0.579
0.683
0.491
0.610
0.208
2
0.342
0.324
0.091
0.836
0.383
0.414
0.458
0.566
0.334
0.458
0.142
0.4
0.176
0.165
0.071
0.769
0.209
0.223
0.253
0.313
0.168
0.229
0.095
B. TF-1 Cell Proliferation Potency Assay
TF-1 is a human erythroleukaemic cell line that expresses human TrkA and proliferates in response to recombinant β NGF. To determine if purified anti-NGF mAbs blocked NGF-induced proliferation, TF-1 cells (ATCC# CRL-2003) were maintained at 37° C. and 5% CO2 in RPMI (Gibco, cat #1 1875-093) media containing recombinant human GM-CSF at 2 ng/mL (R&D Systems, cat #215-GM) and fetal bovine serum (FBS, Hyclone, cat # SH 30070.03). GM-CSF and FBS was removed 24 hours before the assay. On day one of the assay each anti-NGF mAb was titrated (concentrations ranging from 33.3 nM to 1.7 fM) and added to a fixed concentration of recombinant canine NGF (70 pM) and TF-1 cells (2.5×104 cells/well) in RPMI+4% FBS for 72 hours. Cell proliferation was measured using Cell Titer-glo (Promega, cat # G7571). The IC50 values of each anti-NGF mAb on canine NGF-induced TF-1 cell proliferation is shown in Table 10, and the data shows that in the presence of 70 pM canine NGF, most of the anti-NGF antibodies display sub-nM potencies, and some display potencies of less than 50 pM.
TABLE 10
Potency of Mouse Anti-NGF Antibodies on Canine
NGF-induced TF-1 Cell Proliferation
Moniker
Lot
IC50 (nM)
PR-1254970
1734671
0.662
PR-1254971
1734673
1.088
PR-1254972
1734675
0.303
PR-1254973
1734676
0.039
PR-1254974
1734677
0.230
PR-1254977
1734678
0.217
PR-1254978
1734679
0.978
PR-1254979
1734680
0.288
PR-1254980
1734681
0.343
PR-1254981
1734682
0.046
PR-1254982
1734683
0.025
C. SureFire Cellular pERK and PathHunter Assays
To determine if purified mouse anti-NGF mAbs blocked canine NGF-induced cellular responses, purified antibodies were characterized by titration in the SureFire cellular pERK (using 128 pM canine β NGF in each test well) and PathHunter assays (using 441 pM canine NGF in each test well) as described in Example 2 Sections C and D. The IC5o of each anti-NGF mAb on canine β NGF-induced cellular responses is summarized in Table 11, and the data shows that in the presence of 128 pM canine NGF all the anti-NGF antibodies display sub-nM potencies, and some display potencies of less than 50 pM (pERK assay). Also, in the presence of 441 pM canine NGF, all the anti-NGF antibodies display sub-nM potencies, and some display potencies of less than 150 pM (PathHunter assay).
TABLE 11
Summary of pERK and Path Hunter Assay
Data for Purified Anti-NGF mAbs
SureFire pERK
PathHunter
Antibody
IC50 (nM)
IC50 (nM)
PR-1254970
0.02711
0.3346
PR-1254971
0.04750
0.4986
PR-1254972
0.2282
0.3133
PR-1254973
0.01876
0.1428
PR-1254974
0.01561
0.2464
PR-1254977
0.01759
0.1810
PR-1254978
0.02466
0.3559
PR-1254979
0.01627
0.2414
PR-1254980
0.01371
0.3812
PR-1254981
0.02135
0.2794
PR-1254982
0.005804
0.1505
Example 8: Characterization of Purified Anti-NGF Antibodies Following Hybridoma Subcloning
A. Mass Spectrophotometry (MS) and Size Exclusion Chromatography (SEC) Analysis on Anti-NGF Antibodies
The mouse anti-NGF mAbs were reduced using 1M DTT and analyzed using HPLC/MS on a 6224 TOF mass spectrometer and a 1200 HPLC (Agilent technologies) using a Vydac C4, IMM×150 mm column (CN#214TP5115, the Nest Group) at a flow rate of 50 μl/min. Buffer A: 99.9% HPLC water+0.1% FA+0.01% TFA and buffer B: 99.9% ACN+0.1% FA+0.01% TFA. The LC equilibrium and sample desalting was performed using 5% buffer B for 7 min. The separation gradient was performed using 30% to 50% Buffer B for 10 min and a washing step was performed at 95% buffer B for 10 mins. The TOF acquisition parameters were: gas temperature at 350 C and OCT/RF at 750V. The mass range was from 600-3200 m/z and the rate specified was 1.03 spectra/s. Qualitative analysis software (Agilent) was used to deconvolute antibody molecular weights.
The antibodies were analyzed on Shimadzu LC-10AVP system (Shimadzu Scientific). The SEC column used was a Superdex-200 10/300 L (GE Healthcare). The flow rate was 0.75 ml/min and UV280 was used to monitor peaks. The buffer used was Na2SO4+92 mM NaPO4+5 mM NaZ3, pH 7.0. The reagent antibody was injected in 10 μl, (10 μg). The gel protein markers on SEC were from Bio-Rad (CN#151-1901). The MS and SEC results are summarized in Table 12. This data determined the hybridoma-derived antibodies were highly monomeric following purification. In addition, the molecular weights of the heavy and light chains comprising the hybridoma-derived antibodies were determined.
B. Antibody Isotype Determination
The isotype of the anti-NGF mAbs was determined using the Zymed Mouse MonoAb-ID Kit (Invitrogen catalog#90-6550 lot#1407589). The isotyping results are summarized in Table 12. This data indicates that murine IgG1/k, IgG2a/k, and IgG2b/k mouse antibodies are capable of binding and neutralizing NGF.
TABLE 12
Isotyping, Size Exclusion Chromatography, and Mass
Spectrometry Analysis of Anti-NGF Antibodies
Molecular
Molecular
weight (Dal)
weight (Dal)
Heavy
Hybridoma Name
Moniker
Lot
Isotype
% Monomer
Light Chain
Chain
ML129-14G6.3H3
PR-1254970
1734671
IgG1 Kappa
96.9
24221.43
49479.67
ML129-2G11.3B1
PR-1254971
1734673
IgG1 Kappa
96.8
24156.26
49491.69
ML129-20B10.3F4
PR-1254972
1734675
IgG2b Kappa
99.0
24159.34
50329.24
ML129-2B12.5G9
PR-1254973
1734676
IgG2b Kappa
99.4
23539.38
51102.21
ML129-17G6.3E7
PR-1254974
1734677
IgG1 Kappa
98.8
24221.43
49479.45
ML129-21D4.4A11
PR-1254977
1734678
IgG1 Kappa
98.4
24221.46
49479.70
ML129-4B6.4H3
PR-1254978
1734679
IgG1 Kappa
96.7
24170.40
49533.92
ML129-22G3.3F3
PR-1254979
1734680
IgG2a Kappa
99.0
24221.42
50123.17
ML129-23F1.4G3
PR-1254980
1734681
IgG1 Kappa
99.5
24221.42
49493.95
ML130-14A9.5B12
PR-1254981
1734682
IgG1 Kappa
99.1
24180.28
50241.85
ML130-3F7.4A8
PR-1254982
1734683
IgG1 Kappa
99.4
23708.54
50289.13
Example 9: Binding Kinetics of Anti-NGF Antibodies
A biomolecular protein interaction analysis was used to evaluate the binding kinetics of the interaction between the purified anti-NGF hybridoma antibodies and recombinant canine NGF. The antibodies were captured using a goat anti-mouse IgG FC (10000 RU) surface which was directly immobilized to a CM5 chip using an amine coupling procedure according to the manufacturer's instructions (Biacore). A sample size of 5 μl of antibody at a concentration of 1 μg/ml was captured at 10 μl minute. Recombinant canine NGF was used as the antigen. Canine NGF was injected at 75 μl/min (concentration range: 5-0.039 nM) for mouse antibodies. The association rate was monitored for 3.3 minutes and the dissociation rate was monitored for 10 minutes. Aliquots of canine NGF were also simultaneously injected over a reference reaction CM surface to record any nonspecific binding background. The instrument sensitivity for on-rate is 1×107, such that any on-rate that is faster than 1×107 may not be accurately measured; the instrument sensitivity for off-rate is 1×106, such that any off-rate that is slower than 1×10″6 may not be accurately measured. Therefore, an on-rate that is faster than 1×107 is recorded as >1×107 and an off-rate that is slower than 1×10″6 is recorded as <1×10″6. The biomolecular protein interaction analysis results are summarized in Table 13. This data indicates that the isolated murine anti-NGF mAbs have fast on-rates (from greater than 7×106) and slow off-rates (from less than 1×10−3). The overall KDs of the murine anti-NGF mAbs range from about 300 pM to 0.1 pM demonstrating efficient binding of the purified anti-NGF hybridoma antibodies to recombinant canine β NGF.
TABLE 13
Binding Kinetics of Anti-NGF mAbs to Canine NGF
On rate
Off rate
Overall
Antibody
(1/Ms)
(1/s)
affinity (M)
PR-1254972
Expt 1
>1 ×
107
3.14 ×
10−3
<3.14 ×
10−10
lot: 1734675
Expt 2
>1 ×
107
3.21 ×
10−3
<3.21 ×
10−10
Average
>1 ×
107
3.18 ×
10−3
<3.18 ×
10−10
PR-1254973
Expt 1
>1 ×
107
1.21 ×
10−4
<1.21 ×
10−11
lot: 1734676
Expt 2
>1 ×
107
1.38 ×
10−4
<1.38 ×
10−11
Average
>1 ×
107
1.30 ×
10−4
<1.30 ×
10−11
PR-1254977
Expt 1
>1 ×
107
1.39 ×
10−4
<1.39 ×
10−11
lot: 1734678
Expt 2
>1 ×
107
1.60 ×
10−4
<1.6 ×
10−11
Average
>1 ×
107
1.50 ×
10−4
<1.5 ×
10−11
PR-1254980
Expt 1
>1 ×
107
2.37 ×
10−4
<2.37 ×
10−11
lot: 1734681
Expt 2
>1 ×
107
2.25 ×
10−4
<2.25 ×
10−11
Average
>1 ×
107
2.31 ×
10−4
<2.31 ×
10−11
PR-1254981
Expt 1
8.67 ×
106
1.27 ×
10−4
1.47 ×
10−11
lot: 1734682
Expt 2
7.48 ×
106
1.40 ×
10−4
1.87 ×
10−11
Average
8.08 ×
106
1.34 ×
10−4
1.67 ×
10−11
PR-1254982
Expt 1
>1 ×
107
<1 ×
10−6
<1 ×
10−13
lot: 1734683
Expt 2
>1 ×
107
<1 ×
10−6
<1 ×
10−13
Average
>1 ×
107
<1e ×
10−6
<1 ×
10−13
Example 10: Method for Identifying Anti-NGF Antibody Sequences from Hybridomas by Cloning and Sequencing
To identify the nucleotide and amino acid sequence of the six subcloned hybridoma mAbs shown in Table 13, the RNA from individual hybridoma cultures was extracted with Qiagen RNeasy kit (Qiagen, cat #74104). RNA was reverse-transcribed and cDNA antibody sequences amplified using the Qiagen One-Step RT-PCR kit (Qiagen, catalog #210212). Forward primers were degenerate and designed to anneal to the variable regions (heavy chain primers: 1HA, 1HB, 1HC, 1HD, 1HE, 1HF; and light chain primers: 1LA, 1LB, 1LC, 1LD, 1LE, 1LF, 1LG) (EMD4 Biosciences catalog #69896). Reverse primers were also degenerate and made to constant regions of gamma (heavy chains) and kappa (light chains). PCR products of approximately 400-450 base pairs were gel isolated and purified with Qiagen Gel Extraction kit (Qiagen, cat #28706).
Purified PCR products were cloned into TOPO TA cloning vectors (Invitrogen, cat # K4500-01SC). Each topoisomerase reaction mixture was used to transform TOP 10 chemically competent bacteria and plated on LB plates with 75 μg/ml Ampicillin and 60 μl 2% Bluo-Gal (Invitrogen, cat #15519-028). Isolated colonies were picked from the LB plate to inoculate 20 μl LB broth/100 μg/ml carbenicillin. One μl of this mini-culture was used in a PCR reaction with MI 3 forward and reverse primers to amplify the insert in the TOPO vector. PCR products were separated on 2% agarose gels; samples indicating an appropriately-sized insert in the vector were sequenced using an Applied Biosystems model 3730S DNA sequencer. DNA sequences derived from the identification of all murine mAb heavy and light chain variable domains were translated into protein sequence and are shown in FIG. 1 to FIG. 24.
Example 11: Homology Modeling of Murine Anti-NGF Antibodies
The sequences of the heavy and light chain variable regions of each anti-NGF antibody were imported into InsightII (Accelrys, San Diego, Calif.). Each sequence was used as a template for BLAST to find the x-ray crystal structures from the Protein Data Bank (www.rcsb.org) which were closest in identity. One structure for each of the heavy and light chains was selected based both on percent identity and on matching the exact length of all CDR loops. The sequences of each template and each query sequence were aligned and standard homology modeling techniques used to construct homology models of each chain. The complex of both modeled chains was then minimized for 50 cycles of restrained (500 Kcal/Angstrom for all heavy atoms) conjugate gradient minimization using the CVFF force field in the DISCOVER program (Accelrys, San Diego, Calif.).
The likelihood that a given framework residue would impact the binding properties of the antibody depends on its proximity to the CDR residues. Therefore, using the model structures, residues that fell within 5 A of any CDR atom was identified as most important and were recommended to be candidates for retention of the murine residue in the caninized antibody sequences. A change in nucleotide(s) in a mutant gene that restores the original sequence and hence the original phenotype is often referred to as a back mutation. Therefore, we refer to residues that are candidates for retention of the murine residue in the caninized antibody sequences as backmutations.
Example 12: Identification of Canine Heavy and Light Chain Antibody Sequences from Canine PBMCs
To identify canine Ig heavy and lambda light chain antibody variable domain amino acid sequences, RNA was isolated from mongrel canine peripheral blood mononuclear cells (PBMCs) using an RNEasy kit (Qiagen #74104). Canine PBMC mRNA was reverse transcribed (RT) with Superscript III reverse transcriptase (Invitrogen catalog #18080-093) and cDNAs were amplified using the 5′ RACE System (Rapid Amplification of cDNA Ends) (Invitrogen #18374-058). RT and PCR primers (RK323, RK324, RK122, LG010, LG011, LG012) are described in patent publication number: U.S. Pat. No. 7,261,890 B2 entitled Methods for Using Canine Immunoglobulin Variable Domains and Caninized Antibodies). Primers RK323 and RK324 were used for canine IgG reverse transcription followed by nested PCR with RK326 and the Abridged Anchor Primer (AAP) (Invitrogen). LG011 was used for canine lambda light chain RT PCR, followed by nested PCR with LG010 and LG012 and AAP.
The resulting PCR products were separated by agarose gel electrophoresis. The 600 base pair (canine lambda and kappa light chains) and 800 base pair (canine Ig heavy chain) PCR products were purified from the agarose using a Gel Extraction kit (Qiagen #28706) and cloned into the TA site of the pCR2.1 TOPO vectors using the TOPO-TA Cloning system (Invitrogen #K4500-01 SC).
Transformed TOP 10 bacteria were selected and plasmid DNA was isolated using Qiaprep Spin Mini-Prep Kit (Qiagen #27104). Plasmid DNA from 25 heavy chain, 38 kappa light chain and 23 lambda light chain colonies was sequenced to identify the nucleotide and corresponding amino acid sequences. Complete variable domain sequence data were obtained from 25 heavy chain, 38 kappa light chain and 19 lambda light chain clones. Variable domain sequence data including the leader peptide (when identified) are shown in Tables 14, 15 and 16. All derived heavy chain and light chain sequence are unique compared to those disclosed in patent publication number: U.S. Pat. No. 7,261,890 B2.
TABLE 14
Canine Heavy Chain Variable Domain Sequences
Derived from Canine PBMC RNA
Name
Sequence
Ca-1005
EVQLEESGGDLVKPGGSLRLSCVASGFSIGSYGMSWVRQSPGKGLQWVAWIKYDGSR
TFYADAVKGRFTISRDNAKNTLFLQMNSLRAEDTAVYFCVKGPNSSWLPSTYFASWG
QGTLVTVSS
(SEQ ID NO: 178)
Ca-2301
EMQLVESGGDLVRPGGSLRLSCVASGFTFSTYGMTWVRQSPGKGLQWVATIGPGGRN
TYYADAVKGRFTISRDDAENTLFLQMNSLRAEDTAVYYCAQAFDATYYTSFDCWGRG
SLVAVSS
(SEQ ID NO: 86)
Ca-2302
MESVLSWVFLVALLQGIQGEIRLVESGGDLVKPGGSLRLSCVASGFIFGNYDMSWVR
QAPGKGLQWVAAVRYDGSSTYYSDAVKGRITISRDDPGNTVYLQLDSLRAEDTATYY
CVRGGYYSSSFYIGGAFGHWGPGTLITVSS
(SEQ ID NO: 87)
Ca-2303
MECVLGWVFLVAILRGVQGEVQLVESGGDLVKPGGSLRLSCVASGFTFSDYYMSWIR
QAPGKGLQWVADISDGGDGTGYAGAVKGRFTVSRENVKNTLYLQMNDLRAEDTAIYY
CTKAREMYGYRDFDSWGPGTLVTVSS
(SEQ ID NO: 88)
Ca-2304
MESVLGLVALLTILKGVQGEVQLVESGGDLVKPGGSLRLSCVASGFTFSNYYMTWVR
QAPGKGLEWVGYIHNGGTYTYYADAVKGRFTISRDDAKNTLYLEMNSLRAEDTAVYY
CGKMIFDYWGQGTLVTVSS
(SEQ ID NO: 89)
Ca-2305
MESALSWVFLVTILKGVQGEVLLVESGGDLVKPGGSLRLSCLTSGFTFNTYDWGWVR
QAPGKGLQWIAYIKKGGSDVRYADAVKGRFTISRDDAKNTLYLQMNSLRAEDTAVYY
CARSAWDSFDYWGQGTLVTVSS
(SEQ ID NO: 90)
Ca-2306
MESVFCWVFLVAILKGVRGVQGEVQLVESGGDLVKPAGSLRLSCVASGFTFTDYSMN
WVRQAPGKGLQWVATISNDGTSTDYTDAVKGRFTVSRDSARNTVYLQMTSLRADDTA
TYYCVSRHSYSLLADYWGQGTLVTVSS
(SEQ ID NO: 91)
Ca-2307
MQMPWSLLCLLAAPLGVLSEVTLQESGPGLVKPSQTLSLTCAVSGGSVIRNYYWHWI
RQRPGRGLEWMGCWSETTYYSPAFRGRISITIDAATDQFSLHLNSMTTDDTAVYYCA
RALYPTSSWYDGMDYWGHGASVVVSS
(SEQ ID NO: 92)
Ca-2308
EVQLVESGGDLVKPGGSLRLSCESSGFIFSQYAMNWVRQAPGKGLQWVAYIGGAGFI
TYHADDVKGRFTISRDNAKNTLYLQMNSLTINDTAVYYCVRSNSRIPDYWGQGTLVA
VSS
(SEQ ID NO: 93)
Ca-2309
MESVFCWVFLVAILKGVQGEVQLVESGGDLVKPGGSLRLSCVASGFTFSSVYMSWVR
QAPGKGLQWVARITTDGTDTFYADAVKGRFTISRDNVKNMLYLEMNSLRAEDTAIYY
CGDPWQPAYPDLWGQGTMVTVSS
(SEQ ID NO: 94)
Ca-2310
MESVLCWVFLVAILKGVQGEVHLVESGGDLVKPGGTLRLSCVASGFTFSQYDMSWVR
QSPGKGLQWVALSRYHGGGTYYADAVKGRFTISRDNAKNMLYLQMNSLRAEDTAVYY
CVKEGSRWDLRGDYDYWGQGTLVTVSS
(SEQ ID NO: 95)
Ca-2311
MQMPWSLLCLLAAPLGVLSELTLQESGPGLVKPSQTLSLICVVSGGSVISSHYWNWI
RQRPGRGLEWMGYWTGNVNYNPAFQGRISIIGDAAKNQFSLHLSSMTTDDTAVYYCA
RCGIVAPGFLPIGDFDFWGQGTLVTVSS
(SEQ ID NO: 96)
Ca-2312
MESVFCWVFLVAILKGVQGEVQLVESGGDLVKPGGSLRLSCVASGFSFSNYFMFWGR
QAPGKGLQWVARIRSDGGSTYYADAVKGRFTISRDNARNTLYLQMNSLRAEDTATYY
CAKADIIKLPEYRGQGTLVTVSS
(SEQ ID NO: 97)
Ca-2401
ESVLGWIFLATILKGVQGEVQLVESGGDLVKPGGSLRLSCVGSGFTFSSSWMNWVRQ
APGKGLQWIAEISGTGSSTNYADAVKGRFTISRDNDKNTLYLQMNSLRAEDTAMYYC
ARAAYYGNYRNDLDYWGQGTLVTVSS
(SEQ ID NO: 98)
Ca-2402
KPAGSLRLSCVASGFTFSSHSVTWVRQAPGKGLQFVAGITSGGNNRYYTDAVRGRFT
LSRDNAKNTVYLQMNSLRAEDTAMYFCALGSYEWLSGEFDYWGQGTLVTVSS
(SEQ ID NO: 99)
Ca-2403
MESVFCWVFLVAILKGVQGEVQLVESGGDLVKPGGSLRLSCVASGFTLNNYFMYWVR
QAPGKGLQWVARLNSNGDSTFYADAVKGRFTISRDNAKNTLYLQMNSLRAEDTSMYY
CAKDLIYGYTLWGQGTLVTVSS
(SEQ ID NO: 100)
Ca-2404
MASVLSWVFLVAIVKGVQGEVQLVESGGDLVKPGGSLRLSCVASGFIFNKYEVYWVR
QAPGKGLEWVARILESGNPTYYAEAVEGRFTISRDNAKNMAYLQMNSLRADDTAVYY
CATPSVSSTVAIDYWGQGALVTVSS
(SEQ ID NO: 101)
Ca-2405
MQMPWSLLCLLATPLGVLSELTLQESGPGLVKPSQTLSLTCWSRGSVTSDYYWNWIR
QRPGRGLEWMGHWIGSTAYNPAFQGRISITADTAKNQLSLQLRSMTTEDTAVYFCAR
GSSWTPSGDSWGQGTLVTVSS
(SEQ ID NO: 102)
Ca-2406
MASVLKLGFSCRYCKKVSRVRCNXVESGGDLVKPGGSLRLSCVASGFIFNKYEVYWV
RQAPGKGLEWVARILESGNPTYYAEAVEGRFTISRDNAKNMAYLQMNSLRADDTAVY
YCATPSVSSTVAIDYWGQGALVTVSS
(SEQ ID NO: 103)
Ca-2407
MDCSWRIFFLLALATGVHSEVQLVQSAAEVKKPGASVKVSCKTSGYTLTDYYIHWVQ
QAPGTGLHWMGWIDPEXGTTDYAQKFQGXVTLTADTSTNTAYMELSGLRAEDTAVYY
CARFPRSLDYGSFPFDYWGQGTLVTVSS
(SEQ ID NO: 104)
Ca-2408
MESVLCWVFLVAILKGVQGEVRLVESGGDLVKPGGSLRLSCVASGFTFRNYGMSWVR
QRPGKGLQWVAAIRSDGVTYYADDLKVRFTVSRDDARNTLYLQLNSLGAEDTAVYYC
AKAPWGLYDAWGQGTLVTVSS
(SEQ ID NO: 105)
Ca-2409
MESVLSWVFLVAILQGVQGEVQVVESGGDLVKPAGSLRLSCVASGYSISTYTMTWVR
QVPGKGLQLVAGINGDGSSTYYTDAVKGRFTISRDNARNTVYLQMNSLRAEDTAMYY
CLGEYSWFYYWGQGTLVTVSS
(SEQ ID NO: 106)
Ca-2410
MQMPWSLLCLLAAPLGVLSELTLQESGPRLVKPSQTLSLTCAVSGGSVTTTSYWSWI
RQRPGRGLEWVGYWTGTTNYSPAFQGRISISADTAKNQFSLHLSSVTTEDTALYFCA
SKSASTSWYFSLFESWGQGTLVTVSS
(SEQ ID NO: 107)
Ca-2411
MESVLGLVFLLTILKGVQGEVQLVESGGDLVKPGGSLRLSCVASGFTFSSYSMSWVR
QAPGKGLQWVGYIDNGGTSTYYADAVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYY
CGRGSYGMEYWGHGTSLFVSS
(SEQ ID NO: 108)
Ca-2412
MESVLGLLFLVAILKGVQGEIQLVESGGDLLKPGGSLRLSCVASGFTFSGSDMNWIR
QAPGKGLQWVAHITHEGIGTSYVGSVKGRFTISRDNAKNTLYLQMNDLRAEDTAMYY
CAYSPWNYYSFDSWGQGTLVTVSS
(SEQ ID NO: 109)
TABLE 15
Canine Lambda Light Chain Variable Domain Sequences Derived from Canine PBMC RNA
Name
Sequence
Ca-1001
MTSTMAWSPLLLTLLTHCTVSWAQTVLTQSPSVSAVLGRRVTISCTGSDTNIGSHRDVQWYQLVPGKSPKTL
IYGTDNRPSGIPVRFSGSKSGNSGTLTITGIQAEDEADYYCQSYDDDLSMNVFGGGTHLTVLG
(SEQ ID NO: 110)
Ca-1002
MDWVPFYILPFIFSTGFCALPVLTQPTNASASLEESVKLTCTLSSEHSNYIVRWYQQQPGKAPRYLMYVRSD
GSYKRGDGIPSRFSGSSSGADRYLTISNIKSEDEDDYYYCGADYTISGQYGSVFGGGTHLTVLG
(SEQ ID NO: 111)
Ca-1003
LWISGGSALGTPTMAWTHLLLPVLTLCTGSVASSVLTQPPSVSVSLGQTATISCSGESLSKYYAQWFQQKAG
QVPVLVIYKDTERPSGTPDRFSGSSSGNTHTLTISRARAEDEADYYCESEVSTGTYCVRRRHPSNRPRSAQG
LPLGHTLPALL
(SEQ ID NO: 204)
Ca-1006
MTSTMAWSPLLLTLLTHCTGSWAQSVLTQPASLSGSLGQRVTISCTGSSSNIGGYSVNWLQQLPGTGPRTII
YNNSNRPSGVPDRFSGSRSGTTATLTISGLQAEDEADYYCSTWDSNLRTIVFGGGTHLTVLG
(SEQ TD NO: 112)
Ca-1007
MTSTMDWSPLLLTLLAHCTGSWAQSVLTQPASVSGSLGQRVTISCTGSTSNLGTYNVGWLQQVPGTGPRTVI
YTNIYRPSGVPDRFSGSESGSTATLTISDLQAEDEAEYYCTAWDSSLNAYVFGSGTQLTVLG
(SEQ ID NO: 113)
Ca-1008
MTSNMAWCPELLTLLAYCTGSWAQSVLTQPTSVSGSLGQRVTISCSGSTNNIGIVGASWYQQLPGKAPKLLV
YSDGDRPSGVPDRFSGSNSGNSDTLTITGLQAEDEADYYCQSFDTTLDAAVFGGGTHLTVLG
(SEQ ID NO: 114)
Ca-1009
MTSTMAWSPLLLTLLAHCTVSWAQAVETQPPSVSAALGQRVTISCTGSDTNIGSGYEVHWYRQVPGKSPAII
IYGNSNRPSGVPVRFSGSKSGSTATLTITGIEAEDEADYHCQSYDGNLDGGVFGGGTHLTVLG
(SEQ ID NO: 115)
Ca-1010
MTSTMGWFPLILTLLAHCAGSWAQSVLTQPASVSGSLGQRVTISCTGSSPNVGYGDFVAWYQQVPGTSPRTL
IYNTRSRPSGVPDRFSASRSGNTATLTISGLQAEDEADYYCSSYDNTLIGIVEGGGTHLTVLG
(SEQ ID NO: 116)
Ca-1011
MTSTMGWSPLLLTLLAHCTGSWAQSVLTQPASVSGSLGQRVTITCTGSSSNIGRANVAWFQQVPGTGPRTVI
YTSATKRPSGVPDRFSGSKSGSTATITTSGLQAEDEADYYCSSWDNSLDAGVFGGGTHLTVLG
(SEQ ID NO: 117)
Ca-1012
MTSTMGWFPLLLTLLAHSTGSWAQSVLTQPASVSGSLGQRVTITCTGGTSNIGRGFVSWFQQVPGIGPKILI
FDAYRRPSGVPDRFSGSRSGNTATLTISGLQAEDEADYYCAVYDSRLDVGVFGSGSQLTVLS
(SEQ ID NO: 118)
Ca-1202
MTSNMAWCPFLLTLLTYCTGSWARSVLTQPASVSGSPGQKVTIYCSGTMSDIGVLGANWYQQLPGKAPKLLV
DNDGDRPSGVPDRFSASKSGHSDTLTITGLQPEDEGDYYCQSFDSSLDAAIFGEGTHLTVLG
(SEQ ID NO: 119)
Ca-1203
SVASYVLTQSPSQNVTLRQAAHITCEGHNIGTKSVHWYQQKQGQAPVLIIYDDKSRPSGIPERFSGANSGNT
ATLTISGALAEDEADYYCLVWDSSAIWVFGEGTHLTVLG
(SEQ ID NO: 120)
Ca-1204
MTSTMAWSPLLLTLLAHFTGSWAQSVLTQPTSVSGSLGQRVTISCTASSSNIDRDYVAWYQQLPGTRPRALI
YANSNRPSGVPDRFSGSKSGSTATLTISGLQAEDEADYYCSTWDNSLTYVFGSGTQLTVLG
(SEQ ID NO: 121)
Ca-1205
SVASYVLTQVPSVSVNLGKTATITCEGDNVGEKYTHWYQQEYGQAPVLHYEDSRRPSGIPEKFSGSNSGNTA
TLTISGARAEDETDYYCQVAVDDSGNVFGGGTHLTVLG
(SEQ ID NO: 122)
Ca-1206
MTSTMGWFPLILTLLAHCAGSWAQSVLTQPASVSGSLGQRVTISCTGSDSNVGYGDSIAYGDSVAWYQQVPG
TSPRTLIYDVTSRPSGVPDRFSGSRSGTTATLTISGLQAEDEADYYCSSFDKTLNGLIVGGGTHLTVLG
(SEQ ID NO: 123)
Ca-1207
MTSNMAWSPLLLTLLAYCTGSWAQSALTQPTSVSGSLGQRVSISCSGGIHNIGSVGATWYQQLPGKAPKLLV
SSDGDRPSGIPDRFSGSRSGNSVTLTITGLQAEDEAEYYCQSFDSTLGVHVVFGGGTHLTVLG
(SEQ ID NO: 124)
Ca-1208
LCSAVGPPKTESVMTSTMGWSPLLLTLLAHCTGSWAQSVLTQPASVSGSLGQRVTIPCTGSSSNIDRYNVAW
FQQLPGTGPKPSSIVLLTDPQGSLIDSLAPSQAA
(SEQ ID NO: 205)
Ca-1209
MTSTMAWFPLLLTLLAHYTGSWARSDLTQPASVSGSLGQRITISCTGSSSNIGRNYVGWYQQLPGRGPRTVV
YGINSRPSGVPDRFSGSKSGSTVTLTISGLQAEDEADYYCSTWDDSLSVVVFGGGTHLTVLG
(SEQ ID NO: 125)
Ca-1210
MTSTMGWSPLLLTLTHWTGSWAQSVLSQPASMSGSLGLRITICCTGKNSNINNSYVDWNQPLAGTGPRTVIH
DDGDRPSGVPDQFSGSKSGNTATLTISRLQAEDEADYNGASFETSFNAVFGGGTHVTVLG
(SEQ ID NO: 126)
TABLE 16
Canine Kappa Light Chain Variable Domain Sequences Derived from Canine PBMC RNA
Ca Ka016-A1
LSWLRQKPGHSPQRLIHQVSSRDPGVPDRFSGSGSGTDFTLISRVEADDGGVYYCGQGSQSIPTFG
QGTKVEIKR
(SEQ. ID NO. 127)
Ca Ka016-A2
MRFPSQLLGLLMLWIPGSAGDIVMTQTPLSLSVSPGEPASISCKASQSLLHSNGNTYLYWFRQKPGQ
SPQRLIYKVSNRDPGVPDRFSGSGSGTDFTLRISRVETDDAGVYYCGQVIQDPWTFGVGTKLELKR
(SEQ. ID NO. 128)
Ca Ka016-A3
MRFPSQLLSLLMLWIPGSSGDIVMTQTPLSLSVSPGETASISVRASQTLLYSNGKNYLFWYRQKPGQ
SPQRLIDLASNRDPGVPDRFSGSGSGTDFTLRISRVEADDAGVYYCGQGMEIPWTFGAGTKVELKR
(SEQ. ID NO. 129)
Ca Ka016-A4
MKFPSLLLGLLMLWIPGSTGEAVMTQTPLSLAVTPGEVATISCRASQSLLHSDGKSYLNWYLQKPGQ
TPRPLIYEASKRFSGVSDRFSGSGSGTDFTLKINRVEAEDVGVYYCQQSLHFPPTFGPGTKVELKR
(SEQ. ID NO. 130)
Ca Ka016-A5
PDRFSGSGSGTDFTLTISRVEADDAGIYYCGQATQTPPTFGAGTKLDLKR
(SEQ. ID NO. 131)
Ca Ka016-A6
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSVRPGESASISCKASQSLLHSGGGTYLNWFRQRPGQ
SPQRLIYEVSKRDTGVPDRFSGSGSGTDFTLRITRVEADDTGIYYCGQNTQLPLTFGQGTKVEIKR
(SEQ. ID NO. 132)
Ca Ka016-A7
MRFPSQLLGLLMLWIPGSTGDIVMTQTPLSLSVSPGEPASISCKASQSSHSNGNTYLFWLRQKPGQS
PQRLIYRVSNRDPGVPDRFSGSGSGTDFTLRISRVEADDAGVYYCGQRVRSPWTFGAGTKVEVKR
(SEQ. ID NO. 133)
Ca Ka016-A8
MRFPSQLLGLLMLWIPGSAGDIVMTQTPLSLSVSPGEPASISCKASQSLLHSNGNTYLYWFRQKPGQ
SPQRLIYKVSNRDPGVPDRFSGSGSGTDFTLRISRVETDDAGVYYCGQVIQDPWTFGVGTKLELKR
(SEQ. ID NO. 134)
Ca Ka016-A9
MRFPSQLLGLLMLWIPGSSGDVVMAQTPLSLSVSPGETASISCRASQSLLHSNGNTFLFWFRQKPGQ
SPQRLINFLSNRDPGVPDRFSGSGSGTDFTLRINRVEADDAGLYYCGQGLQAPLTFGQGTKLEIKR
(SEQ. ID NO. 135)
Ca Ka016-A10
MRFPSQLLGLLMLWIPGSNGDDVLTQTPLSLSVRPGETVSILCKASESLLHSDGNTYLSWVRQKAGQ
SPQRLMYRVSDRDTGVPDRFSGSGSGTDFTLTISGVEADDAGIYYCGQATHYPLEFGQGTRVEIKR
(SEQ. ID NO. 136)
Ca Ka016-A11
LMLWIPGSTGEIVLTQTPLSLSVSPGEPASISCKASQLLHPNGVTYLYWFRQKPGQSPQRLIYKVSN
RDPGVPDRFSGSGSEIDFTLIISRVEADDGGIYYCGQGIQNPFTFGQGTKLEIKR
(SEQ. ID NO. 137)
Ca Ka016-A12
MRFPSQLLGLLMLWIPGSIGDIVMTQTPLSLSVSPGESASISCKASQSLLHSNGNTYLYWFRQKPGH
SPQRLIHQVSSRDPGVPDRFSGSGSGTDFTLRISRVEADDAGLYYCGQGTQFPFTFGQGTKVEIKR
(SEQ. ID NO. 138)
Ca Ka016-B1
MRFPSQLLGLLMLWIPGSIGDIVMTQTPLSLSVSPGESASISCKASQSLLHSNGNTYLYWFRQKPGH
SPQRLIHQVSSRDPGVPDRFSGSGSGTDFTLRISRVEADDAGLYYCGQGTQFPFTFGQGTKVEIKR
(SEQ. ID NO. 139)
Ca Ka016-B2
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSVSPGETASISCRASQSLLHSNGNTYSFWFRQKPGQ
SPQRLINLVSSRGPGVPDRFSGSGSGTDFTLIISRVEADDAGVYYCGHGKEAPYTFSQGTKLEIKR
(SEQ. ID NO. 140)
Ca Ka016-B3
MRFPSQLLGLLMLWIPGSVGDIVMTQSPMSLSVGPGEASASMSCKANQSLLYSDGITYLSWFLQRPG
QSPQRLIYEVSKRDTGVPGRFIGSGAGTDFTLRISRVEADDAGVYYCGQALFPLTFSQGAKLEIER
(SEQ. ID NO. 141)
Ca Ka016-B4
MRFPSQLLGLLMLWIPGSSGDVVMTQTPLSLSVRPGETASISCRASQSLLHSSGITKLFWYRQKPGQ
SPQRLVYWVSNRDPGVPDRFTGSGSGTDFTLRISRLEADDAGIYYCGHAIGFPLTFGQGTKVEIKR
(SEQ. ID NO. 142)
Ca Ka016-B5
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSVRPGESASISCKASQSLLHSGGGTYLNWFRQRPGQ
SPQRLIYEVSKRDTGVPDRFSGSGSGTDFTLRITRVEADDTGIYYCGQNTQFPLTFGQGTKVEIKR
(SEQ. ID NO. 143)
Ca Ka016-B6
MRFPSQLLGLLMLWIPGSSGGIVMTQTPLSLSVRPGETASISCRASQSLLYSDGNTYLFWFRQKPGQ
SPQRLMYRVSDRDTGVPDRFSGSGSGTDFTLTISGVEADDAGIYYCGQATHYPLEFGQGTXVEIKR
(SEQ. ID NO. 144)
Ca Ka016-B7
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSVRPGESASISCKASQSLLHSGGGTYLNWFRQRPGQ
SPQRLIYEVSKRDTGVPDRFIGSGAGTDFTLRISRVEADDAGVYYCGQGVQGPWTIGAGTKLELQR
(SEQ. ID NO. 145)
Ca Ka016-B8
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSVSVSPGETASISCKASQSLLSHDGNTYLHWFRQKPGQ
SPQRLIYKVSNRDTGVPDRFSGSGSGTDFTLKISRVEADDTGVYYCGQITQDPFTFGQGTKLEIKR
(SEQ. ID NO. 146)
Ca Ka016-B9
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSVSPGETASISCRASQSLLHSNGNTYLFWFRQKPGQ
SPQRLINWVSNRDPGVPDRFGGSGSGTDFTLRISRVEADDAGIYYCGQGIQGPYTFSQGTKLEIKR
(SEQ. ID NO. 147)
Ca Ka016-B10
MRFPSQFLGLLMLWIPGSSGDIAMTQTPLSLSVGPGETASITCKASQSLLHSNGNTYLFWFRQKPGQ
SPQRLIYLVSNRDPGVPDRFSGSGSGTDFTLTISRVEADDAGIYYCGQATQTPPTFGAGTKLKLKR
(SEQ. ID NO. 148)
Ca Ka016-B11
MRFPSQLLGLLMLWIPGSSGDIVMAQTPLSLSVSPGEPASISCKASQSLLHSDGRTCLSWFRQKSGQ
SPQRLIYEVSNRDTGVPDRFSGSGSGTDFTLRISRVEADDTGIYYCGQTVQFPLTFGQGTKLEIKR
(SEQ. ID NO. 149)
Ca Ka016-B12
GQSPQRLIYKVSNRDPGVPDRFSGSGSGTDFTLRISRVEPEDVGVYYCGQGTLNPWTFGAGTKVELK
R
(SEQ. ID NO. 150)
Ca Ka017-1
MRFPSQLLGLLMLWIPGSSGDVVMTQTPLSLSVSPGETASISCRASQSLLHSNGNTFLFWFRQ*PGQ
SPQRLINFVSNRDPGVPDRFSGSGSGTDFTLRISRVEADDAGIYYCGQGLLAPPTFGQGTKVEIRR
(SEQ. ID NO. 151)
NOTE: *INDICATES A STOP CODON
Ca Ka017-2
MRFPSQLLGLLMLWIPGSGGDIVMTQTPPSLSVSPREPASISCKASQSLLRSNGNTYLYWFRQKPGQ
SPEGLIYRVSNRFTGVSDRFSGSGSGTDFTLRISTVEADDAGVYYCGQATQFPSTFSQGTKLEIKR
(SEQ. ID NO. 152)
Ca Ka017-3
MRFPSQLLGLLMLWIPGSXGDIVLTQTPLSLSVSPGEPASISCKASQSLLHSNGITYLNWYRQRPGQ
SPQXLIYKVSNRDTGVPDRFSGSGSGTDFTLRXSKVEADDTGIYYCGQDTQFPLTLGXGTHXEIKR
(SEQ. ID NO. 153)
Ca Ka017-5
MRFPSQLLGLLMLWIPGSTGDIVMTQTPLSLSVSPGEPASIYCKASQSSLHSNGKTFLYWFRQKPGQ
SPQRLIYRVSNRDPGVPDRFSGSGSGTDFTLRISRVEADDAGIYYCGQGIQDPTFGQGTKVEIKR
(SEQ. ID NO. 154)
Ca Ka017-6
MRFPSQLLGLLMLWIPGSGGDIVMTQTPPSLSVSPREAASISCKASQSLLKSNGNTYFYWFRQKPGQ
VSEGLIYKVSSRFTGVSDRFSGSGSGTDFTLRISRVEADDAGVYFCGQALQFPYTFSQGTKLDIKR
(SEQ. ID NO. 155)
Ca Ka017-10
MRFPSQLLGLLMLWIPESGGDVVLTQTPPSLSLSPGETASISCKASRSLLNSDGSTYLDWYLQKPGQ
SPRLLIYLVSNRFSGVSDRFSGSGSGTDFTLTISRVEADDAGVYYCGQGSRVPLTFGQGTKVEIKR
(SEQ. ID NO. 156)
Ca Ka017-11
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSVSPGETASISCRASQSLLHRNGITYLSWFRQRPGQ
SPQRLINLVSNRDPGVPDRFSGSGSGTDFTLRISRVEADDVGVYYCGHGLQTPYTFGQGTSLEIER
(SEQ. ID NO. 157)
Ca Ka017-12
MRFPSQLLGLLVLWIPGSSGDIVMTQTPLSLSVSPGETVSISCRASQSLLYSDGNIYLFWFRRKPGQ
SPQHLINLVSNRDPGVPDRFSGSGSGTDFTLRISRVEADDAGVYYCGQGTQPPYTFSQGTKVEIKR
(SEQ. ID NO. 158)
Ca Ka017-13
MRFPSQLLGLLMLWIPESGGDVVLTQTPPSLSLSPGETASISCKASRSLLNSDGSTYLDWYLQKPGQ
SPRLLIYLVSNRFSGVSDRFSGSGSGTDFTLTISRVEADDAGVYYCGQGSRVPLTFGQGTKVEIKR
(SEQ. ID NO. 159)
Ca Ka017-14
MRFPSQLLGLLMLWIPGSSGDIVMAQTPSLSLVSPGETASISCRASQSLLHSNGITYLFWYRQKPGQ
SPQRLISMVFNRDPGVPDRFGGSGSGTDFTLRISRVEADDAGLYFCGQGTQIPYSFSQGTKLEIKR
(SEQ. ID NO. 160)
Ca Ka017-16
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSISPGETASISCKASQSLLHSGGDTYLNWFRQRPGQ
SPQLLINRVSSRKKGVPDRFSGSGSGTEFTLRISRVEADDAGIYFCGQGTQFPYTFSQGTKLEIKR
(SEQ. ID NO. 160)
Ca Ka017-20
MRFPSQLLGLLMLWIPGSGGDIVMTQTPPSLSVSPGEPASMSCKASQSLLHSNGNTYLYWFRQKPGQ
SPEALIYKVSNRFTGVSDRFSGSGSGTDFTLRINRVEADDVGVYYCGQGIQIPYTFSQGTKLEIKR
(SEQ. ID NO. 162)
Ca Ka017-23
MRFPSQLLGLLMLWIPGSTGEIVLTQTPLSLSVSPGESASISCKASQSLLYSNGNTYLYWFRQKAGQ
SPQRVIYRVSNRDPGVPDRFSGSGSGTDFTLRISSVENDDAGVYYCGQGSEDPPTFGAGTKVELKR
(SEQ. ID NO. 163)
Ca Ka017-24
MRFPSQLLGLLTLWIPGSTGDIVMTQTPLSLSVSPGEPASISCKASQSLLHSNGNTYLYWFRQKPGQ
SPQRLIYKVSNRDPGVPXRFSGSGSGTDFTLRVSXVEADDAGVYYCGQGVQDPFTFGQGTKLEIKR
(SEQ. ID NO. 164)
Example 13: CDR-Grafting to Create Caninized Monoclonal Antibodies
To generate caninized antibody sequences from mouse anti-NGF antibodies, each murine variable heavy chain antibody gene sequence was separately aligned against 36 canine Ig germline variable heavy chain sequences using Vector NTI software. Eleven canine Ig germline variable heavy chain sequences were derived from U.S. Pat. No. 7,261,890 B2, (Methods for Using Canine Immunoglobulin Variable Domains and Caninized Antibodies), the contents of which are herein incorporated by reference, and 25 canine Ig germline variable heavy chain sequences were derived from Table 14 (Canine Heavy Chain Variable Domain Sequences Derived from Canine PBMC RNA). Each murine variable light chain gene sequence was separately aligned against 68 germline variable light chain sequences (derived from U.S. Pat. No. 7,261,890 B2) using Vector NTI software. Canine variable domain sequences having the highest overall homology to the original murine sequences were selected for each heavy chain and light chain sequence to provide the framework sequence. In silico construction of complete CDR grafted antibodies was accomplished by substitution of canine variable domain CDR sequences with murine CDR sequences (derived from the subcloned anti-NGF antibody hybridoma mAbs). To identify residues in each sequence, the first amino acid in each listed sequence was defined as 1, and all remaining residues numbered consecutively thereafter using Kabat numbering system.
The heavy chain CDR sequences from PR-1254972 were grafted in silico onto canine 894 as follows: (1) One N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Six back-mutations (Q3H, V37I, Q46E, D73N, T77N, R83K) were introduced to make the 72.2 VH sequence. (3) One, two, three, four, five, or six of the back-mutations disclosed above could be introduced into 72.2 VH to maintain antibody affinity to NGF after caninization of mAb 72.2. (4) One, two, three, four, five, or six of these back-mutations may be substituted during subsequent affinity maturation of 72.2 VH. 72.3 VH was generated by introducing the back-mutations in 72.2 VH with the addition of H39Q back-mutation. 72.4 VH was generated by introducing back-mutations Q3H, H39Q, Q46E, D73N. The light chain CDR sequences from PR-1254972 were grafted in silico onto canine 1001 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Four back-mutations (12V, V3L, Q45K, S59P) were introduced to make the 72.2 VL sequence. (3) One, two, three, or four of these back-mutations could be introduced into 72.2 VL to maintain antibody affinity to NGF after caninization of mAb 72.2. (4) One, two, three, or four of these back-mutations may be substituted during subsequent affinity maturation of 72.2 VL. 72.4 VL was generated by introducing back-mutations Q45K, and S59P.
The heavy chain CDR sequences from PR-1254973 were grafted in silico onto canine 894 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Eight back-mutations (T24A, M48I, V67A, L69V, T73K, N76S, V78A, A93T) were introduced to make the 73.2 VH sequence. (3) One, two, three, four, five, six, seven, or eight of these back-mutations could be introduced into 73.2 VH to maintain antibody affinity to NGF after caninization of mAb 73.2. (4) One, two, three, four, five, six, seven, or eight of these eight back-mutations may be substituted during subsequent affinity maturation of 73.2 VH. 73.4 VH was generated by introducing back-mutations T24A, T73K, A93T. The light chain CDR sequences from PR-1254973 were grafted in silico onto canine 1034 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Eight back-mutations (I ID, V3Q, S22T, F36H, R46L, I48V, D60S, D70Q) were introduced to make the 73.2 VL sequence. (3) One, two, three, four, five, six, seven, or eight of these back-mutations could be introduced into 73.2 VL to maintain antibody affinity to NGF after caninization of mAb 73.2. (4) One, two, three, four, five, six, seven, or eight of these eight back-mutations may be substituted during subsequent affinity maturation of 73.2 VL. 73.4 VL was generated by introducing back-mutations I1 D, V3Q, F36H, R46L, D60S, D70Q.
The heavy chain CDR sequences from PR-1254977 were grafted in silico onto canine 894 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Eight back-mutations (T24A, Q38K, M48I, R66K, V67A, T68S, L69I, V78A) were introduced to make the 77.2 VH sequence. (3) One, two, three, four, five, six, seven, or eight of these back-mutations could be introduced into 77.2 VH to maintain antibody affinity to NGF after caninization of mAb 77.2. (4) One, two, three, four, five, six, seven, or eight of these back-mutations may be substituted during subsequent affinity maturation of 77.2 VH. 77.3 VH was generated by introducing the back-mutations in 77.2 VH with the addition of R94G back-mutation. 77.4 VH was generated by introducing back-mutations T24A, Q38K, and R94G. The light chain CDR sequences from PR-1254977 were grafted in silico onto canine 997 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Four back-mutations (L2V, F36Y, R46L, S98G) were introduced to make the 77.2 VL sequence. (3) One, two, three, or four of these back-mutations could be introduced into 77.2 VL to maintain antibody affinity to NGF after caninization of mAb 77.2. (4) One, two, three, or four of these back-mutations may be substituted during subsequent affinity maturation of 77.2 VL. 77.4 VL was generated by introducing back-mutations F36Y and R46L.
The heavy chain CDR sequences from PR-1254981 were grafted in silico onto canine 876 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Six back-mutations (Q46E, G49A, T77N, R83K, L91Y, E93T) were introduced to make the 81.2 VH sequence. (3) One, two, three, four, five, or six of these back-mutations could be introduced into 81.2 VH to maintain antibody affinity to NGF after caninization of mAb 81.2. (4) One, two, three, four, five, or six of these six back-mutations may be substituted during subsequent affinity maturation of 81.2 VH. 81.4 VH was generated by introducing back-mutations Q46E, G49A, L91 Y, and E93T.
The light chain CDR sequences from PR-1254981 were grafted in silico onto canine 1011 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs (2) Four back-mutations (V3L, A7T, F36Y, R46L) were introduced to make the 81.2 VL sequence (3) One, two, three, or four of these back-mutations could be introduced into 81.2 VL to maintain antibody affinity to NGF after caninization of mAb 81.2. (4) One, two, three, or four of these back-mutations may be substituted during subsequent affinity maturation of 81.2 VL. 81.4 VL was generated by introducing back-mutations A7T, F36Y, and R46L.
Alternatively, the heavy chain CDR sequences from PR-1254981 were grafted in silico onto canine 1005 VH as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Seven back-mutations (Q46E, T77N, F79Y, R83K, F91Y, V93T, K94R) were introduced to make the 81.5B VH sequence. (3) One, two, three, four, five, six, or seven of these back-mutations could be introduced into 81.5B VH to maintain antibody affinity to NGF after caninization of mAb 81.5B. (4) One, two, three, four, five, six, or seven of these seven back-mutations may be substituted during subsequent affinity maturation of 81.5B VH. 81.6B was generated by introducing back-mutations Q46E, F79Y, F91Y, and V93T. Variants 81.2B and 81.4B were generated by introducing A84K mutation to 81.5B and 81.6B, respectively.
The heavy chain CDR sequences from PR-1254982 were grafted in silico onto canine 892 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Twelve back-mutations (I3Q, I37V, M48L, I67L, T70S, A71K, G73N, N76S, H77Q, L78V, S79F, T93A) were introduced to make the 82.2 VH sequence. (3) One, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve of these back-mutations could be introduced into 82.2 VH to maintain antibody affinity to NGF after caninization of mAb 82.2. (4) One, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve of these back-mutations may be substituted during subsequent affinity maturation of 82.2 VH. 82.4 VH was generated by introducing back-mutations I3Q, A71K, H77Q, S79F, and T93A. The light chain CDR sequences from PR-1254982 were grafted in silico onto canine 1034 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Ten back-mutations (I ID, V3Q, S22T, F36Y, Q45K, R46L, D60S, F71Y, T72S, Y87F) were introduced to make the 82.2 VL sequence. (3) One, two, three, four, five, six, seven, eight, nine, or ten of these back-mutations could be introduced into 82.2 VL to maintain antibody affinity to NGF after caninization of mAb 82.2. (4) One, two, three, four, five, six, seven, eight, nine, or ten of these back-mutations may be substituted during subsequent affinity maturation of 82.2 VL. 82.3 VH was generated by introducing the back-mutations in 82.2 VH with the addition of P44V back-mutation. 82.4 VL was generated by introducing back-mutations I1D, V3Q, F36Y, Q45K, R46L, D60S, F71Y, and Y87F.
Example 14: Isoelectric Point of Canine Framework Amino Acids
The heavy chain framework amino acids (i.e. non-CDR amino acids) of the caninized IgG1 kappa antibodies yield a calculated isoelectric point of less than 8.0. The light chain framework amino acids, when the light chain is kappa, yield a calculated isoelectric point of less than 6.5. The isoelectric point of the caninized antibodies as a whole, i.e. heavy and light chain combined, due to the framework amino acids, and when the light chain is kappa, is less than 8.0. In comparison, the framework amino acids of human IgG1 heavy chains typically yield isoelectric points of greater than 8.0. The framework amino acids of human kappa light chains typically yield isoelectric points of greater than 6.5. The framework amino acids of whole human IgG1/k antibodies typically yield isoelectric points of greater than 8.0.
Example 15: CDR-Grafting to Create Humanized Monoclonal Antibodies
Each murine variable heavy and variable light chain antibody gene sequence (as set forth in Table 16) was separately aligned against 44 human immunoglobulin germline variable heavy chain or 46 germline variable light chain sequences (derived from NCBI Ig Blast website which is well known to those skilled in the art) using Vector NTI software. Human variable domain sequences having the highest overall homology to the original murine sequences were selected for each heavy chain and light chain antibody sequence to provide the framework (FW) 1, 2 and 3 sequences for CDR-grafting purposes. Identification of a suitable human variable heavy and light chain FW4 region (also known as the “joining” region) was accomplished by separately aligning each murine heavy chain and light chain FW4 region with 6 human immunoglobulin germline joining heavy chain and 5 germline joining light chain sequences in the NCBI database. In silico construction of complete CDR grafted variable domains was accomplished by substitution of human variable domain CDR sequences (derived from the NCBI website) with murine CDR sequences (derived from the murine antibodies) with addition of a FW4 region (derived from the NCBI website) to each 3′ end. Further humanization may be accomplished by identification of back-mutations. Full length human Igs may be produced by expressing the variable domains of each CDR-grafted mAb with an in-frame human IgG constant domain. Mouse Anti-NGF mAb CDRs grafted onto human Ig frameworks (CDR-grafted Anti-NGF Abs) produced are those listed in Table 17.
TABLE 17
Mouse Anti-NGF mAb CDRs Humanized by CDR Grafting onto Human Ig Frameworks
Name
Sequence(CDRs are underlined)
HU72 VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMFWVRQATGKGLEWVSTISDGGSYTYYTDNVKG
(CDR GRAFT
RFTTSRENAKNSLYLQMNSLRAGDTAVYYCARDWSDSEGFAYWGQGTLVTVSS
VH3-13/JH5)
(SEQ ID NO: 165)
Hu73 VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGRIDPYGGGTKIINEKFK
(CDR-GRAFT
RRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSGYDYYFDVWGQGTTVTVSS
VH1- 18/1116)
(SEQ ID NO: 166)
HU77 VH
QVQLVQSGAEVKKPGSSVKVSCKASGFNIKDTYIYWVRQAPGQGLEWMGRIDPANGNTIYASKFQG
(CDR-GRAFT
RVTITADKSTSTAYMELSSLRSEDTAVYYCARYGYYAYWGQGTTVTVSS
VIII -69/JH6)
(SEQ ID NO: 167)
HU80 VH
QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIYWVRQAPGQGLEWMGRIDPANGNTIYASKFQG
(CDR GRAFT
RVTMTTDTSTSTAYMELRSLRSDDTAVYYCARYGYYAYWGQGTTVTVSS
V111-18/1116)
(SEQ ID NO: 168)
HU81 VH
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNHYMYWVRQAPGKGLEWVGSISDGGAYTFYPDTVK
(CDR GRAFT
GRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTEESANNGFAFWGQGTLVTVSS
VH3-15/JH1)
(SEQ ID NO: 169)
HU82 VH
QVTLKESGPVLVKPTETLTLTCTVSGFSLTGYNINWIRQPPGKALEWLAMIWGYGDTDYNSALKSR
(CDR-GRAFT
LTISKDTSKSQVVLTMTNMDPVDTATYYCARDHYGGNDWYFDVWGQGTTVTVSS
VH2-26/JH6)
(SEQ ID NO: 170)
HU72 VL
DIVMTQTPLSLPVTPGEPASISCRSSQSIVQSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDR
(CDR GRAFT
FSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPFTFGQGTKLEIKR
01/JK2)
(SEQ ID NO: 171)
HU73 VL
DIQMIQSPSFLSASVGDRVSIICRASENIYSFLAWYLQKPGKSPKLFLYNANTLAEGVSSRFSGRG
(CDR-GRAFT
SGTDFTLTIISLKPEDFAAYYCQHHFGTPFTFGQGTKLEIKR
L22/JK2)
(SEQ ID NO: 172)
HU77 VL
DIVMTQTPLSLPVTPGEPASISCKSTKSLLNGDGFTYLDWYLQKPGQSPQLLIYLVSNRFSGVPDR
(CDR-GRAFT
FSGSGSGTDFTLKISRVEAEDVGVYYCFESNYLFTFGQGTKLEIKR
01/JK2)
(SEQ ID NO: 173)
HU80 VL
DIVMTQTPLSLPVTPGEPASISCKSTKSLLNGDGFTYLDWYLQKPGQSPQLLIYLVSNRFSGVPDR
(CDR-GRAFT
FSGSGSGTDFTLKISRVEAEDVGVYYCFESNYLFTFGQGTKLEIKR
0 I/JK2)
(SEQ ID NO: 174)
HU81 VL
DIVMTQTPLSLPVTPGEPASISCRSSQSILHSNGNTYLEWYLQKPGQSPQLLIYRVSNRFSGVPDR
(CDR GRAFT
FSGSGSGTDFTLKISRVEAEDVGVYYCFQGAHVPFTFGQGTKLEIKR
01/JK2)
(SEQ ID NO: 175)
HU82 VL
DIQMTQSPSSLSASVGDRVTITCRASQDITNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGS
(CDR GRAFT
GSGTDFTFTTSSLQPEDIATYYCQQGKTLPRTFGQGTKLEIKR
08/JK2)
(SEQ ID NO: 176)
Example 16: Method for Constructing Full-Length Mouse/Canine Chimeric and Caninized Antibodies
Using conventional molecular biology techniques, a cDNA fragment encoding the canine IgG1 constant region (which was obtained from the IMGT®, the International ImMunoGeneTics information system, which is the global reference in immunogenetics and immunoinformatics, created in by Marie-Paule Lefranc (Universite Montepellier 2 and CNRS)) was synthesized and ligated to the 3′ end of each of the heavy chain variable domains derived from murine anti-NGF monoclonal antibodies PR-1254972, PR-1254973, PR-1254977, PR-1254981, PR-1254982. For these same anti-NGF mAbs, a cDNA fragment encoding the canine kappa constant region obtained from U.S. Pat. No. 5,852,183 A, (Sequence ID No. 54) was synthesized and ligated to the 3′ end of each of the light chain variable domains. The complete canine IgG heavy chain constant domain nucleotide sequence and amino acid sequence is shown as SEQ ID NO: 51 and SEQ ID NO: 52, respectively. The complete canine kappa light chain constant domain nucleotide sequence and amino acid sequence is shown as SEQ ID NO: 53 and SEQ ID NO: 54, respectively. Complete heavy chain and light chain chimeric cDNAs were ligated into the pHybE expression plasmid; the sequences of these chimeric mAbs are in Table 18 below.
TABLE 18
Mouse/Canine Chimeric Antibody Sequences
Name
Sequence (CDRs are underlined)
PR-1290646 light
DVLMTQTPLSLPSVGDQASISCRSSQSIVQSNGNTYLEWYLQKPGQSPKLLIY
chain amino acid
KVSNRFSGVPDRFSGSGSGTDFTLKISREAEDLGVYYCFQGSHVPFTFGSGTK
sequence
LEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQD
TGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQ
RSECQRVD
(SEQ ID NO: 194)
PR-1290646 heavy
EVHLVESGGGLVKPGGFLILSCAASGFTFSDYYMFWIRQTPGKRLEWVATISD
chain amino acid
GGSYTYYTDNVKGRFTISRDNVKNNLYLQMSHLKSADTAMYYCARDWSDSEGF
sequence
AYWGQGTLVTVSAASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVS
WNSGSLTSGVHTFPSVLQSSGLHSLSSMVTVPSSRWPSETFTCNVVHPASNTK
VDKPVFNECRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLD
LGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKE
FKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIK
DFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGD
PFTCAVMHETLQNHYTDLSLSHSPGK
(SEQ ID NO: 195)
PR-1290654 light
DIQMTQSPASLSAVGETVTVTCRASENIYSFLAWHQQKQGKSPQLLVYNANTL
chain amino acid
AEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHFGTPFTFGSGTKLEIK
sequence
RNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQ
ESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQR SE
CQRVD
(SEQ ID NO: 196)
PR-1290654 heavy
QVQLQQPGAELVKPGASVKLSCKASGYTFTNYWMHWVKQRPGQGLEWIGRIDP
chain amino acid
YGGGTKHNEKFKRKATVTADKSSSTAYILLSSLTSEDSAVYYCTRSGYDYYFD
sequence
VWGTGTTVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSW
NSGSLTSGVHTFPSVLQSSGLHSLSSMVTVPSSRWPSETFTCNVVHPASNTKV
DKPVFNECRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDL
GREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEF
KCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKD
FYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDP
FTCAVMHETLQNHYTDLSLSHSPGV
(SEQ ID NO: 197)
PR-1290656 light
DVVLTQTPLSLPVNIGDQASISCKSTKSLLNGDGFTYLDWYLQKPGQSPQLLI
chain amino acid
YLVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFESNYLFTFGSGT
sequence
KLEMKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQ
DTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSF
QRSECQRVD
(SEQ ID NO: 198)
PR-1290656 heavy
EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYIYWVKQRPEQGLEWIGRIDP
chain amino acid
ANGNTIYASKFQGKASITADTSSNTAYMQLSSLTSGDTAVYYCAGYGYYAYWG
sequence
QGTTLTVSSASTTAPSVFPLAPSCGSTSGSTAVALACLVSGYFPEPVTVSWNS
GSLTSGVHTFPSVLQSSGLHSLSSMVTVPSSRWPSETFTCNVVHPASNTKVDK
PVFNECRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGR
EDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKC
RVNHIDLPSPIERTISKARGRAGKPSVYVLPPSPKELSSSDTVSITCLIKDFY
PPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFT
CAVMHETLQNHYTDLSLSHSPGK
(SEQ ID NO: 199)
PR-1290657 light
DVLMTQTPLSLPVSLGDQASISCRSSQSILHSNGNTYLEWYLQKPGQSPNLLI
chain amino acid
YRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGAHVPFTFGSG
sequence
TKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVI
QDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLI K
SFQRSECQRVD
(SEQ ID NO: 200)
PR-1290657 heavy
EVQLVESGGGAVKPGGSLTLSCAASGFTFSNHYMYWVRQTPEKRLEWVASISD
chain amino acid
GGAYTFYPDTVKGRFTISRDNVNNNLYLQMRHLKSEDTAMYYCTREESANNGF
sequence
AFWGQGTLVTVSAASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVS
WNSGSLTSGVHTFPSVLQSSGLHSLSSMVTVPSSRWPSETRTCNVVHPASNTK
VDKPVFNECRCTDTPPCPVPEPLGGPSVLIFPPKPKSILRITRTPEVTCVVLD
LGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKE
FKCRVNHYIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLI
KDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQG
DPFTCAVMHETLQNHYTDLSLSHSPGV
(SEQ ID NO: 201)
The canine IgG1 constant region nucleotide sequence described above was also ligated to the 3′ end of each of the cDNAs encoding heavy chain variable domains derived from caninized anti-NGF monoclonal antibodies 72.2 VH, 72.3 VH, 72.4 VH, 73.2 VH, 73.4 VH, 77.2 VH, 77.3 VH, 77.4 VH, 81.2 VH, 81.4 VH, 81.2B, 81.4B, 81.5B, 81.6B, 82.2 VH, 82.4 VH. The canine kappa light chain constant domain nucleotide sequence described above was also ligated to the 3′ end of each of the cDNAs encoding light chain variable domains derived from caninized anti-NGF monoclonal antibodies 72.2 VL, 72.4, 73.2 VL, 73.4 VL, 77.2 VL, 77.4 VL, 81.2 VL, 81.4 VL, 82.2 VL.
Full-length chimeric or caninized antibodies were transiently expressed in 293-6E cells by co-transfection of combinations of heavy and light chain pHybE plasmids. Table 20 highlights all possible combination of caninized heavy and light chains that may be combined to produce a caninized antibody per the name in the table (Table 20). In Table 20, the heavy chain plasmids encoding caninized versions of murine heavy chains are listed on the top line and proceed rightward. The light chain plasmids encoding caninized versions of murine light chains are listed on the left-hand column and proceed downward. At each point where these boxes intersect, a name has been indicated to describe a potential resulting caninized antibody.
Example 17: Caninized Monoclonal Antibody Expression and Purification
Selected heavy chain and light chain mouse/canine chimeric and caninized antibody plasmids were co-transfected into 293-6e cells in suspension and allowed to grow for 7-8 days. Cell supernatants were harvested, centrifuged, and filtered. For each expressed antibody, supernatant was mixed with an equal volume of Pierce binding buffer to perform Protein A Sepharose affinity chromatography according to manufacturer's instructions (GE Healthcare #17-1279-04). Although according to several sources canine IgGs bind directly to Protein A moderately well (GE Healthcare Antibody Purification Handbook package insert; Scott, M. A., et. al., Vet Immunol-Immunopatho, 59:205, 1997; Warr, G. W and Hart, I. R., Am J Vet Res, 40:922, 1979; Thermo Scientific Pierce Antibody Production and Purification Technical Handbook) the monoclonal canine mAbs did not quantitatively bind to Protein A and therefore could not be purified from supernatants without modification to the Protein A purification methodology.
To allow quantitative binding of canine IgGs to Protein A, supernatants were concentrated and mixed with an equal volume of Pierce binding buffer (Thermo #21007). To the concentrated and diluted supernatants, NaCl was added to a final concentration of 2.5 M. NaCl-adjusted supernatant was loaded onto Protein A Sepharose by continuous over-night loading, washed with Pierce binding buffer, and eluted using Pierce elution buffer (Thermo #21004). The eluates were neutralized by dropwise addition of 1M Tris pH 8.0; following this the neutralized antibodies were dialyzed into PBS and amounts of antibody were quantified spectrophotometrically by OD28o- The amount purified was mathematically divided by the total volume of cell supernatant purified to determine the overall estimated expression levels in μg/mL. The isolation and purification of theses canine IgG1/k mAbs allowed analytical characterization studies of the mAbs to be completed.
For purification of large-scale cell supernatants (10-15 L), cell supernatants were concentrated, then mixed with Pierce binding buffer A (Thermo, catalog #21001) in a 1 to 1 ratio. To this mixture, 5 M NaCl was added to 1.3 M final concentration. The pH of the mixture was adjusted to 8.5 with 10 N NaOH. The pH-adjusted cell supes were loaded onto a Protein A MabSelect SuRe (GE Healthcare, catalog #17-5438-03) chromatography column and eluted using two steps. The first step of the elution was performed using 20 mM Tris, 25 mM NaCl, pH 8.0, 7.4 ms/cm. Fractions containing antibodies were identified by OD28o and size exclusion chromatography. To quantitatively isolate the remaining antibody bound to the Protein A column, the second step elution was performed using Pierce elution buffer (Thermo, catalog #21004), pH 2.7, 3.7 mS/cm, and fractions containing antibodies were identified by OD28o and size exclusion chromatography. All fractions containing antibodies were neutralized using 2M Tris pH 8.5, and then dialyzed into PBS. The method employed to purify large volumes of cell supernatant containing canine monoclonal antibodies (ex. 10-15 L) differs from the method typically employed to purify human antibodies from large volumes. For human antibodies, Protein A purification is typically accomplished with cell supernatant binding conditions of pH 7.0 to 8.3 and 15 to 20 mS/cm, washing with similar conditions (1×PBS) and a 1 step elution of human antibodies with 0.1 M acetic acid, 0.15 M sodium chloride, pH 2.7 at 15 to 20 mS/cm or Thermo IgG elution buffer, pH 2.7, at 15 mS/cm.
Purified canine antibodies were analyzed by mass spectroscopy (MS) to confirm the expressed antibody protein molecular weight matched the expected weight based on amino acid sequence. In addition, canine antibodies were analyzed by size exclusion chromatography (SEC) to determine the percent monomer. This data indicated that mouse/canine chimeric IgG1/k mAbs may be expressed transiently in 293-6e cells and are 81% or greater monomeric following purification. This data also indicated that caninized IgG1/k mAbs may be expressed transiently in 293-6e cells and in most cases are 80% or greater monomeric following purification. In some cases, expression of protein may not be detected and in some cases purified caninized mAb is between 24 and 34% monomeric. The data is summarized in Tables 19 and 20.
TABLE 19
Mouse/Canine Chimeric Monoclonal Antibody Characterization Data
Estimated
Name of
Moniker of
Expression
Mouse/Canine
Mouse/Canine
Level in Cell
%
Hybridoma
Chimeric
Chimeric
Supernatant
Monomeric
Moniker
Version
Version
(ug/mL)
mAb
PR-1254972
Mu72 Canine
PR-1290646
3.2
97
IgG1/k Chimera
PR-1254973
Mu73 Canine
PR-1290654
7
88.3
IgG1/k Chimera
PR-1254977
Mu77 Canine
PR-1290656
0.3
82.4
IgG1/k Chimera
PR-1254981
Mu81 Canine
PR-1290657
0.9
81
IgG1/k Chimera
PR-1254982
Mu82 Canine
PR-1290658
11.9
92.3
IgG1/k Chimera
TABLE 20
Production of Caninized Antibodies by Combinations of Caninized Heavy and Light Chains
Light chain
Heavy chain
72.2VH
72.3VH
72.4VH
73.2VH
73.4VH
77.2VH
77.3VH
72.2VL
72VHv2/
72.3
72VHv4/
73.5
73VHv4/
77VHv2/
77.5
72VLv2
CaIgG1/k
72VLv2
CaIgG1/k
72VLv2
72VLv2
CaIgG1/k
72.4VL
72VHv2/
72VHv3/
72.4
73VHv2/
73VHv4/
77VHv2/
77VHv3/
72VLv4
72VLv4
CaIgG1/k
72VLv4
72VLv4
72VLv4
72VLv4
73.2VL
72VHv2/
72.5
72VHv4/
73.2
73VHv4/
77VHv2/
77.6
73VLv2
CaIgG/k
73VLv2
CaIgG1/k
73VLv2
73VLv2
CaIgG1/k
73.4VL
72VHv2/
72VHv3/
72VHv4/
73VHv2/
73.4
77VHv2/
77VHv3/
73VLv4
73VLv4
73VLv4
73VLv4
CaIgG/k
73VLv4
73VLv4
77.2VL
72VHv2/
72.6
72VHv4/
73.6
73VHv4/
77VHv2/
77.3
77VLv2
CaIgG/k
77VLv2
CaIgG1/k
77VLv2
77VLv2
CaIgG1/k
77.4VL
72VHv2/
72VHv3/
72VHv4/
73VHv2/
73VHv4/
77VHv2/
77VHv3/
77VLv4
77VLv4
77VLv4
77VLv4
77VLv4
77VLv4
77VLv4
81.2VL
72VHv2/
72.7
72VHv4/
73.7
73VHv4/
77VHv2/
77.7
81VLv2
CaIgG/k
81VLv2
CaIgG1/k
81VLv2
81VLv2
CaIgG1/k
81.4VL
72VHv2/
72VHv3/
72VHv4/
73VHv2/
73VHv4/
77VHv2/
77VHv3/
81VLv4
81VLv4
81VLv4
81VLv4
81VLv4
81VLv4
81VLv4
82.2VL
72VHv2/
72VHv3/
72VHv4/
73VHv2/
73VHv4/
77VHv2/
77VHv3/
82VLv2
82VLv2
82VLv2
82VLv2
82VLv2
82VLv2
82VLv2
82.3VL
72VHv2/
72.8
72VHv4/
73.8
73VHv4/
77VHv2/
77.8
82VLv3
CaIgG/k
82VLv3
CaIgG1/k
82VLv3
82VLv3
CaIgG1/k
82.4VL
72VHv2/
72VHv3/
72VHv4/
73VHv2/
73VHv4/
77VHv2/
77VHv3/
82VLv4
82VLv4
82VLv4
82VLv4
82VLv4
82VLv4
82VLv4
Light chain
Heavy chain
77.4VH
81.2VH
81.4VH
82.2VH
82.4VH
72.2VL
77VHv4/
81.5
81VHv4/
82.5
82VHv4/
72VLv2
CaIgG1/k
72VLv2
CaIgG1/k
72VLv2
72.4VL
77VHv4/
81VHv2/
81VHv4/
82VHv2/
82VHv4/
72VLv4
72VLv4
72VLv4
72VLv4
72VLv4
73.2VL
77VHv4/
81.6
81VHv4/
82.6
82VHv4/
73VLv2
CaIgG1/k
73VLv2
CaIgG1/k
73VLv2
73.4VL
77VHv4/
81VHv2/
81VHv4/
82VHv2/
82VHv4/
73VLv4
73VLv4
73VLv4
73VLv4
73VLv4
77.2VL
77VHv4/
81.7
81VHv4/
82.7
82VHv4/
77VLv2
CaIgG1/k
77VLv2
CaIgG1/k
77VLv2
77.4VL
77.4
81VHv2/
81VHv4/
82VHv2/
82VHv4/
CaIgG1/k
77VLv4
77VLv4
77VLv4
77VLv4
81.2VL
77VHv4/
8.12
81VHv4/
82.8CaIgG1/k
82VHv4/
81VLv2
CaIgG1/k
81VLv2
81VLv2
81.4VL
77VHv4/
81VHv2/
81.4
82VHv2/
82VHv4/
81VLv4
81VLv4
CaIgG1/k
81VLv4
81VLv4
82.2VL
77VHv4/
81VHv2/
81VHv4/
82VHv2/
82VHv4/
82VLv2
82VLv2
82VLv2
82VLv2
82VLv2
82.3VL
77VHv4/
81.8
81VHv4/
82.3
82VHv4/
82VLv3
CaIgG1/k
82VLv3
CaIgG1/k
82VLv3
82.4VL
77VHv4/
81VHv2/
81VHv4/
82VHv2/
82.4
82VLv4
82VLv4
82VLv4
82VLv4
CaIgG1/k
TABLE 21
Caninized Monoclonal Antibody Characterization Data
Estimated
Expression
Level in Cell
%
Supernatants
Monomeric
Name
Moniker
Lot
(ug/mL)
mAb
72.3 Canine IgG1/k
PR-1313524
1804091
2.63
88.3
72.4 Canine IgG1/k
PR-1314949
1805928
1.6
81.5
73.2 Canine IgG1/k
PR-1313520
1810546
13.4
96.5
73.4 Canine IgG1/k
PR-1314950
1805932
1.8
90
77.3 Canine IgG1/k
N/A
N/A
0.7
24.8
77.4 Canine IgG1/k
N/A
N/A
1
34.6
81.2 Canine IgG1/k
N/A
No mAb
No mAb
N/A
detected
detected
81.4 Canine IgG1/k
N/A
No mAb
No mAb
N/A
detected
detected
82.3 Canine IgG1/k
PR-1313519
1810585
4.4
80.7
82.4 Canine IgG1/k
PR-1313521
1816320
9.8
94.2
Example 18: Affinity Analysis of Canine Antibodies
Purified mouse/canine chimeric antibodies and caninized antibodies were analyzed for affinity to canine NGF using a Biacore T100 instrument. Goat anti Canine IgG (Southern Biotech) was immobilized at 5000-10000 RU on a CM5 chip using an amine coupling procedure according to the manufacturer's instructions (Biacore). Canine NGF was injected at 50 uL/min at a concentration range of 50-0.156 nM for the mouse/canine chimeric antibodies or 10-0.156 nM for the caninized antibodies. The association rate was monitored for 5 min and the dissociation rate was monitored for 10-20 min. The chip surface was regenerated using 50-75 ul 10 mM glycine pH 1.5 at a flow rate of 50-100 ul/min. Data was analyzed using Biaevaluation T100 software version 2.0.2, software, GE Healthcare Life Sciences (Piscataway, N.J.). Overall affinity parameters established for mouse/canine chimeric antibodies is summarized in Table 22 and for caninized antibodies in Table 23. This data indicates that the isolated mouse/canine chimeric anti-NGF mAbs have fast on-rates (from greater than 2×106) and slow off-rates (from less than 3×10˜3). The overall KDs of the mouse/canine anti-NGF mAbs range from about 1300 pM to 1.6 pM. This data also indicates that the isolated caninized chimeric anti-NGF mAbs have fast on-rates (from greater than 6×106) and slow off-rates (from less than 2×10˜4). The overall KDs of the caninized anti-NGF mAbs range from about 42 pM to 1.2 pM.
TABLE 22
Affinity Parameters of Mouse/Canine Chimeric Monoclonal
Antibodies to Canine NGF
On-rate
Off-rate
Overall
Name
Moniker
(1/M · S)
(1/S)
Affinity (M)
Mu72 Canine
PR-1290646
2.9 × 106
3.8 × 10−3
1.3 × 10−9
IgG1/k Chimera
Mu73 Canine
PR-1290654
6.3 × 106
9 × 10−5
1.4 × 10−11
IgG1/k Chimera
Mu77 Canine
PR-1290656
9.1 × 106
1.9 × 10−4
2.1 × 10−11
IgG1/k Chimera
Mu81 Canine
PR-1290657
4.2 × 106
3.5 × 10−4
8.2 × 10−11
IgG1/k Chimera
Mu82 Canine
PR-1290658
8.7 × 106
1.4 × 10−5
1.6 × 10−12
IgG1/k Chimera
TABLE 23
Affinity Parameters of Caninized Monoclonal Antibodies to Canine NGF
On-rate
Off-rate
Overall
Name
(1/M · s)
(1/s)
Affinity (M)
73.2 canine IgG1/k
Expt 1
6.3 × 106
2.8 × 10−4
4.4 × 10−11
PR-13113520
Expt 2
6.9 × 106
2.9 × 10−4
4.2 × 10−11
Average
6.6 × 106
2.9 × 10−4
4.3 × 10−11
82.3 canine IgG1/k
Expt 1
8.2 × 106
2 × 10−5
2.4 × 10−12
PR-13113519
Expt 2
8.5 × 106
1.3 × 10−5
1.6 × 10−12
Average
8.4 × 106
1.7 × 10−5
2 × 10−12
82.4 canine IgG1/k
Expt 1
8.6 × 106
1.1 × 10−5
1.2 × 10−12
PR-13113521
Expt 2
7.7 × 106
1.2 × 10−5
1.5 × 10−12
Average
8.2 × 106
1.2 × 10−5
1.4 × 10−12
Example 19: Characterization of Canine Antibodies by the TF-1 Cell Proliferation Potency Assay
Purified mouse/canine chimeric antibodies and caninized antibodies were characterized using the TF-1 Cell Proliferation Potency Assay (described previously) using 70 pM canine NGF in the assay. The summarized potency data is in Tables 20 and 21. The data shows that in the presence of 70 pM canine NGF, all of the mouse/canine chimeric anti-NGF antibodies display sub-nM potencies, and all display potencies of less than 50 pM. The data shows that in the presence of 70 pM canine NGF, some of the caninized anti-NGF antibodies have no neutralization potency on 70 pM canine NGF. Some caninized mAbs have sub-nM potencies, and some have potencies of less than 20 pM.
TABLE 24
Potency of Mouse/Canine Chimeric NGF Monoclonal
Antibodies on Canine NGF-Induced TF-1 Cell Proliferation
Name
Moniker
Lot
IC50 (nM)
Mu72 Canine IgG1/k Chimera
PR-1290646
1785614
0.041
Mu73 Canine IgG1/k Chimera
PR-1290654
1785658
0.008
Mu77 Canine IgG1/k Chimera
PR-1290656
1785699
0.028
Mu81 Canine IgG1/k Chimera
PR-1290657
1778832
0.012
Mu82 Canine IgG1/k Chimera
PR-1290658
1785732
0.007
TABLE 25
Potency of Caninized NGF Monoclonal Antibodies on Canine
NGF-Induced TF-1 Cell Proliferation (N/A = not applicable)
Name
Moniker
Lot
IC50 (nM)
72.3 Canine IgG1/k
PR-1313524
1804091
0
72.4 Canine IgG1/k
PR-1314949
1805928
0
73.2 Canine IgG1/k
PR-1313520
1810546
0.422
73.4 Canine IgG1/k
PR-1314950
1805932
0
77.3 Canine IgG1/k
N/A
N/A
0.625
77.4 Canine IgG1/k
N/A
N/A
0
82.3 Canine IgG1/k
PR-1313519
1810585
0.017
82.4 Canine IgG1/k
PR-1313521
1816320
0.016
Example 20: Characterization of Solubility and Stability of Caninized Anti-NGF Antibodies
Stock solutions of two caninized anti-NGF antibodies (73.2 canine IgG1/k and 82.4 canine IgG1/k) were obtained. The antibodies were formulated in phosphate buffer saline (PBS) at concentrations below 5 mg/ml (PBS contains, but is not limited to, the following ingredients: 15 mM phosphate buffer and 150 mM sodium chloride at pH 7.4).
Solubility:
The solubility of the caninized antibodies at high concentrations in PBS were evaluated by concentrating the antibodies with Amicon 30K molecular weight cutoff centrifuge spin filters. The final concentrations were determined by UV absorbance.
At room temperature, 73.2 canine IgG1/k was soluble to at least 54 mg/ml and 82.4 canine IgG1/k was soluble to at least 83 mg/ml. When stored at 5° C. for 5 hours at those concentrations, 73.2 canines IgG1/k formed a gel layer at the bottom of the container while 82.4 canines IgG1/k remained as a uniform solution. When re-equilibrated to room temperature, 73.2 canines IgG1/k became a uniform solution. When 73.2 canines IgG1/k were diluted to 27 mg/ml, it remained as a uniform solution at 5° C.
In comparison, adalimumab, a human antibody, demonstrated a solubility of at least 150 mg/ml at 5° C. and at room temperature. This was observed in a formulation with a pH of 7 and with a sodium chloride concentration of 150 mM. The observations are described in Table 26.
TABLE 26
Solubility of 73.2 canine IgG1/k, 82.4 canine IgG1/k and
human antibody adalimumab in PBS
Room
temperature
solubility
Antibody
(mg/ml)
Observations when placed at 5° C.
73.2 canine IgG1/k
≥54
Gel layer formed at container bottom *
82.4 canine IgG1/k
≥83
Remained as solution
adalimumab
≥150
Remained as solution
* returned to uniform solution when brought back to room temperature; when diluted to 27 mg/ml, remained as uniform solution at 5° C.
The solubility of 73.2 canine IgG1/k 82.4 canine IgG1/k was also evaluated in 15 mM histidine buffer pH 6.0. This is a buffer typically used to formulate human therapeutic antibodies. The PBS buffer comprising the stock solutions of 73.2 canine IgG1/k and 82.4 canine IgG1/k were exchanged with the histidine buffer using Amicon 3 OK molecular weight cutoff centrifuge spin filters. Following buffer exchange, the antibodies exhibited white precipitation and solubilities of less than 2 mg/ml at room temperature, as determined by UV absorbance. In comparison, the human antibody adalimumab was observed to reach a concentration of at least 150 mg/ml in 15 mM histidine buffer pH 6.0 at room temperature. These observations are summarized in Table 27.
TABLE 27
Solubility of anti-NGF caninized antibodies 73.2 canine IgG1/k, 82.4
canine IgG1/k and human antibody adalimumab in 15 mM histidine
buffer pH 6.0
Room temperature
Antibody
solubility (mg/ml)
Observations
73.2 canine
<2
White precipitate observed
IgG1/k
82.4 canine
<2
White precipitate observed
IgG1/k
adalimumab
≥150
Remained as solution
Freeze-Thaw Stability
An assessment of the freeze-thaw (FT) stability of 73.2 canine IgG1/k and 82.4 canine IgG1/kin PBS, and after dilution with PBS to 1 mg/ml, was performed. Both antibodies were frozen at −80° C. for at least 4 hours. They were then thawed in a 30° C. water bath (this constitutes one freeze-thaw cycle). Stability was assessed for four freeze-thaw cycles by size exclusion HPLC (SEC). The freeze-thaw analysis is summarized in Table 28.
TABLE 28
Freeze-thaw stability of 73.2 canine IgG1/k and 82.4 canine IgG1/k at
1 mg/ml in PBS.
Percentage Species
Post
Post
Post
Antibody
Species
Pre-FT
FT#1
FT#2
FT#4
73.2 canine IgG1/k
Monomer
97.4
97.3
97.3
97.2
Aggregate
1.7
1.8
1.8
1.8
Fragment
0.9
0.9
0.9
1
82.4 canine IgG1/k
Monomer
96.6
96.6
96.6
96.1
Aggregate
2.9
2.9
2.9
3.2
Fragment
0.5
0.5
0.5
0.7
Storage Stability and Accelerated Stability:
The stability of 73.2 canine IgG1/k and 82.4 canine IgG1/k when formulated at a concentration of 10 mg/mL and within a pH range of 5 to 8 and at low (˜7.5 mM) and high (˜150 mM) ionic strengths was assessed. Stability at these conditions was assessed by monitoring the stability of the antibodies in the following buffers and salt concentrations: (A) 15 mM acetate pH 5; (B) 15 mM acetate pH 5+150 mM NaCl; (C) 15 mM histidine pH 6+150 mM NaCl; (D) 15 mM phosphate pH 7.4; (E) PBS pH 7.4; (F) 15 mM Tris pH 7.5; (G) 15 mM Tris pH 8.0. Sodium azide (0.02%) was added to all buffers as an anti-microbial agent.
Stock solutions of 73.2 canine IgG1/k and 82.3 canine IgG1/kin PBS were concentrated up to 15 mg/ml using 3 OK molecular weight cutoff centrifuge spin filters. They were then dialyzed against the buffers listed above for 18 hours using mini-dialysis 1 kD molecular weight cut-off dialysis tubes (GE Healthcare). Following dialysis, samples were diluted with the respective buffers to a final concentration of 10 mg/ml. 150 μl of each sample was aliquoted to cryovials which were then stored at 40° C. or 5° C. Samples were analysed at time=0 hours (TO), at 7 days (T7d), and at 21 days (T21d) and stability was assessed by SEC.
After 21 days at 40° C., accelerated stability testing showed that 73.2 canine IgG1/k and 82.3 canines IgG1/k have much greater fragmentation at pH values below 7.4 than at pH values above 7.4. In comparison, the human antibody adalimumab, exhibited less fragmentation within the pH range 4 to 8 over 21 days at 40° C. In particular, the fragmentation of adalimumab at pH 6 was much less than the fragmentation of 73.2 canine IgG1/k or 82.4 canine IgG1/k at pH 6. Also, adalimumab at the higher stress condition of pH 4 showed equal or less fragmentation compared to 73.2 canine IgG1/k or 82.4 canine IgG1/k at the lower stress condition of pH 5. The results of the stability analyses and fragmentation profiles are shown, respectively, in Tables 25 and 26. These data suggest that canine IgG1/k monoclonal antibodies have a different degradation profile compared to that of human IgG1/k monoclonal antibodies. Specifically, the fragmentation appears to be more extensive for canine IgG1/k antibodies than for human antibodies at pH 6 and below.
TABLE 29
Stability data from SEC for 73.2 canine IgG1/k, 82.4 canine
IgG1/k and human antibody adalimumab in different formulations
at 7 and 21 days at 5° C. and at 40° C.
Percentage
Percentage
Percentage
Monomer
Aggregate
Fragment
Buffer
T0
T7d
T21d
T0
T7d
T21d
T0
T7d
T21d
73.2 canine IgG1/k at 5° C.
A (pH 5)
94.6
91.4
90.3
2.9
3.1
3.4
2.6
5.5
6.2
B (pH 5)
95.2
98.2
98.2
3.4
0.4
0.5
1.4
1.4
1.3
C (pH 6)
93.9
98.0
97.8
4.4
0.5
0.7
1.8
1.5
1.5
D (pH 7.4)
94.3
97.9
97.7
4.7
0.6
0.8
1.0
1.5
1.5
E (pH 7.4)
94.5
97.9
97.8
4.5
0.5
0.8
1.0
1.6
1.5
F (pH 7.5)
94.5
98.0
97.8
4.0
0.5
0.8
1.6
1.5
1.5
G (pH 8.0)
93.7
97.8
97.6
4.6
0.7
0.9
1.7
1.5
1.5
73.2 canine IgG1/k at at 40° C.
A (pH 5)
94.6
81.3
79.0
2.9
4.5
5.1
2.6
14.2
15.9
B (pH 5)
95.2
92.1
90.9
3.4
0.6
1.2
1.4
7.4
7.8
C (pH 6)
93.9
94.4
91.6
4.4
0.6
1.1
1.8
5.0
7.3
D (pH 7.4)
94.3
97.5
96.6
4.7
0.9
1.6
1.0
1.5
1.8
E (pH 7.4)
94.5
98.0
97.3
4.5
0.6
1.1
1.0
1.4
1.6
F (pH 7.5)
94.5
97.5
96.3
4.0
0.9
1.8
1.6
1.6
1.9
G (pH 8.0)
93.7
97.0
95.2
4.6
1.2
2.6
1.7
1.8
2.2
82.4 canine IgG1/k at 5° C.
A (pH 5)
96.7
98.1
98.7
2.3
1.3
.8
1.0
0.5
0.5
B (pH 5)
96.3
97.2
97.3
2.4
2.3
2.3
1.3
0.5
0.4
C (pH 6)
97.0
97.1
97.1
2.6
2.3
2.3
0.4
0.6
0.6
D (pH 7.4)
96.4
97.0
97.1
2.5
2.4
2.5
1.0
0.5
0.4
E (pH 7.4)
96.7
96.8
96.8
2.8
2.5
2.6
0.5
0.7
0.6
F (pH 7.5)
96.8
97.1
96.9
2.9
2.5
2.5
0.2
0.5
0.6
G (pH 8.0)
96.6
96.9
96.9
2.5
2.5
2.5
0.9
0.6
0.6
82.4 canine IgG1/k at 40° C.
A (pH 5)
96.7
93.1
87.8
2.3
2.8
4.2
1.0
4.1
8.0
B (pH 5)
96.3
93.3
91.3
2.4
2.5
3.1
1.3
4.3
5.6
C (pH 6)
97.0
94.1
92.5
2.6
2.3
2.7
0.4
3.5
4.8
D (pH 7.4)
96.4
93.5
93.8
2.5
3.4
3.3
1.0
3.1
2.9
E (pH 7.4)
96.7
95.1
93.6
2.8
2.4
2.6
0.5
2.5
3.8
F (pH 7.5)
96.8
93.4
923.6
2.9
2.6
3.0
0.2
4.0
0.5
G (pH 8.0)
96.6
94.8
92.7
2.5
2.8
3.5
0.9
2.4
0.4
adalimumab at 40° C.
Percentage
Percentage
Percentage
Monomer
Aggregate
Fragment
pH
T0
T7d
T21d
T0
T7d
T21d
T0
T7d
T21d
4
99
98
95
<1
<2
0.5
<1
<2
4.5
6
99
99
99
<1
<1
0.3
<1
<1
0.7
8
99
98
98
<1
<2
1.2
<1
<2
0.8
TABLE 30
Fragmentation profile from SEC for 73.2 canine IgG1/k, 82.4 canine
IgG1/k and human antibody adalimumab in different formulations
at 21 days and at 40° C.
Increase in
Percent
Fragmentation
over 21 days at
Antibody
Buffer
40° C.
73.2 canine IgG1/k
A
(pH 5)
13.3
B
(pH 5)
6.4
C
(pH 6)
5.5
D
(pH 7.4)
0.8
E
(pH 7.4)
0.6
F
(pH 7.5)
0.3
G
(pH 8.0)
0.5
82.4 canine IgG1/k
A
(pH 5)
7.0
B
(pH 5)
4.3
C
(pH 6)
4.4
D
(pH 7.4)
1.9
E
(pH 7.4)
3.3
F
(pH 7.5)
0.3
G
(pH 8.0)
−0.5
adalimumab
pH 4
<4.5
pH 6
<0.7
pH 8
<0.8
Example 21: Canine Single Dose PK Study and Antigen Bridging Assay for PK Serum Sample Analysis
The serum levels of 73.2 canine IgG1/k and 82.4 canine IgG1/k were analyzed following a single dose of 4.5 mg/kg (intravenous or subcutaneous) in mongrel dogs. Following the injection, 13 samples of venous blood were collected over 672 hours. Blood samples were allowed to clot and the serum removed for antibody quantitation.
An NGF bridging assay was developed to quantitate canine anti-NGF mAbs in serum. Streptavidin-coated 96-well plates (MSD #L1 ISA-1) were blocked with Blocker A (MSD #R93BA-4). Canine anti-NGF antibody present in serum (or in PBS) was mixed with equimolar ratios of biotin-tagged NGF and Sulfo-tagged NGF (Sulfo Reagent MSD #R91AN-1) and incubated to form an NGF+antibody complex. The final concentration of the biotin-tagged NGF and sulfo-tagged NGF in the assay was between 1-2 nM. NGF-antibody complexes were added to the streptavidin-coated plate and allowed to bind for 60 minutes. Following incubation, plates were washed with PBS plus 0.05% Tween-20, and bound NGF-antibody complexes were detected in Read Buffer T (MSD #R92TC-1) on an MSD SECTOR Imager 6000. Data was quantitated to estimate the total amount of antibody in μg/mL of a sample liquid and is provided below in Tables 31-34.
TABLE 31
Serum Concentrations of 82.4 canine IgG1/k Following a Single
Subcutaneous Dose
Dog #
1073305
1072602
1072104
1072306
1072105
Hours post injection
ug/mL
0
0.0
0.0
0
0
0.1
0.25
2.6
0.4
0.0
2.24
0.1
1
9.0
2.2
0.9
9.07
0.4
8
31.5
17.7
13.8
32.55
12.8
12
41.1
20.5
18.7
33.31
24.5
24
42.2
25.6
23.2
35.73
23.8
48
52.3
37.2
36.3
41.35
35.3
72
51.2
41.1
34.4
38.91
36.8
144
47.1
42.6
35.2
33.46
36.7
240
39.0
32.4
29.0
26.03
31.7
336
30.9
28.2
24.1
19.99
26.8
504
20.1
18.3
16.0
11.03
18.6
672
16.3
12.8
10.4
5.81
5.4
TABLE 32
Serum Concentrations of 82.4 canine IgG1/k Following a Single
Intravenous Dose
Dog #
1072705
1073804
1073303
1073903
1074502
Hours post injection
ug/mL
0
0
0
0
0
0
0.25
102.6
83.1
84.0
80.1
81.7
1
106.7
70.1
87.4
74.1
81.9
8
88.4
65.7
68.1
67.4
68.9
12
91.5
61.6
62.6
62.1
59.3
24
87.4
60.0
57.2
53.9
53.0
48
76.6
49.9
52.6
50.9
50.2
72
60.3
49.1
46.0
40.8
44.6
144
45.7
43.1
36.1
35.7
36.0
240
37.2
34.4
32.6
24.3
32.6
336
31.7
32.7
24.8
20.6
20.1
504
23.5
17.4
18.5
12.2
12.8
672
15.2
10.7
12.5
7.3
8.3
TABLE 33
Serum Concentrations of 73.2 canine IgG1/k Following a Single
Subcutaneous Dose
Dog #
1072607
1074307
1072606
1074503
Hours post injection
ug/mL
0
0
0
0
0
0.25
0
0
2.1
0
1
1.0
5.9
4.7
0.0
8
18.6
31.3
18.7
7.4
12
22.7
32.5
21.3
8.7
24
26.8
33.2
24.2
12.0
48
33.7
35.9
28.4
16.2
72
35.0
37.6
30.7
19.4
144
34.9
37.4
30.2
21.8
240
31.6
31.8
26.8
22.0
336
24.4
24.7
22.6
16.5
504
15.3
14.3
13.8
10.6
672
6.8
9.2
5.1
5.4
TABLE 34
Serum Concentrations of 73.2 canine IgG1/k Following A Single
Intravenous Dose
Dog #
1072804
1073304
1072604
Hours post injection
ug/mL
0
0
0
0
0.25
93.6
33.2
108.5
1
86.3
30.8
103.1
8
76.9
22.1
85.4
12
72.1
21.4
80.5
24
63.0
17.1
68.0
48
54.6
14.6
56.8
72
49.4
13.5
50.8
144
41.4
10.2
41.4
240
35.4
8.9
30.8
336
30.5
6.3
20.9
504
22.2
4.0
3.4
672
14.8
3.4
0.0
Example 22: Pharmacokinetic Analysis of Serum Concentration Data
Pharmacokinetic parameters for both intravenous (IV) and subcutaneous (SC) dosing routes were calculated for each animal using WinNonlin software (Pharsight Corporation, Mountain View, Calif.) by noncompartmental analysis. Other calculations, e.g. mean, standard deviation (SD), and percent subcutaneous bioavailability (F: %) were carried out using Microsoft Excel software (Microsoft Corporation Redmond, Wash.). The data is shown in Table 35 and 36.
TABLE 35
Pharmacokinetic Analysis of 73.2 canine IgG1/k
Following a Single Intravenous Dose
IV
SC
T½
Vss
CI
T½
Cmax
Tmax
(day)
(mL/kg)
(mL/h/kg)
(day)
(ug/mL)
(day)
% F
14.8*
71
0.15
8.0*
31.3
4.8
51
*Harmonic Mean
TABLE 36
Pharmacokinetic Analysis of 82.4 canine IgG1/k
Following a Single Intravenous Dose
IV
SC
T½
Vss
CI
T½
Cmax
Tmax
(day)
(mL/kg)
(mL/h/kg)
(day)
(ug/mL)
(day)
% F
10.9*
73
0.19
11.6*
41.9
3.0
94
*Harmonic Mean
The data indicates that canine mAbs 73.2 and 82.4 have a half-life of about 8 to about 15 days when dosed IV or SC, suggesting that these molecules exhibit mammalian antibody-like half-lives and overall PK parameters.
Example 23: ELISA for Titering Canine Antibodies
To quantitate canine antibodies in cell supernatants (or other liquids), high-binding EIA plates (Costar #9018) were coated with polyclonal goat anti-dog IgG antibodies (Rockland #604-1102) at 4 μg/ml in PBS. After blocking with 2% non-fat milk in PBS, canine monoclonal antibodies were added to the plates and the plates were washed with PBS plus 0.05% Tween-20. Bound canine mAbs were detected with HRP-tagged goat anti-dog IgG antibodies (Rockland #604-1302) at 0.1 μg/ml. Plates were washed with PBS plus 0.05% Tween-20. Canine mAbs were detected by addition of TMB substrate (Neogen #308177), and the reaction was stopped with IN HC1. Bound canine antibodies were quantitated by absorption at 450 nM to estimate the total amount of antibody in μg/mL of a sample liquid.
The present disclosure incorporates by reference in their entirety techniques well known in the field of molecular biology. These techniques include, but are not limited to, techniques described in the following publications:
Ausubel, F. M. et al. eds., Short Protocols In Molecular Biology (4th Ed. 1999) John Wiley & Sons, NY. (ISBN 0-471-32938-X).
Lu and Weiner eds. Cloning and Expression Vectors for Gene Function Analysis (2001)
BioTechniques Press. Westborough, Mass. 298 pp. (ISBN 1-881299-21-X).
Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).
Old, R. W. & S. B. Primrose, Principles of Gene Manipulation: An Introduction To Genetic Engineering (3d Ed. 1985) Blackwell Scientific Publications, Boston. Studies in Microbiology; V.2:409 pp. (ISBN 0-632-01318-4).
Sambrook, J. et al. eds., Molecular Cloning: A Laboratory Manual (2d Ed. 1989) Cold Spring Harbor Laboratory Press, NY. Vols. 1-3. (ISBN 0-87969-309-6).
Winnacker, E. L. From Genes To Clones: Introduction To Gene Technology (1987) VCH Publishers, NY (translated by Horst Ibelgaufts). 634 pp. (ISBN 0-89573-614-4).
All references, publications, and patent applications disclosed herein are hereby incorporated by reference in their entirety.
Although a number of embodiments, aspects and features have been described above, it will be understood by those skilled in the art that modifications and variations of the described embodiments and features may be made without departing from the present disclosure or the disclosure as defined in the appended claims.
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14797719
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zoetis belgium s.a.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Mar 31st, 2022 03:01PM
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Mar 31st, 2022 03:01PM
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Zoetis
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:zts
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Zoetis
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Oct 9th, 2018 12:00AM
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Jul 14th, 2015 12:00AM
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https://www.uspto.gov?id=US10093725-20181009
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Anti-NGF antibodies and their use
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The present disclosure encompasses NGF binding proteins, specifically to antibodies that are chimeric, CDR grafted and canonized antibodies, and methods of making and uses thereof. The antibodies, or antibody portions, of the disclosure are useful for detecting NGF and for inhibiting NGF activity, e.g., in a mammal subject suffering from a disorder in which NGF activity is detrimental.
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10093725
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1. An isolated anti-NGF antibody that specifically binds to Nerve Growth Factor (NGF) and inhibits the binding of NGF to the TrkA receptor comprising:
a) a variable heavy chain (VH) region comprising:
i) CDR1 comprising SEQ ID NO. 61,
ii) CDR2 comprising SEQ ID NO. 62; and
iii) CDR3 comprising SEQ ID NO. 63; and
b) a variable light chain (VL) region comprising:
i) CDR1 comprising SEQ ID NO. 64,
ii) CDR2 comprising SEQ ID NO. 65; and
iii) CDR3 comprising SEQ ID NO. 66; and
wherein the antibody is a chimeric antibody.
2. An isolated anti-NGF antibody that specifically binds to Nerve Growth Factor (NGF) and inhibits the binding of NGF to the TrkA receptor comprising:
a) a variable heavy chain (VH) region comprising:
i) CDR1 comprising SEQ ID NO. 61,
ii) CDR2 comprising SEQ ID NO. 62; and
iii) CDR3 comprising SEQ ID NO. 63; and
b) a variable light chain (VL) region comprising:
i) CDR1 comprising SEQ ID NO. 64,
ii) CDR2 comprising SEQ ID NO. 65; and
iii) CDR3 comprising SEQ ID NO. 66; and
wherein the antibody is a caninized antibody.
3. An isolated anti-NGF antibody that specifically binds to Nerve Growth Factor (NGF) and inhibits the binding of NGF to the TrkA receptor comprising:
a) a variable heavy chain (VH) region comprising:
i) CDR1 comprising SEQ ID NO. 61,
ii) CDR2 comprising SEQ ID NO. 62; and
iii) CDR3 comprising SEQ ID NO. 63; and
b) a variable light chain (VL) region comprising:
i) CDR1 comprising SEQ ID NO. 64,
ii) CDR2 comprising SEQ ID NO. 65; and
iii) CDR3 comprising SEQ ID NO. 66; and
wherein the antibody is a humanized antibody.
4. A pharmaceutical composition comprising a therapeutically effective amount of any one of the antibodies of claims 1-3 and a pharmaceutically acceptable carrier, diluent or excipient.
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4
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CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of currently pending U.S. application Ser. No. 13/817,721, which is a national stage 371 application of the international application PCT/US2011/048518 filed on Aug. 19, 2011 which claims priority to the U.S. Provisional Application No. 61/375,193, filed Aug. 19, 2010, all contents of which are incorporated by reference in their entirety
TECHNICAL FIELD
The disclosure relates to anti-NGF antibodies and polynucleotides encoding the same, and use of such antibodies and/or polynucleotides in the treatment and/or prevention of pain, including but not limited to post-surgical pain, rheumatoid arthritis pain, cancer pain, and osteoarthritis pain.
BACKGROUND
Nerve growth factor (NGF) is a secreted protein that was discovered over 50 years ago as a molecule that promotes the survival and differentiation of sensory and sympathetic neurons. (See Levi-Montalcini, Science 187: 113 (1975), for a review). The crystal structure of NGF and NGF in complex with the tyrosine kinase A (TrkA) receptor has been determined (McDonald et al., Nature 354: 411 (1991); Wiesmann et al., Nature 401: 184-188 (1999)).
The role of NGF in the development and survival of both peripheral and central neurons has been well characterized. NGF has been shown to be a critical survival and maintenance factor in the development of peripheral sympathetic and embryonic sensory neurons and of basal forebrain cholinergic neurons (see, e.g., Smeyne et al., Nature 368: 246-9 (1994); and Crowley et al., Cell, 76: 1001-11 (1994)). It has been shown to inhibit amyloidogenesis that leads to Alzheimer's disease (Calissano et al., Cell Death and Differentiation, 17: 1126-1133 (2010)). NGF up-regulates expression of neuropeptides in sensory neurons (Lindsay et al., Nature, 337:362-364 (1989)) and its activity is mediated through two different membrane-bound receptors, the TrkA receptor and the p75 common neurotrophin receptor (Chao et al., Science, 232:518-521 (1986); Huang et al., Annu. Rev. Neurosci., 24:677-736 (2001); Bibel et al., Genes Dev., 14:2919-2937 (2000)).
NGF is produced by a number of cell types including mast cells (Leon, et al., Proc. Natl. Acad. Set, 91: 3739-3743 (1994)), B-lymphocytes (Torcia, et al., Cell, 85: 345-356 (1996), keratinocytes (Di Marco, et al., J. Biol. Chem., 268: 22838-22846)), smooth muscle cells (Ueyama, et al., J. Hypertens., 11: 1061-1065 (1993)), fibroblasts (Lindholm, et al., Eur. J. Neurosci., 2: 795-801 (1990)), bronchial epithelial cells (Kassel, et al., Clin, Exp. Allergy, 31: 1432-40 (2001)), renal mesangial cells (Steiner, et al., Am. J. Physiol., 261:F792-798 (1991)) and skeletal muscle myotubes (Schwartz, et al., J Photochem. Photobiol., B66: 195-200 (2002)). In addition, NGF receptors have been found on a variety of cell types outside of the nervous system.
NGF has been implicated in processes outside of the nervous system, e.g., NGF has been shown to enhance vascular permeability (Otten, et al., Eur J Pharmacol., 106: 199-201 (1984)), enhance T- and B-cell immune responses (Otten, et al., Proc. Natl. Acad. Sci., USA 86: 10059-10063 (1989)), induce lymphocyte differentiation and mast cell proliferation and cause the release of soluble biological signals from mast cells (Matsuda, et al., Proc. Natl. Acad. Sci., 85: 6508-6512 (1988); Pearce, et al., J. Physiol, 372:379-393 (1986); Bischoff, et al., Blood, 79: 2662-2669 (1992); Horigome, et al., J. Biol. Chem., 268: 14881-14887 (1993)).
Both local and systemic administrations of NGF have been shown to elicit hyperalgesia and allodynia (Lewin, G. R. et al., Eur. J. Neurosci. 6: 1903-1912 (1994)). Intravenous infusion of NGF in humans produces a whole body myalgia while local administration evokes injection site hyperalgesia and allodynia in addition to the systemic effects (Apfel, S. C. et al., Neurology, 51: 695-702(1998)). Furthermore, in certain forms of cancer, excess NGF facilitates the growth and infiltration of nerve fibers with induction of cancer pain (Zhu, Z. et al., J Clin. Oncol., 17: 241-228 (1999). Although exogenously added NGF has been shown to be capable of having all of these effects, it is important to note that it has only rarely been shown that endogenous NGF is important in any of these processes in vivo (Torcia, et al., Cell, 85(3): 345-56 (1996)).
An elevated level of NGF has been implicated in certain inflammatory conditions in humans and animals, e.g., systemic lupus erythematosus (Bracci-Laudiero, et al., Neuroreport, 4: 563-565 (1993)), multiple sclerosis (Bracci-Laudiero, et al., Neurosci. Lett., 147:9-12 (1992)), psoriasis (Raychaudhuri, et al., Acta Derm. Venereol, 78: 84-86 (1998)), arthritis (Falcim, et al., Ann. Rheum. Dis., 55: 745-748 (1996)), interstitial cystitis (Okragly, et al., J. Urology 6: 438-441 (1999)) and asthma (Braun, et al., Eur. J Immunol., 28:3240-3251 (1998)). The synovium of patients affected by rheumatoid arthritis expresses high levels of NGF while in non-inflamed synovium NGF has been reported to be undetectable (Aloe, et al., Arch. Rheum., 35:351-355 (1992)). Similar results were seen in rats with experimentally induced rheumatoid arthritis (Aloe, et al., Clin. Exp. Rheumatol., 10: 203-204 (1992)). Elevated levels of NGF have been reported in transgenic arthritic mice along with an increase in the number of mast cells (Aloe, et al., Int. J. Tissue Reactions-Exp. Clin. Aspects, 15: 139-143 (1993)). Additionally, elevated levels of expression of canine NGF has been shown in lame dogs (Isola, M., Ferrari, V., Stabile, F., Bernardini, D., Gamier, P., Busetto, R. Nerve growth factor concentrations in the synovial fluid from healthy dogs and dogs with secondary osteoarthritis. Vet. Comp. Orthop. Traumatol. 4: 279 (2011)). PCT Publication No. WO 02/096458 discloses use of anti-NGF antibodies of certain properties in treating various NGF related disorders such as inflammatory condition (e.g., rheumatoid arthritis). It has been reported that a purified anti-NGF antibody injected into arthritic transgenic mice carrying the human tumor necrosis factor (TNF) gene caused reduction in the number of mast cells, as well as a decrease in histamine and substance P levels within the synovium of arthritis mice (Aloe et al., Rheumatol. Int., 14: 249-252 (1995)). It has been shown that exogenous administration of a NGF antibody reduced the enhanced level of TNF occurring in arthritic mice (Manni et al., Rheumatol. Int., 18: 97-102 (1998)).
Increased expression of NGF and high affinity NGF receptor (TrkA) was observed in human osteoarthritis chondrocytes (Iannone et al., Rheumatology, 41: 1413-1418 (2002)). Rodent anti-NGF antagonist antibodies have been reported (Hongo et al., Hybridoma, 19(3):215-227 (2000); Ruberti et al., Cell. Molec. Neurobiol., 13(5): 559-568 (1993)). However, when rodent antibodies are used therapeutically in non-rodent subjects, an anti-murine antibody response develops in significant numbers of treated subjects.
The involvement of NGF in chronic pain has led to considerable interest in therapeutic approaches based on inhibiting the effects of NGF (Saragovi, et al., Trends Pharmacol Sci. 21: 93-98 (2000)). For example, a soluble form of the TrkA receptor was used to block the activity of NGF, which was shown to significantly reduce the formation of neuromas, responsible for neuropathic pain, without damaging the cell bodies of the lesioned neurons (Kryger, et al., J. Hand Surg. (Am.), 26: 635-644 (2001)).
Certain anti-NGF antibodies have been described (PCT Publication Nos. WO 2001/78698, WO 2001/64247, WO 2002/096458, WO 2004/032870, WO 2005/061540, WO 2006/131951, WO 2006/110883; U.S. Publication Nos. US 20050074821, US 20080033157, US 20080182978 and US 20090041717; and U.S. Pat. No. 7,449,616). In animal models of neuropathic pain (e.g., nerve trunk or spinal nerve ligation) systemic injection of neutralizing antibodies to NGF prevents both allodynia and hyperalgesia (Ramer et al., Eur. J. Neurosci., 11: 837-846 (1999); Ro et al., Pain, 79: 265-274 (1999)). Furthermore, treatment with a neutralizing anti-NGF antibody produces significant pain reduction in a murine cancer pain model (Sevcik et al., Pain, 115: 128-141 (2005)). Thus, there is a serious need for anti-NGF antagonist antibodies for humans and animals.
SUMMARY OF THE INVENTION
The present disclosure provides a novel family of binding proteins, CDR grafted antibodies, mammalized (such as bovanized, camelized, caninized, equinized, felinized, humanized etc.) antibodies, and fragments thereof, capable of binding and neutralizing NGF. The disclosure provides a therapeutic means with which to inhibit NGF and provides compositions and methods for treating disease associated with increased levels of NGF, particularly inflammatory disorders.
In one aspect, the present disclosure provides a binding protein, or fragment thereof, comprising hypervariable region sequences wholly or substantially identical to sequences from an antibody from a donor species; and constant region sequences wholly or substantially identical to sequences of antibodies from a target species, wherein the donor and target species are different. The binding protein may for example specifically bind NGF and have a heavy chain having a heavy chain variable region and a light chain having a light chain variable region.
In another aspect, the present disclosure provides a binding protein that specifically binds NGF and which has a heavy chain having a heavy chain variable region and a light chain having a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 206, SEQ ID NO: 207, or an antigen-binding or an immunologically functional immunoglobulin fragment thereof.
In another aspect, the present disclosure provides a binding protein that specifically binds NGF and which has a heavy chain having a heavy chain variable region and a light chain having a light chain variable region, wherein the light chain variable region comprises an amino acid sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 44, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, or an antigen-binding or an immunologically functional immunoglobulin fragment thereof.
A binding protein of the present disclosure may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 50% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 50% to one of said sequences. Alternatively, the binding protein of the present disclosure may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 70% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 70% to one of said sequences. The binding protein of the present disclosure may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 80% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 80% to one of said sequences, binding protein of the present disclosure may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 90% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 90% to one of said sequences.
A binding protein of the present disclosure may comprise a heavy chain human immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. A binding proteins of the present disclosure may alternatively comprise a heavy chain canine immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. A binding protein of the present disclosure may alternatively comprise a heavy chain feline immunoglobulin constant domain. A binding protein of the present disclosure may alternatively comprise a heavy chain equine immunoglobulin constant domain. A binding protein of the present disclosure may further comprise a constant region having an amino acid sequence selected from the group consisting of SEQ ID NO: 52 and SEQ ID NO: 54.
Any of the above binding proteins may be selected from the group consisting of; an immunoglobulin molecule, disulfide linked Fv, monoclonal antibody, scFv, chimeric antibody, single domain antibody, CDR-grafted antibody, diabody, humanized antibody, caninized mAb, canine mAb, feline mAb, felinized mAb, equine mAb, equinized mAb, a multispecific antibody, a Fab, a dual specific antibody, a DVD-Ig, a Fab′, a bispecific antibody, a F(ab′)2, and a Fv.
Any of the above binding proteins may be capable of modulating a biological function of NGF, or neutralizing NGF.
Any of the above binding proteins may be capable of neutralizing NGF with a potency (IC50) of at least about 10 nM, at least about 5 nM, at least about 1 nM, at least about 0.5 nM, at least about 0.1 nM, at least about 0.05 nM, at least about 0.01 nM, or at least about 0.001 nM, as measured in the TF-1 cell proliferation assay or the pERK and Pathhunter assays.
Any of the above binding proteins may have an on rate constant (Kon) for NGF of: at least about 102 M−1s−1, at least about 103 M−1s−1, at least about M−1s−1, at least about 105 M−1s−1, or at least about 106 M−1s−1, or at least about 107 M−1s−1, as measured by surface plasmon resonance.
Any of the above binding proteins may have an off rate constant (Koff) for NGF selected from the group consisting of: at most about 10−3 s−1, at most about 10−4 s−1, at most about 10−5 s−1, at most about 10−6 s−1, and at most about 10−7 s−1, as measured by surface plasmon resonance.
Any of the above binding proteins may have a dissociation constant (KD) for NGF selected from the group consisting of: at most about 10−7 M, at most about 10−8 M, at most about 10−9 M, at most about 10−10 M, at most about 10−11 M at most about 10−12 M, at most about 10−13 M and at most about 10−14 M. The dissociation constant (KD) may be, for example, about 1×10−9 M, about 1×10−10 M, about 3.14×10−10M, about 1×10−11 M, about 2.37×10−11 M, about 1×10−12 M, about 1×10−13 M, and about 3.3×10−14 M.
Any of the above binding proteins may further comprise an agent selected from the group consisting of; an immunoadhension molecule, an imaging agent, a therapeutic agent, and a cytotoxic agent. The agent may be, for example, an imaging agent selected from the group consisting of a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin. The imaging agent may be a radiolabel selected from the group consisting of: 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and 153Sm. Alternatively, the agent may be a therapeutic or cytotoxic agent, such as, for example, an anti-metabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an anti-angiogenic agent, an anti-mitotic agent, an anthracycline, toxin, and an apoptotic agent.
Any of the binding proteins may possess a murine, canine, feline, human or equine glycosylation pattern.
Any of the binding proteins may be a crystallized binding protein. The crystallized binding protein may be a carrier-free pharmaceutical controlled release crystallized binding protein.
In another aspect, the present disclosure provides an isolated nucleic acid encoding any of the above binding proteins. The isolated nucleic acid may comprise RNA or DNA.
In another aspect, the present disclosure provides an isolated nucleic acid comprising or complementary to a nucleic acid sequence that encodes a binding protein that specifically binds NGF having a heavy chain having a heavy chain variable region and a light chain having a light chain variable region, wherein the heavy chain variable region is encoded by a nucleotide sequence having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, and 21.
In another aspect, the present disclosure provides an isolated nucleic acid comprising or complementary to a nucleic acid sequence that encodes a binding protein that specifically binds NGF having a heavy chain having a heavy chain variable region and a light chain having a light chain variable region, wherein the light chain variable region is encoded by a nucleotide sequence having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOS: 3, 7, 11, 15, 19 and 23.
In another aspect, the present disclosure provides a recombinant vector comprising an isolated nucleic acid encoding a binding protein that specifically binds NGF as described herein. A recombinant vector according to the present disclosure may comprise pcDNA, pTT, pTT3, pEFBOS, pBV, pJV or pBJ. Also provided is a host cell comprising such a recombinant vector. The host cell may be for example a eukaryotic cell, or a prokaryotic cell. The host cell may be a protist cell; an animal cell such as but not limited to a mammalian cell, avian cell; an insect cell, such as but not limited to an insect Sf9 cell; a plant cell; or a fungal cell. The host cell may be for example an E. coli cell. The host cell may be a CHO cell, or a COS cell. Also provided is an isolated cell line that produces a binding protein that specifically binds NGF as described herein.
In another aspect, the present disclosure provides a pharmaceutical or diagnostic composition comprising a binding protein that specifically binds NGF as described herein, and a pharmaceutically acceptable carrier, diluent or excipient. A pharmaceutical composition may comprise a therapeutically effective amount of the NGF binding protein.
In another aspect, the present disclosure provides a composition for the release of a binding protein, the composition comprising: (a) a composition comprising a binding protein that specifically binds NGF as described herein, and a pharmaceutically acceptable carrier, excipient or diluent, and (b) at least one polymeric carrier.
In another aspect, the present disclosure provides a method for reducing NGF activity in a subject (for example, a dog, cat, horse, ferret, etc.) suffering from a disorder in which NGF activity is detrimental, comprising administering to the subject a therapeutically effective amount of a binding protein that specifically binds NGF as described herein.
In another aspect, the present disclosure provides a method for making anti-NGF antibodies comprising: (a) production of murine monoclonal antibodies; (b) screening hybridoma supernatants; (c) grafting of donor CDRs into target frameworks; and (d) introducing backmutations in the framework region of the target antibodies, wherein the anti-NGF antibodies comprise hypervariable region sequences wholly or substantially identical to sequences from an antibody from the donor species and constant region sequences wholly or substantially identical to sequences of an antibody from the target species, wherein the donor and the target species are different. In the method, the donor may be, for example, a mouse and the target a non-murine mammal, such as but not limited to a bovine, canine, equine, or feline mammal, or a camel goat, human or sheep.
In another aspect, the present disclosure provides a method for detecting the presence or amount of NGF in a sample, comprising: providing a reagent comprising any of the above binding proteins that specifically bind NGF; combining the binding protein with the sample for a time and under conditions sufficient for the binding protein to bind to any NGF in the sample; and determining the presence or amount of NGF in the sample based on specific binding of the binding protein to NGF. In the method, the binding protein may be immobilized or may be capable of being immobilized on a solid support. In the method, the binding protein may be coupled to a detectable label, such as, for example, an imaging agent such as but not limited to a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin. The imaging agent may be for example a radiolabel selected from the group consisting of: 3H, 14C, 35S, 90Y, 99Tc, 111 In, 125I, 131I, 177Lu, 166Ho, and 153Sm.
In another aspect, the present disclosure provides an immunoassay device for detecting the presence or amount of NGF in a sample, the device comprising any of the above binding proteins that specifically bind NGF, immobilized on a solid support.
In another aspect, the present disclosure provides a kit for detecting the presence or amount of NGF in a sample, the kit comprising: an immunoreagent comprising any of the above binding proteins that specifically bind NGF and instructions for determining the presence or amount of NGF in the sample based on specific binding of the immunoreagent to NGF. In the kit, the binding protein may be immobilized on a solid support.
In still yet another aspect, the present disclosure relates to an antibody or antigen binding fragment thereof comprising:
a heavy chain variable region comprises an amino acid sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO:168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 206, SEQ ID NO: 207, or an antigen-binding or an immunologically functional immunoglobulin fragment thereof; and
a light chain variable region comprises an amino acid sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 44, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, or an antigen-binding or an immunologically functional immunoglobulin fragment thereof.
More specifically, the above-described antibody may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 50% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 50% to one of said sequences. Alternatively, the above-described antibody may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 70% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 70% to one of said sequences. Alternatively, the above-described antibody may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 80% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 80% to one of said sequences. Alternatively, the above-described antibody may comprise at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having a sequence identity of at least 90% to one of said sequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having a sequence identity of at least 90% to one of said sequences.
The above-described antibody may comprise a heavy chain human immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. More specifically, the antibody may comprise a heavy chain canine immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. Alternatively, the antibody comprises a heavy chain feline immunoglobulin constant domain. Still further alternatively, the antibody comprises a heavy chain equine immunoglobulin constant domain. Moreover, the above-described antibody may comprise a constant region having an amino acid sequence selected from the group consisting of SEQ ID NO: 52 and SEQ ID NO: 54. Still further, the above-described antibody is selected from the group consisting of: an immunoglobulin molecule, disulfide linked Fv, monoclonal antibody, scFv, chimeric antibody, single domain antibody, CDR-grafted antibody, diabody, humanized antibody, caninized mAb, canine mAb, feline mAb, felinized mAb, equine mAb, equinized mAb, a multispecific antibody, a Fab, a dual specific antibody, a DVD-Ig, a Fab′, a bispecific antibody, a F(ab′)2, and a Fv.
In another aspect, the above-identified antibody is capable of modulating a biological function of NGF.
In still yet another aspect, the present disclosure relates to an isolated nucleic acid encoding the above-described antibody.
In another aspect, the present invention relates to an antibody or antigen binding fragment thereof having a heavy chain variable region that comprises an amino acid sequence having at least 90% identity with a sequence of SEQ ID NO:37 and a light chain variable region that comprises an amino acid sequence having at least 90% identity with a sequence of SEQ ID NO:38. The above-described antibody may comprise a heavy chain human immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. More specifically, the antibody may comprise a heavy chain canine immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. Alternatively, the antibody comprises a heavy chain feline immunoglobulin constant domain. Still further alternatively, the antibody comprises a heavy chain equine immunoglobulin constant domain. Moreover, the above-described antibody may comprise a constant region having an amino acid sequence selected from the group consisting of SEQ ID NO: 52 and SEQ ID NO: 54. Still further, the above-described antibody is selected from the group consisting of: an immunoglobulin molecule, disulfide linked Fv, monoclonal antibody, scFv, chimeric antibody, single domain antibody, CDR-grafted antibody, diabody, humanized antibody, caninized mAb, canine mAb, feline mAb, felinized mAb, equine mAb, equinized mAb, a multispecific antibody, a Fab, a dual specific antibody, a DVD-Ig, a Fab′, a bispecific antibody, a F(ab′)2, and a Fv.
In another aspect, the above-identified antibody is capable of modulating a biological function of NGF.
In still yet another aspect, the present disclosure relates to an isolated nucleic acid encoding the above-described antibody.
In another aspect, the present invention relates to an antibody or antigen binding fragment thereof having a heavy chain variable region comprises an amino acid sequence having at least 90% identity with a sequence of SEQ ID NO: 192 and the light chain variable region comprises an amino acid sequence having at least 90% identity with a sequence of SEQ ID NO: 193. The above-described antibody may comprise a heavy chain human immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. More specifically, the antibody may comprise a heavy chain canine immunoglobulin constant domain selected from the group consisting of IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgE constant domain, IgG2 constant domain, IgG3 constant domain, and IgA constant domain. Alternatively, the antibody comprises a heavy chain feline immunoglobulin constant domain. Still further alternatively, the antibody comprises a heavy chain equine immunoglobulin constant domain. Moreover, the above-described antibody may comprise a constant region having an amino acid sequence selected from the group consisting of SEQ ID NO: 52 and SEQ ID NO: 54. Still further, the above-described antibody is selected from the group consisting of: an immunoglobulin molecule, disulfide linked Fv, monoclonal antibody, scFv, chimeric antibody, single domain antibody, CDR-grafted antibody, diabody, humanized antibody, caninized mAb, canine mAb, feline mAb, felinized mAb, equine mAb, equinized mAb, a multispecific antibody, a Fab, a dual specific antibody, a DVD-Ig, a Fab′, a bispecific antibody, a F(ab′)2, and a Fv.
In another aspect, the above-identified antibody is capable of modulating a biological function of NGF.
In still yet another aspect, the present disclosure relates to an isolated nucleic acid encoding the above-described antibody.
In still yet another aspect, the present disclosure relates to a pharmaceutical or diagnostic composition comprising at least one of the above-described antibodies, and a pharmaceutically acceptable carrier, diluent or excipient. More specifically, the pharmaceutical or diagnostic composition may comprise a therapeutically effective amount of at least one of the above-described antibodies. In addition, the pharmaceutical or diagnostic composition may comprise at one preservative. An example of at least one preservative that may be used is methylparaben, propylparaben, benzyl alcohol, chlorobutanol or benzalkonium chloride.
The pharmaceutical composition can have a pH of greater than about 7.0. Alternatively, the pharmaceutical composition can have a pH of between about 6.8 and about 8.2. Alternatively; the pharmaceutical composition can have a pH of between about 7.2 and about 7.8. Still further alternatively, the pH of the pharmaceutical composition can be between about 7.4 and about 7.6. Still further alternatively, the pH of the pharmaceutical composition can be about 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1 or 8.2.
The pharmaceutical composition of the present disclosure may have a half-life of from about 8.0 days to about 15.0 days when dosed intravenously or subcutaneously. Alternatively, the pharmaceutical composition of the present invention may have a half-life of from about 10.0 days to about 13.0 days. Still further alternatively, the pharmaceutical composition of the present invention may have a half-life of about 8.0 days, about 8.5 days, about 9.0 days, about 9.5 days, about 10.0 days, about 10.5 days, about 11.0 days, about 11.5 days, about 12.0 days, about 12.5 days, about 13.0 days, about 13.5 days, about 14.0 days, about 14.5 days or about 15.0 days.
In another aspect, the present disclosure relates to a method for reducing NGF activity in a subject suffering from a disorder in which NGF activity is detrimental, comprising administering to the subject a therapeutically effective amount of an antibody of antigen binding fragment thereof of at least one of the above-described antibodies or antigen-binding fragments thereof.
BRIEF DESCRIPTION OF FIGURES
FIG. 1 illustrates PR-1254972 VH nucleotide sequence (SEQ ID NO: 1) of mouse anti-NGF antibody.
FIG. 2 illustrates PR-1254972 VH amino acid sequence (SEQ ID NO: 2) of mouse anti-NGF antibody.
FIG. 3 illustrates PR-1254972 VL nucleotide sequence (SEQ ID NO: 3) of mouse anti-NGF antibody.
FIG. 4 illustrates PR-1254972 VL amino acid (SEQ ID NO: 4) of mouse anti-NGF antibody.
FIG. 5 illustrates PR-1254973 VH nucleotide sequence (SEQ ID NO: 5) of mouse anti-NGF antibody.
FIG. 6 illustrates PR-1254973 VH amino acid (SEQ ID NO: 6) of mouse anti-NGF antibody.
FIG. 7 illustrates PR-1254973 VL nucleotide sequence (SEQ ID NO: 7) of mouse anti-NGF antibody.
FIG. 8 illustrates PR-1254973 VL amino acid (SEQ ID NO: 8) of mouse anti-NGF antibody.
FIG. 9 illustrates PR-1254977 VH nucleotide sequence (SEQ ID NO: 9) of mouse anti-NGF antibody.
FIG. 10 illustrates PR-1254977 VH amino acid (SEQ ID NO: 10) of mouse anti-NGF antibody.
FIG. 11 illustrates PR-1254977 VL nucleotide sequence (SEQ ID NO: 11) of mouse anti-NGF antibody.
FIG. 12 illustrates PR-1254977 VL amino acid (SEQ ID NO: 12) of mouse anti-NGF antibody.
FIG. 13 illustrates PR-1254980 VH nucleotide sequence (SEQ ID NO: 13) of mouse anti-NGF antibody.
FIG. 14 illustrates PR-1254980 VH amino acid (SEQ ID NO: 14) of mouse anti-NGF antibody.
FIG. 15 illustrates PR-1254980 VL nucleotide sequence (SEQ ID NO: 15) of mouse anti-NGF antibody.
FIG. 16 illustrates PR-1254980 VL amino acid (SEQ ID NO: 16) of mouse anti-NGF antibody.
FIG. 17 illustrates PR-1254981 VH nucleotide sequence (SEQ ID NO: 17) of mouse anti-NGF antibody.
FIG. 18 illustrates PR-1254981 VH amino acid (SEQ ID NO: 18) of mouse anti-NGF antibody.
FIG. 19 illustrates PR-1254981 VL nucleotide sequence (SEQ ID NO: 19) of mouse anti-NGF antibody.
FIG. 20 illustrates PR-1254981 VL amino acid (SEQ ID NO: 20) of mouse anti-NGF antibody.
FIG. 21 illustrates PR-1254982 VH nucleotide sequence (SEQ ID NO: 21) of mouse anti-NGF antibody.
FIG. 22 illustrates PR-1254982 VH amino acid (SEQ ID NO: 22) of mouse anti-NGF antibody.
FIG. 23 illustrates PR-1254982 VL nucleotide sequence (SEQ ID NO: 23) of mouse anti-NGF antibody.
FIG. 24 illustrates PR-1254982 VL amino acid (SEQ ID NO: 24) of mouse anti-NGF antibody.
FIG. 25 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 25 (72.1 VH amino acid).
FIG. 26 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 26 (72.1 VL amino acid).
FIG. 27 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined) SEQ ID NO: 27 (73.1 VH amino acid).
FIG. 28 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined) SEQ ID NO: 28 (73.1 VL amino acid).
FIG. 29 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 29 (77.1 VH amino acid).
FIG. 30 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 30 (77.1 VL amino acid).
FIG. 31 A illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 31 (81.1 VH amino acid).
FIG. 31 B illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 177 (81.1B VH amino acid).
FIG. 32 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 32 (81.1 VL amino acid)
FIG. 33 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 33 (82.1 VH amino acid)
FIG. 34 illustrates mouse anti-NGF mAb caninized by CDR grafting onto canine Ig frameworks (CDRs are underlined), SEQ ID NO: 34 (82.1 VL amino acid).
FIG. 35 illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 35 (72.2 VH amino acid).
FIG. 36A illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 36 (72.2 VL amino acid).
FIG. 36B illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 179 (72.3 VH amino acid).
FIG. 36C illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 180 (72.4 VH amino acid).
FIG. 36D illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 181 (72.4 VL amino acid).
FIG. 37 illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 37 (73.2 VH amino acid).
FIG. 38A illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 38 (73.2 VL amino acid).
FIG. 38B illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 182 (73.4 VH amino acid).
FIG. 38C illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 183 (73.4 VL amino acid).
FIG. 39 illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 39 (77.2 VH amino acid).
FIG. 40A illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 40 (77.2 VL amino acid).
FIG. 40B illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 184 (77.3 VH amino acid).
FIG. 40C illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 185 (77.4 VH amino acid).
FIG. 40D illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 186 (77.4 VL amino acid).
FIG. 41 illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 41 (81.2 VH amino acid).
FIG. 42A illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 42 (81.2 VL amino acid).
FIG. 42B illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 187 (81.4 VH amino acid).
FIG. 42C illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 188 (81.4 VL amino acid).
FIG. 42D illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 189 (81.2B VH amino acid).
FIG. 42E illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 190 (81.4B VH amino acid).
FIG. 42F illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold, SEQ ID NO: 206 (81.5B VH amino acid).
FIG. 42G illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold, SEQ ID NO: 207 (81.6B VH amino acid).
FIG. 43 illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 43 (82.2 VH amino acid).
FIG. 44A illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 44 (82.2 VL amino acid).
FIG. 44B illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 191 (82.3 VL amino acid).
FIG. 44C illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 192 (82.4 VH amino acid).
FIG. 44D illustrates caninized anti-NGF antibodies containing back mutation residues (backmutation residues shown in bold), SEQ ID NO: 193 (82.4 VL amino acid).
FIG. 45 illustrates primer sequence to clone canine NGF, SEQ ID NO: 45 (NGF-Dog-S primer).
FIG. 46 illustrates primer sequence to clone canine NGF, SEQ ID NO: 46 (NGF-Dog-AS primer).
FIG. 47 illustrates primer sequence to clone canine NGF, SEQ ID NO: 47 (NGF-d-Ec-S primer).
FIG. 48 illustrates primer sequence to clone canine NGF, SEQ ID NO: 48 (NGF-d-Ec-AS primer).
FIG. 49 illustrates canine NGF C-terminal 6His fusion nucleotide sequence, SEQ ID NO: 49.
FIG. 50 illustrates canine NGF C-terminal 6-His amino acid sequence, SEQ ID NO: 50.
FIG. 51 illustrates canine IgG constant region nucleotide sequence, SEQ ID NO: 51.
FIG. 52 illustrates canine IgG constant region amino acid sequence, SEQ ID NO: 52.
FIG. 53 illustrates canine kappa constant region nucleotide sequence, SEQ ID NO: 53
FIG. 54 illustrates canine kappa constant region amino acid sequence, SEQ ID NO: 54.
FIG. 55 illustrates complementarity determining region, SEQ ID NO: 55 (72.1 VH amino acid; CDR1).
FIG. 56 illustrates complementarity determining region, SEQ ID NO: 56 (72.1 VH amino acid; CDR2).
FIG. 57 illustrates complementarity determining region, SEQ ID NO: 57 (72.1 VH amino acid; CDR3).
FIG. 58 illustrates complementarity determining region, SEQ ID NO: 58 (72.1 VL amino acid; CDR1).
FIG. 59 illustrates complementarity determining region, SEQ ID NO: 59 (72.1 VL amino acid; CDR2).
FIG. 60 illustrates complementarity determining region, SEQ ID NO: 60 (72.1 VL amino acid; CDR3).
FIG. 61 illustrates complementarity determining region, SEQ ID NO: 61 (73.1 VH amino acid; CDR1).
FIG. 62 illustrates complementarity determining region, SEQ ID NO: 62 (73.1 VH amino acid; CDR2).
FIG. 63 illustrates complementarity determining region, SEQ ID NO: 63 (73.1 VH amino acid; CDR3).
FIG. 64 illustrates complementarity determining region, SEQ ID NO: 64 (73.1 VL amino acid; CDR1).
FIG. 65 illustrates complementarity determining region, SEQ ID NO: 65 (73.1 VL amino acid; CDR2).
FIG. 66 illustrates complementarity determining region, SEQ ID NO: 66 (73.1 VL amino acid; CDR3).
FIG. 67 illustrates complementarity determining region, SEQ ID NO: 67 (77.1 VH amino acid; CDR1).
FIG. 68 illustrates complementarity determining region, SEQ ID NO: 68 (77.1 VH amino acid; CDR2).
FIG. 69 illustrates complementarity determining region, SEQ ID NO: 69 (77.1 VH amino acid; CDR3).
FIG. 70 illustrates complementarity determining region, SEQ ID NO: 70 (77.1 VL amino acid; CDR1).
FIG. 71 illustrates complementarity determining region, SEQ ID NO: 71 (77.1 VL amino acid; CDR2).
FIG. 72 illustrates complementarity determining region, SEQ ID NO: 72 (77.1 VL amino acid; CDR3).
FIG. 73 illustrates complementarity determining region, SEQ ID NO: 73 (81.1 VH amino acid; CDR1).
FIG. 74 illustrates complementarity determining region, SEQ ID NO: 74 (81.1 VH amino acid; CDR2).
FIG. 75 illustrates complementarity determining region, SEQ ID NO: 75 (81.1 VH amino acid; CDR3).
FIG. 76 illustrates complementarity determining region, SEQ ID NO: 76 (81.1 VL amino acid; CDR1).
FIG. 77 illustrates complementarity determining region, SEQ ID NO: 77 (81.1 VL amino acid; CDR2).
FIG. 78 illustrates complementarity determining region, SEQ ID NO: 78 (81.1 VL amino acid; CDR3).
FIG. 79 illustrates complementarity determining region, SEQ ID NO: 79 (82.1 VH amino acid; CDR1).
FIG. 80 illustrates complementarity determining region, SEQ ID NO: 80 (82.1 VH amino acid; CDR2).
FIG. 81 illustrates complementarity determining region, SEQ ID NO: 81 (82.1 VH amino acid; CDR3).
FIG. 82 illustrates complementarity determining region, SEQ ID NO: 82 (82.1 VL amino acid; CDR1).
FIG. 83 illustrates complementarity determining region, SEQ ID NO: 83 (82.1 VL amino acid; CDR2).
FIG. 84 illustrates complementarity determining region, SEQ ID NO: 84 (82.1 VL amino acid; CDR3).
FIG. 85 illustrates the sequence of human βNGF (SEQ ID NO: 85).
FIG. 86 illustrates the sequences shown in Table 14 illustrating SEQ ID NOs 178, 86-88 from canine heavy chain variable domain sequences derived from canine PBMC.
FIG. 86A illustrates the sequences shown in Table 14 illustrating SEQ ID NOs 89-93 from canine heavy chain variable domain sequences derived from canine PBMC.
FIG. 86B illustrates the sequences shown in Table 14 illustrating SEQ ID NOs 94-98 from canine heavy chain variable domain sequences derived from canine PBMC.
FIG. 86C illustrates the sequences shown in Table 14 illustrating SEQ ID NOs 99-102 from canine heavy chain variable domain sequences derived from canine PBMC.
FIG. 86D illustrates the sequences shown in Table 14 illustrating SEQ ID NOs 103-107 from canine heavy chain variable domain sequences derived from canine PBMC.
FIG. 86E illustrates the sequences shown in Table 14 illustrating SEQ ID NOs 108-109 from canine heavy chain variable domain sequences derived from canine PBMC.
FIG. 87 illustrates the sequences shown in Table 15 illustrating SEQ ID NOs 110, 111, 204, 112 from canine lambda light chain variable domain sequences derived from canine PBMC RNA.
FIG. 87A illustrates the sequences shown in Table 15 illustrating SEQ ID NOs 113-117 from canine lambda light chain variable domain sequences derived from canine PBMC RNA.
FIG. 87B illustrates the sequences shown in Table 15 illustrating SEQ ID NOs 118-122 from canine lambda light chain variable domain sequences derived from canine PBMC RNA.
FIG. 87C illustrates the sequences shown in Table 15 illustrating SEQ ID NOs 123-126 from canine lambda light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88 illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 127-131 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88A illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 132-136 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88B illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 137-141 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88C illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 142-146 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88D illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 147-151 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88E illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 152-156 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88F illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 157-161 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 88G illustrates the sequences shown in Table 16 illustrating SEQ ID NOs 162-164 from canine kappa light chain variable domain sequences derived from canine PBMC RNA.
FIG. 89 illustrates the sequences shown in Table 17 illustrating SEQ ID NOs 165-168 from mouse anti-NGF CDRs grafted onto Human Ig Frameworks (CDR-grafted Anti-NGF); CDRs underlined.
FIG. 89A illustrates the sequences shown in Table 17 illustrating SEQ ID NOs 169-173 from mouse anti-NGF CDRs grafted onto Human Ig Frameworks (CDR-grafted Anti-NGF); CDRs underlined.
FIG. 89B illustrates the sequences shown in Table 17 illustrating SEQ ID NOs 174-176 from mouse anti-NGF CDRs grafted onto Human Ig Frameworks (CDR-grafted Anti-NGF); CDRs underlined.
FIG. 90 illustrates the sequences shown in Table 18 illustrating SEQ ID NOs 194-196 from Mouse/Canine Chimeric Antibody sequences.
FIG. 90A illustrates the sequences shown in Table 18 illustrating SEQ ID NOs 197-199 from Mouse/Canine Chimeric Antibody sequences.
FIG. 90B illustrates the sequences shown in Table 18 illustrating SEQ ID NOs 200-202 from Mouse/Canine Chimeric Antibody sequences.
FIG. 90C illustrates the sequences shown in Table 18 illustrating SEQ ID NOs 203 from Mouse/Canine Chimeric Antibody sequences.
DETAILED DESCRIPTION OF THE DISCLOSURE
The disclosure describes NGF binding proteins, particularly anti-NGF antibodies, or antigen-binding portions thereof, that bind NGF. Various aspects of the disclosure relate to antibodies and antibody fragments, and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such antibodies and fragments. Methods of using the antibodies of the disclosure to detect human and canine NGF, to inhibit human and canine NGF activity, either in vitro or in vivo; and to regulate gene expression are also encompassed by the disclosure.
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.
Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturers specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
That the present disclosure may be more readily understood, select terms and phrases as used herein are defined below.
Definitions
The terms “acceptor” and “acceptor antibody” refer to the antibody or nucleic acid sequence providing or encoding at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% of the amino acid sequences of one or more of the framework regions. The term “acceptor” encompasses an antibody amino acid or nucleic acid sequence providing or encoding the constant region(s). The term also encompasses the antibody amino acid or nucleic acid sequence providing or encoding one or more of the framework regions and the constant region(s). For example, the term “acceptor” may refer to a human antibody amino acid or nucleic acid sequence that provides or encodes at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequences of one or more of the framework regions. Such an acceptor may contain at least 1, at least 2, at least 3, least 4, at least 5, or at least 10 amino acid residues that does (do) not occur at one or more specific positions of a human antibody. An acceptor framework region and/or acceptor constant region(s) may be, e.g., derived or obtained from a germline antibody gene, a mature antibody gene, a functional antibody (e.g., antibodies well-known in the art, antibodies in development, or antibodies commercially available).
The term “agonist” refers to a modulator that, when contacted with a molecule of interest, causes an increase in the magnitude of a certain activity or function of the molecule compared to the magnitude of the activity or function observed in the absence of the agonist. Particular agonists of interest may include, but are not limited to, NGF polypeptides or polypeptides, nucleic acids, carbohydrates, or any other molecules that bind to NGF.
The term “antagonist” or “inhibitor” refer to a modulator that, when contacted with a molecule of interest causes a decrease in the magnitude of a certain activity or function of the molecule compared to the magnitude of the activity or function observed in the absence of the antagonist. Particular antagonists of interest include those that block or modulate the biological or immunological activity of NGF. Antagonists and inhibitors of NGF may include, but are not limited to, proteins, nucleic acids, carbohydrates, or any other molecules, which bind to NGF.
The term “antibody” broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art Non-limiting examples are discussed herein below.
In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgAl and IgA2) or subclass.
The term “antibody conjugate” refers to a binding protein, such as an antibody, chemically linked to a second chemical moiety, such as a therapeutic or cytotoxic agent. The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials. In one aspect the therapeutic or cytotoxic agents include, but are not limited to, pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
The term “antibody construct” refers to a polypeptide comprising one or more the antigen binding portions linked to a linker polypeptide or an immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Such linker polypeptides are well known in the art (Holliger, et al., Proc. Natl. Acad. Set, 90: 6444-6448 (1993); Poljak, et al., Structure 2: 1121-1123 (1994)). An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences are known in the art; canine, equine, and feline are rarer.
The term “antibody fragments” or “antigen-binding moiety” comprises a portion of a full length antibody, generally the antigen binding or variable domain thereof. Examples of antibody fragments include Fab, Fab′, F(ab)2, Fv, scFv fragments, diabodies, linear antibodies, single-chain antibody molecules.
The term “antigen-binding portion” of an antibody (or simply “antibody portion”) refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., NGF). It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. These may also be bispecific, dual specific, or multi-specific formats; specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature, 341: 544-546 (1989); PCT publication WO 90/05144), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv) (Bird et al., Science, 242: 423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci., 85: 5879-5883 (1988)). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed.
Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (Holliger, et al., Proc. Natl. Acad. Sci., 90: 6444-6448 (1993); Poljak, et al., Structure 2: 1121-1123 (1994)). Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).
Still further, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al., Human Antibodies and Hybridomas, 6: 93-101 (1995)) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, et al., Mol. Immunol., 31: 1047-1058 (1994)). Antibody portions, such as Fab and F(ab′)2 fragments, may be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules may be obtained using standard recombinant DNA techniques, as described herein.
The term “anti-NGF antibody” refers to an antibody which is able to bind to nerve growth factor (NGF) and inhibit NGF biological activity and/or downstream pathway(s) mediated by NGF signaling. An anti-NGF antibody encompasses antibodies that block, antagonize, suppress or reduce (including significantly) NGF biological activity, including downstream pathways mediated by NGF signaling, such as receptor binding and/or elicitation of a cellular response to NGF. Anti-NGF antibodies encompass those that neutralize NGF biological activity, bind NGF and prevent NGF dimerization and/or binding to an NGF receptor (such as p75 and/or trkA), and/or bind NGF and prevent trkA receptor dimerization and/or trkA autophosphorylation. Examples of anti-NGF antibodies are provided herein.
The term “binding protein” refers to a natural or synthetic polypeptide that specifically binds to any portion of a target such as an antigen. The term “binding protein” encompasses antibodies as described herein, including an isolated antibody, antigen-binding portion thereof, or immunologically functional fragment thereof
The term “canine antibody” refers to a naturally-occurring or recombinantly produced immunoglobulin composed of amino acid sequences representative of natural antibodies isolated from canines of various breeds. Canine antibodies are antibodies having variable and constant regions derived from canine germline immunoglobulin sequences. The canine antibodies of the disclosure may include amino acid residues not encoded by canine germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “canine antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto canine framework sequences.
The term “caninization” is defined as a method for transferring non-canine antigen-binding amino acids from a donor antibody to a canine antibody acceptor framework to generate protein therapeutic treatments useful in dogs.
The term “caninized antibody” refers to antibodies which comprise heavy and light chain variable region sequences from a non-canine species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “canine-like”, i.e., more similar to canine germline variable sequences. One type of caninized antibody is a CDR-grafted antibody, in which non-canine CDR sequences are introduced into canine VH and VL sequences to replace the corresponding canine CDR sequences.
Caninized forms of non-canine antibodies provided herein are canine antibodies that contain sequence derived from a non-canine antibody. For the most part, caninized antibodies are canine antibody sequences (“acceptor” or “recipient” antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-canine species (“donor” antibody) such as mouse, rat, rabbit, cat, goat, chicken, bovine, horse, llama, camel, dromedaries, sharks, non-human primates, human, humanized, recombinant sequence, or an engineered sequence having the desired properties. In some instances, framework region (FR) residues of the canine antibody are replaced by corresponding non-canine FR residues. Furthermore, caninized antibodies may include residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. The caninized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc) of a canine antibody. Strategies for caninization of antibodies include, but are not limited to, the strategies disclosed in WO 2003/060080.
The caninized antibody is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a canine antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-canine antibody. A caninized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab′, F(ab′) 2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-canine immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a canine immunoglobulin consensus sequence. A caninized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a canine immunoglobulin. A canine or caninized antibody may contain both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. A caninized antibody may only contain a caninized light chain, or may only contain a caninized heavy chain. An exemplary caninized antibody contains a caninized variable domain of a light chain and a caninized variable domain of a heavy chain.
The term “canonical” residue refers to a residue in a CDR or framework that defines a particular canonical CDR structure as defined by Chothia et al. (J. Mol. Biol., 196:901-907 (1987); Chothia et al., J. Mol. Biol., 227:799 (1992). According to Chothia et al., critical portions of the CDRs of many antibodies have nearly identical peptide backbone conformations despite great diversity at the level of amino acid sequence. Each canonical structure specifies primarily a set of peptide backbone torsion angles for a contiguous segment of amino acid residues forming a loop.
The term “CDR” refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. The term “CDR set” refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as L11, L2 and L3 or H1, H2 and H3 where the “L” and the “H” designates the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan (FASEB J. 9: 133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Still other CDR boundary definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although certain methods described herein use Kabat or Chothia defined CDRs.
The term “CDR-grafted antibody” refers to antibodies which comprise heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of another species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) has been replaced with human CDR sequences.
The term “chimeric antibody” refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human, canine, equine, or feline constant regions. Chimeric antibodies comprise a portion of the heavy and/or light chain that is identical to or homologous with corresponding sequences from antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous with corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, exhibiting the desired biological activity (See e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
The terms “crystal” and “crystallized” refer to an antibody, or antigen binding portion thereof, that exists in the form of a crystal. Crystals are one form of the solid state of matter, which is distinct from other forms such as the amorphous solid state or the liquid crystalline state. Crystals are composed of regular, repeating, three-dimensional arrays of atoms, ions, molecules (e.g., proteins such as antibodies), or molecular assemblies (e.g., antigen/antibody complexes). These three-dimensional arrays are arranged according to specific mathematical relationships that are well-understood in the field. The fundamental unit, or building block, that is repeated in a crystal is called the asymmetric unit. Repetition of the asymmetric unit in an arrangement that conforms to a given, well-defined crystallographic symmetry provides the “unit cell” of the crystal. Repetition of the unit cell by regular translations in all three dimensions provides the crystal. See Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ea., pp. 20 1-16, Oxford University Press, New York, N.Y., (1999).
The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
The terms “donor” and “donor antibody” refer to an antibody providing one or more CDRs. A donor antibody may be an antibody from a species different from the antibody from which the framework regions are obtained or derived. In the context of a humanized antibody, the term “donor antibody” refers to a non-human antibody providing one or more CDRs. In the context of a caninized antibody, the term “donor antibody” refers to a non-canine antibody providing one or more CDRs. In the context of a felinized antibody, the term “donor antibody” refers to a non-feline antibody providing one or more CDRs. In the context of an equinized antibody, the term “donor antibody” refers to a non-equine antibody providing one or more CDRs.
The term “epitope” includes any polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitope determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and may have specific three dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. An antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
The term “equine antibody” refers to a naturally-occurring or recombinantly produced immunoglobulin composed of amino acid sequences representative of natural antibodies isolated from equines of various breeds. Equine antibodies are antibodies having variable and constant regions derived from equine germline immunoglobulin sequences. The equine antibodies of the disclosure may include amino acid residues not encoded by equine germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “equine antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto equine framework sequences.
The term “equalization” is defined as a method for transferring non-equine antigen-binding amino acids from a donor antibody to an equine antibody acceptor framework to generate protein therapeutic treatments useful in horses.
The term “equinized antibody” refers to antibodies which comprise heavy and light chain variable region sequences from a non-equine species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “equine-like”, i.e., more similar to equine germline variable sequences. One type of equinized antibody is a CDR-grafted antibody, in which non-equine CDR sequences are introduced into equine VH and VL sequences to replace the corresponding equine CDR sequences.
Equinized forms of non-equine antibodies provided herein are equine antibodies that contain sequence derived from a non-equine antibody. For the most part, equinized antibodies are equine antibody sequences (“acceptor” or “recipient” antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-equine species (“donor” antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken, bovine, horse, llama, camel, dromedaries, sharks, non-human primates, human, humanized, recombinant sequence, or an engineered sequence having the desired properties. In some instances, framework region (FR) residues of the equine antibody are replaced by corresponding non-equine FR residues. Furthermore, equinized antibodies may include residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. The equinized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc) of an equine antibody.
The equinized antibody is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of an equine antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-equine antibody. An equinized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab′, F(ab′)2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-equine immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of an equine immunoglobulin consensus sequence. An equinized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of an equine immunoglobulin. An equine or equinized antibody for example may contain both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. An equinized antibody may only contain an equinized light chain, or an equinized heavy chain. An exemplary equinized antibody contains an equinized variable domain of a light chain an equinized variable domain of a heavy chain. Equine isotypes include, for example, IgGa, IgGb, IgGc, IgG (T), IgM, and IgA
The term “Fab” refers to antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to readily crystallize. Pepsin treatment yields a binding cross-linking antigen. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteine(s) from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
The term “feline antibody” refers to a naturally-occurring or recombinantly produced immunoglobulin composed of amino acid sequences representative of natural antibodies isolated from felines of various breeds. Feline antibodies are antibodies having variable and constant regions derived from feline germline immunoglobulin sequences. The feline antibodies of the disclosure may include amino acid residues not encoded by feline germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “feline antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto feline framework sequences.
The term “felinization” is defined as a method for transferring non-feline antigen-binding amino acids from a donor antibody to a feline antibody acceptor framework to generate protein therapeutic treatments useful in cats.
The term “felinized antibody” refers to antibodies which comprise heavy and light chain variable region sequences from a non-feline species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “feline-like”, i.e., more similar to feline germline variable sequences. One type of felinized antibody is a CDR-grafted antibody, in which non-feline CDR sequences are introduced into feline VH and VL sequences to replace the corresponding feline CDR sequences.
Felinized forms of non-feline antibodies provided herein are feline antibodies that contain sequence derived from a non-feline antibody. For the most part, felinized antibodies are feline antibody sequences (“acceptor” or “recipient” antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-feline species (“donor” antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken, bovine, horse, llama, camel, dromedaries, sharks, non-human primates, human, humanized, recombinant sequence, or an engineered sequence having the desired properties. In some instances, framework region (FR) residues of the feline antibody are replaced by corresponding non-feline FR residues. Furthermore, felinized antibodies may include residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. The felinized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc) of a feline antibody.
The felinized antibody is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a feline antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-feline antibody. A felinized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab′, F(ab′) 2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-feline immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a feline immunoglobulin consensus sequence. A felinized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a feline immunoglobulin. A feline or felinized antibody may contain both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. A felinized antibody may only contain a felinized light chain or a felinized heavy chain. An exemplary felinized antibody only contains a felinized variable domain of a light chain and a felinized variable domain of a heavy chain.
The term “framework” or “framework sequence” refers to the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence may be determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, -L2, and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy chain) also divide the framework regions on the light chain and the heavy chain into four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying the particular sub-regions as FR1, FR2, FR3 or FR4, a framework region, as referred by others, represents the combined FR's within the variable region of a single, naturally occurring immunoglobulin chain. An FR represents one of the four sub-regions, and FRs represents two or more of the four sub-regions constituting a framework region. Human heavy chain and light chain acceptor sequences are known in the art. Canine heavy chain and light chain acceptor sequences are also known (patent application publication WO03/060080 and U.S. Pat. No. 7,261,890B2).
The term “germline antibody gene” or “gene fragment” refers to an immunoglobulin sequence encoded by non-lymphoid cells that have not undergone the maturation process that leads to genetic rearrangement and mutation for expression of a particular immunoglobulin (Shapiro et al., Crit. Rev. Immunol. 22(3): 183-200 (2002); Marchalonis et al, Adv Exp Med Biol. 484: 13-30 (2001)). One of the advantages provided by the binding proteins of the present disclosure stems from the recognition that germline antibody genes are more likely than mature antibody genes to conserve essential amino acid sequence structures characteristic of individuals in the species, hence less likely to be recognized as from a foreign source when used therapeutically in that species.
The term “Fv” refers to the minimum antibody fragment that contains a complete antigen-recognition and binding site. This region consists of a dimer of one heavy chain and one light chain variable domain.
The term “human antibody” refers to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “human antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
The term “humanized antibody” refers to antibodies which comprise heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “human-like”, i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which non-human CDR sequences are introduced into human VH and VL sequences to replace the corresponding human CDR sequences.
The humanized antibody is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody. A humanized antibody comprises substantially all, or at least one, and typically two, variable domains (Fab, Fab′, F(ab′) 2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. A humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. A humanized or caninized antibody may contain both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Alternatively, a humanized antibody may only contain a humanized light chain, or a humanized heavy chain. An exemplary humanized antibody contains a humanized variable domain of a light chain and a humanized variable domain of a heavy chain.
The bovanized, camelized, caninized, equinized, felinized, or humanized antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including without limitation IgG1, IgG2, IgG3 and IgG4. The bovanized, camelized, caninized, equinized, felinized, or humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions using techniques well-known in the art.
The framework and CDR regions of a bovanized, camelized, caninized, equinized, felinized, or humanized antibody need not correspond precisely to the parental sequences, e.g., the donor antibody CDR or the consensus framework may be mutagenized by substitution, insertion and/or deletion of at least one amino acid residue so that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. Such mutations, however, will not be extensive. Usually, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and at least 95% of the bovanized, camelized, caninized, equinized, felinized, or humanized antibody residues will correspond to those of the parental FR and CDR sequences. The term “consensus framework” refers to the framework region in the consensus immunoglobulin sequence. The term “consensus immunoglobulin sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (See e.g., Winnaker, From Genes to Clones (Veriagsgesellschaft, Weinheim, Germany 1987). In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either may be included in the consensus sequence.
The term “hypervariable region” refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a “complementarity determining region” or “CDR” in the light chain variable domain and in the heavy chain variable domain as defined by Kabat et al., 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and/or as defined by (Chothia and Lesk, Mol. Biol. 196:901-917 (1987) and/or as defined as “AbM loops” by Martin, et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989) and/or as defined by Lefranc et al., Nucleic Acids Res, 27:209-212 (1999) in the international ImMunoGeneTics information systems database. “Framework” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.
The term “identity” refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing their sequences thereof, wherein “identity” refers more specifically to the degree of sequence relatedness between nucleic acid molecules or polypeptides, as determined by the match between strings of two or more nucleotide or two or more amino acid sequences. “Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., “algorithms”). The term “similarity” is used to refer to a related concept with respect to the relationship of two or more nucleic acid molecules or two or more polypeptide molecules. In contrast to “identity,” “similarity” refers to a measure of relatedness, which includes both identical matches and conservative substitution matches. For example, for two polypeptide sequences that have 50/100 identical amino acids, and the remainder are all non-conservative substitutions, then the percent identity and similarity would both be 50%. With respect to the same two sequences, if 25 more positions had conservative substitutions, then the percent identity remains 50%, while percent similarity would be 75% (75/100). Identity and similarity of related nucleic acids and polypeptides may be readily calculated by methods well known and readily available in the art, including but are not limited to, those described in COMPUTATIONAL MOLECULAR BIOLOGY, (Lesk, A. M., ed.), 1988, Oxford University Press, New York; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, (Smith, D. W., ed.), 1993, Academic Press, New York; COMPUTER ANALYSIS OF SEQUENCE DATA, Part 1, (Griffin, A. M., and Griffin, H. G., eds.), 1994, Humana Press, New Jersey; von Heinje, G., SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, 1987, Academic Press; SEQUENCE ANALYSIS PRIMER, (Gribskov, M. and Devereux, J., eds.), 1991, M. Stockton Press, New York; Carillo et al., 1988, SIAM J. Applied Math., 48:1073; and Durbin et al., 1998, BIOLOGICAL SEQUENCE ANALYSIS, Cambridge University Press.
Preferred methods to determine identity are designed to provide the highest match between the compared sequences, and are well described in readily publicly available computer programs. Preferred such computerized methods for determining identity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux et al., 1984, Nucl Acid. Res., 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., 1990, J. Mol Biol., 215:403-410). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., 1990, supra). The well-known Smith Waterman algorithm may also be used to determine identity.
The terms “individual,” “patient,” and “subject” are used interchangeably herein, to refer to mammals, including, but not limited to, humans, murines, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian farm and agricultural animals, mammalian sport animals, and mammalian pets. Exemplary subjects companion animals, such as a dog, cat or horse.
An “isolated antibody” refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds NGF is substantially free of antibodies that specifically bind antigens other than NGF). An isolated antibody that specifically binds NGF may, however, have cross-reactivity to other antigens, such as NGF molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals. The terms “isolated polynucleotide” and “isolated nucleic acid” as used interchangeably herein refer to a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a another polynucleotide to which it is not linked in nature, or is not found in nature within a larger sequence. The term “isolated protein” or “isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation is not associated with naturally associated components that accompany it in its native state; is substantially free of other proteins from the same species; is expressed by a cell from a different species; or does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
The term “Kd” refers to the dissociation constant of a particular antibody-antigen interaction as is known in the art.
The term “Kon” is refers to the on rate constant for association of an antibody to the antigen to form the antibody/antigen complex as is known in the art.
The term “Koff” refers to the off rate constant for dissociation of an antibody from the antibody/antigen complex as is known in the art.
The terms “Kabat numbering”, “Kabat definitions” and “Kabat labeling” are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e. hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al., Ann. NY Acad, Sci., 190:382-391 (1971); and Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991)). For the heavy chain variable region, the hypervariable region ranges from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3. For the light chain variable region, the hypervariable region ranges from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.
The term “key residue” refers to certain residues within the variable region that have more impact on the binding specificity and/or affinity of an antibody, in particular a mammalized antibody such as humanized, caninized, equinized or felinized antibody. A key residue includes, but is not limited to, one or more of the following: a residue that is adjacent to a CDR, a potential glycosylation site (may be either N- or O-glycosylation site), a rare residue, a residue capable of interacting with the antigen, a residue capable of interacting with a CDR, a canonical residue, a contact residue between heavy chain variable region and light chain variable region, a residue within the Vernier zone, and a residue in the region that overlaps between the Chothia definition of a variable heavy chain CDR1 and the Kabat definition of the first heavy chain framework.
The term “labeled binding protein” refers to a protein with a label incorporated that provides for the identification of the binding protein. In one aspect, the label is a detectable marker, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that may be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that may be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 165Ho, 153Sm); fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents, such as gadolinium chelates.
The term “mammalization” refers to a method for transferring donor antigen-binding information to a mammalian antibody acceptor to generate useful therapeutic treatments. More specifically, the invention provides methods for felinization, equinization and caninization of antibodies.
The term “mammalized antibody” refers to antibodies which comprise heavy and light chain variable region sequences from a mammal species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more like “mammal of interest,” see for example, humanized, caninized, equinized or felinized antibodies defined herein. Such mammalized antibodies include, but are not limited to, bovanized, camelized, caninized, equinized, felinized, or humanized antibodies.
The terms “modulate” and “regulate” are used interchangeably and refer to a change or an alteration in the activity of a molecule of interest (e.g., the biological activity of NGF). Modulation may be an increase or a decrease in the magnitude of a certain activity or function of the molecule of interest. Exemplary activities and functions of a molecule include, but are not limited to, binding characteristics, enzymatic activity, cell receptor activation, and signal transduction.
The term “modulator” is a compound capable of changing or altering an activity or function of a molecule of interest (e.g., the biological activity of NGF). For example, a modulator may cause an increase or decrease in the magnitude of a certain activity or function of a molecule compared to the magnitude of the activity or function observed in the absence of the modulator. A modulator may be an inhibitor, which decreases the magnitude of at least one activity or function of a molecule. Exemplary inhibitors include, but are not limited to, proteins, peptides, antibodies, peptibodies, carbohydrates or small organic molecules. Peptibodies are described, e.g., in WO01/83525.
The term “monoclonal antibody” refers to an antibody that is derived from a single done, including any eukaryotic, prokaryotic, or phage done; and not the method by which it is produced and is not limited to antibodies produced through hybridoma technology.
The term “multivalent binding protein” is used in this specification to denote a binding protein comprising two or more antigen binding sites. The multivalent binding protein is engineered to have the three or more antigen binding sites, and is generally not a naturally occurring antibody. The term “multispecific binding protein” refers to a binding protein capable of binding two or more related or unrelated targets. Dual variable domain (DVD) binding proteins are binding proteins that comprise two or more antigen binding sites and are tetravalent or multivalent binding proteins. Such DVDs may be monospecific, i.e. capable of binding one antigen or multispecific, i.e. capable of binding two or more antigens. DVD binding proteins comprising two heavy chain DVD polypeptides and two light chain DVD polypeptides are referred to a DVD Ig. Each half of a DVD Ig comprises a heavy chain DVD polypeptide, and a light chain DVD polypeptide, and two antigen binding sites. Each binding site comprises a heavy chain variable domain and a light chain variable domain with a total of 6 CDRs involved in antigen binding per antigen binding site. DVD binding proteins and methods of making DVD binding proteins are disclosed in U.S. patent application Ser. No. 11/507,050 and incorporated herein by reference.
One aspect of the disclosure pertains to a DVD binding protein comprising binding proteins capable of binding NGF. In another aspect, the DVD binding protein is capable of binding NGF and a second target.
The terms “nerve growth factor” and “NGF” refer to nerve growth factor and variants thereof that retain at least part of the biological activity of NGF. NGF includes all mammalian species of native sequence NGF, including murine, rat, human, rabbit, canine, feline, equine, or bovine.
TABLE 1
Sequence of NGF
Protein
Sequence Identifier
Canine NGF C-terminal 6-His
SEQ ID NO: 50
Human NGF
SEQ ID NO: 85
The term “NGF receptor” refers to a polypeptide that is bound by or activated by NGF. NGF receptors include the TrkA receptor and the p75 receptor of any mammalian species, including, but are not limited to, human, canine, feline, equine, primate, or bovine.
The terms “NGF-related disease” and “NGF-related disorder” encompass any disease or disorder in which the activity of NGF in a subject suffering from the disease or disorder has been shown to be or is suspected of being either responsible for the pathophysiology of the disease or disorder, or a factor that contributes to a worsening of the disease or disorder, which may occur as a result of increased levels of NGF or increased sensitivity of the subject to NGF. Accordingly, an NGF-related disease or NGF-related disorder is a disease or disorder in which reduction of NGF activity is expected to alleviate the symptoms and/or progression of the disease or disorder. Such diseases and disorders may be evidenced, for example, by an increase in the concentration of NGF in a biological fluid of a subject suffering from the disorder (e.g., an increase in the concentration of NGF in serum, plasma, synovial fluid, etc. of the subject), which may be detected, for example, using an anti-NGF antibody as described above. Non-limiting examples of diseases and disorders that may be treated with the antibodies of the disclosure include those diseases and disorders discussed in the section below pertaining to pharmaceutical compositions of the antibodies of the disclosure, and encompass acute pain resulting for example from surgery or other trauma, and chronic pain.
The term “neutralizing” refers to neutralization of biological activity of a NGF when a binding protein specifically binds NGF. A neutralizing binding protein is a neutralizing antibody, who's binding to NGF results in inhibition of a biological activity of NGF. The neutralizing binding protein binds NGF and reduces a biologically activity of NGF by at least about 20%, 40%, 60%, 80%, 85% or more. Inhibition of a biological activity of NGF by a neutralizing binding protein may be assessed by measuring one or more indicators of NGF biological activity well known in the art, including cell proliferation, cell morphology changes, cell signaling, or any detectable cellular response resulting from binding of NGF to the TrkA receptor.
The term “operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. “Operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. The term “expression control sequence” refers to polynucleotide sequences which are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term “control sequences” is intended to include components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
The term “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody or antibody portion.
The term “polynucleotide” means a polymeric form of two or more nucleotides, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA. The term “isolated polynucleotide” shall mean a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or some combination thereof) that, by virtue of its origin, the “isolated polynucleotide” is not associated with all or a portion of a polynucleotide with which the “isolated polynucleotide” is found in nature; is operably linked to a polynucleotide that it is not linked to in nature; or does not occur in nature as part of a larger sequence.
The term “polypeptide” refers to any polymeric chain of amino acids. The terms “peptide” and “protein” are used interchangeably with the term polypeptide and also refer to a polymeric chain of amino acids. The term “polypeptide” encompasses native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. A polypeptide may be monomelic or polymeric.
The term “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.
The term “recombinant host cell” (or simply “host cell”) is intended to refer to a cell into which exogenous DNA has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but, to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell”. In one aspect, host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life. Eukaryotic cells include protist, fungal, plant and animal cells. In another aspect host cells include, but are not limited to, the prokaryotic cell line E. coli; mammalian cell lines CHO, HEK 293 and COS; the insect cell line Sf9; and the fungal cell Saccharomyces cerevisiae.
Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.
The term “recombinant antibody” refers to all species of antibodies or immunoglobulins that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library (Hoogenboom, TIB Tech., 15: 62-70 (1997); Azzazy et al., Clin. Biochem., 35: 425-445 (2002); Gavilondo et al., BioTechniques, 29: 128-145 (2002); Hoogenboom et al., Immunology Today, 21: 371-378 (2000)), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann et al., Current Opinion in Biotechnology, 13: 593-597 (2002); Little et al., Immunology Today, 21: 364-370 (2000)) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies have variable and constant regions derived from species-specific germline immunoglobulin sequences. Such recombinant antibodies may be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to species-specific germline VH and VL sequences, may not naturally exist within the antibody germline repertoire in vivo.
The term “recovering” refers to the process of rendering a chemical species such as a polypeptide substantially free of naturally associated components by isolation, e.g., using protein purification techniques well known in the art.
The term “sample” is used in its broadest sense. A “biological sample” includes, but is not limited to, any quantity of a substance from a living thing or formerly living thing. Such living things include, but are not limited to, humans, mice, rats, monkeys, dogs, rabbits and other animals. Such substances include, but are not limited to, blood, serum, urine, synovial fluid, cells, organs, tissues, bone marrow, lymph nodes and spleen.
The term “single-chainFv” or “scFv” refers to antibody fragments comprising the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).
The terms “specific binding” or “specifically binding” in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.
The term “substantially” in the context of a CDR refers to a CDR having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR.
The term “surface plasmon resonance” refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For further descriptions (Jonsson, et al. Ann. Biol. Clin. 51: 19-26 (1993); Jonsson, et al., Biotechniques 11: 620-627 (1991); Johnsson, et al., J. Mol. Recognit. 8: 125-131 (1995); and Johnnson, B., et al, Anal. Biochem., 198: 268-277 (1991)).
The term “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount may be the amount and/or duration of a therapy which is sufficient to reduce or ameliorate the severity and/or duration of a disorder or one or more symptoms thereof, prevent the advancement of a disorder, cause regression of a disorder, prevent the recurrence, development, onset or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy (e.g., prophylactic or therapeutic agent). A therapeutically effective amount of the antibody or antibody portion may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody, or antibody portion, are outweighed by the therapeutically beneficial effects.
The term “transformation” refers to any process by which exogenous DNA enters a host cell. Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, lipofection, and particle bombardment. Such “transformed” cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. They also include cells which transiently express the inserted DNA or RNA for limited periods of time.
The term “transgenic organism” refers to an organism having cells that contain a transgene, wherein the transgene introduced into the organism (or an ancestor of the organism) expresses a polypeptide not naturally expressed in the organism. A “transgene” is a DNA construct, which is stably and operably integrated into the genome of a cell from which a transgenic organism develops, directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic organism.
The term “vector” is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The term “Vernier zone” refers to a subset of framework residues that may adjust CDR structure and fine-tune the fit to antigen as described by Foote and Winter (1992, J. Mol. Biol. 224:487-499, which is incorporated herein by reference). Vernier zone residues form a layer underlying the CDRs and may impact on the structure of CDRs and the affinity of the antibody.
Anti NGF Binding Proteins
The present disclosure provides a novel family of binding proteins, murine antibodies, CDR grafted antibodies, mammalized (bovanized, camelized, caninized, equinized, felinized, or humanized) antibodies, and fragments thereof, capable of binding and modulating the biological activity or function of NGF, including the capability of neutralizing NGF. The disclosure thus also provides a therapeutic means with which to inhibit NGF and provides compositions and methods for treating disease associated with increased levels of NGF, particularly a disease, condition or disorder where increased levels of NGF, as compared to NGF levels observed in comparable normal subjects, is detrimental.
Binding proteins of the present disclosure may be made by any of a number of techniques known in the art and as described herein, including culturing a host cell described herein in culture medium under conditions sufficient to produce a binding protein capable of binding NGF.
Monoclonal antibodies may be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies may be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981).
Methods for producing and screening for specific antibodies using hybridoma technology are well known in the art. Such methods include, for example, culturing a hybridoma cell secreting an antibody of the disclosure wherein the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the disclosure with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the disclosure. Briefly, for example, mice may be immunized with an NGF antigen. The NGF antigen may be administered, with or without an adjuvant, to stimulate the immune response. Such adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulating complexes). Such adjuvants may protect the polypeptide from rapid dispersal by sequestering it in a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. If a polypeptide is being administered, the immunization schedule will involve two or more administrations of the polypeptide, spread out over several weeks.
After immunization of an animal with an NGF antigen, antibodies and/or antibody-producing cells may be obtained from the animal. An anti-NGF antibody-containing serum may be obtained from the animal by bleeding or sacrificing the animal. The serum may be used as it is obtained from the animal, an immunoglobulin fraction may be obtained from the serum, or the anti-NGF antibodies may be purified from the serum. Serum or immunoglobulins obtained in this manner are polyclonal, thus having a heterogeneous array of properties.
Once an immune response is detected, e.g., antibodies specific for the antigen NGF are detected in the mouse serum, the mouse spleen may be harvested and splenocytes isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, such as, for example, cells from cell line SP20 available from the ATCC. Hybridomas may be selected and cloned by limited dilution. The hybridoma clones may then be assayed by methods known in the art for cells that secrete antibodies capable of binding NGF. Ascites fluid, which generally contains high levels of antibodies, may be generated by immunizing mice with positive hybridoma clones.
Antibody-producing immortalized hybridomas may be prepared from the immunized animal. After immunization, the animal may be sacrificed and the splenic B cells fused to immortalized myeloma cells as is well known in the art (Harlow et al., supra). Alternatively, the myeloma cells may be from a non-secretory cell line and do not secrete immunoglobulin polypeptides. After fusion and antibiotic selection, the hybridomas may be screened using NGF, or a portion thereof, or a cell expressing NGF. Initial screening may be performed, for example, using an enzyme-linked immunoassay (ELISA) or a radioimmunoassay (PJA). An example of ELISA screening is provided in WO 00/37504.
Anti-NGF antibody-producing hybridomas may be selected, cloned and further screened for desirable characteristics, including robust hybridoma growth, high antibody production and desirable antibody characteristics, as discussed further below. Hybridomas may be cultured and expanded in vivo in syngeneic animals, in animals that lack an immune system, e.g., nude mice, or in cell culture in vitro. Methods of selecting, cloning and expanding hybridomas are well known to those of ordinary skill in the art.
An exemplary animal system for preparing hybridomas is the mouse. Hybridoma production in the mouse is very well established, and immunization protocols and techniques for isolation of immunized splenocytes for fusion are well known. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known. Alternatively, the hybridomas may be produced in a non-human, non-mouse species such as a rat, sheep, pig, goat, cattle or horse. Alternatively, human hybridomas may be produced, in which a human non-secretory myeloma is fused with a human cell expressing an anti-NGF antibody.
Antibody fragments that recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab′)2 fragments of the disclosure may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.
Recombinant antibodies may be generated from single, isolated lymphocytes using a procedure referred to in the art as the selected lymphocyte antibody method (SLAM), as described in U.S. Pat. No. 5,627,052, PCT Publication WO 92/02551 and Babcock et al., Proc. Natl. Acad. Sci, 93: 7843-7848 (1996). In this method, single cells secreting antibodies of interest, e.g., lymphocytes derived from any one of the immunized animals described in Section 1, are screened using an antigen-specific hemolytic plaque assay, wherein the antigen NGF, or a fragment thereof, is coupled to sheep red blood cells using a linker, such as biotin, and used to identify single cells that secrete antibodies with specificity for NGF. Following identification of antibody-secreting cells of interest, heavy- and light-chain variable region cDNAs may be rescued from the cells by reverse transcriptase-PCR and these variable regions may then be expressed, in the context of appropriate immunoglobulin constant regions (e.g., human constant regions), in mammalian host cells, such as COS or CHO cells. The host cells transfected with the amplified immunoglobulin sequences, derived from in vivo selected lymphocytes, may then undergo further analysis and selection in vitro, for example by panning the transfected cells to isolate cells expressing antibodies to NGF. The amplified immunoglobulin sequences further may be manipulated in vitro, such as by in vitro affinity maturation methods such as those described in PCT Publication WO 97/29131 and PCT Publication WO 00/56772.
Antibodies may be produced by immunizing a non-human animal comprising some or all of the human immunoglobulin loci with an NGF antigen. For example, human monoclonal antibodies directed against NGF may be generated using transgenic mice carrying parts of the human immune system rather than the mouse system, referred to in the literature and herein as “HuMab” mice, contain a human immunoglobulin gene minilocus that encodes unrearranged human heavy (μ and γ) and K light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and κ chain loci (Lonberg et al., 1994, Nature 368:856-859). These mice exhibit reduced expression of mouse IgM or κ and in response to immunization, and the introduced human heavy chain and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgG K monoclonal antibodies. The preparation of HuMab mice is well described in the literature. (See, e.g., Lonberg et al., 1994, Nature 368:856-859; Lonberg, 1994, Handbook of Exp. Pharmacology 113:49-101; Taylor et al., 1994, International Immunology 6:579-591; Lonberg & Huszar, 1995, Intern. Rev. Immunol. 13:65-93; and Harding & Lonberg, 1995, Ann. N.Y. Acad. Sci 764:536-546). Alternatively, other known mouse strains such as the HCo7, HCo12, and KM transgenic mice strains may be used to generate human anti-NGF antibodies.
Another suitable, though non-limiting example of a transgenic mouse is the XENOMOUSE® transgenic mouse, which is an engineered mouse strain that comprises large fragments of the human immunoglobulin loci and is deficient in mouse antibody production. See, e.g., Green et al. Nature Genetics, 7: 13-21 (1994); and U.S. Pat. Nos. 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598 and 6,130,364; WO 91/10741, WO 94/02602, WO 96/34096, WO 96/33735, WO 98/16654, WO 98/24893, WO 98/50433, WO 99/45031, WO 99/53049, WO 00 09560, and WO 00/037504. The XENOMOUSE® transgenic mouse produces an adult-like human repertoire of fully human antibodies, and generates antigen-specific human mAbs. The XENOMOUSE® transgenic mouse contains approximately 80% of the human antibody repertoire through introduction of megabase sized, germline configuration YAC fragments of the human heavy chain loci and light chain loci (Mendez et al., Nature Genetics 15: 146-156 (1997), Green et al., J. Exp. Med., 188: 483-495 (1998)).
In vitro methods also may be used to make the antibodies of the disclosure, wherein an antibody library is screened to identify an antibody having the desired binding specificity. Methods for such screening of recombinant antibody libraries are well known in the art and include methods described in, for example, U.S. Pat. No. 5,223,409; PCT Publication Nos. WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; Fuchs et al. Bio/Technology, 9: 1370-1372 (1991); Hay et al., Hum Antibody Hybridomas, 3: 81-85 (1992); Huse et al., Science, 246: 1275-1281 (1989); McCafferty et al., Nature, 348: 552-554 (1990); Griffiths et al., EMBO J., 12: 725-734 (1993); Hawkins et al., J o/Biol, 226: 889-896 (1992); Clackson et al., Nature, 352: 624-628 (1991); Gram et al., PNAS, 89: 3576-3580 (1992); Garrad et al., Bio/Technology, 9: 1373-1377 (1991); Hoogenboom et al., Nuc Acid Res, 19: 4133-4137 (1991); and Barbas et al., PNAS, 88: 7978-7982 (1991), US patent application publication 20030186374, and PCT Publication No. WO 97/29131.
The recombinant antibody library may be from a subject immunized with NGF, or a portion of NGF. Alternatively, the recombinant antibody library may be from a naive subject that has not been immunized with NGF, such as a canine antibody library from a canine subject that has not been immunized with canine NGF. Antibodies of the disclosure are selected by screening the recombinant antibody library with the peptide comprising canine NGF to thereby select those antibodies that recognize NGF. Methods for conducting such screening and selection are well known in the art, such as described in the references in the preceding paragraph. To select antibodies of the disclosure having particular binding affinities for hNGF, such as those that dissociate from canine NGF with a particular koff rate constant, the art-known method of surface plasmon resonance may be used to select antibodies having the desired koff rate constant. To select antibodies of the disclosure having a particular neutralizing activity for hNGF, such as those with a particular an IC5o, standard methods known in the art for assessing the inhibition of hNGF activity may be used.
For example, the antibodies of the present disclosure may also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular aspect, such phage may be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e. g., canine, human or murine). Phage expressing an antigen binding domain that binds the antigen of interest may be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and MI 3 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that may be used to make the antibodies of the present disclosure include those disclosed in Brinkman et al., J. Immunol. Methods, 182: 41-50 (1995); Ames et al., J. Immunol. Methods, 184: 177-186 (1995); Kettleborough et al., Eur. J. Immunol, 24:952-958 (1994); Persic et al., Gene, 187: 9-18 (1997); Burton et al., Advances in Immunology, 57: 191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780, 225; 5,658,727; 5,733,743 and 5,969,108.
As described in the above references, after phage selection, the antibody coding regions from the phage may be isolated and used to generate whole antibodies including human antibodies or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments may also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques, 12(6):864-869 (1992); and Sawai et al., AJPJ 34:26-34 (1995); and Better et al., Science, 240: 1041-1043 (1988) (said references incorporated by reference in their entireties).
Examples of techniques which may be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203:46-88 (1991); Shu et al., PNAS, 90:7995-7999 (1993); and Skerra et al., Science, 240: 1038-1040 (1988).
Alternatives to screening of recombinant antibody libraries by phage display are known and include other methodologies for screening large combinatorial libraries which may be applied to the identification of dual specificity antibodies of the disclosure. One type of alternative expression system is one in which the recombinant antibody library is expressed as RNA-protein fusions, as described in PCT Publication No. WO 98/31700, and in Roberts et al., Proc. Natl. Acad. Sci., 94: 12297-12302 (1997). In this system, a covalent fusion is created between a mRNA and the peptide or protein that it encodes by in vitro translation of synthetic mRNAs that carry puromycin, a peptidyl acceptor antibiotic, at their 3′ end. Thus, a specific mRNA may be enriched from a complex mixture of mRNAs (e.g., a combinatorial library) based on the properties of the encoded peptide or protein, e.g., antibody, or portion thereof, such as binding of the antibody, or portion thereof, to the dual specificity antigen. Nucleic acid sequences encoding antibodies, or portions thereof, recovered from screening of such libraries may be expressed by recombinant means as described above (e.g., in mammalian host cells) and, moreover, may be subjected to further affinity maturation by either additional rounds of screening of mRNA-peptide fusions in which mutations have been introduced into the originally selected sequence(s), or by other methods for affinity maturation in vitro of recombinant antibodies, as described above.
In another approach the antibodies of the present disclosure may also be generated or affinity matured using yeast display methods known in the art. In yeast display methods, genetic methods are used to tether antibody domains to the yeast cell wall and display them on the surface of yeast. In particular, such yeast may be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e. g., human or murine). Examples of yeast display methods that may be used to make the antibodies of the present disclosure include those disclosed Wittrup, et al. U.S. Pat. No. 6,699,658 incorporated herein by reference.
The antibodies or antigen binding fragments described herein may also be produced by genetic engineering. For example, the technology for expression of both heavy and light chain genes in E. coli is the subject of the PCT patent applications: publication number WO 901443, WO901443, and WO 9014424 and in Huse et al., 1989 Science 246: 1275-81. The present disclosure thus also encompasses the isolated nucleic acids encoding any of the binding proteins described herein, as well as a recombinant vector comprising such a nucleic acid molecule, and a host cell comprising such a recombinant vector.
A vector is a nucleic acid molecule, which may be a construct, capable of transporting another nucleic acid to which it has been linked. A vector may include any preferred or required operational elements. Preferred vectors are those for which the restriction sites have been described and which contain the operational elements needed for transcription of the nucleic acid sequence. Such operational elements include for example at least one suitable promoter, at least one operator, at least one leader sequence, at least one terminator codon, and any other DNA sequences necessary or preferred for appropriate transcription and subsequent translation of the nucleic acid sequence. Such vectors contain at least one origin of replication recognized by the host organism along with at least one selectable marker and at least one promoter sequence capable of initiating transcription of the nucleic acid sequence. A vector may be a plasmid into which additional DNA segments may be ligated. A vector may be a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as a plasmid is the most commonly used form of vector. However, the present disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. By way of example and not limitation, suitable vectors include pcDNA, pTT (Durocher et al., Nucleic Acids Research, Vol 30, No. 2 (2002)); pTT3 (pTT with additional multiple cloning site, pEFBOS (Mizushima et al., Nucleic acids Research, Vol 18, No. 17 (1990)), pBV, pJV, pBJ, or pHybE (patent publication no.: US 2009/0239259 AI).
Sequences that are operably linked are in a relationship permitting them to function in their intended manner. A control sequence operably linked to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. Operably linked sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences are polynucleotide sequences that are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, such control sequences generally include promoters and transcription termination sequence. Control sequences may include components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
A host cell may be transformed with a vector that introduces exogenous DNA into a host cell. Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, lipofection, calcium-phosphate precipitation, DEAE-dextran transfection, particle bombardment and the like. Transformed cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, and cells which transiently express the inserted DNA or RNA for limited periods of time.
Host organisms such as host cells are cultured under conditions appropriate for amplification of the vector and expression of the protein, as well known in the art. Expressed recombinant proteins may be detected by any of a number of methods also well known in the art.
Suitable host organisms include for example a prokaryotic or eukaryotic cell system. A eukaryotic cell may be a protist cell, animal cell, plant cell or fungal cell. A eukaryotic cell is for example an animal cell which may be a mammalian cell, avian cell, or an insect cell such as an insect Sf9 cell. Cells from established and readily available may be used, such as but not limited to HeLa, MRC-5 or CV-1. The host cell may be an E. coli cell or a yeast cell such as but not limited to Saccharomyces cerevisiae. Mammalian host cells for expressing the recombinant antibodies of the disclosure also include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub et al., Proc. Natl. Acad. Sci., 77: 4216-4220 (1980), used with a DHFR selectable marker, e.g., as described in Kaufman et al., Mol. Biol, 159: 601-621 (1982)), NS0 myeloma cells, COS cells and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells, or by secretion of the antibody into the culture medium in which the host cells are grown. Antibodies may be recovered from the culture medium using standard protein purification methods.
Host cells may also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It will be understood that variations on the above procedure are within the scope of the present disclosure. For example, it may be desirable to transfect a host cell with DNA encoding functional fragments of either the light chain and/or the heavy chain of an antibody of this disclosure. Recombinant DNA technology may also be used to remove some, or all, of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigens of interest. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the disclosure. In addition, bifunctional antibodies may be produced in which one heavy and one light chain are an antibody of the disclosure and the other heavy and light chain are specific for an antigen other than the antigens of interest by crosslinking an antibody of the disclosure to a second antibody by standard chemical crosslinking methods.
In a system for recombinant expression of an antibody, or antigen-binding portion thereof, of the disclosure, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked to CMV enhancer/AdMLP promoter regulatory elements to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium.
Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody from the culture medium. Still further the disclosure provides a method of synthesizing a recombinant antibody of the disclosure by culturing a host cell of the disclosure in a suitable culture medium until a recombinant antibody of the disclosure is synthesized. The method may further comprise isolating the recombinant antibody from the culture medium.
The present disclosure thus provides anti NGF binding proteins that are specific for and substantially neutralize NGF polypeptides, including active human NGF. Also provided are antibody heavy and light chain amino acid sequences which are substantially specific for and substantially neutralize NGF polypeptides when they are bound to them. This specificity enables the anti-human NGF human antibodies and human monoclonal antibodies with like specificity, to be effective immunotherapy for NGF associated diseases.
The present disclosure encompasses anti NGF binding proteins comprising at least one of the amino acid sequences selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 206, SEQ ID NO: 207 and SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 44, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, and which binds an NGF polypeptide epitope with substantially high affinity as described herein and has the capacity to substantially modulate, including substantially reduce, NGF polypeptide activity.
Examples of such binding proteins include binding proteins comprising a variable heavy chain polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 206 and SEQ ID NO: 207; and a variable light chain polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 44, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200 and SEQ ID NO: 202.
Exemplary pairings of a variable heavy chain polypeptide and a variable light chain polypeptide are represented by the following pairings: SEQ ID NO: 2 and SEQ ID NO: 4; SEQ ID NO: 6 and SEQ ID NO: 8; SEQ ID NO: 10 and SEQ ID NO: 12; SEQ ID NO: 14 and SEQ ID NO: 16; SEQ ID NO: 18 and SEQ ID NO: 20; SEQ ID NO: 22 and SEQ ID NO: 24; SEQ ID NO: 25 and SEQ ID NO: 26; SEQ ID NO: 27 and SEQ ID NO: 28; SEQ ID NO: 29 and SEQ ID NO: 30; SEQ ID NO: 31 and SEQ ID NO: 32; SEQ ID NO: 177 and SEQ ID NO: 32; SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO:36; SEQ ID NO: 37 and SEQ ID NO: 38; SEQ ID NO: 39 and SEQ ID NO: 40; SEQ ID NO: 41 and SEQ ID NO: 42; SEQ ID NO: 43 and SEQ ID NO: 44; SEQ ID NO: 180 and SEQ ID NO: 181, SEQ ID NO: 182 and SEQ ID NO: 183; SEQ ID NO: 185 and SEQ ID NO: 186; SEQ ID NO: 187 and SEQ ID NO: 188; and SEQ ID NO: 192 and SEQ ID NO: 193.
Also encompassed in the disclosure are binding proteins that specifically bind NGF as described herein and comprise a heavy chain variable region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 206, SEQ ID NO: 207. Also encompassed are binding proteins that specifically bind NGF as described herein and comprise a light chain variable region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 44, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202.
Exemplary binding proteins that specifically bind NGF as described herein preferably comprise a heavy chain variable region and a light chain variable region as follows:
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 2, or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and the light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 4 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof; a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 6 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 8 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 10 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 12 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 14 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 16 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof; a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 18 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 20 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 22 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 24 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 25 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 26 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 27 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 28 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 29 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 30 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 31 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 32 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 177 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 32 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 33 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 34 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 35 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 36 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 37 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 38 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 39 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 40 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 41 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 42 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 43 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 44 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 180 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 181 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 182 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 183 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 185 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 186 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 187 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 188 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 189 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 42 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 190 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 188 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 206 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 42 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof;
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 207 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 188 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof; and
a heavy chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 192 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof, and a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 193 or an antigen-binding or an immunologically functional immunoglobulin fragment thereof.
Exemplary binding proteins as disclosed herein may include at least one CDR comprising an amino acid sequence selected from: a) heavy chain CDRs consisting of SEQ ID NOS: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81; or modified CDR amino acid sequences having a sequence identity of at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% to one of said sequences; b) light chain CDRs consisting of SEQ ID NOS: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84; or modified CDR amino acid sequences having a sequence identity of at least 50% %, at least 60%, at least 70%, at least 80%, or at least 90% to one of said sequences.
It should be understood that variations are contemplated in any of the nucleic acid and amino acid sequences described herein. Such variations include those that will result in a nucleic acid sequence that is capable of directing production of analogs of the corresponding NGF binding proteins. It will be understood that due to the degeneracy of the genetic code, many substitutions of nucleotides may be made that will lead to a DNA sequence that remains capable of directing production of the corresponding protein or its analogs. All such variant DNA sequences that are functionally equivalent to any of the sequences described herein are encompassed by the present disclosure.
A variant of any of the binding proteins described herein means a protein (or polypeptide) that differs from a given protein (e.g., an anti-NGF antibody) in amino acid sequence by the addition (e.g., insertion), deletion, or conservative substitution of amino acids, but that retains the biological activity of the given protein. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity and degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes may be identified, in part, by considering the hydropathic index of amino acids, as understood in the art (see, e.g., Kyte et al., J. Mol. Biol. 157: 105-132 (1982)). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes may be substituted and still retains protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids also may be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity (see, e.g., U.S. Pat. No. 4,554,101, which is incorporated herein by reference). Substitution of amino acids having similar hydrophilicity values may result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. In one aspect, substitutions are performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties. “Variant” also may be used to describe a polypeptide or fragment thereof that has been differentially processed, such as by proteolysis, phosphorylation, or other post-translational modification, yet retains its biological activity or antigen reactivity, e.g., the ability to bind to NGF. Use of “variant” herein is intended to encompass fragments of a variant unless otherwise contradicted by context.
The binding proteins described herein encompass an immunoglobulin molecule, disulfide linked Fv, scFv, monoclonal antibody, murine antibody, chimeric antibody, single domain antibody, CDR-grafted antibody, diabody, mammalized (bovanized, camelized, caninized, equinized, felinized, or humanized) antibody, a canine antibody, feline antibody, equine antibody, murine antibody, multispecific antibody, Fab, dual specific antibody, DVD, Fab′, bispecific antibody, F(ab′)2, or Fv including a single chain Fv fragment.
A binding protein may comprise a particular heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. An exemplary binding protein includes an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. Furthermore, the antibody may comprise a light chain constant region, such as a kappa light chain constant region or a lambda light chain constant region. An exemplary binding protein comprises a kappa light chain constant region.
Replacements of amino acid residues in the Fc portion to alter antibody effector function are known in the art (Winter, et al. U.S. Pat. Nos. 5,648,260; 5,624,821). The Fc portion of an antibody mediates several important effector functions e.g. cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC) and half-life/clearance rate of antibody and antigen-antibody complexes. In some cases these effector functions are desirable for therapeutic antibody but in other cases might be unnecessary or even deleterious, depending on the therapeutic objectives. Certain human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcyRs and complement C1q, respectively. Neonatal Fc receptors (FcRn) are the critical components determining the circulating half-life of antibodies. At least one amino acid residue may be replaced in the constant region of the antibody, for example the Fc region of the antibody, such that effector functions of the antibody are altered.
Binding proteins according to the present disclosure may comprise a heavy chain immunoglobulin constant domain such as, for example, a human or canine or equine or feline IgM constant domain, a human or canine or equine or feline IgG4 constant domain, a human or canine or equine or feline IgG1 constant domain, a human or canine or equine or feline IgE constant domain, a human or canine or equine or feline IgG2 constant domain, a human or canine or equine or feline IgG3 constant domain, and a human or canine or equine or feline IgA constant domain. A binding protein as described herein may comprise a light chain immunoglobulin constant domain such as but not limited to any of human, canine, equine or feline, kappa or lambda constant domains, or any of canine, equine or feline kappa or lambda equivalent constant domains. An exemplary such binding protein has a constant region having an amino acid sequence of SEQ ID NO: 52 or SEQ ID NO: 54.
Binding proteins as described herein may also encompass an NGF anti-idiotype antibody relative to at least one NGF binding protein of the present disclosure. The anti-idiotype antibody includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule such as, but not limited to, at least one complimentarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or; any portion thereof, which may be incorporated into a binding protein of the present disclosure.
The binding proteins of the disclosure are capable of binding to human and canine NGF with high specificity, and additionally are capable of modulating the biological activity or function of NGF in an organism or a subject, including substantially neutralizing human and canine NGF. Also encompassed by the present disclosure are isolated murine monoclonal antibodies, or antigen-binding portions thereof, that bind to NGF with a substantially high affinity, have a slow off rate and/or have a substantially high neutralizing capacity. An exemplary binding protein as disclosed herein is capable of neutralizing NGF with a potency (IC50) of at least about 10 nM, at least about 5 nM, at least about 1 nM, at least about 0.5 nM, at least about 0.1 nM, at least about 0.05 nM, at least about 0.01 nM, or at least about 0.001 nM, as measured in the TF-1 cell proliferation assay or the pERK and Pathhunter assays. Binding proteins as described herein may have an on rate constant (Kon) to NGF of at least about 102M−1s−1; at least about 103M−1s−1; at least about 104M−1s−1; at least about 105M−1s−1; at least about 106M−1s−1; or at least about 107M−1s−1 as measured by surface plasmon resonance. Binding proteins as described herein may have an off rate constant (Koff) to NGF of at most about 10−3 s−1; at most about 10−4 s−1; at most about 10−5 s−1; at most about 10−6 s−1 or at most about 10−7 s−1, as measured by surface plasmon resonance. Binding proteins as described herein may have a dissociation constant (KD) to NGF of at most about 10−7 M; at most about 10−8 M; at most about 10−9 M; at most about 10−10M; at most about 10−11M; at most about 10−12M; at most about 10−13M, or at most about 10−14M. For example, a binding protein as described herein may have a dissociation constant (KD) of about 1×10−9M, about 1×10−10 M, about 3.14×10−10M, about 1×10−11M, about 2.37×10−11M, about 1×10−12M about 1×10−13M or about 3.3×10−14M.
Binding proteins as described herein including an isolated antibody, or antigen-binding portion thereof, or immunologically functional fragment thereof, may bind NGF and dissociate from NGF with a koff rate constant of about 0.1 s−1 or less, as determined by surface plasmon resonance, or may inhibit NGF activity with an IC50 of about 1×10−6M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from NGF with a koff rate constant of about 1×10−2 s−1 less, as determined by surface plasmon resonance, or may inhibit NGF activity with an IC50 of about 1×10−7 M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from NGF with a koff rate constant of about 1×103 s−1 or less, as determined by surface plasmon resonance, or may inhibit NGF with an IC50 of about 1×10−8M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from NGF with a koff rate constant of about 1×10−4 s−1 or less, as determined by surface plasmon resonance, or may inhibit NGF activity with an IC50 of about 1×10−9M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from NGF with a koff rate constant of about 1×10−5 s−1 or less, as determined by surface plasmon resonance, or may inhibit NGF activity with an IC50 of about 1×10−10M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from NGF with a koff rate constant of about 1×10−5 s−1 less, as determined by surface plasmon resonance, or may inhibit NGF activity with an IC50 of about 1×10−11M or less.
A binding protein as described herein may bind canine NGF, wherein the antibody, or antigen-binding portion thereof, may dissociate from canine NGF with a koff rate constant of about 0.1 s−1 or less, as determined by surface plasmon resonance, or may inhibit canine NGF activity with an IC50 of about 1×10−6M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from canine NGF with a koff rate constant of about 1×10−2 s−1 or less, as determined by surface plasmon resonance, or may inhibit canine NGF activity with an IC50 of about 1×10−7M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from canine NGF with a koff rate constant of about 1×10−3 s−1 or less, as determined by surface plasmon resonance, or may inhibit canine NGF with an IC5o of about 1×10−8M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from canine NGF with a koff rate constant of about 1×10−4 s−1 or less, as determined by surface plasmon resonance, or may inhibit canine NGF activity with an IC50 of about 1×10−10M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from canine NGF with a koff rate constant of about 1×10−5 s−1 or less, as determined by surface plasmon resonance, or may inhibit canine NGF activity with an IC50 of about 1×10−10M or less. Alternatively, the antibody, or an antigen-binding portion thereof, may dissociate from canine NGF with a koff rate constant of about 1×10−5 s−1 or less, as determined by surface plasmon resonance, or may inhibit canine NGF activity with an IC50 of about 1×10−11M or less.
The binding proteins of the disclosure further encompass binding proteins coupled to an immunoadhesion molecule, imaging agent, therapeutic agent, or cytotoxic agent. Non-limiting examples of suitable imaging agents include an enzyme, fluorescent label, luminescent label, bioluminescent label, magnetic label, biotin or a radiolabel including, but not limited to, 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and 153Sm. The therapeutic or cytotoxic agent may be an anti-metabolite, alkylating agent, antibiotic, growth factor, cytokine, anti-angiogenic agent, anti-mitotic agent, anthracydine, toxin, or apoptotic agent. Also provided herein is a labeled binding protein wherein an antibody or antibody portion of the disclosure is derivatized or linked to another functional molecule (e.g., another peptide or protein). For example, a labeled binding protein of the disclosure may be derived by functionally linking an antibody or antibody portion of the disclosed binding protein (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that may mediate associate of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
Useful detectable agents with which an antibody or antibody portion of the disclosure may be derivatized, may include fluorescent compounds. Exemplary fluorescent detectable agents include, for example, fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin and the like. An antibody may also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. When an antibody is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, when the detectable agent horseradish peroxidase is present, addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is detectable. An antibody may also be derivatized with biotin, and detected through indirect measurement of avidin or streptavidin binding.
The binding proteins described herein may be in crystallized form. Crystallized binding proteins according to the present disclosure may be produced according to methods known in the art, as disclosed for example in WO 02072636. Preferably the crystallized binding protein retains biological activity after crystallization. The binding proteins may thus be provided as crystals of whole anti-NGF antibodies or portions or fragments thereof as disclosed herein. Such crystals may be used to prepare formulations and compositions incorporating anti NGF binding proteins, including diagnostic and therapeutic compositions. An exemplary such crystallized binding protein is a carrier-free, controlled release crystallized binding protein. An exemplary crystallized binding protein demonstrates a greater half-life in vivo than the soluble counterpart of the binding protein.
Anti NGF binding proteins as described herein may be glycosylated. The glycosylation may demonstrate, for example, a bovine, camel, canine, murine, equine, feline, or human glycosylation pattern. Glycosylated binding proteins as described herein include the antibody or antigen-binding portion coupled to one or more carbohydrate residues. Nascent in vivo protein production may undergo further processing, known as post-translational modification. Sugar (glycosyl) residues may be added enzymatically, a process known as glycosylation. The resulting proteins bearing covalently linked oligosaccharide side chains are known as glycosylated proteins or glycoproteins. Protein glycosylation depends on the amino acid sequence of the protein of interest, as well as the host cell in which the protein is expressed. Different organisms may produce different glycosylation enzymes (ex., glycosyltransferases and glycosidases), and have different substrates (nucleotide sugars) available. Due to such factors, protein glycosylation pattern, and composition of glycosyl residues, may differ depending on the host system in which the particular protein is expressed. Glycosyl residues useful in the disclosure may include, but are not limited to, glucose, galactose, mannose, fucose, n-acetylglucosamine and sialic acid. The glycosylated binding protein comprises glycosyl residues such that the glycosylation pattern is human, murine, canine, feline, bovine or equine.
It is known to those skilled in the art that differing protein glycosylation may result in differing protein characteristics. For instance, the efficacy of a therapeutic protein produced in a microorganism host, such as yeast, and glycosylated utilizing the host endogenous pathway may be reduced compared to that of the same protein expressed in a mammalian cell, such as a CHO cell line. Such glycoproteins may also be immunogenic in humans and show reduced half-life in vivo after administration. Specific receptors in humans and other animals may recognize specific glycosyl residues and promote the rapid clearance of the protein from the bloodstream. Other adverse effects may include changes in protein folding, solubility, susceptibility to proteases, trafficking, transport, compartmentalization, secretion, recognition by other proteins or factors, antigenicity, or allergenicity. Accordingly, a practitioner may prefer a therapeutic protein with a specific composition and pattern of glycosylation, such as a glycosylation composition and pattern identical, or at least similar, to that produced in human cells or in the species-specific cells of the intended subject animal.
Expressing glycosylated proteins different from that of a host cell may be achieved by genetically modifying the host cell to express heterologous glycosylation enzymes. Using techniques known in the art, a practitioner may generate antibodies or antigen-binding portions thereof exhibiting human protein glycosylation. For example, yeast strains have been genetically modified to express non-naturally occurring glycosylation enzymes such that glycosylated proteins (glycoproteins) produced in these yeast strains exhibit protein glycosylation identical to that of animal cells, especially human cells (U.S. patent applications 20040018590 and 20020137134).
Further, it will be appreciated by those skilled in the art that a protein of interest may be expressed using a library of host cells genetically engineered to express various glycosylation enzymes such that member host cells of the library produce the protein of interest with variant glycosylation patterns. A practitioner may then select and isolate the protein of interest with particular novel glycosylation patterns. The protein having a particularly selected novel glycosylation pattern exhibits improved or altered biological properties.
Anti NGF Chimeric Antibodies
A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a non-murine immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art, see e.g., Morrison, Science, 229: 1202 (1985); Oi et al., BioTechniques, 4: 214 (1986); Gillies et al., J. Immunol. Methods, 125: 191-202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397. In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81: 851-855 (1984); Neuberger et al., Nature, 312:604-608 (1984); Takeda et al., Nature, 314: 452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity may be used.
Anti NGF CDR Grafted Antibodies
CDR-grafted antibodies of the disclosure may comprise heavy and light chain variable region sequences from a non-murine antibody wherein one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of the murine antibodies of the disclosure. A framework sequence from any non-murine antibody may serve as the template for CDR grafting. However, straight chain replacement onto such a framework often leads to some loss of binding affinity to the antigen. The more homologous a non-murine antibody is to the original murine antibody, the less likely the possibility that combining the murine CDRs with the non-murine framework will introduce distortions in the CDRs that could reduce affinity.
A non-murine variable framework that is chosen to replace the murine variable framework apart from the CDRs may have at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity with the murine antibody variable region framework. The non-murine variable framework, apart from the CDRs, that is chosen to replace the murine variable framework, apart from the CDRs, may be a bovine, camel, canine, equine, feline or human variable framework. For example, the non-murine variable framework that is chosen to replace the murine variable framework, apart from the CDRs, is a canine variable framework and has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the murine antibody variable region framework.
Methods for producing CDR-grafted antibodies are known in the art (see EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), and include veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5): 489-498 (1991); Studnicka et al., Protein Engineering, 7(6):805-814 (1994); Roguska et al., PNAS, 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,352).
Anti NGF Humanized Antibodies
The process of modifying a monoclonal antibody from an animal to render it less immunogenic for therapeutic administration to humans (humanization) has been aggressively pursued and has been described in a number of publications (Antibody Engineering: A practical Guide. Carl A. K. Borrebaeck ed. W.H. Freeman and Company, 1992; and references cited above). Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Known human Ig sequences are disclosed in a variety of websites which are available on the Internet (such as the NCBI website, Antibody Resource, and known to those skilled in the art as well as in Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Dept. Health (1983), which is incorporated herein by reference. Additional sequences are shown in Table 1 A below. Such imported sequences may be used to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic of the antibody, as known in the art.
TABLE 1A
Mouse Anti-NGF mAb CDRs Grafted onto Human
Ig Frameworks CDR-Grafted Anti-NGF Abs (This
Table 1A is identical to Table 17 in the Examples)
HU72 VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYM
(CDR GRAFT
FWVRQATGKGLEWVSTISDGGSYTYYTDNVKGRF
VH3-13/JH5)
TISRENAKNSLYLQMNSLRAGDTAVYYCARDWSD
SEGFAYWGQGTLVTVSS (SEQ ID NO: 165)
Hu73 VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWM
(CDR GRAFT
HWVRQAPGQGLEWMGRIDPYGGGTKHNEKFKRRV
VH1-18/JH6)
TMTTDTSTSTAYMELRSLRSDDTAVYYCARSGYD
YYFDVWGQGTTVTVSS (SEQ ID NO: 166)
HU77 VH
QVQLVQSGAEVKKPGSSVKVSCKASGFNIKDTYI
(CDR GRAFT
YWVRQAPGQGLEWMGRIDPANGNTIYASKFQGRV
VH1-69/JH6)
TITADKSTSTAYMELSSLRSEDTAVYYCARYGYY
AYWGQGTTVTVSS (SEQ ID NO: 167)
HU80 VH
QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYI
(CDR GRAFT
YWVRQAPGQGLEWMGRIDPANGNTIYASKFQGRV
VH1-18/JH6)
TMTTDTSTSTAYMELRSLRSDDTAVYYCARYGYY
AYWGQGTTVTVSS (SEQ ID NO: 168)
HU81 VH
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNHYM
(CDR GRAFT
YWVRQAPGKGLEWVGSISDGGAYTFYPDTVKGRF
VH3-15/JH1)
TISRDDSKNTLYLQMNSLKTEDTAVYYCTTEESA
NNGFAFWGQGTLVTVSS (SEQ ID NO: 169)
HU82 VH
QVTLKESGPVLVKPTETLTLTCTVSGFSLTGYNI
(CDR GRAFT
NWIRQPPGKALEWLAMIWGYGDTDYNSALKSRLT
VH2-26/JH6)
ISKDTSKSQVVLTMTNMDPVDTATYYCARDHYGG
NDWYFDVWGQGTTVTVSS (SEQ ID NO: 170)
HU72 VL
DIVMTQTPLSLPVTPGEPASISCRSSQSIVQSNG
(CDR GRAFT
NTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFS
01/JK2)
GSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPFT
FGQGTKLEIKR (SEQ ID NO: 171)
HU73 VL
DIQMIQSPSFLSASVGDRVSIICRASENIYSFLA
(CDR GRAFT
WYLQKPGKSPKLFLYNANTLAEGVSSRFSGRGSG
L22/JK2)
TDFTLTIISLKPEDFAAYYCQHHFGTPFTFGQGT
KLEIKR (SEQ ID NO: 172)
HU77 VL
DIVMTQTPLSLPVTPGEPASISCKSTKSLLNGDG
(CDR GRAFT
FTYLDWYLQKPGQSPQLLIYLVSNRFSGVPDRFS
01/JK2)
GSGSGTDFTLKISRVEAEDVGVYYCFESNYLFTF
GQGTKLEIKR (SEQ ID NO: 173)
HU80 VL
DIVMTQTPLSLPVTPGEPASISCKSTKSLLNGDG
(CDR GRAFT
FTYLDWYLQKPGQSPQLLIYLVSNRFSGVPDRFS
01/JK2)
GSGSGTDFTLKISRVEAEDVGVYYCFESNYLFTF
GQGTKLEIKR (SEQ ID NO: 174)
HU81 VL
DIVMTQTPLSLPVTPGEPASISCRSSQSILHSNG
(CDR GRAFT
NTYLEWYLQKPGQSPQLLIYRVSNRFSGVPDRFS
01/JK2)
GSGSGTDFTLKISRVEAEDVGVYYCFQGAHVPFT
FGQGTKLEIKR (SEQ ID NO: 175)
HU82 VL
DIQMTQSPSSLSASVGDRVTITCRASQDITNYLN
(CDR GRAFT
WYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSG
08/JK2)
TDFTFTISSLQPEDIATYYCQQGKTLPRTFGQGT
KLEIKR (SEQ ID NO: 176)
Framework residues in the human framework regions may be substituted with the corresponding residue from the CDR donor antibody to alter or improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues may be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding. Antibodies may be humanized using a variety of techniques known in the art, such as but not limited to those described in Jones et al., Nature 321:522 (1986); Verhoeyen et al., Science 239: 1534 (1988)), Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994); PCT publication WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596, EP 239,400, U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539; and 4,816,567.
Anti NGF Caninized Antibodies
The process of modifying a monoclonal antibody from an animal to render it less immunogenic for therapeutic administration to canines (caninization) has been described in U.S. Pat. No. 7,261,890 B2 2007). The amino acid sequence of canine IgG1 is provided in GenBank (AF354264). Determination of the amino acid sequence of the variable regions of both a canine IgM and a canine IgA heavy chain (Wasserman et al., Biochem., 16, 3160 (1977), determination of the amino acid sequence of the K light chain from a canine IgA (Wasserman et al., Immunochem., 15, 303 (1978)), complete amino-acid sequence of a canine μ chain was disclosed (McCumber et al., Mol. Immunol, 16, 565 (1979)), a single canine IgG-Aγ chain cDNA and four canine IgG-Aγ chain protein sequences were disclosed (Tang et al., Vet. Immunology Immunopathology, 80, 259 (2001)). It describes PCR amplification of a canine spleen cDNA library with a degenerate oligonucleotide primer designed from the conserved regions of human, mouse, pig, and bovine IgGs. Canine immunoglobulin variable domains, caninized antibodies, and methods for making and using them are disclosed in US Patent Application No. 2004/0181039 and U.S. Pat. Nos. 7,261,890; 6,504,013; 5,852,183; 5,5225,539.
Table 2 below is a list of amino acid sequences of VH and VL regions of selected caninized anti-NGF antibodies of the disclosure.
TABLE 2
SEQ ID NO:
Region
25
72.1 VH
26
72.1 VL
27
73.1 VH
28
73.1 VL
29
77.1 VH
30
77.1 VL
31
81.1 VH
32
81.1 VL
33
82.1 VH
34
82.1 VL
35
72.2 VH
36
72.2 VL
37
73.2 VH
38
73.2 VL
39
77.2 VH
40
77.2 VL
41
81.2 VH
42
81.2 VL
43
82.2 VH
44
82.2 VL
177
81.1B VH
179
72.3 VH
180
72.4 VH
181
72.4 VL
182
73.4 VH
183
73.4 VL
184
77.3 VH
185
77.4 VH
186
77.4 VL
187
81.4 VH
188
81.4 VL
189
81.2B VH
190
81.4B VH
191
82.3 VL
192
82.4 VH
193
82.4 VL
206
81.5B VH
207
81.6B VH
Uses of Anti-NGF Antibodies
Binding proteins as described herein may be used in a method for detecting the presence of NGF in a sample in vivo or in vitro (e.g., in a biological sample, such as serum, plasma, tissue, biopsy). The in vitro method may be used for example to diagnose a disease or disorder, e.g., an NGF-associated disorder. The method includes (i) contacting the sample or a control sample with the anti-NGF antibody or fragment thereof as described herein; and (ii) detecting formation of a complex between the anti-NGF antibody or fragment thereof, and the sample or the control sample, wherein a statistically significant change in the formation of the complex in the sample relative to the control sample is indicative of the presence of the NGF in the sample.
Binding proteins as described herein may be used in a method for detecting the presence of NGF in vivo (e.g., in vivo imaging in a subject). The method may be used to diagnose a disorder, e.g., an NGF-associated disorder. The method includes: (i) administering the anti-NGF antibody or fragment thereof as described herein to a subject or a control subject under conditions that allow binding of the antibody or fragment to NGF; and (ii) detecting formation of a complex between the antibody or fragment and NGF, wherein a statistically significant change in the formation of the complex in the subject relative to the control subject is indicative of the presence of NGF.
Given the ability to bind to NGF, the anti-NGF antibodies, or portions thereof, or combinations thereof, as described herein may be used as immunoreagent(s) to detect NGF (e.g., in a biological sample, such as serum or plasma), in a conventional immunoassay, such as an enzyme linked immunosorbent assays (ELISA), a radioimmunoassay (RIA) or tissue immunohistochemistry. A method for detecting NGF in a biological sample involves contacting a biological sample with an antibody, or antibody portion, of the disclosure and detecting either the antibody (or antibody portion) bound to NGF or unbound antibody (or antibody portion), to thereby detect NGF in the biological sample. The binding protein may be directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include H 14C 5S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, or 15Sm.
NGF may alternatively be assayed in biological fluids by a competition immunoassay utilizing recombinant NGF standards labeled with a detectable substance and an unlabeled anti-NGF antibody. In this assay, the biological sample, the labeled recombinant NGF standards and the anti-NGF antibody are combined and the amount of labeled rNGF standard bound to the unlabeled antibody is determined. The amount of NGF in the biological sample is inversely proportional to the amount of labeled rNGF standard bound to the anti-NGF antibody. Similarly, NGF may also be assayed in biological fluids by a competition immunoassay utilizing rNGF standards labeled with a detectable substance and an unlabeled anti-NGF antibody.
The disclosure thus also contemplates immunoassay reagents, devices and kits including one or more of the presently disclosed binding proteins for detecting the presence or amount of NGF in a sample. It is contemplated for example that an immunoreagent comprising one or more of the presently disclosed binding proteins may be provided in the form of a kit with one or more containers such as vials or bottles, with each container containing a separate reagent such as an anti-NGF binding protein, or a cocktail of anti-NGF binding proteins, detection reagents and washing reagents employed in the assay. The immunoreagent(s) may be advantageously provided in a device in which the immunoreagents(s) is immobilized on a solid support, such as but not limited to a cuvette, tube, microtiter plates or wells, strips, chips or beads. The kit may comprise at least one container for conducting the assay, and/or a buffer, such as an assay buffer or a wash buffer, either one of which may be provided as a concentrated solution, a substrate solution for the detectable label (e.g., an enzymatic label), or a stop solution. Preferably, the kit comprises all components, i.e., reagents, standards, buffers, diluents, etc., which are necessary to perform the assay. The kit may contain instructions for determining the presence or amount of NGF in the sample based on specific binding of the immunoreagent to NGF, in paper form or computer-readable form, such as a disk, CD, DVD, or the like, and/or may be made available online.
The binding proteins in the kit may be labeled with a detectable label such as those described above including a fluorophore, a radioactive moiety, an enzyme, a biotin/avidin label, a chromophore, a chemiluminescent label, or the like; or the kit may include reagents for carrying out detectable labeling. The antibodies, calibrators and/or controls can be provided in separate containers or pre-dispensed into an appropriate assay format, for example, into microtiter plates.
Optionally, the kit includes quality control components (for example, sensitivity panels, calibrators, and positive controls). Preparation of quality control reagents is well-known in the art and is described on insert sheets for a variety of immunodiagnostic products. Sensitivity panel members optionally are used to establish assay performance characteristics, and further optionally are useful indicators of the integrity of the immunoassay kit reagents, and the standardization of assays.
The kit can also optionally include other reagents required to conduct a diagnostic assay or facilitate quality control evaluations, such as buffers, salts, enzymes, enzyme co-factors, enzyme substrates, detection reagents, and the like. Other components, such as buffers and solutions for the isolation and/or treatment of a test sample (e.g., pretreatment reagents), also can be included in the kit. The kit can additionally include one or more other controls. One or more of the components of the kit can be lyophilized, in which case the kit can further comprise reagents suitable for the reconstitution of the lyophilized components.
The various components of the kit optionally are provided in suitable containers as necessary, e.g., a microtiter plate. The kit can further include containers for holding or storing a sample (e.g., a container or cartridge for a urine sample). Where appropriate, the kit optionally also can contain reaction vessels, mixing vessels, and other components that facilitate the preparation of reagents or the test sample. The kit can also include one or more instruments for assisting with obtaining a test sample, such as a syringe, pipette, forceps, measured spoon, or the like. Instructions:
It will be appreciated that the antibodies and antibody portions of the disclosure are capable of substantially neutralizing NGF activity both in vitro and in vivo. Accordingly, such antibodies and antibody portions of the disclosure can also be used to substantially inhibit NGF activity, e.g., in a cell culture containing NGF, in mammalian subjects having NGF with which an antibody of the disclosure cross-reacts. The disclosure thus provides a method for inhibiting NGF activity comprising contacting NGF with an antibody or antibody portion of the disclosure such that NGF activity is substantially inhibited. For example, in a cell culture containing, or suspected of containing NGF, an antibody or antibody portion of the disclosure can be added to the culture medium to inhibit NGF activity in the culture.
Accordingly, the disclosure also provides a method for inhibiting NGF activity comprising contacting NGF with a binding protein such that NGF activity is substantially inhibited. In another aspect, the disclosure provides a method for inhibiting NGF activity in a subject suffering from a disorder in which NGF activity is detrimental, comprising administering to the subject a binding protein disclosed above such that NGF activity in the subject is substantially inhibited and treatment is achieved.
The disclosure also provides a method for reducing NGF activity in a subject, such as a subject suffering from a disease or disorder in which NGF activity is detrimental. The disclosure provides methods for reducing NGF activity in a subject suffering from such a disease or disorder, which method comprises administering to the subject an antibody or antibody portion of the disclosure such that NGF activity in the subject is reduced. The subject can be a mammal expressing an NGF to which an antibody of the disclosure is capable of binding. Still further the subject can be a mammal into which NGF has been introduced (e.g., by administration of NGF or by expression of an NGF transgene). An antibody of the disclosure can be administered to a subject in need thereof for therapeutic purposes.
An antibody of the disclosure can be administered for veterinary purposes to a non-human mammal expressing an NGF with which the antibody is capable of binding. For example, an antibody of the disclosure can be administered for veterinary purposes to a non-human mammal such as a dog, horse, cat, or livestock (beef and dairy cattle, swine, sheep, goats, poultry, etc.) expressing an NGF with which the antibody is capable of binding.
In another aspect, the disclosure provides a method of treating (e.g., curing, suppressing, ameliorating, delaying or preventing or decreasing the risk of the onset, recurrence or relapse of) or preventing an NGF associated disorder, in a subject. The method includes: administering to the subject a disclosed NGF binding protein (particularly an antagonist), e.g., an anti-NGF antibody or fragment thereof as described herein, in an amount sufficient to treat or prevent the NGF associated disorder. The NGF antagonist, e.g., the anti-NGF antibody or fragment thereof, can be administered to the subject, alone or in combination with other therapeutic modalities as described herein.
An antibody of the disclosure can be administered to a non-human mammal expressing an NGF with which the antibody is capable of binding as an animal model of human disease. Such animal models may be useful for evaluating the therapeutic efficacy of antibodies of the disclosure (e.g., testing of dosages and time courses of administration).
In another aspect, the antibodies and binding proteins of the disclosure are useful for treating NGF-related diseases and disorders including or involving acute or chronic pain. Non-limiting examples of NGF-related diseases and disorders include general inflammation, surgical and postsurgical pain including pain from amputation, dental pain, pain from trauma, fracture pain, pain from abscess, neuropathic pain, hyperalgesia and allodynia, neuropathic pain, post-herpetic neuralgia, diabetes including, but not limited to, diabetic neuropathy pain, stroke, thalamic pain syndrome, gout joint pain, osteoarthritis or rheumatoid arthritis pain, rheumatic diseases, lupus, psoriasis, sciatica, pain associated with musculoskeletal diseases including, but not limited to, chronic low back pain, fibromyalgia, sprains, pains associated with sickle cell crises, general headache, migraine, duster headache, tension headache, trigeminal neuralgia, dysmenorrhea, endometriosis, ovarian cysts, visceral pain, prostatitis, cystitis, interstitial cystitis, erythromelalgia or pain caused by pancreatitis or kidney stones, general gastrointestinal disorders including, but not limited to, colitis, gastric ulceration and duodenal ulcers, gastroesophageal reflux, dyspepsia, inflammatory bowel disorders, irritable bowel syndrome, inflammatory bladder disorders, incisional pain, pain from burns and/or wounds, ankylosing spondilitis, periarticular pathologies, cancer pain including, but not limited to, pain from bone metastases and pain from cancer treatment, and pain from HIV or AIDS. Other examples of NGF-related diseases and conditions include malignant melanoma, Sjogren's syndrome, rhinitis, bronchial disorders, and asthma, such as uncontrolled asthma with severe airway hyper-responsiveness, intractable cough; and pain from skin diseases or disorders with an inflammatory component such as, but not limited to, sunburn, allergic skin reactions, dermatitis, pruritis, and vitiligo.
The disclosure also provides a method of treating a subject suffering from a disorder in which NGF is detrimental comprising administering a binding protein before, concurrent, or after the administration of a second agent. In another aspect, the additional therapeutic agent that can be co-administered and/or co-formulated with one or more NGF antagonists, (e.g., anti-NGF antibodies or fragments thereof) include, but are not limited to, TNF antagonists; a soluble fragment of a TNF receptor; ENBREL®; TNF enzyme antagonists; TNF converting enzyme (TACE) inhibitors; muscarinic receptor antagonists; TGF-beta antagonists; interferon gamma; perfenidone; chemotherapeutic agents, methotrexate; leflunomide; sirolimus (rapamycin) or an analog thereof, CCI-779; COX2 or cPLA2 inhibitors; NSAIDs; immunomodulators; p38 inhibitors; TPL-2, MK-2 and NFKB inhibitors; budenoside; epidermal growth factor corticosteroids; cyclosporine; sulfasalazine; aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide; antioxidants; thromboxane inhibitors; anti-IL-6 antibodies; growth factors; elastase inhibitors; pyridinyl-imidazole compounds; antibodies or agonists of TNF, CGRP, substance P, bradykinin, MMP-2, MMP-9, MMP-13, LT, IL-1a, IL-11, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, EMAP-II, GM-CSF, FGF, or PDGF; antibodies of CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands; FK506; rapamycin; mycophenolate mofetil; ibuprofen; prednisolone; phosphodiesterase inhibitors; adenosine agonists; antithrombotic agents; complement inhibitors; adrenergic agents; IRAK, NIK, IKK, p38, or MAP kinase inhibitors; IL-1β converting enzyme inhibitors; TNF converting enzyme inhibitors; T-cell signaling inhibitors; metalloproteinase inhibitors; 6-mercaptopurines; angiotensin converting enzyme inhibitors; soluble cytokine receptors; soluble p55 TNF receptor; soluble p75 TNF receptor; sIL-IRI; sIL-IRII; sIL-6R; anti-inflammatory cytokines; IL-4; IL-10; IL-11; and TGFβ.
Pharmaceutical Compositions
The antibodies and antibody-portions of the disclosure can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises at least one antibody or antibody portion of the disclosure and a pharmaceutically acceptable carrier. Such compositions can be used for example in a method for treating a mammal for a disease or disorder involving increased levels of NGF by administering to the mammal an effective amount of the composition. A pharmaceutical composition may include a therapeutically effective amount of the antibody or antibody portion. The pharmaceutical compositions as described herein may be used for diagnosing, detecting, or monitoring a disorder or one or more symptoms thereof; preventing, treating, managing, or ameliorating a disorder or one or more symptoms thereof; and/or research. As used herein, the phrase “increased levels of NGF” refers to a level of NGF in a subject, such as a mammal, that is greater or higher than an established or predetermined baseline level of NGF such as, for example, a level previously established for said subject or averaged from a group of subjects.
A pharmaceutical composition may comprise, for example, a binding protein and a pharmaceutically acceptable carrier, excipient or diluent. For example, pharmaceutical compositions may comprise a therapeutically effective amount of one or more of the binding proteins as disclosed herein, together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant. The pharmaceutical composition may contain one or more various formulation materials for modifying, maintaining or preserving the composition or properties of the composition, for example, the color, consistency, isotonicity, odor, osmolality, pH, sterility, stability, viscosity and other properties of the composition. Such formulation materials are generally well known and many suitable formulation materials are described for example in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed. (A. R. Gennaro, ed.) 1990, Mack Publishing Company. Non-limiting examples of suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. In addition, the pharmaceutical composition can also contain one or more preservatives. Examples of suitable preservatives that can be used include, but are not limited to, methylparaben, propylparaben, benzyl alcohol, chlorobutanol, and benzalkonium chloride. Optimal pharmaceutical formulations can be readily determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage.
The pharmaceutical composition may comprise at least one additional therapeutic agent for treating a disorder in which NGF activity is detrimental. The additional agent can be, for example, a therapeutic agent, imaging agent, cytotoxic agent, angiogenesis inhibitors, kinase inhibitors, co-stimulation molecule blockers, adhesion molecule blockers, anti-cytokine antibody or functional fragment thereof, methotrexate, cyclosporine, rapamycin, FK506, detectable label or reporter, TNF antagonist, anti-rheumatic, muscle relaxant, narcotic, non-steroid anti-inflammatory drug (NSAID), analgesic, anesthetic, sedative, local anesthetic, neuromuscular blocker, antimicrobial, antipsoriatic, corticosteroid, anabolic steroid, erythropoietin, immunoglobulin, immunosuppressive, growth hormone, hormone replacement drug, radiopharmaceutical, antidepressant, antipsychotic, stimulant, asthma medication, beta agonist, inhaled steroid, oral steroid, epinephrine or analog, cytokine, or a cytokine antagonist.
The pharmaceutical composition of the present disclosure may have a pH greater than about 7.0 or between about 7.0 and about 8.0. Alternatively, the pharmaceutical composition may have a pH of between about 7.2 to about 7.8. Still further alternatively, the pH of the pharmaceutical composition may be between about 7.4 to about 7.6. Still further alternatively, the pH of the pharmaceutical composition may be about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 7.7, 7.8, 7.9 or 8.0. With respect to the pharmaceutical compositions of the present disclosure, there is an increase in degradation, an increase in fragmentation or an increase in degradation and an increase in fragmentation at a pH of 6.0 or less. This finding was surprising as many pharmaceutical compositions comprising humanized antibodies exhibit an increase in degradation, an increase fragmentation or an increase in degradation and an increase in fragmentation at a pH lower than 5.0 and again at a pH higher than about 6.0. Accordingly, most pharmaceutical compositions containing humanized antibodies are stable at a pH between about 5.0 to about 6.0.
A composition for the release of a binding protein may comprise, for example, a formulation including an amount of a crystallized binding protein, crystallized antibody construct or crystallized antibody conjugate as disclosed above. The composition may further comprise an additional ingredient, such as carrier, excipient or diluent, and at least one polymeric carrier. The polymeric carrier can comprise one or more polymers selected from the following: poly (acrylic acid), poly(cyanoacrylates), poly (amino acids), poly (anhydrides), poly (depsipeptide), poly (esters), poly (lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutryate), poly (caprolactone), poly (dioxanone); poly (ethylene glycol), poly ((hydroxypropyl) methacrylamide, poly [(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends and copolymers thereof. The additional ingredient may be, for example, albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-P-cyclodextrin, methoxypolyethylene glycol and polyethylene glycol.
The polymeric carrier may be capable of affecting the release of the binding protein from the composition as described further herein below. Polymeric materials can be used in the formulation of pharmaceutical compositions comprising the disclosed binding proteins to achieve controlled or sustained release of the disclosed binding proteins (Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger et al., J. Macromol. Sci. Rev. Macromol. Chem., 23:61 (1983); Levy et al., Science, 228: 190 (1985); During Qt al, Ann. Neurol., 25: 351 (1989); Howard et al., J. Neurosurg., 7 1: 105 (1989); U.S. Pat. Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; 5,128,326; PCT Publication Nos. WO 99/15154; and WO 99/20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. The polymer used in a sustained release formulation may be inert, free of leachable impurities, stable on storage, sterile, and biodegradable. A controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 1 15-138 (1984)).
Controlled release systems are discussed in the review by Langer (Science, 249: 1527-1533 (1990)). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more therapeutic agents of the disclosure (U.S. Pat. No. 4,526,938; PCT publication Nos. WO 91/05548 and WO 96/20698; Ning et al., Radiotherapy & Oncology, 39: 179-189 (1996), Song et al., PDA Journal of Pharmaceutical Science & Technology, 50: 372-397 (1995); Cleek et al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater., 24: 853-854 (1997); and Lam et al., Proc. Int'l. Symp. Control Rel. Bioact. Mater., 24: 759-760 (1997)).
The binding proteins of the present disclosure can be administered by a variety of methods known in the art. For example, the binding proteins of the present disclosure may be administered by subcutaneous injection, intravenous injection or infusion. Administration can be systemic or local. As will be appreciated by the skilled artisan, the route and/or mode of administration may vary depending upon the desired results. The active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
For example, such pharmaceutical compositions may be administered to a subject by parenteral, intradermal, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal. Methods of administering a prophylactic or therapeutic agent of the disclosure also include, but are not limited to, epidural administration, intratumoral administration, and mucosal administration (e.g., intranasal and oral routes). In addition, pulmonary administration can be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent (U.S. Pat. Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903). The antibodies and antibody portions described herein can be administered for example using Alkermes AIR® pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.). The prophylactic or therapeutic agents may be administered by any convenient route, and may be administered together with other biologically active agents.
Various delivery systems are known and can be used to administer one or more disclosed binding proteins or the combination of one or more disclosed binding proteins and a prophylactic agent or therapeutic agent useful for preventing, managing, treating, or ameliorating a disorder or one or more symptoms thereof, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or antibody fragment, receptor-mediated endocytosis (see, e. g., Wu et al., J. Biol. Chem., 262: 4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. It may be desirable to administer the disclosed binding proteins locally to the area in need of treatment, which may be achieved by, for example, and not by way of limitation, local infusion, by injection, or by means of an implant, said implant being of a porous or non-porous material, including membranes and matrices, such as sialastic membranes, polymers, fibrous matrices (e.g., Tissuel®), or collagen matrices. An effective amount of one or more disclosed binding proteins can be administered locally to the affected area to a subject to prevent, treat, manage, and/or ameliorate a disorder or a symptom thereof. Alternatively, an effective amount of one or more of the disclosed binding proteins is administered locally to the affected area in combination with an effective amount of one or more therapies (e. g., one or more prophylactic or therapeutic agents) other than disclosed binding proteins of a subject to prevent, treat, manage, and/or ameliorate a disorder or one or more symptoms thereof.
The disclosed binding proteins can be delivered in a controlled release or sustained release system such as, for example, an infusion pump device operable to achieve controlled or sustained release of the disclosed binding proteins (see Langer, supra; Sefton, CRC Crit Ref. Biomed. Eng. 14:20 (1987); Buchwald et al., Surgery, 88: 507 (1980); Saudek et al., N. Engl. J. Med., 321: 574 (1989)).
When a composition as described herein comprises a nucleic acid encoding a binding protein as described herein as a prophylactic or therapeutic agent, the nucleic acid can be administered in vivo to promote expression of its encoded prophylactic or therapeutic agent, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (Joliot et al., Proc. Natl. Acad. Sci., 88: 1864-1868 (1991)). Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression by homologous recombination.
A pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. For example, a composition may be formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to human beings and companion animals. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection.
If the compositions of the disclosure are to be administered topically, the compositions can be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art (Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995)). For non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity greater than water are typically employed. Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which are, if desired, sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, such as, for example, osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as Freon) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well-known in the art.
The pharmaceutical composition of the present disclosure can have a half-life of from about 8 days to about 15 days when dosed intravenously or subcutaneously. Alternatively, the pharmaceutical composition of the present invention can have a half-life of from about 10 days to about 13 days. Still further alternatively, the pharmaceutical composition of the present invention can have a half-life of about 8 days, such as about 8.5 days, about 9 days, such as about 9.5 days, about 10 days, such as about 10.5 days, about 11 days, such as about 11.5 days, about 12 days, about 12.5 days, about 13 days, such as about 13.5 days, about 14 days, such as about 14.5 days, or about 15 days.
If the method of the disclosure comprises intranasal administration of a composition, the composition can be formulated in an aerosol form, spray, mist or in the form of drops. In particular, prophylactic or therapeutic agents for use according to the present disclosure can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (composed of, e.g., gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
If the method of the disclosure comprises oral administration, compositions can be formulated orally in the form of tablets, capsules, cachets, gelcaps, solutions, suspensions, and the like. Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excepients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well-known in the art. Liquid preparations for oral administration may take the form of, but not limited to, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of a prophylactic or therapeutic agent(s).
The method of the disclosure may comprise pulmonary administration, e.g., by use of an inhaler or nebulizer, of a composition formulated with an aerosolizing agent (U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903). For example, an antibody of the disclosure, combination therapy, and/or composition of the disclosure may be administered using Alkermes AIR® pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).
The method of the disclosure may comprise administration of a composition formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.
The methods of the disclosure may additionally comprise administration of compositions formulated as depot preparations. Such long acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).
The methods of the disclosure encompass administration of compositions formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions, such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
The ingredients of the disclosed compositions may be supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or a substantially water-free concentrate in a hermetically sealed container such as an ampoule or sachette which may indicate the quantity of active agent. Where the mode of administration is infusion, the disclosed compositions can be dispensed with an infusion solution containing sterile pharmaceutical grade solution such as water or saline. Where the mode of administration is by injection, an ampoule of sterile solution such as water or saline can be provided so that the ingredients may be mixed prior to administration.
In particular, the disclosure also provides that one or more of disclosed binding proteins or pharmaceutical compositions thereof is packaged in a hermetically sealed container such as an ampoule or sachette which may indicate the quantity of the agent. One or more of the disclosed binding proteins or pharmaceutical compositions thereof may be supplied as a dry sterilized lyophilized powder or substantially water-free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. One or more of the disclosed binding proteins or pharmaceutical compositions thereof may be supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least about 0.5 mg, 1 mg, 2 mg, 4 mg, 5 mg, 10 mg, 15 mg, 25 mg, 35 mg, 45 mg, 50 mg, 75 mg, or 100 mg. The lyophilized disclosed binding proteins or pharmaceutical compositions thereof may be stored at any suitable temperature, such as, for example, between about 2° C. and about 8° C. and may be stored in its original container. The disclosed binding proteins or pharmaceutical compositions thereof may be administered within about 1 week, within about 5 days, within about 72 hours, within about 48 hours, within about 24 hours, within about 12 hours, within about 6 hours, within about 5 hours, within about 3 hours, or within about 1 hour after being reconstituted. Alternatively, one or more of the disclosed binding proteins or pharmaceutical compositions thereof may be supplied in liquid form in a hermetically sealed container which may indicate the quantity and concentration of the agent. The liquid form of the administered composition may be supplied in a hermetically sealed container at concentrations of at least about 0.01 mg/mL, at least about 0.05 mg/mL, at least about 0.1 mg/mL, at least about 0.2 mg/mL, at least about 0.25 mg/ml, at least about 0.5 mg/ml, at least about 1 mg/ml, at least about 2.5 mg/ml, at least about 5 mg/ml, at least about 8 mg/ml, at least about 10 mg/ml, at least about 15 mg/kg, at least about 25 mg/ml, at least about 50 mg/ml, at least about 75 mg/ml, or at least about 100 mg/ml. The liquid form may be stored at any suitable temperature such as between about 2° C. and about 8° C. and may be stored in its original container.
The binding proteins of the disclosure can be incorporated into a pharmaceutical composition suitable for parenteral administration. In one aspect, binding proteins are prepared as an injectable solution containing between about 0.1 and about 250 mg/ml antibody. The injectable solution can be composed of either a liquid or lyophilized dosage form in a flint or amber vial, ampoule or pre-filled syringe. The buffer can be any suitable buffer such as L-histidine or a phosphate buffer saline at a concentration of about 1-50 mM, or about 5-10 mM. Other suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate and potassium phosphate. Buffers may be used to modify the toxicity of the pharmaceutical composition. For example, sodium chloride can be used to modify the toxicity of the binding protein solution at a concentration of from about 0.1 and about 300 mM, such as about 150 mM saline to modify the toxicity of a liquid dosage form.
Cryoprotectants, such as sucrose, can be included in a lyophilized dosage form at a concentration of about 0.1 to about 10% or from about 0.5 to about 1.0% may be used. Other suitable cryoprotectants include, but are not limited to, trehalose and lactose. Bulking agents, such as mannitol, can be included in a lyophilized dosage form at a concentration of about 1 to about 10%, or from about 2 to about 4%. Stabilizers, such as L-Methionine, can be used in both liquid and lyophilized dosage forms at a concentration of about 1 to about 50 mM, or about 5 to about 10 mM). Other suitable bulking agents include, but are not limited to, glycine and arginine. Surfactants, such as polysorbate-80, can be included in both liquid and lyophilized dosage forms at a concentration of about 0.001 to about 0.05% or about 0.005 to about 0.01%. Additional surfactants include, but are not limited to, polysorbate 20 and BRIJ surfactants.
An exemplary pharmaceutical formulation or composition of the present disclosure may be a liquid pharmaceutical composition having a pH between about 7.4 to about 8.0. The liquid pharmaceutical composition comprises about 5 mg/ml to about 50 mg/ml of an antibody comprising a heavy chain variable region comprising an amino acid sequence having a sequence of SEQ ID NO: 37 and a light chain variable region comprising an amino acid sequence comprising a sequence of SEQ ID NO: 38. The liquid pharmaceutical composition further comprises at least one buffer (such as, phosphate buffer saline, tris or histidine). The molarity of buffer that can be used can be from about 1 mM to about 60 mM. Optionally, said pharmaceutical composition or formulation can also contain at least one preservative, such as, methylparaben, propylparaben, benzyl alcohol, chlorobutanol or benzalkonium chloride. The amount of preservative that can be used can be from about 0.01 percent by volume to about 5.0% by volume depending on the preservative used.
Another exemplary pharmaceutical formulation or composition of the present disclosure may be a liquid pharmaceutical composition comprising a pH between about 7.4 to about 8.0. The liquid pharmaceutical composition comprises about 5 mg/ml to about 50 mg/ml of an antibody comprising a heavy chain variable region comprising an amino acid sequence having a sequence of SEQ ID NO: 192 and a light chain variable region comprising an amino acid sequence comprising a sequence of SEQ ID NO: 193. The liquid pharmaceutical composition further comprises at least one buffer (such as, phosphate buffer saline, tris or histidine). The molarity of buffer that can be used can be from about 1 mM to about 60 mM. Optionally, said pharmaceutical composition or formulation can also contain at least one preservative, such as, methylparaben, propylparaben, benzyl alcohol, chlorobutanol or benzalkonium chloride. The amount of preservative that can be used can be from about 0.01 percent by volume to about 5.0% by volume depending on the preservative used.
The compositions of this disclosure may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The form of the disclosed composition may depend on the intended mode of administration and therapeutic application. The disclosed compositions may be in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies. The mode of administration may be parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). The disclosed binding proteins may be administered by intravenous infusion or injection, or by intramuscular or subcutaneous injection.
Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other suitable ordered structure such as those suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., antibody or antibody portion) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Dispersions may be prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile, lyophilized powders for the preparation of sterile injectable solutions, the methods of preparation include, but are not limited to, vacuum drying and spray-drying that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption such as, for example, monostearate salts and gelatin.
An antibody or antibody portion of the disclosure may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the disclosure by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.
The disclosed binding proteins may be co-administered with other active compounds which may also be incorporated into the disclosed compositions. An antibody or antibody portion of the disclosure may be co-formulated with and/or co-administered with one or more additional therapeutic agents that are useful for treating disorders in which NGF activity is detrimental. For example, an anti-NGF antibody or antibody portion of the disclosure may be co-formulated and/or co-administered with one or more additional antibodies that bind other targets (e.g., antibodies that bind other cytokines or that bind cell surface molecules). Furthermore, one or more disclosed binding proteins may be used in combination with two or more of the foregoing therapeutic agents. Such combination therapies may, for example, enable the use of lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.
An antibody to NGF or fragment thereof may be formulated with a vehicle that extends the half-life of the binding protein. Suitable vehicles known in the art include, but are not limited to, the Fc domain, polyethylene glycol, and dextran. Such vehicles are described, e.g., in U.S. application Ser. No. 09/428,082 and published PCT Application No. WO 99/25044.
Isolated nucleic acid sequences comprising nucleotide sequences encoding disclosed binding proteins or another prophylactic or therapeutic agent of the disclosure may be administered to treat, prevent, manage, or ameliorate a disorder or one or more symptoms thereof by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid, wherein the nucleic acids produce their encoded antibody or prophylactic or therapeutic agent of the disclosure that mediates a prophylactic or therapeutic effect.
Any of the methods for gene therapy available in the art can be used according to the present disclosure. For general reviews of the methods of gene therapy, see Goldspiel et al., Clinical Pharmacy, 12: 488-505 (1993); Wu et al., Biotherapy, 3: 87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol., 32: 573-596 (1993); Mulligan, Science, 260: 926-932 (1993); and Morgan et al., Ann. Rev. Biochem., 62: 191-217 (1993); TIBTECH, 11(5): 155-215 (1993). Methods commonly known in the art of recombinant DNA technology which can be used are described in, for example, Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990). Detailed descriptions of various methods of gene therapy are disclosed in US20050042664A1.
Antibodies of the disclosure, or antigen binding portions thereof, can be used alone or in combination to treat NGF related diseases. It should be understood that the antibodies of the disclosure or antigen binding portion thereof can be used alone or in combination with an additional agent, e.g., a therapeutic agent, said additional agent being selected by the skilled artisan for its intended purpose. For example, the additional agent can be a therapeutic agent art-recognized as being useful to treat the disease or condition being treated by the antibody of the present disclosure. The additional agent also can be an agent that imparts a beneficial attribute to the therapeutic composition e.g., an agent which affects the viscosity of the composition.
It should further be understood that the combinations which are to be included within this disclosure are those combinations useful for their intended purpose. The agents set forth below are illustrative for purposes and not intended to be limited. The combinations, which are part of this disclosure, can be the antibodies of the present disclosure and at least one additional agent selected from the lists below. The combination can also include more than one additional agent, e.g., two or three additional agents if the combination is such that the formed composition can perform its intended function.
Combinations include non-steroidal anti-inflammatory drug(s) also referred to as NSAIDS which include drugs like ibuprofen. Other combinations are corticosteroids including prednisolone; the well-known side-effects of steroid use can be reduced or even eliminated by tapering the steroid dose required when treating patients in combination with the anti-NGF antibodies of this disclosure. Non-limiting examples of therapeutic agents for rheumatoid arthritis or pain with which an antibody, or antibody portion, of the disclosure can be combined include the following: cytokine suppressive anti-inflammatory drug(s) (CSAIDs); antibodies to or antagonists of cytokines or growth factors, for example, TNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, IL-21, interferons, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of the disclosure, or antigen binding portions thereof, can be combined with antibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, CTLA or their ligands including CD154 (gp39 or CD40L).
Combinations of therapeutic agents may interfere at different points in the autoimmune and subsequent inflammatory cascade; examples include TNF antagonists like chimeric, humanized or human TNF antibodies, D2E7, (PCT Publication No. WO 97/29131), CA2 (Remicade™), CDP 571, and soluble p55 or p75 TNF receptors, derivatives, thereof, (p75TNFR1gG (Enbrel™) or p55TNFR1gG (Lenercept), and also TNFa converting enzyme (TACE) inhibitors; similarly other IL-1 inhibitors (Interleukin-1-converting enzyme inhibitors, IL-IRA etc.) may be effective for the same reason. Other combinations include Interleukin 11.
The antibodies of the disclosure, or antigen binding portions thereof, may also be combined with agents, such as methotrexate, 6-MP, azathioprine sulphasalazine, mesalazine, olsalazine chloroquinine/hydroxychloroquine, pencillamine, aurothiomalate (intramuscular and oral), azathioprine, cochicine, corticosteroids (oral, inhaled and local injection), beta-2 adrenoreceptor agonists (salbutamol, terbutaline, salmeteral), xanthines (theophylline, aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium and oxitropium, cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adensosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, agents which interfere with signaling by proinflammatory cytokines such as TNF or IL-1 (e.g. IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1β converting enzyme inhibitors, TNFa converting enzyme (TACE) inhibitors, T-cell signaling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors and derivatives thereof (e.g. soluble p55 or p75 TNF receptors and the derivatives p75TNFRIgG (Enbrel™ and p55TNFRIgG (Lenercept)), sIL-IRI, sIL-IRII, sIL-6R), anti-inflammatory cytokines (e.g. IL-4, IL-10, IL-11, IL-13 and TGFβ), celecoxib, folic acid, hydroxychloroquine sulfate, rofecoxib, etanercept, infliximab, naproxen, valdecoxib, sulfasalazine, methylprednisolone, meloxicam, methylprednisolone acetate, gold sodium thiomalate, aspirin, triamcinolone acetonide, propoxyphene napsylate/apap, folate, nabumetone, diclofenac, piroxicam, etodolac, diclofenac sodium, oxaprozin, oxycodone hcl, hydrocodone bitartrate/apap, diclofenac sodium/misoprostol, fentanyl, anakinra, human recombinant, tramadol hcl, salsalate, sulindac, cyanocobalamin/fa/pyridoxine, acetaminophen, alendronate sodium, prednisolone, morphine sulfate, lidocaine hydrochloride, indomethacin, glucosamine sulf/chondroitin, amitriptyline hcl, sulfadiazine, oxycodone hcl/acetaminophen, olopatadine hcl, misoprostol, naproxen sodium, omeprazole, cyclophosphamide, rituximab, IL-1 TRAP, MRA, CTLA4-IG, IL-18 BP, anti-IL-18, Anti-IL15, BIRB-796, SCIO-469, VX-702, AMG-548, VX-740, Roflumilast, IC-485, CDC-801, and Mesopram. Other combinations include methotrexate or leflunomide and in moderate or severe rheumatoid arthritis cases, cyclosporine. The antibodies of the disclosure, or antigen binding portions thereof, may also be combined with agents, such as cancer chemotherapeutics, antimicrobials, anti-inflammatories, and anthelmintics used in animals.
The NSAID may be any non-steroidal anti-inflammatory compound. NSAIDs are categorized by virtue of their ability to inhibit cyclooxygenase. Cyclooxygenase 1 and cyclooxygenase 2 are two major isoforms of cyclooxygenase and most standard NSAIDs are mixed inhibitors of the two isoforms. Most standard NSAIDs fall within one of the following five structural categories: (1) propionic acid derivatives, such as ibuprofen, naproxen, naprosyn, diclofenac, and ketoprofen; (2) acetic acid derivatives, such as tolmetin and slindac; (3) fenamic acid derivatives, such as mefenamic acid and meclofenamic acid; (4) biphenylcarboxylic acid derivatives, such as diflunisal and flufenisal; and (5) oxicams, such as piroxim, sudoxicam, and isoxicam. Another class of NSAID has been described which selectively inhibit cyclooxygenase 2. Cox-2 inhibitors have been described (U.S. Pat. Nos. 5,616,601; 5,604,260; 5,593,994; 5,550,142; 5,536,752; 5,521,213; 5,475,995; 5,639,780; 5,604,253; 5,552,422; 5,510,368; 5,436,265; 5,409,944; and 5,130,311). Certain exemplary COX-2 inhibitors include celecoxib (SC-58635), rofecoxib, DUP-697, flosulide (CGP-28238), meloxicam, 6-methoxy-2 naphthylacetic acid (6-MNA), MK-966, nabumetone (prodrug for 6-MNA), nimesulide, NS-398, SC-5766, SC-58215, T-614; or combinations thereof.
The NGF antagonist and/or an additional therapeutic agent, such as NSAID, can be administered to a subject via any suitable route. For example, they can be administered together or separately, and/or simultaneously and/or sequentially, orally, intravenously, sublingually, subcutaneously, intraarterially, intramuscularly, rectally, intraspinally, intrathoracically, intraperitoneally, intraventriculariy, sublingually, transdermally or by inhalation. Administration can be systemic, e.g., intravenous, or localized. The nerve growth factor antagonist and the additional therapeutic agent may be present together with one or more pharmaceutically acceptable carriers or excipients, or they may be present in separate compositions. In another aspect, the invention provides a synergistic composition of an NGF antagonist and an NSAID.
The pharmaceutical compositions of the disclosure may include a “therapeutically effective amount” or a “prophylactically effective amount” of an antibody or antibody portion of the disclosure. Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antibody portion of the disclosure is about 0.001 to about 20 mg/kg or about 0.001 to about 10 mg/kg. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the disclosure described herein are obvious and may be made using suitable equivalents without departing from the scope of the disclosure or the embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting of the disclosure.
EXAMPLES
The following examples are provided for exemplary guidance to make and use the disclosed binding proteins and pharmaceutical compositions thereof according to the inventive subject matter. However, it should be recognized that numerous modifications may be made without departing from the inventive concept presented herein.
Example 1: Immunization of Mice with NGF
To generate mouse anti-NGF mAbs, female A/J mice were immunized subcutaneously with 25 μg of human β NGF (R&D Systems catalog #256-GF/CF) in CFA. Animals were boosted every three weeks with 25 μg human β NGF in IFA. Four days prior to fusion, the mice were boosted with 10 μg of human β NGF in sterile saline intravenously. Spleen cells from the immunized mouse were fused with SP2/0-Agl4 myeloma cells at a 5:1 ratio spleen to SP2/0 cells, using standard techniques. Seven to ten days post fusion, when macroscopic colonies were observed; supernatants were tested in a capture ELISA format for binding to biotinylated human or rat β NGF. ELISA-positive wells were expanded to 24 well plates and tested for binding to biotinylated rat β NGF. Supernatants from hybridoma cell lines testing positive for both human and rat NGF were evaluated in a bioassay format. Cell lines of interest were cloned by limiting dilution to isolate an NGF-specific mouse monoclonal antibody.
Example 2: Screening Hybridoma Supernatants to Identify Secreted Anti-NGF MAbs
A. Indirect Binding ELISA
To determine if anti-NGF mAbs were present in hybridoma supernatants, ELISA plates were coated with goat anti-murine IgG Fc (Jackson ImmunoResearch, cat #115-005-164) and incubated overnight at 4° C. The plates were washed three times with wash buffer. The plates were blocked with 200 μl of 2% milk and incubated for 1 hour at room temperature. The plates were washed as above. Hybridoma supernatants were diluted 5-fold, 25-fold, 125-fold and 1625-fold with PBS and then added to the plate wells and incubated for 1 hour at room temperature. The positive control was crude sera (diluted 1:500 with PBS) isolated from a β NGF immunized mouse and the negative control was hybridoma supernatant derived from a mouse immunized with an antigen other than NGF. The plates were washed and then 50 μl of biotinylated human or rat β NGF at 50 ng/ml was added and incubated for 1 hour at room temperature. The plates were washed. Streptavidin-HRP (Thermo, cat #21 126) conjugate was diluted at 10,000 and added to the plates. The plates were incubated for 30 minutes at room temperature. The plates were washed and then TMB substrate (Invitrogen, catalog #00-2023) was added. The reaction was stopped using 2N H2S04 (VWR, catalog #BDH3500-1). The absorbance at 450 nm was read on a Spectromax 2E plate reader (Molecular Devices); these absorbance readings are shown in Tables 1 and 2. The numerical value indicates binding of mouse anti-NGF antibodies to biotinylated human or rat β NGF. This data indicates that several hybridoma supernatants contained anti-NGF antibodies.
TABLE 3
Biotinylated Human NGF Indirect Binding ELISA data
Supernatant
dilution
(fold)
30F11
23F1
22E1
3C3
16B9
17G6
23H2
25E5
29E6
7H1
19C1
30A1
5
1.066
1.143
1.288
1.137
0.821
1.122
0.913
1.299
1.196
1.155
0.936
1.09
25
1.005
1.171
1.255
1.108
0.644
1.127
0.529
1.254
1.127
1.159
0.555
0.926
125
0.873
0.979
0.772
0.948
0.34
1.017
0.191
0.988
0.889
1.002
0.234
0.507
625
0.436
0.696
0.296
0.571
0.107
0.713
0.085
0.512
0.426
0.673
0.1
0.223
Supernatant
dilution
(fold)
29A7
27A5
26D5
26H12
23D7
22A9
22G3
21D4
3E9
3F9
2G11
1D6
5
1.198
1.116
0.954
0.943
1.087
0.707
0.662
1.154
1.167
0.974
1.038
0.545
25
1.092
0.887
0.903
0.794
1.06
0.549
0.498
1.042
0.996
0.694
0.992
0.457
125
0.762
0.395
0.823
0.381
0.857
0.348
0.24
0.899
0.655
0.323
0.819
0.164
625
0.293
0.174
0.542
0.135
0.489
0.168
0.126
0.543
0.298
0.145
0.486
0.066
Supernatant
dilution
(fold)
4B6
8E4
9E2
9H2
20B10
14G6
12H12
11D1
5
1.252
1.294
1.126
1.167
1.098
1.274
1.222
0.642
25
1.131
1.076
1.085
0.915
0.997
1.206
1.083
0.497
125
0.768
0.595
0.938
0.395
0.576
0.956
0.741
0.275
625
0.341
0.25
0.605
0.171
0.143
0.598
0.363
0.117
Supernatant
dilution
Positive
(fold)
control
4E2
12D6
1D10
2D8
3F7
4F11
4H2
5D8
5G9
6B2
6F10
3
1.018
1.078
0.985
1.105
1.046
1.282
1.192
1.013
0.79
1.052
1.231
1.096
15
0.981
0.991
0.844
0.963
0.868
1.166
1.016
0.8
0.654
0.919
0.939
1.045
75
1.02
0.705
0.501
0.655
0.436
1.049
0.702
0.447
0.42
0.534
0.505
0.999
Supernatant
dilution
Negative
Negative
(fold)
6H2
7C10
7G1
8G9
10A12
10B6
11A9
12A5
12F6
13E3
14A9
control
3
1.322
0.745
0.233
0.849
0.192
1.135
0.056
0.725
1.003
1.003
1.107
0.054
15
1.221
0.378
0.106
0.548
0.089
1.088
0.051
0.401
0.944
0.881
1.082
0.053
75
0.791
0.151
0.066
0.22
0.06
0.872
0.05
0.183
0.681
0.463
0.951
0.051
TABLE 4
Biotinylated Rat NGF Indirect Binding ELISA data
Supernatant
dilution
(fold)
30F11
23F1
22E1
3C3
16B9
17G6
23H2
25E5
29E6
7H1
19C1
30A1
5
0.694
0.764
1.054
0.698
0.443
0.749
0.670
1.091
0.677
0.733
0.660
0.690
25
0.734
0.767
0.936
0.729
0.350
0.758
0.412
1.099
0.655
0.664
0.462
0.681
125
0.603
0.737
0.557
0.628
0.218
0.751
0.176
0.803
0.523
0.603
0.197
0.445
625
0.361
0.528
0.229
0.520
0.094
0.567
0.083
0.396
0.261
0.401
0.088
0.180
Supernatant
dilution
(fold)
29A7
27A5
26D5
26H12
23D7
22A9
22G3
21D4
3E9
3F9
2G11
1D6
5
0.967
0.610
0.611
0.538
0.684
0.508
0.521
0.787
1.098
0.633
0.705
0.327
25
0.907
0.514
0.571
0.368
0.775
0.417
0.384
0.760
0.945
0.502
0.669
0.278
125
0.441
0.236
0.516
0.169
0.654
0.240
0.209
0.671
0.530
0.264
0.588
0.132
625
0.224
0.113
0.413
0.082
0.396
0.117
0.107
0.453
0.219
0.117
0.353
0.063
Supernatant
dilution
(fold)
4B6
8E4
9E2
9H2
20B10
14G6
12H12
11D1
5
0.607
0.685
0.632
0.453
0.472
0.755
0.676
0.122
25
0.508
0.518
0.559
0.310
0.431
0.739
0.571
0.095
125
0.438
0.317
0.529
0.157
0.261
0.665
0.357
0.076
625
0.234
0.150
0.382
0.085
0.108
0.424
0.173
0.060
Supernatant
dilution
Positive
(fold)
control
4E2
12D6
1D10
2D8
3F7
4F11
4H2
5D8
5G9
6B2
6F10
3
0.773
0.777
0.459
1.023
0.590
1.097
0.952
0.945
0.565
0.952
1.122
0.937
15
0.736
0.651
0.379
0.877
0.599
1.125
0.690
0.684
0.467
0.767
0.876
1.005
75
0.760
0.471
0.210
0.548
0.323
1.044
0.576
0.348
0.294
0.406
0.453
0.849
Supernatant
dilution
Negative
(fold)
6H2
7C10
7G1
8G9
10A12
10B6
11A9
12A5
12F6
13E3
14A9
control
3
1.108
0.541
0.197
0.681
0.145
0.440
0.058
0.521
0.904
0.786
0.845
0.055
15
0.860
0.275
0.093
0.396
0.077
0.784
0.052
0.334
0.810
0.737
0.777
0.053
75
0.603
0.115
0.061
0.155
0.060
0.727
0.051
0.153
0.565
0.413
0.582
0.052
B. TrkA Binding ELISA
To determine if anti-NGF mAbs in hybridoma supernatants blocked NGF from binding to the TrkA receptor, ELISA plates were coated with goat anti-human IgG Fc (Jackson ImmunoResearch, cat #109-005-008) at 2 μg/ml in PBS and incubated over night at 4° C. The plates were washed three times with PBS/Tween. The plates were blocked with 200 μl/well of 2% milk in PBS for 1 hour at room temperature. The plates were washed three times as above. Rat TrkA/Fc chimera (R&D Systems, catalog #1056-TK) was added at 1 μg/ml (50 μl/well) in PBS/0.1% BSA and then incubated for 1 hour at room temperature. Biotinylated human NGF was titered and pre-incubated with anti-NGF antibody supernatants diluted 1-fold, 5-fold, and 25-fold, or purified anti-NGF mAbs diluted to 0.08, 0.4, 2, or 10 μg/ml for 1 hour at room temperature on a plate shaker. The negative control was unrelated conditioned supernatant. The positive control was sera from a mouse immunized with NGF. The plates were washed and then 50 μl of each biotinylated NGF/Ab mix was added to the appropriate wells. The plates were incubated for 1 hour at room temperature. The plates were washed. 50 μl of streptavidin-HRP (Thermo, cat #21 126) was added at 10,000 dilution. The plates were incubated for 30 min at room temperature. The plates were washed. 50 μl of TMB (Invitrogen, cat #00-2023) was added and the reaction was stopped using 2N H2S04 (VWR, cat #BDH3500-1). The absorbance at 450 nm was read on a Spectromax 2E plate reader (Molecular Devices), and the absorbance readings are shown in Table 5. The numerical value indicates binding of biotinylated human β NGF to rat TrkA/Fc chimera. This data indicates that several hybridoma supernatants contained anti-NGF receptor-blocking antibodies.
TABLE 5
Rat TrkA Inhibition Binding ELISA Data for Anti-NGF Hybridoma Supernatants
Supernatant
dilution
Negative
Positive
(fold)
control
control
30F11
23F1
22E1
3C3
16B9
17G6
23H2
25E5
29E6
7H1
1
0.465
0.050
0.158
0.108
0.357
0.146
0.142
0.091
0.379
0.304
0.291
0.217
5
0.456
0.055
0.210
0.140
0.429
0.195
0.249
0.123
0.622
0.354
0.600
0.419
25
0.462
0.102
0.331
0.276
0.558
0.345
0.409
0.210
0.418
0.505
0.881
0.758
Supernatant
dilution
(fold)
19C1
30A1
29A7
27A5
26D5
26H12
23D7
22A9
22G3
21D4
3E9
3F9
1
0.285
0.148
0.427
0.444
0.063
0.344
0.131
0.322
0.150
0.133
0.328
0.186
5
0.567
0.281
0.462
0.800
0.076
0.621
0.212
0.362
0.211
0.242
0.416
0.295
25
0.686
0.464
0.502
0.680
0.101
0.665
0.393
0.453
0.337
0.404
0.682
0.498
Supernatant
Dilution
(fold)
Neg
Pos
2G11
1D6
4B6
8E4
9E2
9H2
1
0.372
0.052
0.138
0.226
0.169
0.273
0.103
0.380
5
0.336
0.073
0.205
0.281
0.287
0.669
0.125
0.604
25
0.318
0.228
0.328
0.343
0.424
0.693
0.151
0.521
Supernatant
dilution
(fold)
20B10
14G6
12H12
11D1
19A12
2B12
PBS
PBS
1
0.166
0.113
0.060
0.101
0.100
0.065
0.295
0.315
5
0.200
0.192
0.099
0.152
0.170
0.070
0.334
0.297
25
0.289
0.334
0.190
0.264
0.295
0.095
0.306
0.289
Supernatant
dilution
−ve
+ve
+ve
+ve
(fold)
contrl
contrl
contrl
contrl
13E3
14A9
4E2
12D6
1D10
2D8
3F7
4F11
1
0.386
0.112
0.145
0.104
0.400
0.121
0.283
0.145
0.248
0.359
0.056
0.286
5
0.388
0.164
0.234
0.140
0.383
0.208
0.290
0.211
0.312
0.588
0.083
0.356
25
0.386
0.308
0.488
0.216
0.497
0.376
0.334
0.364
0.447
0.497
0.149
0.541
Supernatant
dilution
(fold)
4H2
5D8
5G9
6B2
6F10
6H2
7C10
8G9
10B6
12A5
12F6
PBS
1
0.396
0.363
0.344
0.096
0.206
0.400
0.230
0.409
0.329
0.306
0.172
0.436
5
0.457
0.398
0.387
0.215
0.212
0.523
0.489
0.473
0.364
0.328
0.227
0.351
25
0.606
0.504
0.473
0.451
0.242
0.738
0.487
0.413
0.399
0.406
0.324
0.338
C. SureFire Cellular Phospho-ERK (pERK) Assay
To determine if anti-NGF mAbs in hybridoma supernatants blocked downstream signaling as a result of blocking NGF from binding to TrkA, Neuroscreen-1 cells (Thermo Fisher Scientific) were grown on collagen I-coated flasks in RPMI medium supplemented with 10% horse serum, 5% FBS, 100 units/ml penicillin/streptomycin, 2 mM L-glutamine, and 10 mM HEPES at 37° C. in a humidified atmosphere at 95% air and 5% CO2. For the ERK phosphorylation assay, 5×104 cells were seeded in each well of a 96-well plate coated with collagen I (Becton Dickinson). Cells were then serum starved for 24 hours before stimulation. 130 pM human β NGF (R&D Systems catalog #256-GF/CF) was mixed into diluted hybridoma supernatants (to achieve a final supernatant dilution (fold) of 10-fold, 100-fold, 500-fold or 1,000-fold) and mixtures were pre-incubated for 15 min at 37° C. before being added to the cells. Each diluted hybridoma supernatant was tested in quadruplicate. After 5 min of stimulation, the medium was removed and replaced with SureFire™ AlphaScreen cell lysis (PerkinElmer). Cell lysates were then processed according to the manufacturer's instructions and fluorescence signals quantified using an En Vision plate reader (PerkinElmer); the fluorescence data is summarized in Table 6. The numerical value indicates ERK phosphorylation due to TrkA signaling in the presence of human β NGF and is expressed as the percentage of signal vs. maximum signal. The maximum signal is defined as 100% response from cells showing ERK phosphorylation in the presence of only β NGF (no hybridoma supernatant). This data indicates that several hybridoma supernatants contained neutralizing anti-NGF antibodies.
TABLE 6
SureFire pERK Assay Data Generated with Anti-NGF mAb Hybridoma Supernatants
Supernatant
dilution
(fold)
23F1
17G6
30F11
3C3
100
5
2
4
2
2
0
1
1
5
2
2
0
5
2
5
3
1000
5
3
4
3
2
2
2
1
4
2
4
3
6
3
5
4
5000
8
7
8
8
13
7
15
8
30
26
28
25
24
25
22
23
10000
35
33
32
32
44
25
43
23
65
45
56
42
57
52
68
62
Supernatant
dilution
(fold)
2B12
21D4
4B6
22G3
100
0
0
−1
0
4
3
5
2
5
4
1
0
13
7
7
2
1000
0
0
0
0
5
3
5
3
1
0
2
1
3
2
4
2
5000
18
16
21
17
11
7
12
8
8
8
7
8
23
18
25
20
10000
51
43
49
41
38
23
37
23
30
34
30
35
51
45
47
43
Supernatant
dilution
(fold)
2G11
14G6
16B9
19A12
100
5
2
6
3
4
3
0
−1
3
3
3
2
2
1
2
1
1000
6
3
6
3
−1
0
0
0
65
57
70
60
3
2
3
2
5000
14
8
14
8
7
7
8
7
72
63
73
62
47
36
46
32
10000
44
30
74
48
38
36
36
36
76
62
77
62
69
55
81
65
Supernatant
dilution
(fold)
30A1
26D5
23D7
23H2
100
0
0
1
0
0
0
0
0
2
2
−1
0
47
41
44
44
1000
1
2
2
2
1
1
1
1
1
0
1
1
86
80
79
82
5000
40
41
42
40
37
30
35
30
59
48
56
54
85
80
85
83
10000
62
67
63
64
64
52
80
71
74
63
70
60
85
84
82
84
Supernatant
dilution
(fold)
9E2
20B10
12H12
11D1
100
3
3
3
4
2
1
1
1
3
3
3
2
69
75
70
78
1000
5
7
5
8
1
1
1
0
30
30
30
32
71
84
72
85
5000
37
57
36
55
20
28
19
29
62
64
60
61
76
78
80
76
10000
55
69
56
67
55
71
73
84
69
78
76
77
89
95
102
101
D. PathHunter Assay
To determine if anti-NGF mAbs in hybridoma supernatants blocked downstream signaling as a result of blocking NGF from binding to TrkA, the PathHunter U20S stable cell line stably expressing the NGF receptor TrkA and the co-activator protein SHC1 fused to complementing fragments of β-galactosidase was purchased from DiscoveRx. Cells were grown in MEM media supplemented with 10% FBS, 100 units/ml penicillin/streptomycin, 2 mM L-glutamine, 500 μg/ml Geneticin G418, and 250 μg/ml Hygromycin at 37° C. in a humidified atmosphere at 95% air and 5% CO2. Sixteen hours before the assay, 2×104 cells were seeded in each well of a 96-well half-volume black plate in 40 μl of MEM media supplemented with 0.5% horse serum. 440 pM human β NGF (R and D Systems catalog #256-GF/CF) was mixed into diluted hybridoma supernatants (to achieve a final supernatant dilution of 10-fold, 100-fold, 500-fold or 1,000-fold) and mixtures were pre-incubated for 15 min at 37° C. before being added to the cells. Cell plates were incubated for 5 min at room temperature before stimulation with 10 μl per well of NGF/antibody mixture. After 3 hours of cell induction at room temperature, 25 μl of PathHunter detection reagent was added to each well according to the manufacturers instructions. The chemiluminescent signal was detected 1 hour later using a TopCount plate reader (PerkinElmer); the chemiluminescence signal data is shown in Table 7. The numerical value indicates β-galactosidase generation due to TrkA signaling in the presence of human β NGF and is expressed as the percentage of signal vs. maximum signal. The maximum signal is defined as 100% response from cells showing in the presence of β-galactosidase generation in the presence of only β NGF (no hybridoma supernatant). This data indicates that several hybridoma supernatants contained neutralizing anti-NGF antibodies.
TABLE 7
PathHunter Data Generated with Hybridoma Supernatants
Supernatant
dilution
(fold)
30F11
23F1
3C3
16B9
17G6
19A12
100
21
22
5
5
16
29
19
24
10
14
25
28
1000
42
37
23
13
40
46
117
114
23
24
21
30
5000
97
99
69
70
71
81
120
127
100
115
93
92
10000
94
93
92
91
78
84
114
129
115
120
89
98
Supernatant
dilution
(fold)
2B12
30A1
26D5
23D7
23H2
22G3
100
89
90
21
24
83
84
31
28
142
176
16
16
1000
64
70
50
57
49
51
54
53
88
134
20
23
5000
128
127
126
139
92
96
112
131
117
120
89
99
10000
128
133
133
129
95
84
124
148
111
136
86
101
Supernatant
dilution
(fold)
21D4
2G11
4B6
9E2
20B10
100
9
10
22
22
24
31
102
104
6
5
1000
16
17
29
27
46
49
77
91
0
4
5000
107
100
66
72
88
94
137
152
25
31
10000
108
112
66
72
102
109
137
143
52
58
Supernatant
dilution
Unrelated
(fold)
14G6
12H12
11D1
26H12
hybridoma
100
18
17
26
23
117
101
106
119
156
185
1000
27
27
46
56
127
118
124
115
136
144
5000
105
83
145
137
109
99
137
166
126
132
10000
113
109
122
145
91
98
151
167
138
137
Example 3: Hybridoma Sub-Cloning
Hybridoma cell lines were subcloned using standard limiting dilution techniques. Cells were diluted to a concentration of 50, 5, or 0.5 cells/mL. 200 ul of the diluted cell suspensions were plated into 96 well tissue culture plates. The plates were incubated at 37° C. with 5% CO2 and −90% relative humidity. The growth was visually checked at day 7 for macroscopic colonies. Supernatants from wells were screened for antibody production when colony growth was visible. Table 8 shows the subclone identification nomenclature and monikers. This data indicates that several anti-NGF antibodies could be isolated from a clonal population of cells.
TABLE 8
Hybridoma Subclone Identification and Monikers
Hybridoma
Supernatant
Subcloned
Name
Hybridoma Name
Monker
Lot #
14G6
ML129-14G6.3H3
PR-1254970
1764671
2G11
ML129-2G11.3B1
PR-1254971
1734673
20B10
ML129-20B10.3F4
PR-1254972
1734675
2B12
ML129-2B12.5G9
PR-1254973
1734676
17G6
ML129-17G6.3E7
PR-1254974
1734677
21D4
ML129-21D4.4A11
PR-1254977
1734678
4B6
ML129-4B6.4H3
PR-1254978
1734679
22G3
ML129-22G3.3F3
PR-1254979
1734680
23F1
ML129-23F1.4G3
PR-1254980
1734681
14A9
ML130-14A9.5B12
PR-1254981
1734682
3F7
ML130-3F7.4A8
PR-1254982
1734683
Example 4: Scale Up and Purification of Monoclonal Antibodies
Subcloned hybridoma cell lines were expanded into Hybridoma SFM (Invitrogen catalog #12045) with 5% Low IgG Fetal bovine serum (Invitrogen catalog #16250-078). Supernatants were harvested, centrifuged and filtered to remove cellular debris, and concentrated. Antibodies were mixed with Pierce binding buffer A (Thermo, catalog #21001) in a 1 ratio. The antibodies were loaded onto a recombinant Protein A sepharose (GE Healthcare, catalog #17-1279-04) chromatography column, eluted using Pierce elution buffer (Thermo, catalog #21004), neutralized using 2M Tris pH 7.5, and then dialyzed into PBS. This work allowed the isolation of anti-NGF mAbs for characterization studies.
Example 5: Cloning of Canine NGF
The coding region of canine NGF was amplified from canine universal cDNA (Biochain Institute, catalog #4734565) using primers of SEQ ID NO: 45 and SEQ ID NO: 46 or primers of SEQ ID NO: 47 and SEQ ID NO: 48 and cloned into a mammalian or bacterial expression vector, respectively. The PCR reactions were set up as recommended by the manufacturer (Novagen, KOD Hot Start Master Mix, catalog #71842-3). The mammalian clone was made as a C-terminal 6-His fusion protein by ligating the PCR product with pTT6 vector (Abbott) at the KpnI/XbaI restriction sites. The bacterial clone was made with the pro-NGF sequence using the mammalian clone as a template and ligated with pET15B (Novagen) at the NdeI/XhoI restriction sites. The DNA sequence and amino acid sequence of the canine NGF isolated are listed as SEQ ID NO: 49 and SEQ ID NO: 50, respectively. This work allowed expression of canine NGF protein for purification.
Example 6: Expression of Canine NGF
The canine NGF clone in the bacterial expression vector was grown at 37° C. in overnight express auto inducing Terrific Broth (Novagen) in Rosetta2 (DE3) E. coli host (EMD Biosciences) in 2 L non-baffled flasks. The cells were centrifuged down and the cell paste was resuspended in 100 mL of lysis buffer (25 mM Tris, 300 mM NaCl, 10% glycerol, 0.1% Triton X 100 pH 8.0) with lysonase and sonicated for 2 min on ice. The sample was centrifuged at 15000 RPM and the pellet was solubilized in 50 mL of 25 mM Tris, 6 M GdHCl pH 8.0. The sample was centrifuged at 15000 rpm for 30 min and the supernatant was loaded on to a 10 ml IMAC resin.
A. IMAC Chromatography
A 10 ml GE-Ni FF column was prepared. Buffer A: 25 mM Tris, 6 M GdHCl pH 8.0, Buffer B: A+500 mM Imidazole. The resin was equilibrated and loaded with recirculation to allow for complete binding (˜10 passes) overnight at 4° C. The column was washed with Buffer A. Batch elution was carried out with 40 ml Buffer A, followed by 30 ml Buffer A. 5 ml fractions were collected and pooled.
B. Refolding by Rapid Dilution
The pooled fraction was reduced by adding 50 mM DTT, and EDTA was added to 10 mM, and incubated for 1 h at RT. The sample was acidified by adding 6M HC1 to pH 4.0 and dialyzed into 6M GdHCl pH 5.0 to remove excess DTT. Refolding was performed by diluting the reduced/acidified sample in 1 L of 100 mM Tris, 1 M Arginine, 5 mM EDTA, 5 mM GSH, and 1 mM GSSG pH 9.5 for 4 h at 4° C. The refolded protein was dialyzed against 25 mM Tris, 200 mM NaCl, 10% Glycerol pH 8.0. Precipitation was cleared by filtration. The clarified sample was concentrated and diafiltered into 25 mM Tris, 200 mM NaCl, 10% Glycerol pH 8.0 using a 10K membrane.
C. Ni-IMAC
Refolded pro-NGF was loaded on a 5 ml Ni-IMAC. Buffer A: 25 mM Tris, 300 mM NaCl, 10% Glycerol pH 8.0. Buffer B: A+500 mM Imidazole. The column was washed with Buffer A. 8 ml fractions were collected. Elution was performed with a linear gradient 0-100% Buffer B. 5 ml fractions were collected. Samples of each fraction were mixed with non-reducing NuPage SLB (Invitrogen) and separated on a 4-12% NuPAGE Novex Bis-Tris Midi gel for analysis. Fractions containing protein were pooled and dialyzed against 20 mM Na Phosphate, 50 mM NaCl, 10% glycerol pH 7.4.
D. Trypsin Digestion
Pro-β NGF was mixed with trypsin in resuspension buffer and incubated on ice for 30 min. Immobilized inhibitor was added and incubated for 15 min and then filtered.
E. Sepharose Cation Exchange Chromatography
The sample was loaded on a 5 ml SP Sepharose high performance chromatography column (GE Healthcare). Buffer A: 20 mM Na Phosphate, 50 mM NaCl, 10% Glycerol pH 7.4, Buffer B: A+1 M NaCl. The column was washed with Buffer A. Elution was performed with linear gradient 0-100% Buffer B. 5 ml fractions were collected. The fractions were separated on a 4-12% Criterion XT Bis-Tris Midi gel for analysis. Fractions containing protein were pooled, dialyzed in PBS pH 7.4, and concentrated.
This work resulted in the production of several milligrams of purified canine NGF for characterization studies and for studies of anti-NGF canine antibodies.
Example 7: Characterization of Subcloned and Purified Hybridoma Antibodies
A. Canine NGF Direct Binding ELISA
To determine if purified mouse anti-NGF mAbs bind to canine β NGF, ELISA plates were coated with 50 μl/well of canine NGF (Abbott Laboratories) at 1 μg/ml in PBS and incubated over night at 4° C. The plates were washed three times with PBS+Tween buffer. The plates were blocked with 200 μl/well of 2% milk in PBS for 1 hour at room temperature. The plates were washed three times as above. Purified antibodies were diluted to 0.4, 2, or 10 μg/ml. 50 μl of each concentration of purified antibody was added to the plates. The plates were incubated for 1 hour at room temperature. The plates were washed. 50 μl of a 5000-fold diluted goat anti-mouse IgG Fc-HRP (Thermo, catalog #31439) was added. The plates were incubated for 1 hour at room temperature. 50 μl of TMB (Invitrogen, catalog #00-2023) was added and the reaction was stopped using 2N H2SO4 (VWR, catalog #BDH3500-1). The absorbance at 450 nm was read on a Spectromax 2E plate reader (Molecular Devices). The results are shown in Table 9, and the numerical value indicates binding of mouse anti-NGF antibodies to canine β NGF.
TABLE 9
Canine NGF Direct Binding ELISA Data Using Purified Anti-NGF mAbs
μg/ml
PR-
PR-
PR-
PR-
PR-
PR-
PR-
PR-
PR-
PR-
PR-
Mab
1254970
1254971
1254972
1254973
1254974
1254977
1254978
1254979
1254980
1254981
1254982
10
0.530
0.497
0.154
0.905
0.552
0.552
0.579
0.683
0.491
0.610
0.208
2
0.342
0.324
0.091
0.836
0.383
0.414
0.458
0.566
0.334
0.458
0.142
0.4
0.176
0.165
0.071
0.769
0.209
0.223
0.253
0.313
0.168
0.229
0.095
B. TF-1 Cell Proliferation Potency Assay
TF-1 is a human erythroleukaemic cell line that expresses human TrkA and proliferates in response to recombinant β NGF. To determine if purified anti-NGF mAbs blocked NGF-induced proliferation, TF-1 cells (ATCC#CRL-2003) were maintained at 37° C. and 5% C02 in RPMI (Gibco, cat #1 1875-093) media containing recombinant human GM-CSF at 2 ng/mL (R&D Systems, cat #215-GM) and fetal bovine serum (FBS, Hyclone, cat #SH 30070.03). GM-CSF and FBS was removed 24 hours before the assay. On day one of the assay each anti-NGF mAb was titrated (concentrations ranging from 33.3 nM to 1.7 fM) and added to a fixed concentration of recombinant canine NGF (70 pM) and TF-1 cells (2.5×10−4 cells/well) in RPMI+4% FBS for 72 hours. Cell proliferation was measured using Cell Titer-glo (Promega, cat #G7571). The IC50 values of each anti-NGF mAb on canine NGF-induced TF-1 cell proliferation is shown in Table 10 and the data shows that in the presence of 70 pM canine NGF, most of the anti-NGF antibodies display sub-nM potencies, and some display potencies of less than 50 pM.
TABLE 10
Potency of Mouse Anti-NGF Antibodies on Canine NGF-induced
TF-1 Cell Proliferation
Moniker
Lot
IC50 (nM)
PR-1254970
1764671
0.662
PR-1254971
1734673
1.088
PR-1254972
1734675
0.303
PR-1254973
1734676
0.039
PR-1254974
1734677
0.230
PR-1254977
1734678
0.217
PR-1254978
1734679
0.978
PR-1254979
1734680
0.288
PR-1254980
1734681
0.343
PR-1254981
1734682
0.046
PR-1254982
1734683
0.025
C. SureFire Cellular pERK and PathHunter Assays
To determine if purified mouse anti-NGF mAbs blocked canine NGF-induced cellular responses, purified antibodies were characterized by titration in the SureFire cellular pERK (using 128 pM canine β NGF in each test well) and PathHunter assays (using 441 pM canine NGF in each test well) as described in Example 2 Sections C and D. The IC5o of each anti-NGF mAb on canine β NGF-induced cellular responses is summarized in Table 11 and the data shows that in the presence of 128 pM canine NGF all the anti-NGF antibodies display sub-nM potencies, and some display potencies of less than 50 pM (pERK assay). Also, in the presence of 441 pM canine NGF, all the anti-NGF antibodies display sub-nM potencies, and some display potencies of less than 150 pM (PathHunter assay).
TABLE 11
Summary of pERK and Path Hunter Assay Data for Purified
Anti-NGF mAbs
SureFire pERK
PathHunter
Antibody
IC50 (nM)
IC50 (nM)
PR-1254970
0.02711
0.3346
PR-1254971
0.04750
0.4986
PR-1254972
0.2282
0.3133
PR-1254973
0.01876
0.1428
PR-1254974
0.01561
0.2464
PR-1254977
0.01759
0.1810
PR-1254978
0.02466
0.3559
PR-1254979
0.01627
0.2414
PR-1254980
0.01371
0.3812
PR-1254981
0.02135
0.2794
PR-1254982
0.005804
0.1505
Example 8: Characterization of Purified Anti-NGF Antibodies Following Hybridoma Subcloning
A. Mass Spectrophotometry (MS) and Size Exclusion Chromatography (SEC) Analysis on Anti-NGF Antibodies
The mouse anti-NGF mAbs were reduced using 1M DTT and analyzed using HPLC/MS on a 6224 TOF mass spectrometer and a 1200 HPLC (Agilent technologies) using a Vydac C4, IMM×150 mm column (CN#214TP5115, the Nest Group) at a flow rate of 50 μl/min. Buffer A: 99.9% HPLC water+0.1% FA+0.01% TFA and buffer B: 99.9% ACN+0.1% FA+0.01% TFA. The LC equilibrium and sample desalting was performed using 5% buffer B for 7 min. The separation gradient was performed using 30% to 50% Buffer B for 10 min and a washing step was performed at 95% buffer B for 10 mins. The TOF acquisition parameters were: gas temperature at 350 C and OCT/RF at 750V. The mass range was from 600-3200 m/z and the rate specified was 1.03 spectra/s. Qualitative analysis software (Agilent) was used to deconvolute antibody molecular weights.
The antibodies were analyzed on Shimadzu LC-10AVP system (Shimadzu Scientific). The SEC column used was a Superdex-200 10/300L (GE Healthcare). The flow rate was 0.75 ml/min and UV280 was used to monitor peaks. The buffer used was Na2S04+92 mM NaP04+5 mM NaZ3, pH 7.0. The reagent antibody was injected in 10 μl, (10 μg). The gel protein markers on SEC were from Bio-Rad (CN#151-1901). The MS and SEC results are summarized in Table 12. This data determined the hybridoma-derived antibodies were highly monomeric following purification. In addition, the molecular weights of the heavy and light chains comprising the hybridoma-derived antibodies were determined.
B. Antibody Isotype Determination
The isotype of the anti-NGF mAbs was determined using the Zymed Mouse MonoAb-ID Kit (Invitrogen catalog#90-6550 lot#1407589). The isotyping results are summarized in Table 12. This data indicates that murine IgG1/k, IgG2a/k, and IgG2b/k mouse antibodies are capable of binding and neutralizing NGF.
TABLE 12
Isotyping, Size Exclusion Chromatography, and Mass
Spectrometry Analysis of Anti-NGF Antibodies
Molecular
Molecular
weight (Dal)
weight (Dal)
Heavy
Hybridoma Name
Moniker
Lot
Isotype
% Monomer
Light Chain
Chain
ML129-14G6.3H3
PR-1254970
1734671
IgG1 Kappa
96.9
24221.43
49479.67
ML129-2G11.3B1
PR-1254971
1734673
IgG1 Kappa
96.8
24156.26
49491.69
ML129-20B10.3F4
PR-1254972
1734675
IgG2b Kappa
99.0
24159.34
50329.24
ML129-2B12.5G9
PR-1254973
1734676
IgG2b Kappa
99.4
23539.38
51102.21
ML129-17G6.3E7
PR-1254974
1734677
IgG1 Kappa
98.8
24221.43
49479.45
ML129-21D4.4A11
PR-1254977
1734678
IgG1 Kappa
98.4
24221.46
49479.70
ML129-4B6.4H3
PR-1254978
1734679
IgG1 Kappa
96.7
24170.40
49533.92
ML129-22G3.3F3
PR-1254979
1734680
IgG2a Kappa
99.0
24221.42
50123.17
ML129-23F1.4G3
PR-1254980
1734681
IgG1 Kappa
99.5
24221.42
49493.95
ML130-14A9.5B12
PR-1254981
1734682
IgG1 Kappa
99.1
24180.28
50241.85
ML130-3F7.4A8
PR-1254982
1734683
IgG1 Kappa
99.4
23708.54
50289.13
Example 9: Binding Kinetics of Anti-NGF Antibodies
A biomolecular protein interaction analysis was used to evaluate the binding kinetics of the interaction between the purified anti-NGF hybridoma antibodies and recombinant canine β NGF. The antibodies were captured using a goat anti-mouse IgG FC (10000 RU) surface which was directly immobilized to a CM5 chip using an amine coupling procedure according to the manufacturers instructions (Biacore). A sample size of 5 μl of antibody at a concentration of 1 μg/ml was captured at 10 μl minute. Recombinant canine NGF was used as the antigen. Canine NGF was injected at 75 μl/min (concentration range: 5-0.039 nM) for mouse antibodies. The association rate was monitored for 3.3 minutes and the dissociation rate was monitored for 10 minutes. Aliquots of canine NGF were also simultaneously injected over a reference reaction CM surface to record any nonspecific binding background. The instrument sensitivity for on-rate is I×IO7, such that any on-rate that is faster than I×IO7 may not be accurately measured; the instrument sensitivity for off-rate is 1×106, such that any off-rate that is slower than 1×10−6 may not be accurately measured. Therefore, an on-rate that is faster than 1×107 is recorded as >1×107 and an off-rate that is slower than 1×10−6 is recorded as <1×10−6 The biomolecular protein interaction analysis results are summarized in Table 13. This data indicates that the isolated murine anti-NGF mAbs have fast on-rates (from greater than 7×106) and slow off-rates (from less than I×IO−3). The overall KDs of the murine anti-NGF mAbs range from about 300 pM to 0.1 pM demonstrating efficient binding of the purified anti-NGF hybridoma antibodies to recombinant canine β NGF.
TABLE 13
Binding Kinetics of Anti-NGF mAbs to Canine NGF
On rate
Off rate
Overall
Antibody
(1/Ms)
(1/s)
affinity(M)
PR-1254972
Expt 1
>1 × 107
3.14 × 10−3
<3.14 × 10−10
lot: 1734675
Expt 2
>1 × 107
3.21 × 10−3
<3.21 × 10−10
Average
>1 × 107
3.18 × 10−3
<3.18 × 10−10
PR-1254973
Expt 1
>1 × 107
1.21 × 10−4
<1.21 × 10−11
lot: 1734676
Expt 2
>1 × 107
1.38 × 10−4
<1.38 × 10−11
Average
>1 × 107
1.30 × 10−4
<1.30 × 10−11
PR-1254977
Expt 1
>1 × 107
1.39 × 10−4
<1.39 × 10−11
lot: 1734678
Expt 2
>1 × 107
1.60 × 10−4
<1.6 × 10−11
Average
>1 × 107
1.50 × 10−4
<1.5 × 10−11
PR-1254980
Expt 1
>1 × 107
2.37 × 10−4
<2.37 × 10−11
lot: 1734681
Expt 2
>1 × 107
2.25 × 10−4
<2.25 × 10−11
Average
>1 × 107
2.31 × 10−4
<2.31 × 10−11
PR-1254981
Expt 1
8.67 × 106
1.27 × 10−4
1.47 × 10−11
lot: 1734682
Expt 2
7.48 × 106
1.40 × 10−4
1.87 × 10−11
Average
8.08 × 106
1.34 × 10−4
1.67 × 10−11
PR-1254982
Expt 1
>1 × 107
>1 × 10−6
>1 × 10−13
lot: 1734683
Expt 2
>1 × 107
>1 × 10−6
>1 × 10−13
Average
>1 × 107
>1e × 10−6
>1 × 10−13
Example 10: Method for Identifying Anti-NGF Antibody Sequences from Hybridomas by Cloning and Sequencing
To identify the nucleotide and amino acid sequence of the six subcloned hybridoma mAbs shown in Table 13, the RNA from individual hybridoma cultures was extracted with Qiagen RNeasy kit (Qiagen, cat #74104). RNA was reverse-transcribed and cDNA antibody sequences amplified using the Qiagen One-Step RT-PCR kit (Qiagen, catalog #210212). Forward primers were degenerate and designed to anneal to the variable regions (heavy chain primers: 1HA, 1HB, 1HC, 1HD, 1HE, 1HF; and light chain primers: 1LA, 1LB, 1LC, 1LD, 1LE, 1LF, 1LG) (EMD4 Biosciences catalog #69896). Reverse primers were also degenerate and made to constant regions of gamma (heavy chains) and kappa (light chains). PCR products of approximately 400-450 base pairs were gel isolated and purified with Qiagen Gel Extraction kit (Qiagen, cat #28706).
Purified PCR products were cloned into TOPO TA cloning vectors (Invitrogen, cat # K4500-01SC). Each topoisomerase reaction mixture was used to transform TOP 10 chemically competent bacteria and plated on LB plates with 75 μg/ml Ampicillin and 60 μl 2% Bluo-Gal (Invitrogen, cat #15519-028). Isolated colonies were picked from the LB plate to inoculate 20 μl LB broth/100 μg/ml carbenicillin. One μl of this mini-culture was used in a PCR reaction with MI 3 forward and reverse primers to amplify the insert in the TOPO vector. PCR products were separated on 2% agarose gels; samples indicating an appropriately-sized insert in the vector were sequenced using an Applied Biosystems model 3730S DNA sequencer. DNA sequences derived from the identification of all murine mAb heavy and light chain variable domains were translated into protein sequence and are shown in FIG. 1 to FIG. 24.
Example 11: Homology Modeling of Murine Anti-NGF Antibodies
The sequences of the heavy and light chain variable regions of each anti-NGF antibody were imported into InsightII (Accelrys, San Diego, Calif.). Each sequence was used as a template for BLAST to find the x-ray crystal structures from the Protein Data Bank (www.rcsb.org) which were closest in identity. One structure for each of the heavy and light chains was selected based both on percent identity and on matching the exact length of all CDR loops. The sequences of each template and each query sequence were aligned and standard homology modeling techniques used to construct homology models of each chain. The complex of both modeled chains was then minimized for 50 cycles of restrained (500 Kcal/Angstrom for all heavy atoms) conjugate gradient minimization using the CVFF force field in the DISCOVER program (Accelrys, San Diego, Calif.).
The likelihood that a given framework residue would impact the binding properties of the antibody depends on its proximity to the CDR residues. Therefore, using the model structures, residues that fell within 5 A of any CDR atom was identified as most important and were recommended to be candidates for retention of the murine residue in the caninized antibody sequences. A change in nucleotide(s) in a mutant gene that restores the original sequence and hence the original phenotype is often referred to as a back mutation. Therefore, we refer to residues that are candidates for retention of the murine residue in the caninized antibody sequences as backmutations.
Example 12: Identification of Canine Heavy and Light Chain Antibody Sequences from Canine PBMCs
To identify canine Ig heavy and lambda light chain antibody variable domain amino acid sequences, RNA was isolated from mongrel canine peripheral blood mononuclear cells (PBMCs) using an RNEasy kit (Qiagen #74104). Canine PBMC mRNA was reverse transcribed (RT) with Superscript III reverse transcriptase (Invitrogen catalog #18080-093) and cDNAs were amplified using the 5′ RACE System (Rapid Amplification of cDNA Ends) (Invitrogen #18374-058). RT and PCR primers (RK323, RK324, RK122, LG010, LG011, LG012) are described in patent publication number: U.S. Pat. No. 7,261,890 B2 entitled Methods for Using Canine Immunoglobulin Variable Domains and Caninized Antibodies). Primers RK323 and RK324 were used for canine IgG reverse transcription followed by nested PCR with RK326 and the Abridged Anchor Primer (AAP) (Invitrogen). LG011 was used for canine lambda light chain RT PCR, followed by nested PCR with LG010 and LG012 and AAP.
The resulting PCR products were separated by agarose gel electrophoresis. The 600 base pair (canine lambda and kappa light chains) and 800 base pair (canine Ig heavy chain) PCR products were purified from the agarose using a Gel Extraction kit (Qiagen #28706) and cloned into the TA site of the pCR2.1 TOPO vectors using the TOPO-TA Cloning system (Invitrogen #K4500-01 SC).
Transformed TOP 10 bacteria were selected and plasmid DNA was isolated using Qiaprep Spin Mini-Prep Kit (Qiagen #27104). Plasmid DNA from 25 heavy chain, 38 kappa light chain and 23 lambda light chain colonies was sequenced to identify the nucleotide and corresponding amino acid sequences. Complete variable domain sequence data were obtained from 25 heavy chain, 38 kappa light chain and 19 lambda light chain clones. Variable domain sequence data including the leader peptide (when identified) are shown in Tables 14, 15 and 16. All derived heavy chain and light chain sequence are unique compared to those disclosed in patent publication number: U.S. Pat. No. 7,261,890 B2.
TABLE 14
Canine Heavy Chain Variable Domain Sequences
Derived from Canine PBMC RNA
Name
Sequence
Ca-1005
EVQLEESGGDLVKPGGSLRLSCVASGFSIGSYGMSW
VRQSPGKGLQWVAWIKYDGSRTFYADAVKGRFTISR
DNAKNTLFLQMNSLRAEDTAVYFCVKGPNSSWLPST
YFASWGQGTLVTVSS (SEQ ID NO: 178)
Ca-2301
EMQLVESGGDLVRPGGSLRLSCVASGFTFSTYGMTW
VRQSPGKGLQWVATIGPGGRNTYYADAVKGRFTISR
DDAENTLFLQMNSLRAEDTAVYYCAQAFDATYYTSF
DCWGRGSLVAVSS (SEQ ID NO: 86)
Ca-2302
MESVLSWVFLVALLQGIQGEIRLVESGGDLVKPGGS
LRLSCVASGFIFGNYDMSWVRQAPGKGLQWVAAVRY
DGSSTYYSDAVKGRITISRDDPGNTVYLQLDSLRAE
DTATYYCVRGGYYSSSFYIGGAFGHWGPGTLITVSS
(SEQ ID NO: 87)
Ca-2303
MECVLGWVFLVAILRGVQGEVQLVESGGDLVKPGGS
LRLSCVASGFTFSDYYMSWIRQAPGKGLQWVADISD
GGDGTGYAGAVKGRFTVSRENVKNTLYLQMNDLRAE
DTAIYYCTKAREMYGYRDFDSWGPGTLVTVSS
(SEQ ID NO: 88)
Ca-2304
MESVLGLVALLTILKGVQGEVQLVESGGDLVKPGGS
LRLSCVASGFTFSNYYMTWVRQAPGKGLEWVGYIHN
GGTYTYYADAVKGRFTISRDDAKNTLYLEMNSLRAE
DTAVYYCGKMIFDYWGQGTLVTVSS
(SEQ ID NO: 89)
Ca-2305
MESALSWVFLVTILKGVQGEVLLVESGGDLVKPGGS
LRLSCLTSGFTFNTYDWGWVRQAPGKGLQWIAYIKK
GGSDVRYADAVKGRFTISRDDAKNTLYLQMNSLRAE
DTAVYYCARSAWDSFDYWGQGTLVTVSS
(SEQ ID NO: 90)
Ca-2306
MESVFCWVFLVAILKGVRGVQGEVQLVESGGDLVKP
AGSLRLSCVASGFTFTDYSMNWVRQAPGKGLQWVAT
ISNDGTSTDYTDAVKGRFTVSRDSARNTVYLQMTSL
RADDTATYYCVSRHSYSLLADYWGQGTLVTVSS
(SEQ ID NO: 91)
Ca-2307
MQMPWSLLCLLAAPLGVLSEVTLQESGPGLVKPSQT
LSLTCAVSGGSVIRNYYWHWIRQRPGRGLEWMGCWS
ETTYYSPAFRGRISITIDAATDQFSLHLNSMTTDDT
AVYYCARALYPTSSWYDGMDYWGHGASVVVSS
(SEQ ID NO: 92)
Ca-2308
EVQLVESGGDLVKPGGSLRLSCESSGFIFSQYAMNW
VRQAPGKGLQWVAYIGGAGFITYHADDVKGRFTISR
DNAKNTLYLQMNSLTINDTAVYYCVRSNSRIPDYWG
QGTLVAVSS (SEQ ID NO: 93)
Ca-2309
MESVFCWVFLVAILKGVQGEVQLVESGGDLVKPGGS
LRLSCVASGFTFSSVYMSWVRQAPGLQWVARITTDG
TDTFYADAVKGRFTISRDNVKNMLYLEMNSLRAEDT
AIYYCGDPWQPAYPDLWGQGTMVTVSS
(SEQ ID NO: 94)
Ca-2310
MESVLCWVFLVAILKGVQGEVHLVESGGDLVKPGGT
LRLSCVASGFTFSQYDMSWVRQSPGKGLQWVALSRY
HGGGTYYADAVKGRFTISRDNAKNMLYLQMNSLRAE
DTAVYYCVKEGSRWDLRGDYDYWGQGTLVTVSS
(SEQ ID NO: 95)
Ca-2311
MQMPWSLLCLLAAPLGVLSELTLQESGPGLVKPSQT
LSLTCVVSGGSVTSSHYWNWIRQRPGRGLEWMGYWT
GNVNYNPAFQGRISIIGDAAKNQFSLHLSSMTTDDT
AVYYCARCGIVAPGFLPIGDFDFWGQGTLVTVSS
(SEQ ID NO: 96)
Ca-2312
MESVFCWVFLVAILKGVQGEVQLVESGGDLVKPGGS
LRLSCVASGFSFSNYFMFWGRQAPGKGLQWVARIRS
DGGSTYYADAVKGRFTISRDNARNTLYLQMNSLRAE
DTATYYCAKADIIKLPEYRGQGTLVTVSS
(SEQ ID NO: 97)
Ca-2401
ESVLGWIFLATILKGVQGEVQLVESGGDLVKPGGSL
RLSCVGSGFTFSSSWMNWVRQAPGKGLQWIAEISGT
GSSTNYADAVKGRFTISRDNDKNTLYLQMNSLRAED
TAMYYCARAAYYGNYRNDLDYWGQGTLVTVSS
(SEQ ID NO: 98)
Ca-2402
KPAGSLRLSCVASGFTFSSHSVTWVRQAPGKGLQFV
AGITSGGNNRYYTDAVRGRFTLSRDNAKNTVYLQMN
SLRAEDTAMYFCALGSYEWLSGEFDYWGQGTLVTVS
S (SEQ ID NO: 99)
Ca-2403
MESVFCWVFLVAILKGVQGEVQLVESGGDLVKPGGS
LRLSCVASGFTLNNYFMYWVRQAPGKGLQWVARLNS
NGDSTFYADAVKGRFTISRDNAKNTLYLQMNSLRAE
DTSMYYCAKDLIYGYTLWGQGTLVTVSS
(SEQ ID NO: 100)
Ca-2404
MASVLSWVFLVAIVKGVQGEVQLVESGGDLVKPGGS
LRLSCVASGFIFNKYEVYWVRQAPGKGLEWVARILE
SGNPTYYAEAVEGRFTISRDNAKNMAYLQMNSLRAD
DTAVYYCATPSVSSTVAIDYWGQGALVTVSS
(SEQ ID NO: 101)
Ca-2405
MQMPWSLLCLLATPLGVLSELTLQESGPGLVKPSQT
LSLTCVVSRGSVTSDYYWNWIRQRPGRGLEWMGHWI
GSTAYNPAFQGRISITADTAKNQLSLQLRSMTTEDT
AVYFCARGSSWTPSGDSWGQGTLVTVSS
(SEQ ID NO: 102)
Ca-2406
MASVLKLGFSCRYCKKVSRVRCNXVESGGDLVKPGG
SLRLSCVASGFIFNKYEVYWVRQAPGKGLEWVARIL
ESGNPTYYAEAVEGRFTISRDNAKNMAYLQMNSLRA
DDTAVYYCATPSVSSTVAIDYWGQGALVTVSS
(SEQ ID NO: 103)
Ca-2407
MDCSWRIFFLLALATGVHSEVQLVQSAAEVKKPGAS
VKVSCKTSGYTLTDYYIHWVQQAPGTGLHWMGWIDP
EXGTTDYAQKFQGXVTLTADTSTNTAYMELSGLRAE
DTAVYYCARFPRSLDYGSFPFDYWGQGTLVTVSS
(SEQ ID NO: 104)
Ca-2408
MESVLCWVFLVAILKGVQGEVRLVESGGDLVKPGGS
LRLSCVASGFTFRNYGMSWVRQRPGKGLQWVAAIRS
DGVTYYADDLKVRFTVSRDDARNTLYLQLNSLGAED
TAVYYCAKAPWGLYDAWGQGTLVTVSS
(SEQ ID NO: 105)
Ca-2409
MESVLSWVFLVAILQGVQGEVQVVESGGDLVKPAGS
LRLSCVASGYSISTYTMTWVRQVPGKGLQLVAGING
DGSSTYYTDAVKGRFTISRDNARNTVYLQMNSLRAE
DTAMYYCLGEYSWFYYWGQGTLVTVSS
(SEQ ID NO: 106)
Ca-2410
MQMPWSLLCLLAAPLGVLSELTLQESGPRLVKPSQT
LSLTCAVSGGSVTTTSYWSWIRQRPGRGLEWVGYWT
GTTNYSPAFQGRISISADTAKNQFSLHLSSVTTEDT
ALYFCASKSASTSWYFSLFESWGQGTLVTVSS
(SEQ ID NO: 107)
Ca-2411
MESVLGLVFLLTILKGVQGEVQLVESGGDLVKPGGS
LRLSCVASGFTFSSYSMSWVRQAPGKGLQWVGYIDN
GGTSTYYADAVKGRFTISRDNAKNTLYLQMNSLRAE
DTAVYYCGRGSYGMEYWGHGTSLFVSS
(SEQ ID NO: 108)
Ca-2412
MESVLGLLFLVAILKGVQGEIQLVESGGDLLKPGGS
LRLSCVASGFTFSGSDMNWIRQAPGKGLQWVAHITH
EGIGTSYVGSVKGRFTISRDNAKNTLYLQMNDLRAE
DTAMYYCAYSPWNYYSFDSWGQGTLVTVSS
(SEQ ID NO: 109)
TABLE 15
Canine Lambda Light Chain Variable
Domain Sequences Derived from Canine PBMC RNA
Name
Sequence
Ca-1001
MTSTMAWSPLLLTLLTHCTVSWAQTVLTQSPSVSAVLG
RRVTISCTGSDTNIGSHRDVQWYQLVPGKSPKTLIYGT
DNRPSGIPVRFSGSKSGNSGTLTITGIQAEDEADYYCQ
SYDDDLSMNVFGGGTHLTVLG (SEQ ID NO: 110)
Ca-1002
MDWVPFYILPFIFSTGFCALPVLTQPTNASASLEESVK
LTCTLSSEHSNYIVRWYQQQPGKAPRYLMYVRSDGSYK
RGDGIPSRFSGSSSGADRYLTISNIKSEDEDDYYYCGA
DYTISGQYGSVFGGGTHLTVLG (SEQ ID NO: 111)
Ca-1003
LWISGGSALGTPTMAWTHLLLPVLTLCTGSVASSVLTQ
PPSVSVSLGQTA TISCSGESLSKYYAQWFQQKAGQVP
VLVIYKDTERPSGIPDRFSGSSSGNTHTLTISRARAED
EADYYCESEVSTGTYCVRRRHPSNRPRSAQGLPLGHTL
PALL (SEQ ID NO: 204)
Ca-1006
MTSTMAWSPLLLTLLTHCTGSWAQSVLTQPASLSGSLG
QRVTISCTGSSSNIGGYSVNWLQQLPGTGPRTIIYNNS
NRPSGVPDRFSGSRSGTTATLTISGLQAEDEADYYCST
WDSNLRTIVFGGGTHLTVLG (SEQ ID NO: 112)
Ca-1007
MTSTMDWSPLLLTLLAHCTGSWAQSVLTQPASVSGSLG
QRVTISCTGSTSNLGTYNVGWLQQVPGTGPRTVIYTNI
YRPSGVPDRFSGSESGSTATLTISDLQAEDEAEYYCTA
WDSSLNAYVFGSGTQLTVLG (SEQ ID NO: 113)
Ca-1008
MTSNMAWCPFLLTLLAYCTGSWAQSVLTQPTSVSGSLG
QRVTISCSGSTNNIGIVGASWYQQLPGKAPKLLVYSDG
DRPSGVPDRFSGSNSGNSDTLTITGLQAEDEADYYCQS
FDTTLDAAVFGGGTHLTVLG (SEQ ID NO: 114)
Ca-1009
MTSTMAWSPLLLTLLAHCTVSWAQAVLTQPPSVSAALG
QRVTISCTGSDTNIGSGYEVHWYRQVPGKSPAIIIYGN
SNRPSGVPVRFSGSKSGSTATLTITGIEAEDEADYHCQ
SYDGNLDGGVFGGGTHLTVLG (SEQ ID NO: 115)
Ca-1010
MTSTMGWFPLILTLLAHCAGSWAQSVLTQPASVSGSLG
QRVTISCTGSSPNVGYGDFVAWYQQVPGTSPRTLIYNT
RSRPSGVPDRFSASRSGNTATLTISGLQAEDEADYYCS
SYDNTLIGIVFGGGTHLTVLG (SEQ ID NO: 116)
Ca-1011
MTSTMGWSPLLLTLLAHCTGSWAQSVLTQPASVSGSLG
QRVTITCTGSSSNIGRANVAWFQQVPGTGPRTVIYTSV
KRPSGVPDRFSGSKSGSTATLTISGLQAEDEADYYCSS
WDNSLDAGVFGGGTHLTVLG (SEQ ID NO: 117)
Ca-1012
MTSTMGWFPLLLTLLAHSTGSWAQSVLTQPASVSGSLG
QRVTITCTGGTSNIGRGFVSWFQQVPGIGPKILIFDAY
RRPSGVPDRFSGSRSGNTATLTISGLQAEDEADYYCAV
YDSRLDVGVFGSGSQLTVLS (SEQ ID NO: 118)
Ca-1202
MTSNMAWCPFLLTLLTYCTGSWARSVLTQPASVSGSPG
QKVTIYCSGTMSDIGVLGANWYQQLPGKAPKLLVDNDG
DRPSGVPDRFSASKSGHSDTLTITGLQPEDEGDYYCQS
FDSSLDAAIFGEGTHLTVLG (SEQ ID NO: 119)
Ca-1203
SVASYVLTQSPSQNVTLRQAAHITCEGHNIGTKSVHWY
QQKQGQAPVLIIYDDKSRPSGIPERFSGANSGNTATLT
ISGALAEDEADYYCLVWDSSAIWVFGEGTHLTVLG
(SEQ ID NO: 120)
Ca-1204
MTSTMAWSPLLLTLLAHFTGSWAQSVLTQPTSVSGSLG
QRVTISCTASSSNIDRDYVAWYQQLPGTRPRALIYANS
NRPSGVPDRFSGSKSGSTATLTISGLQAEDEADYYCST
WDNSLTYVFGSGTQLTVLG (SEQ ID NO: 121)
Ca-1205
SVASYVLTQVPSVSVNLGKTATITCEGDNVGEKYTHWY
QQEYGQAPVLIIYEDSRRPSGIPERFSGSNSGNTATLT
ISGARAEDETDYYCQVWDDSGNVFGGGTHLTVLG
(SEQ ID NO: 122)
Ca-1206
MTSTMGWFPLILTLLAHCAGSWAQSVLTQPASVSGSLG
QRVTISCTGSDSNVGYGDSIAYGDSVAWYQQVPGTSPR
TLIYDVTSRPSGVPDRFSGSRSGTTATLTISGLQAEDE
ADYYCSSFDKTLNGLIVGGGTHLTVLG
(SEQ ID NO: 123)
Ca-1207
MTSNMAWSPLLLTLLAYCTGSWAQSALTQPTSVSGSLG
QRVSISCSGGIHNIGSVGATWYQQLPGKAPKLLVSSDG
DRPSGIPDRFSGSRSGNSVTLTITGLQAEDEAEYYCQS
FDSTLGVHVVFGGGTHLTVLG (SEQ ID NO: 124)
Ca-1208
LCSAVGPPKTESVMTSTMGWSPLLLTLLAHCTGSWAQS
VLTQPASVSGSLGQRVTIPCTGSSSNIDRYNVAWFQQL
PGTGPKPSSIVLLTDPQGSLIDSLAPSQAA
(SEQ ID NO: 205)
Ca-1209
MTSTMAWFPLLLTLLAHYTGSWARSDLTQPASVSGSLG
QRITISCTGSSSNIGRNYVGWYQQLPGRGPRTVVYGIN
SRPSGVPDRFSGSKSGSTVTLTISGLQAEDEADYYCST
WDDSLSVVVFGGGTHLTVLG (SEQ ID NO: 125)
Ca-1210
MTSTMGWSPLLLTLTHWTGSWAQSVLSQPASMSGSLGL
RITICCTGKNSNINNSYVDWNQPLAGTGPRTVIHDDGD
RPSGVPDQFSGSKSGNTATLTISRLQAEDEADYNGASF
ETSFNAVFGGGTHVTVLG (SEQ ID NO: 126)
TABLE 16
Canine Kappa Light Chain Variable Domain
Sequences Derived from Canine PBMC RNA
Ca Ka016-A1
LSWLRQKPGHSPQRLIHQVSSRDPGVPDRFSGS
GSGTDFTLTISRVEADDGGVYYCGQGSQSIPTF
GQGTKVEIKR (SEQ. ID NO. 127)
Ca Ka016-A2
MRFPSQLLGLLMLWIPGSAGDIVMTQTPLSLSV
SPGEPASISCKASQSLLHSKGNTYLYWFRQKPG
QSPQRLIYKVSNRDPGVPDRFSGSGSGTDFTLR
ISRVETDDAGVYYCGQVIQDPWTFGVGTKLELK
R (SEQ. ID NO. 128)
Ca Ka016-A3
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSV
SPGETASISCRASQTLLYSNGKNYLFWYRQKPG
QSPQRLIDLASNRDPGVPDRFSGSGSGTDFTLR
ISRVEADDAGVYYCGQGMEIPWTFGAGTKVELK
R (SEQ. ID NO. 129)
Ca Ka016-A4
MKFPSLLLGLLMLWIPGSTGEAVMTQTPLSLAV
TPGEVATISCRASQSLLHSDGKSYLNWYLQKPG
QTPRPLIYEASKRFSGVSDRFSGSGSGTDFTLK
INRVEAEDVGVYYCQQSLHFPPTFGPGTKVELK
R (SEQ. ID NO. 130)
Ca Ka016-A5
PDRFSGSGSGTDFTLTISRVEADDAGIYYCGQA
TQTPPTFGAGTKLDLKR
(SEQ. ID NO. 131)
Ca Ka016-A6
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSV
RPGESASISCKASQSLLHSGGGTYLNWFRQRPG
QSPQRLIYEVSKRDTGVPDRFSGSGSGTDFTLR
ITRVEADDTGIYYCGQNTQLPLTFGQGTKVEIK
R (SEQ. ID NO. 132)
Ca Ka016-A7
MRFPSQLLGLLMLWIPGSTGDIVMTQTPLSLSV
SPGEPASISCKASQSLLHSNGNTYLFWLROKPG
QSPQRLTYRVSNRDPGVPDRFSGSGSGTDFTLR
ISRVEADDAGVYYCGQRVRSPWTFGAGTKVEVK
R (SEQ. ID NO. 133)
Ca Ka016-A8
MRFPSQLLGLLMLWIPGSAGDIVMTOTPLSLSV
SPGEPASISCKASQSLLHSNGNTYLYWFRQKPG
QSPQRLIYKVSKRDPGVPDRFSGSGSGTDFTLR
ISRVETDDAGVYYCGQVIQDPWTFGVGTKLELK
R (SEQ. ID NO. 134)
Ca Ka016-A9
MRFPSQLLGLLMLWIPGSSGDVVMAQTPLSLSV
SPGETASISCRASQSLLHSNGNTFLFWFRQKPG
QSPQRLINFLSNRDPGVPDRFSGSGSGTDFTLR
INRVEADDAGLYYCGQGLQAPLTFGQGTKLEIK
R (SEQ. ID NO. 135)
Ca Ka016-A10
MRFPSQLLGLLMLWIPGSNGDDVLTQTPLSLSV
RPGETVSILCKASESLLHSDGNTYLSWVRQKAG
QSPQRLMYRVSDRDTGVPDRFSGSGSGTDFTLT
ISGVEADDAGIYYCGQATHYPLEFGQGTRVEIK
R (SEQ. ID NO. 136)
Ca Ka016-A11
LMLWIPGSTGEIVLTQTPLSLSVSPGEPASISC
KASQSLLHPNGVTYLYWFRQKPGQSPQRLIYKV
SNRDPGVPDRFSGSGSEIDFTLIISRVEADDGG
IYYCGQGIQNPFTFGQGTKLEIKR
(SEQ. ID NO. 137)
Ca Ka016-A12
MRFPSQLLGLLMLWIPGSIGDIVMTQTPLSLSV
SPGESASISCKASQSLLHSNGNTYLYWFRQKPG
HSPQRLIHQVSSRDPGVPDRFSGSGSGTDFTLR
ISRVEADDAGLYYCGQGTQFPFTFGQGTKVEIK
R (SEQ. ID NO. 138)
Ca Ka016-B1
MRFPSQLLGLLMLWIPGSIGDIVMTQTPLSLSV
SPGESASISCKASQSLLHSNGNTYLYWFRQKPG
HSPQRLIHQVSSRDPOVPDRFSGSGSGTDFTLR
ISRVEADDAGLYYCGQGTQFPFTFGQGTKVEIK
R (SEQ. ID NO. 139)
Ca Ka016-B2
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSV
SPGETASISCRASQSLLHSNGNTYSFWFRQKPG
QSPQRLINLVSSRGPGVPDRFSGSGSGTDFTLI
ISRVEADDAGVYYCGHGKEAPYTFSQGTKLEIK
R (SEQ. ID NO. 140)
Ca Ka016-B3
MRFPSQLLGLLMLWIPGSVGDIVMTQSPMSLSV
GPGESASMSCKANQSLLYSDGITYLSWFLQRPG
QSPQRLIYEVSKRDTGVPGRFIGSGAGTDFTLR
ISRVEADDAGVYYCGQALQFPLTFSQGAKLEIE
R (SEQ. ID NO. 141)
Ca Ka016-B4
MRFPSQLLGLLMLWIPGSSGDVVMTQTPLSLSV
RPGETASISCRASQSLLHSSGITKLFWYRQKPG
QSPQRLVYWVSKRDPGVPDRFTGSGSGTDFTLR
ISRLEADDAGIYYCGHAIGFPLTFGQGTKVEIK
R (SEQ. ID NO. 142)
Ca Ka016-B5
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSV
RPGESASISCKASQSLLHSGGGTYLNWFRQRPG
QSPQRLIYEVSKRDTGVPDRFSGSGSGTDFTLR
ITRVEADDTGIYYCGQNTQFPLTFGQGTKVEIK
R (SEQ. ID NO. 143)
Ca Ka016-B6
MRFPSQLLGLLMLWIPGSSGGIVMTQTPLSLSV
RPGETASISCRASQSLLYSDGNTYLFWFRQKPG
QSPQRLMYRVSDRDTGVPDRFSGSGSGTDFTLT
ISGVEADDAGIYYCGQATHYPLEFGQGTXVEIK
R (SEQ. ID NO. 144)
Ca Ka016-B7
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSV
RPGESASISCKASQSLLHSGGGTYLNWFRQRPG
QSPQRLIYEVSKRDTGVPDRFIGSGAGTDFTLR
ISRVEADDAGVTYCGQGVQGPWTIGAGTKLELQ
R (SEQ. ID NO. 145)
Ca Ka016-B8
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSVSV
SPGETASISCKASQSLLSHDGNTYLHWFRQKPG
QSPQRLIYKVSNRDTGVPDRFSGSGSGTDFTLK
ISRVEADDTGVYYCGQITQDPFTFGQGTKLEIK
R (SEQ. ID NO. 146)
Ca Ka016-B9
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSV
SPGETASISCRASQSLLHSNGNTYLFWFRQKPG
QSPQRLIKWVSNRDPGVPDRFGGSGSGTDFTLR
ISRVEADDAGIYYCGOGIOGPYTFSQGTKLEIK
R (SEQ. ID NO. 147)
Ca Ka016-B10
MRFPSQFLGLLMLWIPGSSGDIAMTQTPLSLSV
GPGETASITCKASQSLLHSNGKTYLFWFRQKPG
QSPQRLIYLVSNRDPGVPDRFSGSGSGTDFTLT
ISRVEADDAGIYYCGQATQTPPTFGAGTKLDLK
R (SEQ. ID NO. 148)
Ca Ka016-B11
MRFPSQLLGLLMLWIPGSSGDIVMAQTPLSLSV
SPGEPASISCKASQSLLHSDGRTCLSWFRQKSG
QSPQRLIYEVSNRDTGVPDRFSGSGSGTDFTLR
ISRVEADDTGIYYCGQTVQFPLTFGQGTKLEIK
R (SEQ. ID NO. 149)
Ca Ka016-B12
GQSPQRLIYKVSNRDPGVPDRFSGSGSGTDFTL
RISRVEPEDVGVYYCGQGTLNPWTFGAGTKVEL
KR (SEQ. ID NO. 150)
Ca Ka017-1
MRFPSQLLGLLMLWIPGSSGDVVMTQTPLSLSV
SPGETASISCRASQSLLHSNGNTFLFWFRQ*PG
QSPQRLINFVSNRDPGVPDRFSGSGSGTDFTLR
ISRVEADDAGIYYCGQGLLAPPTFGQGTKVEIR
R (SEQ. ID NO. 151)
NOTE: * INDICATES A STOP CODON
Ca Ka017-2
MRFPSQLLGLLMLWIPGSGGDIVMTQTPPSLSV
SPREPASISCKASQSLLRSNGNTYLYWFRQKPG
QSPEGLIYRVSNRFTGVSDRFSGSGSGTDFTLR
ISTVEADDAGVYYCGQATQFPSTFSQGTKLEIK
R (SEQ. ID NO. 152)
Ca Ka017-3
MRFPSQLLGLLMLWIPGSXGDIVLTQTPLSLSV
SPGEPASISCKASQSLLMSNGITYLNWYRQRPG
QSPQXLIYKVSNRDTGVPDRFSGSGSGTDFTLR
XSKVEADDTGIYYCGQDTQFPLTLGXGTHXEIK
R (SEQ. ID NO. 153)
Ca Ka017-5
MRFPSQLLGLLMLWIPGSTGDIVMTQTPLSLSV
SPGEPASIYCKASQSLLHSNGKTFLYWFRQKPG
QSPQRLIYRVSNRDPGVPDRFSGSGSGTDFTLR
ISRVEADDAGIYYCGQGIQDPTFGQGTKVEIKR
(SEQ. ID NO. 154)
Ca Ka017-6
MRFPSQLLGLLMLWIPGSGGDIVMTQTPPSLSV
SPREAASISCKASQSLLKSNGKTYFYWFRQKPG
QVSEGLIYKVSSRFTGVSDRFSGSGSGTDFTLR
ISRVEADDAGVYFCGQALQFPYTFSQGTKLDIK
R (SEQ. ID NO. 155)
Ca Ka017-10
MRFPSQLLGLLMLWIPESGGDVVLTQTPPSLSL
SPGETASISCKASRSLLKSDGSTYLDWYLQKPG
QSPRLLIYLVSNRFSGVSDRFSGSGSGTDFTLT
ISRVEADDAGVYYCGQGSRVPLTFGQGTKVEIK
R (SEQ. ID NO. 156)
Ca Ka017-11
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSV
SPGETASISCRASQSLLHRNGITYLSWFRQRPG
QSPQRLINLVSNRDPGVPDRFSGSGSGTDFTLR
ISRVEADDVGVYYCGHGLQTPYTFGQGTSLEIE
R (SEQ. ID NO. 157)
Ca Ka017-12
MRFPSQLLGLLVLWIPGSSGDIVMTQTPLSLSV
SPGETVSISCRASQSLLYSDGNFYLFWFRRKPG
QSPQHLINLVSNRDPGVPDRFSGSGSGTDFTLR
ISRVEADDAGVYYCGQGTQPPYTFSQGTKVEIK
R (SEQ. ID NO. 158)
Ca Ka017-13
MRFPSQLLGLLMLWIPESGGDVVLTQTPPSLSL
SPGETASISCKASRSLLNSDGSTYLDWYLQKPG
QSPRLLIYLVSNRFSGVSDRFSGSGSGTDFTLT
ISRVEADDAGVYYCGQGSRVPLTFGQGTKVEIK
R (SEQ. ID NO. 159)
Ca Ka017-14
MRFPSQLLGLLMLWIPGSSGDIVMAQTPLSLSV
SPGETASISCRASQSLLHSNGITYLFWYRQKPG
QSPQRLISMVFNRDPGVPDRFGGSGSGTDFTLR
ISRVEADDAGLYFCGHGTQIPYSFSQGTKLEIK
R (SEQ. ID NO. 160)
Ca Ka017-16
MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSI
SPGEPASISCKASQSLLHSGGDTYLNWFRQRPG
QSPQLLINRVSSRKKGVPDRFSGSGSGTEFTLR
ISRVEADDAGIYFCGQGTQFPYTFSQGTKLEIK
R (SEQ. ID NO. 161)
Ca Ka017-20
MRFPSQLLGLLMLWIPGSGGDIVMTQTPPSLSV
SPGEPASMSCKASQSLLHSNGNTYLYWFRQKPG
QSPEALIYKVSNRFTGVSDRFSGSGSGTDFTLR
INRVEADDVGVYYCGQGIQIPYTFSQGTKLEIK
R (SEQ. ID NO. 162)
Ca Ka017-23
MRFPSQLLGLLMLWIPGSTGEIVLTQTPLSLSV
SPGESASTSCKASQSLLYSNGNTYLYWFRQKAG
QSPORVIYRVSKRDPGVPDRFSGSGSGTDFTLR
ISSVENDDAGVYYCGQGSEDPPTFGAGTKVELK
R (SEQ. ID NO. 163)
Ca Ka017-24
MRFPSQLLGLLTLWIPGSTGDIVMTQTPLSLSV
SPGEPASISCKASQSLLHSNGNTYLYWFRQKPG
QSPQRLIYKVSNRDPGVPXRFSGSGSGTDFTLR
VSXVEADDAGVYYCGQGVQDPFTFGQGTKLEIK
R (SEQ. ID NO. 164)
Example 13: CDR-Grafting to Create Caninized Monoclonal Antibodies
To generate caninized antibody sequences from mouse anti-NGF antibodies, each murine variable heavy chain antibody gene sequence was separately aligned against 36 canine Ig germline variable heavy chain sequences using Vector NTI software. Eleven canine Ig germline variable heavy chain sequences were derived from U.S. Pat. No. 7,261,890 B2, (Methods for Using Canine Immunoglobulin Variable Domains and Caninized Antibodies), the contents of which are herein incorporated by reference, and 25 canine Ig germline variable heavy chain sequences were derived from Table 14 (Canine Heavy Chain Variable Domain Sequences Derived from Canine PBMC RNA). Each murine variable light chain gene sequence was separately aligned against 68 germline variable light chain sequences (derived from U.S. Pat. No. 7,261,890 B2) using Vector NTI software. Canine variable domain sequences having the highest overall homology to the original murine sequences were selected for each heavy chain and light chain sequence to provide the framework sequence. In silico construction of complete CDR grafted antibodies was accomplished by substitution of canine variable domain CDR sequences with murine CDR sequences (derived from the subcloned anti-NGF antibody hybridoma mAbs). To identify residues in each sequence, the first amino acid in each listed sequence was defined as 1, and all remaining residues numbered consecutively thereafter using Kabat numbering system.
The heavy chain CDR sequences from PR-1254972 were grafted in silico onto canine 894 as follows: (1) One N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Six back-mutations (Q3H, V37I, Q46E, D73N, T77N, R83K) were introduced to make the 72.2 VH sequence. (3) One, two, three, four, five, or six of the back-mutations disclosed above could be introduced into 72.2 VH to maintain antibody affinity to NGF after caninization of mAb 72.2. (4) One, two, three, four, five, or six of these back-mutations may be substituted during subsequent affinity maturation of 72.2 VH. 72.3 VH was generated by introducing the back-mutations in 72.2 VH with the addition of H39Q back-mutation. 72.4 VH was generated by introducing back-mutations Q3H, H39Q, Q46E, D73N. The light chain CDR sequences from PR-1254972 were grafted in silico onto canine 1001 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Four back-mutations (I2V, V3L, Q45K, S59P) were introduced to make the 72.2 VL sequence. (3) One, two, three, or four of these back-mutations could be introduced into 72.2 VL to maintain antibody affinity to NGF after caninization of mAb 72.2. (4) One, two, three, or four of these back-mutations may be substituted during subsequent affinity maturation of 72.2 VL. 72.4 VL was generated by introducing back-mutations Q45K, and S59P.
The heavy chain CDR sequences from PR-1254973 were grafted in silico onto canine 894 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Eight back-mutations (T24A, M48I, V67A, L69V, T73K, N76S, V78A, A93T) were introduced to make the 73.2 VH sequence. (3) One, two, three, four, five, six, seven, or eight of these back-mutations could be introduced into 73.2 VH to maintain antibody affinity to NGF after caninization of mAb 73.2. (4) One, two, three, four, five, six, seven, or eight of these eight back-mutations may be substituted during subsequent affinity maturation of 73.2 VH. 73.4 VH was generated by introducing back-mutations T24A, T73K, A93T. The light chain CDR sequences from PR-1254973 were grafted in silico onto canine 1034 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Eight back-mutations (I1D, V3Q, S22T, F36H, R46L, I48V, D60S, D70Q) were introduced to make the 73.2 VL sequence. (3) One, two, three, four, five, six, seven, or eight of these back-mutations could be introduced into 73.2 VL to maintain antibody affinity to NGF after caninization of mAb 73.2. (4) One, two, three, four, five, six, seven, or eight of these eight back-mutations may be substituted during subsequent affinity maturation of 73.2 VL. 73.4 VL was generated by introducing back-mutations I1D, V3Q, F36H, R46L, D60S, D70Q.
The heavy chain CDR sequences from PR-1254977 were grafted in silico onto canine 894 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Eight back-mutations (T24A, Q38K, M48I, R66K, V67A, T68S, L69I, V78A) were introduced to make the 77.2 VH sequence. (3) One, two, three, four, five, six, seven, or eight of these back-mutations could be introduced into 77.2 VH to maintain antibody affinity to NGF after caninization of mAb 77.2. (4) One, two, three, four, five, six, seven, or eight of these back-mutations may be substituted during subsequent affinity maturation of 77.2 VH. 77.3 VH was generated by introducing the back-mutations in 77.2 VH with the addition of R94G back-mutation. 77.4 VH was generated by introducing back-mutations T24A, Q38K, and R94G. The light chain CDR sequences from PR-1254977 were grafted in silico onto canine 997 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Four back-mutations (L2V, F36Y, R46L, S98G) were introduced to make the 77.2 VL sequence. (3) One, two, three, or four of these back-mutations could be introduced into 77.2 VL to maintain antibody affinity to NGF after caninization of mAb 77.2. (4) One, two, three, or four of these back-mutations may be substituted during subsequent affinity maturation of 77.2 VL. 77.4 VL was generated by introducing back-mutations F36Y and R46L.
The heavy chain CDR sequences from PR-1254981 were grafted in silico onto canine 876 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Six back-mutations (Q46E, G49A, T77N, R83K, L91Y, E93T) were introduced to make the 81.2 VH sequence. (3) One, two, three, four, five, or six of these back-mutations could be introduced into 81.2 VH to maintain antibody affinity to NGF after caninization of mAb 81.2. (4) One, two, three, four, five, or six of these six back-mutations may be substituted during subsequent affinity maturation of 81.2 VH. 81.4 VH was generated by introducing back-mutations Q46E, G49A, L91 Y, and E93T.
The light chain CDR sequences from PR-1254981 were grafted in silico onto canine 1011 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs (2) Four back-mutations (V3L, A7T, F36Y, R46L) were introduced to make the 81.2 VL sequence (3) One, two, three, or four of these back-mutations could be introduced into 81.2 VL to maintain antibody affinity to NGF after caninization of mAb 81.2. (4) One, two, three, or four of these back-mutations may be substituted during subsequent affinity maturation of 81.2 VL. 81.4 VL was generated by introducing back-mutations A7T, F36Y, and R46L.
Alternatively, the heavy chain CDR sequences from PR-1254981 were grafted in silico onto canine 1005 VH as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Seven back-mutations (Q46E, T77N, F79Y, R83K, F91Y, V93T, K94R) were introduced to make the 81.5B VH sequence. (3) One, two, three, four, five, six, or seven of these back-mutations could be introduced into 81.5B VH to maintain antibody affinity to NGF after caninization of mAb 81.5B. (4) One, two, three, four, five, six, or seven of these seven back-mutations may be substituted during subsequent affinity maturation of 81.5B VH. 81.6B was generated by introducing back-mutations Q46E, F79Y, F91Y, and V93T. Variants 81.2B and 81.4B were generated by introducing A84K mutation to 81.5B and 81.6B, respectively.
The heavy chain CDR sequences from PR-1254982 were grafted in silico onto canine 892 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Twelve back-mutations (I3Q, I37V, M48L, I67L, T70S, A71K, G73N, N76S, H77Q, L78V, S79F, T93A) were introduced to make the 82.2 VH sequence. (3) One, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve of these back-mutations could be introduced into 82.2 VH to maintain antibody affinity to NGF after caninization of mAb 82.2. (4) One, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve of these back-mutations may be substituted during subsequent affinity maturation of 82.2 VH. 82.4 VH was generated by introducing back-mutations I3Q, A71K, H77Q, S79F, and T93A. The light chain CDR sequences from PR-1254982 were grafted in silico onto canine 1034 as follows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found in these proposed constructs. (2) Ten back-mutations (I1D, V3Q, S22T, F36Y, Q45K, R46L, D60S, F71Y, T72S, Y87F) were introduced to make the 82.2 VL sequence. (3) One, two, three, four, five, six, seven, eight, nine, or ten of these back-mutations could be introduced into 82.2 VL to maintain antibody affinity to NGF after caninization of mAb 82.2. (4) One, two, three, four, five, six, seven, eight, nine, or ten of these back-mutations may be substituted during subsequent affinity maturation of 82.2 VL. 82.3 VH was generated by introducing the back-mutations in 82.2 VH with the addition of P44V back-mutation. 82.4 VL was generated by introducing back-mutations I1D, V3Q, F36Y, Q45K, R46L, D60S, F71Y, and Y87F.
Example 14: Isoelectric Point of Canine Framework Amino Acids
The heavy chain framework amino acids (i.e. non-CDR amino acids) of the caninized IgG1 kappa antibodies yield a calculated isoelectric point of less than 8.0. The light chain framework amino acids, when the light chain is kappa, yield a calculated isoelectric point of less than 6.5. The isoelectric point of the caninized antibodies as a whole, i.e. heavy and light chain combined, due to the framework amino acids, and when the light chain is kappa, is less than 8.0. In comparison, the framework amino acids of human IgG1 heavy chains typically yield isoelectric points of greater than 8.0. The framework amino acids of human kappa light chains typically yield isoelectric points of greater than 6.5. The framework amino acids of whole human IgG1/k antibodies typically yield isoelectric points of greater than 8.0.
Example 15: CDR-Grafting to Create Humanized Monoclonal Antibodies
Each murine variable heavy and variable light chain antibody gene sequence (as set forth in Table 16) was separately aligned against 44 human immunoglobulin germline variable heavy chain or 46 germline variable light chain sequences (derived from NCBI Ig Blast website which is well known to those skilled in the art) using Vector NTI software. Human variable domain sequences having the highest overall homology to the original murine sequences were selected for each heavy chain and light chain antibody sequence to provide the framework (FW) 1, 2 and 3 sequences for CDR-grafting purposes. Identification of a suitable human variable heavy and light chain FW4 region (also known as the “joining” region) was accomplished by separately aligning each murine heavy chain and light chain FW4 region with 6 human immunoglobulin germline joining heavy chain and 5 germline joining light chain sequences in the NCBI database. In silico construction of complete CDR grafted variable domains was accomplished by substitution of human variable domain CDR sequences (derived from the NCBI website) with murine CDR sequences (derived from the murine antibodies) with addition of a FW4 region (derived from the NCBI website) to each 3′ end. Further humanization may be accomplished by identification of back-mutations. Full length human Igs may be produced by expressing the variable domains of each CDR-grafted mAb with an in-frame human IgG constant domain. Mouse Anti-NGF mAb CDRs grafted onto human Ig frameworks (CDR-grafted Anti-NGF Abs) produced are those listed in Table 17.
TABLE 17
Mouse Anti-NGF mAb CDRs Humanized by
CDR Grafting onto Human Ig Frameworks
Name
Sequence (CDRs are underlined)
HU72 VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMF
(CDR-GRAFT
WVRQATGKGLEWVSTISDGGSYTYYTDNVKGRFTI
VH3-13/JH5)
SRENAKNSLYLQMNSLRAGDTAVYYCARDWSDSEG
FAYWGQGTLVTVSS (SEQ ID NO: 165)
Hu73 VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMH
(CDR-GRAFT
WVRQAPGQGLEWMGRIDPYGGGTKHNEKFKRRVTM
VH1-18/JH6)
TTDTSTSTAYMELRSLRSDDTAVYYCARSGYDYYF
DVWGQGTTVTVSS (SEQ ID NO: 166)
HU77 VH
QVQLVQSGAEVKKPGSSVKVSCKASGFNIKDTYIY
(CDR-GRAFT
WVROAPGQGLEWMGRIDPANGNTIYASKFQGRVTI
VH1-69/JH6)
TADKSTSTAYMELSSLRSEDTAVYYCARYGYYAYW
GQGTTVTVSS (SEQ ID NO: 167)
HU80 VH
QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIY
(CDR-GRAFT
WVRQAPGQGLEWMGRIDPANGNTIYASKFQGRVTM
VH1-18/JH6)
TTDTSTSTAYMELRSLRSDDTAVYYCARYGYYAYW
GQGTTVTVSS (SEQ ID NO: 168)
HU81 VH
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNHYMY
(CDR-GRAFT
WVRQAPGKGLEWVGSISDGGAYTFYPDTVKGRFTI
VH3-15/JH1)
SRDDSKNTLYLQMNSLKTEDTAVYYCTTEESANNG
FAFWGQGTLVTVSS (SEQ ID NO: 169)
HU82 VH
QVTLKESGPVLVKPTETLTLTCTVSGFSLTGYNIN
(CDR-GRAFT
WIRQPPGKALEWLAMIWGYGDTDYNSALKSRLTIS
VH2-26/JH6)
KDTSKSQVVLTMTNMDPVDTATYYCARDHYGGNDW
YFDVWGQGTTVTVSS (SEQ ID NO: 170)
HU72 VL
DIVMTQTPLSLPVTPGEPASISCRSSQSIVQSNGN
(CDR-GRAFT
TYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGS
01/JK2)
GSGTDFTLKISRVEAEDVGVYYCFQGSHVPFTFGQ
GTKLEIKR (SEQ ID NO: 171)
HU73 VL
DIQMIQSPSFLSASVGDRVSIICRASENIYSFLAW
(CDR-GRAFT
LQKPGKSPKLFLYNANTLAEGVSSRFSGRGSGTDF
L22/JK2)
TLTIISLKPEDFAAYYCQHHFGTPFTFGQGTKLEI
KR (SEQ ID NO: 172)
HU77VL
DIVMTQTPLSLPVTPGEPASISCKSTKSLLNGDGF
(CDR-GRAFT
TYLDWYLQKPGQSPQLLIYLVSNRFSGVPDRFSGS
01/JK2)
GSGTDFTLKISRVEAEDVGVYYCFESNYLFTFGQG
TKLEIKR (SEQ ID NO: 173)
HU80 VL
DIVMTQTPLSLPVTPGEPASISCKSTKSLLNGDGF
(CDR-GRAFT
TYLDWYLQKPGQSPQLLIYLVSNRFSGVPDRFSGS
01/JK2)
GSGTDFTLKISRVEAEDVGVYYCFESNYLFTFGQG
TKLEIKR (SEQ ID NO: 174)
HU81 VL
DIVMTQTPLSLPVTPGEPASISCRSSQSILHSNGN
(CDR-GRAFT
TYLEWYLQKPGQSPQLLIYRVSNRFSGVPDRFSGS
01/JK2)
GSGTDFTLKISRVEAEDVGVYYCFQGAHVPFTFGQ
GTKLEIKR (SEQ ID NO: 175)
HU82 VL
DIQMTQSPSSLSASVGDRVTITCRASQDITNYLNW
(CDR-GRAFT
YQQKPGKAPKLLI YYTSRLHSGVPSRFSGSGSGT
08/JK2)
DFTFTISSLQPEDIATYYCQQGKTLPRTFGQGTKL
EIKR (SEQ ID NO: 176)
Example 16: Method for Constructing Full-Length Mouse/Canine Chimeric and Caninized Antibodies
Using conventional molecular biology techniques, a cDNA fragment encoding the canine IgG1 constant region (which was obtained from the IMGT®, the International ImMunoGeneTics information system, which is the global reference in immunogenetics and immunoinformatics, created in by Marie-Paule Lefranc (Universite Montepellier 2 and CNRS)) was synthesized and ligated to the 3′ end of each of the heavy chain variable domains derived from murine anti-NGF monoclonal antibodies PR-1254972, PR-1254973, PR-1254977, PR-1254981, PR-1254982. For these same anti-NGF mAbs, a cDNA fragment encoding the canine kappa constant region obtained from U.S. Pat. No. 5,852,183 A, (Sequence ID No. 54) was synthesized and ligated to the 3′ end of each of the light chain variable domains. The complete canine IgG heavy chain constant domain nucleotide sequence and amino acid sequence is shown as SEQ ID NO: 51 and SEQ ID NO: 52, respectively. The complete canine kappa light chain constant domain nucleotide sequence and amino acid sequence is shown as SEQ ID NO: 53 and SEQ ID NO: 54, respectively. Complete heavy chain and light chain chimeric cDNAs were ligated into the pHybE expression plasmid; the sequences of these chimeric mAbs are in Table 18 below.
TABLE 18
Mouse/Canine Chimeric Antibody Sequences
Name
Sequence (CDRs are underlined)
PR-1290646 light
DVLMTQTPLSLPVSLGDQASISCRSSQSIVQ
chain amino acid
SNGNTYLEWYLQKPGQSPKLLIYKVSNRFSG
sequence
VPDRFSGSGSGTDFTLKISREAEDLGVYYCF
QGSHVPFTFGSGTKLEIKRNDAOPAVYLFQP
SPDQLHTGSASVVCLLNSFYPKDIKVKWKVD
GVIQDTGIQESVTEQDKDSTYSLSSTLTMSS
TEYLSHELYSCEITHKSLPSTLIKSFQRSEC
QRVD (SEQ ID NO: 194)
PR-1290646 heavy
EVHLVESGGGLVKPGGFLILSCAASGFTFSD
chain amino acid
YYMFWIRQTPGKRLEWVATISDGGSYTYYTD
sequence
NVKGRFTISRDNVKNNLYLQMSHLKSADTAM
YYCARDWSDSEGFAYWGQGTLVTVSAASTTA
PSVFPLAPSCGSTSGSTVALACLVSGYFPEP
VTVSWNSGSLTSGVHTFPSVLQSSGLHSLSS
MVTVPSSRWPSETFTCNVVHPASNTKVDKPV
FNECRCTDTPPCPVPEPLGGPSVLIFPPKPK
DILRITRTPEVTCVVLDLGREDPEVQISWFV
DGKEVHTAKTQSREQQFNGTYRVVSVLPIEH
QDWLTGKEFKCRVNHIDLPSPIERTISKARG
RAHKPSVYVLPPSPKELSSSDTVSITCLIKD
FYPPDIDVEWQSNGQQEPERKHRMTPPQLDE
DGSYFLYSKLSVDKSRWQQGDPFTCAVMHET
LQNHYTDLSLSHSPGK
(SEQ ID NO: 195)
PR-1290654 light
DIQMTQSPASLSASVGETVTVTCRASENIYS
chain amino acid
FLAWHQQKQGKSPQLLVYNANTLAEGVPSRF
sequence
SGSGSGTQFSLKINSLQPEDFGSYYCQHHFG
TPFTFGSGTKLEIKRNDAQPAVYLFQPSPDQ
LHTGSASVVCLLNSFYPKDINVKWKVDGVIQ
DTGIQESVTEQDKDSTYSLSSTLTMSSTEYL
SHELYSCEITHKSLPSTLIKSFQRSECQRVD
(SEQ ID NO: 196)
PR-1290654 heavy
QVQLQQPGAELVKPGASVKLSCKASGYTFTN
chain amino acid
YWMHWVKQRPGQGLEWIGRIDPYGGGTKHNE
sequence
KFKRKATVTADKSSSTAYILLSSLTSEDSAV
YYCTRSGYDYYFDVWGTGTTVTVSSASTTAP
SVFPLAPSCGSTSGSTVALACLVSGYFPEPV
TVSWNSGSLTSGVHTFPSVLQSSGLHSLSSM
VTVPSSRWPSETFTCNVVHPASNTKVDKPVF
NECRCTDTPPCPVPEPLGGPSVLIFPPKPKD
ILRITRTPEVTCVVLDLGREDPEVQISWFVD
GKEVHTAKTQSREQQFNGTYRVVSVLPIEHQ
DWLTGKEFKCRVNHIDLPSPIERTISKARGR
AHKPSVYVLPPSPKELSSSDTVSITCLIKDF
YPPDIDVEWQSNGQQEPERKHRMTPPQLDED
GSYFLYSKLSVDKSRWQQGDPFTCAVMHETL
QNHYTDLSLSHSPGV
(SEQ ID NO: 197)
PR-1290656 light
DVVLTQTPLSLPVNIGDQASISCKSTKSLLN
chain amino acid
GDGFTYLDWYLQKPGQSPQLLIYLVSNRFSG
sequence
VPDRFSGSGSGTDFTLKISRVEAEDLGVYYC
FESNYLFTFGSGTKLEMKRNDAQPAVYLFQP
SPDQLHTGSASVVCLLNSFYPKDINVKWKVD
GVIQDTGIQESVTEQDKDSTYSLSSTLTMSS
TEYLSHELYSCEITHKSLPSTLIKSFQRSEC
QRVD (SEQ ID NO: 198)
PR-1290656 heavy
EVQLQQSGAELVKPGASVKLSCTASGFNIKD
chain amino acid
TYIYWVKQRPEQGLEWIGRIDPANGNTIYAS
sequence
KFQGKASITADTSSNTAYMQLSSLTSGDTAV
YYCAGYGYYAYWGQGTTLTVSSASTTAPSVF
PLAPSCGSTSGSTVALACLVSGYFPEPVTVS
WNSGSLTSGVHTFPSVLQSSGLHSLSSMVTV
PSSRWPSETFTCNVVHPASNTKVDKPVFNEC
RCTDTPPCPVPEPLGGPSVLIFPPKPKDILR
TTRTPEVTCVVLDLGREDPEVQISWFVDGKE
VHTAKTQSREQQFNGTYRVVSVLPIEHQDWL
TGKEFKCRVNHIDLPSPIERTISKARGRAHK
PSVYVLPPSPKELSSSDTVSITCLIKDFYPP
DIDVEWQSNGQQEPERKHRMTPPQLDEDGSY
FLYSKLSVDKSRWQQGDPFTCAVMHETLQNH
YTDLSLSHSPGK (SEQ ID NO: 199)
PR-1290657 light
DVLMTQTPLSLPVSLGDQASISCRSSQSILH
chain amino acid
SNGNTYLEWYLQKPGQSPNLLIYRVSNRFSG
sequence
VPDRFSGSGSGTDFTLKISRVEAEDLGVYYC
RQGAHVPFTFGSGTKLEIKRNDAQPAVYLFQ
PSPDQLHTGSASVVCLLNSFYPKDINVKWKV
DGVIQDTGIQESVTEQDKDSTYSLSSTLTMS
STEYLSHELYSCEITHKSLPSTLIKSFQRSE
CQRVD (SEQ ID NO: 200)
PR-1290657 heavy
EVQLVESGGGAVKPGGSLTLSCAASGFTFSN
chain amino acid
HYMYWVRQTPEKRLEEVASISDGGAYTFYPD
sequence
TVKGRFTISRDNVNNNLYLQMRHLKSEDTAM
YYCTREESANNGFAFWGQGTLVTVSAASTTA
PSVFPLAPSCGSTSGSTVALACLVSGYFPEP
VTVSWNSGSLTSGVHTFPSVLQSSGLHSLSS
MVTVPSSRWPSETFTCNVVHPASNTKVDKPV
FNECRCTDTPPCPVPEPLGGPSVLIFPPKPK
DILRITRTPEVTCVVLDLGREDPEVQISWFV
DGKEVHTAKTQSREQQFNGTYRVVSVLPIEH
QDWLTGKEFKCRVNHIDLPSPIERTISKARG
RAHKPSVYVLPPSPKELSSSDTVSITCLIKD
FYPPDIDVEWQSNGQQEPERKHRMTPPQLDE
DGSYFLYSKLSVDKSRWQQGDPFTCAVMHET
LQNHYTDLSLSHSPGV
(SEQ ID NO: 201)
The canine IgG1 constant region nucleotide sequence described above was also ligated to the 3′ end of each of the cDNAs encoding heavy chain variable domains derived from caninized anti-NGF monoclonal antibodies 72.2 VH, 72.3 VH, 72.4 VH, 73.2 VH, 73.4 VH, 77.2 VH, 77.3 VH, 77.4 VH, 81.2 VH, 81.4 VH, 81.2B, 81.4B, 81.5B, 81.6B, 82.2 VH, 82.4 VH. The canine kappa light chain constant domain nucleotide sequence described above was also ligated to the 3′ end of each of the cDNAs encoding light chain variable domains derived from caninized anti-NGF monoclonal antibodies 72.2 VL, 72.4, 73.2 VL, 73.4 VL, 77.2 VL, 77.4 VL, 81.2 VL, 81.4 VL, 82.2 VL.
Full-length chimeric or caninized antibodies were transiently expressed in 293-6E cells by co-transfection of combinations of heavy and light chain pHybE plasmids. Table 20 highlights all possible combination of caninized heavy and light chains that may be combined to produce a caninized antibody per the name in the table (Table 20). In Table 20, the heavy chain plasmids encoding caninized versions of murine heavy chains are listed on the top line and proceed rightward. The light chain plasmids encoding caninized versions of murine light chains are listed on the left-hand column and proceed downward. At each point where these boxes intersect, a name has been indicated to describe a potential resulting caninized antibody.
Example 17: Caninized Monoclonal Antibody Expression and Purification
Selected heavy chain and light chain mouse/canine chimeric and caninized antibody plasmids were co-transfected into 293-6e cells in suspension and allowed to grow for 7-8 days. Cell supernatants were harvested, centrifuged, and filtered. For each expressed antibody, supernatant was mixed with an equal volume of Pierce binding buffer to perform Protein A Sepharose affinity chromatography according to manufacturer's instructions (GE Healthcare #17-1279-04). Although according to several sources canine IgGs bind directly to Protein A moderately well (GE Healthcare Antibody Purification Handbook package insert; Scott, M. A., et al., Vet Immunol-Immunopatho, 59:205, 1997; Warr, G. W and Hart, I. R., Am J Vet Res, 40:922, 1979; Thermo Scientific Pierce Antibody Production and Purification Technical Handbook) the monoclonal canine mAbs did not quantitatively bind to Protein A and therefore could not be purified from supernatants without modification to the Protein A purification methodology.
To allow quantitative binding of canine IgGs to Protein A, supernatants were concentrated and mixed with an equal volume of Pierce binding buffer (Thermo #21007). To the concentrated and diluted supernatants, NaCl was added to a final concentration of 2.5 M. NaCl-adjusted supernatant was loaded onto Protein A Sepharose by continuous over-night loading, washed with Pierce binding buffer, and eluted using Pierce elution buffer (Thermo #21004). The eluates were neutralized by dropwise addition of 1M Tris pH 8.0; following this the neutralized antibodies were dialyzed into PBS and amounts of antibody were quantified spectrophotometrically by OD28o- The amount purified was mathematically divided by the total volume of cell supernatant purified to determine the overall estimated expression levels in μg/mL. The isolation and purification of theses canine IgG1/k mAbs allowed analytical characterization studies of the mAbs to be completed.
For purification of large-scale cell supernatants (10-15 L), cell supernatants were concentrated, then mixed with Pierce binding buffer A (Thermo, catalog #21001) in a 1 to 1 ratio. To this mixture, 5 M NaCl was added to 1.3 M final concentration. The pH of the mixture was adjusted to 8.5 with 10 N NaOH. The pH-adjusted cell supes were loaded onto a Protein A MabSelect SuRe (GE Healthcare, catalog #17-5438-03) chromatography column and eluted using two steps. The first step of the elution was performed using 20 mM Tris, 25 mM NaCl, pH 8.0, 7.4 ms/cm. Fractions containing antibodies were identified by OD28o and size exclusion chromatography. To quantitatively isolate the remaining antibody bound to the Protein A column, the second step elution was performed using Pierce elution buffer (Thermo, catalog #21004), pH 2.7, 3.7 mS/cm, and fractions containing antibodies were identified by OD28o and size exclusion chromatography. All fractions containing antibodies were neutralized using 2M Tris pH 8.5, and then dialyzed into PBS. The method employed to purify large volumes of cell supernatant containing canine monoclonal antibodies (ex. 10-15 L) differs from the method typically employed to purify human antibodies from large volumes. For human antibodies, Protein A purification is typically accomplished with cell supernatant binding conditions of pH 7.0 to 8.3 and 15 to 20 mS/cm, washing with similar conditions (1×PBS) and a 1 step elution of human antibodies with 0.1 M acetic acid, 0.15 M sodium chloride, pH 2.7 at 15 to 20 mS/cm or Thermo IgG elution buffer, pH 2.7, at 15 mS/cm.
Purified canine antibodies were analyzed by mass spectroscopy (MS) to confirm the expressed antibody protein molecular weight matched the expected weight based on amino acid sequence. In addition, canine antibodies were analyzed by size exclusion chromatography (SEC) to determine the percent monomer. This data indicated that mouse/canine chimeric IgG1/k mAbs may be expressed transiently in 293-6e cells and are 81% or greater monomeric following purification. This data also indicated that caninized IgG1/k mAbs may be expressed transiently in 293-6e cells and in most cases are 80% or greater monomeric following purification. In some cases, expression of protein may not be detected and in some cases purified caninized mAb is between 24 and 34% monomeric. The data is summarized in Tables 19 and 20.
TABLE 19
Mouse/Canine Chimeric Monoclonal Antibody Characterization Data
Moniker
Estimated
of
Expression
Mouse/
Level
%
Hybrid-
Name of
Canine
In Cell
Mono-
oma
Mouse/Canine
Chimeric
Supernatants
meric
Moniker
Chimeric Version
Version
(ug/mL)
aAb
PR-
Mu72 Canine
PR-1290646
3.2
97
1254972
lgG1/k Chimera
PR-
Mu73 Canine
PR-1290654
7
88.3
1254973
lgG1/k Chimera
PR-
Mu77 Canine
PR-1290656
0.3
82.4
1254977
lgG1/k Chimera
PR-
Mu81 Canine
PR-1290657
0.9
81
1254981
lgG1/k Chimera
PR-
Mu82 Canine
PR-1290658
11.9
92.3
1254982
lgG1/k Chimera
TABLE 20
Production of Caninized Antibodies by Combinations of Caninized Heavy and Light Chains
Light chain
Heavy chain
72.2 VH
72.3 VH
72.4 VH
73.2 VH
73.4 VH
77.2 VH
72.2 VL
72VHv2/
72.3
72VHv4/
73.5
73VHv4/
77VHv2/
72VLv2
CaIgG1/k
72VLv2
CaIgG1/k
72VLv2
72VLv2
72.4 VL
72VHv2/
72VHv3/
72.4
73VHv2/
73VHv4/
77VHv2/
72VLv4
72VLv4
CaIgG1/k
72VLv4
72VLv4
72VLv4
73.2 VL
72VHv2/
72.5
72VHv4/
73.2
73VHv4/
77VHv2/
73VLv2
CaIgG/k
73VLv2
CaIgG1/k
73VLv2
73VLv2
73.4 VL
72VHv2/
72VHv3/
72VHv4/
73VHv2/
73.4
77VHv2/
73VLv4
73VLv4
73VLv4
73VLv4
CaIgG/k
73VLv4
77.2 VL
72VHv2/
72.6
72VHv4/
73.6
73VHv4/
77VHv2/
77VLv2
CaIgG/k
77VLv2
CaIgG1/k
77VLv2
77VLv2
77.4 VL
72VHv2/
72VHv3/
72VHv4/
73VHv2/
73VHv4/
77VHv2/
77VLv4
77VLv4
77VLv4
77VLv4
77VLv4
77VLv4
81.2 VL
72VHv2/
72.7
72VHv4/
73.7
73VHv4/
77VHv2/
81VLv2
CaIgG/k
81VLv2
CaIgG1/k
81VLv2
81VLv2
81.4 VL
72VHv2/
72VHv3/
72VHv4/
73VHv2/
73VHv4/
77VHv2/
81VLv4
81VLv4
81VLv4
81VLv4
81VLv4
81VLv4
82.2 VL
72VHv2/
72VHv3/
72VHv4/
73VHv2/
73VHv4/
77VHv2/
82VLv2
82VLv2
82VLv2
82VLv2
82VLv2
82VLv2
82.3 VL
72VHv2/
72.8
72VHv4/
73.8
73VHv4/
77VHv2/
82VLv3
CaIgG/k
82VLv3
CaIgG1/k
82VLv3
82VLv3
82.4 VL
72VHv2/
72VHv3/
72VHv4/
73VHv2/
73VHv4/
77VHv2/
82VLv4
82VLv4
82VLv4
82VLv4
82VLv4
82VLv4
Light chain
Heavy chain
77.3 VH
77.4 VH
81.2 VH
81.4 VH
82.2 VH
82.4 VH
72.2 VL
77.5
77VHv4/
81.5
81VHv4/
82.5
82VHv4/
CaIgG1/k
72VLv2
CaIgG1/k
72VLv2
CaIgG1/k
72VLv2
72.4 VL
77VHv3/
77VHv4/
81VHv2/
81VHv4/
82VHv2/
82VHv4/
72VLv4
72VLv4
72VLv4
72VLv4
72VLv4
72VLv4
73.2 VL
77.6
77VHv4/
81.6
81VHv4/
82.6
82VHv4/
CaIgG1/k
73VLv2
CaIgG1/k
73VLv2
CaIgG1/k
73VLv2
73.4 VL
77VHv3/
77VHv4/
81VHv2/
81VHv4/
82VHv2/
82VHv4/
73VLv4
73VLv4
73VLv4
73VLv4
73VLv4
73VLv4
77.2 VL
77.3
77VHv4/
81.7
81VHv4/
82.7
82VHv4/
CaIgG1/k
77VLv2
CaIgG1/k
77VLv2
CaIgG1/k
77VLv2
77.4 VL
77VHv3/
77.4
81VHv2/
81VHv4/
82VHv2/
82VHv4/
77VLv4
CaIgG1/k
77VLv4
77VLv4
77VLv4
77VLv4
81.2 VL
77.7
77VHv4/
81.2
81VHv4/
82.8CaIgG1/k
82VHv4/
CaIgG1/k
81VLv2
CaIgG1/k
81VLv2
81VLv2
81.4 VL
77VHv3/
77VHv4/
81VHv2/
81.4
82VHv2/
82VHv4/
81VLv4
81VLv4
81VLv4
CaIgG1/k
81VLv4
81VLv4
82.2 VL
77VHv3/
77VHv4/
81VHv2/
81VHv4/
82VHv2/
82VHv4/
82VLv2
82VLv2
82VLv2
82VLv2
82VLv2
82VLv2
82.3 VL
77.8
77VHv4/
81.8
81VHv4/
82.3
82VHv4/
CaIgG1/k
82VLv3
CaIgG1/k
82VLv3
CaIgG1/k
82VLv3
82.4 VL
77VHv3/
77VHv4/
81VHv2/
81VHv4/
82VHv2/
82.4
82VLv4
82VLv4
82VLv4
82VLv4
82VLv4
CaIgG1/k
TABLE 21
Caninized Monoclonal Antibody Characterization Data
Estimated
Expression
Level in
%
Cell Super-
Mono-
natants
meric
Name
Moniker
Lot
(ug/mL)
mAb
72.3 Canine
PR-1313524
1804091
2.63
88.3
lgG1/k
72.4 Canine
PR-1314949
1805928
1.6
81.5
lgG1/k
73.2 Canine
PR-1313520
1810546
13.4
96.5
lgG1/k
73.4 Canine
PR-1314950
1805932
1.8
90
lgG1/k
77.3 Canine
N/A
N/A
0.7
24.8
lgG1/k
77.4 Canine
N/A
N/A
1
34.6
lgG1/k
81.2 Canine
N/A
No mAb
No mAb
N/A
lgG1/k
detected
detected
81.4 Canine
N/A
No mAb
No mAb
N/A
lgG1/k
detected
detected
82.3 Canine
PR-1313519
1810585
4.4
80.7
lgG1/k
82.4 Canine
PR-1313521
1816320
9.8
94.2
lgG1/k
Example 18: Affinity Analysis of Canine Antibodies
Purified mouse/canine chimeric antibodies and caninized antibodies were analyzed for affinity to canine NGF using a Biacore T100 instrument. Goat anti Canine IgG (Southern Biotech) was immobilized at 5000-10000 RU on a CM5 chip using an amine coupling procedure according to the manufacturer's instructions (Biacore). Canine NGF was injected at 50 uL/min at a concentration range of 50-0.156 nM for the mouse/canine chimeric antibodies or 10-0.156 nM for the caninized antibodies. The association rate was monitored for 5 min and the dissociation rate was monitored for 10-20 min. The chip surface was regenerated using 50-75 ul 10 mM glycine pH 1.5 at a flow rate of 50-100 ul/min. Data was analyzed using Biaevaluation T100 software version 2.0.2, software, GE Healthcare Life Sciences (Piscataway, N.J.). Overall affinity parameters established for mouse/canine chimeric antibodies is summarized in Table 22 and for caninized antibodies in Table 23. This data indicates that the isolated mouse/canine chimeric anti-NGF mAbs have fast on-rates (from greater than 2×106) and slow off-rates (from less than 3×10−3). The overall KDs of the mouse/canine anti-NGF mAbs range from about 1300 pM to 1.6 pM. This data also indicates that the isolated caninized chimeric anti-NGF mAbs have fast on-rates (from greater than 6×106) and slow off-rates (from less than 2×10−4). The overall KDs of the caninized anti-NGF mAbs range from about 42 pM to 1.2 pM.
TABLE 22
Affinity Parameters of Mouse/Canine Chimeric
Monoclonal Antibodies to Canine NGF
On-rate
Off-rate
Overall
Name
Moniker
(l/M-S)
(l/S)
Affinity (M)
Mu72 Canine
PR-
2.9 × 106
3.8 × 10−3
1.3 × 10−9
lg1/k Chimera
1290646
Mu73 Canine
PR-
6.3 × 106
9 × 10−3
1.4 × 10−11
lg1/k Chimera
1290654
Mu77 Canine
PR-
9.1 × 106
1.9 × 10−4
2.1 × 10−11
lg1/k Chimera
1290656
Mu81 Canine
PR-
4.2 × 106
3.5 × 10−4
8.2 × 10−11
lg1/k Chimera
1290657
Mu82 Canine
PR-
8.7 × 106
1.4 × 10−5
1.6 × 10−12
lg1/k Chimera
1290658
TABLE 23
Affinity Parameters of Caninized Monoclonal Antibodies to Canine NGF
On-rate
Off-rate
Overall affinity
Name
(l/M-s)
(l/s)
(M)
73.2 canine lgG1/k
Expt 1
6.3 × 106
2.8 × 10−4
4.4 × 10−11
PR-13113520
Expt 2
6.9 × 106
2.9 × 10−4
4.2 × 10−11
Average
6.6 × 106
2.9 × 10−4
4.3 × 10−11
82.3 canine lgG1/k
Expt 1
8.2 × 106
2 × 10−5
2.4 × 10−12
PR-13113519
Expt 2
8.5 × 106
1.3 × 10−5
1.6 × 10−12
Average
8.4 × 106
1.7 × 10−5
2 × 10−12
82.4 canine lgG1/k
Expt 1
8.6 × 106
1.1 × 10−5
1.2 × 10−12
PR-13113521
Expt 2
7.7 × 106
1.2 × 10−5
1.5 × 10−12
Average
8.2 × 106
1.2 × 10−5
1.4 × 10−12
Example 19: Characterization of Canine Antibodies by the TF-1 Cell Proliferation Potency Assay
Purified mouse/canine chimeric antibodies and caninized antibodies were characterized using the TF-1 Cell Proliferation Potency Assay (described previously) using 70 pM canine NGF in the assay. The summarized potency data is in Tables 20 and 21. The data shows that in the presence of 70 pM canine NGF, all of the mouse/canine chimeric anti-NGF antibodies display sub-nM potencies, and all display potencies of less than 50 pM. The data shows that in the presence of 70 pM canine NGF, some of the caninized anti-NGF antibodies have no neutralization potency on 70 pM canine NGF. Some caninized mAbs have sub-nM potencies, and some have potencies of less than 20 pM.
TABLE 24
Potency of Mouse/Canine Chimeric NGF Monoclonal
Antibodies on Canine NGF-induced TF-1 Ce Proliferation
Name
Moniker
Lot
IC50 (nM)
Mu72 Canine
PR-1290646
1785614
0.041
lgG1/k Chimera
Mu73 Canine
PR-1290654
1785658
0.008
lgG1/k Chimera
Mu77 Canine
PR-1290656
1785699
0.028
lgG1/k Chimera
Mu81 Canine
PR-1290657
1778832
0.012
lgG1/k Chimera
Mu82 Canine
PR-1290658
1785732
0.007
lgG1/k Chimera
TABLE 25
Potency of Caninized NGF Monoclonal Antibodies on Canine
NGF-Induced TF-1 Cell Proliferation (N/A = not applicable)
Name
Moniker
Lot
IC50 (nM)
72.3 Canine lgG1/k
PR-1313524
1804091
0
72.4 Canine lgG1/k
PR-1314949
1805928
0
73.2 Canine lgG1/k
PR-1313520
1810546
0.422
73.4 Canine lgG1/k
PR-1314950
1805932
0
77.3 Canine lgG1/k
N/A
N/A
0.625
77.4 Canine lgG1/k
N/A
N/A
0
82.3 Canine lgG1/k
PR-1313519
1810585
0.017
82.4 Canine lgG1/k
PR-1313521
1816320
0.016
Example 20: Characterization of Solubility and Stability of Caninized Anti-NGF Antibodies
Stock solutions of two caninized anti-NGF antibodies (73.2 canine IgG1/k and 82.4 canine IgG1/k) were obtained. The antibodies were formulated in phosphate buffer saline (PBS) at concentrations below 5 mg/ml (PBS contains, but is not limited to, the following ingredients: 15 mM phosphate buffer and 150 mM sodium chloride at pH 7.4).
Solubility:
The solubility of the caninized antibodies at high concentrations in PBS were evaluated by concentrating the antibodies with Amicon 30K molecular weight cutoff centrifuge spin filters. The final concentrations were determined by UV absorbance.
At room temperature, 73.2 canine IgG1/k was soluble to at least 54 mg/ml and 82.4 canine IgG1/k was soluble to at least 83 mg/ml. When stored at 5° C. for 5 hours at those concentrations, 73.2 canines IgG1/k formed a gel layer at the bottom of the container while 82.4 canines IgG1/k remained as a uniform solution. When re-equilibrated to room temperature, 73.2 canines IgG1/k became a uniform solution. When 73.2 canines IgG1/k were diluted to 27 mg/ml, it remained as a uniform solution at 5° C.
In comparison, adalimumab, a human antibody, demonstrated a solubility of at least 150 mg/ml at 5° C. and at room temperature. This was observed in a formulation with a pH of 7 and with a sodium chloride concentration of 150 mM. The observations are described in Table 26
TABLE 26
Solubility of 73.2 canine lgG1/k, 82.4 canine
lgG1/k and human antibody adalimumab in PBS.
Room temperature
Observations
Antibody
solubility (mg/ml)
when placed at 5° C.
73.2 canine
≥54
Gel layer formed at
lg1/k
container bottom*
82.4 canine
≥83
Remained as solution
lgG1/k
adalimumab
≥150
Remained as solution
*returned to uniform solution when brought back to room temperature; when diluted to 27 mg/ml, remained as uniform solution at 5° C.
The solubility of 73.2 canine IgG1/k 82.4 canine IgG1/k was also evaluated in 15 mM histidine buffer pH 6.0. This is a buffer typically used to formulate human therapeutic antibodies. The PBS buffer comprising the stock solutions of 73.2 canine IgG1/k and 82.4 canine IgG1/k were exchanged with the histidine buffer using Amicon 3 OK molecular weight cutoff centrifuge spin filters. Following buffer exchange, the antibodies exhibited white precipitation and solubilities of less than 2 mg/ml at room temperature, as determined by UV absorbance. In comparison, the human antibody adalimumab was observed to reach a concentration of at least 150 mg/ml in 15 mM histidine buffer pH 6.0 at room temperature. These observations are summarized in Table 27.
TABLE 27
Solubility of anti-NGF caninized antibodies 73.2 canine lgG1/k, 82.4 canine
lgG1/k and human antibody adalimumab in 15 mM histidine buffer pH 6.0.
Room temperature
Antibody
solubility (mg/ml)
Observations
73.2 canine lgG1/k
<2
White precipatate observed
82.4 canine lgG1/k
<2
White precipatate observed
adalimumab
≥150
Remained as solution
Freeze-Thaw Stability
An assessment of the freeze-thaw (FT) stability of 73.2 canine IgG1/k and 82.4 canine IgG1/kin PBS, and after dilution with PBS to 1 mg/ml, was performed. Both antibodies were frozen at −80° C. for at least 4 hours. They were then thawed in a 30° C. water bath (this constitutes one freeze-thaw cycle). Stability was assessed for four freeze-thaw cycles by size exclusion HPLC (SEC). The freeze-thaw analysis is summarized in Table 28.
TABLE 28
Freeze-thaw stability of 73.2 canine lgG1/k and
82.4 canine lgG1/k at 1 mg/ml in PBS.
Percentage Species
Post
Post
Post
Antibody
Species
Pre-FT
FT#1
FT#2
FT#4
73.2 canine
Monomer
97.4
97.3
97.3
97.2
lgG1/k
Aggregate
1.7
1.8
1.8
1.8
Fragment
0.9
0.9
0.9
1
82.4 canine
Monomer
96.6
96.6
96.6
96.1
lgG1/k
Aggregate
2.9
2.9
2.9
3.2
Fragment
0.5
0.5
0.5
0.7
Storage Stability and Accelerated Stability:
The stability of 73.2 canine IgG1/k and 82.4 canine IgG1/k when formulated at a concentration of 10 mg/mL and within a pH range of 5 to 8 and at low (˜7.5 mM) and high (˜>150 mM) ionic strengths was assessed. Stability at these conditions was assessed by monitoring the stability of the antibodies in the following buffers and salt concentrations: (A) 15 mM acetate pH 5; (B) 15 mM acetate pH 5+150 mM NaCl; (C) 15 mM histidine pH 6+150 mM NaCl; (D) 15 mM phosphate pH 7.4; (E) PBS pH 7.4; (F) 15 mM Tris pH 7.5; (G) 15 mM Tris pH 8.0. Sodium azide (0.02%) was added to all buffers as an anti-microbial agent.
Stock solutions of 73.2 canine IgG1/k and 82.3 canine IgG1/kin PBS were concentrated up to 15 mg/ml using 3 OK molecular weight cutoff centrifuge spin filters. They were then dialyzed against the buffers listed above for 18 hours using mini-dialysis 1 kD molecular weight cut-off dialysis tubes (GE Healthcare). Following dialysis, samples were diluted with the respective buffers to a final concentration of 10 mg/ml. 150 μl of each sample was aliquoted to cryovials which were then stored at 40° C. or 5° C. Samples were analysed at time=0 hours (TO), at 7 days (T7d), and at 21 days (T21d) and stability was assessed by SEC.
After 21 days at 40° C., accelerated stability testing showed that 73.2 canine IgG1/k and 82.3 canines IgG1/k have much greater fragmentation at pH values below 7.4 than at pH values above 7.4. In comparison, the human antibody adalimumab, exhibited less fragmentation within the pH range 4 to 8 over 21 days at 40° C. In particular, the fragmentation of adalimumab at pH 6 was much less than the fragmentation of 73.2 canine IgG1/k or 82.4 canine IgG1/k at pH 6. Also, adalimumab at the higher stress condition of pH 4 showed equal or less fragmentation compared to 73.2 canine IgG1/k or 82.4 canine IgG1/k at the lower stress condition of pH 5. The results of the stability analyses and fragmentation profiles are shown, respectively, in Tables 29 and 30. These data suggest that canine IgG1/k monoclonal antibodies have a different degradation profile compared to that of human IgG1/k monoclonal antibodies. Specifically, the fragmentation appears to be more extensive for canine IgG1/k antibodies than for human antibodies at pH 6 and below.
TABLE 29
Stability data from SEC for 73.2 canine IgG1/k, 82.4 canine
IgG1/k and human antibody adalimumab in different
formulations at 7 and 21 days at 5° C. and at 40° C.
Percentage
Percentage
Percentage
Monomer
Aggregate
Fragment
Buffer
T0
T7d
T21d
T0
T7d
T21d
T0
T7d
T21d
73.2 canine IgG1/k at 5° C.
A (pH 5)
94.6
91.4
90.3
2.9
3.1
3.4
2.6
5.5
6.2
B (pH 5)
95.2
98.2
98.2
3.4
0.4
0.5
1.4
1.4
1.3
C (pH 6)
93.9
98.0
97.8
4.4
0.5
0.7
1.8
1.5
1.5
D (pH 7.4)
94.3
97.9
97.7
4.7
0.6
0.8
1.0
1.5
1.5
E (pH 7.4)
94.5
97.9
97.8
4.5
0.5
0.8
1.0
1.6
1.5
F (pH 7.5)
94.5
98.0
97.8
4.0
0.5
0.8
1.6
1.5
1.5
G (pH 8.0)
93.7
97.8
97.6
4.6
0.7
0.9
1.7
1.5
1.5
73.2 canine IgG1/k at at 40° C.
A (pH 5)
94.6
81.3
79.0
2.9
4.5
5.1
2.6
14.2
15.9
B (pH 5)
95.2
92.1
90.9
3.4
0.6
1.2
1.4
7.4
7.8
C (pH 6)
93.9
94.4
91.6
4.4
0.6
1.1
1.8
5.0
7.3
D (pH 7.4)
94.3
97.5
96.6
4.7
0.9
1.6
1.0
1.5
1.8
E (pH 7.4)
94.5
98.0
97.3
4.5
0.6
1.1
1.0
1.4
1.6
F (pH 7.5)
94.5
97.5
96.3
4.0
0.9
1.8
1.6
1.6
1.9
G (pH 8.0)
93.7
97.0
95.2
4.6
1.2
2.6
1.7
1.8
2.2
82.4 canine IgG1/k at 5° C.
A (pH 5)
96.7
98.1
98.7
2.3
1.3
.8
1.0
0.5
0.5
B (pH 5)
96.3
97.2
97.3
2.4
2.3
2.3
1.3
0.5
0.4
C (pH 6)
97.0
97.1
97.1
2.6
2.3
2.3
0.4
0.6
0.6
D (pH 7.4)
96.4
97.0
97.1
2.5
2.4
2.5
1.0
0.5
0.4
E (pH 7.4)
96.7
96.8
96.8
2.8
2.5
2.6
0.5
0.7
0.6
F (pH 7.5)
96.8
97.1
96.9
2.9
2.5
2.5
0.2
0.5
0.6
G (pH 8.0)
96.6
96.9
96.9
2.5
2.5
2.5
0.9
0.6
0.6
82.4 canine IgG1/k at 40° C.
A (pH 5)
96.7
93.1
87.8
2.3
2.8
4.2
1.0
4.1
8.0
B (pH 5)
96.3
93.3
91.3
2.4
2.5
3.1
1.3
4.3
5.6
C (pH 6)
97.0
94.1
92.5
2.6
2.3
2.7
0.4
3.5
4.8
D (pH 7.4)
96.4
93.5
93.8
2.5
3.4
3.3
1.0
3.1
2.9
E (pH 7.4)
96.7
95.1
93.6
2.8
2.4
2.6
0.5
2.5
3.8
F (pH 7.5)
96.8
93.4
923.6
2.9
2.6
3.0
0.2
4.0
0.5
G (pH 8.0)
96.6
94.8
92.7
2.5
2.8
3.5
0.9
2.4
0.4
adalimumab at 40° C.
pH
4
99
98
95
<1
<2
0.5
<1
<2
4.5
6
99
99
99
<1
<1
0.3
<1
<1
0.7
8
99
98
98
<1
<2
1.2
<1
<2
0.8
TABLE 30
Fragmentation profile from SEC for 73.2 canine lgG1/k,
82.4 canine lgG1/k and human antibody adalimumab
in different formulations at 21 days and at 40° C.
Increase in
Percent
Fragmentation
over 21 days at
Antibody
Buffer
40° C.
73.2 canine lgG1/k
A (pH 5)
13.3
B (pH 5)
6.4
C (pH 6)
5.5
D (pH 7.4)
0.8
E (pH 7.4)
0.6
F (pH 7.5)
0.3
G (pH 8.0)
0.5
82.4 canine lgG1/k
A (pH 5)
7.0
B (pH 5)
4.3
C (pH 6)
4.4
D (pH 7.4)
1.9
E (pH 7.4)
3.3
F (pH 7.5)
0.3
G (pH 8.0)
−0.5
adalimumab
pH 4
<4.5
pH 6
<0.7
pH 8
<0.8
Example 21: Canine Single Dose PK Study and Antigen Bridging Assay for PK Serum Sample Analysis
The serum levels of 73.2 canine IgG1/k and 82.4 canine IgG1/k were analyzed following a single dose of 4.5 mg/kg (intravenous or subcutaneous) in mongrel dogs. Following the injection, 13 samples of venous blood were collected over 672 hours. Blood samples were allowed to dot and the serum removed for antibody quantitation.
An NGF bridging assay was developed to quantitate canine anti-NGF mAbs in serum. Streptavidin-coated 96-well plates (MSD #L1 ISA-1) were blocked with Blocker A (MSD #R93BA-4). Canine anti-NGF antibody present in serum (or in PBS) was mixed with equimolar ratios of biotin-tagged NGF and Sulfo-tagged NGF (Sulfo Reagent MSD #R91AN-1) and incubated to form an NGF+antibody complex. The final concentration of the biotin-tagged NGF and sulfo-tagged NGF in the assay was between 1-2 nM. NGF-antibody complexes were added to the streptavidin-coated plate and allowed to bind for 60 minutes. Following incubation, plates were washed with PBS plus 0.05% Tween-20, and bound NGF-antibody complexes were detected in Read Buffer T (MSD #R92TC-1) on an MSD SECTOR Imager 6000. Data was quantitated to estimate the total amount of antibody in μg/mL of a sample liquid and is provided below in Tables 27-30.
TABLE 31
Serum Concentrations of 82.4 canine lgG1/k
Following a Single Subcutaneous Dose
Dog #
Hours
1073305
1072602
1072104
1072306
1072105
post injection
ug/mL
0
0.0
0.0
0
0
0.1
0.25
2.6
0.4
0.0
2.24
0.1
1
9.0
2.2
0.9
9.07
0.4
8
31.5
17.7
13.8
32.55
12.8
12
41.1
20.5
18.7
33.31
24.5
24
42.2
25.6
23.2
35.73
23.8
48
52.3
37.2
36.3
41.35
35.3
72
51.2
41.1
34.4
38.91
36.8
144
47.1
42.6
35.2
33.46
36.7
240
39.0
32.4
29.0
26.03
31.7
336
30.9
28.2
24.1
19.99
26.8
TABLE 32
Serum Concentrations of 82.4 canine lgG1/k Following a Single Intravenous Dose
Dog #
Hours post
1072705
1073804
1073303
1073903
1074502
injection
ug/mL
0
0
0
0
0
0
0.25
102.6
83.1
84.0
80.1
81.7
1
106.7
70.1
87.4
74.1
81.9
8
88.4
65.7
68.1
67.4
68.9
12
91.5
61.6
62.6
62.1
59.3
24
87.4
60.0
57.2
53.9
53.0
48
76.6
49.9
52.6
50.9
50.2
72
60.3
49.1
46.0
40.8
44.6
144
45.7
43.1
36.1
35.7
36.0
240
37.2
34.4
32.6
24.3
32.6
336
31.7
32.7
24.8
20.6
20.1
504
23.5
17.4
18.5
12.2
12.8
672
15.2
10.7
12.5
7.3
8.3
TABLE 33
Serum Concentrations of 73.2 canine lgG1/k
Following a Single Subcutaneous Dose
Hours
Dog #
post
1072607
1074307
1072606
1074503
injection
ug/mL
0
0
0
0
0
0.25
0
0
2.1
0
1
1.0
5.9
4.7
0.0
8
18.6
31.3
18.7
7.4
12
22.7
32.5
21.3
8.7
24
26.8
33.2
24.2
12.0
48
33.7
35.9
28.4
16.2
72
35.0
37.6
30.7
19.4
144
34.9
37.4
30.2
21.8
240
31.6
31.8
26.8
22.0
336
24.4
24.7
22.6
16.5
504
15.3
14.3
13.8
10.6
672
6.8
9.2
5.1
5.4
TABLE 34
Serum Concentrations of 73.2 canine lgG1/k
Following a Single Intravenous Dose
Hours
Dog #
post
1072804
1073304
1072604
injection
ug/mL
0
0
0
0
0.25
93.6
33.2
108.5
1
86.3
30.8
103.1
8
76.9
22.1
85.4
12
72.1
21.4
80.5
24
63.0
17.1
68.0
48
54.6
14.6
56.8
72
49.4
13.5
50.8
144
41.4
10.2
41.4
240
35.4
8.9
30.8
336
30.5
6.3
20.9
504
22.2
4.0
3.4
672
14.8
3.4
0.0
Example 22: Pharmacokinetic Analysis of Serum Concentration Data
Pharmacokinetic parameters for both intravenous (IV) and subcutaneous (SC) dosing routes were calculated for each animal using WinNonlin software (Pharsight Corporation, Mountain View, Calif.) by noncompartmental analysis. Other calculations, e.g. mean, standard deviation (SD), and percent subcutaneous bioavailability (F: %) were carried out using Microsoft Excel software (Microsoft Corporation Redmond, Wash.). The data is shown in Table 35 and 36.
TABLE 35
Pharmacokinetic Analysis of 73.2 canine IgG1/k
Following a Single Intravenous Dose
IV
SC
T½
Vss
CI
T½
Cmax
Tmax
(day)
(mL/kg)
(mL/h/kg)
(day)
(ug/mL)
(day)
% F
14.8*
71
0.15
8.0*
31.3
4.8
51
*Harmonic Mean
TABLE 36
Pharmacokinetic Analysis of 82.4 canine IgG1/k
Following a Single Intravenous Dose
IV
SC
T½
Vss
CI
T½
Cmax
Tmax
(day)
(mL/kg)
(mL/h/kg)
(day)
(ug/mL)
(day)
% F
10.9*
73
0.19
11.6*
41.9
3.0
94
*Harmonic Mean
The data indicates that canine mAbs 73.2 and 82.4 have a half-life of about 8 to about 15 days when dosed IV or SC, suggesting that these molecules exhibit mammalian antibody-like half-lives and overall PK parameters.
Example 23: ELISA for Titering Canine Antibodies
To quantitate canine antibodies in cell supernatants (or other liquids), high-binding EIA plates (Costar #9018) were coated with polyclonal goat anti-dog IgG antibodies (Rockland #604-1102) at 4 μg/ml in PBS. After blocking with 2% non-fat milk in PBS, canine monoclonal antibodies were added to the plates and the plates were washed with PBS plus 0.05% Tween-20. Bound canine mAbs were detected with HRP-tagged goat anti-dog IgG antibodies (Rockland #604-1302) at 0.1 μg/ml. Plates were washed with PBS plus 0.05% Tween-20. Canine mAbs were detected by addition of TMB substrate (Neogen #308177), and the reaction was stopped with IN HC1. Bound canine antibodies were quantitated by absorption at 450 nM to estimate the total amount of antibody in μg/mL of a sample liquid.
The present disclosure incorporates by reference in their entirety techniques well known in the field of molecular biology. These techniques include, but are not limited to, techniques described in the following publications:
Ausubel, F. M. et al. eds., Short Protocols in Molecular Biology (4th Ed. 1999) John Wiley & Sons, NY. (ISBN 0-471-32938-X).
Lu and Weiner eds. Cloning and Expression Vectors for Gene Function Analysis (2001)
BioTechniques Press. Westborough, Mass. 298 pp. (ISBN 1-881299-21-X).
Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).
Old, R. W. & S. B. Primrose, Principles of Gene Manipulation: An Introduction To Genetic Engineering (3d Ed. 1985) Blackwell Scientific Publications, Boston. Studies in Microbiology; V.2:409 pp. (ISBN 0-632-01318-4).
Sambrook, J. et al. eds., Molecular Cloning: A Laboratory Manual (2d Ed. 1989) Cold Spring Harbor Laboratory Press, NY. Vols. 1-3. (ISBN 0-87969-309-6).
Winnacker, E. L. From Genes To Clones: Introduction To Gene Technology (1987) VCH Publishers, NY (translated by Horst Ibelgaufts). 634 pp. (ISBN 0-89573-614-4).
All references, publications, and patent applications disclosed herein are hereby incorporated by reference in their entirety.
Although a number of embodiments, aspects and features have been described above, it will be understood by those skilled in the art that modifications and variations of the described embodiments and features may be made without departing from the present disclosure or the disclosure as defined in the appended claims.
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14799182
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zoetis belgium s.a.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Mar 31st, 2022 03:01PM
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Mar 31st, 2022 03:01PM
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Zoetis
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:zts
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Zoetis
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Aug 30th, 2016 12:00AM
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Jan 21st, 2014 12:00AM
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https://www.uspto.gov?id=US09428536-20160830
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Immunostimulatory G, U-containing oligoribonucleotides
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Compositions and methods relating to immunostimulatory RNA oligomers are provided. The immunostimulatory RNA molecules are believed to represent natural ligands of one or more Toll-like receptors, including Toll-like receptor 7 (TLR7) and Toll-like receptor 8 (TLR8). The compositions and methods are useful for stimulating immune activation. Methods useful for screening candidate immunostimulatory compounds are also provided.
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9428536
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1. A synthetic G,U-rich immunostimulatory RNA oligomer which is 15-40 nucleotides long comprising multiples of 5′-GUUGB-3′, wherein B represents U, G, or C; wherein the multiples are linked through a single intervening linking nucleoside, which is selected from the group consisting of G and U.
2. The immunostimulatory RNA oligomer of claim 1 wherein B is U.
3. The immunostimulatory RNA oligomer of claim 1 or 2, wherein the intervening linking nucleoside is U.
4. An immunostimulatory RNA oligomer of claim 1, which comprises multiples of UUG.
5. An immunostimulatory RNA oligomer of claim 1, which is 15-30 nucleotides long.
6. The immunostimulatory RNA oligomer of claim 1, comprising two 5′-GUUGB-3′ motifs.
7. The immunostimulatory RNA oligomer of claim 1, which is at least 90 percent guanine (G) and uracil (U).
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7
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RELATED APPLICATIONS
This application is a continuation and claims the benefit under 35 U.S.C. §120 of U.S. application Ser. No. 11/368,333, now U.S. Pat. No. 8,658,607, issued Feb. 25, 2014, entitled “IMMUNOSTIMULATORY G, U-CONTAINING OLIGORIBONUCLEOTIDES” filed on Mar. 3, 2006, which is a continuation and claims the benefit under 35 U.S.C. §120 of U.S. application Ser. No. 10/407,952, now U.S. Pat. No. 8,153,141, issued Apr. 10, 2012, entitled “IMMUNOSTIMULATORY G, U-CONTAINING OLIGORIBONUCLEOTIDES” filed on Apr. 4, 2003, which claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Ser. No. 60/421,966, entitled “IMMUNOSTIMULATORY G, U-CONTAINING OLIGORIBONUCLEOTIDES” filed on Oct. 29, 2002, and U.S. Provisional Application Ser. No. 60/370,515, entitled “NATURAL LIGANDS OF TLR7 AND TLR8” filed on Apr. 4, 2002, which are herein incorporated by reference in their entirety.
FIELD OF THE INVENTION
The present invention relates generally to the field of immunology and immune stimulation. More particularly, the present invention relates to immunostimulatory ribonucleic acids, homologs of said immunostimulatory ribonucleic acids, and methods of use of said immunostimulatory ribonucleic acids and homologs. Compositions and methods of the invention are believed to be useful for inducing signaling through Toll-like receptor 7 (TLR7) and Toll-like receptor 8 (TLR8).
BACKGROUND OF THE INVENTION
The immune response is conceptually divided into innate immunity and adaptive immunity. Innate immunity is believed to involve recognition of pathogen-associated molecular patterns (PAMPs) shared in common by certain classes of molecules expressed by infectious microorganisms or foreign macromolecules. PAMPs are believed to be recognized by pattern recognition receptors (PRRs) on certain immune cells.
Toll-like receptors (TLRs) are a family of highly conserved polypeptides that play a critical role in innate immunity in mammals. Currently ten family members, designated TLR1-TLR10, have been identified. The cytoplasmic domains of the various TLRs are characterized by a Toll-interleukin 1 (IL-1) receptor (TIR) domain. Medzhitov R et al. (1998) Mol Cell 2:253-8. Recognition of microbial invasion by TLRs triggers activation of a signaling cascade that is evolutionarily conserved in Drosophila and mammals. The TIR domain-containing adapter protein MyD88 has been reported to associate with TLRs and to recruit IL-1 receptor-associated kinase (IRAK) and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) to the TLRs. The MyD88-dependent signaling pathway is believed to lead to activation of NF-kB transcription factors and c-Jun NH2 terminal kinase (Jnk) mitogen-activated protein kinases (MAPKs), critical steps in immune activation and production of inflammatory cytokines. For a review, see Aderem A et al. (2000) Nature 406:782-87.
While a number of specific TLR ligands have been reported, ligands for some TLRs remain to be identified. Ligands for TLR2 include peptidoglycan and lipopeptides. Yoshimura A et al. (1999) J Immunol 163:1-5; Yoshimura A et al. (1999) J Immunol 163:1-5; Aliprantis A O et al. (1999) Science 285:736-9. Viral-derived double-stranded RNA (dsRNA) and poly I:C, a synthetic analog of dsRNA, have been reported to be ligands of TLR3. Alexopoulou L et al. (2001) Nature 413:732-8. Lipopolysaccharide (LPS) is a ligand for TLR4. Poltorak A et al. (1998) Science 282:2085-8; Hoshino K et al. (1999) J Immunol 162:3749-52. Bacterial flagellin is a ligand for TLR5. Hayashi F et al. (2001) Nature 410:1099-1103. Peptidoglycan has been reported to be a ligand not only for TLR2 but also for TLR6. Ozinsky A et al. (2000) Proc Natl Acad Sci USA 97:13766-71; Takeuchi O et al. (2001) Int Immunol 13:933-40. Bacterial DNA (CpG DNA) has been reported to be a TLR9 ligand. Hemmi H et al. (2000) Nature 408:740-5; Bauer S et al. (2001) Proc Natl Acad Sci USA 98, 9237-42. The TLR ligands listed above all include natural ligands, i.e., TLR ligands found in nature as molecules expressed by infectious microorganisms.
The natural ligands for TLR1, TLR7, TLR8 and TLR10 are not known, although recently certain low molecular weight synthetic compounds, the imidazoquinolones imiquimod (R-837) and resiquimod (R-848), were reported to be ligands of TLR7. Hemmi H et al. (2002) Nat Immunol 3:196-200.
SUMMARY OF THE INVENTION
The present invention is based in part on the novel discovery by the inventors of certain immunostimulatory RNA and RNA-like (hereinafter, simply “RNA”) molecules. The immunostimulatory RNA molecules of the invention are believed by the inventors to require a base sequence that includes at least one guanine (G) and at least one uracil (U), wherein optionally the at least one G can be a variant or homolog of G and/or the at least one U can independently be a variant or homolog of U. Surprisingly, the immunostimulatory RNA molecules of the invention can be either single-stranded or at least partially double-stranded. Also surprisingly, the immunostimulatory RNA molecules of the invention do not require a CpG motif in order to exert their immunostimulatory effect. Without meaning to be bound by any particular theory or mechanism, it is the belief of the inventors that the immunostimulatory RNA molecules of the invention signal through an MyD88-dependent pathway, probably through a TLR. Also without meaning to be bound by any particular theory or mechanism, it is the belief of the inventors that the immunostimulatory RNA molecules of the invention interact with and signal through TLR8, TLR7, or some other TLR yet to be identified.
The immunostimulatory RNA molecules of the invention are also believed by the inventors to be representative of a class of RNA molecules, found in nature, which can induce an immune response. Without meaning to be bound by any particular theory or mechanism, it is the belief of the inventors that the corresponding class of RNA molecules found in nature is believed to be present in ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), and viral RNA (vRNA). It is to be noted in this regard that the immunostimulatory RNA molecules of the present invention can be as small as 5-40 nucleotides long. Such short RNA molecules fall outside the range of full length messenger RNAs described to be useful in transfecting dendritic cells in order to induce an immune response to cancer antigens. See, e.g., Boczkowski D et al. (1996) J Exp Med 184:465-72; Mitchell D A et al. (2000) Curr Opin Mol Ther 2:176-81.
It has also been discovered according to the present invention that the immunostimulatory RNA molecules of the invention can be advantageously combined with certain agents which promote stabilization of the RNA, local clustering of the RNA molecules, and/or trafficking of the RNA molecules into the endosomal compartment of cells. In particular, it has been discovered according to the present invention that certain lipids and/or liposomes are useful in this regard. For example, certain cationic lipids, including in particular N-[1-(2,3 dioleoyloxy)-propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP), appear to be especially advantageous when combined with the immunostimulatory RNA molecules of the invention. As another example, covalent conjugation of a cholesteryl moiety to the RNA, for example to the 3′ end of the RNA, promotes the immunostimulatory effect of the RNA, even in the absence of cationic lipid.
The invention provides compositions of matter and methods related to the immunostimulatory RNA molecules of the invention. The compositions and methods are useful, inter alia, for activating immune cells in vivo, in vitro, and ex vivo; treating infection; treating cancer; preparing a pharmaceutical composition; identifying a target receptor for the immunostimulatory RNA; and screening for and characterizing additional immunostimulatory compounds. Furthermore, the compositions of matter and methods related to the immunostimulatory RNA molecules of the instant invention can advantageously be combined with other immunostimulatory compositions of matter and methods related to such other immunostimulatory compositions of matter.
In one aspect the invention provides an immunostimulatory composition. The immunostimulatory composition according to this aspect of the invention includes an isolated RNA oligomer 5-40 nucleotides long having a base sequence having at least one guanine (G) and at least one uracil (U), and optionally a cationic lipid. The RNA oligomer can be of natural or non-natural origin. An RNA oligomer of natural origin can in one embodiment be derived from prokaryotic RNA and in another embodiment can be derived from eukaryotic RNA. In addition, the RNA oligomer of natural origin can include a portion of a ribosomal RNA. An RNA oligomer of non-natural origin can include an RNA molecule synthesized outside of a cell, e.g., using chemical techniques known by those of skill in the art. In one embodiment an RNA oligomer can include a derivative of an RNA oligomer of natural origin.
In one embodiment the isolated RNA oligomer is a G,U-rich RNA as defined below.
In one embodiment the G,U-containing immunostimulatory RNA is an isolated RNA molecule at least 5 nucleotides long which includes a base sequence as provided by 5′-RURGY-3′, wherein R represents purine, U represents uracil, G represents guanine, and Y represents pyrimidine. In one embodiment the G,U-containing immunostimulatory RNA is an isolated RNA molecule at least 5 nucleotides long which includes a base sequence as provided by 5′-GUAGU-3′, wherein A represents adenine. In one embodiment the G,U-containing immunostimulatory RNA is an isolated RNA molecule which includes a base sequence as provided by 5′-GUAGUGU-3′.
In one embodiment the G,U-containing immunostimulatory RNA is an isolated RNA molecule at least 5 nucleotides long which includes a base sequence as provided by 5′-GUUGB-3′, wherein B represents U, G, or C.
In one embodiment the G,U-containing immunostimulatory RNA is an isolated RNA molecule at least 5 nucleotides long which includes a base sequence as provided by 5′-GUGUG-3′.
In other embodiments the isolated RNA molecule can contain multiples of any of the foregoing sequences, combinations of any of the foregoing sequences, or combinations of any of the foregoing sequences including multiples of any of the foregoing sequences. The multiples and combinations can be linked directly or they can be linked indirectly, i.e, through an intervening nucleoside or sequence. In one embodiment the intervening linking nucleoside is G; in one embodiment the intervening linking nucleoside is U.
In one embodiment the base sequence includes 5′-GUGUUUAC-3′. In one embodiment the base sequence is 5′-GUGUUUAC-3′.
In another embodiment the base sequence includes 5′-GUAGGCAC-3′. In one embodiment the base sequence is 5′-GUAGGCAC-3′.
In yet another embodiment the base sequence includes 5′-CUAGGCAC-3′. In one embodiment the base sequence is 5′-CUAGGCAC-3′.
In still another embodiment the base sequence includes 5′-CUCGGCAC-3′. In one embodiment the base sequence is 5′-CUCGGCAC-3′.
In one embodiment the oligomer is 5-12 nucleotides long. In one embodiment the oligomer is 8-12 nucleotides long.
Also according to this aspect of the invention, in one embodiment the base sequence is free of CpG dinucleotide. Thus in this embodiment the immunostimulatory RNA is not a CpG nucleic acid.
In certain embodiments according to this aspect of the invention, the base sequence of the RNA oligomer is at least partially self-complementary. In one embodiment the extent of self-complementarity is at least 50 percent. The extent of self-complementarity can extend to and include 100 percent. Thus for example the base sequence of the at least partially self-complementary RNA oligomer in various embodiments can be at least 50 percent, at least 60 percent, at least 70 percent, at least 80 percent, at least 90 percent, or 100 percent self-complementary. Complementary base pairs include guanine-cytosine (G-C), adenine-uracil (A-U), adenine-thymine (A-T), and guanine-uracil (G-U). G-U “wobble” basepairing, which is fairly common in ribosomal RNA and in RNA retroviruses, is somewhat weaker than traditional Watson-Crick basepairing between G-C, A-T, or A-U. A partially self-complementary sequence can include one or more portions of self-complementary sequence. In an embodiment which involves a partially self-complementary sequence, the RNA oligomer can include a self-complementary portion positioned at and encompassing each end of the oligomer.
In one embodiment according to this aspect of the invention, the oligomer is a plurality of oligomers, i.e., a plurality of RNA oligomers each 6-40 nucleotides long having a base sequence comprising at least one guanine (G) and at least one uracil (U). The plurality of oligomers can, but need not, include sequences which are at least partially complementary to one another. In one embodiment the plurality of oligomers includes an oligomer having a first base sequence and an oligomer having a second base sequence, wherein the first base sequence and the second base sequence are at least 50 percent complementary. Thus for example the at least partially complementary base sequences in various embodiments can be at least 50 percent, at least 60 percent, at least 70 percent, at least 80 percent, at least 90 percent, or 100 percent complementary. As described above, complementary base pairs include guanine-cytosine (G-C), adenine-uracil (A-U), adenine-thymine (A-T), and guanine-uracil (G-U). Partially complementary sequences can include one or more portions of complementary sequence. In an embodiment which involves partially complementary sequences, the RNA oligomers can include a complementary portion positioned at and encompassing at least one end of the oligomers.
In one embodiment the oligomer is a plurality of oligomers which includes an oligomer having a base sequence including 5′-GUGUUUAC-3′ and an oligomer having a base sequence including 5′-GUAGGCAC-3′. In one embodiment the oligomer is a plurality of oligomers which includes an oligomer having a base sequence 5′-GUGUUUAC-3′ and an oligomer having a base sequence 5′-GUAGGCAC-3′.
Further according to this aspect of the invention, in various embodiments the oligomer includes a non-natural backbone linkage, a modified base, a modified sugar, or any combination of the foregoing. The non-natural backbone linkage can be a stabilized linkage, i.e., a linkage which is relatively resistant against RNAse or nuclease degradation, compared with phosphodiester linkage. In one embodiment the non-natural backbone linkage is a phosphorothioate linkage. The oligomer can include one non-natural backbone linkage or a plurality of non-natural backbone linkages, each selected independently of the rest. The modified base can be a modified G, U, A, or C, including the at least one G and the at least one U of the base sequence according to this aspect of the invention. In some embodiments the modified base can be selected from 7-deazaguanosine, 8-azaguanosine, 5-methyluracil, and pseudouracil. The oligomer can include one modified base or a plurality of modified bases, each selected independently of the rest. The modified sugar can be a methylated sugar, arabinose. The oligomer can include one modified sugar or a plurality of modified sugars, each selected independently of the rest.
In one embodiment the cationic lipid is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP). DOTAP is believed to transport RNA oligomer into cells and specifically traffic to the endosomal compartment, where it can release the RNA oligomer in a pH-dependent fashion. Once in the endosomal compartment, the RNA can interact with certain intracellular Toll-like receptor molecules (TLRs), triggering TLR-mediated signal transduction pathways involved in generating an immune response. Other agents with similar properties including trafficking to the endosomal compartment can be used in place of or in addition to DOTAP.
In one embodiment the immunostimulatory composition further includes an antigen. In one embodiment the antigen is an allergen. In one embodiment the antigen is a cancer antigen. In one embodiment the antigen is a microbial antigen.
Also according to this aspect of the invention, in another embodiment the invention is a pharmaceutical composition. The pharmaceutical composition includes an immunostimulatory composition of the invention and a pharmaceutically acceptable carrier. Methods for preparing the pharmaceutical composition are also provided. Such methods entail placing an immunostimulatory composition of the invention in contact with a pharmaceutically acceptable carrier. The pharmaceutical composition can be formulated in a unit dosage for convenience.
In another aspect the invention provides a method of activating an immune cell. The method involves contacting an immune cell with an immunostimulatory composition of the invention, described above, in an effective amount to induce activation of the immune cell. In one embodiment the activation of the immune cell involves secretion of a cytokine by the immune cell. The cytokine in one embodiment is selected from the group consisting of interleukin 6 (IL-6), interleukin 12 (IL-12), an interferon (IFN), and tumor necrosis factor (TNF). In one embodiment the activation of the immune cell includes secretion of a chemokine. In one embodiment the secreted chemokine is interferon-gamma-induced protein 10 (IP-10). In one embodiment the activation of the immune cell includes expression of a costimulatory/accessory molecule by the immune cell. In one embodiment the costimulatory/accessory molecule is selected from the group consisting of intercellular adhesion molecules (ICAMs, e.g., CD54), leukocyte function-associated antigens (LFAs, e.g., CD58), B7s (CD80, CD86), and CD40.
Also according to this aspect of the invention, in one embodiment the activation of the immune cell involves activation of a MyD88-dependent signal transduction pathway. MyD88 is believed to be an adapter molecule that interacts with the Toll/interleukin-1 receptor (TIR) domain of various Toll-like receptor (TLR) molecules and participates in signal transduction pathways that ultimately result in activation of nuclear factor kappa B (NF-κB). Thus in one embodiment the MyD88-dependent signal transduction pathway is associated with a TLR. More particularly, in one embodiment the TLR is TLR8. In another embodiment the TLR is TLR7.
Also according to this aspect of the invention in one embodiment the immune cell is a human immune cell. The immune cell in one embodiment is a myeloid dendritic cell.
In one embodiment of this aspect of the invention the contacting occurs in vitro. In another embodiment the contacting occurs in vivo.
The invention in another aspect provides a method of inducing an immune response in a subject. The method according to this aspect of the invention involves administering to a subject an immunostimulatory composition of the invention in an effective amount to induce an immune response in the subject. It is to be noted that the method according to this aspect of the invention does not involve administration of an antigen to the subject. In one embodiment the subject is a human. In one embodiment the subject has or is at risk of having a cancer. In one embodiment the subject has or is at risk of having an infection with an agent selected from the group consisting of viruses, bacteria, fungi, and parasites. In a particular embodiment the subject has or is at risk of having a viral infection. It is also to be noted that the method according to this aspect of the invention can be used to treat a subject with a suppressed capacity to mount an effective or desirable immune response. For example the subject can have a suppressed immune system due to an infection, a cancer, an acute or chronic disease such as kidney or liver insufficency, surgery, and an exposure to an immunosuppressive agent such as chemotherapy, radiation, certain drugs, or the like. In one embodiment the subject has or is at risk of having an allergy or asthma. Such a subject can be exposed to or at risk of exposure to an allergen that is associated with an allergic response or asthma in the subject.
In yet another aspect the invention provides a method of inducing an immune response in a subject. The method according to this aspect of the invention involves administering an antigen to a subject, and administering to the subject an immunostimulatory composition of the invention in an effective amount to induce an immune response to the antigen. It is to be noted that the antigen can be administered before, after, or concurrently with the immunostimulatory composition of the invention. In addition, both the antigen and the immunostimulatory compound can be administered to the subject more than once.
In one embodiment according to this aspect of the invention the antigen is an allergen. In one embodiment according to this aspect of the invention the antigen is a cancer antigen. The cancer antigen in one embodiment can be a cancer antigen isolated from the subject. In another embodiment the antigen is a microbial antigen. The microbial antigen can be an antigen of a virus, a bacterium, a fungus, or a parasite.
The invention further provides, in yet another aspect, a method of inducing an immune response in a subject. The method according to this aspect of the invention involves isolating dendritic cells of a subject, contacting the dendritic cells ex vivo with an immunostimulatory composition of the invention, contacting the dendritic cells ex vivo with an antigen, and administering the contacted dendritic cells to the subject.
In one embodiment according to this aspect of the invention the antigen is an allergen. In one embodiment according to this aspect of the invention the antigen is a cancer antigen. The cancer antigen in one embodiment can be a cancer antigen isolated from the subject. In another embodiment the antigen is a microbial antigen. The microbial antigen can be an antigen of a virus, a bacterium, a fungus, or a parasite.
An immune response arising from stimulation of one TLR can be modified, enhanced or amplified by stimulation of another TLR, and the combined immunostimulatory effect may be synergistic. For example, TLR9 is reported to respond to bacterial DNA and, more generally, CpG DNA. An immune response arising from TLR9 contacting its natural ligand (or any TLR9 ligand) may be modified, enhanced or amplified by also selectively contacting TLR7 with a TLR7 ligand, or by also selectively contacting TLR8 with a TLR8 ligand, or both. Likewise, an immune response arising from TLR7 contacting a TLR7 ligand may be modified, enhanced or amplified by also selectively contacting TLR8 with a TLR8 ligand, or by also selectively contacting TLR9 with CpG DNA (or any suitable TLR9 ligand), or both. As yet another example, an immune response arising from TLR8 contacting a TLR8 ligand may be modified, enhanced or amplified by also selectively contacting TLR7 with a TLR7 ligand, or by also selectively contacting TLR9 with CpG DNA (or any suitable TLR9 ligand), or both.
The present invention is based in part on the novel discovery by the inventors of what are believed to be natural ligands for TLR7 and TLR8. While naturally occurring ligands derived from microbes have been described for certain TLRs, natural ligands for TLR7 and TLR8 have not previously been described. Certain synthetic small molecules, imidazoquinoline compounds, have been described as ligands for TLR7, but such compounds are to be distinguished from the natural ligands of the present invention. Hemmi H et al. (2002) Nat Immunol 3:196-200.
Isolated natural ligands of TLR7 and TLR8 are useful as compositions that can induce, enhance, and complement an immune response. The natural ligands of TLR7 and TLR8 are useful for preparation of novel compositions that can induce, enhance, and complement an immune response. In addition, the natural ligands of TLR7 and TLR8 are useful for selectively inducing TLR7- and TLR8-mediated signaling and for selectively inducing TLR7- and TLR8-mediated immune responses. Furthermore, the natural ligands of TLR7 and TLR8 are useful in designing and performing screening assays for identification and selection of immunostimulatory compounds.
The present invention is also based in part on the novel discovery according to the invention that human neutrophils strongly express TLR8. This observation is important because neutrophils are very often the first cells to engage infectious pathogens and thus to initiate responses. It is believed that activated neutrophils secrete chemokines and cytokines, which in turn are instrumental in recruiting dendritic cells. TLR9-expressing dendritic cells drawn to the site of the activated neutrophils there become activated, thereby amplifying the immune response.
The present invention is also based in part on the appreciation of the differential expression of various TLRs, including TLR7, TLR8, and TLR9, on various cells of the immune system. This segregation may be of particular significance in humans with respect to TLR7, TLR8, and TLR9. The immune response arising from stimulation of any one of these TLRs may be enhanced or amplified by stimulation of another TLR, and the combined immunostimulatory effect may be synergistic. For example, TLR9 is reported to respond to bacterial DNA and, more generally, CpG DNA. An immune response arising from TLR9 contacting its natural ligand (or any TLR9 ligand) may be enhanced or amplified by also selectively contacting TLR7 with its natural ligand (or any suitable TLR7 ligand), or by also selectively contacting TLR8 with its natural ligand (or any suitable TLR8 ligand), or both. Likewise, an immune response arising from TLR7 contacting its natural ligand (or any TLR7 ligand) may be enhanced or amplified by also selectively contacting TLR8 with its natural ligand (or any suitable TLR8 ligand), or by also selectively contacting TLR9 with CpG DNA (or any suitable TLR9 ligand), or both. As yet another example, an immune response arising from TLR8 contacting its natural ligand (or any TLR8 ligand) may be enhanced or amplified by also selectively contacting TLR7 with its natural ligand (or any suitable TLR7 ligand), or by also selectively contacting TLR9 with CpG DNA (or any suitable TLR9 ligand), or both.
In a further aspect the invention provides a composition including an effective amount of a ligand for TLR8 to induce TLR8 signaling and an effective amount of a ligand for a second TLR selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR9 and TLR10 to induce signaling by the second TLR. In one embodiment the second TLR is TLR3. In one embodiment the second TLR is TLR7. In one embodiment the second TLR is TLR9. In one embodiment the ligand for TLR8 and the ligand for the second TLR are linked. In yet another embodiment the composition further includes a pharmaceutically acceptable carrier.
In another aspect the invention provides a composition including an effective amount of a ligand for TLR7 to induce TLR7 signaling and an effective amount of a ligand for a second TLR selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR8, TLR9, and TLR10 to induce signaling by the second TLR. In one embodiment the second TLR is TLR3. In one embodiment the second TLR is TLR8. In one embodiment the second TLR is TLR9. In one embodiment the ligand for TLR7 and the ligand for the second TLR are linked. In yet another embodiment the composition further includes a pharmaceutically acceptable carrier.
In a further aspect the invention provides a composition including a DNA:RNA conjugate, wherein DNA of the conjugate includes an immunostimulatory motif effective for stimulating TLR9 signaling and wherein RNA of the conjugate includes RNA effective for stimulating signaling by TLR3, TLR7, TLR8, or any combination thereof. In one embodiment the immunostimulatory motif effective for stimulating TLR9 signaling is a CpG motif. In another embodiment the immunostimulatory motif effective for stimulating TLR9 signaling is poly-dT. In yet another embodiment the immunostimulatory motif effective for stimulating TLR9 signaling is poly-dG. In one embodiment the conjugate includes a chimeric DNA:RNA backbone. In one embodiment the chimeric backbone includes a cleavage site between the DNA and the RNA. In one embodiment the conjugate includes a double-stranded DNA:RNA heteroduplex. In yet another embodiment the composition further includes a pharmaceutically acceptable carrier.
In another aspect the invention provides a method for stimulating TLR8 signaling. The method involves contacting TLR8 with an isolated RNA in an effective amount to stimulate TLR8 signaling. In one embodiment the RNA is double-stranded RNA. In one embodiment the RNA is ribosomal RNA. In one embodiment the RNA is transfer RNA. In one embodiment the RNA is messenger RNA. In one embodiment the RNA is viral RNA. In one embodiment the RNA is G,U-rich RNA. In one embodiment the RNA consists essentially of G and U.
In yet another aspect the invention provides a method for stimulating TLR8 signaling. The method according to this aspect involves contacting TLR8 with a mixture of nucleosides consisting essentially of G and U in a ratio between 1G:50U and 10G:1U, in an amount effective to stimulate TLR8 signaling. In one embodiment the nucleosides are ribonucleosides. In one embodiment the nucleosides comprise a mixture of ribonucleosides and deoxyribonucleosides. In one embodiment the G is a guanosine derivative selected from the group consisting of: 8-bromoguanosine, 8-oxoguanosine, 8-mercaptoguanosine, 7-allyl-8-oxoguanosine, guanosine ribonucleoside vanadyl complex, inosine, and nebularine.
A further aspect of the invention provides a method for stimulating TLR8 signaling. The method according to this aspect involves contacting TLR8 with a mixture of ribonucleoside vanadyl complexes. In one embodiment the mixture comprises guanosine ribonucleoside vanadyl complexes.
In another aspect the invention provides a method for stimulating TLR8 signaling. The method according to this aspect involves contacting TLR8 with an isolated G,U-rich oligonucleotide comprising a sequence selected from the group consisting of: UUGUGG, UGGUUG, GUGUGU, and GGGUUU, in an amount effective to stimulate TLR8 signaling. In one embodiment the oligonucleotide is an oligoribonucleotide. In one embodiment the oligonucleotide is 7-50 bases long. In one embodiment the oligonucleotide is 12-24 bases long. In one embodiment the oligonucleotide has a sequence 5′-GUUGUGGUUGUGGUUGUG-3′ (SEQ ID NO:1).
The invention provides in another aspect a method for stimulating TLR8 signaling. The method according to this aspect involves contacting TLR8 with an at least partially double-stranded nucleic acid molecule comprising at least one G-U base pair, in an amount effective to stimulate TLR8 signaling.
In yet another aspect the invention provides a method for supplementing a TLR8-mediated immune response. The method involves contacting TLR8 with an effective amount of a TLR8 ligand to induce a TLR8-mediated immune response, and contacting a TLR other than TLR8 with an effective amount of a ligand for the TLR other than TLR8 to induce an immune response mediated by the TLR other than TLR8.
In a further aspect the invention provides a method for supplementing a TLR8-mediated immune response in a subject. The method according to this aspect involves administering to a subject in need of an immune response an effective amount of a TLR8 ligand to induce a TLR8-mediated immune response, and administering to the subject an effective amount of a ligand for a TLR other than TLR8 to induce an immune response mediated by the TLR other than TLR8. In one embodiment the TLR other than TLR8 is TLR9. In one embodiment the ligand for TLR9 is a CpG nucleic acid. In one embodiment the CpG nucleic acid has a stabilized backbone. In one embodiment the ligand for TLR8 and the ligand for TLR9 are a conjugate. In one embodiment the conjugate comprises a double-stranded DNA:RNA heteroduplex. In one embodiment the conjugate comprises a chimeric DNA:RNA backbone. In one embodiment the chimeric backbone comprises a cleavage site between the DNA and the RNA.
The invention in a further aspect provides a method for stimulating TLR7 signaling. The method according to this aspect involves contacting TLR7 with an isolated guanosine ribonucleoside in an effective amount to stimulate TLR7 signaling. In one embodiment the guanosine ribonucleoside is a guanosine ribonucleoside derivative selected from the group consisting of: 8-bromoguanosine, 8-oxoguanosine, 8-mercaptoguanosine, 7-allyl-8-oxoguanosine, guanosine ribonucleoside vanadyl complex, inosine, and nebularine. In one embodiment the guanosine ribonucleoside derivative is 8-oxoguanosine. In one embodiment the guanosine nucleoside is a ribonucleoside. In one embodiment the guanosine nucleoside comprises a mixture of ribonucleosides and deoxyribonucleosides.
In another aspect the invention further provides a method for stimulating TLR7 signaling. The method according to this aspect involves contacting TLR7 with an isolated nucleic acid comprising a terminal oxidized or halogenized guanosine in an effective amount to stimulate TLR7 signaling. In one embodiment the oxidized or halogenized guanosine is 8-oxoguanosine.
In another aspect the invention provides a method for stimulating TLR7 signaling. The method according to this aspect involves contacting TLR7 with an isolated RNA in an effective amount to stimulate TLR7 signaling. In one embodiment the RNA is double-stranded RNA. In one embodiment the RNA is ribosomal RNA. In one embodiment the RNA is transfer RNA. In one embodiment the RNA is messenger RNA. In one embodiment the RNA is viral RNA. In one embodiment the RNA is G-rich RNA. In one embodiment the RNA is part of a DNA:RNA heteroduplex. In one embodiment the RNA consists essentially of guanosine ribonucleoside.
The invention in yet another aspect provides a method for stimulating TLR7 signaling. The method according to this aspect involves contacting TLR7 with a mixture of nucleosides consisting essentially of G and U in a ratio between 1G:50U and 10G:1U, in an amount effective to stimulate TLR7 signaling.
Provided in yet another aspect of the invention is a method for stimulating TLR7 signaling. The method according to this aspect involves contacting TLR7 with a mixture of ribonucleoside vanadyl complexes. In one embodiment the mixture comprises guanosine ribonucleoside vanadyl complexes.
In a further aspect the invention provides a method for supplementing a TLR7-mediated immune response. The method according to this aspect involves contacting TLR7 with an effective amount of a TLR7 ligand to induce a TLR7-mediated immune response, and contacting a TLR other than TLR7 with an effective amount of a ligand for the TLR other than TLR7 to induce an immune response mediated by the TLR other than TLR7.
In yet another aspect the invention provides a method for supplementing a TLR7-mediated immune response in a subject. The method involves administering to a subject in need of an immune response an effective amount of a TLR7 ligand to induce a TLR7-mediated immune response, and administering to the subject an effective amount of a ligand for a TLR other than TLR7 to induce an immune response mediated by the TLR other than TLR7. In one embodiment the TLR other than TLR7 is TLR9. In one embodiment the ligand for TLR9 is a CpG nucleic acid. In one embodiment the CpG nucleic acid has a stabilized backbone. In one embodiment the ligand for TLR7 and the ligand for TLR9 are a conjugate. In one embodiment the conjugate comprises a double-stranded DNA:RNA heteroduplex. In one embodiment the conjugate comprises a chimeric DNA:RNA backbone. In one embodiment the chimeric backbone comprises a cleavage site between the DNA and the RNA.
The invention in another aspect provides a method for screening candidate immunostimulatory compounds. The method according to this aspect involves measuring a TLR8-mediated reference signal in response to an RNA reference, measuring a TLR8-mediated test signal in response to a candidate immunostimulatory compound, and comparing the TLR8-mediated test signal to the TLR8-mediated reference signal.
In yet another aspect the invention provides a method for screening candidate immunostimulatory compounds, comprising measuring a TLR8-mediated reference signal in response to an imidazoquinoline reference, measuring a TLR8-mediated test signal in response to a candidate immunostimulatory compound, and comparing the TLR8-mediated test signal to the TLR8-mediated reference signal.
Also provided according to yet another aspect of the invention is a method for screening candidate immunostimulatory compounds. The method involves measuring a TLR7-mediated reference signal in response to an imidazoquinoline reference, measuring a TLR7-mediated test signal in response to a candidate immunostimulatory compound, and comparing the TLR7-mediated test signal to the TLR7-mediated reference signal.
In some embodiments the imidazoquinoline is resiquimod (R-848).
In some embodiments the imidazoquinoline is imiquimod (R-837).
In a further aspect the invention also provides a method for screening candidate immunostimulatory compounds. The method according to this aspect involves measuring a TLR7-mediated reference signal in response to a 7-allyl-8-oxoguanosine reference, measuring a TLR7-mediated test signal in response to a candidate immunostimulatory compound, and comparing the TLR7-mediated test signal to the TLR7-mediated reference signal.
Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a bar graph depicting IL-12 p40 secretion by human peripheral blood mononuclear cells (PBMCs) in response to certain stimuli including selected G,U-containing RNA oligonucleotides with or without DOTAP (“with Liposomes” and “without Liposomes”, respectively), as measured by specific enzyme-linked immunosorbent assay (ELISA). The lower case letter “s” appearing in the base sequences signifies phosphorothioate linkage.
FIG. 2 is a bar graph depicting TNF-α secretion by human PBMCs in response to certain stimuli including selected G,U-containing RNA oligonucleotides with or without DOTAP (“with Liposomes” and “without Liposomes”, respectively), as measured by specific ELISA.
FIG. 3 is a bar graph depicting dose-dependence of IL-12 p40 secretion by human PBMCs in response to various concentrations of selected G,U-containing RNA oligonucleotides (with DOTAP), as measured by specific ELISA.
FIG. 4 is a bar graph depicting sequence dependence of TNF-α secretion by human PBMCs in response to various selected RNA oligonucleotides related to the RNA oligonucleotide GUAGGCAC (with DOTAP), as measured by specific ELISA.
FIG. 5 is a bar graph depicting the effect of DOTAP on IL-12 p40 secretion by human PBMCs in response to various stimuli, as measured by specific ELISA.
FIG. 6 is a quartet of bar graphs depicting IL-12 p40 secretion by various types of murine macrophage cells in response to a variety of test and control immunostimulatory compounds, as measured by specific ELISA. Panel A, wild type macrophages in the presence of IFN-γ; Panel B, MyD88-deficient macrophages in the presence of IFN-γ; Panel C, J774 macrophage cell line; Panel D, RAW 264.7 macrophage cell line.
FIG. 7 is a pair of graphs depicting the secretion of (A) TNF-α and (B) IL-12 p40 by human PBMC upon incubation with HIV-1-derived RNA sequences, with and without DOTAP. Circles, 5′-GUAGUGUGUG-3′ (SEQ ID NO:2); Triangles, 5′-GUCUGUUGUGUG-3′ (SEQ ID NO:3). Open symbols, without DOTAP; closed symbols, with DOTAP.
FIG. 8 is a graph depicting apparent relatedness among TLRs.
FIG. 9 depicts nucleic acid binding domains in TLR7, TLR8, and TLR9.
FIG. 10 is a bar graph depicting responsiveness of human PBMC to stringent response factor (SRF).
FIG. 11 is a bar graph depicting responsiveness of human PBMC to the ribonucleoside vanadyl complexes (RVCs). X denotes resiquimod.
FIG. 12 is a series of three bar graphs depicting responsiveness of human TLR7 and human TLR8 to individual ribonucleosides. X denotes resiquimod.
FIG. 13 is a series of three bar graphs depicting responsiveness of TLR7 and TLR8 to mixtures of two ribonucleosides.
FIG. 14 is a bar graph depicting response of human PBMC to a mixture of the ribonucleosides G and U.
FIG. 15 is a bar graph depicting response of human PBMC to G,U-rich RNA, but not DNA, oligonucleotides.
FIG. 16 is a bar graph depicting response of human PBMC to oxidized RNA.
FIG. 17 is a series of three bar graphs depicting human TLR7 and TLR8 responses to oxidized guanosine ribonucleoside. X denotes resiquimod.
FIG. 18 is a pair of bar graphs depicting human TLR7 responses to modified guanosine ribonucleosides.
FIG. 19 is a series of aligned gel images depicting differential expression of TLR1-TLR9 on human CD123+ dendritic cells (CD123+ DC), CD11c+ DC, and neutrophils.
FIG. 20 is a series of three graphs depicting the ability of short, single-stranded G,U-containing RNA oligomers to induce NF-κB in HEK-293 cells stably transfected with expression plasmid for human TLR7 or human TLR8.
DETAILED DESCRIPTION OF THE INVENTION
The invention relates in part to the discovery by the inventors of a number of RNA and RNA-related molecules that are effective as immunostimulatory compounds. Identification of the immunostimulatory compounds arose through a systematic effort aimed at identifying naturally occurring ligands for TLR7 and TLR8. As a result of this effort, it has now been discovered that RNA and RNA-like molecules containing guanine (G) and uracil (U), including specific sequences containing G and U, are immunostimulatory and appear to act through an MyD88-dependent pathway, implicating TLR involvement. Significantly, some of the RNA sequences occur in highly conserved structural features of 5′ untranslated regions of viral RNA that are important to viral replication. The identified immunostimulatory RNA sequences also correspond to or very nearly correspond to other RNAs, including tRNAs derived from bacteria and yeast, as well as rRNA derived from bacteria and possibly some eukaryotes. Importantly, the immunostimulatory RNA of the invention includes single-stranded RNA, in addition to partially or wholly double-stranded RNA, and its effect can be abrogated by RNase treatment. Where the RNA is at least partially double-stranded, it can in one embodiment include a stem-loop structure. As described in greater detail below, it has been discovered according to the invention that single-stranded G,U-rich RNAs as short as 5 nucleotides long can stimulate immune cells to produce large amounts of a number of cytokines and chemokines, including TNF-α, IL-6, IL-12, type 1 interferon (e.g., IFN-α), and IP-10.
It has now been surprisingly discovered by the inventors that certain G,U-containing RNA molecules and their analogs, but not their DNA counterparts, are immunostimulatory. Significantly, the G,U-containing oligoribonucleotides of the invention can be substantially smaller than the messenger RNAs previously described to be useful in preparing dendritic cell vaccines. See, e.g., Boczkowski D et al. (1996) J Exp Med 184:465-72; Mitchell D A et al. (2000) Curr Opin Mol Ther 2:176-81. Although the G,U-containing RNA molecules of the invention can be surrogates for ribosomal RNA and/or viral RNA as found in nature, they can be as small as 5-40 nucleotides long. As described further herein, the G,U-containing oligoribonucleotides of the invention include at least one G and at least one U. Surprisingly, elimination of either G or U from the G,U-containing oligoribonucleotides of the invention essentially abrogates their immunostimulatory effect. The at least one G and at least U can be adjacent to one another, or they can be separated by intervening nucleosides or sequence. Also significantly, the immunostimulatory G,U-containing RNA molecules of the invention do not require a CpG dinucleotide.
In one aspect the invention provides an immunostimulatory composition. The immunostimulatory composition according to this aspect of the invention includes an isolated RNA oligomer 5-40 nucleotides long having a base sequence having at least one guanine (G) and at least one uracil (U). As will be described in greater detail further below, the immunostimulatory RNA oligomer 5-40 nucleotides long having a base sequence having at least one guanine (G) and at least one uracil (U) is advantageously formulated such that the RNA oligomer is stabilized against degradation, concentrated in or on a particle such as a liposome, and/or targeted for delivery to the endosomal compartment of cells. In one formulation, described in the examples below, the RNA oligomer is advantageously combined with the cationic lipid DOTAP, which is believed to assist in trafficking the G,U-containing oligoribonucleotides into the endosomal compartment. Thus, in one aspect the invention is an immunostimulatory composition which includes an RNA oligomer 5-40 nucleotides long having a base sequence having at least one G and at least one U and optionally a cationic lipid.
The RNA oligomer of the invention can be of natural or non-natural origin. RNA as it occurs in nature is a type of nucleic acid that generally refers to a linear polymer of certain ribonucleoside units, each ribonucleoside unit made up of a purine or pyrimidine base and a ribose sugar, linked by internucleoside phosphodiester bonds. In this regard “linear” is meant to describe the primary structure of RNA. RNA in general can be single-stranded or double-stranded, including partially double-stranded.
As used herein, “nucleoside” refers to a single sugar moiety (e.g., ribose or deoxyribose) linked to an exchangeable organic base, which is either a substituted pyrimidine (e.g., cytosine (C), thymidine (T) or uracil (U)) or a substituted purine (e.g., adenine (A) or guanine (G)). As described herein, the nucleoside may be a naturally occurring nucleoside, a modified nucleoside, or a synthetic (artificial) nucleoside.
The terms “nucleic acid” and “oligonucleotide” are used interchangeably to mean multiple nucleotides (i.e., molecules comprising a sugar (e.g., ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g., cytosine (C), thymidine (T) or uracil (U)) or a substituted purine (e.g., adenine (A) or guanine (G)). As used herein, the terms refer to oligoribonucleotides as well as oligodeoxyribonucleotides. The terms shall also include polynucleosides (i.e., a polynucleotide minus the phosphate) and any other organic base-containing polymer. Nucleic acid molecules can be obtained from existing nucleic acid sources (e.g., genomic or cDNA), but are preferably synthetic (e.g., produced by nucleic acid synthesis).
The terms nucleic acid and oligonucleotide also encompass nucleic acids or oligonucleotides with substitutions or modifications, such as in the bases and/or sugars. For example, they include nucleic acids having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3′ position and other than a phosphate group at the 5′ position. Thus modified nucleic acids may include a 2′-O-alkylated ribose group. In addition, modified nucleic acids may include sugars such as arabinose instead of ribose. Thus the nucleic acids may be heterogeneous in backbone composition thereby containing any possible combination of polymer units linked together such as peptide nucleic acids (which have amino acid backbone with nucleic acid bases). In some embodiments, the nucleic acids are homogeneous in backbone composition. Nucleic acids also include substituted purines and pyrimidines such as C-5 propyne modified bases. Wagner R W et al. (1996) Nat Biotechnol 14:840-4. Purines and pyrimidines include but are not limited to adenine, cytosine, guanine, thymidine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, and other naturally and non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties. Other such modifications are well known to those of skill in the art.
A natural nucleoside base can be replaced by a modified nucleoside base, wherein the modified nucleoside base is for example selected from hypoxanthine; dihydrouracil; pseudouracil; 2-thiouracil; 4-thiouracil; 5-aminouracil; 5-(C1-C6)-alkyluracil; 5-(C2-C6)-alkenyluracil; 5-(C2-C6)-alkynyluracil; 5-(hydroxymethyl)uracil; 5-chlorouracil; 5-fluorouracil; 5-bromouracil; 5-hydroxycytosine; 5-(C1-C6)-alkylcytosine; 5-(C2-C6)-alkenylcytosine; 5-(C2-C6)-alkynylcytosine; 5-chlorocytosine; 5-fluorocytosine; 5-bromocytosine; N2-dimethylguanine; 2,4-diamino-purine; 8-azapurine (including, in particular, 8-azaguanine); a substituted 7-deazapurine (including, in particular, 7-deazaguanine), including 7-deaza-7-substituted and/or 7-deaza-8-substituted purine; or other modifications of a natural nucleoside bases. This list is meant to be exemplary and is not to be interpreted to be limiting.
In particular, the at least one guanine base of the immunostimulatory G,U-containing oligoribonucleotide can be a substituted or modified guanine such as 7-deazaguanine; 8-azaguanine; 7-deaza-7-substituted guanine (such as 7-deaza-7-(C2-C6)alkynylguanine); 7-deaza-8-substituted guanine; hypoxanthine; 2,6-diaminopurine; 2-aminopurine; purine; 8-substituted guanine such as 8-hydroxyguanine; and 6-thioguanine. This list is meant to be exemplary and is not to be interpreted to be limiting.
Also in particular, the at least one uracil base of the G,U-containing oligoribonucleotide can be a substituted or modified uracil such as pseudouracil and 5-methyluracil.
For use in the instant invention, the nucleic acids of the invention can be synthesized de novo using any of a number of procedures well known in the art. For example, the β-cyanoethyl phosphoramidite method (Beaucage S L et al. (1981) Tetrahedron Lett 22:1859); nucleoside H-phosphonate method (Garegg et al. (1986) Tetrahedron Lett 27:4051-4; Froehler et al. (1986) Nucl Acid Res 14:5399-407; Garegg et al. (1986) Tetrahedron Lett 27:4055-8; Gaffney et al. (1988) Tetrahedron Lett 29:2619-22). These chemistries can be performed by a variety of automated nucleic acid synthesizers available in the market. These nucleic acids are referred to as synthetic nucleic acids. Alternatively, T-rich and/or TG dinucleotides can be produced on a large scale in plasmids, (see Sambrook T et al., “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor laboratory Press, New York, 1989) and separated into smaller pieces or administered whole. Nucleic acids can be prepared from existing nucleic acid sequences (e.g., genomic or cDNA) using known techniques, such as those employing restriction enzymes, exonucleases or endonucleases. Nucleic acids prepared in this manner are referred to as isolated nucleic acid. An isolated nucleic acid generally refers to a nucleic acid which is separated from components which it is normally associated with in nature. As an example, an isolated nucleic acid may be one which is separated from a cell, from a nucleus, from mitochondria or from chromatin. The term “nucleic acid” encompasses both synthetic and isolated nucleic acid.
For use in vivo, the nucleic acids may optionally be relatively resistant to degradation (e.g., are stabilized). In some embodiments only specific portions of the nucleic acids may optionally be stabilized. A “stabilized nucleic acid molecule” shall mean a nucleic acid molecule that is relatively resistant to in vivo degradation (e.g., via an exo- or endo-nuclease). Stabilization can be a function of length or secondary structure. Nucleic acids that are tens to hundreds of kbs long are relatively resistant to in vivo degradation. For shorter nucleic acids, secondary structure can stabilize and increase their effect. For example, if the 3′ end of an nucleic acid has self-complementarity to an upstream region, so that it can fold back and form a sort of stem loop structure, then the nucleic acid becomes stabilized and therefore exhibits more activity.
In certain embodiments according to this aspect of the invention, the base sequence of the RNA oligomer is at least partially self-complementary. A self-complementary sequence as used herein refers to a base sequence which, upon suitable alignment, may form intramolecular or, more typically, intermolecular basepairing between G-C, A-U, and/or G-U wobble pairs. In one embodiment the extent of self-complementarity is at least 50 percent. For example an 8-mer that is at least 50 percent self-complementary may have a sequence capable of forming 4, 5, 6, 7, or 8 G-C, A-U, and/or G-U wobble basepairs. Such basepairs may but need not necessarily involve bases located at either end of the self-complementary RNA oligomer. Where nucleic acid stabilization may be important to the RNA oligomers, it may be advantageous to “clamp” together one or both ends of a double-stranded nucleic acid, either by basepairing or by any other suitable means. The degree of self-complementarity may depend on the alignment between oligomers, and such alignment may or may not include single- or multiple-nucleoside overhangs. In other embodiments, the degree of self-complementarity is at least 60 percent, at least 70 percent, at least 80 percent, at least 90 percent, or even 100 percent. The foregoing notwithstanding, it should be noted that double-strandedness is not a requirement of the RNA oligomers of the invention.
Similar considerations apply to intermolecular basepairing between RNA oligonucleotides of different base sequence. Thus where a plurality of RNA oligomers are used together, the plurality of oligomers may, but need not, include sequences which are at least partially complementary to one another. In one embodiment the plurality of oligomers includes an oligomer having a first base sequence and an oligomer having a second base sequence, wherein the first base sequence and the second base sequence are at least 50 percent complementary. For example, as between two 8-mers that are at least 50 percent complementary, they may form 4, 5, 6, 7, or 8 G-C, A-U, and/or G-U wobble basepairs. Such basepairs may but need not necessarily involve bases located at either end of the complementary RNA oligomers. The degree of complementarity may depend on the alignment between oligomers, and such alignment may or may not include single- or multiple-nucleoside overhangs. In other embodiments, the degree of complementarity is at least 60 percent, at least 70 percent, at least 80 percent, at least 90 percent, or even 100 percent.
Alternatively, nucleic acid stabilization can be accomplished via phosphate backbone modifications. Preferred stabilized nucleic acids of the instant invention have a modified backbone. It has been demonstrated that modification of the nucleic acid backbone provides enhanced activity of the nucleic acids when administered in vivo. One type of modified backbone is a phosphate backbone modification. Inclusion in immunostimulatory nucleic acids of at least two phosphorothioate linkages at the 5′ end of the oligonucleotide and multiple (preferably five) phosphorothioate linkages at the 3′ end, can in some circumstances provide maximal activity and protect the nucleic acid from degradation by intracellular exo- and endonucleases. Other modified nucleic acids include phosphodiester-modified nucleic acids, combinations of phosphodiester and phosphorothioate nucleic acids, alkylphosphonate and arylphosphonate, alkylphosphorothioate and arylphosphorothioate, methylphosphonate, methylphosphorothioate, phosphorodithioate, p-ethoxy, morpholino, and combinations thereof. Nucleic acids having phosphorothioate linkages provide maximal activity and protect the nucleic acid from degradation by intracellular exo- and endo-nucleases. and combinations thereof. Each of these combinations and their particular effects on immune cells is discussed in more detail with respect to CpG nucleic acids in issued U.S. Pat. Nos. 6,207,646 and 6,239,116, the entire contents of which are hereby incorporated by reference. It is believed that these modified nucleic acids may show more stimulatory activity due to enhanced nuclease resistance, increased cellular uptake, increased protein binding, and/or altered intracellular localization.
Modified backbones such as phosphorothioates may be synthesized using automated techniques employing either phosphoramidate or H-phosphonate chemistries. Aryl- and alkyl-phosphonates can be made, e.g., as described in U.S. Pat. No. 4,469,863; and alkylphosphotriesters (in which the charged oxygen moiety is alkylated as described in U.S. Pat. No. 5,023,243 and European Pat. No. 092,574) can be prepared by automated solid phase synthesis using commercially available reagents. Methods for making other DNA backbone modifications and substitutions have been described. Uhlmann E et al. (1990) Chem Rev 90:544; Goodchild J (1990) Bioconjugate Chem 1:165.
Other stabilized nucleic acids include: nonionic DNA analogs, such as alkyl- and aryl-phosphates (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), phosphodiester and alkylphosphotriesters, in which the charged oxygen moiety is alkylated. Nucleic acids which contain diol, such as tetraethyleneglycol or hexaethyleneglycol, at either or both termini have also been shown to be substantially resistant to nuclease degradation.
Another class of backbone modifications include 2′-O-methylribonucleosides (2′-OMe). These types of substitutions are described extensively in the prior art and in particular with respect to their immunostimulating properties in Zhao et al. (1999) Bioorg Med Chem Lett 9:24:3453-8. Zhao et al. describes methods of preparing 2′-OMe modifications to nucleic acids.
The immunostimulatory G,U-containing RNA oligomers of the invention are typically about 5 to about 40 nucleotides long. Thus in certain distinct embodiments, the G,U-containing RNA oligomer can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides long. In one embodiment the G,U-containing RNA oligomer can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides long. In one embodiment the G,U-containing RNA oligomer can be 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides long. In one embodiment the G,U-containing RNA oligomer can be 8, 9, 10, 11, or 12 nucleotides long.
For example, RNA oligomers with the following base sequences have been discovered to be useful in the compositions and practice of the invention: 5′-GUGUUUAC-3′; 5′-GUAGGCAC-3′; 5′-CUAGGCAC-3′; 5′-CUCGGCAC-3′; and 5′-GUGUUUAC-3′ in combination with 5′-GUAGGCAC-3′.
Because the immunostimulatory effects of the G,U-containing RNA oligomers of the invention have been discovered to be MyD88-dependent, it is the belief of the inventors that the immunostimulatory G,U-containing RNA oligomers of the invention may interact with at least one TLR as a step in exerting their immunostimulatory effect. The immunostimulatory G,U-containing RNA oligomers of the invention may thus represent or mimic at least portions of natural ligands for the at least one TLR. Such natural ligands may include ribosomal RNA, either prokaryotic or eukaryotic, as well as certain viral RNAs. The TLR or TLRs may be TLR8, TLR7, or some yet-to-be defined TLR. Natural ligands for TLR1, TLR7, TLR8, and TLR10 have not previously been described.
The immunostimulatory RNA molecules of the invention have been discovered to occur in nature in all types of RNA, usually in association with highly conserved sequence or key structural feature. In one example, immunostimulatory RNA has been discovered to occur in the context of an internal ribosome entry site (IRES).
An IRES is a minimal cis-acting RNA element contained within a complex structural feature in the 5′ untranslated region (5′ UTR) of viral RNA and other mRNAs that regulates the initiation of translation of the viral genome in a cap-independent manner. Hellen C U et al. (2001) Genes Dev 15:1593-1612. Cap-independent initiation of viral RNA translation was first observed in picornaviruses. Jackson R J et al. (1990) Trends Biochem Sci 15:477-83; Jackson R J et al. (1995) RNA 1:985-1000.
In most eukaryotic cells, mRNA translation initiation commences with recruitment of the cap binding complex eukaryotic initiation factor (eIF)4F, composed of eIF4E (cap binding protein), eIF4A, and eIF4G, to the 5′ capped end of the mRNA. The 40S ribosomal subunit, carrying eIF3, and the ternary initiator complex tRNA-eIF2-GTP are then recruited to the 5′ end of the mRNA through interaction between eIF3 and eIF4G. The 40S subunit then scans the mRNA in a 5′ to 3′ direction until it encounters an appropriate start codon, whereupon the anticodon of initiator methionine-tRNA is engaged, the 60S subunit joins to form an 80S ribosome, and translation commences.
Thus the significance of an IRES, at least in the context of a virus, is believed to be the ability of the IRES to confer a selective advantage to the virus over usual cap-dependent translation in the cell.
The following viruses have been reported to have IRES elements in their genome: all picornaviruses; bovine viral diarrhea virus; classic swine fever virus; cricket paralysis virus; encephalomyocarditis virus; foot-and-mouth disease virus; Friend murine leukemia virus gag mRNA; HCV; human immunodeficiency virus env mRNA; Kaposi's sarcoma-associated herpesvirus; Moloney murine leukemia virus gag mRNA; Plautia stali intestine virus; poliovirus; rhinovirus; Rhopalosiphum padi virus; and Rous sarcoma virus. Hellen C U et al. (2001) Genes Dev 15:1593-1612. This list is not intended to be limiting.
The viral proteins of hepatitis C virus (HCV) are translated from a 9.5 kb single-stranded positive sense RNA which is flanked by 5′ and 3′ UTRs. The highly conserved 5′ UTR includes an IRES present in nt 40-370. Reynolds J E et al. (1996) RNA 2:867-78. The HCV 5′ UTR is believed to have four major structural domains (1-IV), of which domains II and III have subdomains. Subdomain IIId includes a 27 nt stem-loop (nt 253-279) that on the basis of in vivo mutational studies has been reported to be critical in HCV IRES-mediated translation. Kieft J S et al. (1999) J Mol Biol 292:513-29; Klinck R et al. (2000) RNA 6:1423-31. The sequence of the IIId 27-mer is provided by 5′-GCCGAGUAGUGUUGGGUCGCGAAAGGC-3′ (SEQ ID NO:4), wherein the UUGGGU forms the terminal loop. The stem-loop structure is reported to include a number of non-Watson-Crick base pairs, typical of other RNAs, including wobble U∘G, U∘A, G∘A, and A∘A base pairs.
As another example, the immunostimulatory RNA sequences of the invention have been discovered to occur in G,U-rich sequence near the 5′ end of the viral RNA of human immunodeficiency virus type 1 (HIV-1) that is crucial to efficient viral RNA packaging. Russell R S et al. (2002) Virology 303:152-63. Specifically, two key G,U-rich sequences within U5, namely 5′-GUAGUGUGUG-3′ (SEQ ID NO:2) and 5′-GUCUGUUGUGUG-3′ (SEQ ID NO:3), corresponding to nt 99-108 and 112-123 of strain BH10, respectively, have been found according to the present invention to be highly immunostimulatory (see Example 11 below). It will be noted that SEQ ID NO:2 includes both GUAGU and GUGUG, and SEQ ID NO:3 includes GUGUG.
As yet another example, the immunostimulatory RNA sequences of the invention have been found to occur in 5S ribosomal RNA loop E of a large number of species of bacteria.
TLR8 and TLR7 show high sequence homology to TLR9 (FIG. 8). TLR9 is the CpG-DNA receptor and transduces immunostimulatory signals. Two DNA binding motifs have been described in TLR9 (U.S. patent application Ser. No. 09/954,987) that are also present in TLR8 and TLR7 with some modifications (FIG. 9). Despite this similarity, however, TLR7 and TLR8 do not bind CpG-DNA.
It has been discovered according to the present invention that guanosine, particularly guanosine in combination with uracil, and certain guanosine-containing nucleic acids and derivatives thereof, are natural ligands of TLR8. It has been discovered according to the present invention that RNA, oxidized RNA, G,U-rich nucleic acids, and at least partially double-stranded nucleic acid molecules having at least one G-U base pair are TLR8 ligands. In certain preferred embodiments involving guanosine, guanosine derivatives, and G,U-rich nucleic acids, guanosine is the ribonucleoside. Nucleic acid molecules containing GUU, GUG, GGU, GGG, UGG, UGU, UUG, UUU, multiples and any combinations thereof are believed to be TLR8 ligands. In some embodiments the TLR8 ligand is a G,U-rich oligonucleotide that includes a hexamer sequence (UUGUGG)n, (UGGUUG)n, (GUGUGU)n, or (GGGUUU)n where n is an integer from 1 to 8, and preferably n is at least 3. In addition, it has also been discovered according to the present invention that mixtures of ribonucleoside vanadyl complexes (i.e., mixtures of adenine, cytosine, guanosine, and uracil ribonucleoside vanadyl complexes), and guanosine ribonucleoside vanadyl complexes alone, are TLR8 ligands. In addition, it has been discovered according the present invention that certain imidazoquinolines, including resiquimod and imiquimod, are TLR8 ligands.
It has also been discovered according to the present invention that guanosine, and certain guanosine-containing nucleic acids and derivatives thereof, are natural ligands of TLR7. It has been discovered according to the present invention that RNA, oxidized RNA, G-rich nucleic acids, and at least partially double-stranded nucleic acid molecules that are rich in G content are TLR7 ligands. In certain preferred embodiments involving guanosine, guanosine derivatives, and G-rich nucleic acids, guanosine is the ribonucleoside. In addition, it has also been discovered according to the present invention that mixtures of ribonucleoside vanadyl complexes (i.e., mixtures of adenine, cytosine, guanosine, and uracil ribonucleoside vanadyl complexes), and guanosine ribonucleoside vanadyl complexes alone, are TLR7 ligands. In addition, it has been discovered according the present invention that 7-allyl-8-oxoguanosine (loxoribine) is a TLR7 ligand.
In addition to having diverse ligands, the various TLRs are believed to be differentially expressed in various tissues and on various types of immune cells. For example, human TLR7 has been reported to be expressed in placenta, lung, spleen, lymph nodes, tonsil and on plasmacytoid precursor dendritic cells (pDCs). Chuang T-H et al. (2000) Eur Cytokine Netw 11:372-8); Kadowaki N et al. (2001) J Exp Med 194:863-9. Human TLR8 has been reported to be expressed in lung, peripheral blood leukocytes (PBL), placenta, spleen, lymph nodes, and on monocytes. Kadowaki N et al. (2001) J Exp Med 194:863-9; Chuang T-H et al. (2000) Eur Cytokine Netw 11:372-8. Human TLR9 is reportedly expressed in spleen, lymph nodes, bone marrow, PBL, and on pDCs, B cells, and CD123+DCs. Kadowaki N et al. (2001) J Exp Med 194:863-9; Bauer S et al. (2001) Proc Natl Acad Sci USA 98:9237-42; Chuang T-H et al. (2000) Eur Cytokine Netw 11:372-8.
Guanosine derivatives have previously been described as B-cell and NK cell activators, but their receptors and mechanism of action were not understood. Goodman M G et al. (1994) J Pharm Exp Ther 274:1552-57; Reitz A B et al. (1994) J Med Chem 37:3561-78. Such guanosine derivatives include, but are not limited to, 8-bromoguanosine, 8-oxoguanosine, 8-mercaptoguanosine, and 7-allyl-8-oxoguanosine (loxoribine).
Imidazoquinolines are synthetic small molecule immune response modifiers thought to induce expression of several cytokines including interferons (e.g., IFN-α and IFN-γ), tumor necrosis factor alpha (TNF-α) and some interleukins (e.g., IL-1, IL-6 and IL-12). Imidazoquinolines are capable of stimulating a Th1 immune response, as evidenced in part by their ability to induce increases in IgG2a levels. Imidazoquinoline agents reportedly are also capable of inhibiting production of Th2 cytokines such as IL-4, IL-5, and IL-13. Some of the cytokines induced by imidazoquinolines are produced by macrophages and dendritic cells. Some species of imidazoquinolines have been reported to increase NK cell lytic activity and to stimulate B-cell proliferation and differentiation, thereby inducing antibody production and secretion.
As used herein, an imidazoquinoline agent includes imidazoquinoline amines, imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine amines, and 1,2 bridged imidazoquinoline amines. These compounds have been described in U.S. Pat. Nos. 4,689,338, 4,929,624, 5,238,944, 5,266,575, 5,268,376, 5,346,905, 5,352,784, 5,389,640, 5,395,937, 5,494,916, 5,482,936, 5,525,612, 6,039,969 and 6,110,929. Particular species of imidazoquinoline agents include 4-amino-α,α-dimethyl-2-ethoxymethyl-1H-imidazo[4,5-c]quinoline-1-ethanol (resiquimod or R-848 or S-28463; PCT/US01/28764, WO 02/22125); and 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-4-amine (imiquimod or R-837 or S-26308). Imiquimod is currently used in the topical treatment of warts such as genital and anal warts and has also been tested in the topical treatment of basal cell carcinoma.
Nucleotide and amino acid sequences of human and murine TLR3 are known. See, for example, GenBank Accession Nos. U88879, NM_003265, NM_126166, AF355152; and AAC34134, NP_003256, NP_569054, AAK26117. Human TLR3 is reported to be 904 amino acids long and to have a sequence provided in SEQ ID NO:20. A corresponding nucleotide sequence is provided as SEQ ID NO:21. Murine TLR3 is reported to be 905 amino acids long and to have a sequence as provided in SEQ ID NO:22. A corresponding nucleotide sequence is provided as SEQ ID NO:23. TLR3 polypeptide includes an extracellular domain having leucine-rich repeat region, a transmembrane domain, and an intracellular domain that includes a TIR domain.
As used herein a “TLR3 polypeptide” refers to a polypeptide including a full-length TLR3 according to one of the sequences above, orthologs, allelic variants, SNPs, variants incorporating conservative amino acid substitutions, TLR3 fusion proteins, and functional fragments of any of the foregoing. Preferred embodiments include human TLR3 polypeptides having at least 65 percent sequence identity, more preferably at least 80 percent sequence identity, even more preferably with at least 90 percent sequence identity, and most preferably with at least 95 percent sequence identity with the human TLR3 amino acid sequence of SEQ ID NO:20. Preferred embodiments also include murine TLR3 polypeptides having at least 65 percent sequence identity, more preferably at least 80 percent sequence identity, even more preferably with at least 90 percent sequence identity, and most preferably with at least 95 percent sequence identity with the murine TLR3 amino acid sequence of SEQ ID NO:22.
As used herein “TLR3 signaling” refers to an ability of a TLR3 polypeptide to activate the TLR/IL-1R (TIR) signaling pathway, also referred to herein as the TLR signal transduction pathway. Changes in TLR3 activity can be measured by assays such as those disclosed herein, including expression of genes under control of κB-sensitive promoters and enhancers. Such naturally occurring genes include the genes encoding IL-1β, IL-6, IL-8, the p40 subunit of interleukin 12 (IL-12 p40), and the costimulatory molecules CD80 and CD86. Other genes can be placed under the control of such regulatory elements (see below) and thus serve to report the level of TLR3 signaling. Additional nucleotide sequence can be added to SEQ ID NO:21 or SEQ ID NO:23, preferably to the 5′ or the 3′ end of the open reading frame of SEQ ID NO:21, to yield a nucleotide sequence encoding a chimeric polypeptide that includes a detectable or reporter moiety, e.g., FLAG, luciferase (luc), green fluorescent protein (GFP), and others known by those skilled in the art.
SEQ ID NO: 20
Human TLR3 amino acid
MRQTLPCIYF WGGLLPFGML CASSTTKCTV SHEVADCSHL KLTQVPDDLP TNITVLNLTH
60
NQLRRLPAAN FTRYSQLTSL DVGFNTISKL EPELCQKLPM LKVLNLQHNE LSQLSDKTFA
120
FCTNLTELHL MSNSIQKIKN NPFVKQKNLI TLDLSHNGLS STKLGTQVQL ENLQELLLSN
180
NKIQALKSEE LDIFANSSLK KLELSSNQIK EFSPGCFHAI GRLFGLFLNN VQLGPSLTEK
240
LCLELANTSI RNLSLSNSQL STTSNTTFLG LKWTNLTMLD LSYNNLNVVG NDSFAWLPQL
300
EYFFLEYNNI QHLFSHSLHG LFNVRYLNLK RSFTKQSISL ASLPKIDDFS FQWLKCLEHL
360
NMEDNDIPGI KSNMFTGLIN LKYLSLSNSF TSLRTLTNET FVSLAHSPLH ILNLTKNKIS
420
KIESDAFSWL GHLEVLDLGL NEIGQELTGQ EWRGLENIFE IYLSYNKYLQ LTRNSFALVP
480
SLQRLMLRRV ALKNVDSSPS PFQPLRNLTI LDLSNNNIAN INDDMLEGLE KLEILDLQHN
540
NLARLWKHAN PGGPIYFLKG LSHLHILNLE SNGFDEIPVE VFKDLFELKI IDLGLNNLNT
600
LPASVFNNQV SLKSLNLQKN LITSVEKKVF GPAFRNLTEL DMRFNPFDCT CESIAWFVNW
660
INETHTNIPE LSSHYLCNTP PHYHGFPVRL FDTSSCKDSA PFELFFMINT SILLIFIFIV
720
LLIHFEGWRI SFYWNVSVHR VLGFKEIDRQ TEQFEYAAYI IHAYKDKDWV WEHFSSMEKE
780
DQSLKFCLEE RDFEAGVFEL EAIVNSIKRS RKIIFVITHH LLKDPLCKRF KVHHAVQQAI
840
EQNLDSIILV FLEEIPDYKL NHALCLRRGM FKSHCILNWP VQKERIGAFR HKLQVALGSK
900
NSVH
904
SEQ ID NO: 21
Human TLR3 nucleotide
cactttcgag agtgccgtct atttgccaca cacttccctg atgaaatgtc tggatttgga
60
ctaaagaaaa aaggaaaggc tagcagtcat ccaacagaat catgagacag actttgcctt
120
gtatctactt ttgggggggc cttttgccct ttgggatgct gtgtgcatcc tccaccacca
180
agtgcactgt tagccatgaa gttgctgact gcagccacct gaagttgact caggtacccg
240
atgatctacc cacaaacata acagtgttga accttaccca taatcaactc agaagattac
300
cagccgccaa cttcacaagg tatagccagc taactagctt ggatgtagga tttaacacca
360
tctcaaaact ggagccagaa ttgtgccaga aacttcccat gttaaaagtt ttgaacctcc
420
agcacaatga gctatctcaa ctttctgata aaacctttgc cttctgcacg aatttgactg
480
aactccatct catgtccaac tcaatccaga aaattaaaaa taatcccttt gtcaagcaga
540
agaatttaat cacattagat ctgtctcata atggcttgtc atctacaaaa ttaggaactc
600
aggttcagct ggaaaatctc caagagcttc tattatcaaa caataaaatt caagcgctaa
660
aaagtgaaga actggatatc tttgccaatt catctttaaa aaaattagag ttgtcatcga
720
atcaaattaa agagttttct ccagggtgtt ttcacgcaat tggaagatta tttggcctct
780
ttctgaacaa tgtccagctg ggtcccagcc ttacagagaa gctatgtttg gaattagcaa
840
acacaagcat tcggaatctg tctctgagta acagccagct gtccaccacc agcaatacaa
900
ctttcttggg actaaagtgg acaaatctca ctatgctcga tctttcctac aacaacttaa
960
atgtggttgg taacgattcc tttgcttggc ttccacaact agaatatttc ttcctagagt
1020
ataataatat acagcatttg ttttctcact ctttgcacgg gcttttcaat gtgaggtacc
1080
tgaatttgaa acggtctttt actaaacaaa gtatttccct tgcctcactc cccaagattg
1140
atgatttttc ttttcagtgg ctaaaatgtt tggagcacct taacatggaa gataatgata
1200
ttccaggcat aaaaagcaat atgttcacag gattgataaa cctgaaatac ttaagtctat
1260
ccaactcctt tacaagtttg cgaactttga caaatgaaac atttgtatca cttgctcatt
1320
ctcccttaca catactcaac ctaaccaaga ataaaatctc aaaaatagag agtgatgctt
1380
tctcttggtt gggccaccta gaagtacttg acctgggcct taatgaaatt gggcaagaac
1440
tcacaggcca ggaatggaga ggtctagaaa atattttcga aatctatctt tcctacaaca
1500
agtacctgca gctgactagg aactcctttg ccttggtccc aagccttcaa cgactgatgc
1560
tccgaagggt ggcccttaaa aatgtggata gctctccttc accattccag cctcttcgta
1620
acttgaccat tctggatcta agcaacaaca acatagccaa cataaatgat gacatgttgg
1680
agggtcttga gaaactagaa attctcgatt tgcagcataa caacttagca cggctctgga
1740
aacacgcaaa ccctggtggt cccatttatt tcctaaaggg tctgtctcac ctccacatcc
1800
ttaacttgga gtccaacggc tttgacgaga tcccagttga ggtcttcaag gatttatttg
1860
aactaaagat catcgattta ggattgaata atttaaacac acttccagca tctgtcttta
1920
ataatcaggt gtctctaaag tcattgaacc ttcagaagaa tctcataaca tccgttgaga
1980
agaaggtttt cgggccagct ttcaggaacc tgactgagtt agatatgcgc tttaatccct
2040
ttgattgcac gtgtgaaagt attgcctggt ttgttaattg gattaacgag acccatacca
2100
acatccctga gctgtcaagc cactaccttt gcaacactcc acctcactat catgggttcc
2160
cagtgagact ttttgataca tcatcttgca aagacagtgc cccctttgaa ctctttttca
2220
tgatcaatac cagtatcctg ttgattttta tctttattgt acttctcatc cactttgagg
2280
gctggaggat atctttttat tggaatgttt cagtacatcg agttcttggt ttcaaagaaa
2340
tagacagaca gacagaacag tttgaatatg cagcatatat aattcatgcc tataaagata
2400
aggattgggt ctgggaacat ttctcttcaa tggaaaagga agaccaatct ctcaaatttt
2460
gtctggaaga aagggacttt gaggcgggtg tttttgaact agaagcaatt gttaacagca
2520
tcaaaagaag cagaaaaatt atttttgtta taacacacca tctattaaaa gacccattat
2580
gcaaaagatt caaggtacat catgcagttc aacaagctat tgaacaaaat ctggattcca
2640
ttatattggt tttccttgag gagattccag attataaact gaaccatgca ctctgtttgc
2700
gaagaggaat gtttaaatct cactgcatct tgaactggcc agttcagaaa gaacggatag
2760
gtgcctttcg tcataaattg caagtagcac ttggatccaa aaactctgta cattaaattt
2820
atttaaatat tcaattagca aaggagaaac tttctcaatt taaaaagttc tatggcaaat
2880
ttaagttttc cataaaggtg ttataatttg tttattcata tttgtaaatg attatattct
2940
atcacaatta catctcttct aggaaaatgt gtctccttat ttcaggccta tttttgacaa
3000
ttgacttaat tttacccaaa ataaaacata taagcacgta aaaaaaaaaa aaaaaaa
3057
SEQ ID NO: 22
Murine TLR3 amino acid
MKGCSSYLMY SFGGLLSLWI LLVSSTNQCT VRYNVADCSH LKLTHIPDDL PSNITVLNLT
60
HNQLRRLPPT NFTRYSQLAI LDAGFNSISK LEPELCQILP LLKVLNLQHN ELSQISDQTF
120
VFCTNLTELD LMSNSIHKIK SNPFKNQKNL IKLDLSHNGL SSTKLGTGVQ LENLQELLLA
180
KNKILALRSE ELEFLGNSSL RKLDLSSNPL KEFSPGCFQT IGKLFALLLN NAQLNPHLTE
240
KLCWELSNTS IQNLSLANNQ LLATSESTFS GLKWTNLTQL DLSYNNLHDV GNGSFSYLPS
300
LRYLSLEYNN IQRLSPRSFY GLSNLRYLSL KRAFTKQSVS LASHPNIDDF SFQWLKYLEY
360
LNMDDNNIPS TKSNTFTGLV SLKYLSLSKT FTSLQTLTNE TFVSLAHSPL LTLNLTKNHI
420
SKIANGTFSW LGQLRILDLG LNEIEQKLSG QEWRGLRNIF EIYLSYNKYL QLSTSSFALV
480
PSLQRLMLRR VALKNVDISP SPFRPLRNLT ILDLSNNNIA NINEDLLEGL ENLEILDFQH
540
NNLARLWKRA NPGGPVNFLK GLSHLHILNL ESNGLDEIPV GVFKNLFELK SINLGLNNLN
600
KLEPFIFDDQ TSLRSLNLQK NLITSVEKDV FGPPFQNLNS LDMRFNPFDC TCESISWFVN
660
WINQTHTNIF ELSTHYLCNT PHHYYGFPLK LFDTSSCKDS APFELLFIIS TSMLLVFILV
720
VLLIHIEGWR ISFYWNVSVH RILGFKEIDT QAEQFEYTAY IIHAHKDRDW VWEHFSPMEE
780
QDQSLKFCLE ERDFEAGVLG LEAIVNSIKR SRKIIFVITH HLLKDPLCRR FKVHHAVQQA
840
IEQNLDSIIL IFLQNIPDYK LNHALCLRRG MFKSHCILNW PVQKERINAF HHKLQVALGS
900
RNSAH
904
SEQ ID NO: 23
Murine TLR3 nucleotide
tagaatatga tacagggatt gcacccataa tctgggctga atcatgaaag ggtgttcctc
60
ttatctaatg tactcctttg ggggactttt gtccctatgg attcttctgg tgtcttccac
120
aaaccaatgc actgtgagat acaacgtagc tgactgcagc catttgaagc taacacacat
180
acctgatgat cttccctcta acataacagt gttgaatctt actcacaacc aactcagaag
240
attaccacct accaacttta caagatacag ccaacttgct atcttggatg caggatttaa
300
ctccatttca aaactggagc cagaactgtg ccaaatactc cctttgttga aagtattgaa
360
cctgcaacat aatgagctct ctcagatttc tgatcaaacc tttgtcttct gcacgaacct
420
gacagaactc gatctaatgt ctaactcaat acacaaaatt aaaagcaacc ctttcaaaaa
480
ccagaagaat ctaatcaaat tagatttgtc tcataatggt ttatcatcta caaagttggg
540
aacgggggtc caactggaga acctccaaga actgctctta gcaaaaaata aaatccttgc
600
gttgcgaagt gaagaacttg agtttcttgg caattcttct ttacgaaagt tggacttgtc
660
atcaaatcca cttaaagagt tctccccggg gtgtttccag acaattggca agttattcgc
720
cctcctcttg aacaacgccc aactgaaccc ccacctcaca gagaagcttt gctgggaact
780
ttcaaacaca agcatccaga atctctctct ggctaacaac cagctgctgg ccaccagcga
840
gagcactttc tctgggctga agtggacaaa tctcacccag ctcgatcttt cctacaacaa
900
cctccatgat gtcggcaacg gttccttctc ctatctccca agcctgaggt atctgtctct
960
ggagtacaac aatatacagc gtctgtcccc tcgctctttt tatggactct ccaacctgag
1020
gtacctgagt ttgaagcgag catttactaa gcaaagtgtt tcacttgctt cacatcccaa
1080
cattgacgat ttttcctttc aatggttaaa atatttggaa tatctcaaca tggatgacaa
1140
taatattcca agtaccaaaa gcaatacctt cacgggattg gtgagtctga agtacctaag
1200
tctttccaaa actttcacaa gtttgcaaac tttaacaaat gaaacatttg tgtcacttgc
1260
tcattctccc ttgctcactc tcaacttaac gaaaaatcac atctcaaaaa tagcaaatgg
1320
tactttctct tggttaggcc aactcaggat acttgatctc ggccttaatg aaattgaaca
1380
aaaactcagc ggccaggaat ggagaggtct gagaaatata tttgagatct acctatccta
1440
taacaaatac ctccaactgt ctaccagttc ctttgcattg gtccccagcc ttcaaagact
1500
gatgctcagg agggtggccc ttaaaaatgt ggatatctcc ccttcacctt tccgccctct
1560
tcgtaacttg accattctgg acttaagcaa caacaacata gccaacataa atgaggactt
1620
gctggagggt cttgagaatc tagaaatcct ggattttcag cacaataact tagccaggct
1680
ctggaaacgc gcaaaccccg gtggtcccgt taatttcctg aaggggctgt ctcacctcca
1740
catcttgaat ttagagtcca acggcttaga tgaaatccca gtcggggttt tcaagaactt
1800
attcgaacta aagagcatca atctaggact gaataactta aacaaacttg aaccattcat
1860
ttttgatgac cagacatctc taaggtcact gaacctccag aagaacctca taacatctgt
1920
tgagaaggat gttttcgggc cgccttttca aaacctgaac agtttagata tgcgcttcaa
1980
tccgttcgac tgcacgtgtg aaagtatttc ctggtttgtt aactggatca accagaccca
2040
cactaatatc tttgagctgt ccactcacta cctctgtaac actccacatc attattatgg
2100
cttccccctg aagcttttcg atacatcatc ctgtaaagac agcgccccct ttgaactcct
2160
cttcataatc agcaccagta tgctcctggt ttttatactt gtggtactgc tcattcacat
2220
cgagggctgg aggatctctt tttactggaa tgtttcagtg catcggattc ttggtttcaa
2280
ggaaatagac acacaggctg agcagtttga atatacagcc tacataattc atgcccataa
2340
agacagagac tgggtctggg aacatttctc cccaatggaa gaacaagacc aatctctcaa
2400
attttgccta gaagaaaggg actttgaagc aggcgtcctt ggacttgaag caattgttaa
2460
tagcatcaaa agaagccgaa aaatcatttt cgttatcaca caccatttat taaaagaccc
2520
tctgtgcaga agattcaagg tacatcacgc agttcagcaa gctattgagc aaaatctgga
2580
ttcaattata ctgatttttc tccagaatat tccagattat aaactaaacc atgcactctg
2640
tttgcgaaga ggaatgttta aatctcattg catcttgaac tggccagttc agaaagaacg
2700
gataaatgcc tttcatcata aattgcaagt agcacttgga tctcggaatt cagcacatta
2760
aactcatttg aagatttgga gtcggtaaag ggatagatcc aatttataaa ggtccatcat
2820
gaatctaagt tttacttgaa agttttgtat atttatttat atgtatagat gatgatatta
2880
catcacaatc caatctcagt tttgaaatat ttcggcttat ttcattgaca tctggtttat
2940
tcactccaaa taaacacatg ggcagttaaa aacatcctct attaatagat tacccattaa
3000
ttcttgaggt gtatcacagc tttaaagggt tttaaatatt tttatataaa taagactgag
3060
agttttataa atgtaatttt ttaaaactcg agtcttactg tgtagctcag aaaggcctgg
3120
aaattaatat attagagagt catgtcttga acttatttat ctctgcctcc ctctgtctcc
3180
agagtgttgc ttttaagggc atgtagcacc acacccagct atgtacgtgt gggattttat
3240
aatgctcatt tttgagacgt ttatagaata aaagataatt gcttttatgg tataaggcta
3300
cttgaggtaa
3310
Nucleotide and amino acid sequences of human and murine TLR7 are known. See, for example, GenBank Accession Nos. AF240467, AF245702, NM_016562, AF334942, NM_133211; and AAF60188, AAF78035, NP_057646, AAL73191, AAL73192. Human TLR7 is reported to be 1049 amino acids long and to have a sequence provided in SEQ ID NO:24. A corresponding nucleotide sequence is provided as SEQ ID NO:25. Murine TLR7 is reported to be 1050 amino acids long and to have a sequence as provided in SEQ ID NO:26. A corresponding nucleotide sequence is provided as SEQ ID NO:27. TLR7 polypeptide includes an extracellular domain having leucine-rich repeat region, a transmembrane domain, and an intracellular domain that includes a TIR domain.
As used herein a “TLR7 polypeptide” refers to a polypeptide including a full-length TLR7 according to one of the sequences above, orthologs, allelic variants, SNPs, variants incorporating conservative amino acid substitutions, TLR7 fusion proteins, and functional fragments of any of the foregoing. Preferred embodiments include human TLR7 polypeptides having at least 65 percent sequence identity, more preferably at least 80 percent sequence identity, even more preferably with at least 90 percent sequence identity, and most preferably with at least 95 percent sequence identity with the human TLR7 amino acid sequence of SEQ ID NO:24. Preferred embodiments also include murine TLR7 polypeptides having at least 65 percent sequence identity, more preferably at least 80 percent sequence identity, even more preferably with at least 90 percent sequence identity, and most preferably with at least 95 percent sequence identity with the murine TLR7 amino acid sequence of SEQ ID NO:26.
As used herein “TLR7 signaling” refers to an ability of a TLR7 polypeptide to activate the TLR/IL-1R (TIR) signaling pathway, also referred to herein as the TLR signal transduction pathway. Changes in TLR7 activity can be measured by assays such as those disclosed herein, including expression of genes under control of κB-sensitive promoters and enhancers. Such naturally occurring genes include the genes encoding IL-1β, IL-6, IL-8, the p40 subunit of interleukin 12 (IL-12 p40), and the costimulatory molecules CD80 and CD86. Other genes can be placed under the control of such regulatory elements (see below) and thus serve to report the level of TLR7 signaling. Additional nucleotide sequence can be added to SEQ ID NO:25 or SEQ ID NO:27, preferably to the 5′ or the 3′ end of the open reading frame of SEQ ID NO:25, to yield a nucleotide sequence encoding a chimeric polypeptide that includes a detectable or reporter moiety, e.g., FLAG, luciferase (luc), green fluorescent protein (GFP), and others known by those skilled in the art.
SEQ ID NO: 24
Human TLR7 amino acid
MVFPMWTLKR QILILFNIIL ISKLLGARWF PKTLPCDVTL DVPKNHVIVD CTDKHLTEIP
60
GGIPTNTTNL TLTINHIPDI SPASFHRLDH LVEIDFRCNC VPIPLGSKNN MCIKRLQIKP
120
RSFSGLTYLK SLYLDGNQLL EIPQGLPPSL QLLSLEANNI FSIRKENLTE LANIEILYLG
180
QNCYYRNPCY VSYSIEKDAF LNLTKLKVLS LKDNNVTAVP TVLPSTLTEL YLYNNMIAKI
240
QEDDFNNLNQ LQILDLSGNC PRCYNAPFPC APCKNNSPLQ IPVNAFDALT ELKVLRLHSN
300
SLQHVPPRWF KNINKLQELD LSQNFLAKEI GDAKFLHFLP SLIQLDLSFN FELQVYRASM
360
NLSQAFSSLK SLKILRIRGY VFKELKSFNL SPLHNLQNLE VLDLGTNFIK IANLSMFKQF
420
KRLKVIDLSV NKISPSGDSS EVGFCSNART SVESYEPQVL EQLHYFRYDK YARSCRFKNK
480
EASFMSVNES CYKYGQTLDL SKNSIFFVKS SDFQHLSFLK CLNLSGNLIS QTLNGSEFQP
540
LAELRYLDFS NNRLDLLHST AFEELHKLEV LDISSNSHYF QSEGITHMLN FTKNLKVLQK
600
LMMNDNDISS STSRTMESES LRTLEFRGNH LDVLWREGDN RYLQLFKNLL KLEELDISKN
660
SLSFLPSGVF DGMPPNLKNL SLAKNGLKSF SWKKLQCLKN LETLDLSHNQ LTTVPERLSN
720
CSRSLKNLIL KNNQIRSLTK YFLQDAFQLR YLDLSSNKIQ MIQKTSFPEN VLNNLKMLLL
780
HHNRFLCTCD AVWFVWWVNH TEVTIPYLAT DVTCVGPGAH KGQSVISLDL YTCELDLTNL
840
ILFSLSISVS LFLMVMMTAS HLYFWDVWYI YHFCKAKIKG YQRLISPDCC YDAFIVYDTK
900
DPAVTEWVLA ELVAKLEDPR EKHFNLCLEE RDWLPGQPVL ENLSQSIQLS KKTVFVMTDK
960
YAKTENFKIA FYLSHQRLMD EKVDVIILIF LEKPFQKSKF LQLRKRLCGS SVLEWPTNPQ
1020
AHPYFWQCLK NALATDNHVA YSQVFKETV
1049
SEQ ID NO: 25
Human TLR7 nucleotide
actccagata taggatcact ccatgccatc aagaaagttg atgctattgg gcccatctca
60
agctgatctt ggcacctctc atgctctgct ctcttcaacc agacctctac attccatttt
120
ggaagaagac taaaaatggt gtttccaatg tggacactga agagacaaat tcttatcctt
180
tttaacataa tcctaatttc caaactcctt ggggctagat ggtttcctaa aactctgccc
240
tgtgatgtca ctctggatgt tccaaagaac catgtgatcg tggactgcac agacaagcat
300
ttgacagaaa ttcctggagg tattcccacg aacaccacga acctcaccct caccattaac
360
cacataccag acatctcccc agcgtccttt cacagactgg accatctggt agagatcgat
420
ttcagatgca actgtgtacc tattccactg gggtcaaaaa acaacatgtg catcaagagg
480
ctgcagatta aacccagaag ctttagtgga ctcacttatt taaaatccct ttacctggat
540
ggaaaccagc tactagagat accgcagggc ctcccgccta gcttacagct tctcagcctt
600
gaggccaaca acatcttttc catcagaaaa gagaatctaa cagaactggc caacatagaa
660
atactctacc tgggccaaaa ctgttattat cgaaatcctt gttatgtttc atattcaata
720
gagaaagatg ccttcctaaa cttgacaaag ttaaaagtgc tctccctgaa agataacaat
780
gtcacagccg tccctactgt tttgccatct actttaacag aactatatct ctacaacaac
840
atgattgcaa aaatccaaga agatgatttt aataacctca accaattaca aattcttgac
900
ctaagtggaa attgccctcg ttgttataat gccccatttc cttgtgcgcc gtgtaaaaat
960
aattctcccc tacagatccc tgtaaatgct tttgatgcgc tgacagaatt aaaagtttta
1020
cgtctacaca gtaactctct tcagcatgtg cccccaagat ggtttaagaa catcaacaaa
1080
ctccaggaac tggatctgtc ccaaaacttc ttggccaaag aaattgggga tgctaaattt
1140
ctgcattttc tccccagcct catccaattg gatctgtctt tcaattttga acttcaggtc
1200
tatcgtgcat ctatgaatct atcacaagca ttttcttcac tgaaaagcct gaaaattctg
1260
cggatcagag gatatgtctt taaagagttg aaaagcttta acctctcgcc attacataat
1320
cttcaaaatc ttgaagttct tgatcttggc actaacttta taaaaattgc taacctcagc
1380
atgtttaaac aatttaaaag actgaaagtc atagatcttt cagtgaataa aatatcacct
1440
tcaggagatt caagtgaagt tggcttctgc tcaaatgcca gaacttctgt agaaagttat
1500
gaaccccagg tcctggaaca attacattat ttcagatatg ataagtatgc aaggagttgc
1560
agattcaaaa acaaagaggc ttctttcatg tctgttaatg aaagctgcta caagtatggg
1620
cagaccttgg atctaagtaa aaatagtata ttttttgtca agtcctctga ttttcagcat
1680
ctttctttcc tcaaatgcct gaatctgtca ggaaatctca ttagccaaac tcttaatggc
1740
agtgaattcc aacctttagc agagctgaga tatttggact tctccaacaa ccggcttgat
1800
ttactccatt caacagcatt tgaagagctt cacaaactgg aagttctgga tataagcagt
1860
aatagccatt attttcaatc agaaggaatt actcatatgc taaactttac caagaaccta
1920
aaggttctgc agaaactgat gatgaacgac aatgacatct cttcctccac cagcaggacc
1980
atggagagtg agtctcttag aactctggaa ttcagaggaa atcacttaga tgttttatgg
2040
agagaaggtg ataacagata cttacaatta ttcaagaatc tgctaaaatt agaggaatta
2100
gacatctcta aaaattccct aagtttcttg ccttctggag tttttgatgg tatgcctcca
2160
aatctaaaga atctctcttt ggccaaaaat gggctcaaat ctttcagttg gaagaaactc
2220
cagtgtctaa agaacctgga aactttggac ctcagccaca accaactgac cactgtccct
2280
gagagattat ccaactgttc cagaagcctc aagaatctga ttcttaagaa taatcaaatc
2340
aggagtctga cgaagtattt tctacaagat gccttccagt tgcgatatct ggatctcagc
2400
tcaaataaaa tccagatgat ccaaaagacc agcttcccag aaaatgtcct caacaatctg
2460
aagatgttgc ttttgcatca taatcggttt ctgtgcacct gtgatgctgt gtggtttgtc
2520
tggtgggtta accatacgga ggtgactatt ccttacctgg ccacagatgt gacttgtgtg
2580
gggccaggag cacacaaggg ccaaagtgtg atctccctgg atctgtacac ctgtgagtta
2640
gatctgacta acctgattct gttctcactt tccatatctg tatctctctt tctcatggtg
2700
atgatgacag caagtcacct ctatttctgg gatgtgtggt atatttacca tttctgtaag
2760
gccaagataa aggggtatca gcgtctaata tcaccagact gttgctatga tgcttttatt
2820
gtgtatgaca ctaaagaccc agctgtgacc gagtgggttt tggctgagct ggtggccaaa
2880
ctggaagacc caagagagaa acattttaat ttatgtctcg aggaaaggga ctggttacca
2940
gggcagccag ttctggaaaa cctttcccag agcatacagc ttagcaaaaa gacagtgttt
3000
gtgatgacag acaagtatgc aaagactgaa aattttaaga tagcatttta cttgtcccat
3060
cagaggctca tggatgaaaa agttgatgtg attatcttga tatttcttga gaagcccttt
3120
cagaagtcca agttcctcca gctccggaaa aggctctgtg ggagttctgt ccttgagtgg
3180
ccaacaaacc cgcaagctca cccatacttc tggcagtgtc taaagaacgc cctggccaca
3240
gacaatcatg tggcctatag tcaggtgttc aaggaaacgg tctagccctt ctttgcaaaa
3300
cacaactgcc tagtttacca aggagaggcc tggctgttta aattgttttc atatatatca
3360
caccaaaagc gtgttttgaa attcttcaag aaatgagatt gcccatattt caggggagcc
3420
accaacgtct gtcacaggag ttggaaagat ggggtttata taatgcatca agtcttcttt
3480
cttatctctc tgtgtctcta tttgcacttg agtctctcac ctcagctcct gtaaaagagt
3540
ggcaagtaaa aaacatgggg ctctgattct cctgtaattg tgataattaa atatacacac
3600
aatcatgaca ttgagaagaa ctgcatttct acccttaaaa agtactggta tatacagaaa
3660
tagggttaaa aaaaactcaa gctctctcta tatgagacca aaatgtacta gagttagttt
3720
agtgaaataa aaaaccagtc agctggccgg gcatggtggc tcatgcttgt aatcccagca
3780
ctttgggagg ccgaggcagg tggatcacga ggtcaggagt ttgagaccag tctggccaac
3840
atggtgaaac cccgtctgta ctaaaaatac aaaaattagc tgggcgtggt ggtgggtgcc
3900
tgtaatccca gctacttggg aggctgaggc aggagaatcg cttgaacccg ggaggtggag
3960
gtggcagtga gccgagatca cgccactgca atgcagcccg ggcaacagag ctagactgtc
4020
tcaaaagaac aaaaaaaaaa aaacacaaaa aaactcagtc agcttcttaa ccaattgctt
4080
ccgtgtcatc cagggcccca ttctgtgcag attgagtgtg ggcaccacac aggtggttgc
4140
tgcttcagtg cttcctgctc tttttccttg ggcctgcttc tgggttccat agggaaacag
4200
taagaaagaa agacacatcc ttaccataaa tgcatatggt ccacctacaa atagaaaaat
4260
atttaaatga tctgccttta tacaaagtga tattctctac ctttgataat ttacctgctt
4320
aaatgttttt atctgcactg caaagtactg tatccaaagt aaaatttcct catccaatat
4380
ctttcaaact gttttgttaa ctaatgccat atatttgtaa gtatctgcac acttgataca
4440
gcaacgttag atggttttga tggtaaaccc taaaggagga ctccaagagt gtgtatttat
4500
ttatagtttt atcagagatg acaattattt gaatgccaat tatatggatt cctttcattt
4560
tttgctggag gatgggagaa gaaaccaaag tttatagacc ttcacattga gaaagcttca
4620
gttttgaact tcagctatca gattcaaaaa caacagaaag aaccaagaca ttcttaagat
4680
gcctgtactt tcagctgggt ataaattcat gagttcaaag attgaaacct gaccaatttg
4740
ctttatttca tggaagaagt gatctacaaa ggtgtttgtg ccatttggaa aacagcgtgc
4800
atgtgttcaa gccttagatt ggcgatgtcg tattttcctc acgtgtggca atgccaaagg
4860
ctttacttta cctgtgagta cacactatat gaattatttc caacgtacat ttaatcaata
4920
agggtcacaa attcccaaat caatctctgg aataaataga gaggtaatta aattgctgga
4980
gccaactatt tcacaacttc tgtaagc
5007
SEQ ID NO: 26
Murine TLR7 amino acid
MVFSMWTRKR QILIFLNMLL VSRVFGFRWF PKTLPCEVKV NIPEAHVIVD CTDKHLTEIP
60
EGIPTNTTNL TLTINHIPSI SPDSFRRLNH LEEIDLRCNC VPVLLGSKAN VCTKRLQIRP
120
GSFSGLSDLK ALYLDGNQLL EIPQDLPSSL HLLSLEANNI FSITKENLTE LVNIETLYLG
180
QNCYYRNPCN VSYSIEKDAF LVMRNLKVLS LKDNNVTAVP TTLPPNLLEL YLYNNIIKKI
240
QENDFNNLNE LQVLDLSGNC PRCYNVPYPC TPCENNSPLQ IHDNAFNSLT ELKVLRLHSN
300
SLQHVPPTWF KNMRNLQELD LSQNYLAREI EEAKFLHFLP NLVELDFSFN YELQVYHASI
360
TLPHSLSSLE NLKILRVKGY VFKELKNSSL SVLHKLPRLE VLDLGTNFIK IADLNIFKHF
420
ENLKLIDLSV NKISPSEESR EVGFCPNAQT SVDRHGPQVL EALHYFRYDE YARSCRFKNK
480
EPPSFLPLNA DCHIYGQTLD LSRNNIFFIK PSDFQHLSFL KCLNLSGNTI GQTLNGSELW
540
PLRELRYLDF SNNRLDLLYS TAFEELQSLE VLDLSSNSHY FQAEGITHML NFTKKLRLLD
600
KLMMNDNDIS TSASRTMESD SLRILEFRGN HLDVLWRAGD NRYLDFFKNL FNLEVLDISR
660
NSLNSLPPEV FEGMPPNLKN LSLAKNGLKS FFWDRLQLLK HLEILDLSHN QLTKVPERLA
720
NCSKSLTTLI LKHNQIRQLT KYFLEDALQL RYLDISSNKI QVIQKTSFPE NVLNNLEMLV
780
LHHNRFLCNC DAVWFVWWVN HTDVTIPYLA TDVTCVGPGA HKGQSVISLD LYTCELDLTN
840
LILFSVSISS VLFLMVVMTT SHLFFWDMWY IYYFWKAKIK GYQHLQSMES CYDAFIVYDT
900
KNSAVTEWVL QELVAKLEDP REKHFNLCLE ERDWLPGQPV LENLSQSIQL SKKTVFVMTQ
960
KYAKTESFKM AFYLSHQRLL DEKVDVIILI FLEKPLQKSK FLQLRKRLCR SSVLEWPANP
1020
QAHPYFWQCL KNALTTDNHV AYSQMFKETV
1050
SEQ ID NO: 27
Murine TLR7 nucleotide
attctcctcc accagacctc ttgattccat tttgaaagaa aactgaaaat ggtgttttcg
60
atgtggacac ggaagagaca aattttgatc tttttaaata tgctcttagt ttctagagtc
120
tttgggtttc gatggtttcc taaaactcta ccttgtgaag ttaaagtaaa tatcccagag
180
gcccatgtga tcgtggactg cacagacaag catttgacag aaatccctga gggcattccc
240
actaacacca ccaatcttac ccttaccatc aaccacatac caagcatctc tccagattcc
300
ttccgtaggc tgaaccatct ggaagaaatc gatttaagat gcaattgtgt acctgttcta
360
ctggggtcca aagccaatgt gtgtaccaag aggctgcaga ttagacctgg aagctttagt
420
ggactctctg acttaaaagc cctttacctg gatggaaacc aacttctgga gataccacag
480
gatctgccat ccagcttaca tcttctgagc cttgaggcta acaacatctt ctccatcacg
540
aaggagaatc taacagaact ggtcaacatt gaaacactct acctgggtca aaactgttat
600
tatcgaaatc cttgcaatgt ttcctattct attgaaaaag atgctttcct agttatgaga
660
aatttgaagg ttctctcact aaaagataac aatgtcacag ctgtccccac cactttgcca
720
cctaatttac tagagctcta tctttataac aatatcatta agaaaatcca agaaaatgat
780
tttaataacc tcaatgagtt gcaagttctt gacctaagtg gaaattgccc tcgatgttat
840
aatgtcccat atccgtgtac accgtgtgaa aataattccc ccttacagat ccatgacaat
900
gctttcaatt cattgacaga attaaaagtt ttacgtttac acagtaattc tcttcagcat
960
gtgcccccaa catggtttaa aaacatgaga aacctccagg aactagacct ctcccaaaac
1020
tacttggcca gagaaattga ggaggccaaa tttttgcatt ttcttcccaa ccttgttgag
1080
ttggattttt ctttcaatta tgagctgcag gtctaccatg catctataac tttaccacat
1140
tcactctctt cattggaaaa cttgaaaatt ctgcgtgtca aggggtatgt ctttaaagag
1200
ctgaaaaact ccagtctttc tgtattgcac aagcttccca ggctggaagt tcttgacctt
1260
ggcactaact tcataaaaat tgctgacctc aacatattca aacattttga aaacctcaaa
1320
ctcatagacc tttcagtgaa taagatatct ccttcagaag agtcaagaga agttggcttt
1380
tgtcctaatg ctcaaacttc tgtagaccgt catgggcccc aggtccttga ggccttacac
1440
tatttccgat acgatgaata tgcacggagc tgcaggttca aaaacaaaga gccaccttct
1500
ttcttgcctt tgaatgcaga ctgccacata tatgggcaga ccttagactt aagtagaaat
1560
aacatatttt ttattaaacc ttctgatttt cagcatcttt cattcctcaa atgcctcaac
1620
ttatcaggaa acaccattgg ccaaactctt aatggcagtg aactctggcc gttgagagag
1680
ttgcggtact tagacttctc caacaaccgg cttgatttac tctactcaac agcctttgaa
1740
gagctccaga gtcttgaagt tctggatcta agtagtaaca gccactattt tcaagcagaa
1800
ggaattactc acatgctaaa ctttaccaag aaattacggc ttctggacaa actcatgatg
1860
aatgataatg acatctctac ttcggccagc aggaccatgg aaagtgactc tcttcgaatt
1920
ctggagttca gaggcaacca tttagatgtt ctatggagag ccggtgataa cagatacttg
1980
gacttcttca agaatttgtt caatttagag gtattagata tctccagaaa ttccctgaat
2040
tccttgcctc ctgaggtttt tgagggtatg ccgccaaatc taaagaatct ctccttggcc
2100
aaaaatgggc tcaaatcttt cttttgggac agactccagt tactgaagca tttggaaatt
2160
ttggacctca gccataacca gctgacaaaa gtacctgaga gattggccaa ctgttccaaa
2220
agtctcacaa cactgattct taagcataat caaatcaggc aattgacaaa atattttcta
2280
gaagatgctt tgcaattgcg ctatctagac atcagttcaa ataaaatcca ggtcattcag
2340
aagactagct tcccagaaaa tgtcctcaac aatctggaga tgttggtttt acatcacaat
2400
cgctttcttt gcaactgtga tgctgtgtgg tttgtctggt gggttaacca tacagatgtt
2460
actattccat acctggccac tgatgtgact tgtgtaggtc caggagcaca caaaggtcaa
2520
agtgtcatat cccttgatct gtatacgtgt gagttagatc tcacaaacct gattctgttc
2580
tcagtttcca tatcatcagt cctctttctt atggtagtta tgacaacaag tcacctcttt
2640
ttctgggata tgtggtacat ttattatttt tggaaagcaa agataaaggg gtatcagcat
2700
ctgcaatcca tggagtcttg ttatgatgct tttattgtgt atgacactaa aaactcagct
2760
gtgacagaat gggttttgca ggagctggtg gcaaaattgg aagatccaag agaaaaacac
2820
ttcaatttgt gtctagaaga aagagactgg ctaccaggac agccagttct agaaaacctt
2880
tcccagagca tacagctcag caaaaagaca gtgtttgtga tgacacagaa atatgctaag
2940
actgagagtt ttaagatggc attttatttg tctcatcaga ggctcctgga tgaaaaagtg
3000
gatgtgatta tcttgatatt cttggaaaag cctcttcaga agtctaagtt tcttcagctc
3060
aggaagagac tctgcaggag ctctgtcctt gagtggcctg caaatccaca ggctcaccca
3120
tacttctggc agtgcctgaa aaatgccctg accacagaca atcatgtggc ttatagtcaa
3180
atgttcaagg aaacagtcta gctctctgaa gaatgtcacc acctaggaca tgccttgaat
3240
cga
3243
Nucleotide and amino acid sequences of human and murine TLR8 are known. See, for example, GenBank Accession Nos. AF246971, AF245703, NM_016610, XM045706, AY035890, NM_133212; and AAF64061, AAF78036, NP_057694, XP_045706, AAK62677, NP_573475. Human TLR8 is reported to exist in at least two isoforms, one 1041 amino acids long having a sequence provided in SEQ ID NO:28, and the other 1059 amino acids long having a sequence as provided in SEQ ID NO:30. Corresponding nucleotide sequences are provided as SEQ ID NO:29 and SEQ ID NO:31, respectively. The shorter of these two isoforms is believed to be more important. Murine TLR8 is 1032 amino acids long and has a sequence as provided in SEQ ID NO:32. The corresponding nucleotide sequence is provided as SEQ ID NO:33. TLR8 polypeptide includes an extracellular domain having leucine-rich repeat region, a transmembrane domain, and an intracellular domain that includes a TIR domain.
As used herein a “TLR8 polypeptide” refers to a polypeptide including a full-length TLR8 according to one of the sequences above, orthologs, allelic variants, SNPs, variants incorporating conservative amino acid substitutions, TLR8 fusion proteins, and functional fragments of any of the foregoing. Preferred embodiments include human TLR8 polypeptides having at least 65 percent sequence identity, more preferably at least 80 percent sequence identity, even more preferably with at least 90 percent sequence identity, and most preferably with at least 95 percent sequence identity with the human TLR8 amino acid sequence of SEQ ID NO:28. Preferred embodiments also include murine TLR8 polypeptides having at least 65 percent sequence identity, more preferably at least 80 percent sequence identity, even more preferably with at least 90 percent sequence identity, and most preferably with at least 95 percent sequence identity with the murine TLR8 amino acid sequence of SEQ ID NO:32.
As used herein “TLR8 signaling” refers to an ability of a TLR8 polypeptide to activate the TLR/IL-1R (TIR) signaling pathway, also referred to herein as the TLR signal transduction pathway. Changes in TLR8 activity can be measured by assays such as those disclosed herein, including expression of genes under control of κB-sensitive promoters and enhancers. Such naturally occurring genes include the genes encoding IL-1β, IL-6, IL-8, the p40 subunit of interleukin 12 (IL-12 p40), and the costimulatory molecules CD80 and CD86. Other genes can be placed under the control of such regulatory elements (see below) and thus serve to report the level of TLR8 signaling. Additional nucleotide sequence can be added to SEQ ID NO:29 or SEQ ID NO:33, preferably to the 5′ or the 3′ end of the open reading frame of SEQ ID NO:29, to yield a nucleotide sequence encoding a chimeric polypeptide that includes a detectable or reporter moiety, e.g., FLAG, luciferase (luc), green fluorescent protein (GFP), and others known by those skilled in the art.
SEQ ID NO: 28
Human TLR8 amino acid (1041)
MENMFLQSSM LTCIFLLISG SCELCAEENF SRSYPCDEKK QNDSVIAECS NRRLQEVPQT
60
VGKYVTELDL SDNFITHITN ESFQGLQNLT KINLNHNPNV QHQNGNPGIQ SNGLNITDGA
120
FLNLKNLREL LLEDNQLPQI PSGLPESLTE LSLIQNNIYN ITKEGISRLI NLKNLYLAWN
180
CYFNKVCEKT NIEDGVFETL TNLELLSLSF NSLSHVPPKL PSSLRKLFLS NTQIKYISEE
240
DFKGLINLTL LDLSGNCPRC FNAPFPCVPC DGGASINIDR FAFQNLTQLR YLNLSSTSLR
300
KINAAWFKNM PHLKVLDLEF NYLVGEIASG AFLTMLPRLE ILDLSFNYIK GSYPQHINIS
360
RNFSKLLSLR ALHLRGYVFQ ELREDDFQPL MQLPNLSTIN LGINFIKQID FKLFQNFSNL
420
EIIYLSENRI SPLVKDTRQS YANSSSFQRH IRKRRSTDFE FDPHSNFYHF TRPLIKPQCA
480
AYGKALDLSL NSIFFIGPNQ FENLPDIACL NLSANSNAQV LSGTEFSAIP HVKYLDLTNN
540
RLDFDNASAL TELSDLEVLD LSYNSHYFRI AGVTHHLEFI QNFTNLKVLN LSHNNIYTLT
600
DKYNLESKSL VELVFSGNRL DILWNDDDNR YISIFKGLKN LTRLDLSLNR LKHIPNEAFL
660
NLPASLTELH INDNMLKFFN WTLLQQFPRL ELLDLRGNKL LFLTDSLSDF TSSLRTLLLS
720
HNRISHLPSG FLSEVSSLKH LDLSSNLLKT INKSALETKT TTKLSMLELH GNPFECTCDI
780
GDFRRWMDEH LNVKIPRLVD VICASPGDQR GKSIVSLELT TCVSDVTAVI LFFFTFFITT
840
MVMLAALAHH LFYWDVWFIY NVCLAKVKGY RSLSTSQTFY DAYISYDTKD ASVTDWVINE
900
LRYHLEESRD KNVLLCLEER DWDPGLAIID NLMQSINQSK KTVFVLTKKY AKSWNFKTAF
960
YLALQRLMDE NMDVIIFILL EPVLQHSQYL RLRQRICKSS ILQWPDNPKA EGLFWQTLRN
1020
VVLTENDSRY NNMYVDSIKQ Y
1041
SEQ ID NO: 29
Human TLR8 nucleotide
ttctgcgctg ctgcaagtta cggaatgaaa aattagaaca acagaaacat ggaaaacatg
60
ttccttcagt cgtcaatgct gacctgcatt ttcctgctaa tatctggttc ctgtgagtta
120
tgcgccgaag aaaatttttc tagaagctat ccttgtgatg agaaaaagca aaatgactca
180
gttattgcag agtgcagcaa tcgtcgacta caggaagttc cccaaacggt gggcaaatat
240
gtgacagaac tagacctgtc tgataatttc atcacacaca taacgaatga atcatttcaa
300
gggctgcaaa atctcactaa aataaatcta aaccacaacc ccaatgtaca gcaccagaac
360
ggaaatcccg gtatacaatc aaatggcttg aatatcacag acggggcatt cctcaaccta
420
aaaaacctaa gggagttact gcttgaagac aaccagttac cccaaatacc ctctggtttg
480
ccagagtctt tgacagaact tagtctaatt caaaacaata tatacaacat aactaaagag
540
ggcatttcaa gacttataaa cttgaaaaat ctctatttgg cctggaactg ctattttaac
600
aaagtttgcg agaaaactaa catagaagat ggagtatttg aaacgctgac aaatttggag
660
ttgctatcac tatctttcaa ttctctttca cacgtgccac ccaaactgcc aagctcccta
720
cgcaaacttt ttctgagcaa cacccagatc aaatacatta gtgaagaaga tttcaaggga
780
ttgataaatt taacattact agatttaagc gggaactgtc cgaggtgctt caatgcccca
840
tttccatgcg tgccttgtga tggtggtgct tcaattaata tagatcgttt tgcttttcaa
900
aacttgaccc aacttcgata cctaaacctc tctagcactt ccctcaggaa gattaatgct
960
gcctggttta aaaatatgcc tcatctgaag gtgctggatc ttgaattcaa ctatttagtg
1020
ggagaaatag cctctggggc atttttaacg atgctgcccc gcttagaaat acttgacttg
1080
tcttttaact atataaaggg gagttatcca cagcatatta atatttccag aaacttctct
1140
aaacttttgt ctctacgggc attgcattta agaggttatg tgttccagga actcagagaa
1200
gatgatttcc agcccctgat gcagcttcca aacttatcga ctatcaactt gggtattaat
1260
tttattaagc aaatcgattt caaacttttc caaaatttct ccaatctgga aattatttac
1320
ttgtcagaaa acagaatatc accgttggta aaagataccc ggcagagtta tgcaaatagt
1380
tcctcttttc aacgtcatat ccggaaacga cgctcaacag attttgagtt tgacccacat
1440
tcgaactttt atcatttcac ccgtccttta ataaagccac aatgtgctgc ttatggaaaa
1500
gccttagatt taagcctcaa cagtattttc ttcattgggc caaaccaatt tgaaaatctt
1560
cctgacattg cctgtttaaa tctgtctgca aatagcaatg ctcaagtgtt aagtggaact
1620
gaattttcag ccattcctca tgtcaaatat ttggatttga caaacaatag actagacttt
1680
gataatgcta gtgctcttac tgaattgtcc gacttggaag ttctagatct cagctataat
1740
tcacactatt tcagaatagc aggcgtaaca catcatctag aatttattca aaatttcaca
1800
aatctaaaag ttttaaactt gagccacaac aacatttata ctttaacaga taagtataac
1860
ctggaaagca agtccctggt agaattagtt ttcagtggca atcgccttga cattttgtgg
1920
aatgatgatg acaacaggta tatctccatt ttcaaaggtc tcaagaatct gacacgtctg
1980
gatttatccc ttaataggct gaagcacatc ccaaatgaag cattccttaa tttgccagcg
2040
agtctcactg aactacatat aaatgataat atgttaaagt tttttaactg gacattactc
2100
cagcagttcc ctcgtctcga gttgcttgac ttacgtggaa acaaactact ctttttaact
2160
gatagcctat ctgactttac atcttccctt cggacactgc tgctgagtca taacaggatt
2220
tcccacctac cctctggctt tctttctgaa gtcagtagtc tgaagcacct cgatttaagt
2280
tccaatctgc taaaaacaat caacaaatcc gcacttgaaa ctaagaccac caccaaatta
2340
tctatgttgg aactacacgg aaaccccttt gaatgcacct gtgacattgg agatttccga
2400
agatggatgg atgaacatct gaatgtcaaa attcccagac tggtagatgt catttgtgcc
2460
agtcctgggg atcaaagagg gaagagtatt gtgagtctgg agctgacaac ttgtgtttca
2520
gatgtcactg cagtgatatt atttttcttc acgttcttta tcaccaccat ggttatgttg
2580
gctgccctgg ctcaccattt gttttactgg gatgtttggt ttatatataa tgtgtgttta
2640
gctaaggtaa aaggctacag gtctctttcc acatcccaaa ctttctatga tgcttacatt
2700
tcttatgaca ccaaagatgc ctctgttact gactgggtga taaatgagct gcgctaccac
2760
cttgaagaga gccgagacaa aaacgttctc ctttgtctag aggagaggga ttgggacccg
2820
ggattggcca tcatcgacaa cctcatgcag agcatcaacc aaagcaagaa aacagtattt
2880
gttttaacca aaaaatatgc aaaaagctgg aactttaaaa cagcttttta cttggctttg
2940
cagaggctaa tggatgagaa catggatgtg attatattta tcctgctgga gccagtgtta
3000
cagcattctc agtatttgag gctacggcag cggatctgta agagctccat cctccagtgg
3060
cctgacaacc cgaaggcaga aggcttgttt tggcaaactc tgagaaatgt ggtcttgact
3120
gaaaatgatt cacggtataa caatatgtat gtcgattcca ttaagcaata ctaactgacg
3180
ttaagtcatg atttcgcgcc ataataaaga tgcaaaggaa tgacatttct gtattagtta
3240
tctattgcta tgtaacaaat tatcccaaaa cttagtggtt taaaacaaca catttgctgg
3300
cccacagtttt
3311
SEQ ID NO: 30
Human TLR8 amino acid (1059)
MKESSLQNSS CSLGKETKKE NMFLQSSMLT CIFLLISGSC ELCAEENFSR SYPCDEKKQN
60
DSVIAECSNR RLQEVPQTVG KYVTELDLSD NFITHITNES FQGLQNLTKI NLNHNPNVQH
120
QNGNPGIQSN GLNITDGAFL NLKNLRELLL EDNQLPQIPS GLPESLTELS LIQNNIYNIT
180
KEGISRLINL KNLYLAWNCY FNKVCEKTNI EDGVFETLTN LELLSLSFNS LSHVSPKLPS
240
SLRKLFLSNT QIKYISEEDF KGLINLTLLD LSGNCPRCFN APFPCVPCDG GASINIDRFA
300
FQNLTQLRYL NLSSTSLRKI NAAWFKNMPH LKVLDLEFNY LVGEIASGAF LTMLPRLEIL
360
DLSFNYIKGS YPQHINISRN FSKPLSLRAL HLRGYVFQEL REDDFQPLMQ LPNLSTINLG
420
INFIKQIDFK LFQNFSNLEI IYLSENRISP LVKDTRQSYA NSSSFQRHIR KRRSTDFEFD
480
PHSNFYHFTR PLIKPQCAAY GKALDLSLNS IFFIGPNQFE NLPDIACLNL SANSNAQVLS
540
GTEFSAIPHV KYLDLTNNRL DFDNASALTE LSDLEVLDLS YNSHYFRIAG VTHHLEFIQN
600
FTNLKVLNLS HNNIYTLTDK YNLESKSLVE LVFSGNRLDI LWNDDDNRYI SIFKGLKNLT
660
RLDLSLNRLK HIPNEAFLNL PASLTELHIN DNMLKFFNWT LLQQFPRLEL LDLRGNKLLF
720
LTDSLSDFTS SLRTLLLSHN RISHLPSGFL SEVSSLKHLD LSSNLLKTIN KSALETKTTT
780
KLSMLELHGN PFECTCDIGD FRRWMDEHLN VKIPRLVDVI CASPGDQRGK SIVSLELTTC
840
VSDVTAVILF FFTFFITTMV MLAALAHHLF YWDVWFIYNV CLAKIKGYRS LSTSQTFYDA
900
YISYDTKDAS VTDWVINELR YHLEESRDKN VLLCLEERDW DPGLAIIDNL MQSINQSKKT
960
VFVLTKKYAK SWNFKTAFYL ALQRLMDENM DVIIFILLEP VLQHSQYLRL RQRICKSSIL
1020
QWPDNPKAEG LFWQTLRNVV LTENDSRYNN MYVDSIKQY
1059
SEQ ID NO: 31
Human TLR8 nucleotide
ctcctgcata gagggtacca ttctgcgctg ctgcaagtta cggaatgaaa aattagaaca
60
acagaaacgt ggttctcttg acacttcagt gttagggaac atcagcaaga cccatcccag
120
gagaccttga aggaagcctt tgaaagggag aatgaaggag tcatctttgc aaaatagctc
180
ctgcagcctg ggaaaggaga ctaaaaagga aaacatgttc cttcagtcgt caatgctgac
240
ctgcattttc ctgctaatat ctggttcctg tgagttatgc gccgaagaaa atttttctag
300
aagctatcct tgtgatgaga aaaagcaaaa tgactcagtt attgcagagt gcagcaatcg
360
tcgactacag gaagttcccc aaacggtggg caaatatgtg acagaactag acctgtctga
420
taatttcatc acacacataa cgaatgaatc atttcaaggg ctgcaaaatc tcactaaaat
480
aaatctaaac cacaacccca atgtacagca ccagaacgga aatcccggta tacaatcaaa
540
tggcttgaat atcacagacg gggcattcct caacctaaaa aacctaaggg agttactgct
600
tgaagacaac cagttacccc aaataccctc tggtttgcca gagtctttga cagaacttag
660
tctaattcaa aacaatatat acaacataac taaagagggc atttcaagac ttataaactt
720
gaaaaatctc tatttggcct ggaactgcta ttttaacaaa gtttgcgaga aaactaacat
780
agaagatgga gtatttgaaa cgctgacaaa tttggagttg ctatcactat ctttcaattc
840
tctttcacac gtgtcaccca aactgccaag ctccctacgc aaactttttc tgagcaacac
900
ccagatcaaa tacattagtg aagaagattt caagggattg ataaatttaa cattactaga
960
tttaagcggg aactgtccga ggtgcttcaa tgccccattt ccatgcgtgc cttgtgatgg
1020
tggtgcttca attaatatag atcgttttgc ttttcaaaac ttgacccaac ttcgatacct
1080
aaacctctct agcacttccc tcaggaagat taatgctgcc tggtttaaaa atatgcctca
1140
tctgaaggtg ctggatcttg aattcaacta tttagtggga gaaatagcct ctggggcatt
1200
tttaacgatg ctgccccgct tagaaatact tgacttgtct tttaactata taaaggggag
1260
ttatccacag catattaata tttccagaaa cttctctaaa cctttgtctc tacgggcatt
1320
gcatttaaga ggttatgtgt tccaggaact cagagaagat gatttccagc ccctgatgca
1380
gcttccaaac ttatcgacta tcaacttggg tattaatttt attaagcaaa tcgatttcaa
1440
acttttccaa aatttctcca atctggaaat tatttacttg tcagaaaaca gaatatcacc
1500
gttggtaaaa gatacccggc agagttatgc aaatagttcc tcttttcaac gtcatatccg
1560
gaaacgacgc tcaacagatt ttgagtttga cccacattcg aacttttatc atttcacccg
1620
tcctttaata aagccacaat gtgctgctta tggaaaagcc ttagatttaa gcctcaacag
1680
tattttcttc attgggccaa accaatttga aaatcttcct gacattgcct gtttaaatct
1740
gtctgcaaat agcaatgctc aagtgttaag tggaactgaa ttttcagcca ttcctcatgt
1800
caaatatttg gatttgacaa acaatagact agactttgat aatgctagtg ctcttactga
1860
attgtccgac ttggaagttc tagatctcag ctataattca cactatttca gaatagcagg
1920
cgtaacacat catctagaat ttattcaaaa tttcacaaat ctaaaagttt taaacttgag
1980
ccacaacaac atttatactt taacagataa gtataacctg gaaagcaagt ccctggtaga
2040
attagttttc agtggcaatc gccttgacat tttgtggaat gatgatgaca acaggtatat
2100
ctccattttc aaaggtctca agaatctgac acgtctggat ttatccctta ataggctgaa
2160
gcacatccca aatgaagcat tccttaattt gccagcgagt ctcactgaac tacatataaa
2220
tgataatatg ttaaagtttt ttaactggac attactccag cagtttcctc gtctcgagtt
2280
gcttgactta cgtggaaaca aactactctt tttaactgat agcctatctg actttacatc
2340
ttcccttcgg acactgctgc tgagtcataa caggatttcc cacctaccct ctggctttct
2400
ttctgaagtc agtagtctga agcacctcga tttaagttcc aatctgctaa aaacaatcaa
2460
caaatccgca cttgaaacta agaccaccac caaattatct atgttggaac tacacggaaa
2520
cccctttgaa tgcacctgtg acattggaga tttccgaaga tggatggatg aacatctgaa
2580
tgtcaaaatt cccagactgg tagatgtcat ttgtgccagt cctggggatc aaagagggaa
2640
gagtattgtg agtctggagc taacaacttg tgtttcagat gtcactgcag tgatattatt
2700
tttcttcacg ttctttatca ccaccatggt tatgttggct gccctggctc accatttgtt
2760
ttactgggat gtttggttta tatataatgt gtgtttagct aagataaaag gctacaggtc
2820
tctttccaca tcccaaactt tctatgatgc ttacatttct tatgacacca aagatgcctc
2880
tgttactgac tgggtgataa atgagctgcg ctaccacctt gaagagagcc gagacaaaaa
2940
cgttctcctt tgtctagagg agagggattg ggacccggga ttggccatca tcgacaacct
3000
catgcagagc atcaaccaaa gcaagaaaac agtatttgtt ttaaccaaaa aatatgcaaa
3060
aagctggaac tttaaaacag ctttttactt ggctttgcag aggctaatgg atgagaacat
3120
ggatgtgatt atatttatcc tgctggagcc agtgttacag cattctcagt atttgaggct
3180
acggcagcgg atctgtaaga gctccatcct ccagtggcct gacaacccga aggcagaagg
3240
cttgttttgg caaactctga gaaatgtggt cttgactgaa aatgattcac ggtataacaa
3300
tatgtatgtc gattccatta agcaatacta actgacgtta agtcatgatt tcgcgccata
3360
ataaaga
3367
SEQ ID NO: 32
Murine TLR8 amino acid
MENMPPQSWI LTCFCLLSSG TSAIFHKANY SRSYPCDEIR HNSLVIAECN HRQLHEVPQT
60
IGKYVTNIDL SDNAITHITK ESFQKLQNLT KIDLNHNAKQ QHPNENKNGM NITEGALLSL
120
RNLTVLLLED NQLYTIPAGL PESLKELSLI QNNIFQVTKN NTFGLRNLER LYLGWNCYFK
180
CNQTFKVEDG AFKNLIHLKV LSLSFNNLFY VPPKLPSSLR KLFLSNAKIM NITQEDFKGL
240
ENLTLLDLSG NCPRCYNAPF PCTPCKENSS IHIHPLAFQS LTQLLYLNLS STSLRTIPST
300
WFENLSNLKE LHLEFNYLVQ EIASGAFLTK LPSLQILDLS FNFQYKEYLQ FINISSNFSK
360
LRSLKKLHLR GYVFRELKKK HFEHLQSLPN LATINLGINF IEKIDFKAFQ NFSKLDVIYL
420
SGNRIASVLD GTDYSSWRNR LRKPLSTDDD EFDPHVNFYH STKPLIKPQC TAYGKALDLS
480
LNNIFIIGKS QFEGFQDIAC LNLSFNANTQ VFNGTEFSSM PHIKYLDLTN NRLDFDDNNA
540
FSDLHDLEVL DLSHNAHYFS IAGVTHRLGF IQNLINLRVL NLSHNGIYTL TEESELKSIS
600
LKELVFSGNR LDHLWNANDG KYWSIFKSLQ NLIRLDLSYN NLQQIPNGAF LNLPQSLQEL
660
LISGNKLRFF NWTLLQYFPH LHLLDLSRNE LYFLPNCLSK FAHSLETLLL SHNHFSHLPS
720
GFLSEARNLV HLDLSFNTIK MINKSSLQTK MKTNLSILEL HGNYFDCTCD ISDFRSWLDE
780
NLNITIPKLV NVICSNPGDQ KSKSIMSLDL TTCVSDTTAA VLFFLTFLTT SMVMLAALVH
840
HLFYWDVWFI YHMCSAKLKG YRTSSTSQTF YDAYISYDTK DASVTDWVIN ELRYHLEESE
900
DKSVLLCLEE RDWDPGLPII DNLMQSINQS KKTIFVLTKK YAKSWNFKTA FYLALQRLMD
960
ENMDVIIFIL LEPVLQYSQY LRLRQRICKS SILQWPNNPK AENLFWQSLK NVVLTENDSR
1020
YDDLYIDSIR QY
1032
SEQ ID NO: 33
Murine TLR8 nucleotide
attcagagtt ggatgttaag agagaaacaa acgttttacc ttcctttgtc tatagaacat
60
ggaaaacatg ccccctcagt catggattct gacgtgcttt tgtctgctgt cctctggaac
120
cagtgccatc ttccataaag cgaactattc cagaagctat ccttgtgacg agataaggca
180
caactccctt gtgattgcag aatgcaacca tcgtcaactg catgaagttc cccaaactat
240
aggcaagtat gtgacaaaca tagacttgtc agacaatgcc attacacata taacgaaaga
300
gtcctttcaa aagctgcaaa acctcactaa aatcgatctg aaccacaatg ccaaacaaca
360
gcacccaaat gaaaataaaa atggtatgaa tattacagaa ggggcacttc tcagcctaag
420
aaatctaaca gttttactgc tggaagacaa ccagttatat actatacctg ctgggttgcc
480
tgagtctttg aaagaactta gcctaattca aaacaatata tttcaggtaa ctaaaaacaa
540
cacttttggg cttaggaact tggaaagact ctatttgggc tggaactgct attttaaatg
600
taatcaaacc tttaaggtag aagatggggc atttaaaaat cttatacact tgaaggtact
660
ctcattatct ttcaataacc ttttctatgt gccccccaaa ctaccaagtt ctctaaggaa
720
actttttctg agtaatgcca aaatcatgaa catcactcag gaagacttca aaggactgga
780
aaatttaaca ttactagatc tgagtggaaa ctgtccaagg tgttacaatg ctccatttcc
840
ttgcacacct tgcaaggaaa actcatccat ccacatacat cctctggctt ttcaaagtct
900
cacccaactt ctctatctaa acctttccag cacttccctc aggacgattc cttctacctg
960
gtttgaaaat ctgtcaaatc tgaaggaact ccatcttgaa ttcaactatt tagttcaaga
1020
aattgcctcg ggggcatttt taacaaaact acccagttta caaatccttg atttgtcctt
1080
caactttcaa tataaggaat atttacaatt tattaatatt tcctcaaatt tctctaagct
1140
tcgttctctc aagaagttgc acttaagagg ctatgtgttc cgagaactta aaaagaagca
1200
tttcgagcat ctccagagtc ttccaaactt ggcaaccatc aacttgggca ttaactttat
1260
tgagaaaatt gatttcaaag ctttccagaa tttttccaaa ctcgacgtta tctatttatc
1320
aggaaatcgc atagcatctg tattagatgg tacagattat tcctcttggc gaaatcgtct
1380
tcggaaacct ctctcaacag acgatgatga gtttgatcca cacgtgaatt tttaccatag
1440
caccaaacct ttaataaagc cacagtgtac tgcttatggc aaggccttgg atttaagttt
1500
gaacaatatt ttcattattg ggaaaagcca atttgaaggt tttcaggata tcgcctgctt
1560
aaatctgtcc ttcaatgcca atactcaagt gtttaatggc acagaattct cctccatgcc
1620
ccacattaaa tatttggatt taaccaacaa cagactagac tttgatgata acaatgcttt
1680
cagtgatctt cacgatctag aagtgctgga cctgagccac aatgcacact atttcagtat
1740
agcaggggta acgcaccgtc taggatttat ccagaactta ataaacctca gggtgttaaa
1800
cctgagccac aatggcattt acaccctcac agaggaaagt gagctgaaaa gcatctcact
1860
gaaagaattg gttttcagtg gaaatcgtct tgaccatttg tggaatgcaa atgatggcaa
1920
atactggtcc atttttaaaa gtctccagaa tttgatacgc ctggacttat catacaataa
1980
ccttcaacaa atcccaaatg gagcattcct caatttgcct cagagcctcc aagagttact
2040
tatcagtggt aacaaattac gtttctttaa ttggacatta ctccagtatt ttcctcacct
2100
tcacttgctg gatttatcga gaaatgagct gtattttcta cccaattgcc tatctaagtt
2160
tgcacattcc ctggagacac tgctactgag ccataatcat ttctctcacc taccctctgg
2220
cttcctctcc gaagccagga atctggtgca cctggatcta agtttcaaca caataaagat
2280
gatcaataaa tcctccctgc aaaccaagat gaaaacgaac ttgtctattc tggagctaca
2340
tgggaactat tttgactgca cgtgtgacat aagtgatttt cgaagctggc tagatgaaaa
2400
tctgaatatc acaattccta aattggtaaa tgttatatgt tccaatcctg gggatcaaaa
2460
atcaaagagt atcatgagcc tagatctcac gacttgtgta tcggatacca ctgcagctgt
2520
cctgtttttc ctcacattcc ttaccacctc catggttatg ttggctgctc tggttcacca
2580
cctgttttac tgggatgttt ggtttatcta tcacatgtgc tctgctaagt taaaaggcta
2640
caggacttca tccacatccc aaactttcta tgatgcttat atttcttatg acaccaaaga
2700
tgcatctgtt actgactggg taatcaatga actgcgctac caccttgaag agagtgaaga
2760
caaaagtgtc ctcctttgtt tagaggagag ggattgggat ccaggattac ccatcattga
2820
taacctcatg cagagcataa accagagcaa gaaaacaatc tttgttttaa ccaagaaata
2880
tgccaagagc tggaacttta aaacagcttt ctacttggcc ttgcagaggc taatggatga
2940
gaacatggat gtgattattt tcatcctcct ggaaccagtg ttacagtact cacagtacct
3000
gaggcttcgg cagaggatct gtaagagctc catcctccag tggcccaaca atcccaaagc
3060
agaaaacttg ttttggcaaa gtctgaaaaa tgtggtcttg actgaaaatg attcacggta
3120
tgacgatttg tacattgatt ccattaggca atactagtga tgggaagtca cgactctgcc
3180
atcataaaaa cacacagctt ctccttacaa tgaaccgaat
3220
Nucleotide and amino acid sequences of human and murine TLR9 are known. See, for example, GenBank Accession Nos. NM_017442, AF259262, AB045180, AF245704, AB045181, AF348140, AF314224, NM_031178; and NP_059138, AAF 72189, BAB19259, AAF78037, BAB 19260, AAK29625, AAK28488, NP_112455. Human TLR9 is reported to exist in at least two isoforms, one 1032 amino acids long having a sequence provided in SEQ ID NO:34, and the other 1055 amino acids long having a sequence as provided in SEQ ID NO:36. Corresponding nucleotide sequences are provided as SEQ ID NO:35 and SEQ ID NO:37, respectively. The shorter of these two isoforms is believed to be more important. Murine TLR9 is 1032 amino acids long and has a sequence as provided in SEQ ID NO:38. A corresponding nucleotide sequence is provided as SEQ ID NO:39. TLR9 polypeptide includes an extracellular domain having leucine-rich repeat region, a transmembrane domain, and an intracellular domain that includes a TIR domain.
As used herein a “TLR9 polypeptide” refers to a polypeptide including a full-length TLR9 according to one of the sequences above, orthologs, allelic variants, SNPs, variants incorporating conservative amino acid substitutions, TLR9 fusion proteins, and functional fragments of any of the foregoing. Preferred embodiments include human TLR9 polypeptides having at least 65 percent sequence identity, more preferably at least 80 percent sequence identity, even more preferably with at least 90 percent sequence identity, and most preferably with at least 95 percent sequence identity with the human TLR9 amino acid sequence of SEQ ID NO:34. Preferred embodiments also include murine TLR9 polypeptides having at least 65 percent sequence identity, more preferably at least 80 percent sequence identity, even more preferably with at least 90 percent sequence identity, and most preferably with at least 95 percent sequence identity with the murine TLR9 amino acid sequence of SEQ ID NO:38.
As used herein “TLR9 signaling” refers to an ability of a TLR9 polypeptide to activate the TLR/IL-1R (TIR) signaling pathway, also referred to herein as the TLR signal transduction pathway. Without meaning to be held to any particular theory, it is believed that the TLR/IL-1R signaling pathway involves signaling via the molecules myeloid differentiation marker 88 (MyD88) and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), leading to activation of kinases of the IκB kinase complex and the c-jun NH2-terminal kinases (e.g., Jnk 1/2). Häcker H et al. (2000) J Exp Med 192:595-600. Changes in TLR9 activity can be measured by assays such as those disclosed herein, including expression of genes under control of κB-sensitive promoters and enhancers. Such naturally occurring genes include the genes encoding IL-1β, IL-6, IL-8, the p40 subunit of interleukin 12 (IL-12 p40), and the costimulatory molecules CD80 and CD86. Other genes can be placed under the control of such regulatory elements (see below) and thus serve to report the level of TLR9 signaling. Additional nucleotide sequence can be added to SEQ ID NO:35 or SEQ ID NO:39, preferably to the 5′ or the 3′ end of the open reading frame of SEQ ID NO:35, to yield a nucleotide sequence encoding a chimeric polypeptide that includes a detectable or reporter moiety, e.g., FLAG, luciferase (luc), green fluorescent protein (GFP), and others known by those skilled in the art.
SEQ ID NO: 34
Human TLR9 amino acid (1032)
MGFCRSALHP LSLLVQAIML AMTLALGTLP AFLPCELQPH GLVNCNWLFL KSVPHFSMAA
60
PRGNVTSLSL SSNRIHHLHD SDFAHLPSLR HLNLKWNCPP VGLSPMHFPC HMTIEPSTFL
120
AVPTLEELNL SYNNIMTVPA LPKSLISLSL SHTNILMLDS ASLAGLHALR FLFMDGNCYY
180
KNPCRQALEV APGALLGLGN LTHLSLKYNN LTVVPRNLPS SLEYLLLSYN RIVKLAPEDL
240
ANLTALRVLD VGGNCRRCDH APNPCMECPR HFPQLHPDTF SHLSRLEGLV LKDSSLSWLN
300
ASWFRGLGNL RVLDLSENFL YKCITKTKAF QGLTQLRKLN LSFNYQKRVS FAHLSLAPSF
360
GSLVALKELD MHGIFFRSLD ETTLRPLARL PMLQTLRLQM NFINQAQLGI FRAFPGLRYV
420
DLSDNRISGA SELTATMGEA DGGEKVWLQP GDLAPAPVDT PSSEDFRPNC STLNFTLDLS
480
RNNLVTVQPE MFAQLSHLQC LRLSHNCISQ AVNGSQFLPL TGLQVLDLSH NKLDLYHEHS
540
FTELPRLEAL DLSYNSQPFG MQGVGHNFSF VAHLRTLRHL SLAHNNIHSQ VSQQLCSTSL
600
RALDFSGNAL GHMWAEGDLY LHFFQGLSGL IWLDLSQNRL HTLLPQTLRN LPKSLQVLRL
660
RDNYLAFFKW WSLHFLPKLE VLDLAGNQLK ALTNGSLPAG TRLRRLDVSC NSISFVAPGF
720
FSKAKELREL NLSANALKTV DHSWFGPLAS ALQILDVSAN PLHCACGAAF MDFLLEVQAA
780
VPGLPSRVKC GSPGQLQGLS IFAQDLRLCL DEALSWDCFA LSLLAVALGL GVPMLHHLCG
840
WDLWYCFHLC LAWLPWRGRQ SGRDEDALPY DAFVVFDKTQ SAVADWVYNE LRGQLEECRG
900
RWALRLCLEE RDWLPGKTLF ENLWASVYGS RKTLFVLAHT DRVSGLLRAS FLLAQQRLLE
960
DRKDVVVLVI LSPDGRRSRY VRLRQRLCRQ SVLLWPHQPS GQRSFWAQLG MALTRDNHHF
1020
YNRNFCQGPT AE
1032
SEQ ID NO: 35
Human TLR9 nucleotide
ccgctgctgc ccctgtggga agggacctcg agtgtgaagc atccttccct gtagctgctg
60
tccagtctgc ccgccagacc ctctggagaa gcccctgccc cccagcacgg gtttctgccg
120
cagcgccctg cacccgctgt ctctcctggt gcaggccatc atgctggcca tgaccctggc
180
cctgggtacc ttgcctgcct tcctaccctg tgagctccag ccccacggcc tggtgaactg
240
caactggctg ttcctgaagt ctgtgcccca cttctccatg gcagcacccc gtggcaatgt
300
caccagcctt tccttgtcct ccaaccgcat ccaccocctc catgattctg actttgccca
360
cctgcccagc ctgcggcatc tcaacctcaa gtggaactgc ccgccggttg gcctcagccc
420
catgcacttc ccctgccaca tgaccatcga gcccagcacc ttcttggctg tgcccaccct
480
ggaagagcta aacctgagct acaacaacat catgactgtg cctgcgctgc ccaaatccct
540
catatccctg tccctcagcc ataccaacat cctgatgcta gactctgcca gcctcgccgg
600
cctgcatgcc ctgcgcttcc tattcatgga cggcaactgt tattacaaga acccctgcag
660
gcaggcactg gaggtggccc cgggtgccct ccttggcctg ggcaacctca cccacctgtc
720
actcaagtac aacaacctca ctgtggtgcc ccgcaacctg ccttccagcc tggagtatct
780
gctgttgtcc tacaaccgca tcgtcaaact ggcgcctgag gacctggcca atctgaccgc
840
cctgcgtgtg ctcgatgtgg gcggaaattg ccgccgctgc gaccacgctc ccaacccctg
900
catggagtgc cctcgtcact tcccccagct acatcccgat accttcagcc acctgagccg
960
tcttgaaggc ctggtgttga aggacagttc tctctcctgg ctgaatgcca gttggttccg
1020
tgggctggga aacctccgag tgctggacct gagtgagaac ttcctctaca aatgcatcac
1080
taaaaccaag gccttccagg gcctaacaca gctgcgcaag cttaacctgt ccttcaatta
1140
ccaaaagagg gtgtcctttg cccacctgtc tctggcccct tccttcggga gcctggtcgc
1200
cctgaaggag ctggacatgc acggcatctt cttccgctca ctcgatgaga ccacgctccg
1260
gccactggcc cgcctgccca tgctccagac tctgcgtctg cagatgaact tcatcaacca
1320
ggcccagctc ggcatcttca gggccttccc tggcctgcgc tacgtggacc tgtcggacaa
1380
ccgcatcagc ggagcttcgg agctgacagc caccatgggg gaggcagatg gaggggagaa
1440
ggtctggctg cagcctgggg accttgctcc ggccccagtg gacactccca gctctgaaga
1500
cttcaggccc aactgcagca ccctcaactt caccttggat ctgtcacgga acaacctggt
1560
gaccgtgcag ccggagatgt ttgcccagct ctcgcacctg cagtgcctgc gcctgagcca
1620
caactgcatc tcgcaggcag tcaatggctc ccagttcctg ccgctgaccg gtctgcaggt
1680
gctagacctg tcccacaata agctggacct ctaccacgag cactcattca cggagctacc
1740
acgactggag gccctggacc tcagctacaa cagccagccc tttggcatgc agggcgtggg
1800
ccacaacttc agcttcgtgg ctcacctgcg caccctgcgc cacctcagcc tggcccacaa
1860
caacatccac agccaagtgt cccagcagct ctgcagtacg tcgctgcggg ccctggactt
1920
cagcggcaat gcactgggcc atatgtgggc cgagggagac ctctatctgc acttcttcca
1980
aggcctgagc ggtttgatct ggctggactt gtcccagaac cgcctgcaca ccctcctgcc
2040
ccaaaccctg cgcaacctcc ccaagagcct acaggtgctg cgtctccgtg acaattacct
2100
ggccttcttt aagtggtgga gcctccactt cctgcccaaa ctggaagtcc tcgacctggc
2160
aggaaaccag ctgaaggccc tgaccaatgg cagcctgcct gctggcaccc ggctccggag
2220
gctggatgtc agctgcaaca gcatcagctt cgtggccccc ggcttctttt ccaaggccaa
2280
ggagctgcga gagctcaacc ttagcgccaa cgccctcaag acagtggacc actcctggtt
2340
tgggcccctg gcgagtgccc tgcaaatact agatgtaagc gccaaccctc tgcactgcgc
2400
ctgtggggcg gcctttatgg acttcctgct ggaggtgcag gctgccgtgc ccggtctgcc
2460
cagccgggtg aagtgtggca gtccgggcca gctccagggc ctcagcatct ttgcacagga
2520
cctgcgcctc tgcctggatg aggccctctc ctgggactgt ttcgccctct cgctgctggc
2580
tgtggctctg ggcctgggtg tgcccatgct gcatcacctc tgtggctggg acctctggta
2640
ctgcttccac ctgtgcctgg cctggcttcc ctggcggggg cggcaaagtg ggcgagatga
2700
ggatgccctg ccctacgatg ccttcgtggt cttcgacaaa acgcagagcg cagtggcaga
2760
ctgggtgtac aacgagcttc gggggcagct ggaggagtgc cgtgggcgct gggcactccg
2820
cctgtgcctg gaggaacgcg actggctgcc tggcaaaacc ctctttgaga acctgtgggc
2880
ctcggtctat ggcagccgca agacgctgtt tgtgctggcc cacacggacc gggtcagtgg
2940
tctcttgcgc gccagcttcc tgctggccca gcagcgcctg ctggaggacc gcaaggacgt
3000
cgtggtgctg gtgatcctga gccctgacgg ccgccgctcc cgctacgtgc ggctgcgcca
3060
gcgcctctgc cgccagagtg tcctcctctg gccccaccag cccagtggtc agcgcagctt
3120
ctgggcccag ctgggcatgg ccctgaccag ggacaaccac cacttctata accggaactt
3180
ctgccaggga cccacggccg aatagccgtg agccggaatc ctgcacggtg ccacctccac
3240
actcacctca cctctgc
3258
SEQ ID NO: 36
Human TLR9 amino acid (1055)
MPMKWSGWRW SWGPATHTAL PPPQGFCRSA LHPLSLLVQA IMLAMTLALG TLPAFLPCEL
60
QPHGLVNCNW LFLKSVPHFS MAAPRGNVTS LSLSSNRIHH LHDSDFAHLP SLRHLNLKWN
120
CPPVGLSPMH FPCHMTIEPS TFLAVPTLEE LNLSYNNIMT VPALPKSLIS LSLSHTNILM
180
LDSASLAGLH ALRFLFMDGN CYYKNPCRQA LEVAPGALLG LGNLTHLSLK YNNLTVVPRN
240
LPSSLEYLLL SYNRIVKLAP EDLANLTALR VLDVGGNCRR CDHAPNPCME CPRHFPQLHP
300
DTFSHLSRLE GLVLKDSSLS WLNASWFRGL GNLRVLDLSE NFLYKCITKT KAFQGLTQLR
360
KLNLSFNYQK RVSFAHLSLA PSFGSLVALK ELDMHGIFFR SLDETTLRPL ARLPMLQTLR
420
LQMNFINQAQ LGIFRAFPGL RYVDLSDNRI SGASELTATM GEADGGEKVW LQPGDLAPAP
480
VDTPSSEDFR PNCSTLNFTL DLSRNNLVTV QPEMFAQLSH LQCLRLSHNC ISQAVNGSQF
540
LPLTGLQVLD LSHNKLDLYH EHSFTELPRL EALDLSYNSQ PFGMQGVGHN FSFVAHLRTL
600
RHLSLAHNNI HSQVSQQLCS TSLRALDFSG NALGHMWAEG DLYLHFFQGL SGLIWLDLSQ
660
NRLHTLLPQT LRNLPKSLQV LRLRDNYLAF FKWWSLHFLP KLEVLDLAGN QLKALTNGSL
720
PAGTRLRRLD VSCNSISFVA PGFFSKAKEL RELNLSANAL KTVDHSWFGP LASALQILDV
780
SANPLHCACG AAFMDFLLEV QAAVPGLPSR VKCGSPGQLQ GLSIFAQDLR LCLDEALSWD
840
CFALSLLAVA LGLGVPMLHH LCGWDLWYCF HLCLAWLPWR GRQSGRDEDA LPYDAFVVFD
900
KTQSAVADWV YNELRGQLEE CRGRWALRLC LEERDWLPGK TLFENLWASV YGSRKTLFVL
960
AHTDRVSGLL RASFLLAQQR LLEDRKDVVV LVILSPDGRR SRYVRLRQRL CRQSVLLWPH
1020
QPSGQRSFWA QLGMALTRDN HHFYNRNFCQ GPTAE
1055
SEQ ID NO: 37
Human TLR9 nucleotide
atgcccatga agtggagtgg gtggaggtgg agctgggggc cggccactca cacagccctc
60
ccacccccac agggtttctg ccgcagcgcc ctgcacccgc tgtctctcct ggtgcaggcc
120
atcatgctgg ccatgaccct ggccctgggt accttgcctg ccttcctacc ctgtgagctc
180
cagccccacg gcctggtgaa ctgcaactgg ctgttcctga agtctgtgcc ccacttctcc
240
atggcagcac cccgtggcaa tgtcaccagc ctttccttgt cctccaaccg catccaccac
300
ctccatgatt ctgactttgc ccacctgccc agcctgcggc atctcaacct caagtggaac
360
tgcccgccgg ttggcctcag ccccatgcac ttcccctgcc acatgaccat cgagcccagc
420
accttcttgg ctgtgcccac cctggaagag ctaaacctga gctacaacaa catcatgact
480
gtgcctgcgc tgcccaaatc cctcatatcc ctgtccctca gccataccaa catcctgatg
540
ctagactctg ccagcctcgc cggcctgcat gccctgcgct tcctattcat ggacggcaac
600
tgttattaca agaacccctg caggcaggca ctggaggtgg ccccgggtgc cctccttggc
660
ctgggcaacc tcacccacct gtcactcaag tacaacaacc tcactgtggt gccccgcaac
720
ctgccttcca gcctggagta tctgctgttg tcctacaacc gcatcgtcaa actggcgcct
780
gaggacctgg ccaatctgac cgccctgcgt gtgctcgatg tgggcggaaa ttgccgccgc
840
tgcgaccacg ctcccaaccc ctgcatggag tgccctcgtc acttccccca gctacatccc
900
gataccttca gccacctgag ccgtcttgaa ggcctggtgt tgaaggacag ttctctctcc
960
tggctgaatg ccagttggtt ccgtgggctg ggaaacctcc gagtgctgga cctgagtgag
1020
aacttcctct acaaatgcat cactaaaacc aaggccttcc agggcctaac acagctgcgc
1080
aagcttaacc tgtccttcaa ttaccaaaag agggtgtcct ttgcccacct gtctctggcc
1140
ccttccttcg ggagcctggt cgccctgaag gagctggaca tgcacggcat cttcttccgc
1200
tcactcgatg agaccacgct ccggccactg gcccgcctgc ccatgctcca gactctgcgt
1260
ctgcagatga acttcatcaa ccaggcccag ctcggcatct tcagggcctt ccctggcctg
1320
cgctacgtgg acctgtcgga caaccgcatc agcggagctt cggagctgac agccaccatg
1380
ggggaggcag atggagggga gaaggtctgg ctgcagcctg gggaccttgc tccggcccca
1440
gtggacactc ccagctctga agacttcagg cccaactgca gcaccctcaa cttcaccttg
1500
gatctgtcac ggaacaacct ggtgaccgtg cagccggaga tgtttgccca gctctcgcac
1560
ctgcagtgcc tgcgcctgag ccacaactgc atctcgcagg cagtcaatgg ctcccagttc
1620
ctgccgctga ccggtctgca ggtgctagac ctgtcccaca ataagctgga cctctaccac
1680
gagcactcat tcacggagct accacgactg gaggccctgg acctcagcta caacagccag
1720
ccctttggca tgcagggcgt gggccacaac ttcagcttcg tggctcacct gcgcaccctg
1800
cgccacctca gcctggccca caacaacatc cacagccaag tgtcccagca gctctgcagt
1860
acgtcgctgc gggccctgga cttcagcggc aatgcactgg gccatatgtg ggccgaggga
1920
gacctctatc tgcacttctt ccaaggcctg agcggtttga tctggctgga cttgtcccag
1980
aaccgcctgc acaccctcct gccccaaacc ctgcgcaacc tccccaagag cctacaggtg
2040
ctgcgtctcc gtgacaatta cctggccttc tttaagtggt ggagcctcca cttcctgccc
2100
aaactggaag tcctcgacct ggcaggaaac cagctgaagg ccctgaccaa tggcagcctg
2160
cctgctggca cccggctccg gaggctggat gtcagctgca acagcatcag cttcgtggcc
2220
cccggcttct tttccaaggc caaggagctg cgagagctca accttagcgc caacgccctc
2280
aagacagtgg accactcctg gtttgggccc ctggcgagtg ccctgcaaat actagatgta
2340
agcgccaacc ctctgcactg cgcctgtggg gcggccttta tggacttcct gctggaggtg
2400
caggctgccg tgcccggtct gcccagccgg gtgaagtgtg gcagtccggg ccagctccag
2460
ggcctcagca tctttgcaca ggacctgcgc ctctgcctgg atgaggccct ctcctgggac
2520
tgtttcgccc tctcgctgct ggctgtggct ctgggcctgg gtgtgcccat gctgcatcac
2580
ctctgtggct gggacctctg gtactgcttc cacctgtgcc tggcctggct tccctggcgg
2640
gggcggcaaa gtgggcgaga tgaggatgcc ctgccctacg atgccttcgt ggtcttcgac
2700
aaaacgcaga gcgcagtggc agactgggtg tacaacgagc ttcgggggca gctggaggag
2760
tgccgtgggc gctgggcact ccgcctgtgc ctggaggaac gcgactggct gcctggcaaa
2820
accctctttg agaacctgtg ggcctcggtc tatggcagcc gcaagacgct gtttgtgctg
2880
gcccacacgg accgggtcag tggtctcttg cgcgccagct tcctgctggc ccagcagcgc
2940
ctgctggagg accgcaagga cgtcgtggtg ctggtgatcc tgagccctga cggccgccgc
3000
tcccgctatg tgcggctgcg ccagcgcctc tgccgccaga gtgtcctcct ctggccccac
3060
cagcccagtg gtcagcgcag cttctgggcc cagctgggca tggccctgac cagggacaac
3120
caccacttct ataaccggaa cttctgccag ggacccacgg ccgaa
3165
SEQ ID NO: 38
Murine TLR9 amino acid
MVLRRRTLHP LSLLVQAAVL AETLALGTLP AFLPCELKPH GLVDCNWLFL KSVPRFSAAA
60
SCSNITRLSL ISNRIHHLHN SDFVHLSNLR QLNLKWNCPP TGLSPLHFSC HMTIEPRTFL
120
AMRTLEELNL SYNGITTVPR LPSSLVNLSL SHTNILVLDA NSLAGLYSLR VLFMDGNCYY
180
KNPCTGAVKV TPGALLGLSN LTHLSLKYNN LTKVPRQLPP SLEYLLVSYN LIVKLGPEDL
240
ANLTSLRVLD VGGNCRRCDH APNPCIECGQ KSLHLHPETF HHLSHLEGLV LKDSSLHTLN
300
SSWFQGLVNL SVLDLSENFL YESINHTNAF QNLTRLRKLN LSFNYRKKVS FARLHLASSF
360
KNLVSLQELN MNGIFFRSLN KYTLRWLADL PKLHTLHLQM NFINQAQLSI FGTFRALRFV
420
DLSDNRISGP STLSEATPEE ADDAEQEELL SADPHPAPLS TPASKNFMDR CKNFKFTMDL
480
SRNNLVTIKP EMFVNLSRLQ CLSLSHNSIA QAVNGSQFLP LTNLQVLDLS HNKLDLYHWK
540
SFSELPQLQA LDLSYNSQPF SMKGIGHNFS FVAHLSMLHS LSLAHNDIHT RVSSHLNSNS
600
VRFLDFSGNG MGRMWDEGGL YLHFFQGLSG LLKLDLSQNN LHILRPQNLD NLPKSLKLLS
660
LRDNYLSFFN WTSLSFLPNL EVLDLAGNQL KALTNGTLPN GTLLQKLDVS SNSIVSVVPA
720
FFALAVELKE VNLSHNILKT VDRSWFGPIV MNLTVLDVRS NPLHCACGAA FVDLLLEVQT
780
KVPGLANGVK CGSPGQLQGR SIFAQDLRLC LDEVLSWDCF GLSLLAVAVG MVVPILHHLC
840
GWDVWYCFHL CLAWLPLLAR SRRSAQALPY DAFVVFDKAQ SAVADWVYNE LRVRLEERRG
900
RRALRLCLED RDWLPGQTLF ENLWASIYGS RKTLFVLAHT DRVSGLLRTS FLLAQQRLLE
960
DRKDVVVLVI LRPDAHRSRY VRLRQRLCRQ SVLFWPQQPN GQGGFWAQLS TALTRDNRHF
1020
YNQNFCRGPT AE
1032
SEQ ID NO: 39
Murine TLR9 nucleotide
tgtcagaggg agcctcggga gaatcctcca tctcccaaca tggttctccg tcgaaggact
60
ctgcacccct tgtccctcct ggtacaggct gcagtgctgg ctgagactct ggccctgggt
120
accctgcctg ccttcctacc ctgtgagctg aagcctcatg gcctggtgga ctgcaattgg
180
ctgttcctga agtctgtacc ccgtttctct gcggcagcat cctgctccaa catcacccgc
240
ctctccttga tctccaaccg tatccaccac ctgcacaact ccgacttcgt ccacctgtcc
300
aacctgcggc agctgaacct caagtggaac tgtccaccca ctggccttag ccccctgcac
360
ttctcttgcc acatgaccat tgagcccaga accttcctgg ctatgcgtac actggaggag
420
ctgaacctga gctataatgg tatcaccact gtgccccgac tgcccagctc cctggtgaat
480
ctgagcctga gccacaccaa catcctggtt ctagatgcta acagcctcgc cggcctatac
540
agcctgcgcg ttctcttcat ggacgggaac tgctactaca agaacccctg cacaggagcg
600
gtgaaggtga ccccaggcgc cctcctgggc ctgagcaatc tcacccatct gtctctgaag
660
tataacaacc tcacaaaggt gccccgccaa ctgcccccca gcctggagta cctcctggtg
720
tcctataacc tcattgtcaa gctggggcct gaagacctgg ccaatctgac ctcccttcga
780
gtacttgatg tgggtgggaa ttgccgtcgc tgcgaccatg cccccaatcc ctgtatagaa
840
tgtggccaaa agtccctcca cctgcaccct gagaccttcc atcacctgag ccatctggaa
900
ggcctggtgc tgaaggacag ctctctccat acactgaact cttcctggtt ccaaggtctg
960
gtcaacctct cggtgctgga cctaagcgag aactttctct atgaaagcat caaccacacc
1020
aatgcctttc agaacctaac ccgcctgcgc aagctcaacc tgtccttcaa ttaccgcaag
1080
aaggtatcct ttgcccgcct ccacctggca agttccttca agaacctggt gtcactgcag
1140
gagctgaaca tgaacggcat cttcttccgc tcgctcaaca agtacacgct cagatggctg
1200
gccgatctgc ccaaactcca cactctgcat cttcaaatga acttcatcaa ccaggcacag
1260
ctcagcatct ttggtacctt ccgagccctt cgctttgtgg acttgtcaga caatcgcatc
1320
agtgggcctt caacgctgtc agaagccacc cctgaagagg cagatgatgc agagcaggag
1380
gagctgttgt ctgcggatcc tcacccagct ccactgagca cccctgcttc taagaacttc
1440
atggacaggt gtaagaactt caagttcacc atggacctgt ctcggaacaa cctggtgact
1500
atcaagccag agatgtttgt caatctctca cgcctccagt gtcttagcct gagccacaac
1560
tccattgcac aggctgtcaa tggctctcag ttcctgccgc tgactaatct gcaggtgctg
1620
gacctgtccc ataacaaact ggacttgtac cactggaaat cgttcagtga gctaccacag
1680
ttgcaggccc tggacctgag ctacaacagc cagcccttta gcatgaaggg tataggccac
1740
aatttcagtt ttgtggccca tctgtccatg ctacacagcc ttagcctggc acacaatgac
1800
attcataccc gtgtgtcctc acatctcaac agcaactcag tgaggtttct tgacttcagc
1860
ggcaacggta tgggccgcat gtgggatgag gggggccttt atctccattt cttccaaggc
1920
ctgagtggcc tgctgaagct ggacctgtct caaaataacc tgcatatcct ccggccccag
1980
aaccttgaca acctccccaa gagcctgaag ctgctgagcc tccgagacaa ctacctatct
2040
ttctttaact ggaccagtct gtccttcctg cccaacctgg aagtcctaga cctggcaggc
2100
aaccagctaa aggccctgac caatggcacc ctgcctaatg gcaccctcct ccagaaactg
2160
gatgtcagca gcaacagtat cgtctctgtg gtcccagcct tcttcgctct ggcggtcgag
2220
ctgaaagagg tcaacctcag ccacaacatt ctcaagacgg tggatcgctc ctggtttggg
2280
cccattgtga tgaacctgac agttctagac gtgagaagca accctctgca ctgtgcctgt
2340
ggggcagcct tcgtagactt actgttggag gtgcagacca aggtgcctgg cctggctaat
2400
ggtgtgaagt gtggcagccc cggccagctg cagggccgta gcatcttcgc acaggacctg
2460
cggctgtgcc tggatgaggt cctctcttgg gactgctttg gcctttcact cttggctgtg
2520
gccgtgggca tggtggtgcc tatactgcac catctctgcg gctgggacgt ctggtactgt
2580
tttcatctgt gcctggcatg gctacctttg ctggcccgca gccgacgcag cgcccaagct
2640
ctcccctatg atgccttcgt ggtgttcgat aaggcacaga gcgcagttgc ggactgggtg
2700
tataacgagc tgcgggtgcg gctggaggag cggcgcggtc gccgagccct acgcttgtgt
2760
ctggaggacc gagattggct gcctggccag acgctcttcg agaacctctg ggcttccatc
2820
tatgggagcc gcaagactct atttgtgctg gcccacacgg accgcgtcag tggcctcctg
2880
cgcaccagct tcctgctggc tcagcagcgc ctgttggaag accgcaagga cgtggtggtg
2940
ttggtgatcc tgcgtccgga tgcccaccgc tcccgctatg tgcgactgcg ccagcgtctc
3000
tgccgccaga gtgtgctctt ctggccccag cagcccaacg ggcagggggg cttctgggcc
3060
cagctgagta cagccctgac tagggacaac cgccacttct ataaccagaa cttctgccgg
3120
ggacctacag cagaatagct cagagcaaca gctggaaaca gctgcatctt catgcctggt
3180
tcccgagttg ctctgcctgc
3200
Ribonucleoside vanadyl complexes (i.e., mixtures of adenine, cytosine, guanosine, and uracil ribonucleoside vanadyl complexes), are well known by those of skill in the art as RNAse inhibitors. Berger S L et al. (1979) Biochemistry 18:5143; Puskas R S et al. (1982) Biochemistry 21:4602. Ribonucleoside vanadyl complexes are commercially available from suppliers including Sigma-Aldrich, Inc.
In one embodiment, the immunostimulatory G,U-containing RNA oligomer of the invention does not contain a CpG dinucleotide and is not a CpG immunostimulatory nucleic acid. In some embodiments, a CpG immunostimulatory nucleic acid is used in the methods of the invention.
A CpG immunostimulatory nucleic acid is a nucleic acid which contains a CG dinucleotide, the C residue of which is unmethylated. CpG immunostimulatory nucleic acids are known to stimulate Th1-type immune responses. CpG sequences, while relatively rare in human DNA are commonly found in the DNA of infectious organisms such as bacteria. The human immune system has apparently evolved to recognize CpG sequences as an early warning sign of infection and to initiate an immediate and powerful immune response against invading pathogens without causing adverse reactions frequently seen with other immune stimulatory agents. Thus CpG containing nucleic acids, relying on this innate immune defense mechanism can utilize a unique and natural pathway for immune therapy. The effects of CpG nucleic acids on immune modulation have been described extensively in U.S. patents such as U.S. Pat. Nos. 6,194,388 B1, 6,207,646 B1, 6,239,116 B1 and No. 6,218,371 B1, and published patent applications, such as PCT/US98/03678, PCT/US98/10408, PCT/US98/04703, and PCT/US99/09863. The entire contents of each of these patents and patent applications is hereby incorporated by reference.
A CpG nucleic acid is a nucleic acid which includes at least one unmethylated CpG dinucleotide. A nucleic acid containing at least one unmethylated CpG dinucleotide is a nucleic acid molecule which contains an unmethylated cytosine in a cytosine-guanine dinucleotide sequence (i.e., “CpG DNA” or DNA containing a 5′ cytosine followed by 3′ guanosine and linked by a phosphate bond) and activates the immune system. The CpG nucleic acids can be double-stranded or single-stranded. Generally, double-stranded molecules are more stable in vivo, while single-stranded molecules have increased immune activity. Thus in some aspects of the invention it is preferred that the nucleic acid be single stranded and in other aspects it is preferred that the nucleic acid be double stranded. In certain embodiments, while the nucleic acid is single stranded, it is capable of forming secondary and tertiary structures (e.g., by folding back on itself, or by hybridizing with itself either throughout its entirety or at select segments along its length). Accordingly, while the primary structure of such a nucleic acid may be single stranded, its higher order structures may be double or triple stranded. The terms CpG nucleic acid or CpG oligonucleotide as used herein refer to an immunostimulatory CpG nucleic acid unless otherwise indicated. The entire immunostimulatory nucleic acid can be unmethylated or portions may be unmethylated but at least the C of the 5′ CG 3′ must be unmethylated.
In one aspect the invention provides a method of activating an immune cell. The method involves contacting an immune cell with an immunostimulatory composition of the invention, described above, in an effective amount to induce activation of the immune cell. As used herein, an “immune cell” is cell that belongs to the immune system. Immune cells participate in the regulation and execution of inflammatory and immune responses. They include, without limitation, B lymphocytes (B cells), T lymphocytes (T cells), natural killer (NK) cells, dendritic cells, other tissue-specific antigen-presenting cells (e.g., Langerhans cells), macrophages, monocytes, granulocytes (neutrophils, eosinophils, basophils), and mast cells. Splenocytes, thymocytes, and peripheral blood mononuclear cells (PBMCs) include immune cells. Immune cells can be isolated from the blood, spleen, marrow, lymph nodes, thymus, and other tissues using methods well known to those of skill in the art. Immune cells can also include certain cell lines as well as primary cultures maintained in vitro or ex vivo.
In one embodiment the activation of the immune cell involves secretion of a cytokine by the immune cell. In one embodiment the activation of the immune cell involves secretion of a chemokine by the immune cell. In one embodiment the activation of the immune cell involves expression of a costimulatory/accessory molecule by the immune cell. In one embodiment the costimulatory/accessory molecule is selected from the group consisting of intercellular adhesion molecules (ICAMs, e.g., CD54), leukocyte function-associated antigens (LFAs, e.g., CD58), B7s (CD80, CD86), and CD40.
“Activation of an immune cell” shall refer to a transition of an immune cell from a resting or quiescent state to a state of heightened metabolic activity and phenotype associated with immune cell function. Such immune cell function can include, for example, secretion of soluble products such as immunoglobulins, cytokines, and chemokines; cell surface expression of costimulatory/accessory molecules and MHC antigens; immune cell migration; phagocytosis and cytotoxic activity toward target cells; and immune cell maturation. In some instances immune activation can refer to Th1 immune activation; in other instances immune activation can refer to Th2 immune activation.
“Th1 immune activation” as used herein refers to the activation of immune cells to express Th1-like secreted products, including certain cytokines, chemokines, and subclasses of immunoglobulin; and activation of certain immune cells. Th1-like secreted products include, for example, the cytokines IFN-γ, IL-2, IL-12, IL-18, TNF-α, and the chemokine IP-10 (CXCL10). In the mouse, Th1 immune activation stimulates secretion of IgG2a. Th1 immune activation also may include activation of NK cells and dendritic cells, i.e., cells involved in cellular immunity. Th1 immune activation is believed to counter-regulate Th2 immune activation.
“Th2 immune activation” as used herein refers to the activation of immune cells to express Th2-like secreted products, including certain cytokines and subclasses of immunoglobulin. Th2-like secreted products include, for example, the cytokines IL-4 and IL-10. In the mouse, Th2 immune activation stimulates secretion of IgG1 and IgE. Th2 immune activation is believed to counter-regulate Th1 immune activation.
In another aspect, the invention provides a method of inducing an immune response in a subject. The method entails administering to a subject a composition of the invention in an effective amount to induce an immune response in the subject. Thus the compositions of the invention may be used to treat a subject in need of immune activation. A subject in need of immune activation may include a subject in need of Th1-like immune activation.
The compositions and methods of the invention can be used, alone or in conjunction with other agents, to treat a subject in need of Th1-like immune activation. A “subject in need of Th1-like immune activation” is a subject that has or is at risk of developing a disease, disorder, or condition that would benefit from an immune response skewed toward Th1. Such a subject may have or be at risk of having a Th2-mediated disorder that is susceptible to Th1-mediated cross-regulation or suppression. Such disorders include, for example, certain organ-specific autoimmune diseases. Alternatively, such a subject may have or be at risk of having a Th1-deficient state. Such disorders include, for example, tumors, infections with intracellular pathogens, and AIDS.
As used herein, “G,U-rich RNA” shall mean RNA at least 5 nucleotides long that by base composition is at least 60 percent, more preferably at least 80 percent, and most preferably at least 90 percent guanine (G) and uracil (U). Such base composition is measured over the full length of the RNA if it is no more than 10 bases long, and over a stretch of at least 10 contiguous bases if the RNA is more than 10 bases long.
As used herein, “G-rich RNA” shall mean RNA that by base composition is at least 70 percent, more preferably at least 80 percent, even more preferably at least 90 percent, and most preferably at least 95 percent guanine (G). Such base composition is measured over the full length of the RNA if it is no more than 10 bases long, and over a stretch of at least 10 contiguous bases if the RNA is more than 10 bases long.
In some embodiments the compositions of the present invention include a DNA:RNA conjugate. A DNA:RNA conjugate shall mean a molecule or complex that includes at least one deoxyribonucleoside linked to at least one ribonucleoside. The deoxyribonucleoside and ribonucleoside components may be linked by base pair interaction. Alternatively, the deoxyribonucleoside and ribonucleoside components may be linked by covalent linkage between the sugar moieties of the at least one deoxyribonucleoside and the at least one ribonucleoside. The covalent linkage between the sugar moieties may be direct or indirect, for example through a linker. Base pair interactions typically are, but are not limited to, non-covalent Watson-Crick type base pair interactions. Other base pair interactions, including non-covalent (e.g., Hoogstein base pairing) and covalent interactions are contemplated by the invention. Base pair interactions also typically will involve duplex formation involving two strands, but higher order interactions are also contemplated by the invention.
A DNA:RNA conjugate involving a covalent linkage between the sugar moieties of the at least one deoxyribonucleoside and the at least one ribonucleoside is referred to herein as having a chimeric DNA:RNA backbone. The DNA:RNA conjugate having a chimeric DNA:RNA backbone will have primary structure defined by its base sequence, and it may further have a secondary or higher order structure. A secondary or higher order structure will include at least one intramolecular base pair interaction, e.g., a stem-loop structure, or intermolecular base pair interaction.
Heteroduplex base pairing shall refer to intramolecular or intermolecular base pair interaction between DNA and RNA. For example, heteroduplex base pairing may occur between individual complementary single-stranded DNA and RNA molecules. Alternatively, as in the case of suitable DNA:RNA chimeric backbone nucleic acid molecules, heteroduplex base pairing may occur between complementary DNA and RNA regions within the same molecule.
In some embodiments the compositions of the present invention include a chimeric DNA:RNA backbone having a cleavage site between the DNA and RNA. A cleavage site refers to a structural element along the chimeric backbone that is susceptible to cleavage by any suitable means. The cleavage site may be a phosphodiester bond that is relatively susceptible to cleavage by endonuclease. In this instance the DNA and RNA each may include internucleotide linkages that are stabilized, such that the chimeric backbone is most susceptible to endonuclease cleavage at the phosphodiester junction between the stabilized DNA and the stabilized RNA. The cleavage site may be designed so that it is susceptible to cleavage under certain pH conditions, e.g., relatively more stable at higher pH than at lower pH, or vice versa. Such pH sensitivity may be accomplished, for example, by preparation of the chimeric DNA:RNA composition in liposomes. The cleavage site may involve a disulfide linkage. Such disulfide linkage may be relatively more stable under oxidizing conditions than under reducing conditions, e.g., the latter conditions present within an endosome. The cleavage site may also involve a linker that is susceptible to cleavage by an enzyme, pH, redox condition, or the like. In some embodiments the composition may include more than one cleavage site.
Conjugates of the invention permit selection of fixed molar ratios of the components of the conjugates. In the case of DNA:RNA conjugates it may be advantageous or convenient to have a 1:1 ratio of DNA and RNA. Conjugates that are heteroduplex DNA:RNA will commonly have a 1:1 ratio of DNA and RNA. Conjugates that have a chimeric DNA:RNA backbone may also commonly have a 1:1 ratio of DNA and RNA. Conjugates having other DNA:RNA ratios are contemplated by the invention, including, but not limited to, 1:2, 1:3, 1:4, 2:1, 3:1, 4:1, and so on. The conjugation may stabilize one or more components in comparison to the stability of the same component or components alone. Conjugatation may also facilitate delivery of the components into cells at the selected ratio.
Cleavage sites may serve any of several purposes useful in the present invention. Once delivered to a cell of interest, the components joined via the cleavage site (or sites) may be liberated to become independently or optimally active within the cell or in the vicinity of the cell. In some embodiments the cleavage sites may be important to pharmacokinetics of at least one of the components of the conjugate. For instance, the cleavage sites may be designed and selected to confer an extended time release of one of the components.
The invention generally provides efficient methods of identifying immunostimulatory compounds and the compounds and agents so identified. Generally, the screening methods involve assaying for compounds which inhibit or enhance signaling through a particular TLR. The methods employ a TLR, a suitable reference ligand for the TLR, and a candidate immunostimulatory compound. The selected TLR is contacted with a suitable reference compound (TLR ligand) and a TLR-mediated reference signal is measured. The selected TLR is also contacted with a candidate immunostimulatory compound and a TLR-mediated test signal is measured. The test signal and the reference signal are then compared. A favorable candidate immunostimulatory compound may subsequently be used as a reference compound in the assay. Such methods are adaptable to automated, high throughput screening of candidate compounds. Examples of such high throughput screening methods are described in U.S. Pat. Nos. 6,103,479; 6,051,380; 6,051,373; 5,998,152; 5,876,946; 5,708,158; 5,443,791; 5,429,921; and 5,143,854.
The assay mixture comprises a candidate immunostimulatory compound. Typically, a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a different response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration of agent or at a concentration of agent below the limits of assay detection. Candidate immunostimulatory compounds encompass numerous chemical classes, although typically they are organic compounds. Preferably, the candidate immunostimulatory compounds are small organic compounds, i.e., those having a molecular weight of more than 50 yet less than about 2500. Polymeric candidate immunostimulatory compounds can have higher molecular weights, e.g., oligonucleotides in the range of about 2500 to about 12,500. Candidate immunostimulatory compounds comprise functional chemical groups necessary for structural interactions with polypeptides, and may include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups and more preferably at least three of the functional chemical groups. The candidate immunostimulatory compounds can comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups. Candidate immunostimulatory compounds also can be biomolecules such as nucleic acids, peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like. Where the candidate immunostimulatory compound is a nucleic acid, the candidate immunostimulatory compound typically is a DNA or RNA molecule, although modified nucleic acids having non-natural bonds or subunits are also contemplated.
Candidate immunostimulatory compounds are obtained from a wide variety of sources, including libraries of natural, synthetic, or semisynthetic compounds, or any combination thereof. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily modified through conventional chemical, physical, and biochemical means. Further, known pharmacological agents may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc., to produce structural analogs of the candidate immunostimulatory compounds.
Therefore, a source of candidate immunostimulatory compounds are libraries of molecules based on known TLR ligands, e.g., CpG oligonucleotides known to interact with TLR9, in which the structure of the ligand is changed at one or more positions of the molecule to contain more or fewer chemical moieties or different chemical moieties. The structural changes made to the molecules in creating the libraries of analog inhibitors can be directed, random, or a combination of both directed and random substitutions and/or additions. One of ordinary skill in the art in the preparation of combinatorial libraries can readily prepare such libraries based on existing TLR9 ligands.
A variety of other reagents also can be included in the mixture. These include reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc. which may be used to facilitate optimal protein-protein and/or protein-nucleic acid binding. Such a reagent may also reduce non-specific or background interactions of the reaction components. Other reagents that improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, antimicrobial agents, and the like may also be used.
The order of addition of components, incubation temperature, time of incubation, and other parameters of the assay may be readily determined. Such experimentation merely involves optimization of the assay parameters, not the fundamental composition of the assay. Incubation temperatures typically are between 4° C. and 40° C. Incubation times preferably are minimized to facilitate rapid, high throughput screening, and typically are between 1 minute and 10 hours.
After incubation, the level of TLR signaling is detected by any convenient method available to the user. For cell-free binding type assays, a separation step is often used to separate bound from unbound components. The separation step may be accomplished in a variety of ways. For example, separation can be accomplished in solution, or, conveniently, at least one of the components is immobilized on a solid substrate, from which the unbound components may be easily separated. The solid substrate can be made of a wide variety of materials and in a wide variety of shapes, e.g., microtiter plate, microbead, dipstick, resin particle, etc. The substrate preferably is chosen to maximize signal-to-noise ratios, primarily to minimize background binding, as well as for ease of separation and cost.
Separation may be effected for example, by removing a bead or dipstick from a reservoir, emptying or diluting a reservoir such as a microtiter plate well, rinsing a bead, particle, chromatographic column or filter with a wash solution or solvent. The separation step preferably includes multiple rinses or washes. For example, when the solid substrate is a microtiter plate, the wells may be washed several times with a washing solution, which typically includes those components of the incubation mixture that do not participate in specific bindings such as salts, buffer, detergent, non-specific protein, etc. Where the solid substrate is a magnetic bead, the beads may be washed one or more times with a washing solution and isolated using a magnet.
Detection may be effected in any convenient way for cell-based assays such as measurement of an induced polypeptide within, on the surface of, or secreted by the cell. Examples of detection methods useful in cell-based assays include fluorescence-activated cell sorting (FACS) analysis, bioluminescence, fluorescence, enzyme-linked immunosorbent assay (ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR), and the like. Examples of detection methods useful in cell-free assays include bioluminescence, fluorescence, enzyme-linked immunosorbent assay (ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR), and the like.
A subject shall mean a human or animal including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent, e.g., rats and mice, primate, e.g., monkey, and fish or aquaculture species such as fin fish (e.g., salmon) and shellfish (e.g., shrimp and scallops). Subjects suitable for therapeutic or prophylactic methods include vertebrate and invertebrate species. Subjects can be house pets (e.g., dogs, cats, fish, etc.), agricultural stock animals (e.g., cows, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), zoo animals (e.g., lions, giraffes, etc.), but are not so limited. Although many of the embodiments described herein relate to human disorders, the invention is also useful for treating other nonhuman vertebrates.
As used herein, the term “treat”, when used with respect to one of the disorders described herein, refers both to a prophylactic treatment which decreases the likelihood that a subject will develop the disorder as well as to treatment of an established disorder, e.g., to reduce or eliminate the disorder or symptoms of the disorder, or to prevent the disorder or symptoms of the disorder from becoming worse.
A subject that has a disorder refers to a subject that has an objectively measureable manifestation of the disorder. Thus for example a subject that has a cancer is a subject that has detectable cancerous cells. A subject that has an infection is a subject that has been exposed to an infectious organism and has acute or chronic detectable levels of the organism in the body. The infection may be latent (dormant) or active.
A subject at risk of having a disorder is defined as a subject that has a higher than normal risk of developing the disorder. The normal risk is generally the risk of a population of normal individuals that do not have the disorder and that are not identifiably predisposed, e.g., either genetically or environmentally, to developing the disorder. Thus a subject at risk of having a disorder may include, without limitation, a subject that is genetically predisposed to developing the disorder, as well as a subject that is or will be exposed to an environmental agent known or believed to cause the disorder. Environmental agents specifically include, but are not limited to, infectious agents such as viruses, bacteria, fungi, and parasites. Other environmental agents may include, for example, tobacco smoke, certain organic chemicals, asbestos, and the like.
The term “effective amount” of a nucleic acid or other therapeutic agent refers to the amount necessary or sufficient to realize a desired biologic effect. In general, an effective amount is that amount necessary to cause activation of the immune system, resulting potentially in the development of an antigen-specific immune response. In some embodiments, the nucleic acid or other therapeutic agent are administered in an effective amount to stimulate or induce a Th1 immune response or a general immune response. An effective amount to stimulate a Th1 immune response may be defined as that amount which stimulates the production of one or more Th1-type cytokines, such as IL-2, IL-12, TNF-α, and IFN-γ, and/or production of one or more Th1-type antibodies.
In yet another aspect the invention provides a method of inducing an immune response in a subject. The method according to this aspect of the invention involves administering to a subject an antigen, and administering to the subject an immunostimulatory composition of the invention in an effective amount to induce an immune response to the antigen. It is to be noted that the antigen may be administered before, after, or concurrently with the immunostimulatory composition of the invention. In addition, both the antigen and the immunostimulatory compound can be administered to the subject more than once.
The invention further provides, in yet another aspect, a method of inducing an immune response in a subject. The method according to this aspect of the invention involves isolating dendritic cells of a subject, contacting the dendritic cells ex vivo with an immunostimulatory composition of the invention, contacting the dendritic cells ex vivo with an antigen, and administering the contacted dendritic cells to the subject.
The term “antigen” refers to a molecule capable of provoking an immune response. The term antigen broadly includes any type of molecule that is recognized by a host system as being foreign. Antigens include but are not limited to microbial antigens, cancer antigens, and allergens. Antigens include, but are not limited to, cells, cell extracts, proteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, peptide and non-peptide mimics of polysaccharides and other molecules, small molecules, lipids, glycolipids, and carbohydrates. Many antigens are protein or polypeptide in nature, as proteins and polypeptides are generally more antigenic than carbohydrates or fats.
The antigen may be an antigen that is encoded by a nucleic acid vector or it may not be encoded in a nucleic acid vector. In the former case the nucleic acid vector is administered to the subject and the antigen is expressed in vivo. In the latter case the antigen may be administered directly to the subject. An antigen not encoded in a nucleic acid vector as used herein refers to any type of antigen that is not a nucleic acid. For instance, in some aspects of the invention the antigen not encoded in a nucleic acid vector is a peptide or a polypeptide. Minor modifications of the primary amino acid sequences of peptide or polypeptide antigens may also result in a polypeptide which has substantially equivalent antigenic activity as compared to the unmodified counterpart polypeptide. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein as long as antigenicity still exists. The peptide or polypeptide may be, for example, virally derived. The antigens useful in the invention may be any length, ranging from small peptide fragments of a full length protein or polypeptide to the full length form. For example, the antigen may be less than 5, less than 8, less than 10, less than 15, less than 20, less than 30, less than 50, less than 70, less than 100, or more amino acid residues in length, provided it stimulates a specific immune response.
The nucleic acid encoding the antigen is operatively linked to a gene expression sequence which directs the expression of the antigen nucleic acid within a eukaryotic cell. The gene expression sequence is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient transcription and translation of the antigen nucleic acid to which it is operatively linked. The gene expression sequence may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter. Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, β-actin promoter and other constitutive promoters. Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the cytomegalovirus (CMV), simian virus (e.g., SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, the long terminal repeats (LTR) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters are known to those of ordinary skill in the art. The promoters useful as gene expression sequences of the invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those of ordinary skill in the art.
In general, the gene expression sequence shall include, as necessary, 5′ non-transcribing and 5′ non-translating sequences involved with the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Especially, such 5′ non-transcribing sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined antigen nucleic acid. The gene expression sequences optionally include enhancer sequences or upstream activator sequences as desired.
The antigen nucleic acid is operatively linked to the gene expression sequence. As used herein, the antigen nucleic acid sequence and the gene expression sequence are said to be operably linked when they are covalently linked in such a way as to place the expression or transcription and/or translation of the antigen coding sequence under the influence or control of the gene expression sequence. Two DNA sequences are said to be operably linked if induction of a promoter in the 5′ gene expression sequence results in the transcription of the antigen sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the antigen sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a gene expression sequence would be operably linked to an antigen nucleic acid sequence if the gene expression sequence were capable of effecting transcription of that antigen nucleic acid sequence such that the resulting transcript is translated into the desired protein or polypeptide.
The antigen nucleic acid of the invention may be delivered to the immune system alone or in association with a vector. In its broadest sense, a vector is any vehicle capable of facilitating the transfer of the antigen nucleic acid to the cells of the immune system so that the antigen can be expressed and presented on the surface of the immune cell. The vector generally transports the nucleic acid to the immune cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. The vector optionally includes the above-described gene expression sequence to enhance expression of the antigen nucleic acid in immune cells. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antigen nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to, nucleic acid sequences from the following viruses: retrovirus, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and Rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known in the art.
Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, W.H. Freeman Co., New York (1990) and Murray, E. J. Methods in Molecular Biology, vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).
A preferred virus for certain applications is the adeno-associated virus, a double-stranded DNA virus. The adeno-associated virus can be engineered to be replication-deficient and is capable of infecting a wide range of cell types and species. It further has advantages, such as heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, wild-type adeno-associated virus manifest some preference for integration sites into human cellular DNA, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion. Recombinant adeno-associated viruses that lack the replicase protein apparently lack this integration sequence specificity.
Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well-known to those of skill in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been found to be particularly advantageous for delivering genes to cells in vivo because of their inability to replicate within and integrate into a host genome. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRc/CMV, SV40, and pBlueScript. Other plasmids are well-known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA.
It has recently been discovered that gene-carrying plasmids can be delivered to the immune system using bacteria. Modified forms of bacteria such as Salmonella can be transfected with the plasmid and used as delivery vehicles. The bacterial delivery vehicles can be administered to a host subject orally or by other administration means. The bacteria deliver the plasmid to immune cells, e.g., B cells, dendritic cells, likely by passing through the gut barrier. High levels of immune protection have been established using this methodology. Such methods of delivery are useful for the aspects of the invention utilizing systemic delivery of antigen, nucleic acids, and/or other therapeutic agent.
In some aspects of the invention, the nucleic acids are administered along with therapeutic agents such as disorder-specific medicaments. As used herein, a disorder-specific medicament is a therapy or agent that is used predominately in the treatment or prevention of a disorder.
In one aspect, the combination of nucleic acid and disorder-specific medicaments allows for the administration of higher doses of disorder-specific medicaments without as many side effects as are ordinarily experienced at those high doses. In another aspect, the combination of nucleic acid and disorder-specific medicaments allows for the administration of lower, sub-therapeutic doses of either compound, but with higher efficacy than would otherwise be achieved using such low doses. As one example, by administering a combination of an immunostimulatory nucleic acid and a medicament, it is possible to achieve an effective response even though the medicament is administered at a dose which alone would not provide a therapeutic benefit (i.e., a sub-therapeutic dose). As another example, the combined administration achieves a response even though the nucleic acid is administered at a dose which alone would not provide a therapeutic benefit.
The nucleic acids and/or other therapeutic agents can also be administered on fixed schedules or in different temporal relationships to one another. The various combinations have many advantages over the prior art methods of modulating immune responses or preventing or treating disorders, particularly with regard to decreased non-specific toxicity to normal tissues.
Cancer is a disease which involves the uncontrolled growth (i.e., division) of cells. Some of the known mechanisms which contribute to the uncontrolled proliferation of cancer cells include growth factor independence, failure to detect genomic mutation, and inappropriate cell signaling. The ability of cancer cells to ignore normal growth controls may result in an increased rate of proliferation. Although the causes of cancer have not been firmly established, there are some factors known to contribute, or at least predispose a subject, to cancer. Such factors include particular genetic mutations (e.g., BRCA gene mutation for breast cancer, APC for colon cancer), exposure to suspected cancer-causing agents, or carcinogens (e.g., asbestos, UV radiation) and familial disposition for particular cancers such as breast cancer.
The cancer may be a malignant or non-malignant cancer. Cancers or tumors include but are not limited to biliary tract cancer; brain cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; intraepithelial neoplasms; lymphomas; liver cancer; lung cancer (e.g., small cell and non-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer; pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer; testicular cancer; thyroid cancer; and renal cancer, as well as other carcinomas and sarcomas. In one embodiment the cancer is hairy cell leukemia, chronic myelogenous leukemia, cutaneous T-cell leukemia, multiple myeloma, follicular lymphoma, malignant melanoma, squamous cell carcinoma, renal cell carcinoma, prostate carcinoma, bladder cell carcinoma, or colon carcinoma.
A “subject having a cancer” is a subject that has detectable cancerous cells.
A “subject at risk of developing a cancer” is one who has a higher than normal probability of developing cancer. These subjects include, for instance, subjects having a genetic abnormality that has been demonstrated to be associated with a higher likelihood of developing a cancer, subjects having a familial disposition to cancer, subjects exposed to cancer-causing agents (i.e., carcinogens) such as tobacco, asbestos, or other chemical toxins, and subjects previously treated for cancer and in apparent remission.
A “cancer antigen” as used herein is a compound, such as a peptide or protein, associated with a tumor or cancer cell surface and which is capable of provoking an immune response when expressed on the surface of an antigen presenting cell in the context of an MHC molecule. Cancer antigens can be prepared from cancer cells either by preparing crude extracts of cancer cells, for example, as described in Cohen P A et al. (1994) Cancer Res 54:1055-8, by partially purifying the antigens, by recombinant technology, or by de novo synthesis of known antigens. Cancer antigens include but are not limited to antigens that are recombinantly expressed, an immunogenic portion of, or a whole tumor or cancer. Such antigens can be isolated or prepared recombinantly or by any other means known in the art.
The terms “cancer antigen” and “tumor antigen” are used interchangeably and refer to antigens which are differentially expressed by cancer cells and can thereby be exploited in order to target cancer cells. Cancer antigens are antigens which can potentially stimulate apparently tumor-specific immune responses. Some of these antigens are encoded, although not necessarily expressed, by normal cells. These antigens can be characterized as those which are normally silent (i.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation and those that are temporally expressed such as embryonic and fetal antigens. Other cancer antigens are encoded by mutant cellular genes, such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses. Examples of tumor antigens include MAGE, MART-1/Melan-A, gp100, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, Colorectal associated antigen (CRC)—0017-1A/GA733, Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, Smad family of tumor antigens, lmp-1, HA, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2.
Cancers or tumors and tumor antigens associated with such tumors (but not exclusively), include acute lymphoblastic leukemia (etv6; aml1; cyclophilin b), B cell lymphoma (Ig-idiotype), glioma (E-cadherin; α-catenin; β-catenin; γ-catenin; p120ctn), bladder cancer (p21ras), biliary cancer (p21ras), breast cancer (MUC family; HER2/neu; c-erbB-2), cervical carcinoma (p53; p21ras), colon carcinoma (p21ras; HER2/neu; c-erbB-2; MUC family), colorectal cancer (Colorectal associated antigen (CRC)—0017-1A/GA733; APC), choriocarcinoma (CEA), epithelial cell cancer (cyclophilin b), gastric cancer (HER2/neu; c-erbB-2; ga733 glycoprotein), hepatocellular cancer (α-fetoprotein), Hodgkins lymphoma (lmp-1; EBNA-1), lung cancer (CEA; MAGE-3; NY-ESO-1), lymphoid cell-derived leukemia (cyclophilin b), melanoma (p15 protein, gp75, oncofetal antigen, GM2 and GD2 gangliosides), myeloma (MUC family; p21ras), non-small cell lung carcinoma (HER2/neu; c-erbB-2), nasopharyngeal cancer (lmp-1; EBNA-1), ovarian cancer (MUC family; HER2/neu; c-erbB-2), prostate cancer (Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3; PSMA; HER2/neu; c-erbB-2), pancreatic cancer (p21ras; MUC family; HER2/neu; c-erbB-2; ga733 glycoprotein), renal cancer (HER2/neu; c-erbB-2), squamous cell cancers of cervix and esophagus (viral products such as human papilloma virus proteins), testicular cancer (NY-ESO-1), T-cell leukemia (HTLV-1 epitopes), and melanoma (Melan-A/MART-1; cdc27; MAGE-3; p21ras; gp100Pmel117).
For examples of tumor antigens which bind to either or both MHC class I and MHC class II molecules, see the following references: Coulie, Stem Cells 13:393-403, 1995; Traversari et al. J Exp Med 176:1453-1457, 1992; Chaux et al. J Immunol 163:2928-2936, 1999; Fujie et al. Int J Cancer 80:169-172, 1999; Tanzarella et al. Cancer Res 59:2668-2674, 1999; van der Bruggen et al. Eur J Immunol 24:2134-2140, 1994; Chaux et al. J Exp Med 189:767-778, 1999; Kawashima et al. Hum Immunol 59:1-14, 1998; Tahara et al. Clin Cancer Res 5:2236-2241, 1999; Gaugler et al. J Exp Med 179:921-930, 1994; van der Bruggen et al. Eur J Immunol 24:3038-3043, 1994; Tanaka et al. Cancer Res 57:4465-4468, 1997; Oiso et al. Int J Cancer 81:387-394, 1999; Herman et al. Immunogenetics 43:377-383, 1996; Manici et al. J Exp Med 189:871-876, 1999; Duffour et al. Eur J Immunol 29:3329-3337, 1999; Zorn et al. Eur J Immunol 29:602-607, 1999; Huang et al. J Immunol 162:6849-6854, 1999; Boel et al. Immunity 2:167-175, 1995; Van den Eynde et al. J Exp Med 182:689-698, 1995; De Backer et al. Cancer Res 59:3157-3165, 1999; Jäger et al. J Exp Med 187:265-270, 1998; Wang et al. J Immunol 161:3596-3606, 1998; Aarnoudse et al. Int J Cancer 82:442-448, 1999; Guilloux et al. J Exp Med 183:1173-1183, 1996; Lupetti et al. J Exp Med 188:1005-1016, 1998; Wölfel et al. Eur J Immunol 24:759-764, 1994; Skipper et al. J Exp Med 183:527-534, 1996; Kang et al. J Immunol 155:1343-1348, 1995; Morel et al. Int J Cancer 83:755-759, 1999; Brichard et al. Eur J Immunol 26:224-230, 1996; Kittlesen et al. J Immunol 160:2099-2106, 1998; Kawakami et al. J Immunol 161:6985-6992, 1998; Topalian et al. J Exp Med 183:1965-1971, 1996; Kobayashi et al. Cancer Research 58:296-301, 1998; Kawakami et al. J Immunol 154:3961-3968, 1995; Tsai et al. J Immunol 158:1796-1802, 1997; Cox et al. Science 264:716-719, 1994; Kawakami et al. Proc Natl Acad Sci USA 91:6458-6462, 1994; Skipper et al. J Immunol 157:5027-5033, 1996; Robbins et al. J Immunol 159:303-308, 1997; Castelli et al. J Immunol 162:1739-1748, 1999; Kawakami et al. Exp Med 180:347-352, 1994; Castelli et al. J Exp Med 181:363-368, 1995; Schneider et al. Int J Cancer 75:451-458, 1998; Wang et al. J Exp Med 183:1131-1140, 1996; Wang et al. J Exp Med 184:2207-2216, 1996; Parkhurst et al. Cancer Research 58:4895-4901, 1998; Tsang et al. J Natl Cancer Inst 87:982-990, 1995; Correale et al. J Natl Cancer Inst 89:293-300, 1997; Coulie et al. Proc Natl Acad Sci USA 92:7976-7980, 1995; Wölfel et al. Science 269:1281-1284, 1995; Robbins et al. J Exp Med 183:1185-1192, 1996; Brändle et al. J Exp Med 183:2501-2508, 1996; ten Bosch et al. Blood 88:3522-3527, 1996; Mandruzzato et al. J Exp Med 186:785-793, 1997; Guéguen et al. J Immunol 160:6188-6194, 1998; Gjertsen et al. Int J Cancer 72:784-790, 1997; Gaudin et al. J Immunol 162:1730-1738, 1999; Chiari et al. Cancer Res 59:5785-5792, 1999; Hogan et al. Cancer Res 58:5144-5150, 1998; Pieper et al. J Exp Med 189:757-765, 1999; Wang et al. Science 284:1351-1354, 1999; Fisk et al. J Exp Med 181:2109-2117, 1995; Brossart et al. Cancer Res 58:732-736, 1998; Röpke et al. Proc Natl Acad Sci USA 93:14704-14707, 1996; Ikeda et al. Immunity 6:199-208, 1997; Ronsin et al. J Immunol 163:483-490, 1999; Vonderheide et al. Immunity 10:673-679, 1999. These antigens as well as others are disclosed in PCT Application PCT/US98/18601
The compositions and methods of the invention can be used alone or in conjunction with other agents and methods useful for the treatment of cancer. Cancer is currently treated using a variety of modalities including surgery, radiation therapy and chemotherapy. The choice of treatment modality will depend upon the type, location and dissemination of the cancer. For example, surgery and radiation therapy may be more appropriate in the case of solid, well-defined tumor masses and less practical in the case of non-solid tumor cancers such as leukemia and lymphoma. One of the advantages of surgery and radiation therapy is the ability to control to some extent the impact of the therapy, and thus to limit the toxicity to normal tissues in the body. However, surgery and radiation therapy are often followed by chemotherapy to guard against any remaining or radio-resistant cancer cells. Chemotherapy is also the most appropriate treatment for disseminated cancers such as leukemia and lymphoma as well as metastases.
Chemotherapy refers to therapy using chemical and/or biological agents to attack cancer cells. Unlike localized surgery or radiation, chemotherapy is generally administered in a systemic fashion and thus toxicity to normal tissues is a major concern. Because many chemotherapy agents target cancer cells based on their proliferative profiles, tissues such as the gastrointestinal tract and the bone marrow which are normally proliferative are also susceptible to the effects of the chemotherapy. One of the major side effects of chemotherapy is myelosuppression (including anemia, neutropenia and thrombocytopenia) which results from the death of normal hemopoietic precursors.
Many chemotherapeutic agents have been developed for the treatment of cancer. Not all tumors, however, respond to chemotherapeutic agents and others although initially responsive to chemotherapeutic agents may develop resistance. As a result, the search for effective anti-cancer drugs has intensified in an effort to find even more effective agents with less non-specific toxicity.
Cancer medicaments function in a variety of ways. Some cancer medicaments work by targeting physiological mechanisms that are specific to tumor cells. Examples include the targeting of specific genes and their gene products (i.e., proteins primarily) which are mutated in cancers. Such genes include but are not limited to oncogenes (e.g., Ras, Her2, bcl-2), tumor suppressor genes (e.g., EGF, p53, Rb), and cell cycle targets (e.g., CDK4, p21, telomerase). Cancer medicaments can alternately target signal transduction pathways and molecular mechanisms which are altered in cancer cells. Targeting of cancer cells via the epitopes expressed on their cell surface is accomplished through the use of monoclonal antibodies. This latter type of cancer medicament is generally referred to herein as immunotherapy.
Other cancer medicaments target cells other than cancer cells. For example, some medicaments prime the immune system to attack tumor cells (i.e., cancer vaccines). Still other medicaments, called angiogenesis inhibitors, function by attacking the blood supply of solid tumors. Since the most malignant cancers are able to metastasize (i.e., exit the primary tumor site and seed a another site, thereby forming a secondary tumor), medicaments that impede this metastasis are also useful in the treatment of cancer. Angiogenic mediators include basic FGF, VEGF, angiopoietins, angiostatin, endostatin, TNF-α, TNP-470, thrombospondin-1, platelet factor 4, CAI, and certain members of the integrin family of proteins. One category of this type of medicament is a metalloproteinase inhibitor, which inhibits the enzymes used by the cancer cells to exist the primary tumor site and extravasate into another tissue.
Some cancer cells are antigenic and thus can be targeted by the immune system. In one aspect, the combined administration of nucleic acid and cancer medicaments, particularly those which are classified as cancer immunotherapies, is useful for stimulating a specific immune response against a cancer antigen.
The theory of immune surveillance is that a prime function of the immune system is to detect and eliminate neoplastic cells before a tumor forms. A basic principle of this theory is that cancer cells are antigenically different from normal cells and thus elicit immune reactions that are similar to those that cause rejection of immunologically incompatible allografts. Studies have confirmed that tumor cells differ, either qualitatively or quantitatively, in their expression of antigens. For example, “tumor-specific antigens” are antigens that are specifically associated with tumor cells but not normal cells. Examples of tumor specific antigens are viral antigens in tumors induced by DNA or RNA viruses. “Tumor-associated” antigens are present in both tumor cells and normal cells but are present in a different quantity or a different form in tumor cells. Examples of such antigens are oncofetal antigens (e.g., carcinoembryonic antigen), differentiation antigens (e.g., T and Tn antigens), and oncogene products (e.g., HER/neu).
Different types of cells that can kill tumor targets in vitro and in vivo have been identified: natural killer (NK) cells, cytolytic T lymphocytes (CTLs), lymphokine-activated killer cells (LAKs), and activated macrophages. NK cells can kill tumor cells without having been previously sensitized to specific antigens, and the activity does not require the presence of class I antigens encoded by the major histocompatibility complex (MHC) on target cells. NK cells are thought to participate in the control of nascent tumors and in the control of metastatic growth. In contrast to NK cells, CTLs can kill tumor cells only after they have been sensitized to tumor antigens and when the target antigen is expressed on the tumor cells that also express MHC class I. CTLs are thought to be effector cells in the rejection of transplanted tumors and of tumors caused by DNA viruses. LAK cells are a subset of null lymphocytes distinct from the NK and CTL populations. Activated macrophages can kill tumor cells in a manner that is neither antigen-dependent nor MHC-restricted once activated. Activated macrophages are through to decrease the growth rate of the tumors they infiltrate. In vitro assays have identified other immune mechanisms such as antibody-dependent, cell-mediated cytotoxic reactions and lysis by antibody plus complement. However, these immune effector mechanisms are thought to be less important in vivo than the function of NK, CTLs, LAK, and macrophages in vivo (for review see Piessens W F et al. “Tumor Immunology”, In: Scientific American Medicine, Vol. 2, Scientific American Books, N.Y., pp. 1-13, 1996).
The goal of immunotherapy is to augment a patient's immune response to an established tumor. One method of immunotherapy includes the use of adjuvants. Adjuvant substances derived from microorganisms, such as bacillus Calmette-Guérin, heighten the immune response and enhance resistance to tumors in animals.
Immunotherapeutic agents are medicaments which derive from antibodies or antibody fragments which specifically bind or recognize a cancer antigen. Antibody-based immunotherapies may function by binding to the cell surface of a cancer cell and thereby stimulate the endogenous immune system to attack the cancer cell. Another way in which antibody-based therapy functions is as a delivery system for the specific targeting of toxic substances to cancer cells. Antibodies are usually conjugated to toxins such as ricin (e.g., from castor beans), calicheamicin and maytansinoids, to radioactive isotopes such as Iodine-131 and Yttrium-90, to chemotherapeutic agents (as described herein), or to biological response modifiers. In this way, the toxic substances can be concentrated in the region of the cancer and non-specific toxicity to normal cells can be minimized. In addition to the use of antibodies which are specific for cancer antigens, antibodies which bind to vasculature, such as those which bind to endothelial cells, are also useful in the invention. This is because solid tumors generally are dependent upon newly formed blood vessels to survive, and thus most tumors are capable of recruiting and stimulating the growth of new blood vessels. As a result, one strategy of many cancer medicaments is to attack the blood vessels feeding a tumor and/or the connective tissues (or stroma) supporting such blood vessels.
Cancer vaccines are medicaments which are intended to stimulate an endogenous immune response against cancer cells. Currently produced vaccines predominantly activate the humoral immune system (i.e., the antibody-dependent immune response). Other vaccines currently in development are focused on activating the cell-mediated immune system including cytotoxic T lymphocytes which are capable of killing tumor cells. Cancer vaccines generally enhance the presentation of cancer antigens to both antigen presenting cells (e.g., macrophages and dendritic cells) and/or to other immune cells such as T cells, B cells, and NK cells.
Although cancer vaccines may take one of several forms, as discussed infra, their purpose is to deliver cancer antigens and/or cancer associated antigens to antigen presenting cells (APC) in order to facilitate the endogenous processing of such antigens by APC and the ultimate presentation of antigen presentation on the cell surface in the context of MHC class I molecules. One form of cancer vaccine is a whole cell vaccine which is a preparation of cancer cells which have been removed from a subject, treated ex vivo and then reintroduced as whole cells in the subject. Lysates of tumor cells can also be used as cancer vaccines to elicit an immune response. Another form cancer vaccine is a peptide vaccine which uses cancer-specific or cancer-associated small proteins to activate T cells. Cancer-associated proteins are proteins which are not exclusively expressed by cancer cells (i.e., other normal cells may still express these antigens). However, the expression of cancer-associated antigens is generally consistently upregulated with cancers of a particular type. Other cancer vaccines include ganglioside vaccines, heat-shock protein vaccines, viral and bacterial vaccines, and nucleic acid vaccines.
Yet another form of cancer vaccine is a dendritic cell vaccine which includes whole dendritic cells which have been exposed to a cancer antigen or a cancer-associated antigen in vitro. Lysates or membrane fractions of dendritic cells may also be used as cancer vaccines. Dendritic cell vaccines are able to activate APCs directly. A dendritic cell is a professional APC. Dendritic cells form the link between the innate and the acquired immune system by presenting antigens and through their expression of pattern recognition receptors which detect microbial molecules like LPS in their local environment. Dendritic cells efficiently internalize, process, and present soluble specific antigen to which it is exposed. The process of internalizing and presenting antigen causes rapid upregulation of the expression of major histocompatibility complex (MHC) and costimulatory molecules, the production of cytokines, and migration toward lymphatic organs where they are believed to be involved in the activation of T cells.
As used herein, chemotherapeutic agents embrace all other forms of cancer medicaments which do not fall into the categories of immunotherapeutic agents or cancer vaccines. Chemotherapeutic agents as used herein encompass both chemical and biological agents. These agents function to inhibit a cellular activity which the cancer cell is dependent upon for continued survival. Categories of chemotherapeutic agents include alkylating/alkaloid agents, antimetabolites, hormones or hormone analogs, and miscellaneous antineoplastic drugs. Most if not all of these agents are directly toxic to cancer cells and do not require immune stimulation.
An “infectious disease” or, equivalently, an “infection” as used herein, refers to a disorder arising from the invasion of a host, superficially, locally, or systemically, by an infectious organism. Infectious organisms include bacteria, viruses, fungi, and parasites. Accordingly, “infectious disease” includes bacterial infections, viral infections, fungal infections and parasitic infections.
A subject having an infectious disease is a subject that has been exposed to an infectious organism and has acute or chronic detectable levels of the organism in the body. Exposure to the infectious organism generally occurs with the external surface of the subject, e.g., skin or mucosal membranes and/or refers to the penetration of the external surface of the subject by the infectious organism.
A subject at risk of developing an infectious disease is a subject who has a higher than normal risk of exposure to an infection causing pathogen. For instance, a subject at risk may be a subject who is planning to travel to an area where a particular type of infectious agent is found or it may be a subject who through lifestyle or medical procedures is exposed to bodily fluids which may contain infectious organisms or directly to the organism or a subject living in an area where an infectious organism has been identified. Subjects at risk of developing an infectious disease also include general populations to which a medical agency recommends vaccination against a particular infectious organism.
A subject at risk of developing an infectious disease includes those subjects that have a general risk of exposure to a microorganism, e.g., influenza, but that do not have the active disease during the treatment of the invention, as well as subjects that are considered to be at specific risk of developing an infectious disease because of medical or environmental factors that expose the subject to a particular microorganism.
Bacteria are unicellular organisms which multiply asexually by binary fission. They are classified and named based on their morphology, staining reactions, nutrition and metabolic requirements, antigenic structure, chemical composition, and genetic homology. Bacteria can be classified into three groups based on their morphological forms, spherical (coccus), straight-rod (bacillus) and curved or spiral rod (vibrio, campylobacter, spirillum, and spirochaete). Bacteria are also more commonly characterized based on their staining reactions into two classes of organisms, gram-positive and gram-negative. Gram refers to the method of staining which is commonly performed in microbiology labs. Gram-positive organisms retain the stain following the staining procedure and appear a deep violet color. Gram-negative organisms do not retain the stain but take up the counter-stain and thus appear pink.
Infectious bacteria include, but are not limited to, gram negative and gram positive bacteria. Gram positive bacteria include, but are not limited to Pasteurella species, Staphylococci species, and Streptococcus species. Gram negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include but are not limited to: Helicobacter pyloris, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansasii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponema pertenue, Leptospira, Rickettsia, and Actinomyces israelli.
Viruses are small infectious agents which generally contain a nucleic acid core and a protein coat, but are not independently living organisms. Viruses can also take the form of infectious nucleic acids lacking a protein. A virus cannot survive in the absence of a living cell within which it can replicate. Viruses enter specific living cells either by endocytosis or direct injection of DNA (phage) and multiply, causing disease. The multiplied virus can then be released and infect additional cells. Some viruses are DNA-containing viruses and others are RNA-containing viruses. In some aspects, the invention also intends to treat diseases in which prions are implicated in disease progression such as for example bovine spongiform encephalopathy (i.e., mad cow disease, BSE) or scrapie infection in animals, or Creutzfeldt-Jakob disease in humans.
Viruses include, but are not limited to, enteroviruses (including, but not limited to, viruses that the family picornaviridae, such as polio virus, coxsackie virus, echo virus), rotaviruses, adenovirus, hepatitis virus. Specific examples of viruses that have been found in humans include but are not limited to: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papillomaviruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV)); Poxyiridae (variola viruses, vaccinia viruses, pox viruses); Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the etiological agents of spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1=internally transmitted; class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk and related viruses, and astroviruses).
Fungi are eukaryotic organisms, only a few of which cause infection in vertebrate mammals. Because fungi are eukaryotic organisms, they differ significantly from prokaryotic bacteria in size, structural organization, life cycle and mechanism of multiplication. Fungi are classified generally based on morphological features, modes of reproduction and culture characteristics. Although fungi can cause different types of disease in subjects, such as respiratory allergies following inhalation of fungal antigens, fungal intoxication due to ingestion of toxic substances, such as Amanita phalloides toxin and phallotoxin produced by poisonous mushrooms and aflatoxins, produced by aspergillus species, not all fungi cause infectious disease.
Infectious fungi can cause systemic or superficial infections. Primary systemic infection can occur in normal healthy subjects, and opportunistic infections are most frequently found in immunocompromised subjects. The most common fungal agents causing primary systemic infection include Blastomyces, Coccidioides, and Htoplasma. Common fungi causing opportunistic infection in immunocompromised or immunosuppressed subjects include, but are not limited to, Candida albicans, Cryptococcus neoformans, and various Aspergillus species. Systemic fungal infections are invasive infections of the internal organs. The organism usually enters the body through the lungs, gastrointestinal tract, or intravenous catheters. These types of infections can be caused by primary pathogenic fungi or opportunistic fungi.
Superficial fungal infections involve growth of fungi on an external surface without invasion of internal tissues. Typical superficial fungal infections include cutaneous fungal infections involving skin, hair, or nails.
Diseases associated with fungal infection include aspergillosis, blastomycosis, candidiasis, chromoblastomycosis, coccidioidomycosis, cryptococcosis, fungal eye infections, fungal hair, nail, and skin infections, histoplasmosis, lobomycosis, mycetoma, otomycosis, paracoccidioidomycosis, disseminated Penicillium marneffei, phaeohyphomycosis, rhinosporidioisis, sporotrichosis, and zygomycosis.
Parasites are organisms which depend upon other organisms in order to survive and thus must enter, or infect, another organism to continue their life cycle. The infected organism, i.e., the host, provides both nutrition and habitat to the parasite. Although in its broadest sense the term parasite can include all infectious agents (i.e., bacteria, viruses, fungi, protozoa and helminths), generally speaking, the term is used to refer solely to protozoa, helminths, and ectoparasitic arthropods (e.g., ticks, mites, etc.). Protozoa are single-celled organisms which can replicate both intracellularly and extracellularly, particularly in the blood, intestinal tract or the extracellular matrix of tissues. Helminths are multicellular organisms which almost always are extracellular (an exception being Trichinella spp.). Helminths normally require exit from a primary host and transmission into a secondary host in order to replicate. In contrast to these aforementioned classes, ectoparasitic arthropods form a parasitic relationship with the external surface of the host body.
Parasites include intracellular parasites and obligate intracellular parasites. Examples of parasites include but are not limited to Plasmodium falciparum, Plasmodium ovale, Plasmodium malariae, Plasmdodium vivax, Plasmodium knowlesi, Babesia micron, Babesia divergens, Trypanosoma cruzi, Toxoplasma gondii, Trichinella spiralis, Leishmania major, Leishmania donovani, Leishmania braziliensis, Leishmania tropica, Trypanosoma gambiense, Trypanosoma rhodesiense and Schistosoma mansoni.
Other medically relevant microorganisms have been described extensively in the literature, e.g., see C. G. A Thomas, Medical Microbiology, Bailliere Tindall, Great Britain 1983, the entire contents of which is hereby incorporated by reference. Each of the foregoing lists is illustrative and is not intended to be limiting.
The compositions and methods of the invention can be used alone or in conjunction with other agents and methods useful for the treatment of infection. Infection medicaments include but are not limited to anti-bacterial agents, anti-viral agents, anti-fungal agents and anti-parasitic agents. Phrases such as “anti-infective agent”, “antibiotic”, “anti-bacterial agent”, “anti-viral agent”, “anti-fungal agent”, “anti-parasitic agent” and “parasiticide” have well-established meanings to those of ordinary skill in the art and are defined in standard medical texts. Briefly, anti-bacterial agents kill or inhibit bacteria, and include antibiotics as well as other synthetic or natural compounds having similar functions. Anti-viral agents can be isolated from natural sources or synthesized and are useful for killing or inhibiting viruses. Anti-fungal agents are used to treat superficial fungal infections as well as opportunistic and primary systemic fungal infections. Anti-parasite agents kill or inhibit parasites. Many antibiotics are low molecular weight molecules which are produced as secondary metabolites by cells, such as microorganisms. In general, antibiotics interfere with one or more functions or structures which are specific for the microorganism and which are not present in host cells.
One of the problems with anti-infective therapies is the side effects occurring in the host that is treated with the anti-infective agent. For instance, many anti-infectious agents can kill or inhibit a broad spectrum of microorganisms and are not specific for a particular type of species. Treatment with these types of anti-infectious agents results in the killing of the normal microbial flora living in the host, as well as the infectious microorganism. The loss of the microbial flora can lead to disease complications and predispose the host to infection by other pathogens, since the microbial flora compete with and function as barriers to infectious pathogens. Other side effects may arise as a result of specific or non-specific effects of these chemical entities on non-microbial cells or tissues of the host.
Another problem with widespread use of anti-infectants is the development of antibiotic-resistant strains of microorganisms. Already, vancomycin-resistant enterococci, penicillin-resistant pneumococci, multi-resistant S. aureus, and multi-resistant tuberculosis strains have developed and are becoming major clinical problems. Widespread use of anti-infectants will likely produce many antibiotic-resistant strains of bacteria. As a result, new anti-infective strategies will be required to combat these microorganisms.
Antibacterial antibiotics which are effective for killing or inhibiting a wide range of bacteria are referred to as broad-spectrum antibiotics. Other types of antibacterial antibiotics are predominantly effective against the bacteria of the class gram-positive or gram-negative. These types of antibiotics are referred to as narrow-spectrum antibiotics. Other antibiotics which are effective against a single organism or disease and not against other types of bacteria, are referred to as limited-spectrum antibiotics.
Anti-bacterial agents are sometimes classified based on their primary mode of action. In general, anti-bacterial agents are cell wall synthesis inhibitors, cell membrane inhibitors, protein synthesis inhibitors, nucleic acid synthesis or functional inhibitors, and competitive inhibitors. Cell wall synthesis inhibitors inhibit a step in the process of cell wall synthesis, and in general in the synthesis of bacterial peptidoglycan. Cell wall synthesis inhibitors include β-lactam antibiotics, natural penicillins, semi-synthetic penicillins, ampicillin, clavulanic acid, cephalolsporins, and bacitracin.
The β-lactams are antibiotics containing a four-membered β-lactam ring which inhibits the last step of peptidoglycan synthesis. β-lactam antibiotics can be synthesized or natural. The β-lactam antibiotics produced by penicillium are the natural penicillins, such as penicillin G or penicillin V. These are produced by fermentation of Penicillium chrysogenum. The natural penicillins have a narrow spectrum of activity and are generally effective against Streptococcus, Gonococcus, and Staphylococcus. Other types of natural penicillins, which are also effective against gram-positive bacteria, include penicillins F, X, K, and O.
Semi-synthetic penicillins are generally modifications of the molecule 6-aminopenicillanic acid produced by a mold. The 6-aminopenicillanic acid can be modified by addition of side chains which produce penicillins having broader spectrums of activity than natural penicillins or various other advantageous properties. Some types of semi-synthetic penicillins have broad spectrums against gram-positive and gram-negative bacteria, but are inactivated by penicillinase. These semi-synthetic penicillins include ampicillin, carbenicillin, oxacillin, azlocillin, mezlocillin, and piperacillin. Other types of semi-synthetic penicillins have narrower activities against gram-positive bacteria, but have developed properties such that they are not inactivated by penicillinase. These include, for instance, methicillin, dicloxacillin, and nafcillin. Some of the broad spectrum semi-synthetic penicillins can be used in combination with β-lactamase inhibitors, such as clavulanic acids and sulbactam. The β-lactamase inhibitors do not have anti-microbial action but they function to inhibit penicillinase, thus protecting the semi-synthetic penicillin from degradation.
One of the serious side effects associated with penicillins, both natural and semi-synthetic, is penicillin allergy. Penicillin allergies are very serious and can cause death rapidly. In a subject that is allergic to penicillin, the β-lactam molecule will attach to a serum protein which initiates an IgE-mediated inflammatory response. The inflammatory response leads to anaphylaxis and possibly death.
Another type of β-lactam antibiotic is the cephalolsporins. They are sensitive to degradation by bacterial β-lactamases, and thus, are not always effective alone. Cephalolsporins, however, are resistant to penicillinase. They are effective against a variety of gram-positive and gram-negative bacteria. Cephalolsporins include, but are not limited to, cephalothin, cephapirin, cephalexin, cefamandole, cefaclor, cefazolin, cefuroxine, cefoxitin, cefotaxime, cefsulodin, cefetamet, cefixime, ceftriaxone, cefoperazone, ceftazidine, and moxalactam.
Bacitracin is another class of antibiotics which inhibit cell wall synthesis, by inhibiting the release of muropeptide subunits or peptidoglycan from the molecule that delivers the subunit to the outside of the membrane. Although bacitracin is effective against gram-positive bacteria, its use is limited in general to topical administration because of its high toxicity.
Carbapenems are another broad-spectrum β-lactam antibiotic, which is capable of inhibiting cell wall synthesis. Examples of carbapenems include, but are not limited to, imipenems. Monobactams are also broad-spectrum β-lactam antibiotics, and include, eurtreonam. An antibiotic produced by Streptomyces, vancomycin, is also effective against gram-positive bacteria by inhibiting cell membrane synthesis.
Another class of anti-bacterial agents is the anti-bacterial agents that are cell membrane inhibitors. These compounds disorganize the structure or inhibit the function of bacterial membranes. One problem with anti-bacterial agents that are cell membrane inhibitors is that they can produce effects in eukaryotic cells as well as bacteria because of the similarities in phospholipids in bacterial and eukaryotic membranes. Thus these compounds are rarely specific enough to permit these compounds to be used systemically and prevent the use of high doses for local administration.
One clinically useful cell membrane inhibitor is Polymyxin. Polymyxins interfere with membrane function by binding to membrane phospholipids. Polymyxin is effective mainly against Gram-negative bacteria and is generally used in severe Pseudomonas infections or Pseudomonas infections that are resistant to less toxic antibiotics. The severe side effects associated with systemic administration of this compound include damage to the kidney and other organs.
Other cell membrane inhibitors include Amphotericin B and Nystatin which are anti-fungal agents used predominantly in the treatment of systemic fungal infections and Candida yeast infections. Imidazoles are another class of antibiotic that is a cell membrane inhibitor. Imidazoles are used as anti-bacterial agents as well as anti-fungal agents, e.g., used for treatment of yeast infections, dermatophytic infections, and systemic fungal infections. Imidazoles include but are not limited to clotrimazole, miconazole, ketoconazole, itraconazole, and fluconazole.
Many anti-bacterial agents are protein synthesis inhibitors. These compounds prevent bacteria from synthesizing structural proteins and enzymes and thus cause inhibition of bacterial cell growth or function or cell death. In general these compounds interfere with the processes of transcription or translation. Anti-bacterial agents that block transcription include but are not limited to Rifampins and Ethambutol. Rifampins, which inhibit the enzyme RNA polymerase, have a broad spectrum activity and are effective against gram-positive and gram-negative bacteria as well as Mycobacterium tuberculosis. Ethambutol is effective against Mycobacterium tuberculosis.
Anti-bacterial agents which block translation interfere with bacterial ribosomes to prevent mRNA from being translated into proteins. In general this class of compounds includes but is not limited to tetracyclines, chloramphenicol, the macrolides (e.g., erythromycin) and the aminoglycosides (e.g., streptomycin).
The aminoglycosides are a class of antibiotics which are produced by the bacterium Streptomyces, such as, for instance streptomycin, kanamycin, tobramycin, amikacin, and gentamicin Aminoglycosides have been used against a wide variety of bacterial infections caused by Gram-positive and Gram-negative bacteria. Streptomycin has been used extensively as a primary drug in the treatment of tuberculosis. Gentamicin is used against many strains of Gram-positive and Gram-negative bacteria, including Pseudomonas infections, especially in combination with Tobramycin. Kanamycin is used against many Gram-positive bacteria, including penicillin-resistant Staphylococci. One side effect of aminoglycosides that has limited their use clinically is that at dosages which are essential for efficacy, prolonged use has been shown to impair kidney function and cause damage to the auditory nerves leading to deafness.
Another type of translation inhibitor anti-bacterial agent is the tetracyclines. The tetracyclines are a class of antibiotics that are broad-spectrum and are effective against a variety of gram-positive and gram-negative bacteria. Examples of tetracyclines include tetracycline, minocycline, doxycycline, and chlortetracycline. They are important for the treatment of many types of bacteria but are particularly important in the treatment of Lyme disease. As a result of their low toxicity and minimal direct side effects, the tetracyclines have been overused and misused by the medical community, leading to problems. For instance, their overuse has led to widespread development of resistance.
Anti-bacterial agents such as the macrolides bind reversibly to the 50 S ribosomal subunit and inhibit elongation of the protein by peptidyl transferase or prevent the release of uncharged tRNA from the bacterial ribosome or both. These compounds include erythromycin, roxithromycin, clarithromycin, oleandomycin, and azithromycin. Erythromycin is active against most Gram-positive bacteria, Neisseria, Legionella and Haemophilus, but not against the Enterobacteriaceae. Lincomycin and clindamycin, which block peptide bond formation during protein synthesis, are used against gram-positive bacteria.
Another type of translation inhibitor is chloramphenicol. Chloramphenicol binds the 70 S ribosome inhibiting the bacterial enzyme peptidyl transferase thereby preventing the growth of the polypeptide chain during protein synthesis. One serious side effect associated with chloramphenicol is aplastic anemia. Aplastic anemia develops at doses of chloramphenicol which are effective for treating bacteria in a small proportion (1/50,000) of patients. Chloramphenicol which was once a highly prescribed antibiotic is now seldom uses as a result of the deaths from anemia. Because of its effectiveness it is still used in life-threatening situations (e.g., typhoid fever).
Some anti-bacterial agents disrupt nucleic acid synthesis or function, e.g., bind to DNA or RNA so that their messages cannot be read. These include but are not limited to quinolones and co-trimoxazole, both synthetic chemicals and rifamycins, a natural or semi-synthetic chemical. The quinolones block bacterial DNA replication by inhibiting the DNA gyrase, the enzyme needed by bacteria to produce their circular DNA. They are broad spectrum and examples include norfloxacin, ciprofloxacin, enoxacin, nalidixic acid and temafloxacin. Nalidixic acid is a bactericidal agent that binds to the DNA gyrase enzyme (topoisomerase) which is essential for DNA replication and allows supercoils to be relaxed and reformed, inhibiting DNA gyrase activity. The main use of nalidixic acid is in treatment of lower urinary tract infections (UTI) because it is effective against several types of Gram-negative bacteria such as E. coli, Enterobacter aerogenes, K. pneumoniae and Proteus species which are common causes of UTI. Co-trimoxazole is a combination of sulfamethoxazole and trimethoprim, which blocks the bacterial synthesis of folic acid needed to make DNA nucleotides. Rifampicin is a derivative of rifamycin that is active against Gram-positive bacteria (including Mycobacterium tuberculosis and meningitis caused by Neisseria meningitidis) and some Gram-negative bacteria. Rifampicin binds to the beta subunit of the polymerase and blocks the addition of the first nucleotide which is necessary to activate the polymerase, thereby blocking mRNA synthesis.
Another class of anti-bacterial agents is compounds that function as competitive inhibitors of bacterial enzymes. The competitive inhibitors are mostly all structurally similar to a bacterial growth factor and compete for binding but do not perform the metabolic function in the cell. These compounds include sulfonamides and chemically modified forms of sulfanilamide which have even higher and broader antibacterial activity. The sulfonamides (e.g., gantrisin and trimethoprim) are useful for the treatment of Streptococcus pneumoniae, beta-hemolytic streptococci and E. coli, and have been used in the treatment of uncomplicated UTI caused by E. coli, and in the treatment of meningococcal meningitis.
Anti-viral agents are compounds which prevent infection of cells by viruses or replication of the virus within the cell. There are many fewer antiviral drugs than antibacterial drugs because the process of viral replication is so closely related to DNA replication within the host cell, that non-specific antiviral agents would often be toxic to the host. There are several stages within the process of viral infection which can be blocked or inhibited by antiviral agents. These stages include, attachment of the virus to the host cell (immunoglobulin or binding peptides), uncoating of the virus (e.g. amantadine), synthesis or translation of viral mRNA (e.g. interferon), replication of viral RNA or DNA (e.g. nucleoside analogues), maturation of new virus proteins (e.g. protease inhibitors), and budding and release of the virus.
Another category of anti-viral agents are nucleoside analogues. Nucleoside analogues are synthetic compounds which are similar to nucleosides, but which have an incomplete or abnormal deoxyribose or ribose group. Once the nucleoside analogues are in the cell, they are phosphorylated, producing the triphosphate form which competes with normal nucleotides for incorporation into the viral DNA or RNA. Once the triphosphate form of the nucleoside analogue is incorporated into the growing nucleic acid chain, it causes irreversible association with the viral polymerase and thus chain termination. Nucleoside analogues include, but are not limited to, acyclovir (used for the treatment of herpes simplex virus and varicella-zoster virus), gancyclovir (useful for the treatment of cytomegalovirus), idoxuridine, ribavirin (useful for the treatment of respiratory syncitial virus), dideoxyinosine, dideoxycytidine, and zidovudine (azidothymidine).
Another class of anti-viral agents includes cytokines such as interferons. The interferons are cytokines which are secreted by virus-infected cells as well as immune cells. The interferons function by binding to specific receptors on cells adjacent to the infected cells, causing the change in the cell which protects it from infection by the virus. α and β-interferon also induce the expression of Class I and Class II MHC molecules on the surface of infected cells, resulting in increased antigen presentation for host immune cell recognition. α and β-interferons are available as recombinant forms and have been used for the treatment of chronic hepatitis B and C infection. At the dosages which are effective for anti-viral therapy, interferons have severe side effects such as fever, malaise and weight loss.
Immunoglobulin therapy is used for the prevention of viral infection. Immunoglobulin therapy for viral infections is different from bacterial infections, because rather than being antigen-specific, the immunoglobulin therapy functions by binding to extracellular virions and preventing them from attaching to and entering cells which are susceptible to the viral infection. The therapy is useful for the prevention of viral infection for the period of time that the antibodies are present in the host. In general there are two types of immunoglobulin therapies, normal immune globulin therapy and hyper-immune globulin therapy. Normal immune globulin therapy utilizes a antibody product which is prepared from the serum of normal blood donors and pooled. This pooled product contains low titers of antibody to a wide range of human viruses, such as hepatitis A, parvovirus, enterovirus (especially in neonates). Hyper-immune globulin therapy utilizes antibodies which are prepared from the serum of individuals who have high titers of an antibody to a particular virus. Those antibodies are then used against a specific virus. Examples of hyper-immune globulins include zoster immune globulin (useful for the prevention of varicella in immunocompromised children and neonates), human rabies immune globulin (useful in the post-exposure prophylaxis of a subject bitten by a rabid animal), hepatitis B immune globulin (useful in the prevention of hepatitis B virus, especially in a subject exposed to the virus), and RSV immune globulin (useful in the treatment of respiratory syncitial virus infections).
Anti-fungal agents are useful for the treatment and prevention of infective fungi. Anti-fungal agents are sometimes classified by their mechanism of action. Some anti-fungal agents function as cell wall inhibitors by inhibiting glucose synthase. These include, but are not limited to, basiungin/ECB. Other anti-fungal agents function by destabilizing membrane integrity. These include, but are not limited to, imidazoles, such as clotrimazole, sertaconzole, fluconazole, itraconazole, ketoconazole, miconazole, and voriconacole, as well as FK 463, amphotericin B, BAY 38-9502, MK 991, pradimicin, UK 292, butenafine, and terbinafine. Other anti-fungal agents function by breaking down chitin (e.g., chitinase) or immunosuppression (501 cream).
Parasiticides are agents that kill parasites directly. Such compounds are known in the art and are generally commercially available. Examples of parasiticides useful for human administration include but are not limited to albendazole, amphotericin B, benznidazole, bithionol, chloroquine HCl, chloroquine phosphate, clindamycin, dehydroemetine, diethylcarbamazine, diloxanide furoate, eflornithine, furazolidaone, glucocorticoids, halofantrine, iodoquinol, ivermectin, mebendazole, mefloquine, meglumine antimoniate, melarsoprol, metrifonate, metronidazole, niclosamide, nifurtimox, oxamniquine, paromomycin, pentamidine isethionate, piperazine, praziquantel, primaquine phosphate, proguanil, pyrantel pamoate, pyrimethanmine-sulfonamides, pyrimethanmine-sulfadoxine, quinacrine HCl, quinine sulfate, quinidine gluconate, spiramycin, stibogluconate sodium (sodium antimony gluconate), suramin, tetracycline, doxycycline, thiabendazole, tinidazole, trimethroprim-sulfamethoxazole, and tryparsamide.
The compositions and methods of the invention may also find use in the treatment of allergy and asthma.
An “allergy” refers to acquired hypersensitivity to a substance (allergen). Allergic conditions include but are not limited to eczema, allergic rhinitis or coryza, hay fever, allergic conjunctivitis, bronchial asthma, urticaria (hives) and food allergies, other atopic conditions including atopic dermatitis; anaphylaxis; drug allergy; and angioedema. Allergic diseases include but are not limited to rhinitis (hay fever), asthma, urticaria, and atopic dermatitis.
Allergy is a disease associated with the production of antibodies from a particular class of immunoglobulin, IgE, against allergens. The development of an IgE-mediated response to common aeroallergens is also a factor which indicates predisposition towards the development of asthma. If an allergen encounters a specific IgE which is bound to an IgE Fc receptor (FcεR) on the surface of a basophil (circulating in the blood) or mast cell (dispersed throughout solid tissue), the cell becomes activated, resulting in the production and release of mediators such as histamine, serotonin, and lipid mediators.
A subject having an allergy is a subject that is currently experiencing or has previously experienced an allergic reaction in response to an allergen.
A subject at risk of developing an allergy or asthma is a subject that has been identified as having an allergy or asthma in the past but who is not currently experiencing the active disease, as well as a subject that is considered to be at risk of developing asthma or allergy because of genetic or environmental factors. A subject at risk of developing allergy or asthma can also include a subject who has any risk of exposure to an allergen or a risk of developing asthma, i.e., someone who has suffered from an asthmatic attack previously or has a predisposition to asthmatic attacks. For instance, a subject at risk may be a subject who is planning to travel to an area where a particular type of allergen or asthmatic initiator is found or it may even be any subject living in an area where an allergen has been identified. If the subject develops allergic responses to a particular antigen and the subject may be exposed to the antigen, i.e., during pollen season, then that subject is at risk of exposure to the antigen.
The generic name for molecules that cause an allergic reaction is allergen. An “allergen” as used herein is a molecule capable of provoking an immune response characterized by production of IgE. An allergen is a substance that can induce an allergic or asthmatic response in a susceptible subject. Thus, in the context of this invention, the term allergen means a specific type of antigen which can trigger an allergic response which is mediated by IgE antibody. The method and preparations of this invention extend to a broad class of such allergens and fragments of allergens or haptens acting as allergens. The list of allergens is enormous and can include pollens, insect venoms, animal dander, dust, fungal spores, and drugs (e.g., penicillin).
There are numerous species of allergens. The allergic reaction occurs when tissue-sensitizing immunoglobulin of the IgE type reacts with foreign allergen. The IgE antibody is bound to mast cells and/or basophils, and these specialized cells release chemical mediators (vasoactive amines) of the allergic reaction when stimulated to do so by allergens bridging the ends of the antibody molecule. Htamine, platelet activating factor, arachidonic acid metabolites, and serotonin are among the best known mediators of allergic reactions in man. Htamine and the other vasoactive amines are normally stored in mast cells and basophil leukocytes. The mast cells are dispersed throughout animal tissue and the basophils circulate within the vascular system. These cells manufacture and store histamine within the cell unless the specialized sequence of events involving IgE binding occurs to trigger its release.
The symptoms of the allergic reaction vary, depending on the location within the body where the IgE reacts with the antigen. If the reaction occurs along the respiratory epithelium, the symptoms are sneezing, coughing and asthmatic reactions. If the interaction occurs in the digestive tract, as in the case of food allergies, abdominal pain and diarrhea are common. Systemic reactions, for example following a bee sting, can be severe and often life-threatening.
Delayed-type hypersensitivity, also known as type IV allergy reaction, is an allergic reaction characterized by a delay period of at least 12 hours from invasion of the antigen into the allergic subject until appearance of the inflammatory or immune reaction. The T lymphocytes (sensitized T lymphocytes) of individuals in an allergic condition react with the antigen, triggering the T lymphocytes to release lymphokines (macrophage migration inhibitory factor (MIF), macrophage activating factor (MAF), mitogenic factor (MF), skin-reactive factor (SRF), chemotactic factor, neovascularization-accelerating factor, etc.), which function as inflammation mediators, and the biological activity of these lymphokines, together with the direct and indirect effects of locally appearing lymphocytes and other inflammatory immune cells, give rise to the type IV allergy reaction. Delayed allergy reactions include tuberculin type reaction, homograft rejection reaction, cell-dependent type protective reaction, contact dermatitis hypersensitivity reaction, and the like, which are known to be most strongly suppressed by steroidal agents. Consequently, steroidal agents are effective against diseases which are caused by delayed allergy reactions. Long-term use of steroidal agents at concentrations currently being used can, however, lead to the serious side-effect known as steroid dependence. The methods of the invention solve some of these problems, by providing for lower and fewer doses to be administered.
Immediate hypersensitivity (or anaphylactic response) is a form of allergic reaction which develops very quickly, i.e., within seconds or minutes of exposure of the patient to the causative allergen, and it is mediated by IgE antibodies made by B lymphocytes. In nonallergic patients, there is no IgE antibody of clinical relevance; but, in a person suffering with allergic diseases, IgE antibody mediates immediate hypersensitivity by sensitizing mast cells which are abundant in the skin, lymphoid organs, in the membranes of the eye, nose and mouth, and in the respiratory tract and intestines.
Mast cells have surface receptors for IgE, and the IgE antibodies in allergy-suffering patients become bound to them. As discussed briefly above, when the bound IgE is subsequently contacted by the appropriate allergen, the mast cell is caused to degranulate and to release various substances called bioactive mediators, such as histamine, into the surrounding tissue. It is the biologic activity of these substances which is responsible for the clinical symptoms typical of immediate hypersensitivity; namely, contraction of smooth muscle in the airways or the intestine, the dilation of small blood vessels and the increase in their permeability to water and plasma proteins, the secretion of thick sticky mucus, and in the skin, redness, swelling and the stimulation of nerve endings that results in itching or pain.
“Asthma” as used herein refers to a disorder of the respiratory system characterized by inflammation, narrowing of the airways, and increased reactivity of the airways to inhaled agents. Asthma is frequently, although not exclusively, associated with an atopic or allergic condition. Symptoms of asthma include recurrent episodes of wheezing, breathlessness, and chest tightness, and coughing, resulting from airflow obstruction. Airway inflammation associated with asthma can be detected through observation of a number of physiological changes, such as, denudation of airway epithelium, collagen deposition beneath basement membrane, edema, mast cell activation, inflammatory cell infiltration, including neutrophils, inosineophils, and lymphocytes. As a result of the airway inflammation, asthma patients often experience airway hyper-responsiveness, airflow limitation, respiratory symptoms, and disease chronicity. Airflow limitations include acute bronchoconstriction, airway edema, mucous plug formation, and airway remodeling, features which often lead to bronchial obstruction. In some cases of asthma, sub-basement membrane fibrosis may occur, leading to persistent abnormalities in lung function.
Research over the past several years has revealed that asthma likely results from complex interactions among inflammatory cells, mediators, and other cells and tissues resident in the airway. Mast cells, inosineophils, epithelial cells, macrophage, and activated T-cells all play an important role in the inflammatory process associated with asthma. Djukanovic R et al. (1990) Am Rev Respir Dis 142:434-457. It is believed that these cells can influence airway function through secretion of preformed and newly synthesized mediators which can act directly or indirectly on the local tissue. It has also been recognized that subpopulations of T-lymphocytes (Th2) play an important role in regulating allergic inflammation in the airway by releasing selective cytokines and establishing disease chronicity. Robinson D S et al. (1992) N Engl J Med 326:298-304.
Asthma is a complex disorder which arises at different stages in development and can be classified based on the degree of symptoms as acute, subacute or chronic. An acute inflammatory response is associated with an early recruitment of cells into the airway. The subacute inflammatory response involves the recruitment of cells as well as the activation of resident cells causing a more persistent pattern of inflammation. Chronic inflammatory response is characterized by a persistent level of cell damage and an ongoing repair process, which may result in permanent abnormalities in the airway.
A “subject having asthma” is a subject that has a disorder of the respiratory system characterized by inflammation, narrowing of the airways and increased reactivity of the airways to inhaled agents. Asthma is frequently, although not exclusively, associated with atopic or allergic symptoms. An “initiator” as used herein refers to a composition or environmental condition which triggers asthma. Initiators include, but are not limited to, allergens, cold temperatures, exercise, viral infections, SO2.
The compositions and methods of the invention can be used alone or in conjucnction with other agents and methods useful in the treatment of asthma. An “asthma/allergy medicament” as used herein is a composition of matter which reduces the symptoms of, prevents the development of, or inhibits an asthmatic or allergic reaction. Various types of medicaments for the treatment of asthma and allergy are described in the Guidelines For The Diagnosis and Management of Asthma, Expert Panel Report 2, NIH Publication No. 97/4051, Jul. 19, 1997, the entire contents of which are incorporated herein by reference. The summary of the medicaments as described in the NIH publication is presented below. In most embodiments the asthma/allergy medicament is useful to some degree for treating both asthma and allergy.
Medications for the treatment of asthma are generally separated into two categories, quick-relief medications and long-term control medications. Asthma patients take the long-term control medications on a daily basis to achieve and maintain control of persistent asthma. Long-term control medications include anti-inflammatory agents such as corticosteroids, chromolyn sodium and nedocromil; long-acting bronchodilators, such as long-acting β2-agonists and methylxanthines; and leukotriene modifiers. The quick-relief medications include short-acting β2 agonists, anti-cholinergics, and systemic corticosteroids. There are many side effects associated with each of these drugs and none of the drugs alone or in combination is capable of preventing or completely treating asthma.
Asthma medicaments include, but are not limited, PDE-4 inhibitors, bronchodilator/beta-2 agonists, K+ channel openers, VLA-4 antagonists, neurokin antagonists, thromboxane A2 (TXA2) synthesis inhibitors, xanthines, arachidonic acid antagonists, 5 lipoxygenase inhibitors, TXA2 receptor antagonists, TXA2 antagonists, inhibitor of 5-lipox activation proteins, and protease inhibitors.
Bronchodilator/β2 agonists are a class of compounds which cause bronchodilation or smooth muscle relaxation. Bronchodilator/β2 agonists include, but are not limited to, salmeterol, salbutamol, albuterol, terbutaline, D2522/formoterol, fenoterol, bitolterol, pirbuerol methylxanthines and orciprenaline. Long-acting β2 agonists and bronchodilators are compounds which are used for long-term prevention of symptoms in addition to the anti-inflammatory therapies. Long-acting β2 agonists include, but are not limited to, salmeterol and albuterol. These compounds are usually used in combination with corticosteroids and generally are not used without any inflammatory therapy. They have been associated with side effects such as tachycardia, skeletal muscle tremor, hypokalemia, and prolongation of QTc interval in overdose.
Methylxanthines, including for instance theophylline, have been used for long-term control and prevention of symptoms. These compounds cause bronchodilation resulting from phosphodiesterase inhibition and likely adenosine antagonism. Dose-related acute toxicities are a particular problem with these types of compounds. As a result, routine serum concentration must be monitored in order to account for the toxicity and narrow therapeutic range arising from individual differences in metabolic clearance. Side effects include tachycardia, tachyarrhythmias, nausea and vomiting, central nervous system stimulation, headache, seizures, hematemesis, hyperglycemia and hypokalemia. Short-acting β2 agonists include, but are not limited to, albuterol, bitolterol, pirbuterol, and terbutaline. Some of the adverse effects associated with the administration of short-acting β2 agonists include tachycardia, skeletal muscle tremor, hypokalemia, increased lactic acid, headache, and hyperglycemia.
Conventional methods for treating or preventing allergy have involved the use of anti-histamines or desensitization therapies. Anti-histamines and other drugs which block the effects of chemical mediators of the allergic reaction help to regulate the severity of the allergic symptoms but do not prevent the allergic reaction and have no effect on subsequent allergic responses. Desensitization therapies are performed by giving small doses of an allergen, usually by injection under the skin, in order to induce an IgG-type response against the allergen. The presence of IgG antibody helps to neutralize the production of mediators resulting from the induction of IgE antibodies, it is believed. Initially, the subject is treated with a very low dose of the allergen to avoid inducing a severe reaction and the dose is slowly increased. This type of therapy is dangerous because the subject is actually administered the compounds which cause the allergic response and severe allergic reactions can result.
Allergy medicaments include, but are not limited to, anti-histamines, steroids, and prostaglandin inducers. Anti-histamines are compounds which counteract histamine released by mast cells or basophils. These compounds are well known in the art and commonly used for the treatment of allergy. Anti-histamines include, but are not limited to, astemizole, azelastine, betatastine, buclizine, ceterizine, cetirizine analogues, CS 560, desloratadine, ebastine, epinastine, fexofenadine, HSR 609, levocabastine, loratidine, mizolastine, norastemizole, terfenadine, and tranilast.
Prostaglandin inducers are compounds which induce prostaglandin activity. Prostaglandins function by regulating smooth muscle relaxation. Prostaglandin inducers include, but are not limited to, S-5751.
The asthma/allergy medicaments also include steroids and immunomodulators. The steroids include, but are not limited to, beclomethasone, fluticasone, triamcinolone, budesonide, corticosteroids and budesonide.
Corticosteroids include, but are not limited to, beclomethasome dipropionate, budesonide, flunisolide, fluticaosone propionate, and triamcinolone acetonide. Although dexamethasone is a corticosteroid having anti-inflammatory action, it is not regularly used for the treatment of asthma/allergy in an inhaled form because it is highly absorbed and it has long-term suppressive side effects at an effective dose. Dexamethasone, however, can be used according to the invention for the treating of asthma/allergy because when administered in combination with nucleic acids of the invention it can be administered at a low dose to reduce the side effects. Some of the side effects associated with corticosteroid include cough, dysphonia, oral thrush (candidiasis), and in higher doses, systemic effects, such as adrenal suppression, osteoporosis, growth suppression, skin thinning and easy bruising. Barnes & Peterson (1993) Am Rev Respir Dis 148:S1-S26; and Kamada A K et al. (1996) Am J Respir Crit. Care Med 153:1739-48.
Systemic corticosteroids include, but are not limited to, methylprednisolone, prednisolone and prednisone. Cortosteroids are associated with reversible abnormalities in glucose metabolism, increased appetite, fluid retention, weight gain, mood alteration, hypertension, peptic ulcer, and aseptic necrosis of bone. These compounds are useful for short-term (3-10 days) prevention of the inflammatory reaction in inadequately controlled persistent asthma. They also function in a long-term prevention of symptoms in severe persistent asthma to suppress and control and actually reverse inflammation. Some side effects associated with longer term use include adrenal axis suppression, growth suppression, dermal thinning, hypertension, diabetes, Cushing's syndrome, cataracts, muscle weakness, and in rare instances, impaired immune function. It is recommended that these types of compounds be used at their lowest effective dose (guidelines for the diagnosis and management of asthma; expert panel report to; NIH Publication No. 97-4051; July 1997).
The immunomodulators include, but are not limited to, the group consisting of anti-inflammatory agents, leukotriene antagonists, IL-4 muteins, soluble IL-4 receptors, immunosuppressants (such as tolerizing peptide vaccine), anti-IL-4 antibodies, IL-4 antagonists, anti-IL-5 antibodies, soluble IL-13 receptor-Fc fusion proteins, anti-IL-9 antibodies, CCR3 antagonists, CCR5 antagonists, VLA-4 inhibitors, and downregulators of IgE.
Leukotriene modifiers are often used for long-term control and prevention of symptoms in mild persistent asthma. Leukotriene modifiers function as leukotriene receptor antagonists by selectively competing for LTD-4 and LTE-4 receptors. These compounds include, but are not limited to, zafirlukast tablets and zileuton tablets. Zileuton tablets function as 5-lipoxygenase inhibitors. These drugs have been associated with the elevation of liver enzymes and some cases of reversible hepatitis and hyperbilirubinemia. Leukotrienes are biochemical mediators that are released from mast cells, inosineophils, and basophils that cause contraction of airway smooth muscle and increase vascular permeability, mucous secretions and activate inflammatory cells in the airways of patients with asthma.
Other immunomodulators include neuropeptides that have been shown to have immunomodulating properties. Functional studies have shown that substance P, for instance, can influence lymphocyte function by specific receptor-mediated mechanisms. Substance P also has been shown to modulate distinct immediate hypersensitivity responses by stimulating the generation of arachidonic acid-derived mediators from mucosal mast cells. McGillies J et al. (1987) Fed Proc 46:196-9 (1987). Substance P is a neuropeptide first identified in 1931. Von Euler and Gaddum J Physiol (London) 72:74-87 (1931). Its amino acid sequence was reported by Chang et al. in 1971. Chang M M et al. (1971) Nature New Biol 232:86-87. The immunoregulatory activity of fragments of substance P has been studied by Siemion I Z et al. (1990) Molec Immunol 27:887-890 (1990).
Another class of compounds is the down-regulators of IgE. These compounds include peptides or other molecules with the ability to bind to the IgE receptor and thereby prevent binding of antigen-specific IgE. Another type of downregulator of IgE is a monoclonal antibody directed against the IgE receptor-binding region of the human IgE molecule. Thus, one type of downregulator of IgE is an anti-IgE antibody or antibody fragment. Anti-IgE is being developed by Genentech. One of skill in the art could prepare functionally active antibody fragments of binding peptides which have the same function. Other types of IgE downregulators are polypeptides capable of blocking the binding of the IgE antibody to the Fc receptors on the cell surfaces and displacing IgE from binding sites upon which IgE is already bound.
One problem associated with downregulators of IgE is that many molecules do not have a binding strength to the receptor corresponding to the very strong interaction between the native IgE molecule and its receptor. The molecules having this strength tend to bind irreversibly to the receptor. However, such substances are relatively toxic since they can bind covalently and block other structurally similar molecules in the body. Of interest in this context is that the α chain of the IgE receptor belongs to a larger gene family where, e.g., several of the different IgG Fc receptors are contained. These receptors are absolutely essential for the defense of the body against, e.g., bacterial infections. Molecules activated for covalent binding are, furthermore, often relatively unstable and therefore they probably have to be administered several times a day and then in relatively high concentrations in order to make it possible to block completely the continuously renewing pool of IgE receptors on mast cells and basophilic leukocytes.
Chromolyn sodium and nedocromil are used as long-term control medications for preventing primarily asthma symptoms arising from exercise or allergic symptoms arising from allergens. These compounds are believed to block early and late reactions to allergens by interfering with chloride channel function. They also stabilize mast cell membranes and inhibit activation and release of mediators from inosineophils and epithelial cells. A four to six week period of administration is generally required to achieve a maximum benefit.
Anticholinergics are generally used for the relief of acute bronchospasm. These compounds are believed to function by competitive inhibition of muscarinic cholinergic receptors. Anticholinergics include, but are not limited to, ipratropium bromide. These compounds reverse only cholinerigically-mediated bronchospasm and do not modify any reaction to antigen. Side effects include drying of the mouth and respiratory secretions, increased wheezing in some individuals, and blurred vision if sprayed in the eyes.
In addition to standard asthma/allergy medicaments, other methods for treating asthma/allergy have been used either alone or in combination with established medicaments. One preferred, but frequently impossible, method of relieving allergies is allergen or initiator avoidance. Another method currently used for treating allergic disease involves the injection of increasing doses of allergen to induce tolerance to the allergen and to prevent further allergic reactions.
Allergen injection therapy (allergen immunotherapy) is known to reduce the severity of allergic rhinitis. This treatment has been theorized to involve the production of a different form of antibody, a protective antibody which is termed a “blocking antibody”. Cooke R A et al. (1935) Serologic Evidence of Immunity with Coexisting Sensitization in a Type of Human Allergy, Exp Med 62:733. Other attempts to treat allergy involve modifying the allergen chemically so that its ability to cause an immune response in the patient is unchanged, while its ability to cause an allergic reaction is substantially altered. These methods, however, can take several years to be effective and are associated with the risk of side effects such as anaphylactic shock.
The compositions and methods of the invention can be used to modulate an immune response. The ability to modulate an immune response allows for the prevention and/or treatment of particular disorders that can be affected via immune system modulation.
Treatment after a disorder has started aims to reduce, ameliorate, or altogether eliminate the disorder, and/or its associated symptoms, or prevent it from becoming worse. Treatment of subjects before a disorder has started (i.e., prophylactic treatment) aims to reduce the risk of developing the disorder. As used herein, the term “prevent” refers to the prophylactic treatment of patients who are at risk of developing a disorder (resulting in a decrease in the probability that the subject will develop the disorder), and to the inhibition of further development of an already established disorder.
Different doses may be necessary for treatment of a subject, depending on activity of the compound, manner of administration, purpose of the immunization (i.e., prophylactic or therapeutic), nature and severity of the disorder, age and body weight of the subject. The administration of a given dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units. Multiple administration of doses at specific intervals of weeks or months apart is usual for boosting antigen-specific immune responses.
Combined with the teachings provided herein, by choosing among the various active compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and preferred mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is entirely effective to treat the particular subject. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular therapeutic agent being administered (e.g., in the case of an immunostimulatory nucleic acid, the type of nucleic acid, i.e., a CpG nucleic acid, the number of unmethylated CpG motifs or their location in the nucleic acid, the degree of modification of the backbone to the oligonucleotide, etc.), the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular nucleic acid and/or other therapeutic agent without necessitating undue experimentation.
Subject doses of the compounds described herein typically range from about 0.1 μg to 10,000 mg, more typically from about 1 μg/day to 8000 mg, and most typically from about 10 μg to 100 μg. Stated in terms of subject body weight, typical dosages range from about 0.1 μg to 20 mg/kg/day, more typically from about 1 to 10 mg/kg/day, and most typically from about 1 to 5 mg/kg/day.
The pharmaceutical compositions containing nucleic acids and/or other compounds can be administered by any suitable route for administering medications. A variety of administration routes are available. The particular mode selected will depend, of course, upon the particular agent or agents selected, the particular condition being treated, and the dosage required for therapeutic efficacy. The methods of this invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of an immune response without causing clinically unacceptable adverse effects. Preferred modes of administration are discussed herein. For use in therapy, an effective amount of the nucleic acid and/or other therapeutic agent can be administered to a subject by any mode that delivers the agent to the desired surface, e.g., mucosal, systemic.
Administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan. Routes of administration include but are not limited to oral, parenteral, intravenous, intramuscular, intranasal, sublingual, intratracheal, inhalation, subcutaneous, ocular, vaginal, and rectal. For the treatment or prevention of asthma or allergy, such compounds are preferably inhaled, ingested or administered by systemic routes. Systemic routes include oral and parenteral. Inhaled medications are preferred in some embodiments because of the direct delivery to the lung, the site of inflammation, primarily in asthmatic patients. Several types of devices are regularly used for administration by inhalation. These types of devices include metered dose inhalers (MDI), breath-actuated MDI, dry powder inhaler (DPI), spacer/holding chambers in combination with MDI, and nebulizers.
The therapeutic agents of the invention may be delivered to a particular tissue, cell type, or to the immune system, or both, with the aid of a vector. In its broadest sense, a “vector” is any vehicle capable of facilitating the transfer of the compositions to the target cells. The vector generally transports the immunostimulatory nucleic acid, antibody, antigen, and/or disorder-specific medicament to the target cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
In general, the vectors useful in the invention are divided into two classes: biological vectors and chemical/physical vectors. Biological vectors and chemical/physical vectors are useful in the delivery and/or uptake of therapeutic agents of the invention.
Most biological vectors are used for delivery of nucleic acids and this would be most appropriate in the delivery of therapeutic agents that are or that include immunostimulatory nucleic acids.
In addition to the biological vectors discussed herein, chemical/physical vectors may be used to deliver therapeutic agents including immunostimulatory nucleic acids, antibodies, antigens, and disorder-specific medicaments. As used herein, a “chemical/physical vector” refers to a natural or synthetic molecule, other than those derived from bacteriological or viral sources, capable of delivering the nucleic acid and/or other medicament.
A preferred chemical/physical vector of the invention is a colloidal dispersion system. Colloidal dispersion systems include lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system of the invention is a liposome. Liposomes are artificial membrane vessels which are useful as a delivery vector in vivo or in vitro. It has been shown that large unilamellar vesicles (LUVs), which range in size from 0.2-4.0 μm can encapsulate large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form. Fraley et al. (1981) Trends Biochem Sci 6:77.
Liposomes may be targeted to a particular tissue by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein. Ligands which may be useful for targeting a liposome to an immune cell include, but are not limited to: intact or fragments of molecules which interact with immune cell specific receptors and molecules, such as antibodies, which interact with the cell surface markers of immune cells. Such ligands may easily be identified by binding assays well known to those of skill in the art. In still other embodiments, the liposome may be targeted to the cancer by coupling it to a one of the immunotherapeutic antibodies discussed earlier. Additionally, the vector may be coupled to a nuclear targeting peptide, which will direct the vector to the nucleus of the host cell.
Lipid formulations for transfection are commercially available from QIAGEN, for example, as EFFECTENE™ (a non-liposomal lipid with a special DNA condensing enhancer) and SUPERFECT™ (a novel acting dendrimeric technology).
Liposomes are commercially available from Gibco BRL, for example, as LIPOFECTIN™ and LIPOFECTACE™, which are formed of cationic lipids such as N-[1-(2,3 dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB). Methods for making liposomes are well known in the art and have been described in many publications. Liposomes also have been reviewed by Gregoriadis G (1985) Trends Biotechnol 3:235-241.
In one embodiment, the vehicle is a biocompatible microparticle or implant that is suitable for implantation or administration to the mammalian recipient. Exemplary bioerodible implants that are useful in accordance with this method are described in PCT International application no. PCT/US/03307 (Publication No. WO95/24929, entitled “Polymeric Gene Delivery System”. PCT/US/0307 describes a biocompatible, preferably biodegradable polymeric matrix for containing an exogenous gene under the control of an appropriate promoter. The polymeric matrix can be used to achieve sustained release of the therapeutic agent in the subject.
The polymeric matrix preferably is in the form of a microparticle such as a microsphere (wherein the nucleic acid and/or the other therapeutic agent is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the nucleic acid and/or the other therapeutic agent is stored in the core of a polymeric shell). Other forms of the polymeric matrix for containing the therapeutic agent include films, coatings, gels, implants, and stents. The size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix is introduced. The size of the polymeric matrix further is selected according to the method of delivery which is to be used, typically injection into a tissue or administration of a suspension by aerosol into the nasal and/or pulmonary areas. Preferably when an aerosol route is used the polymeric matrix and the nucleic acid and/or the other therapeutic agent are encompassed in a surfactant vehicle. The polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when the matrix is administered to a nasal and/or pulmonary surface that has sustained an injury. The matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time. In some preferred embodiments, the nucleic acid are administered to the subject via an implant while the other therapeutic agent is administered acutely. Biocompatible microspheres that are suitable for delivery, such as oral or mucosal delivery, are disclosed in Chickering et al. (1996) Biotech Bioeng 52:96-101 and Mathiowitz E et al. (1997) Nature 386:410-414 and PCT Pat. Application WO97/03702.
Both non-biodegradable and biodegradable polymeric matrices can be used to deliver the nucleic acid and/or the other therapeutic agent to the subject. Biodegradable matrices are preferred. Such polymers may be natural or synthetic polymers. The polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable, particularly for the nucleic acid agents. The polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multi-valent ions or other polymers.
Bioadhesive polymers of particular interest include bioerodible hydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules, (1993) 26:581-587, the teachings of which are incorporated herein. These include polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).
If the therapeutic agent is a nucleic acid, the use of compaction agents may also be desirable. Compaction agents also can be used alone, or in combination with, a biological or chemical/physical vector. A “compaction agent”, as used herein, refers to an agent, such as a histone, that neutralizes the negative charges on the nucleic acid and thereby permits compaction of the nucleic acid into a fine granule. Compaction of the nucleic acid facilitates the uptake of the nucleic acid by the target cell. The compaction agents can be used alone, i.e., to deliver a nucleic acid in a form that is more efficiently taken up by the cell or, more preferably, in combination with one or more of the above-described vectors.
Other exemplary compositions that can be used to facilitate uptake of a nucleic acid include calcium phosphate and other chemical mediators of intracellular transport, microinjection compositions, electroporation and homologous recombination compositions (e.g., for integrating a nucleic acid into a preselected location within the target cell chromosome).
The compounds may be administered alone (e.g., in saline or buffer) or using any delivery vectors known in the art. For instance the following delivery vehicles have been described: cochleates (Gould-Fogerite et al., 1994, 1996); Emulsomes (Vancott et al., 1998, Lowell et al., 1997); ISCOMs (Mowat et al., 1993, Carlsson et al., 1991, Hu et., 1998, Morein et al., 1999); liposomes (Childers et al., 1999, Michalek et al., 1989, 1992, de Haan 1995a, 1995b); live bacterial vectors (e.g., Salmonella, Escherichia coli, Bacillus calmatte-guerin, Shigella, Lactobacillus) (Hone et al., 1996, Pouwels et al., 1998, Chatfield et al., 1993, Stover et al., 1991, Nugent et al., 1998); live viral vectors (e.g., Vaccinia, adenovirus, Herpes Simplex) (Gallichan et al., 1993, 1995, Moss et al., 1996, Nugent et al., 1998, Flexner et al., 1988, Morrow et al., 1999); microspheres (Gupta et al., 1998, Jones et al., 1996, Maloy et al., 1994, Moore et al., 1995, O'Hagan et al., 1994, Eldridge et al., 1989); nucleic acid vaccines (Fynan et al., 1993, Kuklin et al., 1997, Sasaki et al., 1998, Okada et al., 1997, Ishii et al., 1997); polymers (e.g. carboxymethylcellulose, chitosan) (Hamajima et al., 1998, Jabbal-Gill et al., 1998); polymer rings (Wyatt et al., 1998); proteosomes (Vancott et al., 1998, Lowell et al., 1988, 1996, 1997); sodium fluoride (Hashi et al., 1998); transgenic plants (Tacket et al., 1998, Mason et al., 1998, Haq et al., 1995); virosomes (Gluck et al., 1992, Mengiardi et al., 1995, Cryz et al., 1998); and, virus-like particles (Jiang et al., 1999, Leibl et al., 1998).
The formulations of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.
The term pharmaceutically-acceptable carrier means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal. The term carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.
For oral administration, the compounds (i.e., nucleic acids, antigens, antibodies, and other therapeutic agents) can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any carriers.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The compounds, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long-acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer R (1990) Science 249:1527-1533, which is incorporated herein by reference.
The nucleic acids and optionally other therapeutics and/or antigens may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).
The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the compounds into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the compounds into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. Liquid dose units are vials or ampoules. Solid dose units are tablets, capsules and suppositories.
Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the compounds, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109. Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di-, and tri-glycerides; hydrogel release systems; silastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which an agent of the invention is contained in a form within a matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.
The invention also provides efficient methods of identifying immunostimulatory compounds and optimizing the compounds and agents so identified. Generally, the screening methods involve assaying for compounds which inhibit or enhance signaling through a particular TLR. The methods employ a TLR, a suitable reference ligand for the TLR, and a candidate immunostimulatory compound. The selected TLR is contacted with a suitable reference compound (TLR ligand) and a TLR-mediated reference signal is measured. The selected TLR is also contacted with a candidate immunostimulatory compound and a TLR-mediated test signal is measured. The test signal and the reference signal are then compared. A favorable candidate immunostimulatory compound may subsequently be used as a reference compound in the assay. Such methods are adaptable to automated, high throughput screening of candidate compounds. Examples of such high throughput screening methods are described in U.S. Pat. Nos. 6,103,479; 6,051,380; 6,051,373; 5,998,152; 5,876,946; 5,708,158; 5,443,791; 5,429,921; and 5,143,854.
As used herein “TLR signaling” refers to an ability of a TLR polypeptide to activate the Toll/IL-1R (TIR) signaling pathway, also referred to herein as the TLR signal transduction pathway. Changes in TLR activity can be measured by assays designed to measure expression of genes under control of κB-sensitive promoters and enhancers. Such genes can be naturally occurring genes or they can be genes artificially introduced into a cell. Naturally occurring reporter genes include the genes encoding IL-1β, IL-6, IL-8, the p40 subunit of interleukin 12 (IL-12 p40), and the costimulatory molecules CD80 and CD86. Other genes can be placed under the control of such regulatory elements and thus serve to report the level of TLR signaling.
The assay mixture comprises a candidate immunostimulatory compound. Typically, a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a different response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration of agent or at a concentration of agent below the limits of assay detection. Candidate immunostimulatory compounds may encompass numerous chemical classes, although typically they are organic compounds. In some embodiments, the candidate immunostimulatory compounds are small RNAs or small organic compounds, i.e., organic compounds having a molecular weight of more than 50 yet less than about 2500 Daltons. Polymeric candidate immunostimulatory compounds can have higher molecular weights, e.g., oligonucleotides in the range of about 2500 to about 12,500. Candidate immunostimulatory compounds also may be biomolecules such as nucleic acids, peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like. Where the candidate immunostimulatory compound is a nucleic acid, the candidate immunostimulatory compound typically is a DNA or RNA molecule, although modified nucleic acids having non-natural bonds or subunits are also contemplated.
Candidate immunostimulatory compounds may be obtained from a wide variety of sources, including libraries of natural, synthetic, or semisynthetic compounds, or any combination thereof. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily modified through conventional chemical, physical, and biochemical means. Further, known pharmacological agents may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc., to produce structural analogs of the candidate immunostimulatory compounds.
A variety of other reagents also can be included in the mixture. These include reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc., which may be used to facilitate optimal protein-protein and/or protein-nucleic acid binding. Such a reagent may also reduce non-specific or background interactions of the reaction components. Other reagents that improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, antimicrobial agents, and the like may also be used.
The order of addition of components, incubation temperature, time of incubation, and other parameters of the assay may be readily determined. Such experimentation merely involves optimization of the assay parameters, not the fundamental composition of the assay. Incubation temperatures typically are between 4° C. and 40° C., more typically about 37° C. Incubation times preferably are minimized to facilitate rapid, high throughput screening, and typically are between 1 minute and 10 hours.
After incubation, the level of TLR signaling is detected by any convenient method available to the user. For cell-free binding type assays, a separation step is often used to separate bound from unbound components. The separation step may be accomplished in a variety of ways. For example, separation can be accomplished in solution, or, conveniently, at least one of the components is immobilized on a solid substrate, from which the unbound components may be easily separated. The solid substrate can be made of a wide variety of materials and in a wide variety of shapes, e.g., microtiter plate, microbead, dipstick, resin particle, etc. The substrate preferably is chosen to maximize signal-to-noise ratios, primarily to minimize background binding, as well as for ease of separation and cost.
Separation may be effected, for example, by removing a bead or dipstick from a reservoir, emptying or diluting a reservoir such as a microtiter plate well, rinsing a bead, particle, chromatographic column or filter with a wash solution or solvent. The separation step preferably includes multiple rinses or washes. For example, when the solid substrate is a microtiter plate, the wells may be washed several times with a washing solution, which typically includes those components of the incubation mixture that do not participate in specific bindings such as salts, buffer, detergent, non-specific protein, etc. Where the solid substrate is a magnetic bead, the beads may be washed one or more times with a washing solution and isolated using a magnet.
Detection may be effected in any convenient way for cell-based assays such as measurement of an induced polypeptide within, on the surface of, or secreted by the cell. Examples of detection methods useful in cell-based assays include fluorescence-activated cell sorting (FACS) analysis, bioluminescence, fluorescence, enzyme-linked immunosorbent assay (ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR), and the like. Examples of detection methods useful in cell-free assays include bioluminescence, fluorescence, ELISA, RT-PCR, and the like.
EXAMPLES
Example 1
Responsiveness of Human PBMC to G,U-Containing Oligoribonucleotides
Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors, plated at 3×105 cells/well, stimulated in vitro with various test and control immunostimulatory agents for 16 hours, and then analyzed by enzyme-linked immunosorbent assay (ELISA) using matched antibody pairs from BD-Pharmingen for secreted cytokines IL-12 p40 and TNF-α, performed according to the manufacturer's protocol. Also included were certain negative controls, including medium alone and DOTAP (10 μg/200 μl culture well; “Liposomes”) alone. The control immunostimulatory agents included the imidazoquinolone R-848 (2 μg/ml), lipopolysaccharide (LPS; 1 μg/ml), Pam3Cys (5 μg/ml), poly IC (50 μg/ml), and CpG DNA (50 μg/ml). These are reported ligands for TLR7, TLR4, TLR2, TLR3, and TLR9, respectively. Test immunostimulatory agents included the following RNA molecules, each at 50 μg/ml, with and without DOTAP (10 μg total “with Liposomes” and “without Liposomes”, respectively): GUGUUUAC alone; GUAGGCAC alone; GUGUUUAC in combination with GUAGGCAC; GUAGGA; GAAGGCAC; CUAGGCAC; CUCGGCAC; and CCCCCCCC. These RNA oligonucleotides each contained a phosphorothioate linkage between the penultimate and 3′ terminal nucleoside.
FIG. 1 depicts the responsiveness of human PBMC to the test and control agents listed above, as measured by secreted amounts of IL-12 p40 (pg/ml). As can be seen in FIG. 1, PBMCs were responsive to R-848, LPS, Pam3Cys, and poly IC, while they were unresponsive to DOTAP alone. Significantly, human PBMC secreted large amounts of IL-12 p40 (10-20 ng/ml) in response to G,U-containing RNA oligonucleotides GUGUUUAC alone; GUAGGCAC alone; GUGUUUAC in combination with GUAGGCAC; CUAGGCAC; and CUCGGCAC, each in combination with DOTAP. Also significantly, human PBMC did not secrete significant amounts of IL-12 p40 in response to G,U-free RNA oligonucleotides GAAGGCAC and CCCCCCCC. The immunostimulatory effect of the G,U-containing RNA molecules appeared to be greatly enhanced by the inclusion of DOTAP. In this experiment, the G,U-containing 6-mer RNA GUAGGA appeared to exert little, if any immunostimulatory effect either with or without DOTAP.
FIG. 2 depicts the responsiveness of human PBMC to the test and control agents listed above, as measured by secreted amounts of TNF-α. A similar pattern of results was observed as in FIG. 1, i.e., human PBMC secreted large amounts of TNF-α (40-100 ng/ml) in response to G,U-containing RNA oligonucleotides GUGUUUAC alone; GUAGGCAC alone; GUGUUUAC in combination with GUAGGCAC; CUAGGCAC; and CUCGGCAC, each in combination with DOTAP. Also similar to the results in FIG. 1, human PBMC did not secrete significant amounts of TNF-α in response to G,U-free RNA oligonucleotides GAAGGCAC and CCCCCCCC, or in response to the G,U-containing 6-mer RNA GUAGGA. The immunostimulatory effect of the G,U-containing RNA molecules appeared to be greatly enhanced by the inclusion of DOTAP.
It will be appreciated in this example that the following partial self-complementarity basepairing is possible, where G-U wobble basepairs are shown joined with a dot and G-C and A-U basepairs are shown joined by a line:
Example 2
Dose-Response Behavior of Human PBMC to G,U-Containing Oligoribonucleotides
The experiments described in the preceding example were repeated with varied concentrations of RNA oligonucleotides in order to assess the dose-response behavior of human PBMCs to G,U-containing RNA oligonucleotides of the invention. A total of 10, 3 or 1 μg RNA was added to 10 μg DOTAP and then added to the 200 μl culture wells. After 16 hours IL-12 p40 and TNF-α ELISAs were performed as described in Example 1.
FIG. 3 depicts the dose-response of human PBMC to the various RNAs as measured by secreted amounts of IL-12 p40 (ng/ml). As can be seen from FIG. 3, human PBMC secreted increasing amounts of IL-12 p40 in response to increasing amounts of G,U-containing RNA oligomers GUGUUUAC; GUAGGCAC; CUAGGCAC; and CUCGGCAC, each in combination with DOTAP. Conversely, FIG. 3 also shows that human PBMC appeared not to secrete IL-12 p40 in response to any of the tested amounts of G,U-free RNA oligomers GAAGGCAC or CCCCCCCC.
Corresponding dose-response of human PBMC to the various RNAs was measured by secreted amounts of TNF-α. A similar pattern of results was observed as in FIG. 3, i.e., human PBMC secreted increasing amounts of TNF-α in response to increasing amounts G,U-containing RNA oligonucleotides GUGUUUAC; GUAGGCAC; CUAGGCAC; and CUCGGCAC, each in combination with DOTAP. Also similar to the results in FIG. 3, human PBMC did not appear to secrete significant amounts of TNF-α in response to any of the tested amounts of G,U-free RNA oligonucleotides GAAGGCAC and CCCCCCCC.
Example 3
Base Sequence Sensitivity of RNA Oligomers
Point mutations were made to the RNA oligonucleotide GUAGGCAC by substituting A or C at selected positions. The various oligoribonucleotides included the following: GUAGGCAC; GUAGGA; GAAGGCAC; AUAAACAC; AUAGACAC; AUAAGCAC; GUAAACAC; CUAGGCAC; CUCGGCAC; and GUGUUUAC. The oligonucleotides were titrated onto human PBMC isolated from healthy donors and plated at 3×105 cells/well. A total of 10 μg RNA was added to 10 μg DOTAP and then added to the 200 μl culture wells. Human TNF-α was measured by ELISA using matched antibody pairs from BD-Pharmingen according to the manufacturer's protocol. Results are shown in FIG. 4.
Example 4
Effect of DOTAP on Human PBMC Response to Various Stimuli
In order to characterize further the role of DOTAP in the immunostimulatory effects of the G,U-containing RNA oligomers observed in the previous examples, human PBMCs were isolated from healthy donors, plated at 3×105 cells/well, and stimulated in the presence of known TLR ligands, either with or without DOTAP (“with Liposomes” or “without Liposomes”, respectively). The known TLR ligands examined were total RNA prepared from hyphae (hyphae), total RNA prepared from yeast (yeast), total RNA prepared from promyelocytic cell line HL-60 (HL60), in vitro transcribed ribosomal RNA for E. coli Sp6, in vitro transcribed ribosomal RNA for E. coli T7, LPS, poly IC, Pam3Cys, and R-848. Medium alone and DOTAP alone were used as negative controls. The panel of RNAs from the previous examples, again at 10 μg/ml and without DOTAP, was also included.
Total RNA was isolated from the human promyelocytic cell line HL-60 using Trizol (Sigma). Prior to isolation, cells were treated for 4 hours with 500 μM hydrogen peroxide (H2O2), which induces apoptosis in this cell line (HL60 500). Untreated cells served as control (HL60 0).
Candida albicans RNA was isolated from yeast or hyphae (induced by 4 h incubation with 10% fetal calf serum). Cells from a 100 ml culture were pelleted, washed and resuspended in 10 ml of Tris/EDTA buffer (10 mM, 1 mM). RNA was isolated by extraction with hot acidic phenol according to methods described in Ausubel F M et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, New York.
The genomic fragment of E. coli 16S RNA was amplified with the primers 5′-ATTGAAGAGTTTGATCATGGCTCAGATTGAACG-3′ (SEQ ID NO:5) and 5′-TAAGGAGGTGATCCAACCGCAGGTTCC-3′ (SEQ ID NO:6) from genomic E. coli DNA and cloned into the pGEM T easy vector. In vitro transcription was performed using T7 or Sp6 RNA polymerase. Transcribed RNA was further purified by chloroform/phenol extraction, precipitated, and used at 10 μg.
Following 16 hour incubation, ELISAs were performed as before to assess secretion of IL-12 p40 and TNF-α. Representative results are shown in FIG. 5.
FIG. 5 depicts the effect of DOTAP on the amount of IL-12 p40 secreted by human PBMC following incubation with and without DOTAP. As can be seen from the figure, the following stimuli appeared to exert greater immunostimultory effect in the presence of DOTAP than in its absence: hyphae, yeast, E. coli Sp6, and E. coli T7. The following stimuli appeared to exert reduced immunostimultory effect in the presence of DOTAP than in its absence: LPS, poly IC. The following stimuli appeared to exert about the same immunostimultory effect in the presence or absence of DOTAP: HL60, Pam3Cys and R-848.
Example 5
Immunostimulatory Effect of G,U-Containing RNA Oligomers is Species- and MyD88-Dependent
The following murine cells were isolated and incubated with various RNAs and other known TLR ligands in order to assess species-, cell type-, and signaling pathway-specificity: wild type macrophages in the presence of IFN-γ; MyD88-deficient macrophages in the presence of IFN-γ; J774 (mouse macrophage cell line); and RAW 264.7 (mouse macrophage cell line, e.g., ATCC TIB-71). Murine bone macrophages were generated from wild type or MyD88-deficient C57BL/6 mice by culturing bone marrow cells with 50 ng/ml M-CSF for 5 days. Cells were seeded at 25,000 cells/well and treated with 20 ng/ml IFN-γ for 16 hours. The murine macrophage cell lines RAW and J774 were seeded at 10,000 cells/well.
The following test and control agents were examined: R-848 (2 μg/ml), ODN 1668 (CpG DNA; 5′-TCCATGACGTTCCTGATGCT-3′; SEQ ID NO:7); LPS (1 μg/ml); poly IC (50 μg/ml); Pam3Cys (5 μg/ml); Ionomycin/TPA; the following RNA molecules, each with (“+Lipo”) and without DOTAP (10 μg/200 μl culture well): GUGUUUAC alone (RNA1); GUAGGCAC alone (RNA2); GUGUUUAC in combination with GUAGGCAC (RNA1/2); UCCGCAAUGGACGAAAGUCUGACGGA (RNA6; SEQ ID NO:8); GAGAUGGGUGCGAGAGCGUCAGUAUU (RNA9; SEQ ID NO:9); and the following DNA molecules, corresponding to RNA1, RNA2, and RNA1/2: GTGTTTAC alone (DNA1); GTAGGCAC alone (DNA2); and GTGTTTAC in combination with GTAGGCAC (DNA1/2). These RNA and DNA oligonucleotides each contained a phosphorothioate linkage between the penultimate and 3′ terminal nucleoside. RNA6 and RNA9 each contained in addition a phosphorothioate linkage between the penultimate and 5′ terminal nucleoside. RNA6 corresponds to a ribosomal RNA stem loop derived from Listeria monocytogenes. RNA9 corresponds to a stem loop derived from human immunodeficiency virus (HIV, an RNA retrovirus). The cells were cultured for 12 hours and supernatants were harvested. Murine IL-12 p40, IL-6, and TNF-α were measured by ELISA using matched antibody pairs from BD-Pharmingen according to the manufacturer's protocol. Representative results are shown in FIG. 6.
Panel A of FIG. 6 shows that wild type murine macrophages in the presence of IFN-γ secrete significant amounts of IL-12 p40 in response to R-848; ODN 1668 (CpG DNA); LPS; poly IC; Pam3Cys; and G,U-containing RNA oligomers GUGUUUAC in combination with GUAGGCAC (with DOTAP). In contrast, Panel B of FIG. 6 shows that MyD88-deficient murine macrophages in the presence of IFN-γ secrete little or no IL-12 p40 in response to any of the test and control agents examined, thus demonstrating a dependence on MyD88 for immunostimulatory response to these compounds. Such a result is consistent with participation by a TLR in the immunostimulatory response to any of these compounds, including in particular the G,U-containing RNA oligonucleotides of the invention. Panels C and D of FIG. 6 show generally similar, if somewhat attenuated, response patterns of J774 and RAW 264.7 mouse macrophage cell lines as for wild type murine macrophages in the presence of IFN-γ, as shown in Panel A. Essentially similar results were found in parallel ELISAs measuring IL-6 and TNF-α.
In additional studies involving MyD88 wild-type cells, it was observed that addition of bafilomycin largely or completely abrogated the immunostimulatory effect of the RNA oligomers. Together with the MyD88-dependence, this observation is consistent with involvement of at least one of TLR3, TLR7, TLR8, and TLR9.
Example 6
Use of Cholesteryl Ester in Place of Cationic Lipid
In order to investigate the possibility of using cholesteryl ester-modified RNA oligomer in place of RNA oligomer plus cationic lipid, RNA oligomer GUGUGUGU was prepared with (R 1058) and without (R 1006) a 3′ cholesteryl ester modification. These two RNA oligomers with and without DOTAP, were added over a range of concentrations to overnight cultures of human PBMC. Culture supernatants were harvested, and human TNF-α, IL-12 p40, and IFN-α were measured by ELISA using matched antibody pairs from BD-Pharmingen according to the manufacturer's protocol. Representative results for experiments including DOTAP are shown in Table 1.
TABLE 1
Cholesteryl Ester Modification in Place of DOTAP
TNF-α +
TNF-α −
IFN-α +
IFN-α −
DOTAP
DOTAP
DOTAP
DOTAP
EC50
max
EC50,
max
EC50
max
EC50
max
ID
μM
pg/ml
μM
pg/ml
μM
pg/ml
μM
pg/ml
R 1006
2.8
40000
7.8
2200
4.5
5000
—
—
R 1058
0.2
75000
1.0
3000
0.5
3800
0.5
1500
The results indicate that R 1058, with the cholesteryl ester modification, is more potent than R 1006, having the same base sequence but without cholesterol, both with and without DOTAP.
Example 7
Effect of Oligomer Length
RNA oligomers GUGUGUGU, GUGUGUG, GUGUGU, GUGUG, GUGU, GUG, and GU, with and without DOTAP, were added over a range of concentrations to overnight cultures of human PBMC. Culture supernatants were harvested, and human TNF-α, IL-12 p40, and IFN-α were measured by ELISA using matched antibody pairs from BD-Pharmingen according to the manufacturer's protocol. Representative results for experiments including DOTAP are shown in Table 2.
TABLE 2
Effect of RNA Oligomer Length
TNF-α
IL-12 p40
IFN-α
ID
SEQ
EC50, μM
max pg/ml
EC50, μM
max pg/ml
EC50, μM
max pg/ml
R 1006
GUGUGUGU
2.8
40000
1.6
7000
4.5
5000
R 1048
GUGUGUG
2.2
30000
2.6
10000
4.6
2700
R 1049
GUGUGU
6.7
30000
2.1
8000
4.8
3400
R 1050
GUGUG
7.6
40000
3.9
14000
6.9
400
R 1051
GUGU
—
—
>20
14000
—
—
R 1052
GUG
—
—
>20
6000
5.5
800
R 1053
GU
—
—
>20
5000
—
—
Example 8
Effect of Stabilization of Internucleoside Linkages
GUGUGUGU RNA oligomers were synthesized with specific phosphorothioate and phosphodiester linkages as shown in Table 2, where “*” represents phosphorothioate and “_” represents phosphodiester. RNA oligomers, with and without DOTAP, were added over a range of concentrations to overnight cultures of human PBMC. Culture supernatants were harvested, and human TNF-α, IL-12 p40, and IFN-α were measured by ELISA using matched antibody pairs from BD-Pharmingen according to the manufacturer's protocol. Representative results for experiments including DOTAP are shown in Table 3.
TABLE 3
Effect of Stabilization of Internucleoside
Linkages
TNF-α
IFN-α
EC50,
max,
EC50,
max,
ID
SEQ
μM
pg/ml
μM
pg/ml
R 1006
G*U*G*U*G*U*G*U
2.8
40000
4.5
5000
R 1054
G*U_G*U*G*U*G*U
5.6
40000
6.7
3700
R 1055
G*U_G*U_G*U*G*U
>20
20000
—
—
R 1056
G*U_G*U_G*U_G*U
>20
12000
—
—
R 1057
G_U_G_U_G_U_G_U
—
—
0.1
6000
In like manner, an all-phosphodiester 40-mer capable of forming a stem-loop structure and having a base sequence as provided by 5′-CACACACUGCUUAAGCGCUUGCCUGCUUAAGUAGUGUGUG-3′ (R 1041; SEQ ID NO:10) was synthesized and tested in overnight culture with human PBMC. This RNA oligomer was found to be very potent in its ability to induce IFN-α, with an EC50 of <0.1 μM and a maximum of 5000 pg/ml.
Example 9
DNA:RNA Conjugates
A series of DNA:RNA conjugates, each containing the RNA sequence GUGUGUGU and a poly-dT or a poly-dG sequence, was prepared. The oligomers were as follows, where again “*” represents phosphorothioate and “_” represents phosphodiester:
(R 1060; SEQ ID NO: 11)
G*U*G*U*G*U*G*U_dG_dG*dG*dG*dG*dG
(R 1061; SEQ ID NO: 12)
dG*dG*dG*dG_dG_G*U*G*U*G*U*G*U
(R 1062; SEQ ID NO: 13)
G*U*G*U*G*U*G*U*dT*dT*dT*dT*dT*dT
(R 1063; SEQ ID NO: 14)
dT*dT*dT*dT*dT*G*U*G*U*G*U*G*U
Human PBMC were cultured overnight in the presence of added DNA:RNA conjugate, with and without DOTAP. Culture supernatants were harvested and human TNF-α, IL-6, IL-12 p40, IP-10, and IFN-α were measured by ELISA using matched antibody pairs from BD-Pharmingen according to the manufacturer's protocol. Representative results for experiments including DOTAP are shown in Table 4.
TABLE 4
Immunostimulatory DNA:RNA Conjugates
TNF-α
IL-6
IP-10
EC50,
max
EC50,
max
EC50,
max
ID
μM
pg/ml
μM
pg/ml
μM
pg/ml
R 1060
4.9
20000
—
—
—
—
R 1061
4.3
20000
>20
10000
1.1
180
R 1062
0.3
80000
0.4
28000
0.1
400
R 1063
0.3
60000
0.8
28000
0.1
250
Example 10
Transfer RNA
Human PBMC were cultured overnight in the presence of various concentrations (1, 3, and 10 μg/ml) of tRNA obtained from wheat germ, bovine, yeast, and E. coli sources, added to the culture medium with and without DOTAP. Culture supernatants were harvested and human TNF-α and IL-12 p40 were measured by ELISA using matched antibody pairs from BD-Pharmingen according to the manufacturer's protocol. Yeast and E. coli tRNAs, and to a lesser extent bovine tRNA, induced TNF-α and IL-12 p40 when DOTAP was also present. In addition, E. coli tRNA at 3 and 10 μg/ml induced minor amounts of both cytokines even without DOTAP.
Example 11
HIV RNA
Human PBMC were incubated overnight with either of two key G,U-rich sequences, namely 5′-GUAGUGUGUG-3′ (SEQ ID NO:2) and 5′-GUCUGUUGUGUG-3′ (SEQ ID NO:3), corresponding to nt 99-108 and 112-123 of HIV-1 strain BH10, respectively, each with and without DOTAP. Culture supernatants were harvested, and human IL-12 p40 and TNF-α were measured by ELISA using matched antibody pairs from BD-Pharmingen according to the manufacturer's protocol. Representative results are shown in FIG. 7. The figure shows that both of these RNA molecules, at micromolar concentrations in the presence of DOTAP, induced 50-100 ng/ml of TNF and 50-200 ng/ml of IL-12 p40.
Example 12
Responsiveness of Human PBMC to Stringent Response Factor
When bacteria are starved they enter into a programmed response termed the stringent response. This involves the production of nucleic acid alarmones and ribosomal loss. Bacteria growing at high rates contain 70,000-80,000 ribosomes accounting for as much as 50% of their dry weight. As growth slows, unneeded ribosomes are hydrolyzed. It was hypothesized that rapidly growing cells in their early stationary phase contain large amounts of oligoribonucleotides that are released into the media when the cells enter a neutral pH environment.
FIG. 10 depicts the responsiveness of human PBMC to stringent response factor (SRF). SRF is produced by rapidly growing bacteria (in this case Listeria monocytogenes) in rich media until their late log phase. The bacteria were pelleted and resuspended in an equal volume of PBS for 24 h. The mixture is centrifuged to remove the bacteria. The supernatant is sterilized by passing it through a 0.2 μm filter. The sterilized solution was passed through a molecular filter with a cutoff of 10 kDa. This fraction was separated on a C18 column and the eluant was tested. At a concentration of 5 μg/ml SRF induced TNF from human PBMC. If SRF was treated with any of three RNAses the activity was destroyed. The activity was not due to substances other than RNA because the RNase-treated SRF had near background stimulatory ability. This implied activity was due to RNA.
Example 13
Responsiveness of Human PBMC to Ribonucleoside Vanadyl Complexes
During studies of SRF it was surprisingly determined that the RNAse inhibitor, ribonucleoside vanadyl complexes (RVCs), could stimulate human PBMC to produce TNF (FIG. 11) and IL-6.
FIG. 11 depicts the responsiveness of human PBMC to the ribonucleoside vanadyl complexes (RVCs). It was unexpectedly discovered during testing of RNAse inhibitors that RVCs were stimulatory for human PBMC. 2 mM RVC induced the release of substantial TNF. Also tested was the anti-viral imidazoquinoline, resiquimod (R-848) denoted as X and used at 0.1 μg/ml.
Example 14
Responsiveness of Human TLR7 and Human TLR8 to Ribonucleosides
The observations of Example 13 could be extended to 293 cells genetically reconstituted with TLR7 and TLR8 but not non-transfected 293 cells (FIG. 12). During analysis of individual ribonucleoside vanadyl complexes, it was unexpectedly determined that a mixture of the ribonucleosides A, U, C, and G or the single ribonucleoside G was effective in the absence of vanadate at stimulating PBMC to produce TNF and TLR7 or TLR8 to activate NF-κB (FIG. 12).
FIG. 12 depicts the responsiveness of human TLR7 and human TLR8 to ribonucleosides. It was determined that the response by human PBMC to RNA or RVC was mediated by TLR7 or TLR8 and further that the response could be driven by ribonucleosides only. Human 293 cells were either mock-transfected or transfected with human TLR7 or human TLR8 and monitored for responsiveness to ribonucleosides. The open reading frames of human TLR7 (hTLR7) and human TLR8 (hTLR8) were amplified by PCR from a cDNA library of human PBMC using the following primers pairs: for TLR7, 5′-CACCTCTCATGCTCTGCTCTCTTC-3′ (SEQ ID NO:15) and 5′-GCTAGACCGTTTCCTTGAACACCTG-3′ (SEQ ID NO:16); and for TLR8, 5′-CTGCGCTGCTGCAAGTTACGGAATG-3′ (SEQ ID NO:17) and 5′-GCGCGAAATCATGACTTAACGTCAG-3′ (SEQ ID NO:18). The sequence information for primer selection was obtained from Genbank accession numbers AF240467 and AF245703. All full-length TLR fragments were cloned into pGEM-T Easy vector (Promega, Mannheim, Germany), excised with NotI, cloned into the expression vector pcDNA 3.1(−) (Invitrogen, Karlsruhe, Germany) and sequenced. Sequences of the coding region of hTLR7 and hTLR8 correspond to the accession numbers AF240467 (SEQ ID NO:25) and AF245703, respectively (SEQ ID NO:29).
For monitoring transient NF-κB activation, 3×106 293 HEK cells (ATCC, VA, USA) were electroporated at 200 volt and 960 μF with 1 μg TLR expression plasmid, 20 ng NF-κB luciferase reporter-plasmid and 14 μg of pcDNA3.1(−) plasmid as carrier in 400 μl RPMI medium supplemented with 25% fetal bovine serum (FCS). Cells were seeded at 105 cells per well and after over night culture stimulated with R-848 (denoted in FIG. 12 as X; commercially synthesized by GLSynthesis Inc., Worcester, Mass., USA), RVCs or ribonucleosides for a further 7 hours. Stimulated cells were lysed using reporter lysis buffer (Promega, Mannheim, Germany), and lysate was assayed for luciferase activity using a Berthold luminometer (Wildbad, Germany).
As depicted in FIG. 12, TLR7 transfectants responded to R-848, RVCs, a mixture of ribonucleosides (A, G, C, U at 0.5 mM) and the ribonucleoside guanosine. Likewise TLR8 showed a similar response pattern.
Example 16
Responsiveness of TLR7 and TLR8 to Mixtures of Two Ribonucleosides
FIG. 13 depicts the responsiveness of TLR7 and TLR8 to mixtures of two ribonucleosides. In an experiment conducted as in FIG. 11 it was determined that TLR 8 responded best to a combination of the ribonucleosides G and U, however, TLR7 responded best to G alone. Additionally it can be seen that a minor response was given by a combination of C and U. These data show that ribonucleosides of the proper composition serve as ligands for TLR7 and TLR8. The nonspecific stimulus of TPA served as a control only. X denotes R-848.
Example 17
Human PBMC Respond to a Mixture of the Ribonucleosides G and U
FIG. 14 depicts the response of human PBMC to a mixture of the ribonucleosides G and U. It can be appreciated that the ribonucleosides G and U act synergistically to induce TNF from human PBMC. In this example the ratio of G:U of 1:10 was optimal.
Example 18
Human PBMC Respond to G,U-Rich Oligoribonucleotides
FIG. 15 depicts how human PBMC respond to RNA G,U-rich oligonucleotides. Both RNA and DNA oligonucleotides 5′-GUUGUGGUUGUGGUUGUG-3′ (SEQ ID NOs:1 and 19) were tested at 30 μM on human PBMC and TNF was monitored. Human PBMC were responsive to G,U-rich RNA oligonucleotides and not G,U-rich DNA oligonucleotides.
Example 19
Human PBMC Respond to Oxidized RNA
FIG. 16 depicts the response of human PBMC to oxidized RNA. Ribosomal 16S RNA was isolated from E. coli and subjected to chemical oxidation. The treatments were (mod A) 0.2 mM ascorbic acid plus 0.2 mM CuCl2 for 30 min at 37′C or (mod B) 0.2 mM ascorbic acid plus 0.02 mM CuCl2 for 30 min at 37′C. This treatment induces oxidation at the 8 position of guanosine and also induces strand breaks 3′ of the modified guanosine. It was shown that ribosomal RNA induced TNF production from human PBMC. It was also evident that oxidation of ribosomal RNA greatly potentiates the response.
Example 20
Human TLR7Responds to Oxidized Guanosine Ribonucleoside
FIG. 17 depicts human TLR7 and TLR8 responses to the oxidized guanosine ribonucleoside. Cells mock-transfected or transfected with human TLR 7 or human TLR8, as in Example 14, were tested for responsiveness to 7-allyl-8-oxoguanosine (loxoribine) at 1 mM. It can be clearly shown that human TLR7 is responsive to 7-allyl-8-oxoguanosine. Thus it appears that a ligand for TLR 7 is oxidized nucleic acids.
Example 21
Human TLR7Responds to Other Modified Guanosine Ribonucleoside
FIG. 18 depicts human TLR7 responses to the other modified guanosine ribonucleoside. Cells transfected with human TLR7, as in Example 14, were tested for a dose-dependent response to 7-allyl-8-oxoguanosine (loxoribine). Additionally other modified guanosines were tested. It can be clearly shown that human TLR 7 was responsive to 7-allyl-8-oxoguanosine in a dose-dependent manor. As shown above, human TLR7 was responsive to guanosine; however FIG. 18 also shows that human TLR7 responded mildly to the deoxy form of guanosine as well as to 8-bromo-guanosine.
Example 22
Distribution of Human TLRs
FIG. 19 depicts the distribution of human TLR1-TLR9. Various purified human immune cells were screened by PCR for TLR1 through 9 expression. It was shown that human lymphoid CD123+ dendritic cells (DC) were strongly positive for TLR9 and TLR7 while weaker for TLR8. The converse was shown however for myeloid CD11c+DC. This is very relevant because the two types of DC have very different functions in the immune system. Significantly, FIG. 19 also shows that human neutrophils were strongly positive for human TLR8 while very weak for TLR9 and negative for TLR7. This is also relevant because neutrophils are very often the first cells to engage infectious pathogens and thus believed to initiate responses.
Example 23
HEK-293 cell were stably transfected with human TLR7 or human TLR8. Additionally, the cells were stably transfected with NF-κB-luciferase reporter construct. The cells were titrated with varing amounts of RNA oligonucleotides and cultured for 16 h. Luciferase activity was measured by standard methods and normalizied versus mock-stimulated transfectants. Luciferase activity measured for the mock-stimulated transfectant was set to a value of 1-fold NF-κB induction. Results are shown in FIG. 20, where old NF-κB induced by the stimulating RNA oligonucleotide is plotted versus the concentration of test ribonucleotide. Stimulation with GUGUGUGU is shown for human TLR8. Stimulation with GUAGUCAC is shown for human TLR7 and human TLR8.
Equivalents
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.
All references, patents and patent publications that are recited in this application are incorporated in their entirety herein by reference.
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14159916
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zoetis belgium sa
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Mar 31st, 2022 03:01PM
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Mar 31st, 2022 03:01PM
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Zoetis
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:zts
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Zoetis
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Oct 29th, 2019 12:00AM
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May 27th, 2011 12:00AM
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https://www.uspto.gov?id=US10456463-20191029
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Vaccines comprising cholesterol and CpG as sole adjuvant-carrier molecules
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Described are vaccines having one or more antigens cholesterol and CpG. Aspects of the invention relate to the use of the vaccines of the invention for the treatment and/or prevention of human and animal disorders.
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10456463
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1. A vaccine comprising one or more antigens and an adjuvant, the adjuvant consisting of one or more isolated immunostimulatory oligonucleotides and cholesterol admixed together, and wherein the one or more isolated immunostimulatory oligonucleotides comprises a modified internucleotide linkage or a modified nucleoside base and wherein said adjuvant consisting of one or more isolated immunostimulatory oligonucleotides and cholesterol admixed together is the sole adjuvant in the vaccine.
2. The vaccine of claim 1, wherein the one or more antigens are each independently, a microbial antigen, a self antigen, a tumor antigen, an allergen, or an addictive substance.
3. The vaccine of claim 2, wherein the one or more antigens are each independently, a peptide, a peptide conjugated to a carrier protein, a peptide conjugated to a virus-like particle, a polypeptide, a recombinant protein, a purified protein, whole killed pathogen, live attenuated virus or viral vector expressing an antigen, live attenuated bacteria or a bacterial vector expressing an antigen, a polysaccharide, a polysaccharide conjugated to a carrier protein, a hapten, a hapten conjugated to a carrier protein or a small molecule.
4. The vaccine of claim 3, wherein the antigen is of bacterial origin, viral origin or parasitic origin.
5. The vaccine of claim 4, wherein a) the bacterial antigen is whole killed bacteria, live attenuated bacteria or bacterial purified proteins; or b) the viral antigen is whole killed virus, live attenuated virus or viral purified proteins.
6. The vaccine of claim 3, wherein the carrier protein is a bacterial toxoid or derivative, Pseudomonas exotoxin, KLH or a virus-like particle.
7. The vaccine of claim 6, wherein a) the bacterial toxoid is diphtheria toxoid, or a derivative thereof; or b) the virus-like particle is HBsAg, HBcAg, E. coli bacteriophage Qβ, Norwalk virus or influenza HA.
8. The vaccine of claim 2, wherein a) the addictive substance is nicotine or a nicotine-like molecule; or b) the tumor antigen is one or more of survivin, Her-2, EFGRvIII, PSA, PAP or PMSA.
9. The vaccine of claim 3, wherein the hapten conjugated to a carrier protein is nicotine or a nicotine-like molecule conjugated to diphtheria toxoid or a derivative thereof.
10. The vaccine of claim 1, wherein the amount of cholesterol relative to the amount of antigen is 0.1 to 50 fold greater by weight, 1 to 10 fold greater by weight or equal in weight to the antigen.
11. A vaccine comprising one or more antigens and an adjuvant, the adjuvant consisting of an oil-containing emulsion, one or more isolated immunostimulatory oligonucleotides and cholesterol admixed together, and wherein the one or more isolated immunostimulatory oligonucleotides comprises a modified internucleotide linkage or a modified nucleoside base and wherein said adjuvant consisting of the oil-containing emulsion, said one or more isolated immunostimulatory oligonucleotides and cholesterol admixed together is the sole adjuvant in the vaccine.
12. The vaccine of claim 11, further comprising a pharmaceutical carrier.
13. The vaccine of claim 11, wherein the one or more antigens are each independently, a microbial antigen, a self antigen, a tumor antigen, an allergen, or an addictive substance.
14. The vaccine of claim 11, wherein the one or more antigens are each independently, a peptide, a polypeptide, a recombinant protein, a purified protein, whole killed pathogen, live attenuated virus or viral vector expressing an antigen, live attenuated bacteria or a bacterial vector expressing an antigen, a polysaccharide, a polysaccharide conjugated to a carrier protein, a hapten, a hapten conjugated to a carrier protein, or a small molecule.
15. The vaccine of claim 14, wherein the carrier protein is a bacterial toxoid or a derivative thereof, Pseudomonas exotoxin, KLH or a virus-like particle.
16. The vaccine of claim 15, wherein a) the bacterial toxoid or derivative is diphtheria toxoid or a derivative thereof; or b) the virus-like particle is HBsAg, HBcAg, E. coli bacteriophage Qβ, Norwalk virus or influenza HA.
17. The vaccine of claim 11, wherein the amount of cholesterol relative to the amount of antigen is 0.1 to 50 fold greater by weight, 1 to 10 fold greater by weight or equal in weight to the antigen.
18. The vaccine of claim 11, wherein the one or more immune modulatory molecules are each independently, a TLR agonist, an antimicrobial peptide, a cytokine, a chemokine or a NOD ligand.
19. The vaccine of claim 18, wherein the TLR agonists are each independently, an oligoribonucleotide (ORN), a small molecule that activates TLR 7 and/or TLR 8, or an oligodeoxynucleotide (ODN) that activates through TLR 9.
20. A method of inducing an antigen-specific immune response in a subject in need thereof, comprising administering a vaccine comprising one or more antigens and an adjuvant, the adjuvant consisting of an oil-containing emulsion, one or more isolated immunostimulatory oligonucleotides and cholesterol admixed together, in an effective amount to induce an antigen-specific immune response in the subject, and wherein the one or more isolated immunostimulatory oligonucleotides comprises a modified internucleotide linkage or a modified nucleoside base and wherein said adjuvant consisting of the oil-containing emulsion, said one or more isolated immunostimulatory oligonucleotides and cholesterol admixed together is the sole adjuvant in the vaccine.
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20
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This application claims priority to U.S. provisional patent application No. 61/349,244, filed on May 28, 2010, hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
The invention relates to vaccines having one or more antigens and cholesterol and uses thereof. The invention further relates to vaccines having one or more antigens and one or more immune modulatory molecules and cholesterol and uses thereof.
BACKGROUND OF THE INVENTION
It has been discovered that cholesterol can potentate the activity of immune modulatory molecules and therefore the combination of cholesterol and immune modulatory molecules can be used in the treatment and/or prevention of human and animal disorders. Vaccines comprising one or more antigens and cholesterol, and vaccines comprising one or more antigens, one or more immune modulatory molecules and cholesterol are described.
SUMMARY OF THE INVENTION
In certain aspects, the invention relates to a vaccine comprising one or more antigens and cholesterol. In some aspects, the one or more antigens are each independently, a microbial antigen, a self antigen, a tumor antigen, an allergen, or an addictive substance.
In certain aspects, the invention relates to a vaccine comprising one or more antigens and one or more immune modulatory molecules and cholesterol. In aspects the vaccine further comprises a pharmaceutical carrier. In some aspects, the one or more antigens are each independently, a microbial antigen, a self antigen, a tumor antigen, an allergen, or an addictive substance.
In certain aspects, the invention relates to a method of inducing an antigen-specific immune response in a subject in need thereof, comprising administering a vaccine comprising one or more antigens and cholesterol in an effective amount to induce an antigen-specific immune response in the subject.
In certain aspects, the invention relates to a method of inducing an antigen-specific immune response in a subject in need thereof, comprising administering a vaccine comprising one or more antigens and one or more immune modulatory molecules and cholesterol.
DESCRIPTION OF FIGURES
FIG. 1: Graphs representing antigen specific cytokine secretion by T cells in the presence of no adjuvant, or in the presence of CpG or CpG+cholesterol as an adjuvant. FIG. 1a. Graph of CD4+ T cells secreting single or double cytokines. FIG. 1b. Graph of CD4+ T cells secreting triple cytokines. FIG. 1 c. Graph of CD8+ T cells secreting single or double cytokines. FIG. 1d. Graph of CD8+ T cells secreting triple cytokines.
FIG. 2: Graph of IL-2 (FIG. 2a) and IFN-γ (FIG. 2b) production in the presence of no adjuvant, CpG and CpG+cholesterol.
FIG. 3: Graph of ovalbumin specific CD8+ T cell responses in the presence of no adjuvant or in the presence of CpG or CpG+cholesterol as an adjuvant. FIGS. 3a-3b: cytotoxic T cell responses, FIGS. 3c-3d: antigen-specific CD8+ T cell population.
FIG. 4: Graph of ovalbumin specific antibody titers in the presence of no adjuvant or in the presence of CpG or CpG+cholesterol as an adjuvant. The numbers above each bar represent the ratio of IgG2c/IgG1.
FIG. 5: Transmission Electron Microscopy image of antigen, CpG and cholesterol.
FIG. 6: Graph depicting the injection site reactions in calves immunized with pentavalent inactivated viral vaccine BVDV 1&2, IBRV, PI3V and BRSV in the presence of CpG+cholesterol (at ratios of 1:1 or 1:10 CpG:cholesterol), Advasure-DEAE/Dextran, QCDCR or QCDCR+CpG. Some animals were immunized with commercial vaccine. Placebo animals received sterile saline. Table 1 depicts percentage of calves with clinical disease, fever, leucopenia or viremia following challenge with BVDV-2 post vaccination with pentavalent inactivated viral vaccine BVDV 1&2, IBRV, PI3V and BRSV in the presence of CpG+cholesterol (at ratios of 1:1 or 1:10 CpG:cholesterol), Advasure-DEAE/Dextran, QCDCR or QCDCR+CpG. Some animals were immunized with commercial vaccine. Placebo animals received sterile saline.
FIG. 7: Graph of antigen-specific antibody response in pigs immunized with pertactin (p68) formulated with various adjuvants including CpG+cholesterol.
DESCRIPTION OF SEQUENCES
SEQ ID NO: 1 -
CPG 7909
5′ TCGTCGTTTTGTCGTTTTGTCGTT 3′.
SEQ ID NO: 2 -
CpG 24555
5′ TCGTCGTTTTTCGGTGCTTTT 3′.
SEQ ID NO: 3 -
CPG 10104
5′ TCGTCGTTTCGTCGTTTTGTCGTT 3′.
SEQ ID NO: 4 -
CPG 10101
5′ TCGTCGTTTTCGGCGGCCGCCG 3′.
SEQ ID NO: 5 -
CPG 10109
5′ TCGTC-GTTTTAC-GGCGCC-GTCCCG 3′.
SEQ ID NO: 6 -
CpG 23407
5′ T*C-G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*T
3′.
SEQ ID NO: 7 -
CPG 21798
5′ T*C-G*T*C-G*A*C-G*A*T*C-G*G*C*G*C-G*C*G*C*C*G
3′.
SEQ ID NO: 8 -
CPG 23430
5′ T*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G
3′.
SEQ ID NO: 9 -
CpG 24558
5′ T*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T
3′.
SEQ ID NO: 10 -
CPG 23871
5′ JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G
3′.
SEQ ID NO: 11 -
CPG 23873
5′ JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*
G*T* 3′.
SEQ ID NO: 12 -
CPG 23874
5′ *C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T
3′.
SEQ ID NO: 13 -
CPG 23875
5′ EU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G
3′.
SEQ ID NO: 14 -
CpG 23877
5′ JU*C-G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*G*T
3′.
SEQ ID NO: 15 -
CpG 23878
5′ JU*C*G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*G*T
3′.
SEQ ID NO: 16 -
poly I:
C ODN1a 5′- ICI CIC ICI CIC ICI CIC ICI CIC
IC-3′.
SEQ ID NO: 17 -
5′ GGGGACGACGTCGTGGGGGGG 3′.
SEQ ID NO: 18 -
5′ G*G*G_G_A_C_G_A_C_G_T_C_G_T_G_G*G*G*G*G*G 3'.
SEQ ID NO: 19 -
5′ TCGTCGTTTTGTCGTTTTGTCGTT 3′.
SEQ ID NO: 20 -
5′ TCGTCGTTTTGTCGTTTTTTTCGA 3′.
SEQ ID NO: 21 -
5′ T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*G*C*T*T*T*T 3′.
SEQ ID NO: 22 -
5′ T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T 3′.
SEQ ID NO: 23 -
5′ T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T
3′.
SEQ ID NO: 24 -
5′ T*C*G*T*C*G*T*T*T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T
3′.
SEQ ID NO: 25 -
5′ T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*T*T*T*C*G*A
3′.
SEQ ID NO: 26 -
5′ TCGCGTCGTTCGGCGCGCGCCG 3′.
SEQ ID NO: 27 -
5′ TCGTCGACGTTCGGCGCGCGCCG 3′.
SEQ ID NO: 28 -
5′ TCGGACGTTCGGCGCGCGCCG 3′.
SEQ ID NO: 29 -
5′ TCGGACGTTCGGCGCGCCG 3′.
SEQ ID NO: 30 -
5′ TCGCGTCGTTCGGCGCGCCG 3′.
SEQ ID NO: 31 -
5′ TCGACGTTCGGCGCGCGCCG 3′.
SEQ ID NO: 32 -
5′ TCGACGTTCGGCGCGCGG 3′.
SEQ ID NO: 33 -
5′ TCGCGTCGTTCGGCGCCG 3′.
SEQ ID NO: 34 -
5′ TCGCGACGTTCGGCGCGCGCCG 3′.
SEQ ID NO: 35 -
5′ TCGTCGTTTTCGGCGCGCGCCG 3′.
SEQ ID NO: 36 -
5′ TCGTCGACGATCGGCGCGCGCCG 3′.
SEQ ID NO: 37 -
5′ T*C_G*T*C_G*A*C_G*A*T*C_G*G*C*G*C_G*C*G*C*C*G
3′.
SEQ ID NO: 38 -
5′ T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G
3′.
SEQ ID NO: 39 -
5′ T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G
3′.
SEQ ID NO: 40 -
5′ T*C_G*G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G
3′.
SEQ ID NO: 41 -
5′ T*C_G*G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3′.
SEQ ID NO: 42 -
5′ T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3′.
SEQ ID NO: 43 -
5′ T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3′.
SEQ ID NO: 44 -
5′ T*C_G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3′.
SEQ ID NO: 45 -
5′ T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*C*G 3′.
SEQ ID NO: 46 -
5′ T*C_G*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G
3′.
SEQ ID NO: 47 -
5′ T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G
3′.
SEQ ID NO: 48 -
5′ T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*G*C*C*G*C*C*G
3′.
SEQ ID NO: 49 -
5′ T*C*G*T*C_G*T*T*T*T*A*C_G*G*C*G*C*C_G*T*G*C*C*G
3′.
SEQ ID NO: 50 -
5′ T*C_G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*T
3′.
(*) represents the presence of a stabilized
internucleotide linkage and _ represents a
phosphodiester bond. J represents an iodo
modified nucleotide and E represents an
ethyl modified nucleotide.
DETAILED DESCRIPTION OF THE INVENTION
Aspects of the invention relate to vaccines having one or more antigens and cholesterol, and vaccines having one or more antigens and one or more immune modulatory molecules and cholesterol. In aspects of the invention, methods of inducing an antigen-specific immune response in a subject in need thereof by administering the vaccines of the invention are disclosed. Use of the vaccines in the manufacture of a medicament for the treatment of a disorder are also disclosed.
In aspects of the invention, a one or more antigen(s) are each independently, a microbial antigen, a self antigen, a tumor antigen, an allergen, or an addictive substance. In some aspects, a microbial antigen is of bacterial, viral or parasitic origin. In some aspects, the antigen is a peptide, a peptide conjugated to a carrier protein, a polypeptide, a recombinant protein, a purified protein, whole killed pathogen, live attenuated virus, live attenuated bacteria, antigen expressed within a viral or bacterial vector, a polysaccharide, a polysaccharide conjugated to a carrier protein, protein conjugated to a virus-like particle, a hapten, a hapten conjugated to a carrier protein or a small molecule.
In aspects of the invention, the antigen is of bacterial origin. In some aspects, the bacterial antigen is whole killed bacteria, live attenuated bacteria or bacterial purified proteins.
In aspects of the invention, a bacteria includes, but is not limited to, Aceinetobacter caicoaceticus, Acetobacter paseruianus, Actinobacillus actinomycetemcomitans, Actinobacillus pleuropneumoniae, Actinomyces israelli, Actinomyces viscosus, Aeromonas hydrophila, Aicaliges eutrophus, Alicyclobacillus acidocaldarius, Arhaeglobus fulgidus, Bacillus species, Bacillus antracis, Bacillus pumilus, Bacillus stearothermophillus, Bacillus subtilis, Bacillus thermocatenulatus, Bacteroides species, Bordetella species, Bordetella bronchiseptica, Borrelia burgdorferi, Brucella species, Burkholderia cepacia, Burkholderia glumae, Brachyspira species. Brachyspira hyodysenteria, Brachyspira pilosicoli, Camphylobacter species, Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis, Campylobacter jejuni, Chlamydia psittaci, Chlamydia trachomatis, Chlamydophila species, Chromobacterium viscosum, Clostridium species, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium species, Corynebacterium diphtheriae, Ehrlichia canis, Enterobacter species, Enterobacter aerogenes, Enterococcus species, Erysipelothrix rhusiopathieae, Escherichia species, Escherichia coli, Fusobacterium nucleatum, Haemophilus species, Haemophilus influenzae, Haemophilus somnus, Helicobacter species, Helicobacter pylori, Helicobacter suis, Klebsiella species, Klebsiella pneumoniae, Lactobacillus acidophilis, Lawsonia intracellularis, Legionella species, Legionella pneumophilia, Leptospira species, such as Leptospira canicola, Leptospira grippotyposa, Leptospira hardjo, Leptospira borgpetersenii hardjo-bovis, Leptospira borgpetersenii hardjo-prajitno, Leptospira interrogans, Leptospira icterohaemorrhagiae, Leptospira pomona, Leptospira, Leptospira bratislava, Listeria species, Listeria monocytogenes, Meningococcal bacteria, Moraxella species, Mycobacterium species, Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansaii, Mycobacterium gordonae, Mycoplasma species, such as, Mycoplasma hyopneumoniae, Mycoplasma synoviae, Mycoplasma hyorhinis, Mycoplasma pneumoniae, Mycoplasma mycoides subsp. mycoides LC, Neisseria species, Neisseria gonorrhoeae, Neisseria meningitidis, Odoribacter denticanis, Pasteurella species, Pasteurella (Mannheimia) haemolytica, Pasteurella multocida, Photorhabdus luminescens, Porphyromonas gingivalis, Porphyromonas gulae, Porphyromonas salivosa, Propionibacterium acnes, Proteus species, Proteus vulgaris, Pseudomonas species, Pseudomnas wisconsinensis, Pseudomonas aeruginosa, Pseudomonas fluorescens C9, Pseudomonas fluorescens SIKW1, Pseudomonas fragi, Pseudomonas luteola, Pseudomonas oleovorans, Pseudomonas sp B11-1, Psychrobacter immobilis, Rickettsia spp, Rickettsia prowazekii, Rickettsia rickettsia, Salmonella species, Salmonella bongori, Salmonella choleraeuis, Salmonella dublin, Salmonella enterica, Salmonella newport, Salmonella typhimurium, Salmonella typhi, Serratia marcescens, Shigella species, Spirlina platensis, Staphylococci species, Staphlyoccocus aureus, Staphyloccoccus epidermidis, Staphylococcus hyicus, Streptococcus species, Streptobacillus moniliformis, beta-hemolytic Streptococcus, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguis, Streptomyces albus, Streptomyces cinnamoneus, Streptomyces exfoliates, Streptomyces scabies, Sulfolobus acidocaldarius, Syechocystis sp., Treponena species, Treponema denticola, Treponema minutum, Treponema palladium, Treponema pertenue, Treponema phagedenis, Treponema refringens, Treponema vincentii, Vibrio species, Vibrio cholerae, Yersinia species and combinations thereof.
Polypeptides or polysaccharides of bacterial pathogens include, but are not limited to, an iron-regulated outer membrane protein (IROMP), an outer membrane protein (OMP), and an A-protein of Aeromonis salmonicida which causes furunculosis, p57 protein of Renibacterium salmoninarum which causes bacterial kidney disease (BKD), major surface associated antigen (msa), a surface expressed cytotoxin (mpr), a surface expressed hemolysin (ish), and a flagellar antigen of Yersiniosis; an extracellular protein (ECP), an IROMP, and a structural protein of Pasteurellosis; an OMP and a flagellar protein of Vibrosis anguillarum and V. ordalii; a flagellar protein, an OMP protein, aroA, and purA of Edwardsiellosis ictaluri and E. tarda; and surface antigen of lchthyophthirius; and a structural and regulatory protein of Cytophaga columnari; and a structural and regulatory protein of Rickettsia, IsdA, ClfA, ClfB, Opp3A, HLA and capsular polysaccharides from Staphylococcus aureus.
In aspects of the invention, the antigen is of viral origin. In some aspects, the viral antigen is whole killed or inactivated virus, live attenuated virus or viral purified proteins or peptides.
In some aspects, the virus is one that infects animals including, but not limited to, Avian herpesvirus, Avian influenza, Avian leukosis virus, Avian paramyxoviruses, Border disease virus, Bovine coronavirus, Bovine ephemeral fever virus, Bovine herpes viruses, Bovine immunodeficiency virus, Bovine leukemia virus, Bovine parainfluenza virus 3, Bovine respiratory syncytial virus, Bovine viral diarrhea virus (BVDV), BVDV Type I, BVDV Type II, Canine adenovirus, Canine coronavirus (CCV), Canine distemper virus, Canine herpes viruses, Equine herpes viruses, Canine influenza virus, Canine parainffuenza virus, Canine parvovirus, Canine respiratory coronavirus, Classical swine fever virus, Eastern Equine encephalitis virus (EEE), Equine infectious anemia virus, Equine influenza virus, West nile virus, Feline Calicivirus, Feline enteric coronavirus, Feline immunodeficiency virus, Feline infectious peritonitis virus, Feline herpes Virus, Feline influenza virus, Feline leukemia virus (FeLV), Feline viral rhinotracheitis virus, Lentivirus, Marek's disease virus, Newcastle Disease virus, Ovine herpesviruses, Ovine parainfluenza 3, Ovine progressive pneumonia virus, Ovine pulmonary adenocarcinoma virus, Pantropic CCV, Porcine circovirus (PCV) Type I, PCV Type II, Porcine epidemic diarrhea virus, Porcine hemagglutinating encephalomyletitis virus, Porcine herpesviruses, Porcine parvovirus, Porcine reproductive and respiratory syndrome (PRRS) Virus, Pseudorabies virus, Rabies, Rotovirus, Rhinoviruses, Rinderpest virus, Swine influenza virus, Transmissible gastroenteritis virus, Turkey coronavirus, Venezuelan equine encephalitis virus, Vesicular stomatitis virus, West Nile virus, Western equine encephalitis virus and combinations thereof.
In some aspects, the virus is one that infects humans, including, but not limited to, Adenoviridae (most adenoviruses); Arena viridae (hemorrhagic fever viruses); Astroviruses; Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Calciviridae (e.g., strains that cause gastroenteritis); Coronoviridae (e.g., coronaviruses); Filoviridae (e.g., ebola viruses); Flaviridae (e.g., hepatitis C virus, dengue viruses, encephalitis viruses, yellow fever viruses); Hepadnaviridae (Hepatitis B virus); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus); Iridoviridae (e.g., African swine fever virus); Norwalk and related viruses; Orthomyxoviridae (e.g., influenza viruses); Papovaviridae (papilloma viruses, polyoma viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Parvovirida (parvoviruses); Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Poxyiridae (variola viruses, vaccinia viruses, pox viruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses); Retroviridae (e.g. human immunodeficiency viruses, such as HIV-tor HIV-2 (also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP); Rhabdoviradae (e.g., vesicular stomatitis viruses, rabies viruses); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); and Unclassified viruses (e.g., the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus).
In aspects of the invention, the antigen is of parasitic origin. In some aspects, the parasite is a protein from Anaplasma, Ancylostoma (hookworms), Ascaris, Babesia, Coccidia, Cryptosporidium parvum, Dirofilaria (heartworms), Eimeria species, Fasciola hepatica (liver fluke), Giardia, Hammondia, Isopsora, Leishmania species, Neospora caninum, Sarcocystis, Schistosoma, Strongyloides, Taenia, Toxoplasma gondii, Trichinella species, Trichomonas species or Trypanosoma species. and combinations thereof.
In some aspects, the parasite is an external parasite. In some aspects, an external parasite includes, but is not limited to, ticks, including Ixodes, Rhipicephalus, Dermacentor, Amblyomma, Boophilus, Hyalomma, or Haemaphysalis species, and combinations thereof.
In aspects of the invention, an antigen is a self antigen. In some aspects, a self antigen is an antigen of a subject's own cells or cell products that causes an immune response in a subject. In some aspects, a self antigen includes, but is not limited to, a tumor antigen, an antigen associated with Alzheimer's Disease, an antigen against an antibody, or an antigen that is expressed from human endogenous retroviral elements. An antigen associated with Alzheimer's Disease may be tau or β-amyloid. An antigen against an antibody may be an antigen against a human antibody, for example, in some embodiments the antigen is IgE.
In aspects of the invention, an antigen is a tumor antigen. In some aspects, the tumor antigen is one or more of WT1, MUC1, LMP2, HPV E6 or HPV E7, EGFR or variant form, for example, EGFRvIII, HER-2/neu, Idiotype, MAGE A3, p53 nonmutant, NY-ESO-1, PSMA, GD2, CEA, MelanA/MART1, Ras mutant, gp100, p53 mutant, Proteinase3 (PR1), bcr-abl, Tyrosinase, Survivin, PSA, hTERT, Sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NM 7, PAX3, ALK, Androgen receptor, Cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, GD3, Fucosyl GM1, Mesothelin, PSCA, MAGE A1, sLe (animal), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-beta, MAD-CT-2, or Fos-related antigen 1. Such tumor antigens have been ranked based on criteria such as a) therapeutic function, b) immunogenicity, c) role of the antigen in oncogenicity, d) specificity, expression level and percent of antigen-positive cells, e) stem cell expression, f) number of patients with antigen-positive cancers, g) number of antigenic epitopes and h) cellular location of antigen expression (see Cheever, M. A. et al., Clincal Cancer Research, Sep. 1, 2009, 15(17):5323-5337). In some embodiments, the tumor antigen is one or more of survivin, Her-2, EFGRvIII, PSA, PAP or PMSA.
In aspects of the invention, an antigen is an allergen. An allergen refers to a substance (antigen) that can induce an allergic or asthmatic response in a susceptible subject. The list of allergens is enormous and can include pollens, insect venoms, animal dander dust, fungal spores and drugs (e.g. penicillin). Examples of natural, animal and plant allergens include but are not limited to proteins specific to the following genuses: Agropyron (e.g. Agropyron repens); Agrostis (e.g. Agrostis alba); Alder, Alnus (Alnus gultinoasa); Alternaria (Alternaria alternata); Ambrosia (Ambrosia artemiisfolia; Anthoxanthum (e.g. Anthoxanthum odoratum); Apis (e.g. Apis multiflorum); Arrhenatherum (e.g. Arrhenatherum elatius); Artemisia (Artemisia vulgaris); Avena (e.g. Avena sativa); Betula (Betula verrucosa); Blattella (e.g. Blattella germanica); Bromus (e.g. Bromus inermis); Canine (Canis familfaris); Chamaecyparis (e.g. Chamaecyparis obtuse); Cryptomeria (Cryptomeria japonica); Cupressus (e.g. Cupressus sempervirens, Cupressus arizonica and Cupressus macrocarpa); Dactylis (e.g. Dactylis glomerate); Dermatophagoides (e.g. Dermatophagoides farinae); Fells (Fells domesticus); Festuca (e.g. Festuca elation); Holcus (e.g. Holcus lanatus); Juniperus (e.g. Juniperus sabinoides, Juniperus virginiana, Juniperus communis and Juniperus ashei); Lolium (e.g. Lolium perenne or Lolium multiflorum); Olea (Olea europa); Parietaria (e.g. Parietaria officinalis or Parietaria Judaica); Paspalum (e.g. Paspalum notatum); Periplaneta (e.g. Periplaneta americana); Phalaris (e.g. Phalaris arundinacea); Phleum (e.g. Phleum pretense); Plantago (e.g. Plantago lanceolate); Poa (e.g. Poa pratensis or Poa compressa); Quercus (Quercus alba); Secale (e.g. Secale cereale); Sorghum (e.g. Sorghum halepensis); Thuya (e.g. Thuya orientalis); and Triticum (e.g. Triticum aestivum), and combinations thereof.
In aspects of the invention, an antigen is an addictive substance. An addictive substance is any chemical or biological substance, including synthetic or artificial substances, that cause a subject to develop an addiction to that substance. In some aspects, an addictive substance is nicotine or cocaine. In some embodiments, the antigen in a vaccine against a nicotine addiction is nicotine or a nicotine-like hapten conjugated to a carrier. In some embodiments, the carrier to which nicotine or nicotine-like hapten is conjugated is diphtheria toxoid.
In aspects of the invention, an antigen or a hapten is conjugated to a carrier protein. In some aspects, the carrier protein is a bacterial toxoid or derivative, Pseudomonas exotoxin, KLH or a virus-like particle. In some aspects, a bacterial toxoid is diphtheria toxoid or a derivative thereof. In some aspects, a bacterial toxoid is CRM197. In some aspects, the virus-like particle is HBsAg, HBcAg, E. coli bacteriophage Qβ, Norwalk virus or influenza HA.
Aspects of the invention relate to vaccines having cholesterol. Cholesterol is a white crystalline substance with a chemical formula of C27H45OH. It is a cyclic hydrocarbon alcohol which is classified as a lipid. A lipid is any group of organic compounds, including, but not limited to, the fats, oils, waxes, sterols and triglycerides, that are insoluble in water but are soluble in nonpolar organic solvents, are oily to the touch and together with carbohydrates and proteins are the principal structural material of living cells. Cholesterol is insoluble in water but is soluble in a number of organic solvents.
In aspects of the invention, sterol refers to compounds in animals which are biologically produced from perpenoid precursors. They comprise a steroid ring structure, having a hydroxyl (OH) group. In some aspects, the hydroxyl group may be attached to carbon-3. The hydrocarbon chain of the fatty-acid substituent varies in length. In some aspects, the hydrocarbon chain may be from 16 to 20 carbon atoms. In some aspects, the hydrocarbon chain may be saturated or unsaturated. Sterols can contain one or more double bonds in the ring structure and may also include a variety of substituents attached to the rings. Sterols and their fatty-acid esters may be water insoluble. Fatty-acid esters relate to any of a class of organic compounds corresponding to inorganic salts, which are formed from a condensation reaction in which a molecule of an organic acid unites with a molecule of alcohol with the elimination of a molecule of water. In some aspects, sterols refers to synthetic sterols. In some aspects, synthetic steroids includes, but is not limited to, glucocorticoids (for example, prednisone, dexamethasone, triamcinolone), mineralocorticoid (for example, fludrocortisones), vitamin D (for example, dihydrotachysterol), androgens (for example, oxandrolone, nandrolone, anabolic steroids), estrogens (for example, diethylstilbestrol) and progestins (for example, norethindrone, medroxyprogesterone acetate). In some aspects, a cholate may be used, for example sodium deoxycholate.
In aspects of the invention, sterols include, but are not limited to, natural steroids such as, β-sitosterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol. Such sterols may be purchased commercially. Cholesterol, for example, is disclosed in the Merck Index, 12th Ed., p. 369.
In aspects of the invention, sterols may be used as an adjuvant. In some aspects, the amount of sterol may be about 1 μg to about 5,000 μg per vaccine dose. In some aspects, the amount of sterol may be about 1 μg to about 4,000 μg per vaccine dose, about 1 μg to about 3,000 μg per vaccine dose, about 1 μg to about 2,000 μg per vaccine dose, or about 1 μg to about 1,000 μg per vaccine dose. In some aspects, the amount of sterol may be about 5 μg to about 750 μg per vaccine dose, about 5 μg to about 500 μg per vaccine dose, about 5 μg to about 250 μg per vaccine dose, about 5 μg to about 100 μg per vaccine dose, about 15 μg to about 100 μg per vaccine dose, or about 30 μg to about 75 μg per vaccine dose.
In aspects of the invention, a vaccine has one or more antigens and cholesterol. In some aspects, the amount of cholesterol relative to the amount of antigen is about 0.1 to about 50 fold greater by weight. In some aspects, the amount of cholesterol is about 1 to about 10 fold greater by weight than the antigen. In some aspects, the amount of cholesterol is equal in weight to the antigen.
In aspects of the invention, a vaccine has one or more antigens and one or more immune modulatory molecules and cholesterol. In some aspects, a vaccine further includes a pharmaceutical carrier.
In aspects of the invention, “in conjunction” or “in conjunction with” refers to an admixture, a combination or being in close proximity to one or more antigens, and one or more antigens and one or more immune modulatory molecules. In aspects, the one or more antigens and/or the one or more immune modulatory molecules may be attached to cholesterol by a physical means via one or more linkers. A linker includes, but is not limited to, direct or indirect linkers. In aspects, the one or more antigens and/or one or more immune modulatory molecules and cholesterol may be encapsulated together.
In aspects of the invention, one or more antigens may be admixed with one or more immune modulatory molecules. In aspects, one or more antigens may be admixed with cholesterol. In aspects, one or more immune modulatory molecules may be admixed with cholesterol. In aspects, one or more immune modulatory molecules may be admixed with antigen and cholesterol. In aspects, one or more immune modulatory molecules may be admixed with cholesterol and one or more antigens may be separate. In aspects, one or more antigens may be admixed with cholesterol and one or more immune modulatory molecules may be separate. In aspects, one or more antigens and/or one or more immune modulatory molecules may be in conjunction with cholesterol.
In aspects of the invention, the amount of cholesterol relative to the amount of antigen is greater than the amount of antigen. In some aspects, the amount of cholesterol relative to the amount of antigen is about 0.1 to 50 fold greater by weight than the antigen. In aspects, the amount of cholesterol relative to the amount of antigen is about 10 to about 50 fold, about 20 to about 40 fold, about 30 to about 35 fold greater by weight than the antigen. In aspects, the amount of cholesterol relative to the amount of antigen is about 1 to about 10 fold greater by weight than the antigen. In aspects, the amount of cholesterol relative to the amount of antigen is equal in weight to the antigen. In aspects, the antigen may be one or more antigens and the weight of the antigen is the total weight of the one or more antigens.
In aspects of the invention, an immune modulatory molecule (one or more immune modulatory molecules) is a molecule that modulates immune cells in a subject. This effect may be mediated directly, for example, through a receptor, or indirectly, for example, through cytokines or chemokines released from another immune cell that is modulated directly. An induction of an immune response refers to any increase in number or activity of an immune cell, or an increase in expression or absolute levels of an immune factor, such as a cytokine. Immune cells include, but are not limited to, NK cells, CD4+ T lymphocytes, CD8+ T lymphocytes, B cells, dendritic cells, macrophage and other antigen-presenting cells. Cytokines include, but are not limited to, interleukins, TNF-α, IFN-α,β and γ. In aspects, an immune modulator is a molecule which when used with an antigen enhances antigen specific humoral (for example, antibody) and or cellular (for example, T cell) immune responses.
In some aspects, an immune modulatory molecule is a TLR agonist, an antimicrobial peptide, a cytokine, a chemokine, a NOD ligand or an oligonucleotide. In some aspects, a TLR agonist is an oligoribonucleotide (ORN) or a small molecule that activates TLR 7 and/or TLR 8. In some aspects, a TLR agonist is an oligodeoxynucleotide (ODN) that activates through TLR 9. In some aspects, a TLR 9 agonist is an ODN containing unmethylated CpG motifs, a B-Class oligodeoxynucleotide, a C-Class oligodeoxynucleotide or a P-Class oligodeoxynucleotide. In some aspects, a TLR 9 agonist is IMO-2055, IMO-2125 or IMO-2134 (QAX935). In other aspects, a TLR agonist is a poly I:C that activates TLR 3. In some aspects, the poly I:C is ODN1a having the sequence 5′-ICI CIC ICI CIC ICI CIC ICI CIC IC-3′ (SEQ ID NO:16).
In aspects of the invention, an oligonucleotide can encompass various chemical modifications and substitutions, in comparison to natural RNA and DNA, involving a phosphodiester internucleoside bridge, a β-D-ribose unit and/or a natural nucleoside base (adenine, guanine, cytosine, thymine, uracil). Examples of chemical modifications are known to the skilled person and are described, for example, in Uhlmann E et al. (1990) Chem Rev 90:543; “Protocols for Oligonucleotides and Analogs” Synthesis and Properties & Synthesis and Analytical Techniques, S. Agrawal, Ed, Humana Press, Totowa, USA 1993; Crooke S T et al. (1996) Annu Rev Pharmacol Toxicol 36:107-129; and Hunziker J et al. (1995) Mod Synth Methods 7:331-417. In some aspects, an oligonucleotide may have one or more modifications, wherein each modification is located at a particular phosphodiester internucleoside bridge and/or at a particular β-D-ribose unit and/or at a particular natural nucleoside base position in comparison to an oligonucleotide of the same sequence which is composed of natural DNA or RNA.
In some aspects, the oligonucleotides may comprise one or more modifications and wherein each modification is independently selected from:
a) the replacement of a phosphodiester internucleoside bridge located at the 3′ and/or the 5′ end of a nucleoside by a modified internucleoside bridge,
b) the replacement of phosphodiester bridge located at the 3′ and/or the 5′ end of a nucleoside by a dephospho bridge,
c) the replacement of a sugar phosphate unit from the sugar phosphate backbone by another unit,
d) the replacement of a β-D-ribose unit by a modified sugar unit, and
e) the replacement of a natural nucleoside base by a modified nucleoside base.
In aspects of the invention, the oligonucleotides may include modified internucieotide linkages, such as those described in a) or b) above. These modified linkages may be partially resistant to degradation (e.g., are stabilized). A “stabilized oligonucleotide molecule” is an oligonucleotide that is relatively resistant to in vivo degradation (e.g. via an exo- or endo-nuclease) resulting from such modifications. Oligonucleotides having phosphorothioate linkages, in some aspects, may provide maximal activity and protect the oligonucleotide from degradation by intracellular exo- and endo-nucleases.
A phosphodiester internucleoside bridge located at the 3′ and/or the 5′ end of a nucleoside can be replaced by a modified internucleoside bridge, wherein the modified internucleoside bridge is, for example, selected from phosphorothioate, phosphorodithioate, NR1R2-phosphoramidate, boranophosphate, α-hydroxybenzyl phosphonate, phosphate-(C1-C21)—O-alkyl ester, phosphate-[(C6-C12)aryl-(C1-C21)—O-alkyl]ester, (C1-C8)alkylphosphonate and/or (C6-C12)arylphosphonate bridges, (C7-C12)-α-hydroxymethyl-aryl (e.g., disclosed in WO 95/01363), wherein (C6-C12)aryl, (C6-C20)aryl and (C6-C14)aryl are optionally substituted by halogen, alkyl, alkoxy, nitro, cyano, and where R1 and R2 are, independently of each other, hydrogen, (C1-C18)-alkyl, (C6-C20)-aryl, (C6-C14)-aryl-(C1-C8)-alkyl, preferably hydrogen, (C1-C8)-alkyl, preferably (C1-C4)-alkyl and/or methoxyethyl, or R1 and R2 form, together with the nitrogen atom carrying them, a 5-6-membered heterocyclic ring which can additionally contain a further heteroatom from the group O, S and N.
The replacement of a phosphodiester bridge located at the 3′ and/or the 5′ end of a nucleoside by a dephospho bridge (dephospho bridges are described, for example, in Uhlmann E and Peyman A in “Methods in Molecular Biology”, Vol. 20, “Protocols for Oligonucleotides and Analogs”, S. Agrawal, Ed., Humana Press, Totowa 1993, Chapter 16, pp. 355 ff), wherein a dephospho bridge is for example selected from the dephospho bridges formacetal, 34-hioformacetal, methylhydroxylamine, oxime, methylenedimethyl-hydrazo, dimethylenesulfone and/or silyl groups.
A sugar phosphate unit (i.e., a β-D-ribose and phosphodiester internucleoside bridge together forming a sugar phosphate unit) from the sugar phosphate backbone (i.e., a sugar phosphate backbone is composed of sugar phosphate units) can be replaced by another unit, wherein the other unit is, for example, suitable to build up a “morpholino-derivative” oligomer (as described, for example, in Stirchak E P et al. (1989) Nucleic Acids Res 17:6129-41), that is, e.g., the replacement by a morpholino-derivative unit; or to build up a polyamide nucleic acid (“PNA”: as described for example, in Nielsen P E et al. (1994) Bioconjug Chem 5:3-7), that is, e.g., the replacement by a PNA backbone unit, e.g., by 2-aminoethylglycine. The oligonucleotide may have other carbohydrate backbone modifications and replacements, such as peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), and oligonucleotides having backbone sections with alkyl linkers or amino linkers. The alkyl linker may be branched or unbranched, substituted or unsubstituted, and chirally pure or a racemic mixture.
A β-ribose unit or a β-D-2′-deoxyribose unit can be replaced by a modified sugar unit, wherein the modified sugar unit is, for example, selected from β-D-ribose, α-D-2′-deoxyribose, L-2′-deoxyribose, 2′-F-2′-deoxyribose, 2-F-arabinose, 2′-O—(C1-C6)alkyl-ribose, preferably 2′-O—(C1-C6)alkyl-ribose is 2′-O-methylribose, 2′-O—(C2-C6)alkenyl-ribose, 2′-[O—(C1-C6)alkyl-O—(C1-C6)alkyl]-ribose, 2′-NH2-2′-deoxyribose, β-D-xylo-furanose, α-arabinofuranose, 2,4-dideoxy-β-D-erythro-hexo-pyranose, and carbocyclic (described, for example, in Froehler J (1992) Am Chem Soc 114:8320) and/or open-chain sugar analogs (described, for example, in Vandendriessche et al. (1993) Tetrahedron 49:7223) and/or bicyclosugar analogs (described, for example, in Tarkov M et al. (1993) Helv Chim Acta 76:481).
In some aspects, the sugar is 2′-O-methylribose, for one or both nucleotides linked by a phosphodiester or phosphodiester-like internucleoside linkage.
Nucleic acids also include, but are not limited to, substituted purines and pyrimidines such as C-5 propyne pyrimidine and 7-deaza-7-substituted purine modified bases. Wagner R W et al. (1996) Nat Biotechnol 14:840-4. Purines and pyrimidines include, but are not limited, to adenine, cytosine, guanine, and thymine, and other naturally and non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties.
A modified base is any base which is chemically distinct from the naturally occurring bases typically found in DNA and RNA such as T, C, G, A, and U, but which share basic chemical structures with these naturally occurring bases. The modified nucleoside base may be, for example, selected from hypoxanthine, uracil, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C1-C6)-alkylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 2,4-diamino-purine, 8-azapurine, a substituted 7-deazapurine, preferably 7-deaza-7-substituted and/or 7-deaza-8-substituted purine, 5-hydroxymethylcytosine, N4-alkylcytosine, e.g., N4-ethylcytosine, 5-hydroxydeoxycytidine, 5-hydroxymethyldeoxycytidine, N4-alkyldeoxycytidine, e.g., N4-ethyldeoxycytidine, 6-thiodeoxyguanosine, and deoxyribonucleosides of nitropyrrole, C5-propynylpyrimidine, and diaminopurine e.g., 2,6-diaminopurine, inosine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, hypoxanthine or other modifications of a natural nucleoside bases. This list is meant to be exemplary and is not to be interpreted to be limiting.
In aspects of the invention, for some formulas described herein a set of modified bases is defined. For instance, the letter Y is used to refer to a nucleotide containing a cytosine or a modified cytosine. A modified cytosine is a naturally occurring or non-naturally occurring pyrimidine base analog of cytosine which can replace this base without impairing the immunostimulatory or immune modulatory activity of the oligonucleotide. Modified cytosines include but are not limited to 5-substituted cytosines (e.g. 5-methyl-cytosine, 5-fluoro-cytosine, 5-chloro-cytosine, 5-bromo-cytosine, 5-iodo-cytosine, 5-hydroxy-cytosine, 5-hydroxymethyl-cytosine, 5-difluoromethyl-cytosine, and unsubstituted or substituted 5-alkynyl-cytosine), 6-substituted cytosines, N4-substituted cytosines (e.g. N4-ethyl-cytosine), 5-aza-cytosine, 2-mercapto-cytosine, isocytosine, pseudo-isocytosine, cytosine analogs with condensed ring systems (e.g. N,N′-propylene cytosine or phenoxazine), and uracil and its derivatives (e.g. 5-fluoro-uracil, 5-bromo-uracil, 5-bromovinyl-uracil, 4-thio-uracil, 5-hydroxy-uracil, 5-propynyl-uracil). In some aspects, cytosines include 5-methyl-cytosine, 5-fluoro-cytosine, 5-hydroxy-cytosine, 5-hydroxymethyl-cytosine, and N4-ethyl-cytosine. In some aspects, the cytosine base is substituted by a universal base (e.g. 3-nitropyrrole, P-base), an aromatic ring system (e.g. fluorobenzene or difluorobenzene) or a hydrogen atom (dSpacer).
The letter R is used to refer to guanine or a modified guanine base. A modified guanine is a naturally occurring or non-naturally occurring purine base analog of guanine which can replace this base without impairing the immunostimulatory or immune modulatory activity of the oligonucleotide. Modified guanines include but are not limited to 7-deazaguanine, 7-deaza-7-substituted guanine (such as, 7-deaza-7-(C2-C6)alkynylguanine), 7-deaza-8-substituted guanine, hypoxanthine, N2-substituted guanines (e.g. N2-methyl-guanine), 5-amino-3-methyl-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione, 2,6-diaminopurine, 2-aminopurine, purine, indole, adenine, substituted adenines (e.g. N6-methyl-adenine, 8-oxo-adenine) 8-substituted guanine (e.g. 8-hydroxyguanine and 8-bromoguanine), and 6-thioguanine. In some aspects, the guanine base is substituted by a universal base (e.g. 4-methyl-indole, 5-nitro-indole, and K-base), an aromatic ring system (e.g. benzimidazole or dichloro-benzimidazole, 1-methyl-1H-[1,2,4]triazole-3-carboxylic acid amide) or a hydrogen atom (dSpacer).
In some aspects, other base modifications are also contemplated. For example, the terminal T residues at either end of an oligonucleotide may be replaced by deoxyuridine (U), the G of one or more CpG motifs may be replaced with deoxyinosine (I), and the modification of G residues as 7-deaza deoxyguanosine. In some aspects, the 5′ terminal T of an oligonucleotide may include a halogen substitution. In some aspects, the halogen substitution is ethyl-uridine, bromo-uridine, chloro-uridine or iodo-uridine.
In aspects of the instant invention, the oligonucleotides can be synthesized de novo using any of a number of procedures well known in the art. For example, the b-cyanoethyl phosphoramidite method (Beaucage, S. L., and Caruthers, M. H., Tet. Let. 22:1859, 1981); nucleoside H-phosphonate method (Garegg et al., Tet. Let. 27:4051-4054, 1986; Froehler at al., Nucl. Acid. Res. 14:5399-5407, 1986; Garegg at al., Tet. Let. 27:4055-4058, 1986, Gaffney at al., Tet. Let. 29:2619-2622, 1988). These chemistries can be performed by a variety of automated nucleic acid synthesizers available in the market. These oligonucleotides are referred to as synthetic oligonucleotides. An isolated oligonucleotide generally refers to an oligonucleotide which is separated from components which it is normally associated with in nature. As an example, an isolated oligonucleotide may be one which is separated from a cell, from a nucleus, from mitochondria or from chromatin.
In some aspects of the invention, the internucleotide linkages in the oligonucleotide may be a non-stabilized or stabilized linkage (against nucleases), a phosphodiester (non stabilized), a phosphorothioate (stabilized) or another charged backbone, or a phosphodiester linkage. In some aspects, if the internucleotide linkage at Y—R is a phosphorothioate, the chirality of this linkage may be random, or is preferably a phosphorothioate linkage of Rp configuration.
In aspects of the invention, modified backbones, such as phosphorothioates, may be synthesized using automated techniques employing either phosphoramidate or H-phosphonate chemistries. Aryl- and alkyl-phosphonates can be made, e.g., as described in U.S. Pat. No. 4,469,863; and alkylphosphotriesters (in which the charged oxygen moiety is alkylated as described in U.S. Pat. No. 5,023,243 and European Patent No. 0,092,574) can be prepared by automated solid phase synthesis using commercially available reagents. Methods for making other DNA backbone modifications and substitutions have been described (e.g., Uhlmann, E. and Peyman, A., Chem. Rev. 90:544, 1990; Goodchild, J., Bioconjugate Chem. 1:165, 1990). The symbol * refers to the presence of a stabilized internucleotide linkage and _ refers to the presence of a phosphodiester linkage. In aspects, the one or more immune modulatory molecules may each independently, have a wholly native phosphodiester backbone.
In aspects of the invention, the one or more immune modulatory molecules are oligonucleotides which include at least one unmethylated CpG dinucleotide. An oligonucleotide containing at least one unmethylated CpG dinucleotide is a nucleic acid molecule which contains an unmethylated cytosine-guanine dinucleotide sequence (i.e., “CpG DNA” or DNA containing a 5′ cytosine followed by 3′ guanine and linked by a phosphate bond) and activates the immune system. The entire CpG oligonucleotide can be unmethylated or portions may be unmethylated but at least the C of the 5′ CG 3′ must be unmethylated. CpG The terms CpG oligonucleotide or CpG nucleic acid as used herein refer to an immunostimulatory CpG oligonucleotide or a nucleic acid unless otherwise indicated.
In aspects of the invention, immune modulatory molecules include, but are not limited to, oligonucleotides that are A-Class, B-Class, C-Class, T-Class, P-Class or any Class with an E modification.
A-Class oligonucleotides are potent for inducing IFN-α and NK cell activation but is relatively weak at stimulating B cells. The A-Class oligonucleotides typically have stabilized poly-G sequences at 5′ and 3′ ends and a palindromic phosphodiester CpG dinucleotide-containing sequence of at least 6 nucleotides and form multimeric structures. A-Class oligonucleotides have been described in U.S. Pat. No. 6,949,520, issued Sep. 27, 2005 and published PCT application no. PCT/US00/26527 (WO 01/22990), published on Apr. 5, 2001 The A-Class oligonucleotides do not necessarily contain a hexamer palindrome GACGTC, AGCGCT, or AACGTT, described by Yamamoto and colleagues. Yamamoto S et al. J Immunol 148:4072-6 (1992). In aspects, an “A-Class” CpG oligonucleotide has the following nucleic acid sequence: 5′ GGGGACGACGTCGTGGGGGGG 3′ (SEQ ID NO:17). In aspects, an A-Class oligonucleotide includes, but is not limited to, 5′G*G*G_G_A_C_G_A_C_G_T_C_G_T_G_G*G*G*G*G*G 3′ (SEQ ID NO:18); wherein * refers to a phosphorothioate bond _ and refers to a phosphodiester bond.
B-Class oligonucleotides are potent at activating B cells but are relatively weak in inducing IFN-α and NK cell activation. The B-Class oligonucleotides are monomeric and may be fully stabilized with a wholly phosphorothioate backbone. B-Class oligonucleotides may also have some native phosphodiester linkages, for example, between the C and G of the CpG, in which case they are referred to as semi-soft. In aspects, a B class CpG oligonucleotide may be represented by at least the formula: 5′ X1X2CGX3X4 3′, wherein X1, X2, X3, and X4 are nucleotides. In aspects, X2 is adenine, guanine, or thymine. In aspects, X3 is cytosine, adenine, or thymine. In aspects, a B class CpG oligonucleotide may be represented by at least the formula: 5′ N1X1X2CGX3X4N2 3′, wherein X1, X2, X3, and X4 are nucleotides and N is any nucleotide and N1 and N2 are nucleic acid sequences composed of from about 0-25 N's each. In aspects, X1X2 is a dinucleotide selected from the group consisting of GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT and TpG; and X3X4 is a dinucleotide selected from the group consisting of TpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA, ApA and CpA. In some aspects, X1X2 is GpA or GpT and X3X4 is TpT. in aspects, X1 or X2 or both are purines and X3 or X4 or both are pyrimidines or X1X2 is GpA and X3 or X4 or both are pyrimidines. In some aspects, X1X2 is a dinucleotide selected from the group consisting of TpA, ApA, ApC, ApG and GpG. in some aspects, X3X4 is a dinucleotide selected from the group consisting of TpT, TpA, TpG, ApA, ApG, GpA and CpA. X1X2, in some aspects, is a dinucleotide selected from the group consisting of TpT, TpG, ApT, GpC, CpC, CpT, TpC, GpT and CpG; X3 is a nucleotide selected from the group consisting of A and T, and X4 is a nucleotide, but when X1X2 is TpC, GpT or CpG, X3X4 is not TpC, ApT or ApC. In aspects, the CpG oligonucleotide has the sequence 5′ TCN1TX1X2CGX3X4 3′. The CpG oligonucleotides of the invention, may include, for example, X1X2 selected from the group consisting of GpT, GpG, GpA and ApA and X3X4 selected from the group consisting of TpT, CpT and TpC. B-Class oligonucleotides have been described in U.S. Pat. Nos. 6,194,388 B1 and 6,239,116 B1, issued on Feb. 27, 2001 and May 29, 2001 respectively, and in published PCT application no. WO/1996/002555, published on Feb. 1, 1996 and published PCT application no. WO/1998/018810, published on May 7, 1998. In some aspects, a B-Class oligonucleotide is
CPG 7909
(SEQ ID NO: 1)
5′ TCGTCGTTTTGTCGTTTTGTCGTT 3′,
CpG 24555
(SEQ ID NO: 2)
5′ TCGTCGTTTTTCGGTGCTTTT 3′,
CPG 10104
(SEQ ID NO: 3)
TCGTCGTTTCGTCGTTTTGTCGTT,
(SEQ ID NO: 19)
5′ TCGTCGTTTTGTCGTTTTGTCGTT 3′,
(SEQ ID NO: 20)
5′ TCGTCGTTTTGTCGTTTTTTTCGA 3′,
(SEQ ID NO: 21)
5′ T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*G*C*T*T*T*T 3′,
(SEQ ID NO: 22)
5′ T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T 3′,
(SEQ ID NO: 23)
5′ T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T
3′,
(SEQ ID NO: 24)
5′ T*C*G*T*C*G*T*T*T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T
3′,
or
(SEQ ID NO: 25)
5′ T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*T*T*T*C*G*A
3′
wherein * refers to a phosphorothioate bond.
C-Class oligonucleotides have both a traditional “stimulatory” CpG sequence and a “GC-rich” or “B-cell neutralizing” motif. C-Class CpG oligonucleotides have properties intermediate to A- and B-Classes so activate B cells and NK cells and induce IFN-α (Krieg A M et al. (1995) Nature 374:546-9; Ballas Z K et al. (1996) J Immunol 157:1840-5; Yamamoto S et al. (1992) J Immunol 148:4072-6). The C-Class oligonucleotides, contain a single palindrome such that they can form secondary structures such as stem-loops or tertiary structures such as dimmers. The backbone of C-Class oligonucleotides may have a fully stabilized, chimeric or semi-soft backbone. C-Class oligonucleotides include a B-Class-type sequence and a GC-rich palindrome or near-palindrome. This Class has been described in US published application no. 20030148976, published on Aug. 7, 2003 and in published PCT application no. WO2008/068638, published on Jun. 12, 2008. In some aspects, a C-Class oligonucleotide is CPG 10101 5′ TCGTCGTTTTCGGCGGCCGCCG 3′ (SEQ ID NO:4), CPG 10109 5′ TCGTC-GTTTTAC-GGCGCC-GTCCCG 3′ (SEQ ID NO:5 where dashes represent semi-soft phosphodiester linkages), CpG 23407 5′ TC-GTCGTTTTCGGCGCGCGCCGT 3′ (SEQ ID NO:6 where the dash represents a semi-soft phosphodiester linkage),
(SEQ ID NO: 26)
5′ TCGCGTCGTTCGGCGCGCGCCG 3′,
(SEQ ID NO: 27)
5′ TCGTCGACGTTCGGCGCGCGCCG 3′,
(SEQ ID NO: 28)
5′ TCGGACGTTCGGCGCGCGCCG 3′,
(SEQ ID NO: 29)
5′ TCGGACGTTCGGCGCGCCG 3′,
(SEQ ID NO: 30)
5′ TCGCGTCGTTCGGCGCGCCG 3′,
(SEQ ID NO: 31)
5′ TCGACGTTCGGCGCGCGCCG 3′,
(SEQ ID NO: 32)
5′ TCGACGTTCGGCGCGCCG 3′,
(SEQ ID NO: 33)
5′ TCGCGTCGTTCGGCGCCG 3′,
(SEQ ID NO: 34)
5′ TCGCGACGTTCGGCGCGCGCCG 3′,
or
(SEQ ID NO: 35)
5′ TCGTCGTTTTCGGCGCGCGCCG 3′.
In aspects, a C-Class CpG oligonucleotide is
(SEQ ID NO: 38)
5′ T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3′,
(SEQ ID NO: 39)
5′ T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G
3′,
(SEQ ID NO: 40)
5′ T*C_G*G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3′,
(SEQ ID NO: 41)
5′ T*C_G*G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3′,
(SEQ ID NO: 42)
5′ T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3′,
(SEQ ID NO: 43)
5′ T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3′,
(SEQ ID NO: 44)
5′ T*C_G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3′,
(SEQ ID NO: 45)
5′ T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*C*G 3′,
(SEQ ID NO: 46)
5′ T*C_G*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G
3′,
(SEQ ID NO: 47)
5′ T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G
3′,
(SEQ ID NO: 48)
5′ T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*G*C*C*G*C*C*G 3′,
(SEQ ID NO: 49)
5′ T*C*G*T*C_G*T*T*T*T*A*C_G*G*C*G*C*C_G*T*G*C*C*G
3′
or
(SEQ ID NO: 50)
5′ T*C G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*T
3′
wherein * refers to a phosphorothioate
bond and _ refers to a phosphodiester bond.
In any of these sequences, an ethyl-uridine or a halogen may substitute for the 5′ T; examples of halogen substitutions include, but are not limited to, bromo-uridine or iodo-uridine substitutions.
The P-Class oligonucleotides have the ability in some instances to induce much higher levels of IFN-α secretion than the C-Class oligonucleotides. The P-Class oligonucleotides have the ability to spontaneously self-assemble into concatamers either in vitro and/or in vivo. P-Class oligonucleotides are further disclosed in published PCT application no. WO2008/068638, published on Jun. 12, 2008. In some aspects, a P-Class oligonucleotide is
CpG 21798
(SEQ ID NO: 7)
5′ T*C-G*T*C-G*A*C-G*A*T*C-G*G*C*G*C-G*C*G*C*C*G
3′,
CpG 23430
(SEQ ID NO: 8)
5′ T*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G
3′,
CpG 24558
(SEQ ID NO: 9)
5′ T*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T
3′,
CpG 23871
(SEQ ID NO: 10)
5′ JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G
3′,
CpG 23873
(SEQ ID NO: 11)
5′ JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*
G*T 3′,
CpG 23874
(SEQ ID NO: 12)
5′ JU*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*
G*T 3′,
CpG 23875
(SEQ ID NO: 13)
5′ EU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G
3′,
CpG 23877
(SEQ ID NO: 14)
5′ JU*C-G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*
G*T 3′,
CpG 23878
(SEQ ID NO: 15)
5′ JU*C*G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*
G*T 3′
or
(SEQ ID NO: 37)
5′ T*C_G*T*C_G*A*C_G*A*T*C_G*G*C*G*C_G*C*G*C*C*G
3′.
The T-Class oligonucleotides induce secretion of lower levels of IFN-alpha and IFN-related cytokines and chemokines than B-Class or C-Class oligonucleotides, while retaining the ability to induce levels of IL-10 similar to B-Class oligonucleotides. T-Class oligonucleotides are further disclosed in published PCT application WO2008/068638, published on Jun. 12, 2008.
E modifications can be made on any class of CpG oligonucleotides. These are oligonucleotides with lipophilic substituted nucleotide analogs outside the CpG motif and have enhanced ability to stimulate interferon-α (IFN-α) production and induce TLR9 activation. E modified oligonucleotides are further disclosed in published PCT application WO2008/068638, published on Jun. 12, 2008.
In aspects of the invention, the one or more immune modulatory molecules are in an effective amount to induce or enhance an antigen-specific immune response. In some aspects, the antigen-specific immune response enhanced is a Th1 immune response. In some aspects, the Th1 immune response results in the antigen-specific induction of IFN-γ, or the induction of poly-functional T cells that secrete two or more cytokines. In some aspects, the cytokines include, but are not limited to, IL-2 and IFN-γ or IFN-γ, TNF-α and IL-2.
In aspects of the invention, the amount of immune modulatory molecule is from about 1 μg to about 5 mg per vaccine dose. In some aspects, the amount of immune modulatory molecules is from about 1 μg to about 4 mg per vaccine dose, about 1 μg to about 3 mg per vaccine dose, about 1 μg to about 2 mg per vaccine dose, or about 1 μg to about 1 mg per vaccine dose. In some aspects, the amount of immune modulatory molecules is from about 10 μg to about 750 μg per vaccine dose, about 10 μg to about 500 μg per vaccine dose, about 10 μg to about 250 μg per vaccine dose, about 10 μg to about 100 μg per vaccine dose, about 20 μg to about 100 μg per vaccine dose, or about 30 μg to about 100 μg per vaccine dose. in some aspects, the amount of immune modulatory molecules is about 500 μg per vaccine dose. In some aspects, the amount of immune modulatory molecules is about 250 μg per vaccine dose.
In aspects of the invention, the amount of the immune modulatory molecule relative to the amount of cholesterol is greater than the amount of cholesterol. In aspects of the invention, the ratio of the amount of the immune modulatory molecule to the amount of cholesterol is about 100:1, or about 75:1, or about 50:1, or about 25:1, or about 15:1, or about 10:1, or about 5:1 by weight. In aspects of the invention, the amount of the immune modulatory molecule relative to the amount of cholesterol is about the same as the amount of cholesterol. That is, the amount of the immune modulatory molecule to the amount of cholesterol is in a ratio of about 1:1 by weight. In aspects of the invention, the amount of the immune modulatory molecule relative to the amount of cholesterol is less than the amount of cholesterol. In aspects of the invention, the ratio of the amount of the immune modulatory molecule to the amount of cholesterol is about 1:100, or about 1:75, or about 1:50, or about 1:25, or about 1:15, or about 1:10, or about 1:5 by weight. In one aspect, the ratio of the amount of the immune modulatory molecule to the amount of cholesterol is about 1:10 by weight. One skilled in the art would realize that the ratios given can be as shown or can be approximately as shown.
As used herein, the terms “disorder”, “condition” and “disease” are used interchangeably.
In aspects of the invention, the vaccines are useful as a prophylactic vaccine for the prevention of an infection (e.g., an infectious disease), a disorder associated with a self antigen, or a disorder associated with an addictive substance. Preferably, prophylactic vaccination is used in subjects that are not diagnosed with the condition for which the vaccine is sought, and more preferably the subjects are considered at risk of developing one of these conditions. For example, the subject may be one that is at risk of developing an infection with an infectious organism, or susceptible to a disorder associated with a self-antigen, or susceptible to a disorder associated with an addictive substance.
A subject at risk, as used herein, is a subject who has any risk of exposure to an infection causing pathogen, a subject having or at risk of developing a chronic or treatment-resistant infectious disease, a subject having or at risk of developing cancer, a subject having or at risk of developing an allergy, a subject having or at risk of developing asthma, a subject having or at risk of developing a disorder associated with an addictive substance, a subject having or at risk of developing a disorder involving abnormal protein folding, or a subject having or at risk of developing an autoimmune disorder. A subject at risk also includes subjects that have a predisposition to developing such disorders. Some predispositions can be genetic (and can thereby be identified either by genetic analysis or by family history). Some predispositions are environmental (e.g., prior exposure to infectious agents, self antigens or addictive substances). For a subject at risk of developing an infection, an example of such a subject is a subject living in or expecting to travel to an area where a particular type of infectious agent is or has been found, or it may be a subject who through lifestyle or medical procedures is exposed to an organism either directly or indirectly by contact with bodily fluids that may contain infectious organisms. Subjects at risk of developing infection also include general populations to which a medical agency recommends vaccination for a particular infectious organism.
A subject is a human or a non-human animal treated by veterinarian medicine. Non-human animal subjects include, but are not limited to, a dog, a cat, a bird, a horse, a cow, a pig, a sheep, a goat, a chicken, a non-human primate (e.g., monkey, chimpanzee) and a fish (aquaculture species, e.g., salmon).
An infectious disease, as used herein, is a disease arising from the presence of a foreign microorganism in the body, for example, a bacteria, a virus, a parasite or a fungus.
In aspects of the invention, a bacteria includes, but is not limited to, Aceinetobacter calcoaceticus, Acetobacter paseruianus, Actinobacillus actinomycetemcomitans, Actinobacillus pleuropneumoniae, Actinomyces israelli, Actinomyces viscosus, Aeromonas hydrophila, Alcaliges eutrophus, Alicyclobacillus acidocaldarius, Arhaeglobus fulgidus, Bacillus species, Bacillus antracis, Bacillus pumilus, Bacillus stearothermophillus, Bacillus subtilis, Bacillus thermocatenulatus, Bacteroides species, Bordetella species, Bordetella bronchiseptica, Borrelia burgdorferi, Brucella species, Burkholderia cepacia, Burkholderia glumae, Brachyspira species. Brachyspira hyodysenteria, Brachyspira pilosicoll, Camphylobacter species, Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis, Campylobacter jejuni, Chlamydia psittaci, Chlamydia trachomatis, Chlamydophila species, Chromobacterium viscosum, Clostridium species, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium species, Corynebacterium diphtheriae, Ehrlichia canis, Enterobacter species, Enterobacter aerogenes, Enterococcus species, Erysipelothrix rhusiopathieae, Escherichia species, Escherichia coli, Fusobacterium nucleatum, Haemophilus species, Haemophilus influenzae, Haemophilus somnus, Helicobacter species, Helicobacter pylori, Helicobacter suis, Klebsiella species, Klebsiella pneumoniae, Lactobacillus acidophilis, Lawsonia intracellularis, Legionella species, Legionella pneumophilia, Leptospira species, such as Leptospira canicola, Leptospira grippotyposa, Leptospira hardjo, Leptospira borgpetersenii hardjo-bovis, Leptospira borgpetersenii hardjo-prajitno, Leptospira interrogans, Leptospira icterohaemorrhagiae, Leptospira pomona, Leptospira, Leptospira bratislava, Listeria species, Listeria monocytogenes, Meningococcal bacteria, Moraxella species, Mycobacterium species, Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansaii, Mycobacterium gordonae, Mycoplasma species, such as, Mycoplasma hyopneumoniae, Mycoplasma synoviae, Mycoplasma hyorhinis, Mycoplasma pneumoniae, Mycoplasma mycoides subsp. mycoides LC, Neisseria species, Neisseria gonorrhoeae, Neisseria meningitidis, Odoribacter denticanis, Pasteurella species, Pasteurella (Mannheimia) haemolytica, Pasteurella multocida, Photorhabdus luminescens, Porphyromonas gingivalis, Porphyromonas gulae, Porphyromonas salivosa, Propionibacterium acnes, Proteus species, Proteus vulgaris, Pseudomonas species, Pseudomnas wisconsinensis, Pseudomonas aeruginosa, Pseudomonas fluorescens C9, Pseudomonas fluorescens SIKW1, Pseudomonas Tragi, Pseudomonas luteola, Pseudomonas oleovorans, Pseudomonas sp B11-1, Psychrobacter immobilis, Rickettsia spp, Rickettsia prowazekii, Rickettsia rickettsia, Salmonella species, Salmonella bongori, Salmonella choleraeuis, Salmonella dublin, Salmonella enterica, Salmonella newport, Salmonella typhimurium, Salmonella typhi, Serratia marcescens, Shigella species, Spirlina platensis, Staphylococci species, Staphlyoccocus aureus, Staphyloccoccus epidermidis, Staphylococcus hyicus, Streptococcus species, Streptobacillus moniliformis, beta-hemolytic Streptococcus, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguis, Streptomyces albus, Streptomyces cinnamoneus, Streptomyces exfoliates, Streptomyces scabies, Sulfolobus acidocaldarius, Syechocystis sp., Treponena species, Treponema denticola, Treponema minutum, Treponema palladium, Treponema pertenue, Treponema phagedenis, Treponema refringens, Treponema vincentii, Vibrio species, Vibrio cholerae, Yersinia species and combinations thereof.
In aspects of the invention, a virus includes, but is not limited to, Avian herpesvirus, Avian influenza, Avian leukosis virus, Avian paramyxoviruses, Border disease virus, Bovine coronavirus, Bovine ephemeral fever virus, Bovine herpes viruses, Bovine immunodeficiency virus, Bovine leukemia virus, Bovine parainfluenza virus 3, Bovine respiratory syncytial virus, Bovine viral diarrhea virus (BVDV), BVDV Type I, BVDV Type II, Canine adenovirus, Canine coronavirus (CCV), Canine distemper virus, Canine herpes viruses, Equine herpes viruses, Canine influenza virus, Canine parainfluenza virus, Canine parvovirus, Canine respiratory coronavirus, Classical swine fever virus, Eastern Equine encephalitis virus (EEE), Equine infectious anemia virus, Equine influenza virus, West nile virus, Feline Calicivirus, Feline enteric coronavirus, Feline immunodeficiency virus, Feline infectious peritonitis virus, Feline herpes Virus, Feline influenza virus, Feline leukemia virus (FeLV), Feline viral rhinotracheitis virus, Lentivirus, Marek's disease virus, Newcastle Disease virus, Ovine herpesviruses, Ovine parainfluenza 3, Ovine progressive pneumonia virus, Ovine pulmonary adenocarcinoma virus, Pantropic CCV, Porcine circovirus (PCV) Type I, PCV Type II, Porcine epidemic diarrhea virus, Porcine hemagglutinating encephalomyletitis virus, Porcine herpesviruses, Porcine parvovirus, Porcine reproductive and respiratory syndrome (PRRS) Virus, Pseudorabies virus, Rabies, Rotovirus, Rhinoviruses, Rinderpest virus, Swine influenza virus, Transmissible gastroenteritis virus, Turkey coronavirus, Venezuelan equine encephalitis virus, Vesicular stomatitis virus, West Nile virus, Western equine encephalitis virus and combinations thereof.
In aspects of the invention, a parasite includes, but is not limited to, a protein from Anaplasma, Fasciola hepatica (liver fluke), Coccidia, Eimeria spp., Neospora caninum, Toxoplasma gondii, Giardia, Dirofilaria (heartworms), Ancylostoma (hookworms), Trypanosoma spp., Leishmania spp., Trichomonas spp., Cryptosporidium parvum, Babesia, Schistosoma, Taenia, Strongyloides, Ascaris, Trichinella, Sarcocystis, Hammondia, or Isopsora, and combinations thereof. In aspects, a parasite includes, but is not limited to, ticks, including Ixodes, Rhipicephalus, Dermacentor, Amblyomma, Boophilus, Hyalomma, or Haemaphysalis species, and combinations thereof.
In aspects of the invention, a fungus includes, but is not limited to, spores, molds and yeasts (for example, Candida species).
A chronic or treatment-resistant infectious disease, as used herein, is a disease having a prolonged infection period, sometimes lasting weeks, months and even a lifetime, or an infection that resists other treatments that are usually successful. In some aspects, a chronic or treatment-resistant viral infection includes, but is not limited to, HBV, HCV, HIV, HPV, HSV-1 or HSV-2.
In aspects of the invention, a subject having a cancer is a subject that has detectable cancerous cells. The cancer may be a malignant or non-malignant cancer. Cancers or tumors include, but are not limited to, biliary tract cancer; bladder cancer; brain cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; colorectal cancer; endometrial cancer; esophageal cancer; gastric cancer; gliobastoma; intraepithelial neoplasms; lymphomas (for example, follicular lymphoma); liver cancer; lung cancer (for example, small cell and non-small cell); leukemia (for example, hairy cell leukemia, chronic myelogenous leukemia, cutaneous T-cell leukemia); melanoma (for example, malignant melanoma); multiple myeloma; neuroblastomas; oral cancer; ovarian cancer; pancreas cancer; prostate cancer; rectal cancer; renal cancer; sarcomas; skin cancer; testicular cancer; thyroid cancer; and renal cancer, as well as other carcinomas and sarcomas (for example, squamous cell carcinoma, renal cell carcinoma, prostate carcinoma, bladder cell carcinoma, or colon carcinoma).
In aspects of the invention, a subject having an allergy is a subject that has or is at risk of developing an allergic reaction in response to an allergen. An allergy refers to acquired hypersensitivity to a substance (allergen). Allergic conditions include, but are not limited to, eczema, allergic rhinitis or coryza, hay fever, conjunctivitis, bronchial asthma, urticaria (hives) and food allergies, and other atopic conditions.
Currently, allergic diseases are generally treated by the injection of small doses of antigen followed by subsequent increasing dosage of antigen. It is believed that this procedure induces tolerization to the allergen to prevent further allergic reactions. These methods, however, can take several years to be effective and are associated with the risk of side effects such as anaphylactic shock.
Allergies are generally caused by IgE antibody generation against harmless allergens. The cytokines that are induced by systemic or mucosal administration of immunostimulatory nucleic acids are predominantly of a class called Th1 (examples are IL-12 and IFN-.gamma.) and these induce both humoral and cellular immune responses. The types of antibodies associated with a Th1 response are generally more protective because they have high neutralization and opsonization capabilities. The other major type of immune response, which is associated with the production of IL-4, IL-5 and IL-10 cytokines, is a Th2 immune response. Th2 responses involve predominately antibodies and these have less protective effect against infection and some Th2 isotypes (e.g., IgE) are associated with allergy. In general, it appears that allergic diseases are mediated by Th2 type immune responses while Th1 responses provide the best protection against infection, although excessive Th1 responses are associated with autoimmune disease. Based on the ability of the one or more immune modulatory molecules to shift the immune response in a subject from a Th2 (which is associated with production of IgE antibodies and allergy) to a Th1 response (which is protective against allergic reactions), an effective dose for inducing an immune response of a immune modulatory molecule can be administered to a subject to treat or prevent an allergy.
In aspects of the invention, an allergen refers to a substance (for example, an antigen) that can induce an allergic or asthmatic response in a susceptible subject. Allergens include, but are not limited to, pollens, insect venoms, animal dander dust, fungal spores and drugs (e.g. penicillin). Examples of natural, animal and plant allergens include, but are not limited to, proteins specific to the following genuses: Canine (Canis familiaris); Dermatophagoides (e.g. Dermatophagoides farinae); Felis (Felis domesticus); Ambrosia (Ambrosia artemiisfolia; Lolium (e.g. Lolium perenne or Lolium multiflorum); Cryptomeria (Cryptomeria japonica); Alternaria (Alternaria alternata); Alder; Alnus (Alnus gultinoasa); Betula (Betula verrucose); Quercus (Quercus alba); Olea (Olea europa); Artemisia (Artemisia vulgaris); Plantago (e.g. Plantago lanceolata); Parietaria (e.g. Parietaria officinalls or Parietaria judaica); Blattella (e.g. Blattella germanica); Apis (e.g. Apis multiflorum); Cupressus (e.g. Cupressus sempervirens, Cupressus arizonica and Cupressus macrocarpa); Juniperus (e.g. Juniperus sabinoides, Juniperus virginiana, Juniperus communis and Juniperus ashei); Thuya (e.g. Thuya orientalis); Chamaecyparis (e.g. Chamaecyparis obtusa); Periplaneta (e.g. Periplaneta americana); Agropyron (e.g. Agropyron repens); Secale (e.g. Secale cereale); Triticum (e.g. Triticum aestivum); Dactylis (e.g. Dactylis glomerata); Festuca (e.g. Festuca elafior); Poa (e.g. Poa pratensis or Poa compressa); Avena (e.g. Avena sativa); Holcus (e.g. Holcus lanatus); Anthoxanthum (e.g. Anthoxanthum odoratum); Arrhenatherum (e.g. Arrhenatherum elatius); Agrostis (e.g. Agrostis alba); Phleum (e.g. Phleum pratense); Phalaris (e.g. Phalaris arundinacea); Paspalum (e.g. Paspalum notatum); Sorghum (e.g. Sorghum halepensis); and Bromus (e.g. Bromus inermis).
In aspects of the invention, asthma refers to a disorder of the respiratory system characterized by inflammation, narrowing of the airways and increased reactivity of the airways to inhaled agents. Th2 cytokines, for example, IL-4 and IL-5 are elevated in the airways of asthmatic subjects. These cytokines promote important aspects of the asthmatic inflammatory response, including IgE isotope switching, eosinophil chemotaxis and activation and mast cell growth. Th1 cytokines, especially IFN-.gamma. and IL-12, can suppress the formation of Th2 clones and production of Th2 cytokines. Asthma is frequently, although not exclusively, associated with atopic or allergic symptoms.
In aspects of the invention, a disorder involving abnormal protein folding is a disorder resulting from an associated protein either misfolding or an error in a subject's DNA leading to the incorrect folding of a protein. In aspects, a disorder involving abnormal protein folding is an amyloidose disorder, for example, Alzheimer's disease, MS, or a prion disorder, for example transmissable spongiform encephalopathies (TSEs), which include, but are not limited to, bovine spongiform encephalopathy (BSE, mad cow disease) and Creutzfeld Jakob disease (CJD) in humans. In aspects, a disorder involving an error in a subject's DNA leading to the incorrect folding of a protein includes, but is not limited to, cystic fibrosis and cancers associated with the p53 protein.
In aspects of the invention, an autoimmune disorder is any disorder involving an overactive immune response of the subject's body against substances and tissues (for example, a self antigen) normally present in the subject. In some aspects, an autoimmune disorder is Rheumatoid arthritis (RA), lupus or Crohn's disease.
In aspects of the invention, a disorder associated with a self antigen is any disorder that is caused by an antigen of a subject's own cells or cell products that causes an immune response in said subject. For example, in some embodiments, a self antigen is a tumor antigen, an antigen associated with Alzheimer's Disease, an antigen against an antibody, or an antigen that is expressed from human endogenous retroviral elements. In some aspects, the tumor antigen is one or more of WT1, MUC1, LMP2, HPV E6 or HPV E7, EGFR or variant form thereof, for example, EGFRvIII, HER-2/neu, Idiotype, MAGE A3, p53 non-mutant, NY-ESO-1, PSMA, GD2, CEA, MelanA/MART1, Ras mutant, gp100, p53 mutant, Proteinase3 (PR1), bcr-abl, Tyrosinase, Survivin, PSA, hTERT, Sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, Androgen receptor, Cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, GD3, Fucosyl GM1, Mesothelin, PSCA, MAGE Al, sLe (animal), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-beta, MAD-CT-2, or Fos-related antigen 1. An antigen associated with Alzheimer's Disease may be tau or β-amyloid. An antigen against an antibody may be an antigen against a human antibody, for example, in some embodiments the antigen is IgE.
In aspects of the invention, the vaccines may be used in the prevention of a respiratory viral infection in an animal. In some aspects, the respiratory viral infection is BVDV 1, BVDV 2, IBRV, PI3V or BRSV.
In aspects of the invention, a disorder associated with an addictive substance is any disorder that involves a subject developing an addiction to an addictive chemical or biological substance. For example, in some embodiments, an addictive substance may be nicotine or cocaine. In some embodiments, the vaccine to prevent or treat the addiction contains nicotine or a nicotine-like hapten conjugated to a carrier. In some embodiments, the carrier to which a nicotine or nicotine-like hapten is conjugated is a bacterial toxoid or derivative, Pseudomonas exotoxin, KLH or a virus-like particle. In some aspects, the bacterial toxoid is diphtheria toxoid or a derivative thereof, for example, CRM197. In some aspects, the virus-like particle is HBsAg, HBcAg, E. coli bacteriophage Qβ, Norwalk virus or influenza HA.
As used herein, the term “treat”, “treated” or “treating” when used with respect to an infectious disease refers to a prophylactic treatment which increases the resistance of a subject (a subject at risk of infection) to infection with a pathogen, or in other words, decreases the likelihood that the subject will become infected with the pathogen as well as a treatment after the subject (a subject who has been infected) has become infected in order to fight the infection, e.g., reduce or eliminate the infection or prevent it from becoming worse.
The term “treat”, “treated” or “treating” when used with respect to a cancer refers to a prophylactic treatment which increases the resistance of a subject (a subject at risk of developing cancer) to cancer, or decreases the likelihood that the subject will develop cancer as well as a treatment after the subject (a subject who has or is diagnosed with cancer) has developed such a disorder or begun to develop signs or symptoms of developing such a disorder, to reduce the effect of the disorder, e.g., reduce or eliminate the signs or symptoms associated with the disorder or prevent them from becoming worse.
The term “treat”, “treated” or “treating” when used with respect to asthma or allergy refers to a prophylactic treatment which increases the resistance of a subject (a subject at risk of developing asthma or allergy) to develop such a disorder or decreases the likelihood that the subject will develop asthma or allergy as well as a treatment after the subject (a subject who has or is diagnosed with asthma or allergy) has developed such a disorder or begun to develop signs or symptoms of developing such a disorder, to reduce the effect of the disorder, e.g., reduce or eliminate the signs or symptoms associated with the disorder or prevent them from becoming worse.
The term “treat”, “treated” or “treating” when used with respect to a disorder associated with an addictive substance refers to a prophylactic treatment which increases the resistance of a subject (a subject at risk of a disorder associated with an addictive substance) to develop such a disorder or decreases the likelihood that the subject will develop the disorder associated with an addictive substance as well as treatment after the subject (a subject who has or is diagnosed with a disorder associated with an addictive substance) has developed such a disorder or begun to develop signs or symptoms of developing such a disorder, to reduce the effect of the disorder, e.g., reduce or eliminate the signs or symptoms associated with the disorder or prevent them from becoming worse.
The term “treat”, “treated” or “treating” when used with respect to a disorder associated with abnormal protein folding refers to a prophylactic treatment which increases the resistance of a subject (a subject at risk of a disorder associated with abnormal protein folding) to develop such a disorder or decreases the likelihood that the subject will develop the disorder associated with abnormal protein folding as well as treatment after the subject (a subject who has or is diagnosed with a disorder associated with abnormal protein folding) has developed such a disorder or begun to develop signs or symptoms of developing such a disorder, to reduce the effect of the disorder, e.g., reduce or eliminate the signs or symptoms associated with the disorder or prevent them from becoming worse.
The term “treat”, “treated” or “treating” when used with respect to an autoimmune disorder refers to a prophylactic treatment which increases the resistance of a subject (a subject at risk of an autoimmune disorder) to develop such a disorder or decreases the likelihood that the subject will develop the autoimmune disorder as well as treatment after the subject (a subject at who has or is diagnosed with an autoimmune disorder) has developed such a disorder or begun to develop signs or symptoms of developing such a disorder, to reduce the effect of the disorder, e.g., reduce or eliminate the signs or symptoms associated with the disorder or prevent them from becoming worse.
The term “treat”, “treated” or “treating” when used with respect to a disorder associated with a self antigen refers to a prophylactic treatment which increases the resistance of a subject (a subject at risk of a disorder associated with a self antigen) to develop such a disorder or decreases the likelihood that the subject will develop the disorder associated with a self antigen as well as treatment after the subject (a subject who has or is diagnosed with a disorder associated with a self antigen) has developed such a disorder or begun to develop signs or symptoms of developing such a disorder, to reduce the effect of the disorder, e.g., reduce or eliminate the signs or symptoms associated with the disorder or prevent them from becoming worse.
The treatment of a subject or with the vaccines as described herein, results in the reduction of infection or the complete abolition of the infection, reduction of the signs/symptoms associated with a disorder associated with a self antigen or the complete abolition on the disorder, or reduction of the signs/symptoms associated with a disorder associated with an addictive substance or the complete abolition of the disorder. A subject may be considered as treated if such symptoms related to the infectious disease, cancer, allergy, asthma, disorder associated with abnormal protein folding, autoimmune disorder, disorder associated with a self antigen or disorder associated with an addictive substance, are reduced, are managed or are abolished as a result of such treatment. For an infectious disease, such treatment also encompasses a reduction in the amount of infectious agent present in the subject (e.g., such amounts can be measured using standard assays such as ELISA known to those of ordinary skill in the art). For a cancer, such treatment also encompasses a reduction in the cancerous cells or tissues, and/or a reduction in the signs/symptoms associated with the cancer. For an allergy, such treatment also encompasses a reduction in the signs/symptoms associated with the allergy. For an asthma, such treatment also encompasses a reduction in the signs/symptoms associated with the asthma. For an autoimmune disorder, such treatment also encompasses a reduction in the immune response against the autoimmune disorder, and/or a reduction in the signs/symptoms associated with the disorder. For a disorder associated with abnormal protein folding, such treatment also encompasses a reduction in the amount of abnormal protein, and/or a reduction or reversal in the signs/symptoms associated with the disorder. For a disorder associated with a self antigen, such treatment also encompasses a reduction in the amount of self antigen present in the subject or a reduction in the immune response induced as a result of the self antigen. For a disorder associated with an addictive substance, such treatment also encompasses a reduction in the signs/symptoms associated with addiction to an addictive substance.
The formulations of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients. For certain vaccine formulations using cholesterol, ethanol may be substituted with a pharmaceutically acceptable surfactant and water solution to solubilize the cholesterol into an aqueous formulation.
For use in therapy, an effective amount of the one or more immune modulatory molecules can be administered to a subject by any mode that delivers the immune modulatory molecule to the desired surface. Administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan. Preferred routes of administration include but are not limited to parenteral (for example, intramuscular, subcutaneous, intradermal, intravenous injection), topical to the skin (for example, transdermal) or mucosal (for example, oral, intranasal, intravaginal, intrarectal, trans-buccal, intraocular or sublingual). In the case of treatment of cancers, this may include intra-tumor administrations.
In aspects of the invention, “effective amount” of an immune modulatory molecule refers to the amount necessary or sufficient to realize a desired biologic effect. For example, an effective amount of an immune modulatory molecule for treating a disorder could be that amount necessary to eliminate a microbial infection or a tumor. An effective amount for use as a vaccine adjuvant could be that amount useful for boosting a subject's immune response to a vaccine. An “effective amount” for treating an infectious disease, a cancer, an allergy, asthma, an autoimmune disorder, a disorder associated with abnormal protein folding, a disorder associated with a self antigen or a disorder associated with an addictive substance can be that amount useful for inducing an antigen-specific immune response. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular an immune modulatory molecule being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular an immune modulatory molecule without necessitating undue experimentation.
Subject doses of the compounds described herein for local delivery typically range from about 0.1 μg to about 50 mg per administration which, depending on the application, could be given daily, weekly, or monthly and any other amount of time therebetween. More typically local doses range from about 10 μg to about 10 mg per administration, and optionally from about 100 μg to about 1 mg, with 2-4 administrations being spaced days or weeks apart. More typically, immune stimulant doses range from about 1 μg to about 10 mg per administration, and most typically about 10 μg to about 1 mg, with daily or weekly administrations. Subject doses of the compounds described herein for parenteral delivery for the purpose of inducing an antigen-specific immune response, wherein the compounds are delivered with an antigen but not another therapeutic agent are typically about 5 to about 10,000 times higher than the effective local dose for vaccine adjuvant or immune stimulant applications, and more typically about 10 to about 1,000 times higher, and most typically about 20 to about 100 times higher. Doses of the compounds described herein for parenteral delivery, e.g., for inducing an innate immune response, for increasing ADCC, for inducing an antigen specific immune response when the one or more immune modulatory molecules are administered in combination with other therapeutic agents or in specialized delivery vehicles typically range from about 0.1 μg to about 10 mg per administration which, depending on the application, could be given daily, weekly, or monthly and any other amount of time therebetween. More typically parenteral doses for these purposes range from about 10 μg to about 5 mg per administration, and most typically from about 100 μg to about 1 mg, with 2-4 administrations being spaced days or weeks apart. In some embodiments, however, parenteral doses for these purposes may be used in a range of about 5 to about 10,000 times higher than the typical doses described above.
For any compound described herein the therapeutically effective amount can be initially determined from animal models. A therapeutically effective dose can also be determined from human data for immune modulatory molecules which have been tested in humans (e.g., human clinical trials have been initiated) and for compounds which are known to exhibit similar pharmacological activities, such as other adjuvants, e.g., LT and other antigens for vaccination purposes. Higher doses may be required for parenteral administration. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan.
The one or more immune modulatory molecules either alone or with one or more antigens, cholesterol or other therapeutic agents, may be administered via any route described herein.
The one or more immune modulatory molecules, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the immune modulatory molecules in water-soluble form. Additionally, suspensions of the immune modulatory molecules may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the immune modulatory molecules to allow for the preparation of highly concentrated solutions.
One may dilute or increase the volume of the therapeutic with an inert material. These diluents could include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and/or starch. Certain inorganic salts may be also be used as fillers including calcium triphosphate, magnesium carbonate and/or sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.
To aid dissolution of the therapeutic into the aqueous environment a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and/or dioctyl sodium sultanate. Cationic detergents might be used and could include benzalkonium chloride or benzethomium chloride. The list of potential non-ionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and/or 60, glycerol monostearate, polysorbate 40, 60, 65 and/or 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. In aspects, non-ionic detergents include, but are not limited to, octoxynols, for example, t-octylphenoxy polyethoxyethanol (TRITON X-100™), polyoxyethylene esters, for example, polyoxyethylene sorbitan monooleate (TWEEN 80™, bile salts and cholic acid derivatives, for example sodium deoxycholate or taurodeoxycholate. In aspects, a formulation may comprise 3D-MPL, laureth 9, TRITON X 100™, TWEEN 80™, and sodium deoxycholate. These surfactants could be present in the formulation of the immune modulatory molecules either alone or as a mixture in different ratios.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the immune modulatory molecules in water-soluble form. Additionally, suspensions of the immune modulatory molecules may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the immune modulatory molecules to allow for the preparation of highly concentrated solutions.
Alternatively, the immune modulatory molecules may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
For oral administration, the compounds (for example, immune modulatory molecules alone or with one or more antigens, cholesterol and/or other therapeutic agents) can be formulated readily by combining the immune modulatory molecules with pharmaceutically acceptable carriers well known in the art. Such carriers enable the immune modulatory molecules of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers, i.e. EDTA for neutralizing internal acid conditions or may be administered without any carriers.
Also contemplated are oral dosage forms of the above agents or formulations. The agents or formulations may be chemically modified so that oral delivery of the derivative is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the agent or formulation itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the agent or formulation and increase in circulation time in the body. Examples of such moieties include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. Abuchowski and Davis, 1981, “Soluble Polymer-Enzyme Adducts” In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-383; Newmark, et al., 1982, J. Appl, Biochem. 4:185-189. Other polymers that could be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as indicated above, are polyethylene glycol moieties.
Intranasal delivery of a pharmaceutical composition of the present invention is also contemplated. Intranasal delivery allows the passage of a pharmaceutical composition of the present invention to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran. In aspects, a formulation for intranasal delivery (or mucosal delivery) may comprise 3D-MPL, laureth 9, TRITON X-100™, TWEEN 80™, and sodium deoxycholate. In aspects, such a formulation may be combined with an antigen, for example an influenza virus antigen.
For intranasal administration, a useful device is a small, hard bottle to which a metered dose sprayer is attached. In aspects, the metered dose is delivered by drawing the pharmaceutical composition of the present invention solution into a chamber of defined volume, which chamber has an aperture dimensioned to aerosolize an aerosol formulation by forming a spray when a liquid in the chamber is compressed. The chamber is compressed to administer the pharmaceutical composition of the present invention. In aspects, the chamber is a piston arrangement. Such devices are commercially available.
Alternatively, a plastic squeeze bottle with an aperture or opening dimensioned to aerosolize an aerosol formulation by forming a spray when the bottle is squeezed. The opening is usually found in the top of the bottle, and the top is generally tapered to partially fit in the nasal passages for efficient administration of the aerosol formulation. In aspects, the nasal inhaler will provide a metered amount of the aerosol formulation, for administration of a measured dose of the drug.
For trans-buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
The compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described, the compounds may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer, Science 249:1527-1533, 1990.
The immune modulatory molecules and optionally other therapeutics and/or antigens may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
In aspects of the invention, the formulations may also comprise a bile acid or a derivative thereof. In aspects, this may be in the form of a salt. In aspects, derivatives include, but are not limited to, derivatives of cholic acid and salts thereof. In aspects, sodium salts of cholic acid or cholic acid derivatives are contemplated. In aspects, bile acids and derivatives thereof include, but are not limited to, cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursodeoxycholic acid, hyodeoxycholic acid and derivatives for example, glyco-, tauro-, amidopropyl-1-propanesulfonic-, amidopropyl-2-hydroxy-1-propanesulfonic derivatives of the aforementioned bile acids, or N,N-bis(3D gluconoamidopropyl) deoxycholamide. In aspects, sodium deoxycholate (NaDOC) may be present in a vaccine of the invention.
Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v). Suitable preservatives include benzaikonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).
The pharmaceutical compositions of the invention contain an effective amount of one or more immune modulatory molecules and optionally one or more antigens, cholesterol and/or other therapeutic agents optionally included in a pharmaceutically-acceptable carrier. The term pharmaceutically-acceptable carrier means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal. The term carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.
The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference in their entireties.
EXAMPLES
Example 1
Cholesterol as a Delivery Vehicle for CpG ODN Immunogenicity & Efficacy Data
The use of liposomes containing cationic lipids with cholesterol has shown enhanced efficacy of CpG ODN. The use of cholesterol microspheres, without additional lipids, was tested as an adjuvant for augmenting cellular immunity. C57BI/6 mice (n=5 per group) were immunized intramuscularly on days 0, 14 and 21 with ovalbumin (10 μg), CpG alone (CPG 24555, 10 μg), and CpG (CpG 24555, 10 μg) with cholesterol (1 μg). Antigen specific T cells (CD4+ and CD8+) secreting single, double or triple cytokines (IL-2, IFN-γ and TNF-α) were measured on day 28 using flow cytometry.
Results and Discussion:
CpG+cholesterol enhanced the population of poly-functional CD8+ cells compared to CpG alone. CpG alone and CpG+cholesterol resulted in single and double cytokine producing CD4+ cells (FIG. 1a). CpG+cholesterol resulted in triple cytokine producing CD4+ cells (FIG. 1 b). CpG alone and CpG+cholesterol resulted in single, double and triple cytokine producing CD8+ cells (FIGS. 1c and 1d).
Enhanced secretion of antigen specific IL-2 (FIG. 2a) and IFN-γ (FIG. 2b) (Th1-biased cytokines) was shown with CpG+cholesterol. No enhancement in pro-inflammatory or Th2-biased cytokines was shown.
Cytotoxic T lymphocyte responses to CpG alone and CpG+cholesterol were measured. CpG+cholesterol enhanced ovalbumin specific cytotoxic T cell responses (FIGS. 3a and 3b) and antigen-specific CD8 T cell population was increased (FIGS. 3c and 3d) compared to CpG alone. Cholesterol alone showed similar levels to the no adjuvant control.
Humoral responses to CpG alone and CpG+cholesterol were measured. CpG+cholesterol showed enhanced ovalbumin specific antibody titer and Th1-bias over CpG alone (FIG. 4). The numbers above each bar represent the ratio of IgG2c/IgG1. In mice, a higher IgG2a or 2c is indicative of a Th1 biased immune response whereas a higher IgG1 titer is indicative of a Th2 biased immune response. For CpG and CpG+cholesterol the amount of IgG2c was higher than IgG1 (2.04 and 4.66 respectively) which is indicative of a Th1 biased immune response.
In each case, cholesterol alone showed no significant adjuvant activity.
Co-delivery of antigen and CpG, and antigen with CpG+cholesterol showed no retardation of mobility of CpG in electrophoresis. The same amount of CpG was observed in supernatants of CpG+cholesterol when quantified by UV compared to free CpG. This suggests that there was no strong association of CpG with cholesterol.
Subcutaneous immunization was less effective than intramuscular injection suggesting no evidence for co-delivery. However, co-delivery appears to play some role with a co-formulation of antigen and CpG demonstrating the strongest response.
Without being limited to a particular theory, transmission electron microscopy (TEM) suggested cholesterol formed insoluble helical micelles that may interact with cell membranes to allow the delivery of CpG (FIG. 5).
Example 2
Immunogenicity, Safety and Efficacy of a Pentavalent (IBR, BRSV, PI3, BVDV 1 & 2) Inactivated Vaccine in Calves Against BVDV-2 Challenge
The injection site reactions in calves immunized with a pentavalent inactivated viral vaccine Bovine Virus Diarrhea (BVDV 1&2), Infectious bovine rhinotracheitis (IBRV), Parainfluenza 3 virus (PI3V) and Bovine Respiratory Syncytial virus (BRSV) (respective viral antigen at 15% of 2 ml dose) in the presence of an adjuvant were measured. Adjuvants CpG+cholesterol (at ratios of 1:1 or 1:10 CpG:cholesterol), Advasure-DEAE/Dextran, QCDCR (saponin carrier complex) or QCDCR+CpG were administered. Some animals were immunized with commercial vaccine. Placebo animals received sterile saline. Calves (7/gp; 9-12 months old) were vaccinated at day 0 and day 22 subcutaneously with inactivated 2 ml BVDV 1&2, IBRV, PI3V and BRSV and challenged with 4 ml BVDV-2 (Noncytopathic Bovine Viral Diarrhea Virus Type 2; Strain 24515) intranasally on day 42.
The adjuvants used in the vaccines of each treatment group were as follows: Treatment Group T01 received sterile saline (no adjuvant). Treatment Group T02 received a vaccine in which the adjuvant was the oil-based emulsion contained in a commercial vaccine. Treatment Group T03 received a vaccine in which the adjuvant was CpG-23877 (250 μg)/cholesterol (250 μg), thus providing a ratio of CpG:cholesterol of 1:1. Treatment Group T04 received a vaccine in which the adjuvant was CpG-23877 (250 μg)/cholesterol (2,500 μg), thus providing a ratio of CpG:cholesterol of 1:10. Treatment Group T05 received a vaccine in which the adjuvant was AdvaSure®, an oil-based emulsion containing DEAE-DEXTRAN (100 mg) and ISC (800 μg). Treatment Group T06 received a vaccine in which the adjuvant was Quil A (250 μg), cholesterol (250 μg), dimethyl dioctadecyl ammonium bromide (DDA; 100 μg), Carbopol® (0.0375 μg), and N-(2-Deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N-octadecyldodecanoylamide hydroacetate, also known by the trade name Bay R1005® (1,000 μg). This combination of components is herein referred to as QCDCR. Treatment Group T07 received a vaccine in which the adjuvant was QCDCR (in the amounts given in T06) and CpG 23877 (250 μg).
Blood samples were collected at days 0, 22, 42 and 56 and analysed using ELISA for IgG antibody titers. BVDV ELISA was developed and optimized at PAH using the p67 H fragment of BVDV as antigen. Briefly, NUNC Maxisorp plates were coated with 0.2 ug/ml of recombinant p67 H fragment BVDV antigen in Carbonate-Bicarbonate buffer PH 9.6 and incubated overnight at 4° C. The coating antigen was then discarded and plates damped and blocked using 1% Ovalbumin in PBS-Tween (300 μl/well) for 1 h at 37° C. The blocking buffer was then removed and diluted serum samples added (seven 5-fold serial dilutions; starting at 1:50) and plates incubated for 1 h at 37° C. Plates were washed four times in PBS-T (0.05% Tween 20) before adding 100 μl of sheep anti-bovine IgG-h+I-HRP conjugate in blocking buffer (1:4,000) and incubating for 1 h at room temperature in the dark. Plates were washed again in PBS-T as described above and TMB substrate added (100 μl/well). Following Incubation for 5-10 minutes, the reaction was stopped using 2N Sulfuric acid (50 μl/well) and the Optical Density (OD) measured OD at 450 nM. Results were expressed as Geometric mean titers.
Results and Discussion
The symptoms of BVDV-2 challenge are fever, leukopenia (about a 40% decrease in WBC count from the mean pre-challenge WBC count) from day 3 to 12 after challenge and immune-modulation, thrombocytopenia, respiratory distress, depression, reproduction disorders (abortion) and diarrhea. Protective immunity against BVDV is a Th-1 type immune response. Cell mediated immunity is mediated by CD4+ T cells. CD8+ T cells are important for clearance of the virus and memory responses. IFN-α and IFN-γ are protective against BVDV infection, Vaccines generally should induce CMI and humoral immunity against BVDV. Table 1 depicts the percentage of calves with clinical disease, fever, leukopenia or viremia following challenge with BVDV-2 post vaccination with pentavalent inactivated viral vaccine BVDV 1&2, IBRV, PI3V and BRSV in the presence of CpG+cholesterol (at ratios of 1:1 (T03) or 1:10 (T04) CpG:cholesterol), Advasure-DEAE/Dextran (T05), QCDCR (T06), QCDCR+CpG (T07), commercial vaccine (T02) or sterile saline (T01).
TABLE 1
Percentage of calves with clinical disease,
fever, leukopenia or viremia
% Clinically
Treatment Group
Sick
% Fever
% Leukopenic
% Viremic
Non-vaccinated
42.9
85.7
100
100
control (PBS)
commercial vaccine
57.1
14.3
100
85.7
5V + CpG
14.3
0
42.9
28.6
Cholesterol (1:1)
5V + CpG
0
14.3
71.4
0
Cholesterol (1:10)
5V + Advasure
66.7
33.3
83.3
33.3
DEAE-
Dextran/ISC
5V + QCDCR
57.1
28.6
85.7
42.9
5V + QCDCR-CpG
42.9
14.3
71.4
0
(1:1)
Vaccines administered to calves in groups T02 (commercial vaccine), 103 (CpG:cholesterol 1:1), T04 (CpG:cholesterol 1:10), T05 (Advasure-DEAE/Dextran), T06 (QCDCR) and T07 (QCDCR+CpG) were superior at suppressing fever compared with saline controls. Vaccines administered to calves in T03 (CpG:cholesterol 1:1), T04 (CpG:cholesterol 1:10), T05 (Advasure-DEAE/Dextran), T06 (QCDCR) and T07 (QCDCR+CpG) were superior at suppressing viremia compared with commercial vaccine (T02) and saline groups (T01). T04 and T07 suppressed viremia completely and the commercial vaccine (T02) calves were viremic for a shorter period than the controls. Although leukopenia was not totally prevented in any vaccinated group, there was a vaccine effect noted on multiple days between T03 (CpG:cholesterol 1:1), T04 (CpG:cholesterol 1:10), T05 (Advasure-DEAE/Dextran), T06 (QCDCR) and T07 (QCDCR+CpG) compared to control and commercial vaccine (T02) groups. Calves in groups T03 and T04 experienced less clinical disease compared to the other groups. Overall, the data suggests that vaccines containing CpG's (T03, T04 and T07), such as the E modified P-class CpG, demonstrated an enhanced efficacy and these vaccines were more efficacious than the commercial vaccine.
The injection site reactions that developed following administration of the adjuvants are shown in FIG. 6a. The commercial vaccine (T02) and the Advasure-DEAE/Dextran (T05) vaccines were more reactive than the other vaccines tested. The vaccines containing QCDCR+CpG (T07) and CpG+cholesterol (T03 and T04) were the safest. In calves immunized with CpG+cholesterol, all symptoms of BVDV-2 challenge were reduced compared to non-vaccinated control animals.
The first vaccine dose administered induced low level serum neutralizing antibody titers. All the vaccines tested induced 100% sera-conversion to BVDV 1 and IBRV antigens by day 42. All vaccines tested, except the Advasure-DEAE/Dextran (T05) vaccine, induced a 100% sero-conversion to BVDV 2 by day 42. The Advasure-DEAE/Dextran (T05) vaccine induced 83% sero-conversion on day 42. Following challenge, BVDV 1 and BVDV 2 antibody responses were boosted to significantly higher levels in all groups (Table 2).
TABLE 2
part A
part B
BVDV 2 SN titers
BVDV 1 SN titers
IBR (BHV-1) SN titers
Group
Day 22
42
56
Day 22
42
56
Day 22
42
56
T01
1.4
1.0
109.6
1.2
1.0
5.8
1.0
1.0
1.0
(0/7)
(0/7)
(5/7)
(0/7)
(0/7)
(2/7)
(0/7)
(0/7)
(0/7)
T02
2.9
211.5
18207.5
1.8
76.3
3119.4
3.1
110.4
78.1
(2/7)
(7 of 7)
(7 of 7)
(0 of 7)
(7 of 7)
(7 of 7)
(6 of 7)
(7 of 7)
(7 of 7)
T03
2.3
69.3
4783
3.4
163.9
3530.5
2.0
7.8
7.2
(0 of 7)
(7 of 7)
(7 of 7)
(0 of 7)
(7 of 7)
(7 of 7)
(6 of 7)
(7 of 7)
(7 of 7)
T04
3.2
129.2
11979.8
10.8
371.1
8823.7
2.1
10.3
8.4
(0 of 7)
(7 of 7)
(7 of 7)
(7 of 7)
(7 of 7)
(7 of 7)
(5 of 7)
(7 of 7)
(7 of 7)
T05
1.7
16.6
2598.3
3.4
362.4
8689.6
20.3
100.7
63.3
(0 of 7)
(5 of 6)
(6 of 6)
(2 of 7)
(6 of 6)
(6 of 6)
(7 of 7)
(6 of 6)
(6 of 6)
T06
3.1
153.6
17379.6
15.1
1217.7
24346.5
3.9
67
46.2
(0 of 7)
(7 of 7)
(7 of 7)
(4 of 7)
(7 of 7)
(7 of 7)
(6 of 7)
(7 of 7)
(7 of 7)
T07
6.7
228.3
20162.6
25.5
1103
19972.2
5.0
57.9
41
(3 of 7)
(7 of 7)
(7 of 7)
(7 of 7)
(7 of 7)
(7 of 7)
(7 of 7)
(7 of 7)
(7 of 7)
SN—Serum Neutralization
The respective primary vaccination (T02-T07) primed BVDV-specific IgG antibody responses which were augmented by the booster vaccination and by BVDV2 challenge. There were no significant differences between IgG titers between the groups.
All the vaccine formulations were immunogenic and induced serum neutralization (BVDV 1 and BVDV 2) and IgG BVDV specific antibodies which were boosted by revaccination and challenge. Protection against BVDV challenge is by cell-mediated immunity (CMI; IFNγ secretion and activation of BVDV-specific CD4+ and CD8+ T cells), although antibody can neutralize free virus and hence protect against challenge if present at high levels, e.g. in colostrum fed to calves at birth. CMI (Th-1 type) responses were detected by secretion of IFNγ cytokine in vitro and humoral (Th-2 type) responses were determined by detecting for IL-4.
BVDV 1 and BVDV 2 antigens induced low level IFNγ responses and there were no significant differences (P>0.1) between the treatment groups (data not shown). The BSRV antigen induced IFNγ responses in all groups except in groups T01 (saline) and T03 (CpG:cholesterol 1:1). The IBR antigen induced the strongest IFNγ responses in T05 (Advasure-DEAE/Dextran) and T06 (QCDCR) on all days tested post-vaccination and weak but positive responses in T02 (commercial vaccine), T04 (CpG:cholesterol 1:10), and T07 (QCDCR+CpG). The PI3 antigen induced IFNγ responses in T02 (commercial vaccine), T05 (Advasure-DEAE/Dextran), T06 (QCDCR) and T07 (QCDCR+CpG).
Example 3
Immunogenicity in Swine of a Subunit (Pertactin) Bordetella bronchiseptica Vaccine Formulated with Different Adjuvants
Antigen-specific immune response of pigs immunized with pertactin (p68) formulated with various adjuvants, including CpG+cholesterol, were evaluated.
The Investigational Veterinary Products (IVP) used in the study were as follows: Vaccines were administered in 1-mL doses. Treatment Group T01 received 20 mM phosphate buffered saline. Treatment Group T02 received a vaccine containing Quil A (250 μg), cholesterol (250 μg), dimethyl dioctadecyl ammonium bromide (DDA; 100 μg), Carbopol® (0.075%), N-(2-Deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N-octadecyldodecanoylamide hydroacetate, also known by the trade name Bay R1005® (1,000 μg). This combination of components is herein referred to as QCDCR. The composition also contained CpG 23878 (250 μg) and pertactin (10 μg). Treatment Group T03 received a vaccine in containing cholesterol (2,500 μg), CpG 23877 (250 μg), and pertactin (10 μg). Treatment Group T04 received a vaccine in containing cholesterol (2,500 μg), CpG 23878 (250 μg), and pertactin (10 μgTreatment Group T05 received a vaccine in containing 6% aluminum as Al(OH)3 and pertactin (10 μg).
Sixty-four (64) clinically healthy, high-health status pigs of both sexes were used in the study. Pigs or their dams had no history of vaccination against or exposure to B. bronchiseptica. None of the pigs had a positive pertactin titer (defined as >200) from serum collected on at the farm of origin, or on Day −1.
On Day 0, pigs were vaccinated in the left neck with a 1.0 mL dose given by IM injection. On Day 21, pigs were revaccinated with the same IVP and dose as before, administered into the right neck. Within one hour of each vaccination, pigs were observed by the Investigator or a qualified technician for immediate adverse events related to vaccination.
The primary outcome variable was serum pertactin antibody titers (total IgG). Serum samples were tested for pertactin antibodies using an ELISA. Nunc Maxisorp plates were coated with 50 ng/well of pertactin in carbonate buffer (pH 9.1). Plates were washed and blocked with 1×PBS with 0.05% Tween 20 and 1% non-fat dry milk (1 h, R/T). Serum samples, diluted in blocking buffer, were added to the plates, incubated (1 h, R/T), washed and incubated (1 h, R/T) with HRP conjugate (Bethyl goat anti-pig IgG (h+I)) diluted 1:1250 in blocking buffer. Following a final wash, ABTS (KPL 50-62-00) substrate was added and OD values read after a 12-minute incubation at R/T. Titers were calculated based on a cutoff of 20% of the OD value of a 1:1000 dilution of a positive control serum pool.
Serum samples from T01, T03, T04, T05, and T08 were also tested for pertactin-specific IgG1 and IgG2 antibodies using an ELISA. Nunc Maxisorp plates were coated with 50 ng/well of pertactin in carbonate buffer (pH 9.1). Plates were washed and blocked with 1×PBS with 0.05% Tween 20 and 1% non-fat dry milk (1 h, R/T). Sera samples, diluted in blocking buffer, were added to the plates, incubated (1 h, R/T), washed and incubated (1 h, R/T) with monoclonal antibodies (IgG1-Serotec MCA635 or IgG2-Sertec MCA636) diluted 1:100 in blocking buffer. Plates were incubated (1 h, R/T), washed and an anti-mouse HRP conjugate added (Jackson Laboratories) diluted 1:5000 in blocking buffer. Following a final wash, ABTS (KPL 50-62-00) substrate was added and OD values read after a 20-minute incubation at R/T. IgG1 titers were calculated based on a cutoff of 50% of the OD value of a 1:1000 dilution of a positive control serum pool. IgG2 titers were calculated based on a cutoff of an OD of 0.2.
PBMCs, harvested from the heparin blood samples, were tested for antigen specific IFN-γ production. The IFN-γ response was tested by ELISPOT (Th1) to determine the frequency of INF-γ secreting cells/million cells SFC/106 from PBMCs (after subtracting the background in the medium controls). Additionally, IFN-γ responses were adjusted based on the stimulation index (SI) of the pertactin-stimulated cells compared to the medium control. A stimulation index of at least 2× was required for the sample to be deemed positive.
Results and Discussion
Pigs were vaccinated at day 0 and 21 with the vaccines shown in Table 3.
TABLE 3
Vaccine
Group
Adjuvant (Dose)
Carrier
T01
None
None
T02
CpG23878 (250 μg)
QCDCR
T03
CpG23877 (1:10)
Cholesterol
T04
CpG23878 (1:10)
Cholesterol
T05
Alhydrogel
None
Blood samples for PBMC isolation and serum samples were taken on day −1, day 7, day 20, day 28 and day 35 and analyzed. Serum samples were tested for total IgG, IgG1 and IgG2 antibodies using an ELISA to purified, LPS-free recombinant pertactin. Isotyping antibodies were obtained from Bethyl Labs or AbD Serotec.
Pertactin-specific IgG levels were increased in groups T02 (CpG+QCDCR), T03 (CpG 23877+cholesterol; 1:10) and T04 (CpG 23878+cholesterol; 1:10) compared to the other vaccines tested.
No post-vaccination adverse events were reported for the observation time immediately following vaccination. No observations of reactions being caused by vaccination were recorded. The pigs in treatment group T01 remained negative for pertactin ELISA antibodies throughout the study.
All pigs had negative pertactin-specific ELISA titers 200) on Day −1. The percentage of pigs that ever seroconverted after vaccination was 0% for T01, 100% for T02, T03, T04, and T08. The treatment group (T02) with the CpG #23878 adjuvant and QCDCR carrier, had GMTs of 906.0 and 24728.5 on Days 20 and 35 respectively, and GMTs on both days were significantly higher (P≤0.10) than all other treatment groups (Tables 4 and 5). T01, the negative control, had means that were significantly lower than the other groups on both Days 20 and 35. The GMTs of T08 (formulated with aluminum hydroxide) were significantly lower than the GMTs of the pertactin vaccines formulated with the CpGs using the QCDCR or cholesterol carriers (T02, T03, T04) at both post-vaccination time points. A Graph of antigen-specific antibody response in pigs immunized with pertactin (p68) formulated with various adjuvants including CpG+cholesterol is presented in FIG. 7.
TABLE 4
Pertactin-specific Total IgG ELISA Titers Day 20
Geometric least squares means, standard errors, and ranges of antibody
titers from pigs 20 days after being administered an IVP.
Treat-
Geo-
Stan-
ment
metric
dard
Group
Adjuvant
Carrier
Mean
Error
Range
T01
None
None
25.0a
6.88
25 to 25
T02
CpG #23878
QCDCR
906.0b
265.29
428 to 3188
(250 μg)
T03
CpG#23877
Choles-
367.4c
101.10
57 to 973
(250 μg)
terol
T04
CpG #23878
Choles-
254.2c
69.97
66 to 473
(250 μg)
terol
T05
Alhydrogel
None
46.5d
12.79
25 to 163
a,b,c,dgeometric means with different superscripts are significantly different (P < 0.10)
TABLE 5
Pertactin-specific Total IgG ELISA Titers Day 35
Geometric least squares means, standard errors, and ranges of antibody
titers from pigs 35 days after being administered an IVP.
Treat-
Geo-
Stan-
ment
metric
dard
Group
Adjuvant
Carrier
Mean
Error
Range
T01
None
None
25.0a
5.79
25 to 25
T02
CpG #23878
QCDCR
24728.5b
6077.46
10450 to 46473
(250 μg)
T03
CpG #23877
Choles-
7608.8c
1763.31
4619 to 17106
(250 μg)
terol
T04
CpG #23878
Choles-
4360.1d
1010.43
1968 to 11829
(250 μg)
terol
T05
Alhydrogel
None
625.6e
144.97
233 to 1997
a,b,c,d,egeometric means with different superscripts are significantly different (P ≤ 0.10)
Samples from only selected treatments were tested for isotype-specific pertactin serum antibody titers (Table 6). The IgG2/IgG1 ratio was 0.40 for T03 (CpG 23878 formulated with QCDCR) and 2.64 (CpG23878 formulated with cholesterol).
TABLE 6
Isotype-specific Pertactin Serum Antibody Titers
LS Geometric Mean Titer
Day −1
Day 20
Day 35
Trt
IgG1
IgG2
Ratio*
IgG1
IgG2
Ratio*
IgG1
IgG2
Ratio*
T01
29.7
25.0
0.84
25.0
25.0
1.00
25.0
25.0
1.00
T02
29.3
25.0
0.85
76.9
51.4
0.67
3255.3
1316.1
0.40
T03
36.5
25.0
0.68
33.6
89.3
2.66
767.7
2027.3
2.64
T04
25.0
25.0
1.00
30.1
29.9
0.99
360.8
501.2
1.39
T05
37.1
25.0
0.67
25.0
25.0
1.00
142.1
215.9
1.52
*IgG2/IgG1 ratio
The mean pertactin-specific IFN-γ responses was measured by Stimulation Index (SI) and spot-forming cells (SFC/106). There was considerable variability of the pertactin-specific IFN-γ responses of pigs within groups and between time points, in part because there were only 8 subjects per group. There were significant differences (P≤0.10) between treatments at all time points, including pre-vaccination (Day −1). For all post-vaccination time points (Days 7, 20, 28 and 35) the mean SI for T02 was significantly higher than T01 and T03. In contrast, the SI for T05 were not different from T01 on any post-vaccination time point. The SI for T04 was significantly higher than T01 on Days 20 and 35. The mean SFC/106 for T02 was significantly higher than T01 and T05 at 3 of 4 post-vaccination time points. The IFN-γ (SI and SFC/106) responses of 103 and T08 were not different from T01 at any post-vaccination time point.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
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13699997
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zoetis belgium s.a
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Mar 31st, 2022 03:01PM
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Mar 31st, 2022 03:01PM
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Zoetis
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:zts
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Zoetis
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Oct 27th, 2015 12:00AM
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Sep 2nd, 2011 12:00AM
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https://www.uspto.gov?id=US09168251-20151027
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High dose buprenorphine compositions and use as analgesic
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The present disclosure relates to a method of providing prolonged analgesia to a mammal in need thereof. Specifically, the current disclosure is directed to a method of treating pain in a mammal for a prolonged period of time using a single high dose of a buprenorphine formulation.
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9168251
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1. A method of producing a prolonged analgesic effect in a mammal, the method comprising the step of parenterally administering to a mammal in need of treatment thereof a single high dose of a buprenorphine, wherein said high dose ranges from about 0.12 mg/kg to about 0.3 mg/kg of total mammal body weight, and wherein said dosage provides adequate analgesia to the mammal for a period of at least twelve hours.
2. The method according to claim 1, wherein the mammal comprises canines and felines.
3. The method of claim 1, wherein the buprenorphine is administered by subcutaneous or intramuscular injection as a non-extended release dosage form.
4. The method according to claim 1, wherein the high dose of buprenorphine comprises about 0.24 mg/kg of total mammal body weight of buprenorphine.
5. The method according to claim 1, wherein the high dose buprenorphine provides adequate analgesia to the mammal for a period ranging from 12 hours to about 48 hours.
6. The method according to claim 5, wherein the high dose buprenorphine provides adequate analgesia to the mammal for a period of about 24 hours.
7. The method of claim 6, wherein the buprenorphine is administered once per day, twice per day, every other day, or every two days.
8. The method of claim 6, wherein the parenteral administration comprises subcutaneous administration or intramuscular administration.
9. The method according to claim 6 wherein the high dose of buprenorphine is about 0.24 mg/kg of total mammal body weight.
10. The method according to claim 1 of producing a prolonged analgesic effect in a mammal, wherein said mammal is a feline, the method comprising subcutaneous administering to the feline of a single non-extended release dose of buprenorphine and wherein said dosage provides adequate analgesia to the feline for a period ranging from about 18 hours to about 30 hours.
11. A non-extended release composition for subcutaneous or intramuscular administration to a mammal, the composition comprising: about 0.5 mg/mL to about 3.0 mg/mL buprenorphine or a pharmaceutically acceptable salt thereof, wherein said composition provides a dosage amount of buprenorphine of about 0.12 mg/kg to about 0.24 mg/kg of total body weight; and wherein said composition further comprises about 3% to 5% (v/v) of a tonicity-adjusting agent.
12. The composition of claim 11, wherein the composition comprises about 1.8 mg/mL of buprenorphine or a pharmaceutically acceptable salt thereof.
13. The composition of claim 11, wherein the composition comprises about 2.4 mg/mL of buprenorphine or a pharmaceutically acceptable salt thereof.
14. The composition of claim 11, wherein the tonicity-adjusting agent is dextrose and the composition has a pH range of about 3 to about 5.
15. The composition of claim 11 wherein the composition further comprises from about 0.05 to about 2.5 mg/mL of at least one antimicrobial agent.
16. The composition of claim 11 wherein the composition further comprises from about 5% to about 20% of ethanol.
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16
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CROSS-REFERENCE TO RELATED APPLICATIONS
This is the U.S. national stage entry of International Patent Application No. PCT/US2011/050413, filed on Sep. 2, 2011, which claims priority to U.S. Provisional Patent Application No. 61/379,996, filed on Sep. 3, 2010, the contents of which are fully incorporated herein by reference.
TECHNICAL FIELD
The present disclosure relates to pharmaceutical compositions and methods of providing analgesia to a mammal in need thereof. Specifically, the current disclosure is directed to pharmaceutical compositions and methods of treating pain in a mammal using a high dose of buprenorphine.
BACKGROUND
Buprenorphine is a semi-synthetic thebaine derivative which acts as a partial μ-opioid receptor agonist and κ-opioid receptor antagonist—an opioid. Opioid receptors are found principally in the central nervous system and the gastrointestinal tract. As a result of the partial agonist activity of buprenorphine at μ-opioid receptors, buprenorphine has a powerful analgesic effect—approximately twenty to forty times more potent as morphine.
Buprenorphine is available in various dosage forms, including sublingual and other oral formulations as well as parenteral dosage forms. The treatment of certain mammals, such as cats and dogs, with a sublingual medication that relies on continuous exposure to the oral mucosa of the mammal's mouth can be difficult to administer, resulting in poor pain control for the mammal. In addition, parenteral dosage forms may be given to mammals by several different routes of administration. Specifically, buprenorphine may be administered intravenously (“IV”) and intramuscularly (“IM”). The dosage varies depending on the route of administration. For example, when administered IV, the buprenorphine dose generally ranges from approximately 0.01-0.02 mg/kg of mammal body weight. See U. Krotscheck, D. V. M, D M Boothe D. V. M., and A A Little, D. V. M, Pharmacokinetics of buprenorphine following intravenous administration in dogs, AJVR, Vol. 69, No. 6, June 2008. Similarly, the dosing of buprenorphine for IM administration also ranges from approximately 0.01-0.02 mg/kg of mammal body weight. See L S Slingby, P M Taylor, and A E Waterman-Pearson, Effects of two doses of buprenorphine four or six hours apart on nociceptive thresholds, pain and sedation in dogs after castration, THE VETERINARY RECORD, Nov. 18, 2006, pp. 705-711; and S Dobbins, N O Brown, F S Shofer, Comparison of the Effects of Burprenorphine, Oxymorphone Hydrochloride, and Ketoprofen for Postoperative Analgesia After Onychectomy or Onychectomy and Sterilization in Cats; JOURNAL OF THE AMERICAN ANIMAL HOSPITAL ASSOCIATION; Vol. 38, November/December 2002, pp. 507-514. Subcutaneous (“SQ”) administration of buprenorphine has also been disclosed at doses up to approximately 0.02 mg/kg of mammal body weight. See P V Steagall et al., Effects of subcutaneous methadone, morphine, buprenorphine or saline on thermal and pressure thresholds in cats; JOURNAL OF VETERINARY PHARMACOLOGY AND THERAPEUTICS, 2006, Vol. 29, pp. 531-537.
Regardless of the route of administration, all forms of administration of buprenorphine are known to require dosing of the mammal every 2-8 hours for adequate pain control, depending on the route of administration and the pain threshold of the mammal. However, continuous administration of buprenorphine to the mammal by injection can be difficult to perform and stressful to the mammal, further complicating the pain control process. Companion animals, in particular, can be difficult to medicate, so analgesics that provide 24 hours of effect after a single dose would be advantageous. According to CVM-FDA policy, new veterinary pain medications are required to provide 72 hours of post-operative analgesia. Currently, no veterinary product is approved by CVM-FDA that will provide 24 hours of analgesia following a single injection, and can be administered for three consecutive days.
Additionally, the use of higher doses of buprenorphine would be expected to result in adverse effects to the mammal. Specifically, adverse effects associated with high dose buprenorphine include excessive sedation, respiratory depression, excessive salivation, and nausea. Due to the seriousness of such effects, commercially available buprenorphine products, such as the Vetergesic® buprenorphine injection, warns that dosing should not exceed to 10-20 micrograms per kg (0.01-0.02 mg/kg) for analgesia in dogs and cats, repeated if necessary after 2-6 hours.
Extended-release buprenorphine formulations have been developed to prolong the duration of pain control in a mammal. Injectable extended-release formulations, for example, include injectable microparticles, polymer matrix systems, fat emulsions, microspheres, and oil in water emulsions. However, the manufacturing of such formulations is complex and costly, and typically incorporates the use of organic solvents which could introduce potential toxicity if not completely removed. Additionally, it can be difficult to achieve sterility of microparticles and other oil solutions because terminal sterilization is not always possible. It is also difficult to appropriately control the release of a drug such as buprenorphine in an injectable dosage form in order to achieve the desired onset and duration of analgesic effects in the target species. Therefore, it would be desirable to have compositions and less complex methods of providing prolonged pain control to a mammal while minimizing the number of administrations/doses that must be given to the mammal.
SUMMARY
The present disclosure is directed to compositions and methods which include a single high dose non-extended release buprenorphine formulation administered in mammals for prolonged periods of pain control for at least 24 hours without the adverse effects generally expected from high dose treatment.
In embodiments, a method of producing a prolonged analgesic effect in a mammal is provided. The method comprises parenteral administration to a mammal in need thereof of single high dose non-extended release buprenorphine formulation wherein said dose provides adequate analgesia to the mammal for at least twelve hours.
In embodiments, a pharmaceutical composition producing a prolonged analgesic effect in a mammal is provided. The composition comprises a high of buprenorphine wherein said dose provides adequate analgesia to the mammal for at least twelve hours.
The mammals may be companion animals. The companion animals may be canines and felines. In embodiments, the companion animal is feline.
The route of parenteral administration may include subcutaneous, intramuscular, intravenous, intraarterial, intracerebral, intradermal, intrathecal, and intracerebral.
The high dose of buprenorphine may range from about 0.04 mg/kg to about 2 mg/kg of total mammal body weight, from about 0.05 mg/kg to about 1.5 mg/kg of total mammal weight, from about 0.1 mg/kg to about 0.5 mg/kg of total mammal body weight, and from about 0.12 mg/kg to about 0.3 mg/kg of total body weight. In embodiments, the high dose of buprenorphine is about 0.12 mg of buprenorphine per kg of total mammal body weight. In embodiments, the high dose of buprenorphine is about 0.24 mg of buprenorphine per kg of total mammal body weight.
The single high dose non-extended release buprenorphine formulation may provide analgesia to the mammal for a period ranging from about 12 hours to about 48 hours, from about 18 hours to about 30 hours, or for about 24 hours. The single high dose non-extended release buprenorphine formulation may be administered twice per day, once per day, every other day, every two days, or every 48 hours.
The pharmaceutical composition may comprise or consist essentially of a high dose of buprenorphine (such as from about 0.04 mg/kg to about 2.0 mg/kg total mammal body weight). The pharmaceutical composition may be a non-extended release formulation. In embodiments, the pharmaceutical composition is a non-extended release formulation comprising or consisting essentially of buprenorphine in the amount of about 0.12 mg/kg of total mammal body weight. In embodiments, the pharmaceutical composition is a non-extended release formulation comprising or consisting essentially of buprenorphine in the amount of about 0.24 mg/kg of total mammal body weight. The pharmaceutical composition may include a tonicity-adjusting agent and/or at least one antimicrobial agent. In embodiments, the pharmaceutical composition may comprise from about 5% to about 20% of a co-solvent such as ethanol. The pharmaceutical composition may include a tonicity-adjusting agent, at least one antimicrobial agent and/or a co-solvent such as ethanol.
The pharmaceutical composition may comprise or consist essentially of buprenorphine concentrations such as from about 0.5 mg/mL to about 3 mg/mL administered to provide a single high dose of buprenorphine (such as from about 0.12 mg/kg to about 0.24 mg/kg total mammal body weight). The pharmaceutical composition may be a non-extended release formulation. In embodiments, the pharmaceutical composition can also comprise about 3% to about 5% (w/w) of a tonicity-adjusting agent. In embodiments, the pharmaceutical composition may comprise from about 0.05 to about 2.5 mg/mL of at least one antimicrobial agent. In embodiments, the pharmaceutical composition may comprise from about 5% to about 20% of a co-solvent such as ethanol. The pharmaceutical composition may include a tonicity-adjusting agent, at least one antimicrobial agent and/or a co-solvent such as ethanol.
In embodiments, the pharmaceutical composition is a non-extended release formulation comprising or consisting essentially of buprenorphine at 1.8 mg/mL to 2.4 mg/mL to provide the amount of about 0.12 mg/kg of total mammal body weight. In embodiments, the pharmaceutical composition is a non-extended release formulation comprising or consisting essentially of buprenorphine at 1.8 mg/mL to 2.4 mg/mL to provide the amount of about 0.24 mg/kg of total mammal body weight.
One or more buffers are added to adjust the pH of the formulation to a range of about 3 to about 5. In embodiments, 5-15 mM buffer is added to adjust the pH of the formulation to about 4.0.
The pharmaceutical composition may be administered twice per day, once per day, every other day, every two days, or every 48 hours. In embodiments, the pharmaceutical composition is administered once per day.
DESCRIPTION OF DRAWINGS
FIG. 1 is a graph illustrating the buprenorphine plasma concentration over time for multiple doses of buprenorphine administered intravenously and subcutaneously, including 0.02 mg/kg intravenous dosing, 0.02 mg/kg subcutaneous dosing, 0.06 mg/kg subcutaneous dosing, 0.12 mg/kg subcutaneous dosing, and 0.24 mg/kg subcutaneous dosing.
FIG. 2 is a graph illustrating mean buprenorphine plasma concentration following subcutaneous administration of saline.
FIG. 3 is a graph illustrating the mean thermal threshold following subcutaneous administration of saline.
FIG. 4 is a graph illustrating the mean buprenorphine plasma concentration following subcutaneous administration of Buprenex® at 0.02 mg/kg (0.3 mg/mL).
FIG. 5 is a graph illustrating the mean thermal threshold following subcutaneous administration of Buprenex® at 0.02 mg/kg (0.3 mg/mL).
FIG. 6 is a graph illustrating the mean buprenorphine plasma concentration following subcutaneous administration of preserved buprenorphine solution at 0.06 mg/kg (1.2 mg/mL).
FIG. 7 is a graph illustrating the mean thermal threshold following subcutaneous administration on preserved buprenorphine solution at 0.06 mg/kg (1.2 mg/mL).
FIG. 8 is a graph illustrating the mean buprenorphine plasma concentration following subcutaneous administration of preserved buprenorphine solution at 0.12 mg/kg (1.2 mg/mL).
FIG. 9 is a graph illustrating the mean thermal threshold following subcutaneous administration of preserved buprenorphine solution at 0.12 mg/kg (1.2 mg/mL).
FIG. 10 is a graph illustrating the mean buprenorphine plasma concentration following subcutaneous administration of preserved buprenorphine solution at 0.24 mg/kg (1.2 mg/mL).
FIG. 11 is a graph illustrating the mean thermal threshold following subcutaneous administration of preserved buprenorphine solution at 0.24 mg/kg (1.2 mg/mL).
FIG. 12 is a graph illustrating the mean buprenorphine plasma concentration following subcutaneous administration of Buprenex® at 0.12 mg/kg (0.3 mg/mL).
FIG. 13 is a graph illustrating the mean thermal threshold following subcutaneous administration of Buprenex® at 0.12 mg/kg (0.3 mg/mL).
FIG. 14 is a graph illustrating the mean buprenorphine plasma concentration following subcutaneous administration of buprenorphine solution at 0.12 mg/kg (0.6 mg/mL).
FIG. 15 is a graph illustrating the mean thermal threshold following subcutaneous administration of buprenorphine solution at 0.12 mg/kg (0.6 mg/mL).
FIG. 16 is a graph illustrating the mean buprenorphine plasma concentration following subcutaneous administration of buprenorphine solution at 0.12 mg/kg (1.2 mg/mL).
FIG. 17 is a graph illustrating the mean thermal threshold following subcutaneous administration of buprenorphine solution at 0.12 mg/kg (1.2 mg/mL).
DETAILED DESCRIPTION
The present disclosure is directed to compositions and methods of providing prolonged analgesia to a mammal. Specifically, the compositions and methods of the present disclosure include a single high dose of buprenorphine which is administered to mammals to provide adequate analgesia for at least twelve hours. As used herein, “adequate analgesia” refers to a pain controlled state of a mammal that is assessed by an animal caregiver according to routine techniques or established criteria to make the assessment. For example, adequate analgesia may be assessed by clinical observations such as assessing whether the mammal appears to be comfortable; whether the mammal appears content and quiet when unattended; whether the mammal appears interested in or curious in its surroundings; whether the mammal is interested in the assessor when approaching its cage; whether the mammal seeks attention when the cage is approached and the door opened; whether the mammal exhibits minimal body tension when stroked; whether the mammal exhibits a normal or mild response when palpated at a surgery site; or whether the mammal is not bothered by palpation at a surgery site or palpation at any other location on its body.
Alternatively, or in addition to clinical observation, adequate analgesia may be assessed using thermal threshold techniques which determine whether a mammal is able to tolerate an increase in its reaction skin temperature when compared to its baseline skin temperature (which is known as its thermal threshold). The thermal threshold of a mammal can be determined using routine techniques known in the art. For example, in felines, thermal threshold can be determined using a device as described by Dixon in “A thermal threshold testing device for evaluation of analgesics in cats.” Res Vet Sci 2002; 72 (3): 205-210 which is incorporated herein by reference.
As used herein, the term “buprenorphine” means an opioid drug having the chemical name, 9α-cyclopropylmethyl-4,5-epoxy-6,14-ethano-3-hydroxy-6-methoxymorphinan-7-yl]-3,3-dimethylbutan-2-ol, or salts or derivatives thereof. Buprenorphine may comprise the free base or pharmaceutically acceptable salts, such as an acid addition salt or a salt with a base. Suitable examples of pharmaceutically acceptable salts include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, monopotassium maleate, mucate, napsylate, nitrate, N-methylglucamine, oxalate, pamoate(embonate), palmitate, pantothenate, phosphate/diphosphate, polygalacturonate, potassium, salicylate, sodium, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate, triethiodide, trimethylammonium, and valerate salts. In embodiments, the pharmaceutically acceptable salt includes hydrochloride, sulfate, methane sulfonate, stearate, tartrate, and lactate salts. In embodiments, buprenorphine comprises the acid addition salt, buprenorphine hydrochloride.
The disclosed compositions and methods comprise a high dose of buprenorphine. Generally, the term “high-dose” refers to any dose of buprenorphine greater than conventional doses of about 0.01 mg/kg to about 0.02 mg/kg of total body weight of the mammal. Accordingly, a high-dose of buprenorphine constitutes a dose of buprenorphine in the range of about 0.04 mg/kg to about 2.0 mg/kg of total mammal weight. In embodiments, the high dose of buprenorphine is in the range of about 0.1 mg/kg to about 1 mg/kg of total mammal weight, or in the range of about 0.15 mg/kg to about 0.5 mg/kg of total mammal weight, or in the range of about 0.2 mg/kg to about 0.3 mg/kg of total mammal weight. In embodiments, high dose buprenorphine is about 0.1 mg/kg of total mammal body weight. In embodiments, high dose buprenorphine is about 0.2 mg/kg of total mammal body weight. In embodiments, high dose buprenorphine is about 0.3 mg/kg of total mammal body weight. In embodiments, high dose buprenorphine is about 0.4 mg/kg of total mammal body weight. In embodiments, high dose buprenorphine is about 0.5 mg/kg of total mammal body weight. In embodiments, high dose buprenorphine is about 0.6 mg/kg of total mammal body weight. In embodiments, high dose buprenorphine is about 0.7 mg/kg of total mammal body weight. In embodiments, high dose buprenorphine is about 0.8 mg/kg of total mammal body weight. In embodiments, high dose buprenorphine is about 0.9 mg/kg of total mammal body weight. In embodiments, high dose buprenorphine is about 1.0 mg/kg of total mammal body weight. In embodiments, high dose buprenorphine is about 1.1 mg/kg of total mammal body weight. In embodiments, high dose buprenorphine is about 1.2 mg/kg of total mammal body weight. In embodiments, high dose buprenorphine is about 1.3 mg/kg of total mammal body weight. In embodiments, high dose buprenorphine is about 1.4 mg/kg of total mammal body weight. In embodiments, high dose buprenorphine is about 1.5 mg/kg of total mammal body weight. In embodiments, high dose buprenorphine is about 1.6 mg/kg of total mammal body weight. In embodiments, high dose buprenorphine is about 1.7 mg/kg of total mammal body weight. In embodiments, high dose buprenorphine is about 1.8 mg/kg of total mammal body weight. In embodiments, high dose buprenorphine is about 1.9 mg/kg of total mammal body weight. In embodiments, high dose buprenorphine is about 2.0 mg/kg of total mammal body weight.
Although veterinarians would expect initial plasma levels of buprenorphine administered at a high dose to be greater than plasma levels of buprenorphine administered at a lower dose, Applicants have surprisingly found that administration of high dose buprenorphine retains therapeutic plasma levels for a longer period of time than expected. In particular, Applicants have surprisingly found that administration of high dose buprenorphine retains therapeutic plasma levels for a period of at least 12 hours rather than periods of only 2-8 hours.
In addition, veterinarians would expect a high dose of buprenorphine to cause adverse side effects in mammals such as severe sedation, respiratory depression, cardiovascular effects, anorexia, dysphoria, and hyperexcitability, and therefore, would refrain from administering a high dose. Applicants, however, have surprisingly found that administration of high dose buprenorphine does not cause observable side effects in mammals and have demonstrated its safe use in mammals for the treatment of pain. Additionally, the disclosed compositions and methods enable less frequent administration of buprenorphine resulting in less stress and agitation to the mammal.
As used herein, the term “mammal” may generally be defined as a class of vertebrates, in which the females are characterized by the possession of mammary glands, and both males and females are characterized by sweat glands, hair and/or fur, three middle ear bones used in hearing, and a neocortex region in the brain. The methods of the current disclosure are typically directed to the administration of buprenorphine to domestic and companion animals. Examples of mammals that may be treated with the current disclosure include, but are not limited to canines, felines, pigs, cows, horses, sheep, donkeys, and mules. In a one embodiment, the method of the current disclosure is directed to the treatment of companion animals such as canines and felines. In another embodiment, the method comprises treatment of felines.
The disclosed methods include administering the high dose buprenorphine by a parenteral route of administration. Parenteral administration generally comprises all routes of administration wherein the active ingredient is absorbed systemically by means of piercing the skin or through a mucous membrane, and does not encompass rectal absorption or absorption through the digestive tract (i.e., oral administration). Non-limiting examples of parenteral routes of administration include intradermal, subcutaneous, intercavernous, intravitreal, transscleral, intravenous, intramuscular, intracardiac, intraosseous, and intraperitoneal administrations. In embodiments, the high dose buprenorphine is administered to the mammal by subcutaneous administration. In embodiments, the high dose buprenorphine is administered to the mammal by intramuscular administration.
In embodiments, a single high dose of buprenorphine is administered to a mammal during a period ranging from about 12 hours to about 72 hours providing adequate analgesia to the mammal for a period ranging from about 12 hours to about 48 hours. The disclosed methods include administering a single high dose of buprenorphine to a mammal twice per day, once per day, every 36 hours, every other day, every two days, every 48 hours, every three days, or every 72 hours. In embodiments, the disclosed methods include administering a single high dose of buprenorphine once per day. In embodiments, the disclosed methods include administering a single high dose of buprenorphine once every 36 hours. In embodiments, the disclosed methods include administering a single high dose of buprenorphine once every 48 hours. In embodiments, the disclosed methods include administering a single high dose of buprenorphine once every 72 hours.
The present disclosure is also directed to pharmaceutical compositions or formulations that comprise or consist essentially of buprenorphine and at least one pharmaceutically acceptable excipient. In embodiments, a single composition comprises the single high dose of buprenorphine. In embodiments, a single composition consists essentially of the single high dose of buprenorphine.
Pharmaceutically acceptable excipients used in the composition or formulations of the present invention may be excipients involved in enabling buprenorphine to be administered by a parenteral route of administration. Pharmaceutically acceptable excipients may include, but are not limited to, solvent systems, solubilization agents, stabilization agents, tonicity-adjusting agents, antimicrobial preservative agents and combinations thereof.
Suitable solvent systems may include solvents and oils. Suitable solvents may include, but are not limited to, propylene glycol, glycerin, ethanol, polyethylene glycol 300, polyethylene glycol 400, sorbitol, dimethylacetamide, Cremophor EL, dimethylacetamide, N-methyl-2-pyrrolidone, and mixtures thereof. Suitable examples of oils include sesame, soybean, corn, castor, cottonseed, peanut, arachis, ethyl oleate, isopropyl myristate, glycofurol, petrolatum, and combinations thereof.
Solubilization agents may include surfactants and complexation agents. Non-limiting examples of surfactants include polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monooleate, polyoxyethylene sorbitan monolaurate (Tween 20), lecithin, polyoxyethylene-poloxypropylene copolymers (Pluronics®), and combinations thereof. Suitable examples of complexation agents include, but are not limited to, hydroxypropyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin (Captisol®), polyvinylpyrrolidone, arginine, lysine, histidine, and combinations thereof.
Stabilization agents may comprise buffers, antioxidants, chelating agents and combinations thereof. Suitable buffers may include, for example, acetate, citrate, tartrate, phosphate, triethanolamine (TRIS) and combinations thereof. One or more buffers are added to adjust the pH of the formulation to a range of about 3 to about 5. In embodiments, 5-15 mM buffer is added to adjust the pH of the formulation to about 4.0. Suitable antioxidants may include, for example, ascorbic acid, acetylcysteine, sulfurous acid salts, such as bisulfite and metabisulfite, monothioglycerol, and combinations thereof. Suitable chelating agents may include ethylenediaminetetraacetic acid (EDTA), sodium citrate and combinations thereof.
Tonicity-adjusting agent may be used in the disclosed compositions to reduce irritation to the body tissue at the injection site. Suitable tonicity-adjusting agents include, for example, sodium chloride, glycerin, mannitol, dextrose and combinations thereof. Dextrose anhydrous, for example, may be present in the formulation in the amount of about 10 mg/mL to about 100 mg/mL or about 30 mg/mL to about 70 mg/mL or about 1% to about 10% or about 3% to about 7%.
Antimicrobial agents may be used in the disclosed compositions to prevent microbial growth in the aqueous environment of the formulation. Suitable antimicrobial agents may include, for example, phenol, meta-cresol, benzyl alcohol, parabens, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acids such as acetate, borate, and nitrate, and combinations thereof. Antimicrobial agents may be present in the formulation at any suitable amount. Methyl paraben, for example, may be present in the formulation in the amount of about 0.1 mg/ml to about 3.0 mg/ml or about 0.4 mg/mL to about 2.4 mg/mL or about 0.02 to about 0.3% or about 0.04 to about 0.24%. Propyl paraben, for example, may be present in the formulation in the amount of about 0.01 mg/ml to about 0.5 mg/ml or about 0.05 mg/mL to about 0.3 mg/mL or about 0.005% to about 0.05% or about 0.01% to about 0.03%. Chlorocresol, for example, may be present in the formulation in the amount of about about 0.36 mg/mL to about 1.5%. Benzyl alcohol, for example, may be present in the formulation in the amount of about 0.5% to about 3% or about 0.9% to about 2%.
Solvents may be used in the disclosed compositions as co-solvents and/or preservatives. Suitable solvents may include water and alcohols such as, for example, ethanol. Solvents may be present in the formulation at any suitable amount. Ethanol, for example, may be present in the formulation in the amount of from about 2 to about 30% (v/v) or from about 5 to about 20% (v/v). In embodiments, the amount of ethanol is about 10%.
In embodiments, the pharmaceutical composition comprises buprenorphine, a tonicity-adjusting agent (such as dextrose) in an amount of from about 3% to about 7% (v/v), and at least one antimicrobial agent (such as methylparaben, propylparaben or combinations thereof) in an amount of from about 0.05 mg/mL to about 2.5 mg/mL. In embodiments, the pharmaceutical composition comprises buprenorphine, a tonicity-adjusting agent (such as dextrose) in an amount of from about 3% to about 5% (v/v), and at least one antimicrobial agent (such as methylparaben, propylparaben or combinations thereof) in an amount of from about 0.05 mg/mL to about 2.5 mg/mL.
The disclosed compositions and formulations may be “non-extended” or “immediate” release formulations of buprenorphine. Non-extended release formulations refer to formulations that do not rely on other excipients to delay the release of the buprenorphine from the composition or formulation. The non-extended release formulations do not include components of sustained release formulations such as microparticles, polymer matrix systems, fat emulsions, microspheres, oil in water emulsions, and the like.
The buprenorphine and formulation excipients described above may be present in a formulation in any suitable amount. Tables 1A and 1B list non-limiting examples of suitable amounts of buprenorphine and particular excipients as concentration and percentage by volume of the formulation.
TABLE 1A
Components
Concentration
Percent (v/v)
Function
Buprenorphine HCl
0.8 mg/mL-2.4 mg/mL
0.08-0.24%
active substance
Dextrose anhydrous
30-70
mg/mL
3-7%
tonicity agent
Methyl paraben
0.4-2.4
mg/mL
0.04-0.24%
preservative
Propyl paraben
0.05-0.3
mg/mL
0.01-0.03%
preservative
Sodium Acetate Trihydrate
5-15
mM
buffer
Acetic Acid
buffer
Ethanol
50-20
mg/mL
5-20%
co-solvent/preservative
HCl or NaOH
as needed
as needed
pH adjustment
Water for Injection
solvent
The following formulation is a non-limiting example of a suitable high dose buprenorphine formulation:
TABLE 1B
Components
Amount, mg
Percent
Function
Buprenorphine HCl
1.8
0.18
active agent
(free base equivalent)
Dextrose anhydrous
50.0
5.0
tonicity agent
Methyl paraben
1.8
0.18
preservative
Propyl paraben
0.2
0.02
preservative
Sodium acetate trihydrate
0.2
0.02
buffer
Acetic acid
0.5
0.05
buffer
Ethanol
100.0
10.0
co-solvent/preservative
HCl or NaOH
as needed
as needed
pH adjustment to
approximately pH 4.0
Water
up to 1 ml
solvent
The dose volume of buprenorphine in a formulation may depend on the buprenorphine solution concentration and the buprenorphine dose. For example, in order to control the volume administered to the mammal when higher doses of buprenorphine are administered, the buprenorphine concentration may be increased. As presented in Table 1C below, a dose volume of 0.075 mL/kg may be used to deliver a dose of 0.06 mg/kg at a concentration of 0.8 mg/mL; a dose volume of 0.033 mL/kg may be used to deliver a dose of 0.06 mg/kg at a concentration of 1.8 mg/mL; a dose volume of 0.025 mL/kg may be used to deliver a dose of 0.06 mg/kg at a concentration of 2.4 mg/mL; a dose volume of 0.3 mL/kg may be used to deliver a dose of 0.24 mg/kg at a concentration of 0.8 mg/mL; a dose volume of 0.13 mL/kg may be used to deliver a dose of 0.24 mg/kg at a concentration of 1.8 mg/mL; a dose volume of 0.1 mL/kg may be used to deliver a dose of 0.24 mg/kg at a concentration of 2.4 mg/mL; a dose volume of 0.2 mL/kg may be used to deliver a dose of 0.48 mg/kg at a concentration of 2.4 mg/mL; a dose volume of 0.3 mL/kg may be used to deliver a dose of 0.72 mg/kg at a concentration of 2.4 mg/mL; a dose volume of 1.25 mL/kg may be used to deliver a dose of 1.0 mg/kg at a concentration of 0.8 mg/mL; a dose volume of 0.56 mL/kg may be used to deliver a dose of 1.0 mg/kg at a concentration of 1.8 mg/mL; and a dose volume of 0.42 mL/kg may be used to deliver a dose of 1.0 mg/kg at a concentration of 2.4 mg/mL. In embodiments a buprenorphine dose volume of 0.13 mL/kg may be used to deliver a buprenorphine dose of 0.24 mg/kg at a concentration of 1.8 mg/mL.
TABLE 1C
Dose Volume (mL/kg)
Dose (mg/kg)
Concentration (mg/mL)
0.075
0.06
0.8
0.033
0.06
1.8
0.025
0.06
2.4
0.3
0.24
0.8
0.13
0.24
1.8
0.1
0.24
2.4
0.2
0.48
2.4
0.3
0.72
2.4
1.25
1.0
0.8
0.56
1.0
1.8
0.42
1.0
2.4
The disclosed compositions may be administered during a period ranging from about 12 hours to about 48 hours. The disclosed compositions may be administered twice per day, once per day, every other day, every two days, or every 48 hours. In embodiments, the disclosed composition is administered once per day.
The disclosed compositions and methods provide adequate analgesia over a prolonged period of time. The term “prolonged” refers to a period of at least about 12 hours, a period of from about 12 hours to about 72 hours, or a period of from about 24 hours to about 48 hours. In embodiments, the disclosed compositions and methods provide adequate analgesia for up to about 72 hours. It should be appreciated that the duration of pain relief will vary depending on the pain tolerance of the mammal in need of pain relief.
It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the disclosure described herein are obvious and may be made using suitable equivalents without departing from the scope of the disclosure or the embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting of the disclosure.
EXAMPLES
In the examples below, the mammals studied were adult cats. The cats were housed individually. Fresh food and water was provided ad libitum. Cats were weighed prior to dosing. Body weights were used to calculate dose volumes administered. Cats were dosed on a per kilogram basis.
A number of different buprenorphine formulations were used in the examples. One buprenorphine formulation used was Buprenex® (buprenorphine), which is a commercially available non-buffered injectable 0.3 mg/mL solution labeled for human use (Reckitt Benckiser Pharmaceuticals Inc.). Other non-extended release buprenorphine formulations used include formulations containing buprenorphine as well as 5% dextrose, 2.3 mg/mL methlyparaben and 0.3 mg/mL propylparaben at a pH of 5.2. Another formulation contained buprenorphine, 5% dextrose, 10% ethanol, 1.8 mg/mL methlyparaben, 0.2 mg/mL propylparaben and an 10 mM acetate buffer at a pH of 5.2. Other formulations contained buprenorphine as well as 5% dextrose at a pH of 4.7, 5.4, 6.11 or 6.28.
Example 1
Buprenorphine Plasma Concentration Over Time for Cats
Buprenex® was used in the following examples. A syringe with a 22 g needle was filled with the appropriate volume of buprenorphine or control and was administered by subcutaneous injection at the base of the neck between the shoulder blades.
Blood samples were collected prior to buprenorphine administration and at approximately 0.25, 0.5, 1, 2, 4, 6, 8, 12, 20, 24, 36, 48 and 72 hours following dosing. Blood samples were collected by direct jugular venipuncture into a vacutainer containing lithium heparin. Volume of each sample was approximately 1.5 mL. Blood samples were placed immediately on ice and centrifuged at approximately 4° C. for approximately 15 minutes at approximately 3000 rpm for plasma preparation. Plasma samples were immediately frozen on dry ice and stored at −70° C. until analysis.
As set forth in Table 2, six treatment groups were monitored to determine the plasma profile for various routes of administration and dosing.
TABLE 2
Number of
Group
Animals
Route of Admin
Formulation
1
6
SQ
placebo (saline)
2
3
IM
0.02
mg buprenorphine
3
3
SQ
0.02
mg buprenorphine
4
6
SQ
0.06
mg buprenorphine
5
6
SQ
0.12
mg buprenorphine
6
6
SQ
0.24
mg buprenorphine
The first group consisted of a control group, in which six cats were administered placebo (saline) subcutaneously (hereinafter “control SQ”). In the second group, a total of three cats were administered 0.02 mg of buprenorphine per kilogram of body weight by the intramuscular route of administration (hereinafter “0.02 mg/kg IM”). In the third group, a total of three cats were administered 0.02 mg of buprenorphine per kilogram of body weight by the intramuscular route of administration (hereinafter “0.02 mg/kg SQ”). In the fourth group, a total of six cats were administered 0.06 mg of buprenorphine per kilogram of body weight by the intramuscular route of administration (hereinafter “0.06 mg/kg SQ”). In the fifth group, a total of six cats were administered 0.12 mg of buprenorphine per kilogram of body weight by the intramuscular route of administration (hereinafter “0.12 mg/kg SQ”). In the final group, a total of six cats were administered 0.24 mg of buprenorphine per kilogram of body weight by the intramuscular route of administration (hereinafter “0.24 mg/kg SQ”).
Regardless of the treatment group, plasma was drawn from each cat in the group every four hours, for a period of 48 hours, and tested to determine the buprenorphine concentration in mg/mL. The results of the plasma profile study are set forth in FIG. 1 demonstrating the effectiveness of the disclosed methods of treatment. In particular, FIG. 1 illustrates that the Control SQ, 0.02 mg/kg IM, and 0.02 mg/kg SQ treatment groups showed buprenorphine plasma concentrations that were negligible after approximately 8 hours. In contrast, however, the 0.06 mg/kg SQ, 0.12 mg/kg, and 0.24 mg/kg SQ treatment groups showed effective buprenorphine plasma concentrations 24 hours after the initial dosing. No significant side effects were noted in the study animals, such as a change in heart and respiration rates.
Example 2A
Dose Characterization
Studies were conducted to evaluate the efficacy on duration of analgesia of a non-extended release buprenorphine injectable solution administered at higher than conventional doses. Thermal threshold studies and surgical studies were performed to evaluate the efficacy of the non-extended release high dose buprenorphine injectable solution. Five studies were conducted in which eight treatment groups were evaluated.
The primary objective of these studies was to determine analgesic effects of buprenorphine injectable solutions in cats at subcutaneous doses of: 0 (saline), 0.02, 0.06, 0.12, and 0.24 mg/kg. Additional studies were done to determine if the concentration of the buprenorphine injectable solution influenced analgesia. The concentration of the buprenorphine injectable solution was increased from 0.3 mg/mL to 1.2 mg/mL, and the dose volume was maintained at about 0.03 to 0.2 mL/Kg. Furthermore, the dose of 0.12 mg/kg was evaluated at the concentrations of: 0.3 mg/mL, 0.6 mg/mL and 1.2 mg/mL.
Treatment groups are summarized in Table 4. Treatment Group 1 is included because 0.02 mg/kg is the most common dose recommended in the literature for post-operative pain in cats. Cats were administered a subcutaneous dose of buprenorphine or control article. Effectiveness was evaluated by determining the thermal threshold of each cat at prescribed time points. Blood samples were collected at prescribed time points for determination of buprenorphine plasma concentrations.
TABLE 4
Treatment Groups
Buprenorphine
Number
Dose Level
Concentration
Group
of Cats
(mg/kg)
(mg/mL)
Formulation
1
6
Saline
N/A
Physiologic Saline
2
3
0.02
0.3
Buprenex ®
3
6
0.06
1.2
Buprenorphine HCl in
5% dextrose
2.3 mg/mL methlyparaben
0.3 mg/mL propylparaben
pH = 5.2
4
6
0.12
1.2
Buprenorphine HCl in
5% dextrose
2.3 mg/mL methlyparaben
0.3 mg/mL propylparaben
pH = 5.2
5
6
0.24
1.2
Buprenorphine HCl in
5% dextrose
2.3 mg/mL methlyparaben
0.3 mg/mL propylparaben
pH = 5.2
6
3
0.12
0.3
Buprenex ®
3
7
3
0.12
0.6
Buprenorphine HCl in
5% dextrose
pH = 6.28
8
3
0.12
1.2
Buprenorphine HCl in
5% dextrose
pH = 6.11
Thermal threshold studies were conducted and buprenorphine plasma concentrations were determined at prescribed time points. Thermal threshold was determined using the device as described by Dixon in “A thermal threshold testing device for evaluation of analgesics in cats.” Res Vet Sci 2002; 72 (3): 205-210 which is incorporated herein by reference. Thermal stimulation was provided by a probe that was held in position on the cat's shaved thorax by a pressure bladder connected to an elastic band. This ensured there was consistent contact between the probe and the cat's skin. The temperature rise of the probe was 0.6° C./second with a safety cut-off at 55° C. Each cat received five baseline threshold stimulations prior to dose administration, and following administration at: 0.5, 1, 2, 3, 4, 5, 6, 8, 12, 16, 20, 24, 30, 36, 48, 60, and 72 hours. Starting skin temperature, reaction skin temperature and the type of reaction indicating a response to the increased temperature was recorded at each threshold evaluation.
Mean buprenorphine plasma concentrations and mean thermal threshold data are presented in FIGS. 2-17. Thermal threshold data is presented as the difference between the starting and reaction skin temperature. If the test article provided analgesia, the temperature at which the cat reacted was increased compared to baseline. A horizontal line, which corresponds to the baseline temperature that the cat reacted to, is included in the figures so differences between baseline and results after test article administration are easier to distinguish. Three to six cats were used in each treatment group. Results are presented as the mean±standard deviation.
As illustrated in FIGS. 3, 5, 7, 9, 11, 13, 15 and 17, thermal threshold studies indicated the non-extended release high dose buprenorphine injectable solution provided at least 24 hours of analgesia.
As illustrated in Table 5, as the dose increased from 0.02 mg/kg to 0.24 mg/kg, mean peak plasma concentration increased, and buprenorphine remained detectable in the plasma for longer periods of time. It should be appreciated that any variability in the mean buprenorphine plasma levels may be attributed to differences in rates of buprenorphine metabolism or clearance among the study animals.
TABLE 5
Comparison of Mean Buprenorphine Plasma Concentrations of Different Doses
Buprenorphine
Estimated Peak
Estimated Time
Estimated Duration of
Concentration of
Plasma
to Peak Plasma
Detectable Plasma
Dose
Test Article
Concentration
Concentration
Concentration
(mg/kg)
(mg/mL)
(ng/mL)
(Hours)
(Hours)
0.02
0.3
1.3
1
12
0.06
1.2
3.2
0.5
48
0.12
1.2
9.3
1
72
0.24
1.2
13.6
0.5
72
In addition, different buprenorphine injectable solution concentrations (0.3, 0.6, and 1.2 mg/mL) at a dose of 0.12 mg/kg did not appear to affect thermal threshold.
TABLE 6
Comparison of Mean Buprenorphine Plasma Concentrations
of Different Test Article Buprenorphine Concentrations
Buprenorphine
Estimated Peak
Estimated Time
Estimated Duration of
Concentration
Plasma
to Peak Plasma
Detectable Plasma
Dose
of Test Article
Concentration
Concentration
Concentration
(mg/kg)
(mg/mL)
(ng/mL)
(Hours)
(Hours)
0.12
0.3
6.6
0.5
48
0.12
0.6
14.8
1
72
0.12
1.2
9.3
1
72
Thermal threshold data indicated that higher doses of buprenorphine appear to result in longer analgesia. Buprenorphine administered at 0.02 mg/kg appeared to provide analgesia for approximately 20 hours in this model. When doses were increased, duration of analgesia appeared to increase to 24-28 hours, but there did not appear to be a clear difference between the three higher doses (0.06 mg/kg, 0.12 mg/kg, and 0.24 mg/kg) in this model. All three treatment groups had similar thermal threshold curves (FIGS. 9, 13, and 15). Therefore, a single high dose of a non-extended release buprenorphine formulation appears to have a 24 to 28 hour duration of adequate analgesia.
Example 2B
Administration of Buprenorphine in Cats for Post-Operative Pain Management
Based on foregoing results, and an intended use for high dose buprenorphine for post-operative pain in mammals, surgical studies were performed. The cats studied underwent an ovariohysterectomy or orthopedic (onychectomy) procedure and were monitored to determine the effectiveness of post-operative pain control. The objectives of these studies were to evaluate the ability of a single high dose of a non-extended release buprenorphine formulation to provide at least 24 hours of analgesia following the surgical procedure and to evaluate the ability of buprenorphine to control post-operative pain over 72 hours with three administrations of the formulation 24 hours apart.
Three studies were performed. Study personnel were blinded to treatment. Studies evaluated the ability of non-extended release buprenorphine injectable solution to control post-operative soft tissue or orthopedic pain for 24 hours with a single dose of buprenorphine administered one hour before surgery. Injectable solution was administered one hour prior to induction of anesthesia because thermal threshold data indicated a one hour onset of analgesia following administration. Efficacy of two additional doses, at 24 and 48 hours, to control post-operative pain until 72 hours after the first injection, was also evaluated. Buprenorphine injectable solution was administered at several different doses.
Female and male cats were obtained from local shelters and were identified by ID number provided by the shelter. Cats enrolled in the study were between six months and four years of age, weighed between two and ten kilograms, were non-pregnant and non-lactating, were generally in good health, and had a good disposition that allowed study procedures to be performed. Additionally, the cats had not received any medications within 30 days before study start, or during the study (except antibiotics).
Cats were single-housed in the feline ward of the Veterinary Specialty Center in standard Snyder cages (26 inches deep×21 inches wide×25 inches tall). Fluorescent lighting was on from 8 AM-6 PM. Temperature was controlled remotely and maintained at 65-72° F. Food and water were offered in stainless steel bowls. Cats were allowed to be acclimated to their new surroundings for 24 hours prior to procedures being performed. Prior to administration of medications and surgery, a medical history was provided and a physical exam was performed on each cat.
Three separate groups of cats were administered buprenorphine after an ovariohysterectomy procedure and monitored to determine the effectiveness of post-operative pain control. Specifically, each of the three groups consisted of nine female cats.
Each of the three treatment groups was administered a different dose of buprenorphine by subcutaneous administration. Increased doses of buprenorphine (0.06, 0.12, and 0.24 mg/kg) at a solution concentration of 1.2 or 2.4 mg/mL were used at a dosing interval of 24 hours. The first group received 0.06 mg/kg of total body weight as the total daily dosage amount, the second group received 0.12 mg/kg of total body weight as the total daily dosage amount, and the third group received 0.24 mg/kg of total body weight as the total daily dosage amount. A total of three doses of buprenorphine were administered to each cat in the respective treatment groups at 24-hour intervals for a total of 72 hours.
Buprenorphine and premedications (acepromazine, 0.05 mg/kg SQ and atropine, 0.04 mg/kg SQ) were administered one hour before induction of anesthesia. Buprenorphine and premedications were administered as separate injections. Two more doses of buprenorphine where administered 24 and 48 hours after the first dose. Additionally, a group in which buprenorphine was administered at the conventional dose of 0.02 mg/kg every eight hours for 72 hours was included for comparison. Treatment groups are summarized in Table 4.
Anesthesia was induced with propofol, 4-6 mg/kg intravenously, slowly to effect and the cat was intubated. Anesthesia was maintained with sevoflurane. Sevoflurane concentration was maintained at the appropriate setting to provide a surgical plane of anesthesia throughout surgery. Balanced electrolyte fluids were administered at 10 mL/kg/hr during the surgery to maintain blood pressure. Ovariohysterectomy was performed following current standards of practice using a midline abdominal approach. Onychectomy was performed following current standards of practice, using a scalpel or laser. Each method was used in five cats. If the cat was neutered in addition to the onychectomy, it was done following current standards of practice.
Baseline heart rate and respiratory rate were measured immediately prior to buprenorphine administration and three minutes after anesthesia induction. Heart rate, electrocardiogram, respiratory rate, end-tidal CO2, hemoglobin saturation (SpO2), body temperature, and either indirect or direct blood pressure were monitored at five minute intervals during surgery.
Sedation, excitation, and analgesia/pain were evaluated using a sedation, excitation, & pain scoring procedure. A single person did all assessments for an individual cat. If an assessment time point and drug administration time point coincided the assessment was done prior to the dose being administered. These time points were: 24 and 48 hours.
Baseline (immediately prior to buprenorphine administration) sedation, excitation, and pain/analgesia were measured in each cat. Following surgery, the cat was placed on a towel and continuously monitored in the immediate postoperative period until extubation. The treatment groups were continuously monitored over the course of the 72-hour period to determine if the 24-hour regimen for each group was successful in relieving the pain of the cats. Cats were monitored for sedation, excitation, and postoperative pain/analgesia within 30 minutes of extubation, 2, 4, 8, 12, 16, 20, 24, 32, 48, 56, and 72 hours after test article administration. Free choice cat food and water were offered to the cats four hours after recovery. If at any time during the study (not just the predetermined time points) the assessor thought the cat may be painful, an assessment was performed.
After finishing the sedation, excitation, and pain assessment, the assessor used his/her clinical judgment to determine if the cat needed to be rescued. The first rescue medication administered was meloxicam (0.1 mg/kg SQ). If the first rescue did not provide adequate analgesia, a second rescue was administered (hydromorphone 0.3 mg/kg SQ).
Treatment was considered successful if the cat made it through the entire 72-hour period without needing additional pain medications, other than the three doses of buprenorphine. Conversely, if a cat required rescue during the study period, it was considered a treatment failure. Buprenorphine formulations used in the surgical studies are summarized in Table 7.
TABLE 7
Buprenorphine
Formulation
1
Buprenex ®
2
Buprenorphine HCl in 5% Dextrose,
pH = 4.7-5.4
3
Buprenorphine HCl in 5% Dextrose,
pH = 5.4
4
Buprenorphine HCl in
5% Dextrose
10% Ethanol
1.8 mg/mL methlyparaben
0.2 mg/mL propylparaben
10 mM Acetate buffer
pH = 5.2
The results of the study are included in Table 8 below. The results shown below also include the treatment success rates for cats administered the typical low dose of 0.02 mg/kg subcutaneously.
TABLE 8
Success Rate for 24-Hour Treatment with Subcutaneous Buprenorphine
No. of Study
Surgical
Dose
Volume
Frequency
Success Rate
Group
Animals
Procedure
Formulation
(mg/kg)
(mg/ml)
(hrs)
(percent)
1
3
OHE
1
0.02
0.3
8
33
2
9
OHE
2
0.06
1.2
24
78
3
9
OHE
2
0.12
1.2
24
56
4
9
OHE
2
0.24
1.2
24
100
5
10
OYN
3
0.24
1.2
24
70
6
6
OHE
4
0.24
2.4
24
83
OHE=Ovariohysterectomy
OYN=Onychectomy
In this study, the dose of 0.24 mg/kg was the most effective (100% [0.24 mg/kg], vs. 33% [0.02 mg/kg], 56% [0.12 mg/kg], and 78% [0.06 mg/kg]). The conventionally used dose of buprenorphine of 0.02 mg/kg administered every eight hours, had a success rate of 33%, and results were included for comparison. Based on the 100% success rate in controlling soft tissue pain, the 0.24 mg/kg dose was evaluated in an orthopedic surgical model (onychectomy) in cats. Success rate in this study was 70%. The success rate was considered acceptable and was not unexpected because onychectomies are more painful than ovariohysterectomies.
A third study was performed because the formulation of the buprenorphine was changed (preservative was added and concentration increased). Efficacy of the new formulation needed to be confirmed. A soft tissue surgery (ovariohysterectomy) was used. Success rate of the dose 0.24 mg/kg was 83%.
As can be seen in Table 8, the higher doses of buprenorphine resulted in higher success rates compared to the traditional low dose of 0.02 mg/kg. These finding are consistent with the ability of a high dose non-extended release buprenorphine formulation to provide prolonged adequate analgesia to a mammal.
Example 3
Safety Studies
The following buprenorphine formulation was tested at various dosages to determine the safety of a high dose non-extended release buprenorphine formulation.
Component
Amount
buprenorphine
2.4
mg/mL
dextrose
5%
methyl paraben
1.8
mg/mL
propyl paraben
0.2
mg/mL
acetate buffer
10
mM
ethanol
10%
The pH of the formulation was adjusted to about 4.0.
This study consisted of five treatment groups in which there were four cats (two males, and two females) per treatment group. Treatment groups were designated by dosage. The cats were randomized into one of the treatment groups. The three treatment groups that were administered the test article were Group 1 (5×, 1.2 mg/kg), Group 4 (1×, 0.24 mg/kg), and Group 5 (control). Groups 2 and 3 were not dosed.
The buprenorphine solution was administered by subcutaneous injection for nine consecutive days to three groups of cats (four cats per group). Nine consecutive doses were administered daily to each cat. All doses were administered subcutaneously intrascapularly. At each administration, the cats were observed for pain on injection (e.g. meowing or growling). Body weight from the previous day was used for each dose calculation. Each group of four cats received one of the treatments outlined below in Table 9. For heart rate and respiration rate, each group of cats was evaluated at 30 minutes and at 1, 2, 4, and 7 hours post daily dose.
TABLE 9
Treatment Groups
Number of
Dose
Concentration
Dose Volume
Group
Study Animals
Compound
(mg/kg)
(mg/mL)
(mL/kg)
1
4
Buprenorphine
1.2
2.4
0.5
Formulation
2*
4
Buprenorphine
0.72
2.4
0.3
Formulation
3*
4
Buprenorphine
0.48
2.4
0.2
Formulation
4
4
Buprenorphine
0.24
2.4
0.1
Formulation
5
4
Saline
0
NA
0.1
*Group 2 and 3 were not dosed.
Reactions to the injections were in different cats at different times during the study, including one control cat treated with saline. All injection site observations were normal during the study. This indicated that the administration of buprenorphine was well-tolerated and did not result in injection site reactions.
No dose of buprenorphine had an effect on body weight over the study period. This was most likely due to the fact that the treatment with buprenorphine did not have an effect on food or water consumption.
The induction of constipation is a concern with opioid administration. In this study, while there was variability in the evidence of defecation, no dose had an effect on the frequency of defecation and all feces notes in the study were normal. Therefore, constipation was not an issue associated with administration of buprenorphine in this study.
Frequency of urination did not appear to be affected by either dose. Additionally, there were no significant findings in the urinalysis, indicating that buprenorphine did not have detrimental effects on the urinary system o the treated cats.
Behavioral side-effects associated with opioid administration are sedation, dysphoria (manic behavior), and euphoria (rubbing, purring, rolling, kneading, etc). Sedation and dysphoria are considered negative behavioral effects, while euphoria is usually considered a positive attribute. Only one cat in the 5× group had evidence of slight sedation and this was only at a single timepoint. The same cat showed signs of dysphoria, several times during the study, which varied in intensity. All groups, including control-treated cats, showed signs of euphoria during the study with no apparent difference between groups. Results indicate buprenorphine has minimal negative effects on behavior and that this only occurred at the 5× dose.
Pupil dilation occurred in both buprenorphine treatment groups but this is a well-known side-effect of all opioids including buprenorphine administered at much lower doses (i.e., 0.02 mg/kg) and is not considered a detrimental side-effect.
Neither dose of the formulated buprenorphine had a clinically relevant effect on clinical pathology parameters. Several cats showed a stress lukogram (leukocytosis with a mature neutrophilia) but this is atypical response for cats that are “stressed” (i.e., being handled during a study). Two different cats showed signs of mild dehydration during the study, but they were limited to one or two timepoints throughout the study. Due to the mild nature and limited duration of dehydration it is not considered clinically relevant. Mild hyperkalemia observed in several cats is suspected to have been an artifact because there were no other hematological abnormalities. The presence of blood and protein in the urine is most likely a result of inflammation induced by repeated cystocentesis. In summary, none of the mild clinical pathology changes are clinically relevant.
Body temperature was within acceptable limits throughout the study in all treatment groups, indicating that formulated buprenorphine did not affect the study cats' ability to thermoregulate.
None of the buprenorphine doses had an effect on heart rate, respiration rate, or mean blood pressure during the study. All heart and lung auscultations were normal.
Therefore, based on these findings, high dose non-extended release buprenorphine formulations are safe, even when administered at five times the dose of 0.24 mg/kg, and for three times as long as the intended duration of administration.
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13818013
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zoetis belgium s.a
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Mar 31st, 2022 03:01PM
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Mar 31st, 2022 03:01PM
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Zoetis
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:zts
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Zoetis
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Dec 8th, 2020 12:00AM
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Jun 21st, 2013 12:00AM
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https://www.uspto.gov?id=US10859571-20201208
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Method and a system for quantitative or qualitative determination of a target component
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A method and a system for quantitative or qualitative determination of a target component in a liquid sample includes i) providing a plurality of magnetic particles including one or more capture sites for the target component on their respective surfaces; ii) providing a plurality of fluorophores configured to bind to the capture sites of the magnetic particles; iii) bringing the liquid sample into contact with the fluorophores and the magnetic particles in a flow channel of a micro fluidic device including a transparent window; and iv) at least temporally immobilizing the magnetic particles adjacent to the transparent window using a magnet, emitting exciting electromagnetic beam towards the immobilized magnetic particles, reading signals emitted from fluorophores captured by the immobilized magnetic particles and performing a quantitative or qualitative determination of the target component based on the read signal.
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10859571
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1. A method for quantitative or qualitative determination of at least one target component in a liquid sample, the method comprises:
providing a plurality of magnetic particles comprising one or more capture sites for the target component on the respective surfaces of the magnetic particles;
providing a plurality of fluorophores coupled to a component which is identical or homolog to the at least one target component so that the component is also able to bind to said capture sites of the magnetic particles;
arranging the fluorophores and said magnetic particles at a distance from each other inside a flow channel of a micro fluidic device comprising a transparent window to the flow channel, and temporarily immobilizing the fluorophores and said magnetic particles in the flow channel of the micro fluidic device, such that they cannot bind to each other prior to feeding the liquid sample to the flow channel, the flow channel is defined by a groove or walls;
feeding said liquid sample suspected of containing the at least one target component into said flow channel to resuspend the fluorophores and the magnetic particles, wherein the feeding of said liquid sample into said flow channel comprises using an actuator for moving a flexible wall section of the flow channel or of a sink section in fluid connection with the flow channel;
mixing said liquid sample suspected of containing the at least one target component, said fluorophores, and said magnetic particles simultaneously inside said flow channel while allowing said at least one target component, if present, to compete with the fluorophores with the capture sites of the magnetic particles; and
at least temporally immobilizing said magnetic particles adjacent to said transparent window using a magnet, exciting the fluorophores bound to the immobilized magnetic particles with an electromagnetic beam, reading signals emitted from fluorophores captured by said immobilized magnetic particles and performing a quantitative or qualitative determination of said at least one target component based on the read signals.
2. The method of claim 1, wherein the liquid sample comprises a biological fluid or a fraction of a biological fluid.
3. The method of claim 1, wherein the liquid sample comprises human, animal, or vegetable fluids selected from at least one of blood, saliva, urine, milk, cytosol, intracellular fluid, interstitial fluid, tissue fluid, and one or more fractions or mixtures thereof, or suspended biological solids.
4. The method of claim 1, wherein the at least one target component comprises a microorganism selected from bacterial pathogens, viral pathogens, or fungal pathogens.
5. The method of claim 1, wherein the at least one target component comprises at least one of one or more of the groups cells, proteins, nucleotides, carbohydrates, or lipids.
6. The method of claim 1, wherein the magnetic particles are coated magnetic particles comprising a coating comprising the captures sites for the at least one target component.
7. The method of claim 1, wherein the fluorophores are comprised of at least one of quantum dots, aromatic probes or conjugated probes.
8. The method of claim 1, wherein the fluorophores are quantum dots that emit one or more discrete frequencies of light when stimulated by a light source and wherein each quantum dot comprises a core of an excitable material and an organic coating which is coupled to the component which can bind to the capture sites of the magnetic particles.
9. The method of claim 1, wherein the actuator is a step motor driven actuator.
10. The method of claim 1, wherein said at least temporally immobilized magnetic particles are subjected to said electromagnetic beam such that at least a part of possible fluorophores captured by said capture sites of the magnetic particles are excited, and subsequently the emitted signal from any captured fluorophores is read and a quantitative or qualitative determination of said at least one target component based on the read signal is performed.
11. The method of claim 1, wherein said at least temporally immobilized magnetic particles are released from magnetic forces applied by the magnet prior to being subjected to said electromagnetic beam.
12. The method of claim 1, wherein the quantitative or qualitative determination of at least one target component in the liquid sample is performed by comparing the read signal(s) with signals obtained from liquid samples of known composition.
13. The method of claim 1, wherein the quantitative or qualitative determination of the at least one target component in the liquid sample is performed by multiplexing the read signals from different groups of fluorophores using reference fluorophores with a different exciting wavelength.
14. The method of claim 1, wherein the plurality of fluorophores are configured to bind directly to said capture sites of the magnetic particles.
15. The method of claim 1, wherein the micro fluidic device comprises a substrate with a groove for the flow channel and a foil covering the flow channel.
16. The method of claim 1, wherein the micro fluidic device comprises an inlet for the liquid sample.
17. The method of claim 1, wherein the arranging and immobilizing of the fluorophores and the magnetic particles at a distance from each other inside the flow channel comprises temporally immobilizing of the fluorophores and the magnetic particles in respectively a first zone and a second zone separate from the first zone, wherein the first zone and the second zone are arranged between an inlet zone and a reading zone where said magnetic particles are at least temporally immobilized adjacent to the transparent window.
18. The method of claim 1, wherein the liquid sample is brought into the flow channel by moving a flexible wall section of the flow channel or a sink section in fluid communication with the flow channel to press out air of the flow channel and there after sucking the liquid sample into the flow channel to resuspend and mixing with the temporally immobilized fluorophores and magnetic particles.
19. The method of claim 1, wherein said method comprises performing two or more parallel assays on the liquid sample for quantitative or qualitative determination of the at least one target component, each assay comprises:
bringing a part of the liquid sample into contact with said fluorophores and said magnetic particles in the flow channel of the micro fluidic device comprising the transparent window; and
at least temporally immobilizing said magnetic particles adjacent to said transparent window using the magnet, emitting exciting electromagnetic beam towards said immobilized magnetic particles, and reading signals emitted from fluorophores captured by said immobilized magnetic particles,
wherein the respective parallel assays are performed in respective flow channels using respective magnetic particles and respective fluorophores.
20. The method of claim 19, wherein the fluorophores and/or the magnetic particles used in one of the two or more parallel assays differ from the fluorophores and/or the magnetic particles used in another one of the two or more parallel assays.
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TECHNICAL FIELD
The present invention relates to a method and a system for quantitative or qualitative determination of a target component in a liquid sample, in particular a biologic target component or optionally several target components in the same liquid sample.
BACKGROUND ART
A plurality of methods and devices for quantitative or qualitative determination of a target component in a liquid sample are known from the prior art. Many of these prior art methods comprise complicated or time consuming steps, such as washing steps. For many years new and improved methods have constantly been developed, and in particular methods using optical labeling and read out systems.
The use of magnetic particles with capture probes has also been explored in several methods.
A fluorimetric immunological assay with magnetic particles is described in U.S. Pat. No. 4,731,337. In this document it was suggested to perform a test using plastic pearls comprising a magnetic substance and carrying antigen for an antibody to be studied in a transparent test tube. The pearls are put into the test tube together with a sample containing the antibody to be studied as well as an antibody marked with a fluorescent molecule. Upon completion of the reaction, the quantity of the marked antibody adhering to the antigen on the solid phase is measured in a fluorometer, in which both the excitation radiation is passed into the sample and the fluorescent radiation is collected to the detector through the bottom of the measurement vessel. Moreover, the fluorometer is provided with means for generating a magnetic field, and by its means the pearls are pulled against the bottom of the measurement vessel for the time of the measurement. Moreover, before the measurement, a coloring agent is added to the sample to absorb intensively at the wavelength of the excitation radiation or of the emission radiation in order to reduce interference by the excess tracer remaining in the liquid phase or by the background radiation with the measurement.
The above method has, however, never been used in practice.
WO 2010/042242 discloses for example a use of magnetic particles in combination with a fluidic device. The fluidic device disclosed herein has a main channel, wherein a first inlet fluidly connects to an upstream end of the main channel and the method comprises introduction of magnetic beads into the channel. The magnetic beads are configured to bind to a target. A magnet is applied to magnetically move the magnetic beads through various sections of the fluidic device to allow the magnetic beads to capture a target, to a washing step and other steps required for obtaining a quantification of the target captured on the magnetic beads.
WO 2008/109675 discloses a device and method for the capture of magnetic beads in a rotary magnetic bead trap. The device allows capture, washing, elution and ejection of beads in an automated system. Analyte is eluted in a small volume in a capillary-scale fluid system compatible with LC-MS/MS analysis.
US 2010/0248258 discloses a microfluidic chip and method for rapid detection of different target proteins. The microfluidic chip utilizes antibody-conjugated magnetic beads to bind to the target proteins to form a magnetic complex, and then use the signal labeled-antibodies that can recognize the magnetic complex. The method comprises purifying the magnetic complex by the micro-magnetic field on biochip, and introducing the purified magnetic complex into the fluorescent detection area on the chip to detect the amount of the target protein in the purified complex immediately.
U.S. Pat. No. 4,347,312 discloses a method for detecting the presence of antibiotics in milk which comprises the steps of: (a) contacting a solid matrix having attached thereto a purified immobilized antibody with a volume of milk and an enzyme-labeled antibiotic, the antibody being specific to the antibiotic; (b) separating the matrix from the milk and rinsing the matrix with water to remove excess milk and enzyme-labeled antibiotic; (c) contacting the rinsed matrix with a substrate, the substrate in the presence of the enzyme-labeled antibiotic exhibiting a color change the amount of which is quantitatively related to the amount of enzyme-labeled antibiotic; and (d) measuring the amount of antibiotic present in the milk by comparing the color change in the substrate with a standard. Also provided is a method for producing purified antibodies for use in the foregoing detection method by: (a) covalently conjugating an antibiotic having a lactam-ring in the molecule to a protein capable of binding thereto through the lactam-ring; (b) injecting into a host animal capable of raising antibodies specific to the antibiotic the conjugate obtained in step (a) so as to raise the specific antibodies; (c) covalently conjugating the same antibiotic in step (a) to a second protein capable of binding thereto through the lactam-ring and different than the protein used in step (a) to form a second conjugate; (d) covalently binding the second conjugate to a solid matrix to form an affinity matrix for purifying the antibodies; (e) isolating and purifying the specific antibodies raised in step (b) by contacting the host animal serum with the affinity matrix; and (f) recovering the specific antibodies in a pure form.
DISCLOSURE OF INVENTION
An object of the present invention is to provide a new method for quantitative or qualitative determination of a target component in a liquid sample, which method is simple and fast and where it is possible to perform determinations of two or more target components simultaneously.
An object of the present invention is further to provide a system for performing quantitative or qualitative determination of a target component in a liquid sample where the cost for each determination is relatively low.
In an embodiment of the invention it is further an object to provide a method which does not require time consuming washing steps and preferably where it is possible to perform a determination relatively fast e.g. in a few minutes.
These objects have been achieved by the present invention and embodiments hereof as defined in the claims and described below.
The invention has shown to provide a completely new approach for quantitative or qualitative determination of a target component in a liquid.
Further it has been found that very good and accurate results can be obtained in a fast and simple way.
The method of the invention for quantitative or qualitative determination of a target component in a liquid sample, comprises
providing a plurality of magnetic particles comprising one or more capture sites for the target component on their respective surfaces;
providing a plurality of fluorophores configured to bind to the capture sites of the magnetic particles;
bringing the liquid sample, the fluorophores and the magnetic particles into a flow channel of a micro fluidic device comprising a transparent window; and
at least temporally immobilizing the magnetic particles adjacent to the transparent window using a magnet, emitting exciting electromagnetic beam(s) towards the immobilized magnetic particles, reading signals emitted from fluorophores captured by the immobilized magnetic particles and performing a quantitative or qualitative determination of the target component based on the read signal.
The method of the invention is highly suitable for qualitative determination of one or more target components and it has been found that the method can provide highly accurate results. However, the method of the invention has also shown to be very suitable for qualitative determinations e.g. for screening purposes. Examples of this will be described further below.
The “method” is also referred to by the term “test” and a liquid which is subjected to the method of the invention is tested/subjected to a test.
The term “at least temporally immobilizing the magnetic particles adjacent to the transparent window” should be taken to mean that the magnetic particles should be immobilized for a sufficient time to excite possible fluorophores captured on the magnetic particles and read signals emitted from such fluorophores. This will be further described below.
The liquid sample, the fluorophores and the magnetic particles are fed into the flow channel of the micro fluidic device, such that they are in contact with each other. In an embodiment two or all of the liquid sample, the fluorophores and the magnetic particles are brought into contact before being fed into the liquid channel. In an embodiment the liquid sample, the fluorophores and the magnetic particles are brought into contact within the liquid channel
The transparent window is for example in the form of a transparent wall section of the flow channel.
By performing the test using a micro fluid device, the method becomes very fast and only a small amount of sample is required.
Heretofore when performing tests using magnetic particle for capturing marked components, it has been an ordinary requirement that some kind of washing was required or at least a masking of false positive was required in order to obtain a useful determination. By using micro fluidic device for performing the test it has surprisingly been found that washing is not required and not even desired. In fact it is preferred that after the magnetic particles are immobilized, no further liquid should be added and in a preferred embodiment the liquid in the flow channel is at standstill, meaning that there is no flow and no turbulence within the flow channel. Further it is desired that no light absorbing elements are added beyond what is inherently in the sample. In an embodiment the sample comprises no added light absorbing elements beyond the fluorophores and the magnetic particles and what is inherent in the liquid sample. In an embodiment the sample comprises no elements absorbing light emitted by the fluorophores beyond the fluorophores and the magnetic particles.
The method has shown to give surprisingly reliable results even while determining two or more target components simultaneously.
Preferred micro fluidic devices will be described further below.
The term “liquid sample” means any liquid containing sample including liquid sample comprising solid parts, such ad dispersions and suspensions. The sample comprises liquid at the time of performing the method.
In principle any liquid sample can be applied, including but not limited to liquid samples comprising particles, such as dispersed particles. The liquid sample is in one embodiment crushed food or tissue optionally blended with water or it may be an extract thereof. Thus, the method of the invention can for example be applied for performing quantitative and/or qualitative tests on tissue, vegetables, meat and etc.
In an embodiment the liquid sample comprises human or animal faeces e.g. in an aqueous suspension.
In an embodiment the liquid sample comprises waste water or water from a nature source e.g. a lake or a river.
The liquid sample should preferably have a sufficiently low viscosity for being mixed with the fluorophores and the magnetic particles preferably such as to allow possible present target components to bind to the magnetic particles. If the viscosity is too high, the sample may be diluted with liquid, such as water prior to testing. In an embodiment of the invention the liquid sample is tested in several dilutions and e.g. in undiluted condition for improving the accuracy of the determination.
In an embodiment the liquid sample comprises a biological fluid or a fraction of a biological fluid. Examples of such biological fluids include human or animal or vegetable fluids, such as blood, saliva, urine, milk, cytosol (intracellular fluid), interstitial fluid (tissue fluid) and/or one or more fractions and/or mixtures thereof.
The term “a target component” means one or more molecules of a specific type of components. The term “two or more or several target components” means two or more or several different types of target components”
A type of components comprises components which can be captured by a specific capture site on the magnetic particles. The components included in a type of components can be identical components or it can be components with a certain similarity including that they can be captured by a specific capture site on the magnetic particles.
In the following the term “target component” is for simplification mainly used in singular, but it should also include the plural version of the term “target components” unless otherwise specified.
The term “target component” and “target analytes” are used interchangeably.
The target component can in principle be any kind of target component which can be determined in a binding assay. The skilled person can in a simple manner use knowledge from other types of binding assays to select suitable target components and corresponding capture sites to be provided on the magnetic particles.
In the following it should be understood that the method for quantitative and/or qualitative determination can be performed on one or more target components simultaneously, and unless otherwise specified the singular term ‘target component’ should be interpreted to also include the plural term ‘target components’.
In an embodiment the target component is a biomolecule, such as a single organic molecule or a structure of organic molecules e.g. an organic organism. Since there are high needs in the industry e.g. the health care industry, the food industry, the method of the invention is highly suitable for use in quantitative and/or qualitative determinations of biomolecules, in particular because the method of the invention is both very fast and highly reliable.
In an embodiment of the invention, the target component may for example be a mutant variant of a molecule or an organic organism, such as a microorganism.
In an embodiment, the target component is or comprises a microorganism such as at least one of bacterial, viral or fungal pathogens, e.g. E-coli E. coli, Citrobacter spp, Aeromonas spp., Pasteurella spp., non-serogroup DI Salmonella, Camphylobacter Staphylococcus spp and combinations thereof.
In an embodiment, the target component is or comprises a cell, such as a blood cell, a stem cell or a tumor cell.
In an embodiment, the target component is or comprises proteins, nucleotides, carbohydrates, or lipids, in particular an enzyme, an antigen or an antibody.
In an embodiment the target component is or comprises a “hapten”. A hapten is a small molecule that can elicit an immune response only when attached to a large carrier such as a protein; the carrier may be one that does not elicit an immune response by itself. The hapten may for example be a steroid, a hormone, an antibiotic or an inorganic constituent.
The skilled person will realize that the target component can be any kind of component for which a capture cite can be provided.
Target components and corresponding capture sites are well known in the art. Also it is well known to immobilize such capture sites on a surface such as magnetic particles.
Examples of capture sites and magnetic particles comprising such capture sites for microorganism target component can for example be found in WO2012016107.
The capture sites are selected in relation to at least one target component, such that the capture sites bind to at least one target component.
In an embodiment of the method of the invention, the magnetic particles are coated magnetic particles comprising a coating comprising the captures sites, wherein the capture sites are selected to be capture sites for the target component, the coating for example comprises the capture sites in the form of antigen, antibody, avidine, biotin or Goat anti-Mouse IgG.
In an embodiment the capture sites are specific for the target component.
If there are two or more target components there may be several groups of capture sites or there may be one type of capture sites that are capture sites for all target components.
In an embodiment where there are two or more target components, the capture sites are specific for the two or more target components e.g. for a group of similar but not identical target components.
In an embodiment of the invention, the capture sites are specific for a group of components comprising one or more target components.
In an embodiment the capture sites are for example binding sites for protein, the protein content can thereby be determined. In an embodiment the capture sites are binding sites for a pre-selected type or group of proteins.
In an embodiment the binding sites are binding sites for haptens such as small organic molecules e.g. steroid hormones, antibiotics or even other hapten molecules of other origin.
The magnetic particles may in principle have any size which is suitable for handling in the micro fluidic device.
The use of magnetic particle technology in quantitative and qualitative biological tests, particularly antibody-coated magnetic beads and immunomagnetic beads have become widely used and magnetic particles for such test are commercially available in many forms.
Methods for constructing immunomagnetic particles are generally known in the art (e.g. Safarik, I. and Safarikova, M. “Magnetic techniques for the isolation and purification of proteins and peptides.: BioMagn. Res. Technol. 2 (2004)).
The magnetic particles are preferably of micro or nano size. Preferably the magnetic particles have an average size of up to about 50 μm, more preferably up to about 25 μm, such as from about 1 to about 20 μm.
The magnetic particles may be spherical or non-spherical. Some examples of magnetic particles include Cortex Megacell™-Streptavidin magnetic particles, Cortex Megabeads™-Streptavidin CM3454 (8.8 μm particle size and coated with magnetizable polystyrene/iron oxide particles), Cortex Megabeads™-Streptavidin CTM-C M019 (15.6 μm particle size and coated with polystyrene copolymer/iron oxide particles), Dynabeads™ M-280-Streptavidin (3-4 μm particle size), and Genpoint BugTrap™ magnetic beads.
Other examples of suitable magnetic particles are the magnetic particles available from Spherotech, Inc. US. Ademtech, France including for example smaller size magnetic beads size of 200-500 nm, i.e. functionalized with streptavidin, protein A or/and G plus a number of different antibodies. Product 03152 MasterBeads StreptAvidin (Mean Diameter: ˜500 nm); product 03231 Bio-Adembeads StreptAvidin plus (Mean Diameter: ˜300 nm); product 03221 Bio-Adembeads streptavidin plus product (Mean Diameter: ˜200 nm); 02650 Active-Masterbeads (Mean Diameter: ˜500 nm) for direct protein conjugation.
Banglabs Inc, US, BM549 BioMag® Goat anti-Mouse IgG (Mean Diameter: ˜1.5 μm); BM551/10272 BioMag® Streptavidin (Mean Diameter: ˜1.5 μm); BM553/9750 BioMag® Protein G (Mean Diameter: ˜1.5 μm); PMS3N/10098 ProMag™ 3 Series●Streptavidin (Mean Diameter: ˜3.28 μm); CM01N COMPEL™ Magnetic Streptavidin modified (Mean Diameter: ˜8 μm).
Fluorophores are well known in the art and are widely used within the technology of quantitative and qualitative assays.
A fluorophore (also called a fluorochrome or a florescent chromophore) is a molecule which can be excited by absorbing light energy and re-emits energy at a specific wavelength. The wavelength, amount, and time before emission of the emitted energy depend on both the fluorophore and its chemical environment as the molecule in its excited state may interact with surrounding molecules.
The excitation energy may be a very narrow or a broader band of energy, or it may be all energies beyond a cut-off level. The emission energy and wavelength is usually more specific than the excitation energy, and it is usually of a longer wavelength or lower energy. Excitation energies range from ultraviolet through the visible spectrum, and emission energies may continue from visible light into the near infrared region.
Generally it is desired to select fluorophores with a relatively specific emission wavelength and energy for a simpler qualitative or quantitative determination of the target component. In particular it is desired that the emission wavelength is relatively specific, i.e. it should preferably have a wavelength band which in the method of determination is sufficiently narrow to be distinguished from other emissions.
The term “relative specific wavelength” means that the wavelength can be distinguished from other emitting wavelengths in the test.
In particular in situations where there are several different fluorophores and optionally several target components it is preferred that the fluorophores have relatively specific emission wavelengths such that emission from the respective fluorophores can be distinguished from each other.
The fluorophores can be any type of fluorophores which can be configured to bind to the capture sites of the magnetic particles. Fluorophores are well known to the skilled person and are commercially available.
Examples of quantum dots are described in U.S. Pat. No. 7,498,177 and the quantum dots available from Life Technologies Europe BV. include more than 150 different product configurations with emission wavelength spanning in a broad wavelength range for examples quantum dots with the respective emission wavelengths: 525, 545, 565, 585, 605, 625, 655 and IR 705 and 800 nm. In an embodiment StreptAvidin, Biotin, antibodies and a number of different functionalities have been conjugated in the Invitrogen/life Technologies portfolio of Quantum dot products.
Examples of quantum dots also include quantum dots available from Ocean NanoTech, Springdale, Ark. 72764, including more than 40 different product configurations with emission wavelength spanning in nm and a functionalized outer core of PEG or other biological compatible coating, for example with the respective emission wavelengths: 530, 550, 580, 590, 600, 610, 620 and 630 nm. The quantum dots from Ocean NanoTech include quantum dots with different functional groups e.g. amine, COOH, phenylboronic acid (PBA), as well as quantum dots with amphiphilic polymer and PEG coating. Other examples of quantum dots available from Ocean NanoTech are quantum dots with a sole core e.g. provided in toluene and with only an octadecylamine coat or with amphiphilic polymer and PEG coating.
In an embodiment the fluorophores are quantum dots or aromatic probes and/or conjugated probes, such as fluorescein, derivatives of benzene, metal-chalcogenide fluorophores or combinations thereof.
The fluorophores are preferably configured to bind to the capture sites of the magnetic particles by being coupled to a component which can bind to the capture sites of the magnetic particles.
In an embodiment the component is identical to the target component. In most situations it is most simple to configure the fluorophores to bind to the capture sites by coupling the fluorophores to a component which is identical to a target component.
In an embodiment the component is homolog to the target component. If for example the target component is a pathogen, an expensive or rare component or in certain other situations it may be very beneficial to configure the fluorophores to bind to the capture sites by coupling the fluorophores to a target component homolog.
The term “a component homolog to a target component” means herein that the component should have a homology to the target component such that at least some of the homolog components will bind to the capture sites of the magnetic particles when applied in a competitive assay of equal molar amount of the target component and the homolog component.
In a preferred embodiment of the invention the fluorophores are quantum dots that emit one or more discrete frequencies of light when stimulated by a light source. In this embodiment several different quantum dots can be excited with the same wavelength or at least with a light beam having a relatively small band width.
Preferably each quantum dot comprises a core of an excitable material, such as a semiconductor nanoparticle or a rare earth doped oxide colloidal nanoparticle.
In an embodiment, the quantum dots comprise each a core with a size of up to about 25 nm, such as from 2-10 nm. The quantum dots preferably are coated with an organic coating, such as a polymer coating. Preferably the coating is coupled to a component which can bind to the capture sites of the magnetic particles e.g. such as described above.
In an embodiment of the invention, each of the quantum dots comprises a core of a binary semiconductor alloy, such as cadmium-selenide, cadmium-sulphide, indium-arsenide or indium-phosphide, covered with a transparent shell optionally comprising or consisting of Zinc sulphide.
The liquid sample, one or more of the fluorophores and one or more of the magnetic particles may in principle be brought into contact with each other in any order as well as outside the micro fluidic device or in the flow channel of the micro fluidic device.
In an embodiment the liquid sample is brought into contact with one or more of said fluorophores and one or more of said magnetic particles outside the flow channel of the micro fluidic device and thereafter the liquid sample is fed to the flow channel of the micro fluidic device.
In an embodiment of the invention, the liquid sample is brought into contact with one or more of the fluorophores and one or more of the magnetic particles outside the micro fluidic device in the form of a micro fluidic device and thereafter the liquid sample is fed to a flow channel of the micro fluidic device.
In an embodiment the magnetic particles and the liquid sample are brought into contact with each other e.g. in a syringe or a secondary test tube prior to application of the liquid sample in the flow channel of the micro fluidic device, where after the liquid sample with the magnetic particles is fed into the flow channel of the micro fluidic device and the liquid sample is mixed with the fluorophores in the flow channel of the micro fluidic device. In this situation the target component—if present in a sufficient amount—will be captured on essentially all capture sites of the magnetic particles and there will not be any or almost none detectable emission from fluorophores. If on the other hand a large emission signal is detected, it can be concluded that the target component is not present in the liquid sample.
In an embodiment the fluorophores and the liquid sample are brought into contact with each other e.g. in a syringe or a test tube prior to application of the liquid sample into the flow channel of the micro fluidic device and the liquid sample is mixed with the magnetic particles in the flow channel of the micro fluidic device. In this situation the target component—if present—will compete with the fluorophores about the capture sites of the magnetic particles and the detected emission signal will provide information about the amount of the target component.
In an embodiment the fluorophores, the magnetic particles and the liquid sample are brought into contact with each other e.g. in a syringe or a test tube prior to application of the liquid sample into the flow channel of the micro fluidic device and the liquid sample is fed to the flow channel of the micro fluidic device for determination. In this situation the target component—if present—will compete with the fluorophores about the capture sites of the magnetic particles and the reactions (competition) will initiate outside the micro fluidic device, and no further mixing or reaction time (incubation) in the flow channel of the micro fluidic device may be required.
In a preferred embodiment the liquid sample is brought into contact with one or more of the fluorophores and the magnetic particles in the flow channel of the micro fluidic device.
In an embodiment the fluorophores and the magnetic particles are arranged in the flow channel of the micro fluidic device, and the method comprises feeding the liquid sample into the flow channel, the fluorophores and the magnetic particles are preferably arranged in the flow channel at a distance from each other.
In an embodiment the fluorophores and the magnetic particles are arranged in the flow channel of the micro fluidic device and the method of the invention comprises feeding the liquid sample into the flow channel, the fluorophores and the magnetic particles are preferably arranged in the flow channel at a distance from each other.
The fluorophores and the magnetic particles should preferably be applied in the flow channel of the micro fluidic device such that the capture sites of the magnetic particles do not capture any substantial amount of fluorophores prior to intermixing with the liquid sample. The fluorophores and the magnetic particles may be applied by any method e.g. by drying out in sections of the flow channel. In an embodiment the fluorophores and/or the magnetic particles are applied by producing the micro fluidic device in a substrate with a groove for the flow channel and a lid and the fluorophores and/or the magnetic particles are applied prior to adding the lid to the micro fluidic device.
In an embodiment the fluorophores and the magnetic particles are temporally immobilized in the flow channel of the micro fluidic device of the micro fluidic device such that they cannot bind to each other prior to the feeding of the liquid sample to the flow channel.
It is well known to temporally immobilize components in micro fluidic devices and any of such well known methods can be applied in the method of the invention.
The magnetic particles may for example be temporally immobilized by magnetic forces.
In an embodiment the fluorophores and/or the magnetic particles are temporally immobilized by being dried in the flow channel.
When the liquid sample comes in contact with the temporally immobilized fluorophores and/or the temporally immobilized magnetic particles the fluorophores and/or the magnetic particles are resuspended. The term “resuspended” is herein used to mean that the fluorophores/magnetic particles are dissolved or suspended in the liquid.
In an embodiment the magnetic particles are permanently immobilized by magnetic forces and the liquid sample is forced to flow into contact with the magnetic particles. Since only a small amount of possible target components will come into reach of the capture sites of the magnetic particles in this embodiment, it is generally desired that the magnetic particles are free to intermix with the liquid sample, i.e. the magnetic particles should preferably not be permanently immobilized.
The liquid sample may be fed into the flow channel by any method and means, e.g. by pipetting, by using a syringe, by dripping into the inlet or by being sucked into the flow channel of the micro fluidic device in the form of a fluidic device.
In an embodiment of the invention the liquid sample is fed into the flow channel of the micro fluidic device by being sucked into the flow channel, the suction is provided by an actuator. The actuator is advantageously arranged to move a flexible wall section of the flow channel or of a sink section in fluid connection with the flow channel.
The actuator may preferably be arranged to move the flexible wall section of the flow channel or the sink section to provide a suction to suck the liquid sample into the flow channel. The flow channel preferably comprises a first feeding end and a second actuator end comprising the flexible wall section. In an embodiment the flow channel in combination with a sink section in fluid connection comprises a first feeding end and a second actuator end comprising the flexible wall section, where the second actuator end is a part of or all of the sink section.
In order to obtain an accurate quantitative determination, the method preferably comprises allowing the capture sites of the magnetic particles to capture possible target component in the liquid sample and/or fluorophores. The reaction time is usually very short e.g. from seconds to a few minutes, such as about 10 minutes or less. The reaction time is preferably about 1 minute or less. In a preferred embodiment, the method comprises stirring and/or pulsating the liquid sample in the flow channel. Such pulsation can for example be provided using an actuator. After a certain pre-selected reaction time the magnetic particles are at least temporally immobilized adjacent to the transparent window using a magnet.
In an embodiment where the magnet is a permanent magnet, the magnetic particles will immediately be attracted to the magnet after the sample is introduced into the flow channel. If the magnetic force is of a suitable strength e.g. as described below, the magnetic particles will be pulled towards the transparent window at a suitable speed such that the reaction with possible target components and fluorophores will have taken place before immobilization for at least a part of the capture sites of the magnetic particles and preferably for most or substantially all of the capture sites. If the test is a quantitative test, the reaction time can usually be shorter than when the test is a qualitative test.
The transparent window is advantageously a window of the flow channel of the micro fluidic device.
The term “transparent” means herein that the window is transparent for the excitation and emission wavelengths of the fluorophores. Accordingly the windows need not be transparent for visual inspection, however, generally it is desired that the window or preferably the whole flow channel is transparent for visual inspection.
Generally it is known to produce micro fluidic devices and examples of general production methods and materials can be found in e.g. US 2010/0254858 and EP 1 827 693.
In an embodiment a wall section or the whole wall of the flow channel is preferably transparent.
Generally it is desired that the magnet has a magnetic field sufficiently strong to at least temporally immobilize the magnetic particles adjacent to the transparent window.
The magnet may in principle be any type of magnet with a suitable strength. In an embodiment the magnet is selected to generate a magnetic field adjacent to the transparent window for immobilizing the magnetic particles, which magnet field is from about 0.05 to about 1 tesla, such as from about 0.1 to about 0.5 tesla, such as from about 0.15 to about 0.3 tesla.
The magnetic field need not be homogeneous. In an embodiment the magnet provides a magnetic field of from about 0.2 to about 0.3 tesla in a distance of about 1 mm and a magnetic field of from about 0.01 to about 0.2 tesla in a distance of about 2.5 mm. In an embodiment the magnet provides a magnetic field in a distance of 2.5 mm which is about from ⅓ to ½ the strength of the magnetic field in a distance of about 1 mm.
If the magnet is undesirably strong it may provide a too fast immobilization of the magnetic particles i.e. the capture sites of the magnetic particles may not have suitable time to bind target component or fluorophores prior to immobilization of the magnetic particles. This is of course only relevant if the magnet is a permanent magnet. If the magnet is undesirably weak, it may not be able to immobilize a sufficient amount of magnetic particles. By a few tests the skilled person can find a suitable magnet strength adapted to a specific method of the invention.
The magnet may preferably be a permanent magnet, for the reason of simplification and low cost. However, in an embodiment an electromagnet, such as an adjustable electromagnet may be suitable e.g. if magnetic forces are applied in the mixing of the liquid sample with the fluorophores and/or the magnetic particles.
The magnet may be movable or stationary depending on the setup for performing the method of the invention. For a simple structure it is desired that the magnet is stationary arranged to immobilize the magnetic particles for excitation and read out adjacent to the transparent window.
In a preferred embodiment the at least temporally immobilized magnetic particles are subjected to the electromagnetic beam such that at least a part of possible fluorophores captured by the capture sites of the magnetic particles are excited, where after the emitted signal from the possibly captured fluorophores are read and a quantitative or qualitative determination of the target component based on the read signal is performed.
It has been found that where the fluid in the flow channel after the magnetic particles are immobilized using the magnet is at standstill, the at least temporally immobilized magnetic particles remain immobilized at least for a time such as up to several minutes after releasing the magnetic particles from influence of the magnetic force of the magnet. Thereby the magnet can be removed from the transparent window and making room for an emitter to excite the fluorophores and a reader to read out any signal from the fluorophores. Thereby the emitter and/or reader can be positioned where the magnet previously was positioned which has shown to provide extremely reliable results. In an embodiment the emitter is arranged to emit the electromagnetic beam via one or more emitting optical fibers comprising output ends arranged immediately adjacent to the transparent window (e.g. where the magnet was arranged when immobilizing the magnetic particles) In an embodiment the receiver is arranged to receive the signal from the fluorophores via one or more receiver optical fibers comprising input ends arranged immediately adjacent to the transparent window (e.g. where the magnet was arranged when immobilizing the magnetic particles).
The output ends of the emitting optical fibers and the input ends of the receiver optical fibers are advantageously arranged in a pattern. In an embodiment the output ends of the emitting optical fibers are arranged in a circle surrounding the input ends of the receiver optical fibers. In an embodiment the input ends of the receiver optical fibers are arranged in a circle surrounding the output ends of the emitting optical fibers. In an embodiment one or more lenses are arranged to collect the signal and direct it to the input ends of the receiver optical fibers.
Methods of excitation of fluorophores are well known in the art. The exciting wavelength is preferably adjusted to the excitation peak of the fluorophores. In an embodiment the excitation light is a relative band emission and preferably relatively low energy, such that the excitation light does not result in an undesired heating of the liquid sample or the elements therein.
In an embodiment the micro fluidic device is kept at a controlled temperature to optimize the bonding and ensuring that an undesired temperature does not interfere with the binding assay.
In an embodiment of the invention, the plurality of fluorophores is substantially identical.
If only one target component is to be determined, it is often desired that the fluorophores applied are substantially identical with respect to excitation and emitting, however, it should be understood that one target component can in principle be detected using different types of fluorophores in the same test.
In a preferred embodiment of the invention, the plurality of fluorophores comprises two or more groups of fluorophores, wherein the two or more groups of fluorophores differ from each other with respect to types, sizes, coatings, shape and/or amounts.
If two or more groups of fluorophores are present they may in principle be selected independently of each other, but preferably such that they emit at different wavelengths. In this situation it is particularly preferred to apply quantum dots as fluorophores, because quantum dots at different sizes emit at different wavelengths while they are excited at substantially the same wavelength.
The amount of fluorophores is preferably selected to provide an estimated competition for possible target components on the liquid sample. The amount of fluorophores can be determined by a few tests. A possible outset for selecting the amount of fluorophores is to select an amount of about 0.02 to about 100 times the amount which corresponds to the maximal estimated amount of target component in the liquid sample, such as an amount of about 1 to about 50 times or an amount of about 10 to about 50 times the amount which corresponds to the maximal estimated amount of target component in the liquid sample. For improved accuracy of the result, it is often desired to repeat the determination of a target component in a liquid sample using different amounts of fluorophores.
The amount of fluorephore may for example vary from about 0.02 to about 50 nM (nano mol), preferably from about 0.1 to about 10 nM.
The magnetic particles may be equal or different from each other. In an embodiment the magnetic particles are substantially identical with respect to capture sites and optionally with respect to number of capture sites and/or size.
In an embodiment the plurality of magnetic particles comprises two or more groups of magnetic particles, wherein the two or more groups of magnetic particles differ from each other with respect to e.g. with respect to size, capture sites number and/or type.
In an embodiment of the invention where the method comprises quantitative or qualitative determination of two or more target components in a liquid sample, the magnetic particle comprises one or more types of capture sites for the two or more target components, the capture sites for one target component preferably differs from the capture sites for another target component. For example one group of magnetic particles can comprise one type of capture sites and another group of magnetic particles can comprise another type of capture sites.
The plurality of fluorophores may e.g. comprise at least one group of fluorophores configured to bind to one capture site for one target component and at least another group of fluorophores configured to bind to the capture site for another target component.
By a few examples and based on the teaching herein the skilled person can find suitable fluorophores and magnetic particles for a given test according to the method of the invention.
In a preferred embodiment of the invention, the method comprises performing two or more parallel assays on the liquid sample for quantitative or qualitative determination of the target component(s), each assay comprises
bringing a part of the liquid sample into contact with the fluorophores and the magnetic particles in a micro fluidic device comprising a transparent window; and
at least temporally immobilizing the magnetic particles adjacent to the transparent window using a magnet, emitting exciting electromagnetic beam(s) towards the immobilized magnetic particles, reading signals emitted from fluorophores captured by the immobilized magnetic particles.
Preferably the fluorophores used in one of the two or more parallel assays differ from the fluorophores used in another one of the two or more parallel assays.
For example the fluorophores used in one of the two or more parallel assays differ from the fluorophores used in another one of the two or more parallel assays with respect to types, sizes, coatings, shape and/or amounts.
In an embodiment of the invention the magnetic particles used in one of the two or more parallel assays differ from the magnetic particles used in another one of the two or more parallel assays. For example the magnetic particles used in one of the two or more parallel assays differ from the magnetic particles in another one of the two or more parallel assays with respect to types, sizes, coatings, shape and/or amounts.
In an embodiment the two or more parallel assays are performed simultaneously in the same micro fluidic device. The two or more parallel assays may e.g. be performed in respective flow channels, such as in parallel flow channels of the same micro fluidic device.
In an embodiment of the invention, the quantitative or qualitative determination of target component(s) in a liquid sample is performed by comparing the read signal(s) with a reference schedule.
The reference schedule can be any type of reference schedule which can be applied for calibrating the read signal, for example such as it is generally known in the art.
In an embodiment of the invention, the quantitative or qualitative determination of target component(s) in a liquid sample is performed by comparing the read signal(s) with signals obtained from liquid samples with a known composition, e.g. by using an artificial intelligent processor.
In an embodiment of the invention, the quantitative or qualitative determination of target component(s) in a liquid sample is performed by multiplexing the read signal(s) from different groups of fluorophores e.g. from the same assay, from fluorophores from parallel assays and/or from fluorophores in reference tests of known or unknown liquid samples.
The invention also comprises a system for quantitative or qualitative determination of a target component in a liquid sample.
The system of the invention for quantitative or qualitative determination is specifically suitable for use for performing the method of the invention, and accordingly the system for quantitative or qualitative determination and embodiments thereof has at least some of the above mentioned benefits.
The system for quantitative or qualitative determination of a target component in a liquid sample comprises
a micro fluidic device comprising at least one flow channel with a transparent window and an inlet for the liquid sample;
a plurality of magnetic particles comprising one or more capture sites for the target component on their respective surfaces;
a plurality of fluorophores configured to bind to the capture sites of the magnetic particles;
a magnet arranged to at least temporally immobilize the magnetic particle adjacent to the transparent window;
an emitter for exciting the fluorophores, and
a reader for reading signals emitted from the fluorophores.
Advantageously the micro fluidic device is as described elsewhere herein. The magnetic particles, the fluorophores and the magnet may e.g. be as described above.
In an embodiment the micro fluidic device is of polymer and or glass.
In an embodiment the micro fluidic device comprises a substrate with a groove for the flow channel and a foil covering the flow channel.
The micro fluidic device comprises preferably an excitation and read out zone which is also referred to as a reading zone and which is provided in the form of the transparent window, which is preferably transparent for at least the excitation and emitting wavelengths of the fluorophores.
In an embodiment the excitation and read out zone is identical to a zone where the magnet is positioned when it is acting on the magnetic particle to immobilize the magnetic particles. After the magnetic particles have been immobilized using the magnet and any liquid in the flow channel is at standstill, the magnet is removed while the magnetic particles remain immobilized at least for a sufficient time to excite the fluorophore and read the signal emitted from the fluorophore.
The emitter and the reader are advantageously a common emitter and reader unit.
In an embodiment the emitter comprises emitting optical fibers comprising output ends and the receiver comprises one or more receiver optical fibers comprising input ends. Advantageously fiber sections comprising the optical fiber output ends of the emitter and fiber sections comprising the optical fiber input ends of the reader are connected to each other to form a common emitting-reading fiber bundle. The output ends of the emitting optical fibers and the input ends of the receiver optical fibers are advantageously arranged in a pattern e.g. as described above.
The flow channel of the micro fluidic device may in principle have any shape. In an embodiment the flow channel comprises an elongate flow section and one or more chamber sections (chamber sections that have a substantially larger cross-section than the flow section). The fluorophores and the magnetic particles may for example be temporally immobilized in such chamber sections.
In an embodiment of the system of the invention for quantitative or qualitative determination the micro fluidic device is of polymer and or glass or a combination thereof. In a preferred embodiment the micro fluidic device is of polymer. The polymer micro fluidic device is easy and cost-effective to produce. The micro fluidic device preferably comprises a substrate with a groove for the flow channel and a foil covering the flow channel.
In an embodiment the micro fluidic device comprises an inlet to the flow channel. The inlet is for example an opening for suction, a capillary inlet or a membrane covered inlet.
In an embodiment of the invention the inlet of the micro fluidic device is a membrane covered inlet. In this embodiment the liquid sample can for example be introduced into the flow channel using a syringe or similar needle assisted device which can be used to penetrate the membrane. The membrane may e.g. simultaneously provide an escape for gas in the flow channel.
In an embodiment of the invention the inlet of the micro fluidic device is a capillary inlet, meaning that the liquid sample can be drawn into the flow channel using capillary forces. In this embodiment it is desired that the inner surfaces of the flow channel, in particular adjacent to the inlet, have a relatively high surface tension and have sufficiently small dimensions for providing the capillary forces. It is well known in the art to provide micro fluidic devices with a capillary inlet. Most polymers have a relatively low surface tension and often it is required to treat the surface of polymer micro fluidic device where the inner surfaces of the flow channel should provide capillary forces for a liquid e.g. an aqueous liquid.
In an embodiment of the invention the inlet of the micro fluidic device is an opening for suction, i.e. the micro fluidic device is configured such that the liquid sample is adapted to be sucked into the flow channel. In this embodiment the inner surfaces of the flow channel need not provide capillary forces and even when the micro fluidic device is of a material with a low surface tension, such surface need not be treated for increasing the surface tension. The micro fluid device with a suction inlet is therefore very simple to produce and can be provided at relatively low cost.
In an embodiment of the invention the micro fluidic device comprises an inlet for suction in the liquid sample. Advantageously the
micro fluidic device comprises a flexible wall section and the system can beneficially comprise an actuator, where the actuator is arranged to move the flexible wall section. The actuator is e.g. a step motor driven actuator.
In an embodiment of the invention where system comprises an actuator and where the inlet of the micro fluidic device comprises a flexible wall section and the inlet is an opening for suction, the inlet and the actuator are arranged such that the upon activation of the actuator, the flexible wall section will be moved and air will be pressed out of the flow channel where after the flexible wall will return to its initial position and the liquid sample will be sucked into the flow channel. Thereby a simple and effective suction of the liquid sample into the flow channel and a simple and effective mixing of the sample with the fluorophores and magnetic particles can be obtained.
In an embodiment of the invention the micro fluidic device comprises a sink section and the flow channel is in fluid communication with the sink section.
The sink section of the micro fluidic device is a section which is applied in a distance from the transparent window where the magnetic particle is at least temporally immobilized for excitation and read out. The sink section is in an embodiment applied to collect the sample or most of the sample during or after the test has been completed. By collecting the sample or most of the sample while simultaneously immobilizing the magnetic particles adjacent to the transparent window for excitation and read out, the risk of obtaining false signals due to fluorophores in the sample can be highly reduced.
Advantageously the sink section is positioned remotely to the inlet to the flow channel. Preferably the fluid introduced via the inlet must pass the transparent window where the magnetic particle is at least temporally immobilized for excitation and read out before the liquid reaches the sink section.
The terms “sink” and “sink section” are used interchangeably
The flow channel may have any shape and is preferably adapted for performing determinations on relatively small volumes of liquid sample, such as from about 1 μl to about 1 ml, preferably from about 5 μl to about 0.5 ml. The flow channel can have any shape e.g. with a cross sectional shape selected from round, ellipsoidal, semi ellipsoidal, quadrilateral polygonal, square, rectangular and trapezoidal shapes, where any edges optionally being rounded. In one embodiment the microfluidic device comprises two or more distinct flow channel sections, e.g. a channel section for mixing the liquid sample with the fluorophores and/or the magnetic particles and a channel section with a transparent window for at least temporally immobilizing the magnetic particles for excitation of optional captured fluorophores and for reading out possible emission energy.
In an embodiment of the invention the microfluidic device comprises at least one gas escape opening for allowing gas to escape from the flow channel. The gas escape opening may be of any type and shape e.g. as known from prior art microfluidic devices. The gas escape opening may for example be arranged to allow gas to escape completely out of the microfluidic device or it may allow the gas to escape into a gas collecting chamber e.g. in the form of an inflatable unit.
In an embodiment of the invention the microfluidic device comprises a flexible wall section which can be used to create a suction at the inlet of the flow channel. In this embodiment the inlet can function as a gas escape opening.
In an embodiment the micro fluidic device comprises two or more flow channels, and the two or more flow channels comprise a common section or are in fluid connection with a common sink section, where the common section or the common sink section comprises a flexible wall section which can be used to create a suction at the inlets of the flow channels.
In an embodiment the micro fluidic device comprises two or more flow channels, and the two or more flow channels or one or more sink sections in fluid connection comprise each a flexible wall section which can be used to create a suction at the respective inlets of the flow channels. In this embodiment suction can be applied individually in the respective flow channels. The flexible wall section is in an embodiment applied as a wall section of a sink section where the sample or parts thereof can be collected after performing the test. The sink section is e.g. as described above.
In an embodiment the flexible wall section is used to pump out the sample after the magnetic particles have been mixed with the sample and the fluorophores while simultaneously immobilizing the magnetic particles adjacent to the transparent window for excitation and read out, thereby reducing the risk of obtaining false signals due to fluorophores remaining in the sample.
In an embodiment where the micro fluidic device comprises a flow channel in fluid connection with a sink for collecting sample and optionally a flexible wall suitable for pumping the sample into and out of the sink, a sample modifier such as a surfactant is applied in the sink. When the sample is pumped into the sink using the flexible wall section or other pumping means the sample will be intermixed with the surfactant and accordingly the surface tension of the sample is reduced. By reintroducing the sample into the area of the flow channel comprising the transparent window, the magnetic particles immobilized adjacent to the transparent window will be washed with the modified sample. The modifier applied in the sink may e.g. be in a dry form such that it will not be mixed with the sample until the sample is introduced into the sink.
In an embodiment the flexible wall section is used to pump out the sample after termination of the test. Whether it is preferred to collect the sample in the sink section, to pump out the sample or to let the sample be distributed in the flow channel depends largely on the kind and the toxicity of the sample applied. If for example there is risk of undesired contamination or if the sample potentially comprises elements that are undesirable to spread e.g. bacteria, viruses or similar, it may be desired to collect the sample in the sink section during or after performing the test.
In an embodiment the flow channel of the micro fluidic device comprises one or more chambers, e.g. for mixing the liquid sample with the fluorophores and/or the magnetic. In general it is desired that the flow channel comprises at least one liquid flow channel section which has at least one dimension (often the width dimension) of at least about 100 μm, such as at least 500 μm. In practice it can be as wide as handling will allow. The other dimension(s), e.g. the depth of the channel, is preferably smaller than the width, such as half the width or e.g. down to about 25 μm or down to about 10 μm, if desired.
In this context a chamber of a flow channel means a subsection of the flow channel that has at least 25%, such as at least 50%, larger cross-sectional area than an adjacent section of the flow channel. The chamber may for example have a larger cross sectional area than an adjacent channel section by being wider. The depth of the flow channel may be substantially constant or it may vary.
In an embodiment of the system of the invention for quantitative or qualitative determination, the micro fluidic device comprises an excitation and read out zone in the form of a length section comprising the transparent window, the window is transparent for at least the exciting and emitting wavelengths of the fluorophores.
In an embodiment of the system of the invention for quantitative or qualitative determination, the micro fluidic device comprises an excitation and read out section that has a length dimension of at least about 1 mm, such as at least about 3 mm, such as at least about 5 mm.
The read out section is preferably formed as a narrowed part of the flow channel or as an expanded part of the flow channel in order to provide a simple positioning of the micro fluidic device in relation to the emitter and reader.
According to the system of the invention at least the window of the micro fluidic device is of a transparent material.
In an embodiment the whole flow channel is visible due to transparency of the material. In an embodiment the whole micro fluidic device is of a transparent material
In a preferred embodiment at least the transparent window is transparent to the exciting wavelength(s) and emitting wavelength(s) of the fluorophores. In an embodiment at least the transparent window is transparent to at least one wavelength selected from Infrared light (about 700 nm to about 1000 μm), visible light (about 400 nm to about 700 nm), UV light (about 400 nm to about 10 nm) about and X-ray light (about 10 nm to about 0.01 nm).
It is in an embodiment desired to apply short wave light for the determination, i.e. the fluorophores are preferably excitable by short wavelength energy where the heat generation is relatively small and will not interfere with the determination.
Examples of materials which may be used for the micro fluidic device comprise materials selected from glass and polymer, preferably polymers selected from cyclic olefin copolymers (COC), acrylonitrile-butadiene-styrene copolymer, polycarbonate, polydimethyl-siloxane (PDMS), polyethylene (PE), polymethylmethacrylate (PMMA), polymethylpentene, polypropylene, polystyrene, polysulfone, polytetra-fluoroethylene (PTFE), polyurethane (PU), polyvinylchloride (PVC), polyvinylidene chloride (PVDC), polyvinylidine fluoride, styrene-acryl copolymers polyisoprene, polybutadiene, polychloroprene, polyisobutylene, poly(styrene-butadiene-styrene), silicones, epoxy resins, Poly ether block amide, polyester, acrylonitrile butadiene styrene (ABS), acrylic, celluloid, cellulose acetate, ethylene-vinyl acetate (EVA), ethylene vinyl alcohol (EVAL), fluoroplastics, polyacetal (POM), polyacrylates (acrylic), polyacrylonitrile (PAN) polyamide (PA), polyamide-imide (PAI), polyaryletherketone (PAEK), polybutadiene (PBD), polybutylene (PB), polybutylene terephthalate (PBT), polyethylene terephthalate (PET), polycyclohexylene dimethylene terephthalate (PCT), polyketone (PK), polyester/polythene/polyethene, polyetheretherketone (PEEK), polyetherimide (PEI), polyethersulfone (PES), polyethylenechlorinates (PEC), polyimide (PI), polylactic acid (PLA), polymethylpentene (PMP), polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polyphthalamide (PPA), and mixtures thereof.
In an embodiment the micro fluidic device is provided from a base part of a rigid material—e.g. produced by injection molding or by laser carving in a substrate. The base part is covered with a foil which is bonded to the base part to form the flow channel and optionally sink section.
In an embodiment of the invention the micro fluidic device comprises two or more flow channels optionally for performing parallel tests, the two or more flow channels optionally have a common inlet.
In an embodiment of the system of the invention for quantitative or qualitative determination, the system comprises a temperature regulator for regulating the temperature of the liquid sample in the flow channel. For some tests the reaction between possible target component and capture sites is temperature sensitive and accordingly it can be desirable to regulate the temperature. The temperature regulator can for example comprise a peltier element, a thin film heating element and/or other resistive heating elements.
In an embodiment of the system of the invention for quantitative or qualitative determination, the magnetic particles are coated magnetic particles comprising a coating comprising the captures sites, wherein the capture sites are selected to be capture sites for the target component, such as a biomolecule.
In an embodiment of the system of the invention for quantitative or qualitative determination, the fluorophores are quantum dots or aromatic probes and/or conjugated probes, the fluorophores are preferably quantum dots.
In an embodiment of the system of the invention for quantitative or qualitative determination, the fluorophores are configured to bind to the capture sites of the magnetic particles by being coupled to a component which can bind to the capture sites of the magnetic particles, the component is preferably identical or homolog to the target component.
In an embodiment of the system of the invention for quantitative or qualitative determination, the fluorophores and the magnetic particles are temporally immobilized in the flow channel of the micro fluidic device of the micro fluidic device such that they cannot bind to each other prior to the feeding of a liquid sample to the flow channel.
In an embodiment of the system of the invention for quantitative or qualitative determination, the magnet is arranged to at least temporally immobilize the magnetic particle adjacent to the transparent wall section for a sufficient time to excite at least a part of possible fluorophores captured by the capture sites of the magnetic particles by the emitter and to read out possibly emitted signal from possibly captured fluorophores.
Emitters and readers for electromagnetic waves are well known in the art, and when the fluorophores have been selected the skilled person will in a simple manner be able to select an emitter and reader that are useful in combination with the fluorophores.
In an embodiment of the system of the invention for quantitative or qualitative determination, the emitter is a light emitting diode or a laser which is capable of emitting electromagnetic radiation comprising the exciting wavelength of the fluorophores.
In an embodiment of the system of the invention for quantitative or qualitative determination, the emitter is configured to emit electromagnetic radiation directed at the transparent window, the window has a planar surface, the emitter is preferably configured to emit electromagnetic radiation directed at the transparent window with an angle to the surface of the window which is from about 20° to about 170°, such as from about 30° to about 150°. Preferably the surface of the window is substantially plane in order to optimize the exciting and emitting functions.
In an embodiment of the system of the invention for quantitative or qualitative determination, the reader is configured for reading signals emitted from fluorophores captured by magnetic particles which are temporally immobilized adjacent to the window.
The emitter and the reader are advantageously a common emitter and reader unit.
In an embodiment the emitter comprises emitting optical fibers comprising output ends and the receiver comprises one or more receiver optical fibers comprising input ends. Advantageously fiber sections comprising the optical fiber output ends of the emitter and fiber sections comprising the optical fiber input ends of the reader are connected to each other to form a common emitting-reading fiber bundle. The output ends of the emitting optical fibers and the input ends of the receiver optical fibers are advantageously arranged in a pattern e.g. as described above.
In an embodiment of the system of the invention for quantitative or qualitative determination, the system comprises a computer for performing the quantitative or qualitative determination of target component(s) in a liquid sample based on the read signal(s). The computer is preferably programmed to perform a quantitative and/or qualitative determination of the target component in the liquid sample based on the read signal.
In an embodiment the computer comprises a memory for storing of read signal(s) and/or quantitative or qualitative determinations performed.
In an embodiment the computer comprises a memory, which memory comprises a reference schedule for comparing the read signal(s) to perform the determination. The reference schedule may preferably comprise sets of a quantitative or qualitative determination with read signal(s), for example a set of data comprises a) the result(s) of read signal(s) for a liquid sample with a known content of the target component and b) the known content of the target component.
In an embodiment the computer is an artificial intelligent processor, programmed to compare read signal(s) with stored signals obtained from liquid samples with known compositions.
In an embodiment the system comprises a signal processor comprising the computer wherein the signal processor is configured to multiplex signals from different groups of fluorophores, from fluorophores from parallel assays and/or from fluorophores in reference tests of known or unknown liquid samples.
In an embodiment the system comprises a signal processor comprising the computer wherein the signal processor is configured to multiplex signals from different groups of fluorophores applied in same assay.
Multiplexing of signals is well known in the art and has also been applied in the art of analyzing test samples to quantify two or more targets labeled with fluorophores emitting different wavelengths. Reference is made to for example US 2009/0270269 and WO 2010/141105 and further information about multiplexing can also be found in “Luminescent quantum dots for multiplexed biological detection and imaging” Chan et al. Current Opinion in Biotechnology 2002, 13:40-46, Elsvier Science.
When performing the quantitative or qualitative determination using multiplexing it is preferred that the fluorophores applied are quantum dots. According to the invention it has been found that by using quantum dots as fluorophores and multiplexing the signals it is possible to quantitatively determine a plurality of target components simultaneously, e.g. 10 or more or even 50 or more.
The emitter, the reader and the signal processor comprising the computer are advantageously in form of a common or at least interconnected unit.
The invention also relates to a kit for preparing a liquid sample for being analyzed by the method of the invention as described above.
The kit of the invention is adapted for preparing a liquid sample for optical analysis for quantitative or qualitative determination of a plurality of target components in the sample. The kit comprises
a plurality of magnetic particles comprising a type of capture sites for each of the target components on their surfaces; and
a plurality of groups of fluorophores, each group of fluorophores is configured to bind to one of the types of capture sites of the magnetic particles.
The magnetic particles and the groups of fluorophores are as described above.
In an embodiment where the kit for preparing a liquid sample for optical analysis for quantitative or qualitative determination of N different target components in the sample, where N is an integer of 2 or more, the kit comprises
a plurality of magnetic particles comprising N types of capture sites, one type of capture sites for each of the target components; and
N groups of fluorophores, each group of fluorophores is configured to bind to one of the types of capture sites of the magnetic particles.
In an embodiment where the kit for preparing a liquid sample for optical analysis for quantitative or qualitative determination of N different target components in the sample, where N is an integer of 2 or more, the kit comprises
N groups of magnetic particles, each group of magnetic particles comprises one type of capture sites for one target component; and
N groups of fluorophores, each group of fluorophores is configured to bind to one of the types of capture sites of the magnetic particles;
In an embodiment two or more groups of fluorophores are provided in one single solution. The magnetic particles are advantageously provided in the form of one solution or suspension for simple handling.
The two or more groups of fluorophores are as described above and preferably the two or more groups of fluorophores are quantum dots capable of being excited by electromagnetic waves of the same wavelength.
In an embodiment the kit further comprises a micro fluidic device and/or a magnet. The micro fluidic device and/or magnet can for example be as described above.
In principle N can be as high as the number available of different fluorophores such as quantum dots. In an embodiment N is an integer from 2 to 10.
The invention also relates to a micro fluidic device for use in preparing a liquid sample for optical analysis for quantitative or qualitative determination of a of target component in the sample. The micro fluidic device comprises at least one flow channel with a transparent window and an inlet for the liquid sample, the micro fluidic device further comprises in its flow channel
a plurality of magnetic particles comprising capture sites for the target component on their surfaces; and
a plurality of fluorophores configured to bind the capture sites of the magnetic particles.
The micro fluidic device is preferably a micro fluidic device as described above.
The invention further relates to a micro fluidic device for use in preparing a liquid sample for optical analysis for quantitative or qualitative determination of a of target component in the sample which micro fluidic device comprises a substrate with a groove for a flow channel and a foil covering the flow channel, the flow channel comprises a transparent window and an inlet for suction in the liquid sample. The micro fluidic device comprises a flexible wall section of the flow channel or of a sink section in fluid connection with the flow channel. The flexible wall section can be moved such that air will be pressed out of the flow channel where after the flexible wall will return to its initial position.
In an embodiment of the invention the micro fluidic device comprises a sink section and the flow channel is in fluid communication with the sink section.
The sink section of the micro fluidic device is a section which is applied in a distance from the transparent window where the magnetic particle is at least temporally immobilized for excitation and read out. The sink section is in an embodiment applied to collect the sample or most of the sample during or after the test has been completed. Advantageously the sink section is positioned remotely to the inlet to the flow channel.
Further preferred embodiments of the micro fluidic device are as described above.
In an further aspect a modification of the invention relates to a sandwich-type assay for quantitative or qualitative determination of a target component in a liquid sample. The assay comprises providing a plurality of magnetic particles comprising one or more capture sites for the target component on their respective surfaces;
providing a plurality of fluorophores comprising one or more capture sites for the target component;
bringing the liquid sample, said fluorophores and said magnetic particles into a flow channel of a micro fluidic device comprising a transparent window into the flow channel; and
at least temporally immobilizing said magnetic particles adjacent to said transparent window using a magnet, emitting exciting electromagnetic beam towards said immobilized magnetic particles, reading signals emitted from fluorophores captured by said immobilized magnetic particles via said target components and performing a quantitative or qualitative determination of said target component based on the read signal.
The magnetic particles are advantageously as described above.
The fluorophores are advantageously as described above with the modification that the fluorophores are not configured to bind to the capture sites of the magnetic particle, but instead the fluorophores comprise one or more capture sites for the target component.
The liquid sample, the fluorophores and the magnetic are advantageously brought into contact using the methods described above.
The micro fluidic device is advantageously as described above.
The at least temporally immobilizing of the magnetic particles, the exciting and read out are advantageously as described above.
The sandwich-type assay is specifically advantageous to use where the target component is a relatively large component comprising two or more capture sites such that it can be sandwiched between the magnetic particle and the fluorophore.
It should be emphasized that the term “comprises/comprising” when used herein is to be interpreted as an open term, i.e. it should be taken to specify the presence of specifically stated feature(s), such as element(s), unit(s), integer(s), step(s) component(s) and combination(s) thereof, but does not preclude the presence or addition of one or more other stated features.
All features of the inventions including ranges and preferred ranges can be combined in various ways within the scope of the invention, unless there are specific reasons for not combining such features.
BRIEF DESCRIPTION OF DRAWINGS AND EXAMPLES
The invention will be explained more fully below in connection with examples and preferred embodiments and with reference to the drawings in which:
FIG. 1a is a schematic top view of a micro titer plate suitable for performing the method of the invention.
FIG. 1b is a schematic cross sectional view seen in the line A-A′ of FIG. 1.
FIG. 2 is a schematic top view of a micro fluidic device suitable for performing the method of the invention.
FIG. 3 is a schematic sectional side view seen in the line B-B′ of FIG. 2.
FIG. 4 is a schematic top view of micro fluidic device suitable for performing the method of the invention and with temporally immobilized magnetic particles and temporally immobilized fluorophores.
FIG. 5 is a schematic illustration of the system of the invention comprising a micro fluidic device, an emitter and a reader.
FIG. 6 is a schematic illustration of a fluorophore in the form of a quantum dot suitable for use in the invention.
FIGS. 7a, 7b and 7c are schematic illustrations of a performance of the method of the invention.
FIGS. 8a, 8b and 8c are schematic illustrations of another performance of the method of the invention.
The figures are schematic and may be simplified for clarity. Throughout, the same reference numerals are used for identical or corresponding parts.
FIGS. 1a and 1b show a test plate suitable for being applied in the present invention. The shown test plate is a micro titer plate with 12×8 wells 1.
FIG. 9 is a schematic side view of an emitter-reader assembly.
Micro titer plates are well known in the art under many names, such as well plates and micro plates. A micro titer plate is a generally flat plate with multiple “wells” used as small test tubes. The shown micro titer plate comprises a thin cover film 2, which is peeled of prior to use of the titer plate. The cover film 2 can be divided into sections, such that it can be peeled off in sections, e.g. such that only one or only a number less than all wells are uncovered by removal of a section of the cover film 2. The micro titer plate has an edge 4 for reducing spill.
Each well 1 of a micro plate typically holds somewhere between tens of nanoliters to several milliliters of liquid. Wells of a suitable micro titer plate can in principle have any shape, such as circular or square, and their respective bottom parts can be rounded or plane. In the shown micro titer plate, the wells 1 are round and with plane bottom parts 3. The round bottom parts 2 of the respective wells 1 constitute the transparent window usable for exciting and reading out. In use the fluorophores and the magnetic particles can be pre-arranged in the wells e.g. in dry form and e.g. in temporally immobilized form. Alternatively the fluorophores and the magnetic particles can be added to the well immediately before, simultaneously with or after adding the liquid sample. After a selected incubating time e. g. on a shaking board, the micro titer plate is placed on a magnet for temporally immobilizing the magnetic particles adjacent to the transparent window, namely at the bottom part 3. An emitter is arranged to emitting exciting electromagnetic beam towards the immobilized magnetic particles, and a reader is arranged to read signals emitted from fluorophores captured by the immobilized magnetic particles. The read signals are used to perform a quantitative or qualitative determination of the target component. For reducing noise, the liquid can be removed from the respective wells, and optionally the wells are washed e.g. with water prior to reading out signals. The incubating time is usually very short e.g. a few minutes.
FIGS. 2 and 3 show a test plate suitable for being applied in the present invention. The shown test plate is a micro fluidic device. Although any micro fluidic devices in principle could be applied in the present invention, the micro fluidic device shown is particularly designed for the purpose and provides additional benefits to the present invention as described herein.
The micro fluidic device comprises a substrate 12 with three flow channels 11. The channels 11 are provided in the form of grooves covered with a foil 11a. Each channel 11 comprises an inlet 13 and the channels 11 is in fluid connection with a common sink 14.
The inlet 13 is in the form of a well shaped inlet.
The common sink 14 of the micro fluidic device comprises a flexible wall section 15. The flexible wall section 15 can be moved e.g. using a not shown actuator as described above.
By pressing the flexible wall section 15 it will be moved and air will be pressed out of the channels 11 where after the flexible wall section 15 will return to its initial position and a liquid sample arranged in the inlet will be sucked into the channel to a desired position. By further manipulating the flexible wall section the liquid sample can be drawn further into the channels 11 or it can be pulsated in the channels. Finally the flexible wall section 15 can be manipulated to collect the sample in the sink and to reflush the sample into the channels, if desired. The flexible wall section 15 thereby provides a simple and cheap method of controlling the liquid sample in the micro fluidic device.
The micro fluidic device also comprises an indent which provides a read out section 16 for the channels 11. In the read out sections 16 of the channels 11, the channels comprise a transparent window and the magnetic particles can be temporally immobilized using a not shown magnet.
FIG. 4 shows another preferred micro fluidic device suitable for use in the invention.
The micro fluidic device comprises a substrate 22 with five flow channels 21. Each channel 21 comprises an inlet 23 and is in fluid connection with a sink 24 with a not shown flexible wall section.
The micro fluidic device also comprises an indent which provides a read out section 26 for the channels 21, where the channels comprise a transparent window and the magnetic particles can be temporally immobilized using a not shown magnet.
Each channel 21 comprises temporally immobilized magnetic particles and temporally immobilized fluorophores. The micro fluidic device is divided into zones comprising zone 0 which is the inlet zone, zone 1 and zone 2 which comprise temporally immobilized fluorophores and magnetic particles 17 arranged such that they do not react until they are in contact with the liquid sample, zone 3 which is the read out zone and zone 4 which is the sink zone.
In an embodiment zone 1 comprises temporally immobilized fluorophores and zone 2 comprises temporally immobilized magnetic particles.
In an embodiment zone 1 comprises temporally immobilized magnetic particles and zone 2 comprises temporally immobilized fluorophores.
The micro fluidic device could comprise several subzones of zone 1 and zone 2, if desired.
In use the liquid sample is fed to the inlet 23, the sample is sucked into zone 1 of the channels using the flexible wall section. Optionally the liquid sample is pulsated in zone 1 to dissolve or resuspend the immobilized elements 17 in zone 1. Thereafter the liquid sample is drawn further into the channels 21 to zone 2 for dissolving or resuspending the immobilized elements 17 in zone 2. After a preselected incubation time the liquid sample is drawn fully into the sinks 24. The magnetic particles are immobilized in the read out zone 3. If desired, the liquid sample can be reintroduced into the channels 21 by using the flexible wall of the sinks 24 and the immobilized magnetic particles can be flushed using the liquid sample to remove not immobilized fluorophores and other elements that could potentially provide noise.
FIG. 5 shows a system of the invention comprising a support element 32 supporting a micro fluidic device 31, an emitter 38 and a reader 39 coupled to a computer 34. The micro fluidic device comprises a read out section 36. The support element 32 comprises a temperature control element 35 for maintaining the liquid sample at a desired temperature during the test. The support element 32 further comprises a magnet 33. The micro fluidic device is arranged such that the magnet is located adjacent the read out section 36 to thereby temporally immobilize the magnetic particles in the read out section 36. The emitter 38 is configured to emit electromagnetic radiation directed at the read out section 36 to thereby excite fluorophores on the immobilized magnetic particles. The reader 39 is configured to read signals emitted from fluorophores captures by the immobilized magnetic particles and the read signals are transmitted to the computer 34 for processing to quantitative and/or qualitative determination of target compound(s).
FIG. 6 shows a fluorophore in the form of a quantum dot suitable for use in the invention. The quantum dot comprises a core 41 of a binary semiconductor alloy covered by a transparent shell 42 which is at least transparent for the wavelength emitted by the core. The shell 42 is further covered by an organic coating 43, such as a polymer coating which is coupled to one or more not shown components which can bind to the capture sites of the magnetic particles e.g. such as described above.
FIGS. 7a, 7b and 7c show a performance of the method of the invention in three steps. Step 1 is illustrated in FIG. 7a. Sample with the target component 51 is mixed with fluorophores 52 coupled to homologue target component 53. The relative amount of target component 51 to fluorophores 52 coupled to homologue target component 53 is relatively low. Step 2 is illustrated in FIG. 7b. The mixture of target component 51 and fluorophores 52 coupled to homologue target component 53 is further mixed with magnetic particles 54 carrying capture sites 55 for the target component 51 and the homologue target component 53. Step 3 is illustrated in FIG. 7c. Target component 51 and the homologue target component 53 are captured by the capture sites 55 carried by the magnetic particles 54. In the illustration shown, only the homologue target component 53 is captured by the capture sites 55. This is shown to illustrate that the amount of captured homologue target component 53 is relatively high and accordingly the amount of immobilized fluorophores 52 is relatively high. When the magnetic particles 54 are immobilized using a magnet arranged adjacent to the transparent window, and the fluorophores 52 are excited, the emitted signal from the fluorophores 52 is relatively high, and the amount of target component 51 can be determined.
FIGS. 8a, 8b and 8c show another performance of the method of the invention in three steps. Step 1 is illustrated in FIG. 8a. Sample with the target component 61 is mixed with fluorophores 62 coupled to homologue target component 63. The relative amount of target component 61 to fluorophores 62 coupled to homologue target component 63 is relatively high. Step 2 is illustrated in FIG. 8b. The mixture of target component 61 and fluorophores 62 coupled to homologue target component 63 is further mixed with magnetic particles 64 carrying capture sites 65 for the target component 61 and the homologue target component 63. Step 3 is illustrated in FIG. 8c. Target component 61 and the homologue target component 63 are captured by the capture sites 65 carried by the magnetic particles 64. In the illustration only the target component 61 captured by the capture sites 65 is shown to illustrate that the amount of captured target component 61 is relatively high and accordingly the amount of immobilized fluorophores 62 is relatively low or there may be none at all and when the magnetic particles 64 are immobilized using a magnet adjacent to the transparent window and the fluorophores 62 have been excited, the emitted signal from the fluorophores 62 is relatively low or absent, and the amount of target component 61 can be determined.
The emitter-reader assembly shown in FIG. 9 is comprises a casing 90 comprising a plurality of not shown diodes with respective center wavelengths for exciting the respective wavelengths of the fluorophores. The emitter-reader assembly further comprises an emitter fiber bundle 91 comprising a plurality of optical fibers in light connection with the respective diodes for guiding the light towards not shown fluorophores bound to temporally immobilized magnetic particles in a micro fluidic device. The emitter fiber bundle 91 has a length section 92 adjacent to emitter output ends 93 of the optical fibers from where the light 99 is emitted.
In the length section 92 the emitter bundle 91 is merged with a reader fiber bundle 96 such that the length section is a common emitter-reader length section 92. The common emitter-reader length section 92 is held together by a sleeve 94. The reader fiber bundle 96 comprises a plurality of optical fibers having reader input ends 95 arranged to receive the light signal 99 from the fluorophores. The reader fiber bundle 96 is fixed to a connector 97 where it is connected to a not shown reading unit—e.g. a spectroscope, via a waveguide 98 e.g. in form of another fiber bundle.
The emitter output ends 93 and the reader input ends 95 are advantageously arranged in a predetermined pattern. The predetermined pattern is advantageously selected such as to obtain high exciting rate and high reading rate. The emitter output ends 93 and the reader input ends 95 are advantageously positioned immediately adjacent to the transparent window, e.g. where the magnet was arranged when immobilizing the magnetic particles and/or immediately adjacent to the magnet.
EXAMPLES
Example 1
Screening Tests
Milk samples are screened for the target analyte Ampecillin.
A system as shown in FIG. 5 is used. The micro fluidic device is in the form of a cartridge similar to the micro fluidic device of FIG. 4, but with the difference that the 5 flow channels each have their respective inlet with an inlet-well. The magnet applied is a permanent magnet arranged to immobilize magnetic particles in the reading zone.
The channels are in fluid connection to sink sections 4 and have together with the sink section 5 zones, an inlet zone 0, a zone with temporally immobilized magnetic particles 1, a zone with temporally immobilized fluorophores 2, a reading zone with a transparent window 3 and a zone with flexible wall and sink sections 4.
By having 5 separate flow channels with separate inlets it is possible to screen 5 different samples simultaneously.
The temporally immobilized magnetic particles are 1.5 μm Biomag Protein G magnetic particles from Qiagen with Ampicillin antibody loaded onto Protein G. 1 μL of 0.4% by weight of the magnetic particles solution in buffer is deposited in the channel (zone 1) and dried down.
The temporally immobilized fluorophores are Qdot 655 Biotin Conjugate from Invitrogen loaded with Ampicillin. 1 μL of 15 nM buffer solution of the Qdot 655 is deposited in the channel (zone 2) and dried down.
As an internal reference signal Bio-Adembeads Streptavidin magnetic beads from Ademtech are labeled with Qdot 605 biotin conjugate from Invitrogen.
The Bio-Adembeads Streptavidin magnetic beads are deposited in the fluorophores zone (zone 2).
The tests are performed as follows:
5 different milk samples are loaded in the 5 inlet-wells on the cartridge. Each sample is drawn into the respective channel of the cartridge and re-suspends the magnetic particle in zone 1. Incubation is done by cycling the flow for 20 seconds over the site comprising the immobilized magnetic particles to re-suspend these and allow the magnetic particles to catch target analytes in the exposed sample volume. The sample is then drawn further into the channels of the cartridge to zone 2 and re-suspends the Qdots. Again incubation is done by cycling the flow for 20 seconds. Finally the sample is drawn into the sink section 4 whereby the magnetic particles approaching the magnet while the sample is passing are immobilized in the reading zone.
The magnetic particles are subjected to exciting wavelength(s) and the emitted signal is recorded.
The signals recorded at 655 nm can be normalized with the signal recorded at 605 nm. The resulting signal will show whether the respective sample comprises the target analyte.
Example 2
Quantitative Determination of One Target Analyte
Mouse serum is tested for Mouse IgG. The samples are prepared by dilution of the Mouse serum in buffer.
A system as shown in FIG. 5 is used. The micro fluidic device is in the form of a cartridge similar to the micro fluidic device of FIG. 4 but with the difference that the micro fluidic device comprises 2 flow channels with a common inlet with an inlet-well and the micro fluidic device comprises a common sink section in fluid connection with the flow channels. The micro fluidic device further comprises a flexible wall section which is common for the flow channels. In this example it is important that the flow channels and the deposition in the flow channels are essentially identical.
The magnet applied is a permanent magnet arranged to immobilize magnetic particles in the reading zone.
The channels in flow connection with the sink sections 4 have 5 zones, a common inlet zone 0, a zone with temporally immobilized fluorophores 1, a zone with temporally immobilized magnetic particles 2, a reading zone with a transparent window 3 and a common zone with flexible wall and sink section 4. It should be observed that the magnetic particles zone and the fluorophores zone in this example are reversed compared to the order thereof in example 1.
By having 5 separate flow channels with separate inlet it is possible to screen 5 different samples simultaneously.
The temporally immobilized magnetic particles are 1.5 μm Biomag Protein G magnetic particles from Qiagen with mouse IgG loaded onto Protein G. 1 μL of 0.4% by weight of the magnetic particles solution in buffer is deposited in the channel (zone 2) and dried down.
The temporally immobilized fluorophores are Qdot 655 Goat F(ab′)2 anti-Mouse IgG Conjugate (H+L) from Invitrogen. 1 μL of 15 nM buffer solution of the Qdot 655 is deposited in the channel (zone 1) and dried down.
Additionally a surfactant in the form of a detergent is applied in the sink section.
The tests are performed as follows:
Sample is applied in the well and drawn into the channels of the cartridge and re-suspends Qdots in zone 1. Incubation is done by cycling the flow for 20 seconds over the site for the immobilized Qdots to re-suspend these. The sample is then drawn further into the channels of the cartridge and re-suspends the immobilized magnetic particles in zone 2 and simultaneously the magnetic particles will catch analytes and Qdots. The analytes and Qdots will compete about the capture sites of the magnetic particles. Again incubation is done by cycling the flow for 20 seconds. Finally the sample is drawn into the sink section whereby the magnetic particles approaching the magnet while the sample is passing are immobilized in the reading zone. In the sink section the dried down detergent is dissolved and thereby the surface tension of the sample is lowered. To reduce background noise, the sample is finally pushed back into the channels where it is flushing the reading zone of non-immobilized sample but leaving the magnetic particles with the signal at the reading site. The detergent improves the flushing of the fluidic system.
The magnetic particles are subjected to exciting wavelength(s) and the emitted signal is recorded.
By comparing the obtained signals by a reference schedule as described above, e.g. a calibration curve, the quantitative determination can be obtained.
Example 3
Quantitative Determination of Two Target Analytes
Milk sample tested for the target analyte Ampecillin and the target analyte Tetracyclin.
A system as shown in FIG. 5 is used. The micro fluidic device is in the form of a cartridge similar to the micro fluidic device of FIG. 4 but with the difference that the micro fluidic device comprises 2 flow channels with a common inlet with an inlet-well, a common flexible wall section and in fluid connection with a common sink section. In this example it is desired that the flow channels and the deposition in the flow channels are essentially identical for improved precision.
The magnet applied is a permanent magnet arranged to immobilize magnetic particles in the reading zone.
The channels in fluid connection with the sink section 4 have 5 zones, a common inlet zone 0, a zone with temporally immobilized magnetic particles 1, a zone with temporally immobilized fluorophores 2, a reading zone with a transparent window 3 and a common zone with flexible wall and sink 4.
The temporally immobilized magnetic particles are 1.5 μm Biomag Protein G magnetic particles from Qiagen with Ampicillin antibody loaded onto Protein G and 1.5 μm Biomag Protein G magnetic particles from Qiagen with Tetracyclin antibody loaded onto Protein G. 1 μL of 0.2% by weight of each of the magnetic particles solution in buffer is deposited in the channel (zone 1) and dried down.
The temporally immobilized fluorophores are Qdot 655 Biotin Conjugate from Invitrogen loaded with Ampicillin and Qdot 605 Biotin Conjugate from Invitrogen loaded with Tetracyclin. 1 μL 7.5 nM buffer solutions of both Qdots are deposited in the channel (zone 2) and dried down.
The tests are performed as follows:
Sample is applied in the well and drawn into channels of the cartridge and re-suspends magnetic beads in zone 1. Incubation is done by cycling the flow for 20 seconds over the site for the immobilized magnetic particles to re-suspend these and allow the magnetic particles to catch target analytes in the exposed sample volume. The sample is then drawn further into the channels of the cartridge and re-suspends the Qdots in zone 2. Again incubation is done by cycling the flow for 20 seconds. Finally the sample is drawn into the sink section whereby the magnetic particles approaching the magnet while the sample is passing are immobilized in the reading zone. The magnetic particles are subjected to exciting wavelength(s) and the emitted signal is recorded.
The recorded signal at 655 nm is related to the content of Ampicillin in the sample. The recorded signal at 605 nm is related to the content of Tetracyclin in the sample.
Example 4
Quantitative Determination of One Target Analyte in Whole Blood
Whole blood is tested for CRP. The sample is undiluted.
A system as shown in FIG. 5 is used. The micro fluidic device is in the form of a cartridge similar to the micro fluidic device of FIG. 4 but with the difference that the micro fluidic device comprises 2 flow channels with a common inlet with an inlet-well and with a common flexible wall section and in fluid communication with a common sink section. In this example it is important that the flow channels and the deposition in the flow channels are essentially identical.
The magnet applied is a permanent magnet arranged to immobilize magnetic particles in the reading zone.
The channels in fluid connection with a sink section 4 have 5 zones, a common inlet zone 0, a zone with temporally immobilized fluorophores 1, a zone with temporally immobilized magnetic particles 2, a reading zone with a transparent window 3 and a common zone with flexible wall and sink section 4. By having 5 separate flow channels with separate inlets it is possible to screen 5 different samples simultaneously.
The temporally immobilized magnetic particles are 1.5 μm Biomag Protein G magnetic particles from Qiagen with CPR loaded onto Protein G. 1 μL of 0.4% by weight of the magnetic particles solution in buffer is deposited in the channel (zone 2) and dried down.
The temporally immobilized fluorophores are Qdot 655 Biotin Conjugate from Invitrogen loaded with CRP antibody. 1 μL of 15 nM buffer solution of the Qdot 655 is deposited in the channel (zone 1) and dried down.
The tests are performed as follows:
Sample is applied in the well and is drawn into cartridge and re-suspends Qdots in zone 1. Incubation is done by cycling the flow for 40 seconds over the site for the immobilized Qdots. The sample is then drawn further into channels of the cartridge and re-suspends the immobilized magnetic beads and simultaneously the magnetic particles will catch analytes and Qdots. The analytes and Qdots will compete about the capture sites of the magnetic particles. Again incubation is done by cycling the flow for 40 seconds. Finally, the sample is drawn into the sink section whereby the magnetic particles approaching the magnet while the sample is passing are immobilized in the reading zone.
The magnetic particles are subjected to exciting wavelength(s) and the emitted signal is recorded.
By comparing the obtained signals by a reference schedule as described above, e.g. a calibration curve, the quantitative determination can be obtained.
Example 5
Example 1 is repeated using an extract of crushed beef diluted with water. Samples with different degree of dilution are applied.
Example 6
Example 2 is repeated with the difference that the sample is mixed with the magnetic particles and the fluorophores before applying the sample to the well and drawing it into the channels of the cartridge.
The mouse serum is diluted in a buffer and mixed with magnetic particle solution and q-dot solution in a vial and is incubated for 5 minutes prior to application in the well and introduction into the channels.
The sample can immediately be drawn into the sink section whereby the magnetic particles approaching the magnet while the sample is passing are immobilized in the reading zone.
Example 7
Example 2 is repeated with the difference that the system is flushed with the sample by pushing the sample from the sink section into the channels to flush the reading zone from non-immobilized sample but leaving the magnetic particles with the signal at the reading site.
When the system has been flushed, a read-out module is positioned above one channel. The Qdots are excited using a 420 LED and the emitted spectrum is recorded. An algorithm running on a PC finds and records the peak light intensity at 655 nm and 605 nm. The read-out module is then positioned above the next channel.
Some preferred embodiments have been shown in the foregoing, but it should be stressed that the invention is not limited to these, but may be embodied in other ways within the subject-matter defined in the following claims.
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14409703
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zoetis denmark aps
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Mar 31st, 2022 03:01PM
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Mar 31st, 2022 03:01PM
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Zoetis
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:zts
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Zoetis
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Feb 25th, 2014 12:00AM
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Mar 3rd, 2006 12:00AM
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https://www.uspto.gov?id=US08658607-20140225
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Immunostimulatory G, U-containing oligoribonucleotides
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Compositions and methods relating to immunostimulatory RNA oligomers are provided. The immunostimulatory RNA molecules are believed to represent natural ligands of one or more Toll-like receptors, including Toll-like receptor 7 (TLR7) and Toll-like receptor 8 (TLR8). The compositions and methods are useful for stimulating immune activation. Methods useful for screening candidate immunostimulatory compounds are also provided.
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8658607
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1. A method of inducing an immune response in a subject to an antigen, the method comprising:
administering a composition comprising an isolated single-stranded RNA (ssRNA) G,U-rich oligoribonucleotide 5-40 nucleotides long having at least 80 percent G and U to a subject via a route of administration selected from the group consisting of mucosal, oral, intranasal, sublingual, ocular, vaginal, rectal, buccal, and by inhalation, wherein the G,U-rich oligoribonucleotide is free of CpG dinucleotide, wherein the administration requires cationic lipids, wherein the ssRNA is a ligand of Toll-like receptor 7 (TLR7) or TLR8, wherein the composition further comprises an antigen, and wherein the composition comprises an effective amount of the ssRNA G,U-rich oligoribonucleotide to induce an immune response to the antigen.
2. The method of claim 1, wherein TLR7- mediated activation by the ssRNA G,U-rich oligoribonucleotide leads to IFN-α secretion from plasmacytoid precursor dendritic cells (pDCs) and TLR8- mediated activation by the ssRNA G,U-rich oligoribonucleotide leads to TNF-α secretion from monocytes.
3. The method of claim 1, wherein the antigen is selected from the group consisting of an allergen, an antigen of a bacterium, an antigen of a virus, an antigen of a fungus, an antigen of a parasite, and a cancer antigen.
4. The method of claim 1, wherein the antigen comprises a peptide or a polypeptide.
5. The method of claim 1, wherein the antigen comprises a cell.
6. The method of claim 1, wherein the composition is administered to the subject more than once.
7. The method of claim 1, wherein the immune response comprises a Th1 immune response.
8. The method of claim 1, wherein the ssRNA G,U-rich oligoribonucleotide is a ligand of both TLR7 and TLR8.
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8
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RELATED APPLICATIONS
This application is a continuation of currently pending U.S. patent application Ser. No. 10/407,952, filed on Apr. 4, 2003, the entire contents of which are incorporated herein by reference, and which claims benefit under 35 U.S.C. §119(e) of U.S. 60/421,966, filed Oct. 29, 2002, and U.S. 60/370,515, filed Apr. 4, 2002, the contents of which are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
The present invention relates generally to the field of immunology and immune stimulation. More particularly, the present invention relates to immunostimulatory ribonucleic acids, homologs of said immunostimulatory ribonucleic acids, and methods of use of said immunostimulatory ribonucleic acids and homologs. Compositions and methods of the invention are believed to be useful for inducing signaling through Toll-like receptor 7 (TLR7) and Toll-like receptor 8 (TLR8).
BACKGROUND OF THE INVENTION
The immune response is conceptually divided into innate immunity and adaptive immunity. Innate immunity is believed to involve recognition of pathogen-associated molecular patterns (PAMPs) shared in common by certain classes of molecules expressed by infectious microorganisms or foreign macromolecules. PAMPs are believed to be recognized by pattern recognition receptors (PRRs) on certain immune cells.
Toll-like receptors (TLRs) are a family of highly conserved polypeptides that play a critical role in innate immunity in mammals. Currently ten family members, designated TLR1-TLR10, have been identified. The cytoplasmic domains of the various TLRs are characterized by a Toll-interleukin 1 (IL-1) receptor (TIR) domain. Medzhitov R et al. (1998) Mol Cell 2:253-8. Recognition of microbial invasion by TLRs triggers activation of a signaling cascade that is evolutionarily conserved in Drosophila and mammals. The TIR domain-containing adapter protein MyD88 has been reported to associate with TLRs and to recruit IL-1 receptor-associated kinase (IRAK) and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) to the TLRs. The MyD88-dependent signaling pathway is believed to lead to activation of NF-kB transcription factors and c-Jun NH2 terminal kinase (Jnk) mitogen-activated protein kinases (MAPKs), critical steps in immune activation and production of inflammatory cytokines. For a review, see Aderem A et al. (2000) Nature 406:782-87.
While a number of specific TLR ligands have been reported, ligands for some TLRs remain to be identified. Ligands for TLR2 include peptidoglycan and lipopeptides. Yoshimura A et al. (1999) J Immunol 163:1-5; Yoshimura A et al. (1999) J Immunol 163:1-5; Aliprantis A O et al. (1999) Science 285:736-9. Viral-derived double-stranded RNA (dsRNA) and poly I:C, a synthetic analog of dsRNA, have been reported to be ligands of TLR3. Alexopoulou L et al. (2001) Nature 413:732-8. Lipopolysaccharide (LPS) is a ligand for TLR4. Poltorak A et al. (1998) Science 282:2085-8; Hoshino K et al. (1999) J Immunol 162:3749-52. Bacterial flagellin is a ligand for TLR5. Hayashi F et al. (2001) Nature 410:1099-1103. Peptidoglycan has been reported to be a ligand not only for TLR2 but also for TLR6. Ozinsky A et al. (2000) Proc Natl Acad Sci USA 97:13766-71; Takeuchi O et al. (2001) Int Immunol 13:933-40. Bacterial DNA (CpG DNA) has been reported to be a TLR9 ligand. Hemmi H et al. (2000) Nature 408:740-5; Bauer S et al. (2001) Proc Natl Acad Sci USA 98, 9237-42. The TLR ligands listed above all include natural ligands, i.e., TLR ligands found in nature as molecules expressed by infectious microorganisms.
The natural ligands for TLR1, TLR7, TLR8 and TLR10 are not known, although recently certain low molecular weight synthetic compounds, the imidazoquinolones imiquimod (R-837) and resiquimod (R-848), were reported to be ligands of TLR7. Hemmi H et al. (2002) Nat Immunol 3:196-200.
SUMMARY OF THE INVENTION
The present invention is based in part on the novel discovery by the inventors of certain immunostimulatory RNA and RNA-like (hereinafter, simply “RNA”) molecules. The immunostimulatory RNA molecules of the invention are believed by the inventors to require a base sequence that includes at least one guanine (G) and at least one uracil (U), wherein optionally the at least one G can be a variant or homolog of G and/or the at least one U can independently be a variant or homolog of U. Surprisingly, the immunostimulatory RNA molecules of the invention can be either single-stranded or at least partially double-stranded. Also surprisingly, the immunostimulatory RNA molecules of the invention do not require a CpG motif in order to exert their immunostimulatory effect. Without meaning to be bound by any particular theory or mechanism, it is the belief of the inventors that the immunostimulatory RNA molecules of the invention signal through an MyD88-dependent pathway, probably through a TLR. Also without meaning to be bound by any particular theory or mechanism, it is the belief of the inventors that the immunostimulatory RNA molecules of the invention interact with and signal through TLR8, TLR7, or some other TLR yet to be identified.
The immunostimulatory RNA molecules of the invention are also believed by the inventors to be representative of a class of RNA molecules, found in nature, which can induce an immune response. Without meaning to be bound by any particular theory or mechanism, it is the belief of the inventors that the corresponding class of RNA molecules found in nature is believed to be present in ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), and viral RNA (vRNA). It is to be noted in this regard that the immunostimulatory RNA molecules of the present invention can be as small as 5-40 nucleotides long. Such short RNA molecules fall outside the range of full length messenger RNAs described to be useful in transfecting dendritic cells in order to induce an immune response to cancer antigens. See, e.g., Boczkowski D et al. (1996) J Exp Med 184:465-72; Mitchell D A et al. (2000) Curr Opin Mol Ther 2:176-81.
It has also been discovered according to the present invention that the immunostimulatory RNA molecules of the invention can be advantageously combined with with certain agents which promote stabilization of the RNA, local clustering of the RNA molecules, and/or trafficking of the RNA molecules into the endosomal compartment of cells. In particular, it has been discovered according to the present invention that certain lipids and/or liposomes are useful in this regard. For example, certain cationic lipids, including in particular N-[1-(2,3 dioleoyloxy)-propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP), appear to be especially advantageous when combined with the immunostimulatory RNA molecules of the invention. As another example, covalent conjugation of a cholesteryl moiety to the RNA, for example to the 3′ end of the RNA, promotes the immunostimulatory effect of the RNA, even in the absence of cationic lipid.
The invention provides compositions of matter and methods related to the immunostimulatory RNA molecules of the invention. The compositions and methods are useful, inter alia, for activating immune cells in vivo, in vitro, and ex vivo; treating infection; treating cancer; preparing a pharmaceutical composition; identifying a target receptor for the immunostimulatory RNA; and screening for and characterizing additional immunostimulatory compounds. Furthermore, the compositions of matter and methods related to the immunostimulatory RNA molecules of the instant invention can advantageously be combined with other immunostimulatory compositions of matter and methods related to such other immunostimulatory compositions of matter.
In one aspect the invention provides an immunostimulatory composition. The immunostimulatory composition according to this aspect of the invention includes an isolated RNA oligomer 5-40 nucleotides long having a base sequence having at least one guanine (G) and at least one uracil (U), and optionally a cationic lipid. The RNA oligomer can be of natural or non-natural origin. An RNA oligomer of natural origin can in one embodiment be derived from prokaryotic RNA and in another embodiment can be derived from eukaryotic RNA. In addition, the RNA oligomer of natural origin can include a portion of a ribosomal RNA. An RNA oligomer of non-natural origin can include an RNA molecule synthesized outside of a cell, e.g., using chemical techniques known by those of skill in the art. In one embodiment an RNA oligomer can include a derivative of an RNA oligomer of natural origin.
In one embodiment the isolated RNA oligomer is a G,U-rich RNA as defined below.
In one embodiment the G,U-containing immunostimulatory RNA is an isolated RNA molecule at least 5 nucleotides long which includes a base sequence as provided by 5′-RURGY-3′, wherein R represents purine, U represents uracil, G represents guanine, and Y represents pyrimidine. In one embodiment the G,U-containing immunostimulatory RNA is an isolated RNA molecule at least 5 nucleotides long which includes a base sequence as provided by 5′-GUAGU-3′, wherein A represents adenine. In one embodiment the G,U-containing immunostimulatory RNA is an isolated RNA molecule which includes a base sequence as provided by 5′-GUAGUGU-3′.
In one embodiment the G,U-containing immunostimulatory RNA is an isolated RNA molecule at least 5 nucleotides long which includes a base sequence as provided by 5′-GUUGB-3′, wherein B represents U, G, or C.
In one embodiment the G,U-containing immunostimulatory RNA is an isolated RNA molecule at least 5 nucleotides long which includes a base sequence as provided by 5′-GUGUG-3′.
In other embodiments the isolated RNA molecule can contain multiples of any of the foregoing sequences, combinations of any of the foregoing sequences, or combinations of any of the foregoing sequences including multiples of any of the foregoing sequences. The multiples and combinations can be linked directly or they can be linked indirectly, i.e, through an intervening nucleoside or sequence. In one embodiment the intervening linking nucleoside is G; in one embodiment the intervening linking nucleoside is U.
In one embodiment the base sequence includes 5′-GUGUUUAC-3′. In one embodiment the base sequence is 5′-GUGUUUAC-3′.
In another embodiment the the base sequence includes 5′-GUAGGCAC-3′. In one embodiment the the base sequence is 5′-GUAGGCAC-3′.
In yet another embodiment the base sequence includes 5′-CUAGGCAC-3′. In one embodiment the base sequence is 5′-CUAGGCAC-3′.
In still another embodiment the base sequence includes 5′-CUCGGCAC-3′. In one embodiment the base sequence is 5′-CUCGGCAC-3′.
In one embodiment the oligomer is 5-12 nucleotides long. In one embodiment the oligomer is 8-12 nucleotides long.
Also according to this aspect of the invention, in one embodiment the base sequence is free of CpG dinucleotide. Thus in this embodiment the immunostimulatory RNA is not a CpG nucleic acid.
In certain embodiments according to this aspect of the invention, the base sequence of the RNA oligomer is at least partially self-complementary. In one embodiment the extent of self-complementarity is at least 50 percent. The extent of self-complementarity can extend to and include 100 percent. Thus for example the base sequence of the at least partially self-complementary RNA oligomer in various embodiments can be at least 50 percent, at least 60 percent, at least 70 percent, at least 80 percent, at least 90 percent, or 100 percent self-complementary. Complementary base pairs include guanine-cytosine (G-C), adenine-uracil (A-U), adenine-thymine (A-T), and guanine-uracil (G-U). G-U “wobble” basepairing, which is fairly common in ribosomal RNA and in RNA retroviruses, is somewhat weaker than traditional Watson-Crick basepairing between G-C, A-T, or A-U. A partially self-complementary sequence can include one or more portions of self-complementary sequence. In an embodiment which involves a partially self-complementary sequence, the RNA oligomer can include a self-complementary portion positioned at and encompassing each end of the oligomer.
In one embodiment according to this aspect of the invention, the oligomer is a plurality of oligomers, i.e., a plurality of RNA oligomers each 6-40 nucleotides long having a base sequence comprising at least one guanine (G) and at least one uracil (U). The plurality of oligomers can, but need not, include sequences which are at least partially complementary to one another. In one embodiment the plurality of oligomers includes an oligomer having a first base sequence and an oligomer having a second base sequence, wherein the first base sequence and the second base sequence are at least 50 percent complementary. Thus for example the at least partially complementary base sequences in various embodiments can be at least 50 percent, at least 60 percent, at least 70 percent, at least 80 percent, at least 90 percent, or 100 percent complementary. As described above, complementary base pairs include guanine-cytosine (G-C), adenine-uracil (A-U), adenine-thymine (A-T), and guanine-uracil (G-U). Partially complementary sequences can include one or more portions of complementary sequence. In an embodiment which involves partially complementary sequences, the RNA oligomers can include a complementary portion positioned at and encompassing at least one end of the oligomers.
In one embodiment the oligomer is a plurality of oligomers which includes an oligomer having a base sequence including 5′-GUGUUUAC-3′ and an oligomer having a base sequence including 5′-GUAGGCAC-3′. In one embodiment the oligomer is a plurality of oligomers which includes an oligomer having a base sequence 5′-GUGUUUAC-3′ and an oligomer having a base sequence 5′-GUAGGCAC-3′.
Further according to this aspect of the invention, in various embodiments the oligomer includes a non-natural backbone linkage, a modified base, a modified sugar, or any combination of the foregoing. The non-natural backbone linkage can be a stabilized linkage, i.e., a linkage which is relatively resistant against RNAse or nuclease degradation, compared with phosphodiester linkage. In one embodiment the non-natural backbone linkage is a phosphorothioate linkage. The oligomer can include one non-natural backbone linkage or a plurality of non-natural backbone linkages, each selected independently of the rest. The modified base can be a modified G, U, A, or C, including the at least one G and the at least one U of the base sequence according to this aspect of the invention. In some embodiments the modified base can be selected from 7-deazaguanosine, 8-azaguanosine, 5-methyluracil, and pseudouracil. The oligomer can include one modified base or a plurality of modified bases, each selected independently of the rest. The modified sugar can be a methylated sugar, arabinose. The oligomer can include one modified sugar or a plurality of modified sugars, each selected independently of the rest.
In one embodiment the cationic lipid is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP). DOTAP is believed to transport RNA oligomer into cells and specifically traffic to the endosomal compartment, where it can release the RNA oligomer in a pH-dependent fashion. Once in the endosomal compartment, the RNA can interact with certain intracellular Toll-like receptor molecules (TLRs), triggering TLR-mediated signal transduction pathways involved in generating an immune response. Other agents with similar properties including trafficking to the endosomal compartment can be used in place of or in addition to DOTAP.
In one embodiment the immunostimulatory composition further includes an antigen. In one embodiment the antigen is an allergen. In one embodiment the antigen is a cancer antigen. In one embodiment the antigen is a microbial antigen.
Also according to this aspect of the invention, in another embodiment the invention is a pharmaceutical composition. The pharmaceutical composition includes an immunostimulatory composition of the invention and a pharmaceutically acceptable carrier. Methods for preparing the pharmaceutical composition are also provided. Such methods entail placing an immunostimulatory composition of the invention in contact with a pharmaceutically acceptable carrier. The pharmaceutical composition can be formulated in a unit dosage for convenience.
In another aspect the invention provides a method of activating an immune cell. The method involves contacting an immune cell with an immunostimulatory composition of the invention, described above, in an effective amount to induce activation of the immune cell. In one embodiment the activation of the immune cell involves secretion of a cytokine by the immune cell. The cytokine in one embodiment is selected from the group consisting of interleukin 6 (IL-6), interleukin 12 (IL-12), an interferon (IFN), and tumor necrosis factor (TNF). In one embodiment the activation of the immune cell includes secretion of a chemokine. In one embodiment the secreted chemokine is interferon-gamma-induced protein 10 (IP-10). In one embodiment the activation of the immune cell includes expression of a costimulatory/accessory molecule by the immune cell. In one embodiment the costimulatory/accessory molecule is selected from the group consisting of intercellular adhesion molecules (ICAMs, e.g., CD54), leukocyte function-associated antigens (LFAs, e.g., CD58), B7s (CD80, CD86), and CD40.
Also according to this aspect of the invention, in one embodiment the activation of the immune cell involves activation of a MyD88-dependent signal transduction pathway. MyD88 is believed to be an adapter molecule that interacts with the Toll/interleukin-1 receptor (TIR) domain of various Toll-like receptor (TLR) molecules and participates in signal transduction pathways that ultimately result in activation of nuclear factor kappa B (NF-κB). Thus in one embodiment the MyD88-dependent signal transduction pathway is associated with a TLR. More particularly, in one embodiment the TLR is TLR8. In another embodiment the TLR is TLR7.
Also according to this aspect of the invention in one embodiment the immune cell is a human immune cell. The immune cell in one embodiment is a myeloid dendritic cell.
In one embodiment of this aspect of the invention the contacting occurs in vitro. In another embodiment the contacting occurs in vivo.
The invention in another aspect provides a method of inducing an immune response in a subject. The method according to this aspect of the invention involves administering to a subject an immunostimulatory composition of the invention in an effective amount to induce an immune response in the subject. It is to be noted that the method according to this aspect of the invention does not involve administration of an antigen to the subject. In one embodiment the subject is a human. In one embodiment the subject has or is at risk of having a cancer. In one embodiment the subject has or is at risk of having an infection with an agent selected from the group consisting of viruses, bacteria, fungi, and parasites. In a particular embodiment the subject has or is at risk of having a viral infection. It is also to be noted that the method according to this aspect of the invention can be used to treat a subject with a suppressed capacity to mount an effective or desirable immune response. For example the subject can have a suppressed immune system due to an infection, a cancer, an acute or chronic disease such as kidney or liver insufficency, surgery, and an exposure to an immunosuppressive agent such as chemotherapy, radiation, certain drugs, or the like. In one embodiment the subject has or is at risk of having an allergy or asthma. Such a subject can be exposed to or at risk of exposure to an allergen that is associated with an allergic response or asthma in the subject.
In yet another aspect the invention provides a method of inducing an immune response in a subject. The method according to this aspect of the invention involves administering an antigen to a subject, and administering to the subject an immunostimulatory composition of the invention in an effective amount to induce an immune response to the antigen. It is to be noted that the antigen can be administered before, after, or concurrently with the immunostimulatory composition of the invention. In addition, both the antigen and the immunostimulatory compound can be administered to the subject more than once.
In one embodiment according to this aspect of the invention the antigen is an allergen. In one embodiment according to this aspect of the invention the antigen is a cancer antigen. The cancer antigen in one embodiment can be a cancer antigen isolated from the subject. In another embodiment the antigen is a microbial antigen. The microbial antigen can be an antigen of a virus, a bacterium, a fungus, or a parasite.
The invention further provides, in yet another aspect, a method of inducing an immune response in a subject. The method according to this aspect of the invention involves isolating dendritic cells of a subject, contacting the dendritic cells ex vivo with an immunostimulatory composition of the invention, contacting the dendritic cells ex vivo with an antigen, and administering the contacted dendritic cells to the subject.
In one embodiment according to this aspect of the invention the antigen is an allergen. In one embodiment according to this aspect of the invention the antigen is a cancer antigen. The cancer antigen in one embodiment can be a cancer antigen isolated from the subject. In another embodiment the antigen is a microbial antigen. The microbial antigen can be an antigen of a virus, a bacterium, a fungus, or a parasite.
An immune response arising from stimulation of one TLR can be modified, enhanced or amplified by stimulation of another TLR, and the combined immunostimulatory effect may be synergistic. For example, TLR9 is reported to respond to bacterial DNA and, more generally, CpG DNA. An immune response arising from TLR9 contacting its natural ligand (or any TLR9 ligand) may be modified, enhanced or amplified by also selectively contacting TLR7 with a TLR7 ligand, or by also selectively contacting TLR8 with a TLR8 ligand, or both. Likewise, an immune response arising from TLR7 contacting a TLR7 ligand may be modified, enhanced or amplified by also selectively contacting TLR8 with a TLR8 ligand, or by also selectively contacting TLR9 with CpG DNA (or any suitable TLR9 ligand), or both. As yet another example, an immune response arising from TLR8 contacting a TLR8 ligand may be modified, enhanced or amplified by also selectively contacting TLR7 with a TLR7 ligand, or by also selectively contacting TLR9 with CpG DNA (or any suitable TLR9 ligand), or both.
The present invention is based in part on the novel discovery by the inventors of what are believed to be natural ligands for TLR7 and TLR8. While naturally occurring ligands derived from microbes have been described for certain TLRs, natural ligands for TLR7 and TLR8 have not previously been described. Certain synthetic small molecules, imidazoquinoline compounds, have been described as ligands for TLR7, but such compounds are to be distinguished from the natural ligands of the present invention. Hemmi H et al. (2002) Nat Immunol 3:196-200.
Isolated natural ligands of TLR7 and TLR8 are useful as compositions that can induce, enhance, and complement an immune response. The natural ligands of TLR7 and TLR8 are useful for preparation of novel compositions that can induce, enhance, and complement an immune response. In addition, the natural ligands of TLR7 and TLR8 are useful for selectively inducing TLR7- and TLR8-mediated signaling and for selectively inducing TLR7- and TLR8-mediated immune responses. Furthermore, the natural ligands of TLR7 and TLR8 are useful in designing and performing screening assays for identification and selection of immunostimulatory compounds.
The present invention is also based in part on the novel discovery according to the invention that human neutrophils strongly express TLR8. This observation is important because neutrophils are very often the first cells to engage infectious pathogens and thus to initiate responses. It is believed that activated neutrophils secrete chemokines and cytokines, which in turn are instrumental in recruiting dendritic cells. TLR9-expressing dendritic cells drawn to the site of the activated neutrophils there become activated, thereby amplifying the immune response.
The present invention is also based in part on the appreciation of the differential expression of various TLRs, including TLR7, TLR8, and TLR9, on various cells of the immune system. This segregation may be of particular significance in humans with respect to TLR7, TLR8, and TLR9. The immune response arising from stimulation of any one of these TLRs may be enhanced or amplified by stimulation of another TLR, and the combined immunostimulatory effect may be synergistic. For example, TLR9 is reported to respond to bacterial DNA and, more generally, CpG DNA. An immune response arising from TLR9 contacting its natural ligand (or any TLR9 ligand) may be enhanced or amplified by also selectively contacting TLR7 with its natural ligand (or any suitable TLR7 ligand), or by also selectively contacting TLR8 with its natural ligand (or any suitable TLR8 ligand), or both. Likewise, an immune response arising from TLR7 contacting its natural ligand (or any TLR7 ligand) may be enhanced or amplified by also selectively contacting TLR8 with its natural ligand (or any suitable TLR8 ligand), or by also selectively contacting TLR9 with CpG DNA (or any suitable TLR9 ligand), or both. As yet another example, an immune response arising from TLR8 contacting its natural ligand (or any TLR8 ligand) may be enhanced or amplified by also selectively contacting TLR7 with its natural ligand (or any suitable TLR7 ligand), or by also selectively contacting TLR9 with CpG DNA (or any suitable TLR9 ligand), or both.
In a further aspect the invention provides a composition including an effective amount of a ligand for TLR8 to induce TLR8 signaling and an effective amount of a ligand for a second TLR selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR9 and TLR10 to induce signaling by the second TLR. In one embodiment the second TLR is TLR3. In one embodiment the second TLR is TLR7. In one embodiment the second TLR is TLR9. In one embodiment the ligand for TLR8 and the ligand for the second TLR are linked. In yet another embodiment the composition further includes a pharmaceutically acceptable carrier.
In another aspect the invention provides a composition including an effective amount of a ligand for TLR7 to induce TLR7 signaling and an effective amount of a ligand for a second TLR selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR8, TLR9, and TLR10 to induce signaling by the second TLR. In one embodiment the second TLR is TLR3. In one embodiment the second TLR is TLR8. In one embodiment the second TLR is TLR9. In one embodiment the ligand for TLR7 and the ligand for the second TLR are linked. In yet another embodiment the composition further includes a pharmaceutically acceptable carrier.
In a further aspect the invention provides a composition including a DNA:RNA conjugate, wherein DNA of the conjugate includes an immunostimulatory motif effective for stimulating TLR9 signaling and wherein RNA of the conjugate includes RNA effective for stimulating signaling by TLR3, TLR7, TLR8, or any combination thereof. In one embodiment the immunostimulatory motif effective for stimulating TLR9 signaling is a CpG motif. In another embodiment the immunostimulatory motif effective for stimulating TLR9 signaling is poly-dT. In yet another embodiment the immunostimulatory motif effective for stimulating TLR9 signaling is poly-dG. In one embodiment the conjugate includes a chimeric DNA:RNA backbone. In one embodiment the chimeric backbone includes a cleavage site between the DNA and the RNA. In one embodiment the conjugate includes a double-stranded DNA:RNA heteroduplex. In yet another embodiment the composition further includes a pharmaceutically acceptable carrier.
In another aspect the invention provides a method for stimulating TLR8 signaling. The method involves contacting TLR8 with an isolated RNA in an effective amount to stimulate TLR8 signaling. In one embodiment the RNA is double-stranded RNA. In one embodiment the RNA is ribosomal RNA. In one embodiment the RNA is transfer RNA. In one embodiment the RNA is messenger RNA. In one embodiment the RNA is viral RNA. In one embodiment the RNA is G,U-rich RNA. In one embodiment the RNA consists essentially of G and U.
In yet another aspect the invention provides a method for stimulating TLR8 signaling. The method according to this aspect involves contacting TLR8 with a mixture of nucleosides consisting essentially of G and U in a ratio between 1G:50U and 10G:1U, in an amount effective to stimulate TLR8 signaling. In one embodiment the nucleosides are ribonucleosides. In one embodiment the nucleosides comprise a mixture of ribonucleosides and deoxyribonucleosides. In one embodiment the G is a guanosine derivative selected from the group consisting of: 8-bromoguanosine, 8-oxoguanosine, 8-mercaptoguanosine, 7-allyl-8-oxoguanosine, guanosine ribonucleoside vanadyl complex, inosine, and nebularine.
A further aspect of the invention provides a method for stimulating TLR8 signaling. The method according to this aspect involves contacting TLR8 with a mixture of ribonucleoside vanadyl complexes. In one embodiment the mixture comprises guanosine ribonucleoside vanadyl complexes.
In another aspect the invention provides a method for stimulating TLR8 signaling. The method according to this aspect involves contacting TLR8 with an isolated G,U-rich oligonucleotide comprising a sequence selected from the group consisting of: UUGUGG, UGGUUG, GUGUGU, and GGGUUU, in an amount effective to stimulate TLR8 signaling. In one embodiment the oligonucleotide is an oligoribonucleotide. In one embodiment the oligonucleotide is 7-50 bases long. In one embodiment the oligonucleotide is 12-24 bases long. In one embodiment the oligonucleotide has a sequence
5′-GUUGUGGUUGUGGUUGUG-3′.
(SEQ ID NO:1)
The invention provides in another aspect a method for stimulating TLR8 signaling. The method according to this aspect involves contacting TLR8 with an at least partially double-stranded nucleic acid molecule comprising at least one G-U base pair, in an amount effective to stimulate TLR8 signaling.
In yet another aspect the invention provides a method for supplementing a TLR8-mediated immune response. The method involves contacting TLR8 with an effective amount of a TLR8 ligand to induce a TLR8-mediated immune response, and contacting a TLR other than TLR8 with an effective amount of a ligand for the TLR other than TLR8 to induce an immune response mediated by the TLR other than TLR8.
In a further aspect the invention provides a method for supplementing a TLR8-mediated immune response in a subject. The method according to this aspect involves administering to a subject in need of an immune response an effective amount of a TLR8 ligand to induce a TLR8-mediated immune response, and administering to the subject an effective amount of a ligand for a TLR other than TLR8 to induce an immune response mediated by the TLR other than TLR8. In one embodiment the TLR other than TLR8 is TLR9. In one embodiment the ligand for TLR9 is a CpG nucleic acid. In one embodiment the CpG nucleic acid has a stabilized backbone. In one embodiment the ligand for TLR8 and the ligand for TLR9 are a conjugate. In one embodiment the conjugate comprises a double-stranded DNA:RNA heteroduplex. In one embodiment the conjugate comprises a chimeric DNA:RNA backbone. In one embodiment the chimeric backbone comprises a cleavage site between the DNA and the RNA.
The invention in a further aspect provides a method for stimulating TLR7 signaling. The method according to this aspect involves contacting TLR7 with an isolated guanosine ribonucleoside in an effective amount to stimulate TLR7 signaling. In one embodiment the guanosine ribonucleoside is a guanosine ribonucleoside derivative selected from the group consisting of: 8-bromoguanosine, 8-oxoguanosine, 8-mercaptoguanosine, 7-allyl-8-oxoguanosine, guanosine ribonucleoside vanadyl complex, inosine, and nebularine. In one embodiment the guanosine ribonucleoside derivative is 8-oxoguanosine. In one embodiment the guanosine nucleoside is a ribonucleoside. In one embodiment the guanosine nucleoside comprises a mixture of ribonucleosides and deoxyribonucleosides.
In another aspect the invention further provides a method for stimulating TLR7 signaling. The method according to this aspect involves contacting TLR7 with an isolated nucleic acid comprising a terminal oxidized or halogenized guanosine in an effective amount to stimulate TLR7 signaling. In one embodiment the oxidized or halogenized guanosine is 8-oxoguanosine.
In another aspect the invention provides a method for stimulating TLR7 signaling. The method according to this aspect involves contacting TLR7 with an isolated RNA in an effective amount to stimulate TLR7 signaling. In one embodiment the RNA is double-stranded RNA. In one embodiment the RNA is ribosomal RNA. In one embodiment the RNA is transfer RNA. In one embodiment the RNA is messenger RNA. In one embodiment the RNA is viral RNA. In one embodiment the RNA is G-rich RNA. In one embodiment the RNA is part of a DNA:RNA heteroduplex. In one embodiment the RNA consists essentially of guanosine ribonucleoside.
The invention in yet another aspect provides a method for stimulating TLR7 signaling. The method according to this aspect involves contacting TLR7 with a mixture of nucleosides consisting essentially of G and U in a ratio between 1G:50U and 10G:1U, in an amount effective to stimulate TLR7 signaling.
Provided in yet another aspect of the invention is a method for stimulating TLR7 signaling. The method according to this aspect involves contacting TLR7 with a mixture of ribonucleoside vanadyl complexes. In one embodiment the mixture comprises guanosine ribonucleoside vanadyl complexes.
In a further aspect the invention provides a method for supplementing a TLR7-mediated immune response. The method according to this aspect involves contacting TLR7 with an effective amount of a TLR7 ligand to induce a TLR7-mediated immune response, and contacting a TLR other than TLR7 with an effective amount of a ligand for the TLR other than TLR7 to induce an immune response mediated by the TLR other than TLR7.
In yet another aspect the invention provides a method for supplementing a TLR7-mediated immune response in a subject. The method involves administering to a subject in need of an immune response an effective amount of a TLR7 ligand to induce a TLR7-mediated immune response, and administering to the subject an effective amount of a ligand for a TLR other than TLR7 to induce an immune response mediated by the TLR other than TLR7. In one embodiment the TLR other than TLR7 is TLR9. In one embodiment the ligand for TLR9 is a CpG nucleic acid. In one embodiment the CpG nucleic acid has a stabilized backbone. In one embodiment the ligand for TLR7 and the ligand for TLR9 are a conjugate. In one embodiment the conjugate comprises a double-stranded DNA:RNA heteroduplex. In one embodiment the conjugate comprises a chimeric DNA:RNA backbone. In one embodiment the chimeric backbone comprises a cleavage site between the DNA and the RNA.
The invention in another aspect provides a method for screening candidate immunostimulatory compounds. The method according to this aspect involves measuring a TLR8-mediated reference signal in response to an RNA reference, measuring a TLR8-mediated test signal in response to a candidate immunostimulatory compound, and comparing the TLR8-mediated test signal to the TLR8-mediated reference signal.
In yet another aspect the invention provides a method for screening candidate immunostimulatory compounds, comprising measuring a TLR8-mediated reference signal in response to an imidazoquinoline reference, measuring a TLR8-mediated test signal in response to a candidate immunostimulatory compound, and comparing the TLR8-mediated test signal to the TLR8-mediated reference signal.
Also provided according to yet another aspect of the invention is a method for screening candidate immunostimulatory compounds. The method involves measuring a TLR7-mediated reference signal in response to an imidazoquinoline reference, measuring a TLR7-mediated test signal in response to a candidate immunostimulatory compound, and comparing the TLR7-mediated test signal to the TLR7-mediated reference signal.
In some embodiments the imidazoquinoline is resiquimod (R-848).
In some embodiments the imidazoquinoline is imiquimod (R-837).
In a further aspect the invention also provides a method for screening candidate immunostimulatory compounds. The method according to this aspect involves measuring a TLR7-mediated reference signal in response to a 7-allyl-8-oxoguanosine reference, measuring a TLR7-mediated test signal in response to a candidate immunostimulatory compound, and comparing the TLR7-mediated test signal to the TLR7-mediated reference signal.
Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a bar graph depicting IL-12 p40 secretion by human peripheral blood mononuclear cells (PBMCs) in response to certain stimuli including selected G,U-containing RNA oligonucleotides with or without DOTAP (“with Liposomes” and “without Liposomes”, respectively), as measured by specific enzyme-linked immunosorbent assay (ELISA). The lower case letter “s” appearing in the base sequences signifies phosphorothioate linkage.
FIG. 2 is a bar graph depicting TNF-α secretion by human PBMCs in response to certain stimuli including selected G,U-containing RNA oligonucleotides with or without DOTAP (“with Liposomes” and “without Liposomes”, respectively), as measured by specific ELISA.
FIG. 3 is a bar graph depicting dose-dependence of IL-12 p40 secretion by human PBMCs in response to various concentrations of selected G,U-containing RNA oligonucleotides (with DOTAP), as measured by specific ELISA.
FIG. 4 is a bar graph depicting sequence dependence of TNF-α secretion by human PBMCs in response to various selected RNA oligonucleotides related to the RNA oligonucleotide GUAGGCAC (with DOTAP), as measured by specific ELISA.
FIG. 5 is a bar graph depicting the effect of DOTAP on IL-12 p40 secretion by human PBMCs in response to various stimuli, as measured by specific ELISA.
FIG. 6 is a quartet of bar graphs depicting IL-12 p40 secretion by various types of murine macrophage cells in response to a variety of test and control immunostimulatory compounds, as measured by specific ELISA. Panel A, wild type macrophages in the presence of IFN-γ; Panel B, MyD88-deficient macrophages in the presence of IFN-γ; Panel C, J774 macrophage cell line; Panel D, RAW 264.7 macrophage cell line.
FIG. 7 is a pair of graphs depicting the secretion of (A) TNF-α and (B) IL-12 p40 by human PBMC upon incubation with HIV-1-derived RNA sequences, with and without DOTAP. Circles, 5′-GUAGUGUGUG-3′ (SEQ ID NO:2); Triangles, 5′-GUCUGUUGUGUG-3′ (SEQ ID NO:3). Open symbols, without DOTAP; closed symbols, with DOTAP.
FIG. 8 is a graph depicting apparent relatedness among TLRs.
FIG. 9 depicts nucleic acid binding domains in TLR7, TLR8, and TLR9.
FIG. 10 is a bar graph depicting responsiveness of human PBMC to stringent response factor (SRF).
FIG. 11 is a bar graph depicting responsiveness of human PBMC to the ribonucleoside vanadyl complexes (RVCs). X denotes resiquimod.
FIG. 12 is a series of three bar graphs depicting responsiveness of human TLR7 and human TLR8 to individual ribonucleosides. X denotes resiquimod.
FIG. 13 is a series of three bar graphs depicting responsiveness of TLR7 and TLR8 to mixtures of two ribonucleosides.
FIG. 14 is a bar graph depicting response of human PBMC to a mixture of the ribonucleosides G and U.
FIG. 15 is a bar graph depicting response of human PBMC to G,U-rich RNA, but not DNA, oligonucleotides.
FIG. 16 is a bar graph depicting response of human PBMC to oxidized RNA.
FIG. 17 is a series of three bar graphs depicting human TLR7 and TLR8 responses to oxidized guanosine ribonucleoside. X denotes resiquimod.
FIG. 18 is a pair of bar graphs depicting human TLR7 responses to modified guanosine ribonucleosides.
FIG. 19 is a series of aligned gel images depicting differential expression of TLR1-TLR9 on human CD123+ dendritic cells (CD123+ DC), CD11c+ DC, and neutrophils.
FIG. 20 is a series of three graphs depicting the ability of short, single-stranded G,U-containing RNA oligomers to induce NF-κB in HEK-293 cells stably transfected with expression plasmid for human TLR7 or human TLR8.
DETAILED DESCRIPTION OF THE INVENTION
The invention relates in part to the discovery by the inventors of a number of RNA and RNA-related molecules that are effective as immunostimulatory compounds. Identification of the immunostimulatory compounds arose through a systematic effort aimed at identifying naturally occurring ligands for TLR7 and TLR8. As a result of this effort, it has now been discovered that RNA and RNA-like molecules containing guanine (G) and uracil (U), including specific sequences containing G and U, are immunostimulatory and appear to act through an MyD88-dependent pathway, implicating TLR involvement. Significantly, some of the RNA sequences occur in highly conserved structural features of 5′ untranslated regions of viral RNA that are important to viral replication. The identified immunostimulatory RNA sequences also correspond to or very nearly correspond to other RNAs, including tRNAs derived from bacteria and yeast, as well as rRNA derived from bacteria and possibly some eukaryotes. Importantly, the immunostimulatory RNA of the invention includes single-stranded RNA, in addition to partially or wholly double-stranded RNA, and its effect can be abrogated by RNase treatment. Where the RNA is at least partially double-stranded, it can in one embodiment include a stem-loop structure. As described in greater detail below, it has been discovered according to the invention that single-stranded G,U-rich RNAs as short as 5 nucleotides long can stimulate immune cells to produce large amounts of a number of cytokines and chemokines, including TNF-α, IL-6, IL-12, type 1 interferon (e.g., IFN-α), and IP-10.
It has now been surprisingly discovered by the inventors that certain G,U-containing RNA molecules and their analogs, but not their DNA counterparts, are immunostimulatory. Significantly, the G,U-containing oligoribonucleotides of the invention can be substantially smaller than the messenger RNAs previously described to be useful in preparing dendritic cell vaccines. See, e.g., Boczkowski D et al. (1996) J Exp Med 184:465-72; Mitchell D A et al. (2000) Curr Opin Mol Ther 2:176-81. Although the G,U-containing RNA molecules of the invention can be surrogates for ribosomal RNA and/or viral RNA as found in nature, they can be as small as 5-40 nucleotides long. As described further herein, the G,U-containing oligoribonucleotides of the invention include at least one G and at least one U. Surprisingly, elimination of either G or U from the G,U-containing oligoribonucleotides of the invention essentially abrogates their immunostimulatory effect. The at least one G and at least U can be adjacent to one another, or they can be separated by intervening nucleosides or sequence. Also significantly, the immunostimulatory G,U-containing RNA molecules of the invention do not require a CpG dinucleotide.
In one aspect the invention provides an immunostimulatory composition. The immunostimulatory composition according to this aspect of the invention includes an isolated RNA oligomer 5-40 nucleotides long having a base sequence having at least one guanine (G) and at least one uracil (U). As will be described in greater detail further below, the immunostimulatory RNA oligomer 5-40 nucleotides long having a base sequence having at least one guanine (G) and at least one uracil (U) is advantageously formulated such that the RNA oligomer is stabilized against degradation, concentrated in or on a particle such as a liposome, and/or targeted for delivery to the endosomal compartment of cells. In one formulation, described in the examples below, the RNA oligomer is advantageously combined with the cationic lipid DOTAP, which is believed to assist in trafficking the G,U-containing oligoribonucleotides into the endosomal compartment. Thus, in one aspect the invention is an immunostimulatory composition which includes an RNA oligomer 5-40 nucleotides long having a base sequence having at least one G and at least one U and optionally a cationic lipid.
The RNA oligomer of the invention can be of natural or non-natural origin. RNA as it occurs in nature is a type of nucleic acid that generally refers to a linear polymer of certain ribonucleoside units, each ribonucleoside unit made up of a purine or pyrimidine base and a ribose sugar, linked by internucleoside phosphodiester bonds. In this regard “linear” is meant to describe the primary structure of RNA. RNA in general can be single-stranded or double-stranded, including partially double-stranded.
As used herein, “nucleoside” refers to a single sugar moiety (e.g., ribose or deoxyribose) linked to an exchangeable organic base, which is either a substituted pyrimidine (e.g., cytosine (C), thymidine (T) or uracil (U)) or a substituted purine (e.g., adenine (A) or guanine (G)). As described herein, the nucleoside may be a naturally occuring nucleoside, a modified nucleoside, or a synthetic (artificial) nucleoside.
The terms “nucleic acid” and “oligonucleotide” are used interchangeably to mean multiple nucleotides (i.e., molecules comprising a sugar (e.g., ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g., cytosine (C), thymidine (T) or uracil (U)) or a substituted purine (e.g., adenine (A) or guanine (G)). As used herein, the terms refer to oligoribonucleotides as well as oligodeoxyribonucleotides. The terms shall also include polynucleosides (i.e., a polynucleotide minus the phosphate) and any other organic base-containing polymer. Nucleic acid molecules can be obtained from existing nucleic acid sources (e.g., genomic or cDNA), but are preferably synthetic (e.g., produced by nucleic acid synthesis).
The terms nucleic acid and oligonucleotide also encompass nucleic acids or oligonucleotides with substitutions or modifications, such as in the bases and/or sugars. For example, they include nucleic acids having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3′ position and other than a phosphate group at the 5′ position. Thus modified nucleic acids may include a 2′-O-alkylated ribose group. In addition, modified nucleic acids may include sugars such as arabinose instead of ribose. Thus the nucleic acids may be heterogeneous in backbone composition thereby containing any possible combination of polymer units linked together such as peptide nucleic acids (which have amino acid backbone with nucleic acid bases). In some embodiments, the nucleic acids are homogeneous in backbone composition. Nucleic acids also include substituted purines and pyrimidines such as C-5 propyne modified bases. Wagner R W et al. (1996) Nat Biotechnol 14:840-4. Purines and pyrimidines include but are not limited to adenine, cytosine, guanine, thymidine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, and other naturally and non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties. Other such modifications are well known to those of skill in the art.
A natural nucleoside base can be replaced by a modified nucleoside base, wherein the modified nucleoside base is for example selected from hypoxanthine; dihydrouracil; pseudouracil; 2-thiouracil; 4-thiouracil; 5-aminouracil; 5-(C1-C6)-alkyluracil; 5-(C2-C6)-alkenyluracil; 5-(C2-C6)-alkynyluracil; 5-(hydroxymethyl)uracil; 5-chlorouracil; 5-fluorouracil; 5-bromouracil; 5-hydroxycytosine; 5-(C1-C6)-alkylcytosine; 5-(C2-C6)-alkenylcytosine; 5-(C2-C6)-alkynylcytosine; 5-chlorocytosine; 5-fluorocytosine; 5-bromocytosine; N2-dimethylguanine; 2,4-diamino-purine; 8-azapurine (including, in particular, 8-azaguanine); a substituted 7-deazapurine (including, in particular, 7-deazaguanine), including 7-deaza-7-substituted and/or 7-deaza-8-substituted purine; or other modifications of a natural nucleoside bases. This list is meant to be exemplary and is not to be interpreted to be limiting.
In particular, the at least one guanine base of the immunostimulatory G,U-containing oligoribonucleotide can be a substituted or modified guanine such as 7-deazaguanine; 8-azaguanine; 7-deaza-7-substituted guanine (such as 7-deaza-7-(C2-C6)alkynylguanine); 7-deaza-8-substituted guanine; hypoxanthine; 2,6-diaminopurine; 2-aminopurine; purine; 8-substituted guanine such as 8-hydroxyguanine; and 6-thioguanine. This list is meant to be exemplary and is not to be interpreted to be limiting.
Also in particular, the at least one uracil base of the G,U-containing oligoribonucleotide can be a substituted or modified uracil such as pseudouracil and 5-methyluracil.
For use in the instant invention, the nucleic acids of the invention can be synthesized de novo using any of a number of procedures well known in the art. For example, the β-cyanoethyl phosphoramidite method (Beaucage S L et al. (1981) Tetrahedron Lett 22:1859); nucleoside H-phosphonate method (Garegg et al. (1986) Tetrahedron Lett 27:4051-4; Froehler et al. (1986) Nucl Acid Res 14:5399-407; Garegg et al. (1986) Tetrahedron Lett 27:4055-8; Gaffney et al. (1988) Tetrahedron Lett 29:2619-22). These chemistries can be performed by a variety of automated nucleic acid synthesizers available in the market. These nucleic acids are referred to as synthetic nucleic acids. Alternatively, T-rich and/or TG dinucleotides can be produced on a large scale in plasmids, (see Sambrook T et al., “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor laboratory Press, New York, 1989) and separated into smaller pieces or administered whole. Nucleic acids can be prepared from existing nucleic acid sequences (e.g., genomic or cDNA) using known techniques, such as those employing restriction enzymes, exonucleases or endonucleases. Nucleic acids prepared in this manner are referred to as isolated nucleic acid. An isolated nucleic acid generally refers to a nucleic acid which is separated from components which it is normally associated with in nature. As an example, an isolated nucleic acid may be one which is separated from a cell, from a nucleus, from mitochondria or from chromatin. The term “nucleic acid” encompasses both synthetic and isolated nucleic acid.
For use in vivo, the nucleic acids may optionally be relatively resistant to degradation (e.g., are stabilized). In some embodiments only specific portions of the nucleic acids may optionally be stabilized. A “stabilized nucleic acid molecule” shall mean a nucleic acid molecule that is relatively resistant to in vivo degradation (e.g., via an exo- or endo-nuclease). Stabilization can be a function of length or secondary structure. Nucleic acids that are tens to hundreds of kbs long are relatively resistant to in vivo degradation. For shorter nucleic acids, secondary structure can stabilize and increase their effect. For example, if the 3′ end of an nucleic acid has self-complementarity to an upstream region, so that it can fold back and form a sort of stem loop structure, then the nucleic acid becomes stabilized and therefore exhibits more activity.
In certain embodiments according to this aspect of the invention, the base sequence of the RNA oligomer is at least partially self-complementary. A self-complementary sequence as used herein refers to a base sequence which, upon suitable alignment, may form intramolecular or, more typically, intermolecular basepairing between G-C, A-U, and/or G-U wobble pairs. In one embodiment the extent of self-complementarity is at least 50 percent. For example an 8-mer that is at least 50 percent self-complementary may have a sequence capable of forming 4, 5, 6, 7, or 8 G-C, A-U, and/or G-U wobble basepairs. Such basepairs may but need not necessarily involve bases located at either end of the self-complementary RNA oligomer. Where nucleic acid stabilization may be important to the RNA oligomers, it may be advantageous to “clamp” together one or both ends of a double-stranded nucleic acid, either by basepairing or by any other suitable means. The degree of self-complementarity may depend on the alignment between oligomers, and such alignment may or may not include single- or multiple-nucleoside overhangs. In other embodiments, the degree of self-complementarity is at least 60 percent, at least 70 percent, at least 80 percent, at least 90 percent, or even 100 percent. The foregoing notwithstanding, it should be noted that double-strandedness is not a requirement of the RNA oligomers of the invention.
Similar considerations apply to intermolecular basepairing between RNA oligonucleotides of different base sequence. Thus where a plurality of RNA oligomers are used together, the plurality of oligomers may, but need not, include sequences which are at least partially complementary to one another. In one embodiment the plurality of oligomers includes an oligomer having a first base sequence and an oligomer having a second base sequence, wherein the first base sequence and the second base sequence are at least 50 percent complementary. For example, as between two 8-mers that are at least 50 percent complementary, they may form 4, 5, 6, 7, or 8 G-C, A-U, and/or G-U wobble basepairs. Such basepairs may but need not necessarily involve bases located at either end of the complementary RNA oligomers. The degree of complementarity may depend on the alignment between oligomers, and such alignment may or may not include single- or multiple-nucleoside overhangs. In other embodiments, the degree of complementarity is at least 60 percent, at least 70 percent, at least 80 percent, at least 90 percent, or even 100 percent.
Alternatively, nucleic acid stabilization can be accomplished via phosphate backbone modifications. Preferred stabilized nucleic acids of the instant invention have a modified backbone. It has been demonstrated that modification of the nucleic acid backbone provides enhanced activity of the nucleic acids when administered in vivo. One type of modified backbone is a phosphate backbone modification. Inclusion in immunostimulatory nucleic acids of at least two phosphorothioate linkages at the 5′ end of the oligonucleotide and multiple (preferably five) phosphorothioate linkages at the 3′ end, can in some circumstances provide maximal activity and protect the nucleic acid from degradation by intracellular exo- and endonucleases. Other modified nucleic acids include phosphodiester-modified nucleic acids, combinations of phosphodiester and phosphorothioate nucleic acids, alkylphosponate and arylphosphonate, alkylphosphorothioate and arylphosphorothioate, methylphosphonate, methylphosphorothioate, phosphorodithioate, p-ethoxy, morpholino, and combinations thereof. Nucleic acids having phosphorothioate linkages provide maximal activity and protect the nucleic acid from degradation by intracellular exo- and endo-nucleases. and combinations thereof. Each of these combinations and their particular effects on immune cells is discussed in more detail with respect to CpG nucleic acids in issued U.S. Pat. Nos. 6,207,646 and 6,239,116, the entire contents of which are hereby incorporated by reference. It is believed that these modified nucleic acids may show more stimulatory activity due to enhanced nuclease resistance, increased cellular uptake, increased protein binding, and/or altered intracellular localization.
Modified backbones such as phosphorothioates may be synthesized using automated techniques employing either phosphoramidate or H-phosphonate chemistries. Aryl- and alkyl-phosphonates can be made, e.g., as described in U.S. Pat. No. 4,469,863; and alkylphosphotriesters (in which the charged oxygen moiety is alkylated as described in U.S. Pat. No. 5,023,243 and European Pat. No. 092,574) can be prepared by automated solid phase synthesis using commercially available reagents. Methods for making other DNA backbone modifications and substitutions have been described. Uhlmann E et al. (1990) Chem Rev 90:544; Goodchild J (1990) Bioconjugate Chem 1:165.
Other stabilized nucleic acids include: nonionic DNA analogs, such as alkyl- and aryl-phosphates (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), phosphodiester and alkylphosphotriesters, in which the charged oxygen moiety is alkylated. Nucleic acids which contain diol, such as tetraethyleneglycol or hexaethyleneglycol, at either or both termini have also been shown to be substantially resistant to nuclease degradation.
Another class of backbone modifications include 2′-O-methylribonucleosides (2′-OMe). These types of substitutions are described extensively in the prior art and in particular with respect to their immunostimulating properties in Zhao et al. (1999) Bioorg Med Chem Lett 9:24:3453-8. Zhao et al. describes methods of preparing 2′-OMe modifications to nucleic acids.
The immunostimulatory G,U-containing RNA oligomers of the invention are typically about 5 to about 40 nucleotides long. Thus in certain distinct embodiments, the G,U-containing RNA oligomer can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides long. In one embodiment the G,U-containing RNA oligomer can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides long. In one embodiment the G,U-containing RNA oligomer can be 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides long. In one embodiment the G,U-containing RNA oligomer can be 8, 9, 10, 11, or 12 nucleotides long.
For example, RNA oligomers with the following base sequences have been discovered to be useful in the compositions and practice of the invention: 5′-GUGUUUAC-3′; 5′-GUAGGCAC-3′; 5′-CUAGGCAC-3′; 5′-CUCGGCAC-3′; and 5′-GUGUUUAC-3′ in combination with 5′-GUAGGCAC-3′.
Because the immunostimulatory effects of the G,U-containing RNA oligomers of the invention have been discovered to be MyD88-dependent, it is the belief of the inventors that the immunostimulatory G,U-containing RNA oligomers of the invention may interact with at least one TLR as a step in exerting their immunostimulatory effect. The immunostimulatory G,U-containing RNA oligomers of the invention may thus represent or mimic at least portions of natural ligands for the at least one TLR. Such natural ligands may include ribosomal RNA, either prokaryotic or eukaryotic, as well as certain viral RNAs. The TLR or TLRs may be TLR8, TLR7, or some yet-to-be defined TLR. Natural ligands for TLR1, TLR7, TLR8, and TLR10 have not previously been described.
The immunostimulatory RNA molecules of the invention have been discovered to occur in nature in all types of RNA, usually in association with highly conserved sequence or key structural feature. In one example, immunostimulatory RNA has been discovered to occur in the context of an internal ribosome entry site (IRES).
An IRES is a minimal cis-acting RNA element contained within a complex structural feature in the 5′ untranslated region (5′ UTR) of viral RNA and other mRNAs that regulates the initiation of translation of the viral genome in a cap-independent manner. Hellen C U et al. (2001) Genes Dev 15:1593-1612. Cap-independent initiation of viral RNA translation was first observed in picornaviruses. Jackson R J et al. (1990) Trends Biochem Sci 15:477-83; Jackson R J et al. (1995) RNA 1:985-1000.
In most eukaryotic cells, mRNA translation initiation commences with recruitment of the cap binding complex eukaryotic initiation factor (eIF)4F, composed of eIF4E (cap binding protein), eIF4A, and eIF4G, to the 5′ capped end of the mRNA. The 40S ribosomal subunit, carrying eIF3, and the ternary initiator complex tRNA-eIF2-GTP are then recruited to the 5′ end of the mRNA through interaction between eIF3 and eIF4G. The 40S subunit then scans the mRNA in a 5′ to 3′ direction until it encounters an appropriate start codon, whereupon the anticodon of initiator methionine-tRNA is engaged, the 60S subunit joins to form an 80S ribosome, and translation commences.
Thus the significance of an IRES, at least in the context of a virus, is believed to be the ability of the IRES to confer a selective advantage to the virus over usual cap-dependent translation in the cell.
The following viruses have been reported to have IRES elements in their genome: all picornaviruses; bovine viral diarrhea virus; classic swine fever virus; cricket paralysis virus; encephalomyocarditis virus; foot-and-mouth disease virus; Friend murine leukemia virus gag mRNA; HCV; human immunodeficiency virus env mRNA; Kaposi's sarcoma-associated herpesvirus; Moloney murine leukemia virus gag mRNA; Plautia stali intestine virus; poliovirus; rhinovirus; Rhopalosiphum padi virus; and Rous sarcoma virus. Hellen C U et al. (2001) Genes Dev 15:1593-1612. This list is not intended to be limiting.
The viral proteins of hepatitis C virus (HCV) are translated from a 9.5 kb single-stranded positive sense RNA which is flanked by 5′ and 3′ UTRs. The highly conserved 5′ UTR includes an IRES present in nt 40-370. Reynolds J E et al. (1996) RNA 2:867-78. The HCV 5′ UTR is believed to have four major structural domains (I-IV), of which domains II and III have subdomains. Subdomain IIId includes a 27 nt stem-loop (nt 253-279) that on the basis of in vivo mutational studies has been reported to be critical in HCV IRES-mediated translation. Kieft J S et al. (1999) J Mol Biol 292:513-29; Klinck R et al. (2000) RNA 6:1423-31. The sequence of the IIId 27-mer is provided by 5′-GCCGAGUAGUGUUGGGUCGCGAAAGGC-3′(SEQ ID NO:4), wherein the UUGGGU forms the terminal loop. The stem-loop structure is reported to include a number of non-Watson-Crick base pairs, typical of other RNAs, including wobble U∘G, U∘A, G∘A, and A∘A base pairs.
As another example, the immunostimulatory RNA sequences of the invention have been discovered to occur in G,U-rich sequence near the 5′ end of the viral RNA of human immunodeficiency virus type 1 (HIV-1) that is crucial to efficient viral RNA packaging. Russell R S et al. (2002) Virology 303:152-63. Specifically, two key G,U-rich sequences within U5, namely 5′-GUAGUGUGUG-3′ (SEQ ID NO:2) and 5′-GUCUGUUGUGUG-3′ (SEQ ID NO:3), corresponding to nt 99-108 and 112-123 of strain BH10, respectively, have been found according to the present invention to be highly immunostimulatory (see Example 11 below). It will be noted that SEQ ID NO:2 includes both GUAGU and GUGUG, and SEQ ID NO:3 includes GUGUG.
As yet another example, the immunostimulatory RNA sequences of the invention have been found to occur in 5S ribosomal RNA loop E of a large number of species of bacteria.
TLR8 and TLR7 show high sequence homology to TLR9 (FIG. 8). TLR9 is the CpG-DNA receptor and transduces immunostimulatory signals. Two DNA binding motifs have been described in TLR9 (U.S. patent application Ser. No. 09/954,987) that are also present in TLR8 and TLR7 with some modifications (FIG. 9). Despite this similarity, however, TLR7 and TLR8 do not bind CpG-DNA.
It has been discovered according to the present invention that guanosine, particularly guanosine in combination with uracil, and certain guanosine-containing nucleic acids and derivatives thereof, are natural ligands of TLR8. It has been discovered according to the present invention that RNA, oxidized RNA, G,U-rich nucleic acids, and at least partially double-stranded nucleic acid molecules having at least one G-U base pair are TLR8 ligands. In certain preferred embodiments involving guanosine, guanosine derivatives, and G,U-rich nucleic acids, guanosine is the ribonucleoside. Nucleic acid molecules containing GUU, GUG, GGU, GGG, UGG, UGU, UUG, UUU, multiples and any combinations thereof are believed to be TLR8 ligands. In some embodiments the TLR8 ligand is a G,U-rich oligonucleotide that includes a hexamer sequence (UUGUGG)n, (UGGUUG)n, (GUGUGU)n, or (GGGUUU)n where n is an integer from 1 to 8, and preferably n is at least 3. In addition, it has also been discovered according to the present invention that mixtures of ribonucleoside vanadyl complexes (i.e., mixtures of adenine, cytosine, guanosine, and uracil ribonucleoside vanadyl complexes), and guanosine ribonucleoside vanadyl complexes alone, are TLR8 ligands. In addition, it has been discovered according the present invention that certain imidazoquinolines, including resiquimod and imiquimod, are TLR8 ligands.
It has also been discovered according to the present invention that guanosine, and certain guanosine-containing nucleic acids and derivatives thereof, are natural ligands of TLR7. It has been discovered according to the present invention that RNA, oxidized RNA, G-rich nucleic acids, and at least partially double-stranded nucleic acid molecules that are rich in G content are TLR7 ligands. In certain preferred embodiments involving guanosine, guanosine derivatives, and G-rich nucleic acids, guanosine is the ribonucleoside. In addition, it has also been discovered according to the present invention that mixtures of ribonucleoside vanadyl complexes (i.e., mixtures of adenine, cytosine, guanosine, and uracil ribonucleoside vanadyl complexes), and guanosine ribonucleoside vanadyl complexes alone, are TLR7 ligands. In addition, it has been discovered according the present invention that 7-allyl-8-oxoguanosine (loxoribine) is a TLR7 ligand.
In addition to having diverse ligands, the various TLRs are believed to be differentially expressed in various tissues and on various types of immune cells. For example, human TLR7 has been reported to be expressed in placenta, lung, spleen, lymph nodes, tonsil and on plasmacytoid precursor dendritic cells (pDCs). Chuang T-H et al. (2000) Eur Cytokine Netw 11:372-8); Kadowaki N et al. (2001) J Exp Med 194:863-9. Human TLR8 has been reported to be expressed in lung, peripheral blood leukocytes (PBL), placenta, spleen, lymph nodes, and on monocytes. Kadowaki N et al. (2001) J Exp Med 194:863-9; Chuang T-H et al. (2000) Eur Cytokine Netw 11:372-8. Human TLR9 is reportedly expressed in spleen, lymph nodes, bone marrow, PBL, and on pDCs, B cells, and CD123+ DCs. Kadowaki N et al. (2001) J Exp Med 194:863-9; Bauer S et al. (2001) Proc Natl Acad Sci USA 98:9237-42; Chuang T-H et al. (2000) Eur Cytokine Netw 11:372-8.
Guanosine derivatives have previously been described as B-cell and NK cell activators, but their receptors and mechanism of action were not understood. Goodman M G et al. (1994) J Pharm Exp Ther 274:1552-57; Reitz A B et al. (1994) J Med Chem 37:3561-78. Such guanosine derivatives include, but are not limited to, 8-bromoguanosine, 8-oxoguanosine, 8-mercaptoguanosine, and 7-allyl-8-oxoguanosine (loxoribine).
Imidazoquinolines are synthetic small molecule immune response modifiers thought to induce expression of several cytokines including interferons (e.g., IFN-α and IFN-γ), tumor necrosis factor alpha (TNF-α) and some interleukins (e.g., IL-1, IL-6 and IL-12). Imidazoquinolines are capable of stimulating a Th1 immune response, as evidenced in part by their ability to induce increases in IgG2a levels. Imidazoquinoline agents reportedly are also capable of inhibiting production of Th2 cytokines such as IL-4, IL-5, and IL-13. Some of the cytokines induced by imidazoquinolines are produced by macrophages and dendritic cells. Some species of imidazoquinolines have been reported to increase NK cell lytic activity and to stimulate B-cell proliferation and differentiation, thereby inducing antibody production and secretion.
As used herein, an imidazoquinoline agent includes imidazoquinoline amines, imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine amines, and 1,2 bridged imidazoquinoline amines. These compounds have been described in U.S. Pat. Nos. 4,689,338, 4,929,624, 5,238,944, 5,266,575, 5,268,376, 5,346,905, 5,352,784, 5,389,640, 5,395,937, 5,494,916, 5,482,936, 5,525,612, 6,039,969 and 6,110,929. Particular species of imidazoquinoline agents include 4-amino-α,α-dimethyl-2-ethoxymethyl-1H-imidazo[4,5-c]quinoline-1-ethanol (resiquimod or R-848 or S-28463; PCT/US01/28764, WO 02/22125); and 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-4-amine (imiquimod or R-837 or S-26308). Imiquimod is currently used in the topical treatment of warts such as genital and anal warts and has also been tested in the topical treatment of basal cell carcinoma.
Nucleotide and amino acid sequences of human and murine TLR3 are known. See, for example, GenBank Accession Nos. U88879, NM—003265, NM—126166, AF355152; and AAC34134, NP—003256, NP—569054, AAK26117. Human TLR3 is reported to be 904 amino acids long and to have a sequence provided in SEQ ID NO:20. A corresponding nucleotide sequence is provided as SEQ ID NO:21. Murine TLR3 is reported to be 905 amino acids long and to have a sequence as provided in SEQ ID NO:22. A corresponding nucleotide sequence is provided as SEQ ID NO:23. TLR3 polypeptide includes an extracellular domain having leucine-rich repeat region, a transmembrane domain, and an intracellular domain that includes a TIR domain.
As used herein a “TLR3 polypeptide” refers to a polypeptide including a full-length TLR3 according to one of the sequences above, orthologs, allelic variants, SNPs, variants incorporating conservative amino acid substitutions, TLR3 fusion proteins, and functional fragments of any of the foregoing. Preferred embodiments include human TLR3 polypeptides having at least 65 percent sequence identity, more preferably at least 80 percent sequence identity, even more preferably with at least 90 percent sequence identity, and most preferably with at least 95 percent sequence identity with the human TLR3 amino acid sequence of SEQ ID NO:20. Preferred embodiments also include murine TLR3 polypeptides having at least 65 percent sequence identity, more preferably at least 80 percent sequence identity, even more preferably with at least 90 percent sequence identity, and most preferably with at least 95 percent sequence identity with the murine TLR3 amino acid sequence of SEQ ID NO:22.
As used herein “TLR3 signaling” refers to an ability of a TLR3 polypeptide to activate the TLR/IL-1R (TIR) signaling pathway, also referred to herein as the TLR signal transduction pathway. Changes in TLR3 activity can be measured by assays such as those disclosed herein, including expression of genes under control of κB-sensitive promoters and enhancers. Such naturally occurring genes include the genes encoding IL-1β, IL-6, IL-8, the p40 subunit of interleukin 12 (IL-12 p40), and the costimulatory molecules CD80 and CD86. Other genes can be placed under the control of such regulatory elements (see below) and thus serve to report the level of TLR3 signaling. Additional nucleotide sequence can be added to SEQ ID NO:21 or SEQ ID NO:23, preferably to the 5′ or the 3′ end of the open reading frame of SEQ ID NO:21, to yield a nucleotide sequence encoding a chimeric polypeptide that includes a detectable or reporter moiety, e.g., FLAG, luciferase (luc), green fluorescent protein (GFP), and others known by those skilled in the art.
SEQ ID NO:20 Human TLR3 amino acid
MRQTLPCIYF WGGLLPFGML CASSTTKCTV SHEVADCSHL KLTQVPDDLP TNITVLNLTH
60
NQLRRLPAAN FTRYSQLTSL DVGFNTISKL EPELCQKLPM LKVLNLQHNE LSQLSDKTFA
120
FCTNLTELHL MSNSIQKIKN NPFVKQKNLI TLDLSHNGLS STKLGTQVQL ENLQELLLSN
180
NKIQALKSEE LDIFANSSLK KLELSSNQIK EFSPGCFHAI GRLFGLFLNN VQLGPSLTEK
240
LCLELANTSI RNLSLSNSQL STTSNTTFLG LKWTNLTMLD LSYNNLNVVG NDSFAWLPQL
300
EYFFLEYNNI QHLFSHSLHG LFNVRYLNLK RSFTKQSISL ASLPKIDDFS FQWLKCLEHL
360
NMEDNDIPGI KSNMFTGLIN LKYLSLSNSF TSLRTLTNET FVSLAHSPLH ILNLTKMKIS
420
KIESDAFSWL GHLEVLDLGL NEIGQELTGQ EWRGLENIFE IYLSYNKYLQ LTRNSFALVP
480
SLQRLMLRRV ALKNVDSSPS PFQPLRNLTI LDLSNNNIAN INDDMLEGLE KLEILDLQHN
540
NLARLWKHAN PGGPIYFLKG LSHLHILNLE SNGFDEIPVE VFKDLFELKI IDLGLNNLNT
600
LPASVFNNQV SLKSLNLQKN LITSVEKKVF GPAFRNLTEL DMRFNPFDCT CESIAWFVNW
660
INETHTNIPE LSSHYLCNTP PHYHGFPVRL FDTSSCKDSA PFELFFMINT SILLIFIFIV
720
LLIHFEGWRI SFYWNVSVHR VLGFKEIDRQ TEQFEYAAYI IHAYKDKDWV WEHFSSMEKE
780
DQSLKFCLEE RDFEAGVFEL EAIVNSIKRS RKIIFVITHH LLKDPLCKRF KVHHAVQQAI
840
EQNLDSIILV FLEEIPDYKL NHALCLRRGM FKSHCILNWP VQKERIGAFR HKLQVALGSK
900
NSVH
904
SEQ ID NO:21 Human TLR3 nucleotide
cactttcgag agtgccgtct atttgccaca cacttccctg atgaaatgtc tggatttgga
60
ctaaagaaaa aaggaaaggc tagcagtcat ccaacagaat catgagacag actttgcctt
120
gtatctactt ttgggggggc cttttgccct ttgggatgct gtgtgcatcc tccaccacca
180
agtgcactgt tagccatgaa gttgctgact gcagccacct gaagttgact caggtacccg
240
atgatctacc cacaaacata acagtgttga accttaccca taatcaactc agaagattac
300
cagccgccaa cttcacaagg tatagccagc taactagctt ggatgtagga tttaacacca
360
tctcaaaact ggagccagaa ttgtgccaga aacttcccat gttaaaagtt ttgaacctcc
420
agcacaatga gctatctcaa ctttctgata aaacctttgc cttctgcacg aatttgactg
480
aactccatct catgtccaac tcaatccaga aaattaaaaa taatcccttt gtcaagcaga
540
agaatttaat cacattagat ctgtctcata atggcttgtc atctacaaaa ttaggaactc
600
aggttcagct ggaaaatctc caagagcttc tattatcaaa caataaaatt caagcgctaa
660
aaagtgaaga actggatatc tttgccaatt catctttaaa aaaattagag ttgtcatcga
720
atcaaattaa agagttttct ccagggtgtt ttcacgcaat tggaagatta tttggcctct
780
ttctgaacaa tgtccagctg ggtcccagcc ttacagagaa gctatgtttg gaattagcaa
840
acacaagcat tcggaatctg tctctgagta acagccagct gtccaccacc agcaatacaa
900
ctttcttggg actaaagtgg acaaatctca ctatgctcga tctttcctac aacaacttaa
960
atgtggttgg taacgattcc tttgcttggc ttccacaact agaatatttc ttcctagagt
1020
ataataatat acagcatttg ttttctcact ctttgcacgg gcttttcaat gtgaggtacc
1080
tgaatttgaa acggtctttt actaaacaaa gtatttccct tgcctcactc cccaagattg
1140
atgatttttc ttttcagtgg ctaaaatgtt tggagcacct taacatggaa gataatgata
1200
ttccaggcat aaaaagcaat atgttcacag gattgataaa cctgaaatac ttaagtctat
1260
ccaactcctt tacaagtttg cgaactttga caaatgaaac atttgtatca cttgctcatt
1320
ctcccttaca catactcaac ctaaccaaga ataaaatctc aaaaatagag agtgatgctt
1380
tctcttggtt gggccaccta gaagtacttg acctgggcct taatgaaatt gggcaagaac
1440
tcacaggcca ggaatggaga ggtctagaaa atattttcga aatctatctt tcctacaaca
1500
agtacctgca gctgactagg aactcctttg ccttggtccc aagccttcaa cgactgatgc
1560
tccgaagggt ggcccttaaa aatgtggata gctctccttc accattccag cctcttcgta
1620
acttgaccat tctggatcta agcaacaaca acatagccaa cataaatgat gacatgttgg
1680
agggtcttga gaaactagaa attctcgatt tgcagcataa caacttagca cggctctgga
1740
aacacgcaaa ccctggtggt cccatttatt tcctaaaggg tctgtctcac ctccacatcc
1800
ttaacttgga gtccaacggc tttgacgaga tcccagttga ggtcttcaag gatttatttg
1860
aactaaagat catcgattta ggattgaata atttaaacac acttccagca tctgtcttta
1920
ataatcaggt gtctctaaag tcattgaacc ttcagaagaa tctcataaca tccgttgaga
1980
agaaggtttt cgggccagct ttcaggaacc tgactgagtt agatatgcgc tttaatccct
2040
ttgattgcac gtgtgaaagt attgcctggt ttgttaattg gattaacgag acccatacca
2100
acatccctga gctgtcaagc cactaccttt gcaacactcc acctcactat catgggttcc
2160
cagtgagact ttttgataca tcatcttgca aagacagtgc cccctttgaa ctctttttca
2220
tgatcaatac cagtatcctg ttgattttta tctttattgt acttctcatc cactttgagg
2280
gctggaggat atctttttat tggaatgttt cagtacatcg agttcttggt ttcaaagaaa
2340
tagacagaca gacagaacag tttgaatatg cagcatatat aattcatgcc tataaagata
2400
aggattgggt ctgggaacat ttctcttcaa tggaaaagga agaccaatct ctcaaatttt
2460
gtctggaaga aagggacttt gaggcgggtg tttttgaact agaagcaatt gttaacagca
2520
tcaaaagaag cagaaaaatt atttttgtta taacacacca tctattaaaa gacccattat
2580
gcaaaagatt caaggtacat catgcagttc aacaagctat tgaacaaaat ctggattcca
2640
ttatattggt tttccttgag gagattccag attataaact gaaccatgca ctctgtttgc
2700
gaagaggaat gtttaaatct cactgcatct tgaactggcc agttcagaaa gaacggatag
2760
gtgcctttcg tcataaattg caagtagcac ttggatccaa aaactctgta cattaaattt
2820
atttaaatat tcaattagca aaggagaaac tttctcaatt taaaaagttc tatggcaaat
2880
ttaagttttc cataaaggtg ttataatttg tttattcata tttgtaaatg attatattct
2940
atcacaatta catctcttct aggaaaatgt gtctccttat ttcaggccta tttttgacaa
3000
ttgacttaat tttacccaaa ataaaacata taagcacgta aaaaaaaaaa aaaaaaa
3057
SEQ ID NO:22 Murine TLR3 amino acid
MKGCSSYLMY SFGGLLSLWI LLVSSTNQCT VRYNVADCSH LKLTHIPDDL PSNITVLNLT
60
HNQLRRLPPT NFTRYSQLAI LDAGFNSISK LEPELCQILP LLKVLNLQHN ELSQISDQTF
120
VFCTNLTELD SMSNSIHKIK SNPFKNQKNL IKLDLSHNGL SSTKLGTGVQ LENLQELLLA
180
KNKILALRSE ELEFLGNSSL RKLDLSSNPL KEFSPGCFQT IGKLFALLLN NAQLNPHLTE
240
KLCWELSNTS IQNLSLANNQ LLATSESTFS GLKWTNLTQL DLSYNNLHDV GNGSFSYLPS
300
LRYLSLEYNN IQRLSPRSFY GLSNLRYLSL KRAFTKQSVS LASHPNIDDF SFQWLKYLEY
360
LNMDDNNIPS TKSNTFTGLV SLKYLSLSKT FTSLQTLTNE TFVSLAHSPL LTLNLTKNHI
420
SKIANGTFSW LGQLRILDLG LNEIEQKLSG QEWRGLRNIF EIYLSYNKYL QLSTSSFALV
480
PSLQRLMLRR VALKNVDISP SPFRPLRNLT ILDLSNNNIA NINEDLLEGL ENLEILDFQH
540
NNLARLWKRA NPGGPVNFLK GLSHLHILNL ESNGLDEIPV GVFKNLFELK SINLGLNNLN
600
KLEPFIFDDQ TSLRSLNLQK NLITSVEKDV FGPPFQNLNS LDMRFNPFDC TCESISWFVN
660
WINQTHTNIF ELSTHYLCNT PHHYYGFPLK LFDTSSCKDS APFELLFIIS TSMLLVFILV
720
VLLIHIEGWR ISFYWNVSVH RILGFKBIDT QAEQFEYTAY IIHAHKDRDW VWEHFSPMEE
780
QDQSLKFCLE ERDFEAGVLG LEAIVNSIKR SRKIIFVITH HLLKDPLCRR FKVHHAVQQA
840
IEQNLDSIIL IFLQNIPDYK LNHALCLRRG MFKSHCILNW PVQKERINAF HHKLQVALGS
900
RNSAH
904
SEQ ID NO:23 Murine TLR3 nucleotide
tagaatatga tacagggatt gcacccataa tctgggctga atcatgaaag ggtgttcctc
60
ttatctaatg tactcctttg ggggactttt gtccctatgg attcttctgg tgtcttccac
120
aaaccaatgc actgtgagat acaacgtagc tgactgcagc catttgaagc taacacacat
180
acctgatgat cttccctcta acataacagt gttgaatctt actcacaacc aactcagaag
240
attaccacct accaacttta caagatacag ccaacttgct atcttggatg caggatttaa
300
ctccatttca aaactggagc cagaactgtg ccaaatactc cctttgttga aagtattgaa
360
cctgcaacat aatgagctct ctcagatttc tgatcaaacc tttgtcttct gcacgaacct
420
gacagaactc gatctaatgt ctaactcaat acacaaaatt aaaagcaacc ctttcaaaaa
480
ccagaagaat ctaatcaaat tagatttgtc tcataatggt ttatcatcta caaagttggg
540
aacgggggtc caactggaga acctccaaga actgctctta gcaaaaaata aaatccttgc
600
gttgcgaagt gaagaacttg agtttcttgg caattcttct ttacgaaagt tggacttgtc
660
atcaaatcca cttaaagagt tctccccggg gtgtttccag acaattggca agttattcgc
720
cctcctcttg aacaacgccc aactgaaccc ccacctcaca gagaagcttt gctgggaact
780
ttcaaacaca agcatccaga atctctctct ggctaacaac cagctgctgg ccaccagcga
840
gagcactttc tctgggctga agtggacaaa tctcacccag ctcgatcttt cctacaacaa
900
cctccatgat gtcggcaacg gttccttctc ctatctccca agcctgaggt atctgtctct
960
ggagtacaac aatatacagc gtctgtcccc tcgctctttt tatggactct ccaacctgag
1020
gtacctgagt ttgaagcgag catttactaa gcaaagtgtt tcacttgctt cacatcccaa
1080
cattgacgat ttttcctttc aatggttaaa atatttggaa tatctcaaca tggatgacaa
1140
taatattcca agtaccaaaa gcaatacctt cacgggattg gtgagtctga agtacctaag
1200
tctttccaaa actttcacaa gtttgcaaac tttaacaaat gaaacatttg tgtcacttgc
1260
tcattctccc ttgctcactc tcaacttaac gaaaaatcac atctcaaaaa tagcaaatgg
1320
tactttctct tggttaggcc aactcaggat acttgatctc ggccttaatg aaattgaaca
1380
aaaactcagc ggccaggaat ggagaggtct gagaaatata tttgagatct acctatccta
1440
taacaaatac ctccaactgt ctaccagttc ctttgcattg gtccccagcc ttcaaagact
1500
gatgctcagg agggtggccc ttaaaaatgt ggatatctcc ccttcacctt tccgccctct
1560
tcgtaacttg accattctgg acttaagcaa caacaacata gccaacataa atgaggactt
1620
gctggagggt cttgagaatc tagaaatcct ggattttcag cacaataact tagccaggct
1680
ctggaaacgc gcaaaccccg gtggtcccgt taatttcctg aaggggctgt ctcacctcca
1740
catcttgaat ttagagtcca acggcttaga tgaaatccca gtcggggttt tcaagaactt
1800
attcgaacta aagagcatca atctaggact gaataactta aacaaacttg aaccattcat
1860
ttttgatgac cagacatctc taaggtcact gaacctccag aagaacctca taacatctgt
1920
tgagaaggat gttttcgggc cgccttttca aaacctgaac agtttagata tgcgcttcaa
1980
tccgttcgac tgcacgtgtg aaagtatttc ctggtttgtt aactggatca accagaccca
2040
cactaatatc tttgagctgt ccactcacta cctctgtaac actccacatc attattatgg
2100
cttccccctg aagcttttcg atacatcatc ctgtaaagac agcgccccct ttgaactcct
2160
cttcataatc agcaccagta tgctcctggt ttttatactt gtggtactgc tcattcacat
2220
cgagggctgg aggatctctt tttactggaa tgtttcagtg catcggattc ttggtttcaa
2280
ggaaatagac acacaggctg agcagtttga atatacagcc tacataattc atgcccataa
2340
agacagagac tgggtctggg aacatttctc cccaatggaa gaacaagacc aatctctcaa
2400
attttgccta gaagaaaggg actttgaagc aggcgtcctt ggacttgaag caattgttaa
2460
tagcatcaaa agaagccgaa aaatcatttt cgttatcaca caccatttat taaaagaccc
2520
tctgtgcaga agattcaagg tacatcacgc agttcagcaa gctattgagc aaaatctgga
2580
ttcaattata ctgatttttc tccagaatat tccagattat aaactaaacc atgcactctg
2640
tttgcgaaga ggaatgttta aatctcattg catcttgaac tggccagttc agaaagaacg
2700
gataaatgcc tttcatcata aattgcaagt agcacttgga tctcggaatt cagcacatta
2760
aactcatttg aagatttgga gtcggtaaag ggatagatcc aatttataaa ggtccatcat
2820
gaatctaagt tttacttgaa agttttgtat atttatttat atgtatagat gatgatatta
2880
catcacaatc caatctcagt tttgaaatat ttcggcttat ttcattgaca tctggtttat
2940
tcactccaaa taaacacatg ggcagttaaa aacatcctct attaatagat tacccattaa
3000
ttcttgaggt gtatcacagc tttaaagggt tttaaatatt tttatataaa taagactgag
3060
agttttataa atgtaatttt ttaaaactcg agtcttactg tgtagctcag aaaggcctgg
3120
aaattaatat attagagagt catgtcttga acttatttat ctctgcctcc ctctgtctcc
3180
agagtgttgc ttttaagggc atgtagcacc.acacccagct atgtacgtgt gggattttat
3240
aatgctcatt tttgagacgt ttatagaata aaagataatt gcttttatgg tataaggcta
3300
cttgaggtaa
3310
Nucleotide and amino acid sequences of human and murine TLR7 are known. See, for example, GenBank Accession Nos. AF240467, AF245702, NM—016562, AF334942, NM—133211; and AAF60188, AAF78035, NP—057646, AAL73191, AAL73192. Human TLR7 is reported to be 1049 amino acids long and to have a sequence provided in SEQ ID NO:24. A corresponding nucleotide sequence is provided as SEQ ID NO:25. Murine TLR7 is reported to be 1050 amino acids long and to have a sequence as provided in SEQ ID NO:26. A corresponding nucleotide sequence is provided as SEQ ID NO:27. TLR7 polypeptide includes an extracellular domain having leucine-rich repeat region, a transmembrane domain, and an intracellular domain that includes a TIR domain.
As used herein a “TLR7 polypeptide” refers to a polypeptide including a full-length TLR7 according to one of the sequences above, orthologs, allelic variants, SNPs, variants incorporating conservative amino acid substitutions, TLR7 fusion proteins, and functional fragments of any of the foregoing. Preferred embodiments include human TLR7 polypeptides having at least 65 percent sequence identity, more preferably at least 80 percent sequence identity, even more preferably with at least 90 percent sequence identity, and most preferably with at least 95 percent sequence identity with the human TLR7 amino acid sequence of SEQ ID NO:24. Preferred embodiments also include murine TLR7 polypeptides having at least 65 percent sequence identity, more preferably at least 80 percent sequence identity, even more preferably with at least 90 percent sequence identity, and most preferably with at least 95 percent sequence identity with the murine TLR7 amino acid sequence of SEQ ID NO:26.
As used herein “TLR7 signaling” refers to an ability of a TLR7 polypeptide to activate the TLR/IL-1R (TIR) signaling pathway, also referred to herein as the TLR signal transduction pathway. Changes in TLR7 activity can be measured by assays such as those disclosed herein, including expression of genes under control of κB-sensitive promoters and enhancers. Such naturally occurring genes include the genes encoding IL-1β, IL-6, IL-8, the p40 subunit of interleukin 12 (IL-12 p40), and the costimulatory molecules CD80 and CD86. Other genes can be placed under the control of such regulatory elements (see below) and thus serve to report the level of TLR7 signaling. Additional nucleotide sequence can be added to SEQ ID NO:25 or SEQ ID NO:27, preferably to the 5′ or the 3′ end of the open reading frame of SEQ ID NO:25, to yield a nucleotide sequence encoding a chimeric polypeptide that includes a detectable or reporter moiety, e.g., FLAG, luciferase (luc), green fluorescent protein (GFP), and others known by those skilled in the art.
SEQ ID NO:24 Human TLR7 amino acid
MVFPMWTLKR QILILFNIIL ISKLLGARWF PKTLPCDVTL DVPKNHVIVD CTDKHLTEIP
60
GGIPTNTTNL TLTINHIPDI SPASFHRLDH LVEIDFRCNC VPIPLGSKNN MCIKRLQIKP
120
RSFSGLTYLK SLYLDGNQLL EIPQGLPPSL QLLSLEANNI FSIRKENLTE LANIEILYLG
180
QNCYYRNPCY VSYSIEKDAF LNLTKLKVLS LKDNNVTAVP TVLPSTLTEL YLYNNMIAKI
240
QEDDFNNLNQ LQILDLSGNC PRCYNAPFPC APCKNNSPLQ IPVNAFDALT ELKVLRLHSN
300
SLQEVPPRWF KNINKLQELD LSQNFLAKEI GDAKFLHFLP SLIQLDLSFN FELQVYRASM
360
NLSQAFSSLK SLKILRIRGY VFKELKSFNL SPLHNLQNLE VLDLGTNFIK IANLSMFKQF
420
KRLKVIDLSV NKISPSGDSS EVGFCSNART SVESYEPQVL EQLHYFRYDK YARSCRFKNK
480
EASFMSVNES CYKYGQTLDL SKNSIFFVKS SDFQHLSFLK CLNLSGNLIS QTLNGSEFQP
540
LAELRYLDFS NNRLDLLHST AFEELHKLEV LDISSNSHYF QSEGITHMLN FTKNLKVLQK
600
LMMNDNDISS STSRTMESES LRTLEFRGNH LDVLWREGDN RYLQLFKNLL KLEELDISKN
660
SLSFLPSGVF DGMPPNLKNL SLAKNGLKSF SWKKLQCLKN LETLDLSHNQ LTTVPERLSN
720
CSRSLKNLIL KNNQIRSLTK YFLQDAFQLR YLDLSSNKIQ MIQKTSFPEN VLNNLKMLLL
780
HHNRFLCTCD AVWFVWWVNH TEVTIPYLAT DVTCVGPGAH KGQSVISLDL YTCELDLTNL
840
ILFSLSISVS LFLMVMMTAS HLYFWDVWYI YHFCKAKIKG YQRLISPDCC YDAFIVYDTK
900
DPAVTEWVLA ELVAKLEDPR EKHFNLCLEE RDWLPGQPVL ENLSQSIQLS KKTVFVMTDK
960
YAKTENFKIA FYLSHQRLMD EKVDVIILIF LEKPFQKSKF LQLRKRLCGS SVLEWPTNPQ
1020
AHPYFWQCLK NALATDNHVA YSQVFKETV
1049
SEQ ID NO:25 Human TLR7 nucleotide
actccagata taggatcact ccatgccatc aagaaagttg atgctattgg gcccatctca
60
agctgatctt ggcacctctc atgctctgct ctcttcaacc agacctctac attccatttt
120
ggaagaagac taaaaatggt gtttccaatg tggacactga agagacaaat tcttatcctt
180
tttaacataa tcctaatttc caaactcctt ggggctagat ggtttcctaa aactctgccc
240
tgtgatgtca ctctggatgt tccaaagaac catgtgatcg tggactgcac agacaagcat
300
ttgacagaaa ttcctggagg tattcccacg aacaccacga acctcaccct caccattaac
360
cacataccag acatctcccc agcgtccttt cacagactgg accatctggt agagatcgat
420
ttcagatgca actgtgtacc tattccactg gggtcaaaaa acaacatgtg catcaagagg
480
ctgcagatta aacccagaag ctttagtgga ctcacttatt taaaatccct ttacctggat
540
ggaaaccagc tactagagat accgcagggc ctcccgccta gcttacagct tctcagcctt
600
gaggccaaca acatcttttc catcagaaaa gagaatctaa cagaactggc caacatagaa
660
atactctacc tgggccaaaa ctgttattat cgaaatcctt gttatgtttc atattcaata
720
gagaaagatg ccttcctaaa cttgacaaag ttaaaagtgc tctccctgaa agataacaat
780
gtcacagccg tccctactgt tttgccatct actttaacag aactatatct ctacaacaac
840
atgattgcaa aaatccaaga agatgatttt aataacctca accaattaca aattcttgac
900
ctaagtggaa attgccctcg ttgttataat gccccatttc cttgtgcgcc gtgtaaaaat
960
aattctcccc tacagatccc tgtaaatgct tttgatgcgc tgacagaatt aaaagtttta
1020
cgtctacaca gtaactctct tcagcatgtg cccccaagat ggtttaagaa catcaacaaa
1080
ctccaggaac tggatctgtc ccaaaacttc ttggccaaag aaattgggga tgctaaattt
1140
ctgcattttc tccccagcct catccaattg gatctgtctt tcaattttga acttcaggtc
1200
tatcgtgcat ctatgaatct atcacaagca ttttcttcac tgaaaagcct gaaaattctg
1260
cggatcagag gatatgtctt taaagagttg aaaagcttta acctctcgcc attacataat
1320
cttcaaaatc ttgaagttct tgatcttggc actaacttta taaaaattgc taacctcagc
1380
atgtttaaac aatttaaaag actgaaagtc atagatcttt cagtgaataa aatatcacct
1440
tcaggagatt caagtgaagt tggcttctgc tcaaatgcca gaacttctgt agaaagttat
1500
gaaccccagg tcctggaaca attacattat ttcagatatg ataagtatgc aaggagttgc
1560
agattcaaaa acaaagaggc ttctttcatg tctgttaatg aaagctgcta caagtatggg
1620
cagaccttgg atctaagtaa aaatagtata ttttttgtca agtcctctga ttttcagcat
1680
ctttctttcc tcaaatgcct gaatctgtca ggaaatctca ttagccaaac tcttaatggc
1740
agtgaattcc aacctttagc agagctgaga tatttggact tctccaacaa ccggcttgat
1800
ttactccatt caacagcatt tgaagagctt cacaaactgg aagttctgga tataagcagt
1860
aatagccatt attttcaatc agaaggaatt actcatatgc taaactttac caagaaccta
1920
aaggttctgc agaaactgat gatgaacgac aatgacatct cttcctccac cagcaggacc
1980
atggagagtg agtctcttag aactctggaa ttcagaggaa atcacttaga tgttttatgg
2040
agagaaggtg ataacagata cttacaatta ttcaagaatc tgctaaaatt agaggaatta
2100
gacatctcta aaaattccct aagtttcttg ccttctggag tttttgatgg tatgcctcca
2160
aatctaaaga atctctcttt ggccaaaaat gggctcaaat ctttcagttg gaagaaactc
2220
cagtgtctaa agaacctgga aactttggac ctcagccaca accaactgac cactgtccct
2280
gagagattat ccaactgttc cagaagcctc aagaatctga ttcttaagaa taatcaaatc
2340
aggagtctga cgaagtattt tctacaagat gccttccagt tgcgatatct ggatctcagc
2400
tcaaataaaa tccagatgat ccaaaagacc agcttcccag aaaatgtcct caacaatctg
2460
aagatgttgc ttttgcatca taatcggttt ctgtgcacct gtgatgctgt gtggtttgtc
2520
tggtgggtta accatacgga ggtgactatt ccttacctgg ccacagatgt gacttgtgtg
2580
gggccaggag cacacaaggg ccaaagtgtg atctccctgg atctgtacac ctgtgagtta
2640
gatctgacta acctgattct gttctcactt tccatatctg tatctctctt tctcatggtg
2700
atgatgacag caagtcacct ctatttctgg gatgtgtggt atatttacca tttctgtaag
2760
gccaagataa aggggtatca gcgtctaata tcaccagact gttgctatga tgcttttatt
2820
gtgtatgaca ctaaagaccc agctgtgacc gagtgggttt tggctgagct ggtggccaaa
2880
ctggaagacc caagagagaa acattttaat ttatgtctcg aggaaaggga ctggttacca
2940
gggcagccag ttctggaaaa cctttcccag agcatacagc ttagcaaaaa gacagtgttt
3000
gtgatgacag acaagtatgc aaagactgaa aattttaaga tagcatttta cttgtcccat
3060
cagaggctca tggatgaaaa agttgatgtg attatcttga tatttcttga gaagcccttt
3120
cagaagtcca agttcctcca gctccggaaa aggctctgtg ggagttctgt ccttgagtgg
3180
ccaacaaacc cgcaagctca cccatacttc tggcagtgtc taaagaacgc cctggccaca
3240
gacaatcatg tggcctatag tcaggtgttc aaggaaacgg tctagccctt ctttgcaaaa
3300
cacaactgcc tagtttacca aggagaggcc tggctgttta aattgttttc atatatatca
3360
caccaaaagc gtgttttgaa attcttcaag aaatgagatt gcccatattt caggggagcc
3420
accaacgtct gtcacaggag ttggaaagat ggggtttata taatgcatca agtcttcttt
3480
cttatctctc tgtgtctcta tttgcacttg agtctctcac ctcagctcct gtaaaagagt
3540
ggcaagtaaa aaacatgggg ctctgattct cctgtaattg tgataattaa atatacacac
3600
aatcatgaca ttgagaagaa ctgcatttct acccttaaaa agtactggta tatacagaaa
3660
tagggttaaa aaaaactcaa gctctctcta tatgagacca aaatgtacta gagttagttt
3720
agtgaaataa aaaaccagtc agctggccgg gcatggtggc tcatgcttgt aatcccagca
3780
ctttgggagg ccgaggcagg tggatcacga ggtcaggagt ttgagaccag tctggccaac
3840
atggtgaaac cccgtctgta ctaaaaatac aaaaattagc tgggcgtggt ggtgggtgcc
3900
tgtaatccca gctacttggg aggctgaggc aggagaatcg cttgaacccg ggaggtggag
3960
gtggcagtga gccgagatca cgccactgca atgcagcccg ggcaacagag ctagactgtc
4020
tcaaaagaac aaaaaaaaaa aaacacaaaa aaactcagtc agcttcttaa ccaattgctt
4080
ccgtgtcatc cagggcccca ttctgtgcag attgagtgtg ggcaccacac aggtggttgc
4140
tgcttcagtg cttcctgctc tttttccttg ggcctgcttc tgggttccat agggaaacag
4200
taagaaagaa agacacatcc ttaccataaa tgcatatggt ccacctacaa atagaaaaat
4260
atttaaatga tctgccttta tacaaagtga tattctctac ctttgataat ttacctgctt
4320
aaatgttttt atctgcactg caaagtactg tatccaaagt aaaatttcct catccaatat
4380
ctttcaaact gttttgttaa ctaatgccat atatttgtaa gtatctgcac acttgataca
4440
gcaacgttag atggttttga tggtaaaccc taaaggagga ctccaagagt gtgtatttat
4500
ttatagtttt atcagagatg acaattattt gaatgccaat tatatggatt cctttcattt
4560
tttgctggag gatgggagaa gaaaccaaag tttatagacc ttcacattga gaaagcttca
4620
gttttgaact tcagctatca gattcaaaaa caacagaaag aaccaagaca ttcttaagat
4680
gcctgtactt tcagctgggt ataaattcat gagttcaaag attgaaacct gaccaatttg
4740
ctttatttca tggaagaagt gatctacaaa ggtgtttgtg ccatttggaa aacagcgtgc
4800
atgtgttcaa gccttagatt ggcgatgtcg tattttcctc acgtgtggca atgccaaagg
4860
ctttacttta cctgtgagta cacactatat gaattatttc caacgtacat ttaatcaata
4920
agggtcacaa attcccaaat caatctctgg aataaataga gaggtaatta aattgctgga
4980
gccaactatt tcacaacttc tgtaagc
5007
SEQ ID NO:26 Murine TLR7 amino acid
MVFSMWTRKR QILIFLNMLL VSRVFGFRWF PKTLPCEVKV NIPEAHVIVD CTDKHLTEIP
60
EGIPTNTTNL TLTINHIPSI SPDSFRRLNH LEEIDLRCNC VPVLLGSKAN VCTKRLQIRP
120
GSFSGLSDLK ALYLDGNQLL EIPQDLPSSL HLLSLEANNI FSITKENLTE LVNIETLYLG
180
QNCYYRNPCN VSYSIEKDAF LVMRNLKVLS LKDNNVTAVP TTLPPNLLEL YLYNNIIKKI
240
QENDFNNLNE LQVLDLSGNC PRCYNVPYPC TPCENNSPLQ IHDNAFNSLT ELKVLRLHSN
300
SLQHVPPTWF KNMRNLQELD LSQNYLAREI EEAKFLHFLP NLVELDFSFN YELQVYHASI
360
TLPHSLSSLE NLKILRVKGY VFKELKNSSL SVLHKLPRLE VLDLGTNFIK IADLNIFKHF
420
ENLKLIDLSV NKISPSEESR EVGFCPNAQT SVDRHGPQVL EALHYFRYDE YARSCRFKNK
480
EPPSFLPLNA DCHIYGQTLD LSRNNIFFIK PSDFQHLSFL KCLNLSGNTI GQTLNGSELW
540
PLRELRYLDF SNNRLDLLYS TAFEELQSLE VLDLSSNSHY FQAEGITHML NFTKKLRLLD
600
KLMMNDNDIS TSASRTMESD SLRILEFRGN HLDVLWRAGD NRYLDFFKNL FNLEVLDISR
660
NSLNSLPPEV FEGMPPNLKN LSLAKNGLKS FFWDRLQLLK HLEILDLSHN QLTKVPERLA
720
NCSKSLTTLI LKHNQIRQLT KYFLEDALQL RYLDISSNKI QVIQKTSFPE NVLNNLEMLV
780
LHHNRFLCNC DAVWFVWWVN ETDVTIPYLA TDVTCVGPGA HKGQSVISLD LYTCELDLTN
840
LILFSVSISS VLFLMVVMTT SHLFFWDMWY IYYFWKAKIK GYQHLQSMES CYDAFIVYDT
900
KNSAVTEWVL QELVAKLEDP REKHFNLCLE ERDWLPGQPV LENLSQSIQL SKKTVFVMTQ
960
KYAKTESFKM AFYLSHQRLL DEKVDVIILI FLEKPLQKSK FLQLRKRLCR SSVLEWPANP
1020
QAHPYFWQCL KNALTTDNHV AYSQMFKETV
1050
SEQ ID NO:27 Murine TLR7 nucleotide
attctcctcc accagacctc ttgattccat tttgaaagaa aactgaaaat ggtgttttcg
60
atgtggacac ggaagagaca aattttgatc tttttaaata tgctcttagt ttctagagtc
120
tttgggtttc gatggtttcc taaaactcta ccttgtgaag ttaaagtaaa tatcccagag
180
gcccatgtga tcgtggactg cacagacaag catttgacag aaatccctga gggcattccc
240
actaacacca ccaatcttac ccttaccatc aaccacatad caagcatctc tccagattcc
300
ttccgtaggc tgaaccatct ggaagaaatc gatttaagat gcaattgtgt acctgttcta
360
ctggggtcca aagccaatgt gtgtaccaag aggctgcaga ttagacctgg aagctttagt
420
ggactctctg acttaaaagc cctttacctg gatggaaacc aacttctgga gataccacag
480
gatctgccat ccagcttaca tcttctgagc cttgaggcta acaacatctt ctccatcacg
540
aaggagaatc taacagaact ggtcaacatt gaaacactct acctgggtca aaactgttat
600
tatcgaaatc cttgcaatgt ttcctattct attgaaaaag atgctttcct agttatgaga
660
aatttgaagg ttctctcact aaaagataac aatgtcacag ctgtccccac cactttgcca
720
cctaatttac tagagctcta tctttataac aatatcatta agaaaatcca agaaaatgat
780
tttaataacc tcaatgagtt gcaagttctt gacctaagtg gaaattgccc tcgatgttat
840
aatgtcccat atccgtgtac accgtgtgaa aataattccc ccttacagat ccatgacaat
900
gctttcaatt cattgacaga attaaaagtt ttacgtttac acagtaattc tcttcagcat
960
gtgcccccaa catggtttaa aaacatgaga aacctccagg aactagacct ctcccaaaac
1020
tacttggcca gagaaattga ggaggccaaa tttttgcatt ttcttcccaa ccttgttgag
1080
ttggattttt ctttcaatta tgagctgcag gtctaccatg catctataac tttaccacat
1140
tcactctctt cattggaaaa cttgaaaatt ctgcgtgtca aggggtatgt ctttaaagag
1200
ctgaaaaact ccagtctttc tgtattgcac aagcttccca ggctggaagt tcttgacctt
1260
ggcactaact tcataaaaat tgctgacctc aacatattca aacattttga aaacctcaaa
1320
ctcatagacc tttcagtgaa taagatatct ccttcagaag agtcaagaga agttggcttt
1380
tgtcctaatg ctcaaacttc tgtagaccgt catgggcccc aggtccttga ggccttacac
1440
tatttccgat acgatgaata tgcacggagc tgcaggttca aaaacaaaga gccaccttct
1500
ttcttgcctt tgaatgcaga ctgccacata tatgggcaga ccttagactt aagtagaaat
1560
aacatatttt ttattaaacc ttctgatttt cagcatcttt cattcctcaa atgcctcaac
1620
ttatcaggaa acaccattgg ccaaactctt aatggcagtg aactctggcc gttgagagag
1680
ttgcggtact tagacttctc caacaaccgg cttgatttac tctactcaac agcctttgaa
1740
gagctccaga gtcttgaagt tctggatcta agtagtaaca gccactattt tcaagcagaa
1800
ggaattactc acatgctaaa ctttaccaag aaattacggc ttctggacaa actcatgatg
1860
aatgataatg acatctctac ttcggccagc aggaccatgg aaagtgactc tcttcgaatt
1920
ctggagttca gaggcaacca tttagatgtt ctatggagag ccggtgataa cagatacttg
1980
gacttcttca agaatttgtt caatttagag gtattagata tctccagaaa ttccctgaat
2040
tccttgcctc ctgaggtttt tgagggtatg ccgccaaatc taaagaatct ctccttggcc
2100
aaaaatgggc tcaaatcttt cttttgggac agactccagt tactgaagca tttggaaatt
2160
ttggacctca gccataaCca gctgacaaaa gtacctgaga gattggccaa ctgttccaaa
2220
agtctcacaa cactgattCt taagcataat caaatcaggc aattgacaaa atattttcta
2280
gaagatgctt tgcaattgCg ctatctagac atcagttcaa ataaaatcca ggtcattcag
2340
aagactagct tcccagaaaa tgtcctcaac aatctggaga tgttggtttt acatcacaat
2400
cgctttcttt gcaactgtga tgctgtgtgg tttgtctggt gggttaacca tacagatgtt
2460
actattccat acctggccac tgatgtgact tgtgtaggtc caggagcaca caaaggtcaa
2520
agtgtcatat cccttgatct gtatacgtgt gagttagatc tcacaaacct gattctgttc
2580
tcagtttcca tatcatcagt cctctttctt atggtagtta tgacaacaag tcacctcttt
2640
ttctgggata tgtggtacat ttattatttt tggaaagcaa agataaaggg gtatcagcat
2700
ctgcaatcca tggagtcttg ttatgatgct tttattgtgt atgacactaa aaactcagct
2760
gtgacagaat gggttttgca ggagctggtg gcaaaattgg aagatccaag agaaaaacac
2820
ttcaatttgt gtctagaaga aagagactgg ctaccaggac agccagttct agaaaacctt
2880
tcccagagca tacagctcag caaaaagaca gtgtttgtga tgacacagaa atatgctaag
2940
actgagagtt ttaagatggc attttatttg tctcatcaga ggctcctgga tgaaaaagtg
3000
gatgtgatta tcttgatatt cttggaaaag cctcttcaga agtctaagtt tcttcagctc
3060
aggaagagac tctgcaggag ctctgtcctt gagtggcctg caaatccaca ggctcaccca
3120
tacttctggc agtgcctgaa aaatgccctg accacagaca atcatgtggc ttatagtcaa
3180
atgttcaagg aaacagtcta gctctctgaa gaatgtcacc acctaggaca tgccttgaat
3240
cga
3243
Nucleotide and amino acid sequences of human and murine TLR8 are known. See, for example, GenBank Accession Nos. AF246971, AF245703, NM—016610, XM—045706, AY035890, NM—133212; and AAF64061, AAF78036, NP 057694, XP—045706, AAK62677, NP—573475. Human TLR8 is reported to exist in at least two isoforms, one 1041 amino acids long having a sequence provided in SEQ ID NO:28, and the other 1059 amino acids long having a sequence as provided in SEQ ID NO:30. Corresponding nucleotide sequences are provided as SEQ ID NO:29 and SEQ ID NO:31, respectively. The shorter of these two isoforms is believed to be more important. Murine TLR8 is 1032 amino acids long and has a sequence as provided in SEQ ID NO:32. The corresponding nucleotide sequence is provided as SEQ ID NO:33. TLR8 polypeptide includes an extracellular domain having leucine-rich repeat region, a transmembrane domain, and an intracellular domain that includes a TIR domain.
As used herein a “TLR8 polypeptide” refers to a polypeptide including a full-length TLR8 according to one of the sequences above, orthologs, allelic variants, SNPs, variants incorporating conservative amino acid substitutions, TLR8 fusion proteins, and functional fragments of any of the foregoing. Preferred embodiments include human TLR8 polypeptides having at least 65 percent sequence identity, more preferably at least 80 percent sequence identity, even more preferably with at least 90 percent sequence identity, and most preferably with at least 95 percent sequence identity with the human TLR8 amino acid sequence of SEQ ID NO:28. Preferred embodiments also include murine TLR8 polypeptides having at least 65 percent sequence identity, more preferably at least 80 percent sequence identity, even more preferably with at least 90 percent sequence identity, and most preferably with at least 95 percent sequence identity with the murine TLR8 amino acid sequence of SEQ ID NO:32.
As used herein “TLR8 signaling” refers to an ability of a TLR8 polypeptide to activate the TLR/IL-1R (TIR) signaling pathway, also referred to herein as the TLR signal transduction pathway. Changes in TLR8 activity can be measured by assays such as those disclosed herein, including expression of genes under control of κB-sensitive promoters and enhancers. Such naturally occurring genes include the genes encoding IL-1β, IL-6, IL-8, the p40 subunit of interleukin 12 (IL-12 p40), and the costimulatory molecules CD80 and CD86. Other genes can be placed under the control of such regulatory elements (see below) and thus serve to report the level of TLR8 signaling. Additional nucleotide sequence can be added to SEQ ID NO:29 or SEQ ID NO:33, preferably to the 5′ or the 3′ end of the open reading frame of SEQ ID NO:29, to yield a nucleotide sequence encoding a chimeric polypeptide that includes a detectable or reporter moiety, e.g., FLAG, luciferase (luc), green fluorescent protein (GFP), and others known by those skilled in the art.
SEQ ID NO:28 Human TLR8 amino acid (1041)
MENMFLQSSM LTCIFLLISG SCELCAEENF SRSYPCDEKK QNDSVIAECS NRRLQEVPQT
60
VGKYVTELDL SDNFITHITN ESFQGLQNLT KINLNHNPNV QHQNGNPGIQ SNGLNITDGA
120
FLNLKNLREL LLEDNQLPQI PSGLPESLTE LSLIQNNIYN ITKEGISRLI NLKNLYLAWN
180
CYFNKVCEKT NIEDGVFETL TNLELLSLSF NSLSHVPPKL PSSLRKLFLS NTQIKYISEE
240
DFKGLINLTL LDLSGNCPRC FNAPFPCVPC DGGASINIDR FAFQNLTQLR YLNLSSTSLR
300
KINAAWFKNM PHLKVLDLEF NYLVGEIASG AFLTMLPRLE ILDLSFNYIK GSYPQHINIS
360
RNFSKLLSLR ALHLRGYVFQ ELREDDFQPL MQLPNLSTIN LGINFIKQID FKLFQNFSNL
420
EIIYLSENRI SPLVKDTRQS YANSSSFQRH IRKRRSTDFE FDPHSNFYHF TRPLIKPQCA
480
AYGKALDLSL NSIFFIGPNQ FENLPDIACL NLSANSNAQV LSGTEFSAIP HVKYLDLTNN
540
PLDFDNASAL TELSDLEVLD LSYNSHYFRI AGVTHHLEFI QNFTNLKVLN LSHNNIYTLT
600
DKYNLESKSL VELVFSGNRL DILWNDDDNR YISIFKGLKN LTRLDLSLNR LKHIPNEAFL
660
NLPASLTELH INDNMLKFFN WTLLQQFPRL ELLDLRGNKL LFLTDSLSDF TSSLRTLLLS
720
HNRISHLPSG FLSEVSSLKH LDLSSNLLKT INKSALETKT TTKLSMLELH GNPFECTCDI
780
GDFRRWMDEH LNVKIPRLVD VICASPGDQR GKSIVSLELT TCVSDVTAVI LFFFTFFITT
840
MVMLAALAHH LFYWDVWFIY NVCLAKVKGY RSLSTSQTFY DAYISYDTKD ASVTDWVINE
900
LRYHLEESRD KNVLLCLEER DWDPGLAIID NLMQSINQSK KTVFVLTKKY AKSWNFKTAF
960
YLALQRLMDE NNDVIIFILL EPVLQHSQYL RLRQRICKSS ILQWPDNPKA EGLFWQTLRN
1020
VVLTENDSRY NNMYVDSIKQ Y
1041
SEQ ID NO:29 Human TLR8 nucleotide
ttctgcgctg ctgcaagtta cggaatgaaa aattagaaca acagaaacat ggaaaacatg
60
ttccttcagt cgtcaatgct gacctgcatt ttcctgctaa tatctggttc ctgtgagtta
120
tgcgccgaag aaaatttttc tagaagctat ccttgtgatg agaaaaagca aaatgactca
180
gttattgcag agtgcagcaa tcgtcgacta caggaagttc cccaaacggt gggcaaatat
240
gtgacagaac tagacctgtc tgataatttc atcacacaca taacgaatga atcatttcaa
300
gggctgcaaa atctcactaa aataaatcta aaccacaacc ccaatgtaca gcaccagaac
360
ggaaatcccg gtatacaatc aaatggcttg aatatcacag acggggcatt cctcaaccta
420
aaaaacctaa gggagttact gcttgaagac aaccagttac cccaaatacc ctctggtttg
480
ccagagtctt tgacagaact tagtctaatt caaaacaata tatacaacat aactaaagag
540
ggcatttcaa gacttataaa cttgaaaaat ctctatttgg cctggaactg ctatttt4ac
600
aaagtttgcg agaaaactaa catagaagat ggagtatttg aaacgctgac aaatttggag
660
ttgctatcac tatctttcaa ttctctttca cacgtgccac ccaaactgcc aagctcccta
720
cgcaaacttt ttctgagcaa cacccagatc aaatacatta gtgaagaaga tttcaaggga
780
ttgataaatt taacattact agatttaagc gggaactgtc cgaggtgctt caatgcccca
840
tttccatgcg tgccttgtga tggtggtgct tcaattaata tagatcgttt tgcttttcaa
900
aacttgaccc aacttcgata cctaaacctc tctagcactt ccctcaggaa gattaatgct
960
gcctggttta aaaatatgcc tcatctgaag gtgctggatc ttgaattcaa ctatttagtg
1020
ggagaaatag cctctggggc atttttaacg atgctgcccc gcttagaaat acttgacttg
1080
tcttttaact atataaaggg gagttatcca cagcatatta atatttccag aaacttctct
1140
aaacttttgt ctctacgggc attgcattta agaggttatg tgttccagga actcagagaa
1200
gatgatttcc agcccctgat gcagcttcca aacttatcga ctatcaactt gggtattaat
1260
tttattaagc aaatcgattt caaacttttc caaaatttct ccaatctgga aattatttac
1320
ttgtcagaaa acagaatatc accgttggta aaagataccc ggcagagtta tgcaaatagt
1380
tcctcttttc aacgtcatat ccggaaacga cgctcaacag attttgagtt tgacccacat
1440
tcgaactttt atcatttcac ccgtccttta ataaagccac aatgtgctgc ttatggaaaa
1500
gccttagatt taagcctcaa cagtattttc ttcattgggc caaaccaatt tgaaaatctt
1560
cctgacattg cctgtttaaa tctgtctgca aatagcaatg ctcaagtgtt aagtggaact
1620
gaattttcag ccattcctca tgtcaaatat ttggatttga caaacaatag actagacttt
1680
gataatgcta gtgctcttac tgaattgtcc gacttggaag ttctagatct cagctataat
1740
tcacactatt tcagaatagc aggcgtaaca catcatctag aatttattca aaatttcaca
1800
aatctaaaag ttttaaactt gagccacaac aacatttata ctttaacaga taagtataac
1860
ctggaaagca agtccctggt agaattagtt ttcagtggca atcgccttga cattttgtgg
1920
aatgatgatg acaacaggta tatctccatt ttcaaaggtc tcaagaatct gacacgtctg
1980
gatttatccc ttaataggct gaagcacatc ccaaatgaag cattccttaa tttgccagcg
2040
agtctcactg aactacatat aaatgataat atgttaaagt tttttaactg gacattactc
2100
cagcagttcc ctcgtctcga gttgcttgac ttacgtggaa acaaactact ctttttaact
2160
gatagcctat ctgactttac atcttccctt cggacactgc tgctgagtca taacaggatt
2220
tcccacctac cctctggctt tctttctgaa gtcagtagtc tgaagcacct cgatttaagt
2280
tccaatctgc taaaaacaat caacaaatcc gcacttgaaa ctaagaccac caccaaatta
2340
tctatgttgg aactacacgg aaaccccttt gaatgcacct gtgacattgg agatttccga
2400
agatggatgg atgaacatct gaatgtcaaa attcccagac tggtagatgt catttgtgcc
2460
agtcctgggg atcaaagagg gaagagtatt gtgagtctgg agctgacaac ttgtgtttca
2520
gatgtcactg cagtgatatt atttttcttc acgttcttta tcaccaccat ggttatgttg
2580
gctgccctgg ctcaccattt gttttactgg gatgtttggt ttatatataa tgtgtgttta
2640
gctaaggtaa aaggctacag gtctctttcc acatcccaaa ctttctatga tgcttacatt
2700
tcttatgaca ccaaagatgc ctctgttact gactgggtga taaatgagct gcgctaccac
2760
cttgaagaga gccgagacaa aaacgttctc ctttgtctag aggagaggga ttgggacccg
2820
ggattggcca tcatcgacaa cctcatgcag agcatcaacc aaagcaagaa aacagtattt
2880
gttttaacca aaaaatatgc aaaaagctgg aactttaaaa cagcttttta cttggctttg
2940
cagaggctaa tggatgagaa catggatgtg attatattta tcctgctgga gccagtgtta
3000
cagcattctc agtatttgag gctacggcag cggatctgta agagctccat cctccagtgg
3060
cctgacaacc cgaaggcaga aggcttgttt tggcaaactc tgagaaatgt ggtcttgact
3120
gaaaatgatt cacggtataa caatatgtat gtcgattcca ttaagcaata ctaactgacg
3180
ttaagtcatg atttcgcgcc ataataaaga tgcaaaggaa tgacatttct gtattagtta
3240
tctattgcta tgtaacaaat tatcccaaaa cttagtggtt taaaacaaca catttgctgg
3300
cccacagttt t
3311
SEQ ID NO:30 Human TLR8 amino acid (1059)
MKESSLQNSS CSLGKETKKE NNFLQSSMLT CIFLLISGSC ELCAEENFSR SYPCDEKKQN
60
DSVIAECSNR RLQEVPQTVG KYVTELDLSD NFITHITNES FQGLQNLTKI NLNHNPNVQH
120
QNGNPGIQSN GLNITDGAFL NLKNLRELLL EDNQLPQIPS GLPESLTELS LIQNNIYNIT
180
KEGISRLINL KNLYLAWNCY FNKVCEKTNI EDGVFETLTN LELLSLSFNS LSHVSPKLPS
240
SLRKLFLSNT QIKYISEEDF KGLThLTLLD LSGNCPRCFN APFPCVPCDG GASINIDRFA
300
FQNLTQLRYL NLSSTSLRKI NAAWFKNMPH LKVLDLEFNY LVGEIASGAF LTMLPRLEIL
360
DLSFNYIKGS YPQHINISRN FSKPLSLRAL HLRGYVFQEL REDDFQPLMQ LPNLSTINLG
420
INFIKQIDFK LFQNFSNLEI IYLSENRISP LVKDTRQSYA NSSSFQRHIR KRRSTDFEFD
480
PHSNFYHFTR PLIKPQCAAY GKALDLSLNS IFFIGPNQFE NLPDIACLNL SANSNAQVLS
540
GTEFSAIPHV KYLDLTNNRL DFDNASALTE LSDLEVLDLS YNSHYFRIAG VTHHLEFIQN
600
FTNLKVLNLS RNNIYTLTDK YNLESKSLVE LVFSGNRLDI LWNDDDNRYI SIFKGLKNLT
660
RLDLSLNRLK HIPNEAFLNL PASLTELHIN DNNLKFFNWT LLQQFPRLEL LDLRGNKLLF
720
LTDSLSDFTS SLRTLLLSHN RISHLPSGFL SEVSSLKHLD LSSNLLKTIN KSALETKTTT
780
KLSMLELHGN PFECTCDIGD FRRWNDEHLN VKIPRLVDVI CASPGDQRGK SIVSLELTTC
840
VSDVTAVILF FFTFFITTMV MLAALAHHLF YWDVWFIYNV CLAKIKGYRS LSTSQTFYDA
900
YISYDTKDAS VTDWVINELR YHLEESRDKN VLLCLEERDW DPGLAIIDNL MQSINQSKKT
960
VFVLTKKYAK SWNFKTAFYL ALQRLMDENM DVIIFILLEP VLQHSQYLRL RQRICKSSIL
1020
QWPDNPKAEG LFWQTLRNVV LTENDSRYNN MYVDSIKQY
1059
SEQ ID NO:31 Human TLR8 nucleotide
ctcctgcata gagggtacca ttctgcgctg ctgcaagtta cggaatgaaa aattagaaca
60
acagaaacgt ggttctcttg acacttcagt gttagggaac atcagcaaga cccatcccag
120
gagaccttga aggaagcctt tgaaagggag aatgaaggag tcatctttgc aaaatagctc
180
ctgcagcctg ggaaaggaga ctaaaaagga aaacatgttc cttcagtcgt caatgctgac
240
ctgcattttc ctgctaatat ctggttcctg tgagttatgc gccgaagaaa atttttctag
300
aagctatcct tgtgatgaga aaaagcaaaa tgactcagtt attgcagagt gcagcaatcg
360
tcgactacag gaagttcccc aaacggtggg caaatatgtg acagaactag acctgtctga
420
taatttcatc acacacataa cgaatgaatc atttcaaggg ctgcaaaatc tcactaaaat
480
aaatctaaac cacaacccca atgtacagca ccagaacgga aatcccggta tacaatcaaa
540
tggcttgaat atcacagacg gggcattcct caacctaaaa aacctaaggg agttactgct
600
tgaagacaac cagttacccc aaataccctc tggtttgcca gagtctttga cagaacttag
660
tctaattcaa aacaatatat acaacataac taaagagggc atttcaagac ttataaactt
720
gaaaaatctc tatttggcct ggaactgcta ttttaacaaa gtttgcgaga aaactaacat
780
agaagatgga gtatttgaaa cgctgacaaa tttggagttg ctatcactat ctttcaattc
840
tctttcacac gtgtcaccca aactgccaag ctccctacgc aaactttttc tgagcaacac
900
ccagatcaaa tacattagtg aagaagattt caagggattg ataaatttaa cattactaga
960
tttaagcggg aactgtccga ggtgcttcaa tgccccattt ccatgcgtgc cttgtgatgg
1020
tggtgcttca attaatatag atcgttttgc ttttcaaaac ttgacccaac ttcgatacct
1080
aaacctctct agcacttccc tcaggaagat taatgctgcc tggtttaaaa atatgcctca
1140
tctgaaggtg ctggatcttg aattcaacta tttagtggga gaaatagcct ctggggcatt
1200
tttaacgatg ctgccccgct tagaaatact tgacttgtct tttaactata taaaggggag
1260
ttatccacag catattaata tttccagaaa cttctctaaa cctttgtctc tacgggcatt
1320
gcatttaaga ggttatgtgt tccaggaact cagagaagat gatttccagc ccctgatgca
1380
gcttccaaac ttatcgacta tcaacttggg tattaatttt attaagcaaa tcgatttcaa
1440
acttttccaa aatttctcca atctggaaat tatttacttg tcagaaaaca gaatatcacc
1500
gttggtaaaa gatacccggc agagttatgc aaatagttcc tcttttcaac gtcatatccg
1560
gaaacgacgc tcaacagatt ttgagtttga cccacattcg aacttttatc atttcacccg
1620
tcctttaata aagccacaat gtgctgctta tggaaaagcc ttagatttaa gcctcaacag
1680
tattttcttc attgggccaa accaatttga aaatcttcct gacattgcct gtttaaatct
1740
gtctgcaaat agcaatgctc aagtgttaag tggaactgaa ttttcagcca ttcctcatgt
1800
caaatatttg gatttgacaa acaatagact agactttgat aatgctagtg ctcttactga
1860
attgtccgac ttggaagttc tagatctcag ctataattca cactatttca gaatagcagg
1920
cgtaacacat catctagaat ttattcaaaa tttcacaaat ctaaaagttt taaacttgag
1980
ccacaacaac atttatactt taacagataa gtataacctg gaaagcaagt ccctggtaga
2040
attagttttc agtggcaatc gccttgacat tttgtggaat gatgatgaca acaggtatat
2100
ctccattttc aaaggtctca agaatctgac acgtctggat ttatccctta ataggctgaa
2160
gcacatccca aatgaagcat tccttaattt gccagcgagt ctcactgaac tacatataaa
2220
tgataatatg ttaaagtttt ttaactggac attactccag cagtttcctc gtctcgagtt
2280
gcttgactta cgtggaaaca aactactctt tttaactgat agcctatctg actttacatc
2340
ttcccttcgg acactgctgc tgagtcataa caggatttcc cacctaccct ctggctttct
2400
ttctgaagtc agtagtctga agcacctcga tttaagttcc aatctgctaa aaacaatcaa
2460
caaatccgca cttgaaacta agaccaccac caaattatct atgttggaac tacacggaaa
2520
cccctttgaa tgcacctgtg acattggaga tttccgaaga tggatggatg aacatctgaa
2580
tgtcaaaatt cccagactgg tagatgtcat ttgtgccagt cctggggatc aaagagggaa
2640
gagtattgtg agtctggagc taacaacttg tgtttcagat gtcactgcag tgatattatt
2700
tttcttcacg ttctttatca ccaccatggt tatgttggct gccctggctc accatttgtt
2760
ttactgggat gtttggttta tatataatgt gtgtttagct aagataaaag gctacaggtc
2820
tctttccaca tcccaaactt tctatgatgc ttacatttct tatgacacca aagatgcctc
2880
tgttactgac tgggtgataa atgagctgcg ctaccacctt gaagagagcc gagacaaaaa
2940
cgttctcctt tgtctagagg agagggattg ggacccggga ttggccatca tcgacaacct
3000
catgcagagc atcaaccaaa gcaagaaaac agtatttgtt ttaaccaaaa aatatgcaaa
3060
aagctggaac tttaaaacag ctttttactt ggctttgcag aggctaatgg atgagaacat
3120
ggatgtgatt atatttatcc tgctggagcc agtgttacag cattctcagt atttgaggct
3180
acggcagcgg atctgtaaga gctccatcct ccagtggcct gacaacccga aggcagaagg
3240
cttgttttgg caaactctga gaaatgtggt cttgactgaa aatgattcac ggtataacaa
3300
tatgtatgtc gattccatta agcaatacta actgacgtta agtcatgatt tcgcgccata
3360
ataaaga
3367
SEQ ID NO:32 Murine TLR8 amino acid
MENMPPQSWO LTCFCLLSSG TSAIFHKANY SRSYPCDEIR HNSLVIAECN HRQLHEVPQT
60
IGKYVTNIDL SDNAITHITK ESFZKLQNLT KIDLNHNAKQ QHPNENKNGM NITEGALLSL
120
RNLTVLLLED NQLYTIPAGL PESLKELSLI QNNIFQVTKN NTFGLRNLER LYLGWNCYFK
180
CNQTFKVEDG AFKNLIHLKV LSLSFNNLFY VPPKLPSSLR KLFLSNAKIM NITQEDFKGL
240
ENLTLLDLSG NCPRCYNAPF PCTPCKENSS INIHPLAFQS LTQLLYLNLS STSLRTIPST
300
WFENLSNLKE LELEFNYLVQ EIASGAFLTK LPSLQILDLS FNFQYKEYLQ FINISSNFSK
360
LRSLKKLHLR GYVFRELKKK HFIFLQSLPN LATINLGINF IEKIDFKAFQ NFSKLDVIYL
420
SGNRIASVLD GTDYSSWRNR LRKPLSTDDD EFDPHVNFYH STKPLIKPQC TAYGKALDLS
480
LNNIFIIGKS QFEGFQDIAC LNLSFNANTQ VFNGTEFSSM PHIKYLDLTN NRLDFDDNNA
540
FSDLHDLEVL DLSHNAHYFS IAGVTHRLGF IQNLINLRVL NLSHNGIYTL TEESELKSIS
600
LKELVFSGNR LDHLWNANDG KYWSIFKSLQ NLIRLDLSYN NLQQIPNGAF LNLPQSLQEL
660
LISGNKLRFF NWTLLQYFPH LHLLDLSRNE LYFLPNCLSK FAHSLETLLL SHNHFSHLPS
720
GFLSEARNLV HLDLSFNTIK MINKSSLQTK MKTNLSILEL HGNYFDCTCD ISDFRSWLDE
780
NLNITIPKLV NVICSNPGDQ KSKSIMSLDL TTCVSDTTAA VLFFLTFLTT SMVMLAALVH
840
HLFYWDVWFI YHMCSAKLKG YRTSSTSQTF YDAYISYDTK DASVTDWVIN ELRYHLEESE
900
DKSVLLCLEE RDWDPGLPII DNLMQSINQS KKTIFVLTKK YAKSWNFKTA FYLALQRLMD
960
ENMDVIIFIL LEPVLQYSQY LRLRQRICKS SILQWPNNPK AENLFWQSLK NVVLTENDSR
1020
YDDLYIDSIR QY
1032
SEQ ID NO:33 Murine TLR8 nucleotide
attcagagtt ggatgttaag agagaaacaa acgttttacc ttcctttgtc tatagaacat
60
ggaaaacatg ccccctcagt catggattct gacgtgcttt tgtctgctgt cctctggaac
120
cagtgccatc ttccataaag cgaactattc cagaagctat ccttgtgacg agataaggca
180
caactccctt gtgattgcag aatgcaacca tcgtcaactg catgaagttc cccaaactat
240
aggcaagtat gtgacaaaca tagacttgtc agacaatgcc attacacata taacgaaaga
300
gtcctttcaa aagctgcaaa acctcactaa aatcgatctg aaccacaatg ccaaacaaca
360
gcacccaaat gaaaataaaa atggtatgaa tattacagaa ggggcacttc tcagcctaag
420
aaatctaaca gttttactgc tggaagacaa ccagttatat actatacctg ctgggttgcc
480
tgagtctttg aaagaactta gcctaattca aaacaatata tttcaggtaa ctaaaaacaa
540
cacttttggg cttaggaact tggaaagact ctatttgggc tggaactgct attttaaatg
600
taatcaaacc tttaaggtag aagatggggc atttaaaaat cttatacact tgaaggtact
660
ctcattatct ttcaataacc ttttctatgt gccccccaaa ctaccaagtt ctctaaggaa
720
actttttctg agtaatgcca aaatcatgaa catcactcag gaagacttca aaggactgga
780
aaatttaaca ttactagatc tgagtggaaa ctgtccaagg tgttacaatg ctccatttcc
840
ttgcacacct tgcaaggaaa actcatccat ccacatacat cctctggctt ttcaaagtct
900
cacccaactt ctctatctaa acctttccag cacttccctc aggacgattc cttctacctg
960
gtttgaaaat ctgtcaaatc tgaaggaact ccatcttgaa ttcaactatt tagttcaaga
1020
aattgcctcg ggggcatttt taacaaaact acccagttta caaatccttg atttgtcctt
1080
caactttcaa tataaggaat atttacaatt tattaatatt tcctcaaatt tctctaagct
1140
tcgttctctc aagaagttgc acttaagagg ctatgtgttc cgagaactta aaaagaagca
1200
tttcgagcat ctccagagtc ttccaaactt ggcaaccatc aacttgggca ttaactttat
1260
tgagaaaatt gatttcaaag ctttccagaa tttttccaaa ctcgacgtta tctatttatc
1320
aggaaatcgc atagcatctg tattagatgg tacagattat tcctcttggc gaaatcgtct
1380
tcggaaacct ctctcaacag acgatgatga gtttgatcca cacgtgaatt tttaccatag
1440
caccaaacct ttaataaagc cacagtgtac tgcttatggc aaggccttgg atttaagttt
1500
gaacaatatt ttcattattg ggaaaagcca atttgaaggt tttcaggata tcgcctgctt
1560
aaatctgtcc ttcaatgcca atactcaagt gtttaatggc acagaattct cctccatgcc
1620
ccacattaaa tatttggatt taaccaacaa cagactagac tttgatgata acaatgcttt
1680
cagtgatctt cacgatctag aagtgctgga cctgagccac aatgcacact atttcagtat
1740
agcaggggta acgcaccgtc taggatttat ccagaactta ataaacctca gggtgttaaa
1800
cctgagccac aatggcattt acaccctcac agaggaaagt gagctgaaaa gcatctcact
1860
gaaagaattg gttttcagtg gaaatcgtct tgaccatttg tggaatgcaa atgatggcaa
1920
atactggtcc atttttaaaa gtctccagaa tttgatacgc ctggacttat catacaataa
1980
ccttcaacaa atcccaaatg gagcattcct caatttgcct cagagcctcc aagagttact
2040
tatcagtggt aacaaattac gtttctttaa ttggacatta ctccagtatt ttcctcacct
2100
tcacttgctg gatttatcga gaaatgagct gtattttcta cccaattgcc tatctaagtt
2160
tgcacattcc ctggagacac tgctactgag ccataatcat ttctctcacc taccctctgg
2220
cttcctctcc gaagccagga atctggtgca cctggatcta agtttcaaca caataaagat
2280
gatcaataaa tcctccctgc aaaccaagat gaaaacgaac ttgtctattc tggagctaca
2340
tgggaactat tttgactgca cgtgtgacat aagtgatttt cgaagctggc tagatgaaaa
2400
tctgaatatc acaattccta aattggtaaa tgttatatgt tccaatcctg gggatcaaaa
2460
atcaaagagt atcatgagcc tagatctcac gacttgtgta tcggatacca ctgcagctgt
2520
cctgtttttc ctcacattcc ttaccacctc catggttatg ttggctgctc tggttcacca
2580
cctgttttac tgggatgttt ggtttatcta tcacatgtgc tctgctaagt taaaaggcta
2640
caggacttca tccacatccc aaactttcta tgatgcttat atttcttatg acaccaaaga
2700
tgcatctgtt actgactggg taatcaatga actgcgctac caccttgaag agagtgaaga
2760
caaaagtgtc ctcctttgtt tagaggagag ggattgggat ccaggattac ccatcattga
2820
taacctcatg cagagcataa accagagcaa gaaaacaatc tttgttttaa ccaagaaata
2880
tgccaagagc tggaacttta aaacagcttt ctacttggcc ttgcagaggc taatggatga
2940
gaacatggat gtgattattt tcatcctcct ggaaccagtg ttacagtact cacagtacct
3000
gaggcttcgg cagaggatct gtaagagctc catcctccag tggcccaaca atcccaaagc
3060
agaaaacttg ttttggcaaa gtctgaaaaa tgtggtcttg actgaaaatg attcacggta
3120
tgacgatttg tacattgatt ccattaggca atactagtga tgggaagtca cgactctgcc
3180
atcataaaaa cacacagctt ctccttacaa tgaaccgaat
3220
Nucleotide and amino acid sequences of human and murine TLR9 are known. See, for example, GenBank Accession Nos. NM—017442, AF259262, AB045180, AF245704, AB045181, AF348140, AF314224, NM—031178; and NP—059138, AAF 72189, BAB19259, AAF78037, BAB19260, AAK29625, AAK28488, NP—112455. Human TLR9 is reported to exist in at least two isoforms, one 1032 amino acids long having a sequence provided in SEQ ID NO:34, and the other 1055 amino acids long having a sequence as provided in SEQ ID NO:36. Corresponding nucleotide sequences are provided as SEQ ID NO:35 and SEQ ID NO:37, respectively. The shorter of these two isoforms is believed to be more important. Murine TLR9 is 1032 amino acids long and has a sequence as provided in SEQ ID NO:38. A corresponding nucleotide sequence is provided as SEQ ID NO:39. TLR9 polypeptide includes an extracellular domain having leucine-rich repeat region, a transmembrane domain, and an intracellular domain that includes a TIR domain.
As used herein a “TLR9 polypeptide” refers to a polypeptide including a full-length TLR9 according to one of the sequences above, orthologs, allelic variants, SNPs, variants incorporating conservative amino acid substitutions, TLR9 fusion proteins, and functional fragments of any of the foregoing. Preferred embodiments include human TLR9 polypeptides having at least 65 percent sequence identity, more preferably at least 80 percent sequence identity, even more preferably with at least 90 percent sequence identity, and most preferably with at least 95 percent sequence identity with the human TLR9 amino acid sequence of SEQ ID NO:34. Preferred embodiments also include murine TLR9 polypeptides having at least 65 percent sequence identity, more preferably at least 80 percent sequence identity, even more preferably with at least 90 percent sequence identity, and most preferably with at least 95 percent sequence identity with the murine TLR9 amino acid sequence of SEQ ID NO:38.
As used herein “TLR9 signaling” refers to an ability of a TLR9 polypeptide to activate the TLR/IL-1R (TIR) signaling pathway, also referred to herein as the TLR signal transduction pathway. Without meaning to be held to any particular theory, it is believed that the TLR/IL-1R signaling pathway involves signaling via the molecules myeloid differentiation marker 88 (MyD88) and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), leading to activation of kinases of the IκB kinase complex and the c-jun NH2-terminal kinases (e.g., Jnk 1/2). Häcker H et al. (2000) J Exp Med 192:595-600. Changes in TLR9 activity can be measured by assays such as those disclosed herein, including expression of genes under control of κB-sensitive promoters and enhancers. Such naturally occurring genes include the genes encoding IL-1β, IL-6, IL-8, the p40 subunit of interleukin 12 (IL-12 p40), and the costimulatory molecules CD80 and CD86. Other genes can be placed under the control of such regulatory elements (see below) and thus serve to report the level of TLR9 signaling. Additional nucleotide sequence can be added to SEQ ID NO:35 or SEQ ID NO:39, preferably to the 5′ or the 3′ end of the open reading frame of SEQ ID NO:35, to yield a nucleotide sequence encoding a chimeric polypeptide that includes a detectable or reporter moiety, e.g., FLAG, luciferase (luc), green fluorescent protein (GFP), and others known by those skilled in the art.
SEQ ID NO:34 Human TLR9 amino acid (1032)
MGFCRSALHP LSLLVQAIML AMTLALGTLP AFLPCELQPH GLVNCNWLFL KSVPHFSMAA
60
PRGNVTSLSL SSNRIHHLHD SDFAKLPSLR HLNLKWNCPP VGLSPMHFPC HMTIEPSTFL
120
AVPTLEELNL SYNNIMTVPA LPKSLISLSL SHTNILMLDS ASLAGLHALR FLFMDGNCYY
180
KNPCRQALEV APGALLGLGN LTHLSLKYNN LTVVPRNLPS SLEYLLLSYN RIVKLAPEDL
240
ANLTALRVLD VGGNCRRCDH APNPCMECPR HFPQLHPDTF SHLSRLEGLV LKDSSLSWLN
300
ASWFRGLGNL RVLDLSENFL YKCITKTKAF QGLTQLRKLN LSFNYQKRVS FAHLSLAPSF
360
GSLVALKELD MHGIFFRSLD ETTLRPLARL PMLQTLRLQM NFINQAQLGI FRAFPGLRYV
420
DLSDNRISGA SELTATMGEA DGGEKVWLQP GDLAPAPVDT PSSEDFRPNC STLNFTLDLS
480
RNNLVTVQPE MFAQLSHLQC LRLSHNCISQ AVNGSQFLPL TGLQVLDLSH NKLDLYHEHS
540
FTELPRLEAL DLSYNSQPFG MQGVGHWFSF VAHLRTLRHL SLAHNNIHSQ VSQQLCSTSL
600
RALDFSGNAL GHMWAEGDLY LHFFQGLSGL IWLDLSQNRL HTLLPQTLRN LPKSLQVLRL
660
RDNYLAFFKW WSLHFLPKLE VLDLAGNQLK ALTNGSLPAG TRLRRLDVSC NSISFVAPGF
720
FSKAKELREL NLSANALKTV DHSWFGPLAS ALQILDVSAN PLHCACGAAF MDFLLEVQAA
780
VPGLPSRVKC GSPGQLQGLS IFAQDLRLCL DEALSWDCFA LSLLAVALGL GVPMLHHLCG
840
WDLWYCFHLC LAWLPWRGRQ SGRDEDALPY DAFVVFDKTQ SAVADWVYNE LRGQLEECRG
900
RWALRLCLEE RDWLPGKTLF ENLWASVYGS RKTLFVLAHT DRVSGLLRAS FLLAQQRLLE
960
DRKDVVVLVI SLPDGRRSRY VRLRQRLCRQ SVLLWPHQPS GQRSFWAQLG MALTRDNHHF
1020
YNRNFCQGPT AE
1032
SEQ ID NO:35 Human TLR9 nucleotide
ccgctgctgc ccctgtggga agggacctcg agtgtgaagc atccttccct gtagctgctg
60
tccagtctgc ccgccagacc ctctggagaa gcccctgccc cccagcatgg gtttctgccg
120
cagcgccctg cacccgctgt ctctcctggt gcaggccatc atgctggcca tgaccctggc
180
cctgggtacc ttgcctgcct tcctaccctg tgagctccag ccccacggcc tggtgaactg
240
caactggctg ttcctgaagt ctgtgcccca cttctccatg gcagcacccc gtggcaatgt
300
caccagcctt tccttgtcct ccaaccgcat ccaccacctc catgattctg actttgccca
360
cctgcccagc ctgcggcatc tcaacctcaa gtggaactgc ccgccggttg gcctcagccc
420
catgcacttc ccctgccaca tgaccatcga gcccagcacc ttcttggctg tgcccaccct
480
ggaagagcta aacctgagct acaacaacat catgactgtg cctgcgctgc ccaaatccct
540
catatccctg tccctcagcc ataccaacat cctgatgcta gactctgcca gcctcgccgg
600
cctgcatgcc ctgcgcttcc tattcatgga cggcaactgt tattacaaga acccctgcag
660
gcaggcactg gaggtggccc cgggtgccct ccttggcctg ggcaacctca cccacctgtc
720
actcaagtac aacaacctca ctgtggtgcc ccgcaacctg ccttccagcc tggagtatct
780
gctgttgtcc tacaaccgca tcgtcaaact ggcgcctgag gacctggcca atctgaccgc
840
cctgcgtgtg ctcgatgtgg gcggaaattg ccgccgctgc gaccacgctc ccaacccctg
900
catggagtgc cctcgtcact tcccccagct acatcccgat accttcagcc acctgagccg
960
tcttgaaggc ctggtgttga aggacagttc tctctcctgg ctgaatgcca gttggttccg
1020
tgggctggga aacctccgag tgctggacct gagtgagaac ttcctctaca aatgcatcac
1080
taaaaccaag gccttccagg gcctaacaca gctgcgcaag cttaacctgt ccttcaatta
1140
ccaaaagagg gtgtcctttg cccacctgtc tctggcccct tccttcggga gcctggtcgc
1200
cctgaaggag ctggacatgc acggcatctt cttccgctca ctcgatgaga ccacgctccg
1260
gccactggcc cgcctgccca tgctccagac tctgcgtctg cagatgaact tcatcaacca
1320
ggcccagctc ggcatcttca gggccttccc tggcctgcgc tacgtggacc tgtcggacaa
1380
ccgcatcagc ggagcttcgg agctgacagc caccatgggg gaggcagatg gaggggagaa
1440
ggtctggctg cagcctgggg accttgctcc ggccccagtg gacactocca gctctgaaga
1500
cttcaggccc aactgcagca ccctcaactt caccttggat ctgtcacgga acaacctggt
1560
gaccgtgcag ccggagatgt ttgcccagct ctcgcacctg cagtgcctgc gcctgagcca
1620
caactgcatc tcgcaggcag tcaatggctc ccagttcctg ccgctgaccg gtctgcaggt
1680
gctagacctg tcccacaata agctggacct ctaccacgag cactcattca cggagctacc
1740
acgactggag gccctggacc tcagctacaa cagccagccc tttggcatgc agggcgtggg
1800
ccacaacttc agcttcgtgg ctcacctgcg caccctgcgc cacctcagcc tggcccacaa
1860
caacatccac agccaagtgt cccagcagct ctgcagtacg tcgctgcggg ccctggactt
1920
cagcggcaat gcactgggcc atatgtgggc cgagggagac ctctatctgc acttcttcca
1980
aggcctgagc ggtttgatct ggctggactt gtcccagaac cgcctgcaca ccctcctgcc
2040
ccaaaccctg cgcaacctcc ccaagagcct acaggtgctg cgtctccgtg acaattacct
2100
ggccttcttt aagtggtgga gcctccactt cctgcccaaa ctggaagtcc tcgacctggc
2160
aggaaaccag ctgaaggccc tgaccaatgg cagcctgcct gctggcaccc ggctccggag
2220
gctggatgtc agctgcaaca gcatcagctt cgtggccccc ggcttctttt ccaaggccaa
2280
ggagctgcga gagctcaacc ttagcgccaa cgccctcaag acagtggacc actcctggtt
2340
tgggcccctg gcgagtgccc tgcaaatact agatgtaagc gccaaccctc tgcactgcgc
2400
ctgtggggcg gcctttatgg acttcctgct ggaggtgcag gctgccgtgc ccggtctgcc
2460
cagccgggtg aagtgtggca gtccgggcca gctccagggc ctcagcatct ttgcacagga
2520
cctgcgcctc tgCctggatg aggccctctc ctgggactgt ttcgccctct cgctgctggc
2580
tgtggctctg ggcctgggtg tgCccatgct gcatcacctc tgtggctggg acctctggta
2640
ctgcttccac Ctgtgcctgg cctggcttcc ctggcggggg cggcaaagtg ggcgagatga
2700
ggatgccctg ccCtaCgatg ccttcgtggt cttcgacaaa acgcagagcg cagtggcaga
2760
ctgggtgtaC aacgagcttc gggggcagct ggaggagtgc cgtgggcgct gggcactccg
2820
cctgtgcctg gaggaacgcg actggctgcc tggcaaaacc ctctttgaga acctgtgggc
2880
ctcggtctat ggcagccgca.agacgctgtt tgtgctggcc cacacggacc gggtcagtgg
2940
tctcttgcgc gccagcttcc tgctggccca gcagcgcctg ctggaggacc gcaaggacgt
3000
cgtggtgctg gtgatcctga gccctgacgg ccgccgctcc cgctacgtgc ggctgcgcca
3060
gcgcctctgc cgccagagtg tcctcctctg gccccaccag cccagtggtc agcgcagctt
3120
ctgggcccag ctgggcatgg ccctgaccag ggacaaccac cacttctata accggaactt
3180
ctgccaggga cccacggccg aatagccgtg agccggaatc ctgcacggtg ccacctccac
3240
actcacctca cctctgc
3258
SEQ ID NO:36 Human TLR9 amino acid (1055)
MPMKWSGWRW SWGPATHTAL PPPQGFCRSA LHPLSLLVQA IMLAMTLALG TLPAFLPCEL
60
QPHGLVNCNW LFLKSVPHFS MAAPRGNVTS LSLSSNRIHH LRDSDFAHLP SLRHLNLKWN
120
CPPVGLSPMH FPCHMTIEPS TFLAVPTLEE LNLSYNNIMT VPALPKSLIS LSLSHTNILM
180
LDSASLAGLH ALRFLFMDGN CYYKNPCRQA LEVAPGALLG LGNLTHLSLK YNNLTVVPRN
240
LPSSLEYLLL SYNRIVKLAP EDLANLTALR VLDVGGNCRR CDHAPNPCME CPRHFPQLHP
300
DTFSHLSRLE GLVLKDSSLS WLNASWFRGL GNLRVLDLSE NFLYKCITKT KAFQGLTQLR
360
KLNLSFNYQK RVSFAHLSLA PSFGSLVALK ELDMHGIFFR SLDETTLRPL ARLPMLQTLR
420
LQMNFINQAQ LGIFRAFPGL RYVDLSDNRI SGASELTATM GEADGGEKVW LQPGDLAPAP
480
VDTPSSEDFR PNCSTLNFTL DLSRNNLVTV QPEMFAQLSH LQCLRLSHNC ISQAVNGSQF
540
LPLTGLQVLD LSHNKLDLYH EHSFTELPRL EALDLSYNSQ PFGMQGVGHN FSFVAHLRTL
600
RHLSLAHNNI HSQVSQQLCS TSLRALDFSG NALGHMWAEG DLYLHFFQGL SGLIWLDLSQ
660
NRLHTLLPQT LRNLPKSLQV LRLRDNYLAF FKWWSLHFLP KLEVLDLAGN QLKALTNGSL
720
TAGTRLRRLD VSCNSISFVA PGFFSKAKEL RELNLSANAL KTVDHSWFGP LASALQILDV
780
SANPLHCACG AAFMDFLLEV QAAVPGLPSR VKCGSPGQLQ GLSIFAQDLR LCLDEALSWD
840
CFALSLLAVA LGLGVPMLHH LCGWDLWYCF HLCLAWLPWR GRQSGRDEDA LPYDAFVVFD
900
KTQSAVADWV YNELRGQLEE CRGRWALRLC LEERDWLPGK TLFENLWASV YGSRKTLFVL
960
AHTDRVSGLL PASFLLAQQR LLEDRKDVVV LVILSPDGRR SRYVRLRQRL CRQSVLLWPH
1020
QPSGQRSFWA QLGMALTRDN HHFYNRNFCQ GPTAE
1055
SEQ ID NO:37 Human TLR9 nucleotide
atgcccatga agtggagtgg gtggaggtgg agctgggggc cggccactca cacagccctc
60
ccacccccac agggtttctg ccgcagcgcc ctgcacccgc tgtctctcct ggtgcaggcc
120
atcatgctgg ccatgaccct ggccctgggt accttgcctg ccttcctacc ctgtgagctc
180
cagccccacg gcctggtgaa ctgcaactgg ctgttcctga agtctgtgcc ccacttctcc
240
atggcagcac cccgtggcaa tgtcaccagc ctttccttgt cctccaaccg catccaccac
300
ctccatgatt ctgactttgc ccacctgccc agcctgcggc atctcaacct caagtggaac
360
tgcccgccgg ttggcctcag ccccatgcac ttcccctgcc acatgaccat cgagcccagc
420
accttcttgg ctgtgcccac cctggaagag ctaaacctga gctacaacaa catcatgact
480
gtgcctgcgc tgcccaaatc cctcatatcc ctgtccctca gccataccaa catcctgatg
540
ctagactctg ccagcctcgc cggcctgcat gccctgcgct tcctattcat ggacggcaac
600
tgttattaca agaacccctg caggcaggca ctggaggtgg ccccgggtgc cctccttggc
660
ctgggcaacc tcacccacct gtcactcaag tacaacaacc tcactgtggt gccccgcaac
720
ctgccttcca gcctggagta tctgctgttg tcctacaacc gcatcgtcaa actggcgcct
780
gaggacctgg ccaatctgac cgccctgcgt gtgctcgatg tgggcggaaa ttgccgccgc
840
tgcgaccacg ctcccaaccc ctgcatggag tgccctcgtc acttccccca gctacatccc
900
gataccttca gccacctgag ccgtcttgaa ggcctggtgt tgaaggacag ttctctctcc
960
tggctgaatg ccagttggtt ccgtgggctg ggaaacctcc gagtgctgga cctgagtgag
1020
aacttcctct acaaatgcat cactaaaacc aaggccttcc agggcctaac acagctgcgc
1080
aagcttaacc tgtccttcaa ttaccaaaag agggtgtcct ttgcccacct gtctctggcc
1140
ccttccttcg ggagcctggt cgccctgaag gagctggaca tgcacggcat cttcttccgc
1200
tcactcgatg agaccacgct ccggccactg gcccgcctgc ccatgctcca gactctgcgt
1260
ctgcagatga acttcatcaa ccaggcccag ctcggcatct tcagggcctt ccctggcctg
1320
cgctacgtgg acctgtcgga caaccgcatc agcggagctt cggagctgac agccaccatg
1380
ggggaggcag atggagggga gaaggtctgg ctgcagcctg gggaccttgc tccggcccca
1440
gtggacactc ccagctctga agacttcagg cccaactgca gcaccctcaa cttcaccttg
1500
gatctgtcac ggaacaacct ggtgaccgtg cagccggaga tgtttgccca gctctcgcac
1560
ctgcagtgcc tgcgcctgag ccacaactgc atctcgcagg cagtcaatgg ctcccagttc
1620
ctgccgctga ccggtctgca ggtgctagac ctgtcccaca ataagctgga cctctaccac
1680
gagcactcat tcacggagct accacgactg gaggccctgg acctcagcta caacagccag
1720
ccctttggca tgcagggcgt gggccacaac ttcagcttcg tggctcacct gcgcaccctg
1800
cgccacctca gcctggccca caacaacatc cacagccaag tgtcccagca gctctgcagt
1860
acgtcgctgc gggccctgga cttcagcggc aatgcactgg gccatatgtg ggccgaggga
1920
gacctctatc tgcacttctt ccaaggcctg agcggtttga tctggctgga cttgtcccag
1980
aaccgcctgc acaccctcct gccccaaacc ctgcgcaacc tccccaagag cctacaggtg
2040
ctgcgtctcc gtgacaatta cctggccttc tttaagtggt ggagcctcca cttcctgccc
2100
aaactggaag tcctcgacct ggcaggaaac cagctgaagg ccctgaccaa tggcagcctg
2160
cctgctggca cccggctccg gaggctggat gtcagctgca acagcatcag cttcgtggcc
2220
cccggcttct tttccaaggc caaggagctg cgagagctca accttagcgc caacgccctc
2280
aagacagtgg accactcctg gtttgggccc ctggcgagtg ccctgcaaat actagatgta
2340
agcgccaacc ctctgcactg cgcctgtggg gcggccttta tggacttcct gctggaggtg
2400
caggctgccg tgcccggtct gcccagccgg gtgaagtgtg gcagtccggg ccagctccag
2460
ggcctcagca tctttgcaca ggacctgcgc ctctgcctgg atgaggccct ctcctgggac
2520
tgtttcgccc tctcgctgct ggctgtggct ctgggcctgg gtgtgcccat gctgcatcac
2580
ctctgtggct gggacctctg gtactgcttc cacctgtgcc tggcctggct tccctggcgg
2640
gggcggcaaa gtgggcgaga tgaggatgcc ctgccctacg atgccttcgt ggtcttcgac
2700
aaaacgcaga gcgcagtggc agactgggtg tacaacgagc ttcgggggca gctggaggag
2760
tgccgtgggc gctgggcact ccgcctgtgc ctggaggaac gcgactggct gcctggcaaa
2820
accctctttg agaacctgtg ggcctcggtc tatggcagcc gcaagacgct gtttgtgctg
2880
gcccacacgg accgggtcag tggtctcttg cgcgccagct tcctgctggc ccagcagcgc
2940
ctgctggagg accgcaagga cgtcgtggtg ctggtgatcc tgagccctga cggccgccgc
3000
tcccgctatg tgcggctgcg ccagcgcctc tgccgccaga gtgtcctcct ctggccccac
3060
cagcccagtg gtcagcgcag cttctgggcc cagctgggca tggccctgac cagggacaac
3120
caccacttct ataaccggaa cttctgccag ggacccacgg ccgaa
3165
SEQ ID NO:38 Murine TLR9 amino acid
MVLRRRTLHP LSLLVQAAVL AETLALGTLP AFLPCELKPH GLVDCNWLFL KSVPRFSAAA
60
SCSNITRLSL ISNRIHHLHN SDFVHLSNLR QLNLKWNCPP TGLSPLHFSC HMTIEPRTFL
120
AMRTLEELNL SYNGITTVPR LPSSLVNLSL SRTNILVLDA NSLAGLYSLR VLFMDGNCYY
180
KNPCTGAVKV TPGALLGLSN LTHLSLKYNN LTKVPRQLPP SLEYLLVSYN LIVKLGPEDL
240
ANLTSLRVLD VGGNCRRCDH APHPCIECGQ KSLHLHPETF HHLSHLEGLV LKDSSLHTLN
300
SSWFQGLVNL SVLDLSENFL YESINHTNAF QNLTRLRKLN LSFNYRKKVS FARLHLASSF
360
KNLVSLQELN MNGIFFRSLN KYTLRWLADL PKLHTLHLQM NFINQAQLSI FGTFRALRFV
420
DLSDNRISGP STLSEATPEE ADDAEQEELL SADPHPAPLS TPASKNFMDR CKNFKFTMDL
480
SRNNLVTIKP EMFVNLSRLQ CLSLSHNSIA QAVNGSQFLP LTNLQVLDLS HNKLDLYHWK
540
SFSELPQLQA LDLSYNSQPF SMKGIGHNFS FVAHLSMLHS LSLAHNDIHT RVSSHLNSNS
600
VRFLDFSGNG MGRMWDEGGL YLHFFQGLSG LLKLDLSQNN LHILRPQNLD NLPKSLKLLS
660
LRDNYLSFFN WTSLSFLPNL EVLDLAGNQL KALTNGTLPN GTLLQKLDVS SNSIVSVVPA
720
FFALAVELKE VNLSHNILKT VDRSWFGPIV NMLTVLDVRS NPLHCACGAA FVDLLLEVQT
780
KVPGLANGVK CGSPGQLQGR SIFAQDLRLC LDEVLSWDCF GLSLLAVAVG MVVPILHHLC
840
GWDVWYCFHL CLAWLPLLAR SRRSAQALPY DAFVVFDKAQ SAVADWVYNE LRVRLEERRG
900
RRALRLCLED RDWLPGQTLF ENLWASIYGS RKTLFVLAHT DRVSGLLRTS FLLAQQRLLE
960
DRKDVVVLVI LRPDAHRSRY VRLRQRLCRQ SVLFWPQQPN GQGGFWAQLS TALTRDNRHF
1020
YNQNFCRGPT AE
1032
SEQ ID NO:39 Murine TLR9 nucleotide
tgtcagaggg agcctcggga gaatcctcca tctcccaaca tggttctccg tcgaaggact
60
ctgcacccct tgtccctcct ggtacaggct gcagtgctgg ctgagactct ggccctgggt
120
accctgcctg ccttcctacc ctgtgagctg aagcCtCatg gcctggtgga ctgcaattgg
180
ctgttcctga agtctgtacc ccgtttctct gcggcagcat cctgctccaa catcacccgc
240
ctctccttga tctccaaccg tatccaccac ctgcacaact ccgacttcgt ccacctgtcc
300
aacctgcggc agctgaacct caagtggaac tgtccaccca ctggccttag ccccctgcac
360
ttctcttgcc acatgaccat tgagcccaga accttcctgg ctatgcgtac actggaggag
420
ctgaacctga gctataatgg tatcaccact gtgccccgac tgcccagctc cctggtgaat
480
ctgagcctga gccacaccaa catcctggtt ctagatgcta acagcctcgc cggcctatac
540
agcctgcgcg ttctcttcat ggacgggaac tgctactaca agaacccctg cacaggagcg
600
gtgaaggtga ccccaggcgc cctcctgggc ctgagcaatc tcacccatct gtctctgaag
660
tataacaacc tcacaaaggt gccccgccaa ctgcccccca gcctggagta cctcctggtg
720
tcctataacc tcattgtcaa gctggggcct gaagacctgg ccaatctgac ctcccttcga
780
gtacttgatg tgggtgggaa ttgccgtcgc tgcgaccatg cccccaatcc ctgtatagaa
840
tgtggccaaa agtccctcca cctgcaccct gagaccttcc atcacctgag ccatctggaa
900
ggcctggtgc tgaaggacag ctctctdcat acactgaact cttcctggtt ccaaggtctg
960
gtcaacctct cggtgctgga cctaagcgag aactttctct atgaaagcat caaccacacc
1020
aatgcctttc agaacctaac ccgcctgcgc aagctcaacc tgtccttcaa ttaccgcaag
1080
aaggtatcct ttgcccgcct ccacctggca agttccttca agaacctggt gtcactgcag
1140
gagctgaaca tgaacggcat cttcttccgc tcgctcaaca agtacacgct cagatggctg
1200
gccgatctgc ccaaactcca cactctgcat cttcaaatga acttcatcaa ccaggcacag
1260
ctcagcatct ttggtacctt ccgagccctt-cgctttgtgg acttgtcaga caatcgcatc
1320
agtgggcctt caacgctgtc agaagccacc cctgaagagg cagatgatgc agagcaggag
1380
gagctgttgt ctgcggatcc tcacccagct ccactgagca cccctgcttc taagaacttc
1440
atggacaggt gtaagaactt caagttcacc atggacctgt ctcggaacaa cctggtgact
1500
atcaagccag agatgtttgt caatctctca cgcctccagt gtcttagcct gagccacaac
1560
tccattgcac aggctgtcaa tggctctcag ttcctgccgc tgactaatct gcaggtgctg
1620
gacctgtccc ataacaaact ggacttgtac cactggaaat cgttcagtga gctaccacag
1680
ttgcaggccc tggacctgag ctacaacagc cagcccttta gcatgaaggg tataggccac
1740
aatttcagtt ttgtggccca tctgtccatg ctacacagcc ttagcctggc acacaatgac
1800
attcataccc gtgtgtcctc acatctcaac agcaactcag tgaggtttct tgacttcagc
1860
ggcaacggta tgggccgcat gtgggatgag gggggccttt atctccattt cttccaaggc
1920
ctgagtggcc tgctgaagct ggacctgtct caaaataacc tgcatatcct ccggccccag
1980
aaccttgaca acctccccaa gagcctgaag ctgctgagcc tccgagacaa ctacctatct
2040
ttctttaact ggaccagtct gtccttcctg cccaacctgg aagtcctaga cctggcaggc
2100
aaccagctaa aggccctgac caatggcacc ctgcctaatg gcaccctcct ccagaaactg
2160
gatgtcagca gcaacagtat cgtctctgtg gtcccagcct tcttcgctct ggcggtcgag
2220
ctgaaagagg tcaacctcag ccacaacatt ctcaagacgg tggatcgctc ctggtttggg
2280
cccattgtga tgaacctgac agttctagac gtgagaagca accctctgca ctgtgcctgt
2340
ggggcagcct tcgtagactt actgttggag gtgcagacca aggtgcctgg cctggctaat
2400
ggtgtgaagt gtggcagccc cggccagctg cagggccgta gcatcttcgc acaggacctg
2460
cggctgtgcc tggatgaggt cctctcttgg gactgctttg gcctttcact cttggctgtg
2520
gccgtgggca tggtggtgcc tatactgcac catctctgcg gctgggacgt ctggtactgt
2580
tttcatctgt gcctggcatg gctacctttg ctggcccgca gccgacgcag cgcccaagct
2640
ctcccctatg atgccttcgt ggtgttcgat aaggcacaga gcgcagttgc ggactgggtg
2700
tataacgagc tgcgggtgcg gctggaggag cggcgcggtc gccgagccct acgcttgtgt
2760
ctggaggacc gagattggct gcctggccag acgctcttcg agaacctctg ggcttccatc
2820
tatgggagcc gcaagactct atttgtgctg gcccacacgg accgcgtcag tggcctcctg
2880
cgcaccagct tcctgctggc tcagcagcgc ctgttggaag accgcaagga cgtggtggtg
2940
ttggtgatcc tgcgtccgga tgcccaccgc tcccgctatg tgcgactgcg ccagcgtctc
3000
tgccgccaga gtgtgctctt ctggccccag cagcccaacg ggcagggggg cttctgggcc
3060
cagctgagta cagccctgac tagggacaac cgccacttct ataaccagaa cttctgccgg
3120
ggacctacag cagaatagct cagagcaaca gctggaaaca gctgcatctt catgcctggt
3180
tcccgagttg ctctgcctgc
3200
Ribonucleoside vanadyl complexes (i.e., mixtures of adenine, cytosine, guanosine, and uracil ribonucleoside vanadyl complexes), are well known by those of skill in the art as RNAse inhibitors. Berger S L et al. (1979) Biochemistry 18:5143; Puskas R S et al. (1982) Biochemistry 21:4602. Ribonucleoside vanadyl complexes are commercially available from suppliers including Sigma-Aldrich, Inc.
In one embodiment, the immunostimulatory G,U-containing RNA oligomer of the invention does not contain a CpG dinucleotide and is not a CpG immunostimulatory nucleic acid. In some embodiments, a CpG immunostimulatory nucleic acid is used in the methods of the invention.
A CpG immunostimulatory nucleic acid is a nucleic acid which contains a CG dinucleotide, the C residue of which is unmethylated. CpG immunostimulatory nucleic acids are known to stimulate Th1-type immune responses. CpG sequences, while relatively rare in human DNA are commonly found in the DNA of infectious organisms such as bacteria. The human immune system has apparently evolved to recognize CpG sequences as an early warning sign of infection and to initiate an immediate and powerful immune response against invading pathogens without causing adverse reactions frequently seen with other immune stimulatory agents. Thus CpG containing nucleic acids, relying on this innate immune defense mechanism can utilize a unique and natural pathway for immune therapy. The effects of CpG nucleic acids on immune modulation have been described extensively in U.S. patents such as U.S. Pat. Nos. 6,194,388 B1, 6,207,646 B1, 6,239,116 B1 and 6,218,371 B1, and published patent applications, such as PCT/US98/03678, PCT/US98/10408, PCT/US98/04703, and PCT/US99/09863. The entire contents of each of these patents and patent applications is hereby incorporated by reference.
A CpG nucleic acid is a nucleic acid which includes at least one unmethylated CpG dinucleotide. A nucleic acid containing at least one unmethylated CpG dinucleotide is a nucleic acid molecule which contains an unmethylated cytosine in a cytosine-guanine dinucleotide sequence (i.e., “CpG DNA” or DNA containing a 5′ cytosine followed by 3′ guanosine and linked by a phosphate bond) and activates the immune system. The CpG nucleic acids can be double-stranded or single-stranded. Generally, double-stranded molecules are more stable in vivo, while single-stranded molecules have increased immune activity. Thus in some aspects of the invention it is preferred that the nucleic acid be single stranded and in other aspects it is preferred that the nucleic acid be double stranded. In certain embodiments, while the nucleic acid is single stranded, it is capable of forming secondary and tertiary structures (e.g., by folding back on itself, or by hybridizing with itself either throughout its entirety or at select segments along its length). Accordingly, while the primary structure of such a nucleic acid may be single stranded, its higher order structures may be double or triple stranded. The terms CpG nucleic acid or CpG oligonucleotide as used herein refer to an immunostimulatory CpG nucleic acid unless otherwise indicated. The entire immunostimulatory nucleic acid can be unmethylated or portions may be unmethylated but at least the C of the 5′ CG 3′ must be unmethylated.
In one aspect the invention provides a method of activating an immune cell. The method involves contacting an immune cell with an immunostimulatory composition of the invention, described above, in an effective amount to induce activation of the immune cell. As used herein, an “immune cell” is cell that belongs to the immune system. Immune cells participate in the regulation and execution of inflammatory and immune responses. They include, without limitation, B lymphocytes (B cells), T lymphocytes (T cells), natural killer (NK) cells, dendritic cells, other tissue-specific antigen-presenting cells (e.g., Langerhans cells), macrophages, monocytes, granulocytes (neutrophils, eosinophils, basophils), and mast cells. Splenocytes, thymocytes, and peripheral blood mononuclear cells (PBMCs) include immune cells. Immune cells can be isolated from the blood, spleen, marrow, lymph nodes, thymus, and other tissues using methods well known to those of skill in the art. Immune cells can also include certain cell lines as well as primary cultures maintained in vitro or ex vivo.
In one embodiment the activation of the immune cell involves secretion of a cytokine by the immune cell. In one embodiment the activation of the immune cell involves secretion of a chemokine by the immune cell. In one embodiment the activation of the immune cell involves expression of a costimulatory/accessory molecule by the immune cell. In one embodiment the costimulatory/accessory molecule is selected from the group consisting of intercellular adhesion molecules (ICAMs, e.g., CD54), leukocyte function-associated antigens (LFAs, e.g., CD58), B7s (CD80, CD86), and CD40.
“Activation of an immune cell” shall refer to a transition of an immune cell from a resting or quiescent state to a state of heightened metabolic activity and phenotype associated with immune cell function. Such immune cell function can include, for example, secretion of soluble products such as immunoglobulins, cytokines, and chemokines; cell surface expression of costimulatory/accessory molecules and MHC antigens; immune cell migration; phagocytosis and cytotoxic activity toward target cells; and immune cell maturation. In some instances immune activation can refer to Th1 immune activation; in other instances immune activation can refer to Th2 immune activation.
“Th1 immune activation” as used herein refers to the activation of immune cells to express Th1-like secreted products, including certain cytokines, chemokines, and subclasses of immunoglobulin; and activation of certain immune cells. Th1-like secreted products include, for example, the cytokines IFN-γ, IL-2, IL-12, IL-18, TNF-α, and the chemokine IP-10 (CXCL10). In the mouse, Th1 immune activation stimulates secretion of IgG2a. Th1 immune activation also may include activation of NK cells and dendritic cells, i.e., cells involved in cellular immunity. Th1 immune activation is believed to counter-regulate Th2 immune activation.
“Th2 immune activation” as used herein refers to the activation of immune cells to express Th2-like secreted products, including certain cytokines and subclasses of immunoglobulin. Th2-like secreted products include, for example, the cytokines IL-4 and IL-10. In the mouse, Th2 immune activation stimulates secretion of IgG1 and IgE. Th2 immune activation is believed to counter-regulate Th1 immune activation.
In another aspect, the invention provides a method of inducing an immune response in a subject. The method entails administering to a subject a composition of the invention in an effective amount to induce an immune response in the subject. Thus the compositions of the invention may be used to treat a subject in need of immune activation. A subject in need of immune activation may include a subject in need of Th1-like immune activation.
The compositions and methods of the invention can be used, alone or in conjunction with other agents, to treat a subject in need of Th1-like immune activation. A “subject in need of Th1-like immune activation” is a subject that has or is at risk of developing a disease, disorder, or condition that would benefit from an immune response skewed toward Th1. Such a subject may have or be at risk of having a Th2-mediated disorder that is susceptible to Th1-mediated cross-regulation or suppression. Such disorders include, for example, certain organ-specific autoimmune diseases. Alternatively, such a subject may have or be at risk of having a Th1-deficient state. Such disorders include, for example, tumors, infections with intracellular pathogens, and AIDS.
As used herein, “G,U-rich RNA” shall mean RNA at least 5 nucleotides long that by base composition is at least 60 percent, more preferably at least 80 percent, and most preferably at least 90 percent guanine (G) and uracil (U). Such base composition is measured over the full length of the RNA if it is no more than 10 bases long, and over a stretch of at least 10 contiguous bases if the RNA is more than 10 bases long.
As used herein, “G-rich RNA” shall mean RNA that by base composition is at least 70 percent, more preferably at least 80 percent, even more preferably at least 90 percent, and most preferably at least 95 percent guanine (G). Such base composition is measured over the full length of the RNA if it is no more than 10 bases long, and over a stretch of at least 10 contiguous bases if the RNA is more than 10 bases long.
In some embodiments the compositions of the present invention include a DNA:RNA conjugate. A DNA:RNA conjugate shall mean a molecule or complex that includes at least one deoxyribonucleoside linked to at least one ribonucleoside. The deoxyribonucleoside and ribonucleoside components may be linked by base pair interaction. Alternatively, the deoxyribonucleoside and ribonucleoside components may be linked by covalent linkage between the sugar moieties of the at least one deoxyribonucleoside and the at least one ribonucleoside. The covalent linkage between the sugar moieties may be direct or indirect, for example through a linker. Base pair interactions typically are, but are not limited to, non-covalent Watson-Crick type base pair interactions. Other base pair interactions, including non-covalent (e.g., Hoogstein base pairing) and covalent interactions are contemplated by the invention. Base pair interactions also typically will involve duplex formation involving two strands, but higher order interactions are also contemplated by the invention.
A DNA:RNA conjugate involving a covalent linkage between the sugar moieties of the at least one deoxyribonucleoside and the at least one ribonucleoside is referred to herein as having a chimeric DNA:RNA backbone. The DNA:RNA conjugate having a chimeric DNA:RNA backbone will have primary structure defined by its base sequence, and it may further have a secondary or higher order structure. A secondary or higher order structure will include at least one intramolecular base pair interaction, e.g., a stem-loop structure, or intermolecular base pair interaction.
Heteroduplex base pairing shall refer to intramolecular or intermolecular base pair interaction between DNA and RNA. For example, heteroduplex base pairing may occur between individual complementary single-stranded DNA and RNA molecules. Alternatively, as in the case of suitable DNA:RNA chimeric backbone nucleic acid molecules, heteroduplex base pairing may occur between complementary DNA and RNA regions within the same molecule.
In some embodiments the compositions of the present invention include a chimeric DNA:RNA backbone having a cleavage site between the DNA and RNA. A cleavage site refers to a structural element along the chimeric backbone that is susceptible to cleavage by any suitable means. The cleavage site may be a phosphodiester bond that is relatively susceptible to cleavage by endonuclease. In this instance the DNA and RNA each may include internucleotide linkages that are stabilized, such that the chimeric backbone is most susceptible to endonuclease cleavage at the phosphodiester junction between the stabilized DNA and the stabilized RNA. The cleavage site may be designed so that it is susceptible to cleavage under certain pH conditions, e.g., relatively more stable at higher pH than at lower pH, or vice versa. Such pH sensitivity may be accomplished, for example, by preparation of the chimeric DNA:RNA composition in liposomes. The cleavage site may involve a disulfide linkage. Such disulfide linkage may be relatively more stable under oxidizing conditions than under reducing conditions, e.g., the latter conditions present within an endosome. The cleavage site may also involve a linker that is susceptible to cleavage by an enzyme, pH, redox condition, or the like. In some embodiments the composition may include more than one cleavage site.
Conjugates of the invention permit selection of fixed molar ratios of the components of the conjugates. In the case of DNA:RNA conjugates it may be advantageous or convenient to have a 1:1 ratio of DNA and RNA. Conjugates that are heteroduplex DNA:RNA will commonly have a 1:1 ratio of DNA and RNA. Conjugates that have a chimeric DNA:RNA backbone may also commonly have a 1:1 ratio of DNA and RNA. Conjugates having other DNA:RNA ratios are contemplated by the invention, including, but not limited to, 1:2, 1:3, 1:4, 2:1, 3:1, 4:1, and so on. The conjugation may stabilize one or more components in comparison to the stability of the same component or components alone. Conjugatation may also facilitate delivery of the components into cells at the selected ratio.
Cleavage sites may serve any of several purposes useful in the present invention. Once delivered to a cell of interest, the components joined via the cleavage site (or sites) may be liberated to become independently or optimally active within the cell or in the vicinity of the cell. In some embodiments the cleavage sites may be important to pharmacokinetics of at least one of the components of the conjugate. For instance, the cleavage sites may be designed and selected to confer an extended time release of one of the components.
The invention generally provides efficient methods of identifying immunostimulatory compounds and the compounds and agents so identified. Generally, the screening methods involve assaying for compounds which inhibit or enhance signaling through a particular TLR. The methods employ a TLR, a suitable reference ligand for the TLR, and a candidate immunostimulatory compound. The selected TLR is contacted with a suitable refernce compound (TLR ligand) and a TLR-mediated reference signal is measured. The selected TLR is also contacted with a candidate immunostimulatory compound and a TLR-mediated test signal is measured. The test signal and the reference signal are then compared. A favorable candidate immunostimulatory compound may subsequently be used as a reference compound in the assay. Such methods are adaptable to automated, high throughput screening of candidate compounds. Examples of such high throughput screening methods are described in U.S. Pat. Nos. 6,103,479; 6,051,380; 6,051,373; 5,998,152; 5,876,946; 5,708,158; 5,443,791; 5,429,921; and 5,143,854.
The assay mixture comprises a candidate immunostimulatory compound. Typically, a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a different response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration of agent or at a concentration of agent below the limits of assay detection. Candidate immunostimulatory compounds encompass numerous chemical classes, although typically they are organic compounds. Preferably, the candidate immunostimulatory compounds are small organic compounds, i.e., those having a molecular weight of more than 50 yet less than about 2500. Polymeric candidate immunostimulatory compounds can have higher molecular weights, e.g., oligonucleotides in the range of about 2500 to about 12,500. Candidate immunostimulatory compounds comprise functional chemical groups necessary for structural interactions with polypeptides, and may include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups and more preferably at least three of the functional chemical groups. The candidate immunostimulatory compounds can comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups. Candidate immunostimulatory compounds also can be biomolecules such as nucleic acids, peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like. Where the candidate immunostimulatory compound is a nucleic acid, the candidate immunostimulatory compound typically is a DNA or RNA molecule, although modified nucleic acids having non-natural bonds or subunits are also contemplated.
Candidate immunostimulatory compounds are obtained from a wide variety of sources, including libraries of natural, synthetic, or semisynthetic compounds, or any combination thereof. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily modified through conventional chemical, physical, and biochemical means. Further, known pharmacological agents may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc., to produce structural analogs of the candidate immunostimulatory compounds.
Therefore, a source of candidate immunostimulatory compounds are libraries of molecules based on known TLR ligands, e.g., CpG oligonucleotides known to interact with TLR9, in which the structure of the ligand is changed at one or more positions of the molecule to contain more or fewer chemical moieties or different chemical moieties. The structural changes made to the molecules in creating the libraries of analog inhibitors can be directed, random, or a combination of both directed and random substitutions and/or additions. One of ordinary skill in the art in the preparation of combinatorial libraries can readily prepare such libraries based on existing TLR9 ligands.
A variety of other reagents also can be included in the mixture. These include reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc. which may be used to facilitate optimal protein-protein and/or protein-nucleic acid binding. Such a reagent may also reduce non-specific or background interactions of the reaction components. Other reagents that improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, antimicrobial agents, and the like may also be used.
The order of addition of components, incubation temperature, time of incubation, and other parameters of the assay may be readily determined. Such experimentation merely involves optimization of the assay parameters, not the fundamental composition of the assay. Incubation temperatures typically are between 4° C. and 40° C. Incubation times preferably are minimized to facilitate rapid, high throughput screening, and typically are between 1 minute and 10 hours.
After incubation, the level of TLR signaling is detected by any convenient method available to the user. For cell-free binding type assays, a separation step is often used to separate bound from unbound components. The separation step may be accomplished in a variety of ways. For example, separation can be accomplished in solution, or, conveniently, at least one of the components is immobilized on a solid substrate, from which the unbound components may be easily separated. The solid substrate can be made of a wide variety of materials and in a wide variety of shapes, e.g., microtiter plate, microbead, dipstick, resin particle, etc. The substrate preferably is chosen to maximize signal-to-noise ratios, primarily to minimize background binding, as well as for ease of separation and cost.
Separation may be effected for example, by removing a bead or dipstick from a reservoir, emptying or diluting a reservoir such as a microtiter plate well, rinsing a bead, particle, chromatographic column or filter with a wash solution or solvent. The separation step preferably includes multiple rinses or washes. For example, when the solid substrate is a microtiter plate, the wells may be washed several times with a washing solution, which typically includes those components of the incubation mixture that do not participate in specific bindings such as salts, buffer, detergent, non-specific protein, etc. Where the solid substrate is a magnetic bead, the beads may be washed one or more times with a washing solution and isolated using a magnet.
Detection may be effected in any convenient way for cell-based assays such as measurement of an induced polypeptide within, on the surface of, or secreted by the cell. Examples of detection methods useful in cell-based assays include fluorescence-activated cell sorting (FACS) analysis, bioluminescence, fluorescence, enzyme-linked immunosorbent assay (ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR), and the like. Examples of detection methods useful in cell-free assays include bioluminescence, fluorescence, enzyme-linked immunosorbent assay (ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR), and the like.
A subject shall mean a human or animal including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent, e.g., rats and mice, primate, e.g., monkey, and fish or aquaculture species such as fin fish (e.g., salmon) and shellfish (e.g., shrimp and scallops). Subjects suitable for therapeutic or prophylactic methods include vertebrate and invertebrate species. Subjects can be house pets (e.g., dogs, cats, fish, etc.), agricultural stock animals (e.g., cows, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), zoo animals (e.g., lions, giraffes, etc.), but are not so limited. Although many of the embodiments described herein relate to human disorders, the invention is also useful for treating other nonhuman vertebrates.
As used herein, the term “treat”, when used with respect to one of the disorders described herein, refers both to a prophylactic treatment which decreases the likelihood that a subject will develop the disorder as well as to treatment of an established disorder, e.g., to reduce or eliminate the disorder or symptoms of the disorder, or to prevent the disorder or symptoms of the disorder from becoming worse.
A subject that has a disorder refers to a subject that has an objectively measureable manifestation of the disorder. Thus for example a subject that has a cancer is a subject that has detectable cancerous cells. A subject that has an infection is a subject that has been exposed to an infectious organism and has acute or chronic detectable levels of the organism in the body. The infection may be latent (dormant) or active.
A subject at risk of having a disorder is defined as a subject that has a higher than normal risk of developing the disorder. The normal risk is generally the risk of a population of normal individuals that do not have the disorder and that are not identifiably predisposed, e.g., either genetically or environmentally, to developing the disorder. Thus a subject at risk of having a disorder may include, without limitation, a subject that is genetically predisposed to developing the disorder, as well as a subject that is or will be exposed to an environmental agent known or believed to cause the disorder. Environmental agents specifically include, but are not limited to, infectious agents such as viruses, bacteria, fungi, and parasites. Other environmental agents may include, for example, tobacco smoke, certain organic chemicals, asbestos, and the like.
The term “effective amount” of a nucleic acid or other therapeutic agent refers to the amount necessary or sufficient to realize a desired biologic effect. In general, an effective amount is that amount necessary to cause activation of the immune system, resulting potentially in the development of an antigen-specific immune response. In some embodiments, the nucleic acid or other therapeutic agent are administered in an effective amount to stimulate or induce a Th1 immune response or a general immune response. An effective amount to stimulate a Th1 immune response may be defined as that amount which stimulates the production of one or more Th1-type cytokines, such as IL-2, IL-12, TNF-α, and IFN-γ, and/or production of one or more Th1-type antibodies.
In yet another aspect the invention provides a method of inducing an immune response in a subject. The method according to this aspect of the invention involves administering to a subject an antigen, and administering to the subject an immunostimulatory composition of the invention in an effective amount to induce an immune response to the antigen. It is to be noted that the antigen may be administered before, after, or concurrently with the immunostimulatory composition of the invention. In addition, both the antigen and the immunostimulatory compound can be administered to the subject more than once.
The invention further provides, in yet another aspect, a method of inducing an immune response in a subject. The method according to this aspect of the invention involves isolating dendritic cells of a subject, contacting the dendritic cells ex vivo with an immunostimulatory composition of the invention, contacting the dendritic cells ex vivo with an antigen, and administering the contacted dendritic cells to the subject.
The term “antigen” refers to a molecule capable of provoking an immune response. The term antigen broadly includes any type of molecule that is recognized by a host system as being foreign. Antigens include but are not limited to microbial antigens, cancer antigens, and allergens. Antigens include, but are not limited to, cells, cell extracts, proteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, peptide and non-peptide mimics of polysaccharides and other molecules, small molecules, lipids, glycolipids, and carbohydrates. Many antigens are protein or polypeptide in nature, as proteins and polypeptides are generally more antigenic than carbohydrates or fats.
The antigen may be an antigen that is encoded by a nucleic acid vector or it may not be encoded in a nucleic acid vector. In the former case the nucleic acid vector is administered to the subject and the antigen is expressed in vivo. In the latter case the antigen may be administered directly to the subject. An antigen not encoded in a nucleic acid vector as used herein refers to any type of antigen that is not a nucleic acid. For instance, in some aspects of the invention the antigen not encoded in a nucleic acid vector is a peptide or a polypeptide. Minor modifications of the primary amino acid sequences of peptide or polypeptide antigens may also result in a polypeptide which has substantially equivalent antigenic activity as compared to the unmodified counterpart polypeptide. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein as long as antigenicity still exists. The peptide or polypeptide may be, for example, virally derived. The antigens useful in the invention may be any length, ranging from small peptide fragments of a full length protein or polypeptide to the full length form. For example, the antigen may be less than 5, less than 8, less than 10, less than 15, less than 20, less than 30, less than 50, less than 70, less than 100, or more amino acid residues in length, provided it stimulates a specific immune response.
The nucleic acid encoding the antigen is operatively linked to a gene expression sequence which directs the expression of the antigen nucleic acid within a eukaryotic cell. The gene expression sequence is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient transcription and translation of the antigen nucleic acid to which it is operatively linked. The gene expression sequence may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter. Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, β-actin promoter and other constitutive promoters. Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the cytomegalovirus (CMV), simian virus (e.g., SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, the long terminal repeats (LTR) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters are known to those of ordinary skill in the art. The promoters useful as gene expression sequences of the invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those of ordinary skill in the art.
In general, the gene expression sequence shall include, as necessary, 5′ non-transcribing and 5′ non-translating sequences involved with the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Especially, such 5′ non-transcribing sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined antigen nucleic acid. The gene expression sequences optionally include enhancer sequences or upstream activator sequences as desired.
The antigen nucleic acid is operatively linked to the gene expression sequence. As used herein, the antigen nucleic acid sequence and the gene expression sequence are said to be operably linked when they are covalently linked in such a way as to place the expression or transcription and/or translation of the antigen coding sequence under the influence or control of the gene expression sequence. Two DNA sequences are said to be operably linked if induction of a promoter in the 5′ gene expression sequence results in the transcription of the antigen sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the antigen sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a gene expression sequence would be operably linked to an antigen nucleic acid sequence if the gene expression sequence were capable of effecting transcription of that antigen nucleic acid sequence such that the resulting transcript is translated into the desired protein or polypeptide.
The antigen nucleic acid of the invention may be delivered to the immune system alone or in association with a vector. In its broadest sense, a vector is any vehicle capable of facilitating the transfer of the antigen nucleic acid to the cells of the immune system so that the antigen can be expressed and presented on the surface of the immune cell. The vector generally transports the nucleic acid to the immune cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. The vector optionally includes the above-described gene expression sequence to enhance expression of the antigen nucleic acid in immune cells. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antigen nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to, nucleic acid sequences from the following viruses: retrovirus, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and Rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known in the art.
Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, W. H. Freeman Co., New York (1990) and Murray, E. J. Methods in Molecular Biology, vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).
A preferred virus for certain applications is the adeno-associated virus, a double-stranded DNA virus. The adeno-associated virus can be engineered to be replication-deficient and is capable of infecting a wide range of cell types and species. It further has advantages, such as heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, wild-type adeno-associated virus manifest some preference for integration sites into human cellular DNA, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion. Recombinant adeno-associated viruses that lack the replicase protein apparently lack this integration sequence specificity.
Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well-known to those of skill in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been found to be particularly advantageous for delivering genes to cells in vivo because of their inability to replicate within and integrate into a host genome. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRc/CMV, SV40, and pBlueScript. Other plasmids are well-known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA.
It has recently been discovered that gene-carrying plasmids can be delivered to the immune system using bacteria. Modified forms of bacteria such as Salmonella can be transfected with the plasmid and used as delivery vehicles. The bacterial delivery vehicles can be administered to a host subject orally or by other administration means. The bacteria deliver the plasmid to immune cells, e.g., B cells, dendritic cells, likely by passing through the gut barrier. High levels of immune protection have been established using this methodology. Such methods of delivery are useful for the aspects of the invention utilizing systemic delivery of antigen, nucleic acids, and/or other therapeutic agent.
In some aspects of the invention, the nucleic acids are administered along with therapeutic agents such as disorder-specific medicaments. As used herein, a disorder-specific medicament is a therapy or agent that is used predominately in the treatment or prevention of a disorder.
In one aspect, the combination of nucleic acid and disorder-specific medicaments allows for the administration of higher doses of disorder-specific medicaments without as many side effects as are ordinarily experienced at those high doses. In another aspect, the combination of nucleic acid and disorder-specific medicaments allows for the administration of lower, sub-therapeutic doses of either compound, but with higher efficacy than would otherwise be achieved using such low doses. As one example, by administering a combination of an immunostimulatory nucleic acid and a medicament, it is possible to achieve an effective response even though the medicament is administered at a dose which alone would not provide a therapeutic benefit (i.e., a sub-therapeutic dose). As another example, the combined administration achieves a response even though the nucleic acid is administered at a dose which alone would not provide a therapeutic benefit.
The nucleic acids and/or other therapeutic agents can also be administered on fixed schedules or in different temporal relationships to one another. The various combinations have many advantages over the prior art methods of modulating immune responses or preventing or treating disorders, particularly with regard to decreased non-specific toxicity to normal tissues.
Cancer is a disease which involves the uncontrolled growth (i.e., division) of cells. Some of the known mechanisms which contribute to the uncontrolled proliferation of cancer cells include growth factor independence, failure to detect genomic mutation, and inappropriate cell signaling. The ability of cancer cells to ignore normal growth controls may result in an increased rate of proliferation. Although the causes of cancer have not been firmly established, there are some factors known to contribute, or at least predispose a subject, to cancer. Such factors include particular genetic mutations (e.g., BRCA gene mutation for breast cancer, APC for colon cancer), exposure to suspected cancer-causing agents, or carcinogens (e.g., asbestos, UV radiation) and familial disposition for particular cancers such as breast cancer.
The cancer may be a malignant or non-malignant cancer. Cancers or tumors include but are not limited to biliary tract cancer; brain cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; intraepithelial neoplasms; lymphomas; liver cancer; lung cancer (e.g., small cell and non-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer; pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer; testicular cancer; thyroid cancer; and renal cancer, as well as other carcinomas and sarcomas. In one embodiment the cancer is hairy cell leukemia, chronic myelogenous leukemia, cutaneous T-cell leukemia, multiple myeloma, follicular lymphoma, malignant melanoma, squamous cell carcinoma, renal cell carcinoma, prostate carcinoma, bladder cell carcinoma, or colon carcinoma.
A “subject having a cancer” is a subject that has detectable cancerous cells.
A “subject at risk of developing a cancer” is one who has a higher than normal probability of developing cancer. These subjects include, for instance, subjects having a genetic abnormality that has been demonstrated to be associated with a higher likelihood of developing a cancer, subjects having a familial disposition to cancer, subjects exposed to cancer-causing agents (i.e., carcinogens) such as tobacco, asbestos, or other chemical toxins, and subjects previously treated for cancer and in apparent remission.
A “cancer antigen” as used herein is a compound, such as a peptide or protein, associated with a tumor or cancer cell surface and which is capable of provoking an immune response when expressed on the surface of an antigen presenting cell in the context of an MHC molecule. Cancer antigens can be prepared from cancer cells either by preparing crude extracts of cancer cells, for example, as described in Cohen P A et al. (1994) Cancer Res 54:1055-8, by partially purifying the antigens, by recombinant technology, or by de novo synthesis of known antigens. Cancer antigens include but are not limited to antigens that are recombinantly expressed, an immunogenic portion of, or a whole tumor or cancer. Such antigens can be isolated or prepared recombinantly or by any other means known in the art.
The terms “cancer antigen” and “tumor antigen” are used interchangeably and refer to antigens which are differentially expressed by cancer cells and can thereby be exploited in order to target cancer cells. Cancer antigens are antigens which can potentially stimulate apparently tumor-specific immune responses. Some of these antigens are encoded, although not necessarily expressed, by normal cells. These antigens can be characterized as those which are normally silent (i.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation and those that are temporally expressed such as embryonic and fetal antigens. Other cancer antigens are encoded by mutant cellular genes, such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses. Examples of tumor antigens include MAGE, MART-1/Melan-A, gp100, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, Colorectal associated antigen (CRC)—C017-1A/GA733, Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, Smad family of tumor antigens, lmp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2.
Cancers or tumors and tumor antigens associated with such tumors (but not exclusively), include acute lymphoblastic leukemia (etv6; aml1; cyclophilin b), B cell lymphoma (Ig-idiotype), glioma (E-cadherin; α-catenin; β-catenin; γ-catenin; p120ctn), bladder cancer (p21ras), biliary cancer (p21ras), breast cancer (MUC family; HER2/neu; c-erbB-2), cervical carcinoma (p53; p21ras), colon carcinoma (p21ras; HER2/neu; c-erbB-2; MUC family), colorectal cancer (Colorectal associated antigen (CRC)—C017-1A/GA733; APC), choriocarcinoma (CEA), epithelial cell cancer (cyclophilin b), gastric cancer (HER2/neu; c-erbB-2; ga733 glycoprotein), hepatocellular cancer (α-fetoprotein), Hodgkins lymphoma (lmp-1; EBNA-1), lung cancer (CEA; MAGE-3; NY-ESO-1), lymphoid cell-derived leukemia (cyclophilin b), melanoma (p15 protein, gp75, oncofetal antigen, GM2 and GD2 gangliosides), myeloma (MUC family; p21ras), non-small cell lung carcinoma (HER2/neu; c-erbB-2), nasopharyngeal cancer (lmp-1; EBNA-1), ovarian cancer (MUC family; HER2/neu; c-erbB-2), prostate cancer (Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3; PSMA; HER2/neu; c-erbB-2), pancreatic cancer (p21ras; MUC family; HER2/neu; c-erbB-2; ga733 glycoprotein), renal cancer (HER2/neu; c-erbB-2), squamous cell cancers of cervix and esophagus (viral products such as human papilloma virus proteins), testicular cancer (NY-ESO-1), T-cell leukemia (HTLV-1 epitopes), and melanoma (Melan-A/MART-1; cdc27; MAGE-3; p21ras; gp100Pmel117).
For examples of tumor antigens which bind to either or both MHC class I and MHC class II molecules, see the following references: Coulie, Stem Cells 13:393-403, 1995; Traversari et al. J Exp Med 176:1453-1457, 1992; Chaux et al. J Immunol 163:2928-2936, 1999; Fujie et al. Int J Cancer 80:169-172, 1999; Tanzarella et al. Cancer Res 59:2668-2674, 1999; van der Bruggen et al. Eur J Immunol 24:2134-2140, 1994; Chaux et al. J Exp Med 189:767-778, 1999; Kawashima et al. Hum Immunol 59:1-14, 1998; Tahara et al. Clin Cancer Res 5:2236-2241, 1999; Gaugler et al. J Exp Med 179:921-930, 1994; van der Bruggen et al. Eur J Immunol 24:3038-3043, 1994; Tanaka et al. Cancer Res 57:4465-4468, 1997; Oiso et al. Int J Cancer 81:387-394, 1999; Herman et al. Immunogenetics 43:377-383, 1996; Manici et al. J Exp Med 189:871-876, 1999; Duffour et al. Eur J Immunol 29:3329-3337, 1999; Zorn et al. Eur J Immunol 29:602-607, 1999; Huang et al. J Immunol 162:6849-6854, 1999; Boël et al. Immunity 2:167-175, 1995; Van den Eynde et al. J Exp Med 182:689-698, 1995; De Backer et al. Cancer Res 59:3157-3165, 1999; Jäger et al. J Exp Med 187:265-270, 1998; Wang et al. J Immunol 161:3596-3606, 1998; Aarnoudse et al. Int J Cancer 82:442-448, 1999; Guilloux et al. J Exp Med 183:1173-1183, 1996; Lupetti et al. J Exp Med 188:1005-1016, 1998; Wölfel et al. Eur J Immunol 24:759-764, 1994; Skipper et al. J Exp Med 183:527-534, 1996; Kang et al. J Immunol 155:1343-1348, 1995; Morel et al. Int J Cancer 83:755-759, 1999; Brichard et al. Eur J Immunol 26:224-230, 1996; Kittlesen et al. J Immunol 160:2099-2106, 1998; Kawakami et al. J Immunol 161:6985-6992, 1998; Topalian et al. J Exp Med 183:1965-1971, 1996; Kobayashi et al. Cancer Research 58:296-301, 1998; Kawakami et al. J Immunol 154:3961-3968, 1995; Tsai et al. J Immunol 158:1796-1802, 1997; Cox et al. Science 264:716-719, 1994; Kawakami et al. Proc Natl Acad Sci USA 91:6458-6462, 1994; Skipper et al. J Immunol 157:5027-5033, 1996; Robbins et al. J Immunol 159:303-308, 1997; Castelli et al. J Immunol 162:1739-1748, 1999; Kawakami et al. J Exp Med 180:347-352, 1994; Castelli et al. J Exp Med 181:363-368, 1995; Schneider et al. Int J Cancer 75:451-458, 1998; Wang et al. J Exp Med 183:1131-1140, 1996; Wang et al. J Exp Med 184:2207-2216, 1996; Parkhurst et al. Cancer Research 58:4895-4901, 1998; Tsang et al. J Natl Cancer Inst 87:982-990, 1995; Correale et al. J Natl Cancer Inst 89:293-300, 1997; Coulie et al. Proc Natl Acad Sci USA 92:7976-7980, 1995; Wölfel et al. Science 269:1281-1284, 1995; Robbins et al. J Exp Med 183:1185-1192, 1996; Brändle et al. J Exp Med 183:2501-2508, 1996; ten Bosch et al. Blood 88:3522-3527, 1996; Mandruzzato et al. J Exp Med 186:785-793, 1997; Guéguen et al. J Immunol 160:6188-6194, 1998; Gjertsen et al. Int J Cancer 72:784-790, 1997; Gaudin et al. J Immunol 162:1730-1738, 1999; Chiari et al. Cancer Res 59:5785-5792, 1999; Hogan et al. Cancer Res 58:5144-5150, 1998; Pieper et al. J Exp Med 189:757-765, 1999; Wang et al. Science 284:1351-1354, 1999; Fisk et al. J Exp Med 181:2109-2117, 1995; Brossart et al. Cancer Res 58:732-736, 1998; Röpke et al. Proc Natl Acad Sci USA 93:14704-14707, 1996; Ikeda et al. Immunity 6:199-208, 1997; Ronsin et al. J Immunol 163:483-490, 1999; Vonderheide et al. Immunity 10:673-679, 1999. These antigens as well as others are disclosed in PCT Application PCT/US98/18601
The compositions and methods of the invention can be used alone or in conjunction with other agents and methods useful for the treatment of cancer. Cancer is currently treated using a variety of modalities including surgery, radiation therapy and chemotherapy. The choice of treatment modality will depend upon the type, location and dissemination of the cancer. For example, surgery and radiation therapy may be more appropriate in the case of solid, well-defined tumor masses and less practical in the case of non-solid tumor cancers such as leukemia and lymphoma. One of the advantages of surgery and radiation therapy is the ability to control to some extent the impact of the therapy, and thus to limit the toxicity to normal tissues in the body. However, surgery and radiation therapy are often followed by chemotherapy to guard against any remaining or radio-resistant cancer cells. Chemotherapy is also the most appropriate treatment for disseminated cancers such as leukemia and lymphoma as well as metastases.
Chemotherapy refers to therapy using chemical and/or biological agents to attack cancer cells. Unlike localized surgery or radiation, chemotherapy is generally administered in a systemic fashion and thus toxicity to normal tissues is a major concern. Because many chemotherapy agents target cancer cells based on their proliferative profiles, tissues such as the gastrointestinal tract and the bone marrow which are normally proliferative are also susceptible to the effects of the chemotherapy. One of the major side effects of chemotherapy is myelosuppression (including anemia, neutropenia and thrombocytopenia) which results from the death of normal hemopoietic precursors.
Many chemotherapeutic agents have been developed for the treatment of cancer. Not all tumors, however, respond to chemotherapeutic agents and others although initially responsive to chemotherapeutic agents may develop resistance. As a result, the search for effective anti-cancer drugs has intensified in an effort to find even more effective agents with less non-specific toxicity.
Cancer medicaments function in a variety of ways. Some cancer medicaments work by targeting physiological mechanisms that are specific to tumor cells. Examples include the targeting of specific genes and their gene products (i.e., proteins primarily) which are mutated in cancers. Such genes include but are not limited to oncogenes (e.g., Ras, Her2, bcl-2), tumor suppressor genes (e.g., EGF, p53, Rb), and cell cycle targets (e.g., CDK4, p21, telomerase). Cancer medicaments can alternately target signal transduction pathways and molecular mechanisms which are altered in cancer cells. Targeting of cancer cells via the epitopes expressed on their cell surface is accomplished through the use of monoclonal antibodies. This latter type of cancer medicament is generally referred to herein as immunotherapy.
Other cancer medicaments target cells other than cancer cells. For example, some medicaments prime the immune system to attack tumor cells (i.e., cancer vaccines). Still other medicaments, called angiogenesis inhibitors, function by attacking the blood supply of solid tumors. Since the most malignant cancers are able to metastasize (i.e., exit the primary tumor site and seed a another site, thereby forming a secondary tumor), medicaments that impede this metastasis are also useful in the treatment of cancer. Angiogenic mediators include basic FGF, VEGF, angiopoietins, angiostatin, endostatin, TNF-α, TNP-470, thrombospondin-1, platelet factor 4, CAI, and certain members of the integrin family of proteins. One category of this type of medicament is a metalloproteinase inhibitor, which inhibits the enzymes used by the cancer cells to exist the primary tumor site and extravasate into another tissue.
Some cancer cells are antigenic and thus can be targeted by the immune system. In one aspect, the combined administration of nucleic acid and cancer medicaments, particularly those which are classified as cancer immunotherapies, is useful for stimulating a specific immune response against a cancer antigen.
The theory of immune surveillance is that a prime function of the immune system is to detect and eliminate neoplastic cells before a tumor forms. A basic principle of this theory is that cancer cells are antigenically different from normal cells and thus elicit immune reactions that are similar to those that cause rejection of immunologically incompatible allografts. Studies have confirmed that tumor cells differ, either qualitatively or quantitatively, in their expression of antigens. For example, “tumor-specific antigens” are antigens that are specifically associated with tumor cells but not normal cells. Examples of tumor specific antigens are viral antigens in tumors induced by DNA or RNA viruses. “Tumor-associated” antigens are present in both tumor cells and normal cells but are present in a different quantity or a different form in tumor cells. Examples of such antigens are oncofetal antigens (e.g., carcinoembryonic antigen), differentiation antigens (e.g., T and Tn antigens), and oncogene products (e.g., HER/neu).
Different types of cells that can kill tumor targets in vitro and in vivo have been identified: natural killer (NK) cells, cytolytic T lymphocytes (CTLs), lymphokine-activated killer cells (LAKs), and activated macrophages. NK cells can kill tumor cells without having been previously sensitized to specific antigens, and the activity does not require the presence of class I antigens encoded by the major histocompatibility complex (MHC) on target cells. NK cells are thought to participate in the control of nascent tumors and in the control of metastatic growth. In contrast to NK cells, CTLs can kill tumor cells only after they have been sensitized to tumor antigens and when the target antigen is expressed on the tumor cells that also express MHC class I. CTLs are thought to be effector cells in the rejection of transplanted tumors and of tumors caused by DNA viruses. LAK cells are a subset of null lymphocytes distinct from the NK and CTL populations. Activated macrophages can kill tumor cells in a manner that is neither antigen-dependent nor MHC-restricted once activated. Activated macrophages are through to decrease the growth rate of the tumors they infiltrate. In vitro assays have identified other immune mechanisms such as antibody-dependent, cell-mediated cytotoxic reactions and lysis by antibody plus complement. However, these immune effector mechanisms are thought to be less important in vivo than the function of NK, CTLs, LAK, and macrophages in vivo (for review see Piessens W F et al. “Tumor Immunology”, In: Scientific American Medicine, Vol. 2, Scientific American Books, N.Y., pp. 1-13, 1996).
The goal of immunotherapy is to augment a patient's immune response to an established tumor. One method of immunotherapy includes the use of adjuvants. Adjuvant substances derived from microorganisms, such as bacillus Calmette-Guérin, heighten the immune response and enhance resistance to tumors in animals.
Immunotherapeutic agents are medicaments which derive from antibodies or antibody fragments which specifically bind or recognize a cancer antigen. Antibody-based immunotherapies may function by binding to the cell surface of a cancer cell and thereby stimulate the endogenous immune system to attack the cancer cell. Another way in which antibody-based therapy functions is as a delivery system for the specific targeting of toxic substances to cancer cells. Antibodies are usually conjugated to toxins such as ricin (e.g., from castor beans), calicheamicin and maytansinoids, to radioactive isotopes such as Iodine-131 and Yttrium-90, to chemotherapeutic agents (as described herein), or to biological response modifiers. In this way, the toxic substances can be concentrated in the region of the cancer and non-specific toxicity to normal cells can be minimized. In addition to the use of antibodies which are specific for cancer antigens, antibodies which bind to vasculature, such as those which bind to endothelial cells, are also useful in the invention. This is because solid tumors generally are dependent upon newly formed blood vessels to survive, and thus most tumors are capable of recruiting and stimulating the growth of new blood vessels. As a result, one strategy of many cancer medicaments is to attack the blood vessels feeding a tumor and/or the connective tissues (or stroma) supporting such blood vessels.
Cancer vaccines are medicaments which are intended to stimulate an endogenous immune response against cancer cells. Currently produced vaccines predominantly activate the humoral immune system (i.e., the antibody-dependent immune response). Other vaccines currently in development are focused on activating the cell-mediated immune system including cytotoxic T lymphocytes which are capable of killing tumor cells. Cancer vaccines generally enhance the presentation of cancer antigens to both antigen presenting cells (e.g., macrophages and dendritic cells) and/or to other immune cells such as T cells, B cells, and NK cells.
Although cancer vaccines may take one of several forms, as discussed infra, their purpose is to deliver cancer antigens and/or cancer associated antigens to antigen presenting cells (APC) in order to facilitate the endogenous processing of such antigens by APC and the ultimate presentation of antigen presentation on the cell surface in the context of MHC class I molecules. One form of cancer vaccine is a whole cell vaccine which is a preparation of cancer cells which have been removed from a subject, treated ex vivo and then reintroduced as whole cells in the subject. Lysates of tumor cells can also be used as cancer vaccines to elicit an immune response. Another form cancer vaccine is a peptide vaccine which uses cancer-specific or cancer-associated small proteins to activate T cells. Cancer-associated proteins are proteins which are not exclusively expressed by cancer cells (i.e., other normal cells may still express these antigens). However, the expression of cancer-associated antigens is generally consistently upregulated with cancers of a particular type. Other cancer vaccines include ganglioside vaccines, heat-shock protein vaccines, viral and bacterial vaccines, and nucleic acid vaccines.
Yet another form of cancer vaccine is a dendritic cell vaccine which includes whole dendritic cells which have been exposed to a cancer antigen or a cancer-associated antigen in vitro. Lysates or membrane fractions of dendritic cells may also be used as cancer vaccines. Dendritic cell vaccines are able to activate APCs directly. A dendritic cell is a professional APC. Dendritic cells form the link between the innate and the acquired immune system by presenting antigens and through their expression of pattern recognition receptors which detect microbial molecules like LPS in their local environment. Dendritic cells efficiently internalize, process, and present soluble specific antigen to which it is exposed. The process of internalizing and presenting antigen causes rapid upregulation of the expression of major histocompatibility complex (MHC) and costimulatory molecules, the production of cytokines, and migration toward lymphatic organs where they are believed to be involved in the activation of T cells.
As used herein, chemotherapeutic agents embrace all other forms of cancer medicaments which do not fall into the categories of immunotherapeutic agents or cancer vaccines. Chemotherapeutic agents as used herein encompass both chemical and biological agents. These agents function to inhibit a cellular activity which the cancer cell is dependent upon for continued survival. Categories of chemotherapeutic agents include alkylating/alkaloid agents, antimetabolites, hormones or hormone analogs, and miscellaneous antineoplastic drugs. Most if not all of these agents are directly toxic to cancer cells and do not require immune stimulation.
An “infectious disease” or, equivalently, an “infection” as used herein, refers to a disorder arising from the invasion of a host, superficially, locally, or systemically, by an infectious organism. Infectious organisms include bacteria, viruses, fungi, and parasites. Accordingly, “infectious disease” includes bacterial infections, viral infections, fungal infections and parasitic infections.
A subject having an infectious disease is a subject that has been exposed to an infectious organism and has acute or chronic detectable levels of the organism in the body. Exposure to the infectious organism generally occurs with the external surface of the subject, e.g., skin or mucosal membranes and/or refers to the penetration of the external surface of the subject by the infectious organism.
A subject at risk of developing an infectious disease is a subject who has a higher than normal risk of exposure to an infection causing pathogen. For instance, a subject at risk may be a subject who is planning to travel to an area where a particular type of infectious agent is found or it may be a subject who through lifestyle or medical procedures is exposed to bodily fluids which may contain infectious organisms or directly to the organism or a subject living in an area where an infectious organism has been identified. Subjects at risk of developing an infectious disease also include general populations to which a medical agency recommends vaccination against a particular infectious organism.
A subject at risk of developing an infectious disease includes those subjects that have a general risk of exposure to a microorganism, e.g., influenza, but that do not have the active disease during the treatment of the invention, as well as subjects that are considered to be at specific risk of developing an infectious disease because of medical or environmental factors that expose the subject to a particular microorganism.
Bacteria are unicellular organisms which multiply asexually by binary fission. They are classified and named based on their morphology, staining reactions, nutrition and metabolic requirements, antigenic structure, chemical composition, and genetic homology. Bacteria can be classified into three groups based on their morphological forms, spherical (coccus), straight-rod (bacillus) and curved or spiral rod (vibrio, campylobacter, spirillum, and spirochaete). Bacteria are also more commonly characterized based on their staining reactions into two classes of organisms, gram-positive and gram-negative. Gram refers to the method of staining which is commonly performed in microbiology labs. Gram-positive organisms retain the stain following the staining procedure and appear a deep violet color. Gram-negative organisms do not retain the stain but take up the counter-stain and thus appear pink.
Infectious bacteria include, but are not limited to, gram negative and gram positive bacteria. Gram positive bacteria include, but are not limited to Pasteurella species, Staphylococci species, and Streptococcus species. Gram negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include but are not limited to: Helicobacter pyloris, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansasii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponema pertenue, Leptospira, Rickettsia, and Actinomyces israelli.
Viruses are small infectious agents which generally contain a nucleic acid core and a protein coat, but are not independently living organisms. Viruses can also take the form of infectious nucleic acids lacking a protein. A virus cannot survive in the absence of a living cell within which it can replicate. Viruses enter specific living cells either by endocytosis or direct injection of DNA (phage) and multiply, causing disease. The multiplied virus can then be released and infect additional cells. Some viruses are DNA-containing viruses and others are RNA-containing viruses. In some aspects, the invention also intends to treat diseases in which prions are implicated in disease progression such as for example bovine spongiform encephalopathy (i.e., mad cow disease, BSE) or scrapie infection in animals, or Creutzfeldt-Jakob disease in humans.
Viruses include, but are not limited to, enteroviruses (including, but not limited to, viruses that the family picornaviridae, such as polio virus, coxsackie virus, echo virus), rotaviruses, adenovirus, hepatitis virus. Specific examples of viruses that have been found in humans include but are not limited to: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papillomaviruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV)); Poxviridae (variola viruses, vaccinia viruses, pox viruses); Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the etiological agents of spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1=internally transmitted; class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk and related viruses, and astroviruses).
Fungi are eukaryotic organisms, only a few of which cause infection in vertebrate mammals. Because fungi are eukaryotic organisms, they differ significantly from prokaryotic bacteria in size, structural organization, life cycle and mechanism of multiplication. Fungi are classified generally based on morphological features, modes of reproduction and culture characteristics. Although fungi can cause different types of disease in subjects, such as respiratory allergies following inhalation of fungal antigens, fungal intoxication due to ingestion of toxic substances, such as Amanita phalloides toxin and phallotoxin produced by poisonous mushrooms and aflatoxins, produced by aspergillus species, not all fungi cause infectious disease.
Infectious fungi can cause systemic or superficial infections. Primary systemic infection can occur in normal healthy subjects, and opportunistic infections are most frequently found in immunocompromised subjects. The most common fungal agents causing primary systemic infection include Blastomyces, Coccidioides, and Htoplasma. Common fungi causing opportunistic infection in immunocompromised or immunosuppressed subjects include, but are not limited to, Candida albicans, Cryptococcus neoformans, and various Aspergillus species. Systemic fungal infections are invasive infections of the internal organs. The organism usually enters the body through the lungs, gastrointestinal tract, or intravenous catheters. These types of infections can be caused by primary pathogenic fungi or opportunistic fungi.
Superficial fungal infections involve growth of fungi on an external surface without invasion of internal tissues. Typical superficial fungal infections include cutaneous fungal infections involving skin, hair, or nails.
Diseases associated with fungal infection include aspergillosis, blastomycosis, candidiasis, chromoblastomycosis, coccidioidomycosis, cryptococcosis, fungal eye infections, fungal hair, nail, and skin infections, histoplasmosis, lobomycosis, mycetoma, otomycosis, paracoccidioidomycosis, disseminated Penicillium marneffei, phaeohyphomycosis, rhinosporidioisis, sporotrichosis, and zygomycosis.
Parasites are organisms which depend upon other organisms in order to survive and thus must enter, or infect, another organism to continue their life cycle. The infected organism, i.e., the host, provides both nutrition and habitat to the parasite. Although in its broadest sense the term parasite can include all infectious agents (i.e., bacteria, viruses, fungi, protozoa and helminths), generally speaking, the term is used to refer solely to protozoa, helminths, and ectoparasitic arthropods (e.g., ticks, mites, etc.). Protozoa are single-celled organisms which can replicate both intracellularly and extracellularly, particularly in the blood, intestinal tract or the extracellular matrix of tissues. Helminths are multicellular organisms which almost always are extracellular (an exception being Trichinella spp.). Helminths normally require exit from a primary host and transmission into a secondary host in order to replicate. In contrast to these aforementioned classes, ectoparasitic arthropods form a parasitic relationship with the external surface of the host body.
Parasites include intracellular parasites and obligate intracellular parasites. Examples of parasites include but are not limited to Plasmodium falciparum, Plasmodium ovale, Plasmodium malariae, Plasmdodium vivax, Plasmodium knowlesi, Babesia microti, Babesia divergens, Trypanosoma cruzi, Toxoplasma gondii, Trichinella spiralis, Leishmania major, Leishmania donovani, Leishmania braziliensis, Leishmania tropica, Trypanosoma gambiense, Trypanosoma rhodesiense and Schistosoma mansoni.
Other medically relevant microorganisms have been described extensively in the literature, e.g., see C. G. A Thomas, Medical Microbiology, Bailliere Tindall, Great Britain 1983, the entire contents of which is hereby incorporated by reference. Each of the foregoing lists is illustrative and is not intended to be limiting.
The compositions and methods of the invention can be used alone or in conjunction with other agents and methods useful for the treatment of infection. Infection medicaments include but are not limited to anti-bacterial agents, anti-viral agents, anti-fungal agents and anti-parasitic agents. Phrases such as “anti-infective agent”, “antibiotic”, “anti-bacterial agent”, “anti-viral agent”, “anti-fungal agent”, “anti-parasitic agent” and “parasiticide” have well-established meanings to those of ordinary skill in the art and are defined in standard medical texts. Briefly, anti-bacterial agents kill or inhibit bacteria, and include antibiotics as well as other synthetic or natural compounds having similar functions. Anti-viral agents can be isolated from natural sources or synthesized and are useful for killing or inhibiting viruses. Anti-fungal agents are used to treat superficial fungal infections as well as opportunistic and primary systemic fungal infections. Anti-parasite agents kill or inhibit parasites. Many antibiotics are low molecular weight molecules which are produced as secondary metabolites by cells, such as microorganisms. In general, antibiotics interfere with one or more functions or structures which are specific for the microorganism and which are not present in host cells.
One of the problems with anti-infective therapies is the side effects occurring in the host that is treated with the anti-infective agent. For instance, many anti-infectious agents can kill or inhibit a broad spectrum of microorganisms and are not specific for a particular type of species. Treatment with these types of anti-infectious agents results in the killing of the normal microbial flora living in the host, as well as the infectious microorganism. The loss of the microbial flora can lead to disease complications and predispose the host to infection by other pathogens, since the microbial flora compete with and function as barriers to infectious pathogens. Other side effects may arise as a result of specific or non-specific effects of these chemical entities on non-microbial cells or tissues of the host.
Another problem with widespread use of anti-infectants is the development of antibiotic-resistant strains of microorganisms. Already, vancomycin-resistant enterococci, penicillin-resistant pneumococci, multi-resistant S. aureus, and multi-resistant tuberculosis strains have developed and are becoming major clinical problems. Widespread use of anti-infectants will likely produce many antibiotic-resistant strains of bacteria. As a result, new anti-infective strategies will be required to combat these microorganisms.
Antibacterial antibiotics which are effective for killing or inhibiting a wide range of bacteria are referred to as broad-spectrum antibiotics. Other types of antibacterial antibiotics are predominantly effective against the bacteria of the class gram-positive or gram-negative. These types of antibiotics are referred to as narrow-spectrum antibiotics. Other antibiotics which are effective against a single organism or disease and not against other types of bacteria, are referred to as limited-spectrum antibiotics.
Anti-bacterial agents are sometimes classified based on their primary mode of action. In general, anti-bacterial agents are cell wall synthesis inhibitors, cell membrane inhibitors, protein synthesis inhibitors, nucleic acid synthesis or functional inhibitors, and competitive inhibitors. Cell wall synthesis inhibitors inhibit a step in the process of cell wall synthesis, and in general in the synthesis of bacterial peptidoglycan. Cell wall synthesis inhibitors include β-lactam antibiotics, natural penicillins, semi-synthetic penicillins, ampicillin, clavulanic acid, cephalolsporins, and bacitracin.
The β-lactams are antibiotics containing a four-membered β-lactam ring which inhibits the last step of peptidoglycan synthesis. β-lactam antibiotics can be synthesized or natural. The β-lactam antibiotics produced by penicillium are the natural penicillins, such as penicillin G or penicillin V. These are produced by fermentation of Penicillium chrysogenum. The natural penicillins have a narrow spectrum of activity and are generally effective against Streptococcus, Gonococcus, and Staphylococcus. Other types of natural penicillins, which are also effective against gram-positive bacteria, include penicillins F, X, K, and O.
Semi-synthetic penicillins are generally modifications of the molecule 6-aminopenicillanic acid produced by a mold. The 6-aminopenicillanic acid can be modified by addition of side chains which produce penicillins having broader spectrums of activity than natural penicillins or various other advantageous properties. Some types of semi-synthetic penicillins have broad spectrums against gram-positive and gram-negative bacteria, but are inactivated by penicillinase. These semi-synthetic penicillins include ampicillin, carbenicillin, oxacillin, azlocillin, mezlocillin, and piperacillin. Other types of semi-synthetic penicillins have narrower activities against gram-positive bacteria, but have developed properties such that they are not inactivated by penicillinase. These include, for instance, methicillin, dicloxacillin, and nafcillin. Some of the broad spectrum semi-synthetic penicillins can be used in combination with β-lactamase inhibitors, such as clavulanic acids and sulbactam. The β-lactamase inhibitors do not have anti-microbial action but they function to inhibit penicillinase, thus protecting the semi-synthetic penicillin from degradation.
One of the serious side effects associated with penicillins, both natural and semi-synthetic, is penicillin allergy. Penicillin allergies are very serious and can cause death rapidly. In a subject that is allergic to penicillin, the β-lactam molecule will attach to a serum protein which initiates an IgE-mediated inflammatory response. The inflammatory response leads to anaphylaxis and possibly death.
Another type of β-lactam antibiotic is the cephalolsporins. They are sensitive to degradation by bacterial β-lactamases, and thus, are not always effective alone. Cephalolsporins, however, are resistant to penicillinase. They are effective against a variety of gram-positive and gram-negative bacteria. Cephalolsporins include, but are not limited to, cephalothin, cephapirin, cephalexin, cefamandole, cefaclor, cefazolin, cefuroxine, cefoxitin, cefotaxime, cefsulodin, cefetamet, cefixime, ceftriaxone, cefoperazone, ceftazidine, and moxalactam.
Bacitracin is another class of antibiotics which inhibit cell wall synthesis, by inhibiting the release of muropeptide subunits or peptidoglycan from the molecule that delivers the subunit to the outside of the membrane. Although bacitracin is effective against gram-positive bacteria, its use is limited in general to topical administration because of its high toxicity.
Carbapenems are another broad-spectrum β-lactam antibiotic, which is capable of inhibiting cell wall synthesis. Examples of carbapenems include, but are not limited to, imipenems. Monobactams are also broad-spectrum β-lactam antibiotics, and include, euztreonam. An antibiotic produced by Streptomyces, vancomycin, is also effective against gram-positive bacteria by inhibiting cell membrane synthesis.
Another class of anti-bacterial agents is the anti-bacterial agents that are cell membrane inhibitors. These compounds disorganize the structure or inhibit the function of bacterial membranes. One problem with anti-bacterial agents that are cell membrane inhibitors is that they can produce effects in eukaryotic cells as well as bacteria because of the similarities in phospholipids in bacterial and eukaryotic membranes. Thus these compounds are rarely specific enough to permit these compounds to be used systemically and prevent the use of high doses for local administration.
One clinically useful cell membrane inhibitor is Polymyxin. Polymyxins interfere with membrane function by binding to membrane phospholipids. Polymyxin is effective mainly against Gram-negative bacteria and is generally used in severe Pseudomonas infections or Pseudomonas infections that are resistant to less toxic antibiotics. The severe side effects associated with systemic administration of this compound include damage to the kidney and other organs.
Other cell membrane inhibitors include Amphotericin B and Nystatin which are anti-fungal agents used predominantly in the treatment of systemic fungal infections and Candida yeast infections. Imidazoles are another class of antibiotic that is a cell membrane inhibitor. Imidazoles are used as anti-bacterial agents as well as anti-fungal agents, e.g., used for treatment of yeast infections, dermatophytic infections, and systemic fungal infections. Imidazoles include but are not limited to clotrimazole, miconazole, ketoconazole, itraconazole, and fluconazole.
Many anti-bacterial agents are protein synthesis inhibitors. These compounds prevent bacteria from synthesizing structural proteins and enzymes and thus cause inhibition of bacterial cell growth or function or cell death. In general these compounds interfere with the processes of transcription or translation. Anti-bacterial agents that block transcription include but are not limited to Rifampins and Ethambutol. Rifampins, which inhibit the enzyme RNA polymerase, have a broad spectrum activity and are effective against gram-positive and gram-negative bacteria as well as Mycobacterium tuberculosis. Ethambutol is effective against Mycobacterium tuberculosis.
Anti-bacterial agents which block translation interfere with bacterial ribosomes to prevent mRNA from being translated into proteins. In general this class of compounds includes but is not limited to tetracyclines, chloramphenicol, the macrolides (e.g., erythromycin) and the aminoglycosides (e.g., streptomycin).
The aminoglycosides are a class of antibiotics which are produced by the bacterium Streptomyces, such as, for instance streptomycin, kanamycin, tobramycin, amikacin, and gentamicin. Aminoglycosides have been used against a wide variety of bacterial infections caused by Gram-positive and Gram-negative bacteria. Streptomycin has been used extensively as a primary drug in the treatment of tuberculosis. Gentamicin is used against many strains of Gram-positive and Gram-negative bacteria, including Pseudomonas infections, especially in combination with Tobramycin. Kanamycin is used against many Gram-positive bacteria, including penicillin-resistant Staphylococci. One side effect of aminoglycosides that has limited their use clinically is that at dosages which are essential for efficacy, prolonged use has been shown to impair kidney function and cause damage to the auditory nerves leading to deafness.
Another type of translation inhibitor anti-bacterial agent is the tetracyclines. The tetracyclines are a class of antibiotics that are broad-spectrum and are effective against a variety of gram-positive and gram-negative bacteria. Examples of tetracyclines include tetracycline, minocycline, doxycycline, and chlortetracycline. They are important for the treatment of many types of bacteria but are particularly important in the treatment of Lyme disease. As a result of their low toxicity and minimal direct side effects, the tetracyclines have been overused and misused by the medical community, leading to problems. For instance, their overuse has led to widespread development of resistance.
Anti-bacterial agents such as the macrolides bind reversibly to the 50 S ribosomal subunit and inhibit elongation of the protein by peptidyl transferase or prevent the release of uncharged tRNA from the bacterial ribosome or both. These compounds include erythromycin, roxithromycin, clarithromycin, oleandomycin, and azithromycin. Erythromycin is active against most Gram-positive bacteria, Neisseria, Legionella and Haemophilus, but not against the Enterobacteriaceae. Lincomycin and clindamycin, which block peptide bond formation during protein synthesis, are used against gram-positive bacteria.
Another type of translation inhibitor is chloramphenicol. Chloramphenicol binds the 70 S ribosome inhibiting the bacterial enzyme peptidyl transferase thereby preventing the growth of the polypeptide chain during protein synthesis. One serious side effect associated with chloramphenicol is aplastic anemia. Aplastic anemia develops at doses of chloramphenicol which are effective for treating bacteria in a small proportion (1/50,000) of patients. Chloramphenicol which was once a highly prescribed antibiotic is now seldom uses as a result of the deaths from anemia. Because of its effectiveness it is still used in life-threatening situations (e.g., typhoid fever).
Some anti-bacterial agents disrupt nucleic acid synthesis or function, e.g., bind to DNA or RNA so that their messages cannot be read. These include but are not limited to quinolones and co-trimoxazole, both synthetic chemicals and rifamycins, a natural or semi-synthetic chemical. The quinolones block bacterial DNA replication by inhibiting the DNA gyrase, the enzyme needed by bacteria to produce their circular DNA. They are broad spectrum and examples include norfloxacin, ciprofloxacin, enoxacin, nalidixic acid and temafloxacin. Nalidixic acid is a bactericidal agent that binds to the DNA gyrase enzyme (topoisomerase) which is essential for DNA replication and allows supercoils to be relaxed and reformed, inhibiting DNA gyrase activity. The main use of nalidixic acid is in treatment of lower urinary tract infections (UTI) because it is effective against several types of Gram-negative bacteria such as E. coli, Enterobacter aerogenes, K. pneumoniae and Proteus species which are common causes of UTI. Co-trimoxazole is a combination of sulfamethoxazole and trimethoprim, which blocks the bacterial synthesis of folic acid needed to make DNA nucleotides. Rifampicin is a derivative of rifamycin that is active against Gram-positive bacteria (including Mycobacterium tuberculosis and meningitis caused by Neisseria meningitidis) and some Gram-negative bacteria. Rifampicin binds to the beta subunit of the polymerase and blocks the addition of the first nucleotide which is necessary to activate the polymerase, thereby blocking mRNA synthesis.
Another class of anti-bacterial agents is compounds that function as competitive inhibitors of bacterial enzymes. The competitive inhibitors are mostly all structurally similar to a bacterial growth factor and compete for binding but do not perform the metabolic function in the cell. These compounds include sulfonamides and chemically modified forms of sulfanilamide which have even higher and broader antibacterial activity. The sulfonamides (e.g., gantrisin and trimethoprim) are useful for the treatment of Streptococcus pneumoniae, beta-hemolytic streptococci and E. coli, and have been used in the treatment of uncomplicated UTI caused by E. coli, and in the treatment of meningococcal meningitis.
Anti-viral agents are compounds which prevent infection of cells by viruses or replication of the virus within the cell. There are many fewer antiviral drugs than antibacterial drugs because the process of viral replication is so closely related to DNA replication within the host cell, that non-specific antiviral agents would often be toxic to the host. There are several stages within the process of viral infection which can be blocked or inhibited by antiviral agents. These stages include, attachment of the virus to the host cell (immunoglobulin or binding peptides), uncoating of the virus (e.g. amantadine), synthesis or translation of viral mRNA (e.g. interferon), replication of viral RNA or DNA (e.g. nucleoside analogues), maturation of new virus proteins (e.g. protease inhibitors), and budding and release of the virus.
Another category of anti-viral agents are nucleoside analogues. Nucleoside analogues are synthetic compounds which are similar to nucleosides, but which have an incomplete or abnormal deoxyribose or ribose group. Once the nucleoside analogues are in the cell, they are phosphorylated, producing the triphosphate form which competes with normal nucleotides for incorporation into the viral DNA or RNA. Once the triphosphate form of the nucleoside analogue is incorporated into the growing nucleic acid chain, it causes irreversible association with the viral polymerase and thus chain termination. Nucleoside analogues include, but are not limited to, acyclovir (used for the treatment of herpes simplex virus and varicella-zoster virus), gancyclovir (useful for the treatment of cytomegalovirus), idoxuridine, ribavirin (useful for the treatment of respiratory syncitial virus), dideoxyinosine, dideoxycytidine, and zidovudine (azidothymidine).
Another class of anti-viral agents includes cytokines such as interferons. The interferons are cytokines which are secreted by virus-infected cells as well as immune cells. The interferons function by binding to specific receptors on cells adjacent to the infected cells, causing the change in the cell which protects it from infection by the virus. α and β-interferon also induce the expression of Class I and Class II MHC molecules on the surface of infected cells, resulting in increased antigen presentation for host immune cell recognition. α and β-interferons are available as recombinant forms and have been used for the treatment of chronic hepatitis B and C infection. At the dosages which are effective for anti-viral therapy, interferons have severe side effects such as fever, malaise and weight loss.
Immunoglobulin therapy is used for the prevention of viral infection. Immunoglobulin therapy for viral infections is different from bacterial infections, because rather than being antigen-specific, the immunoglobulin therapy functions by binding to extracellular virions and preventing them from attaching to and entering cells which are susceptible to the viral infection. The therapy is useful for the prevention of viral infection for the period of time that the antibodies are present in the host. In general there are two types of immunoglobulin therapies, normal immune globulin therapy and hyper-immune globulin therapy. Normal immune globulin therapy utilizes a antibody product which is prepared from the serum of normal blood donors and pooled. This pooled product contains low titers of antibody to a wide range of human viruses, such as hepatitis A, parvovirus, enterovirus (especially in neonates). Hyper-immune globulin therapy utilizes antibodies which are prepared from the serum of individuals who have high titers of an antibody to a particular virus. Those antibodies are then used against a specific virus. Examples of hyper-immune globulins include zoster immune globulin (useful for the prevention of varicella in immunocompromised children and neonates), human rabies immune globulin (useful in the post-exposure prophylaxis of a subject bitten by a rabid animal), hepatitis B immune globulin (useful in the prevention of hepatitis B virus, especially in a subject exposed to the virus), and RSV immune globulin (useful in the treatment of respiratory syncitial virus infections).
Anti-fungal agents are useful for the treatment and prevention of infective fungi. Anti-fungal agents are sometimes classified by their mechanism of action. Some anti-fungal agents function as cell wall inhibitors by inhibiting glucose synthase. These include, but are not limited to, basiungin/ECB. Other anti-fungal agents function by destabilizing membrane integrity. These include, but are not limited to, imidazoles, such as clotrimazole, sertaconzole, fluconazole, itraconazole, ketoconazole, miconazole, and voriconacole, as well as FK 463, amphotericin B, BAY 38-9502, MK 991, pradimicin, UK 292, butenafine, and terbinafine. Other anti-fungal agents function by breaking down chitin (e.g., chitinase) or immunosuppression (501 cream).
Parasiticides are agents that kill parasites directly. Such compounds are known in the art and are generally commercially available. Examples of parasiticides useful for human administration include but are not limited to albendazole, amphotericin B, benznidazole, bithionol, chloroquine HCl, chloroquine phosphate, clindamycin, dehydroemetine, diethylcarbamazine, diloxamide furoate, eflornithine, furazolidaone, glucocorticoids, halofantrine, iodoquinol, ivermectin, mebendazole, mefloquine, meglumine antimoniate, melarsoprol, metrifonate, metronidazole, niclosamide, nifurtimox, oxamniquine, paromomycin, pentamidine isethionate, piperazine, praziquantel, primaquine phosphate, proguanil, pyrantel pamoate, pyrimethanmine-sulfonamides, pyrimethanmine-sulfadoxine, quinacrine HCl, quinine sulfate, quinidine gluconate, spiramycin, stibogluconate sodium (sodium antimony gluconate), suramin, tetracycline, doxycycline, thiabendazole, tinidazole, trimethroprim-sulfamethoxazole, and tryparsamide.
The compositions and methods of the invention may also find use in the treatment of allergy and asthma.
An “allergy” refers to acquired hypersensitivity to a substance (allergen). Allergic conditions include but are not limited to eczema, allergic rhinitis or coryza, hay fever, allergic conjunctivitis, bronchial asthma, urticaria (hives) and food allergies, other atopic conditions including atopic dermatitis; anaphylaxis; drug allergy; and angioedema. Allergic diseases include but are not limited to rhinitis (hay fever), asthma, urticaria, and atopic dermatitis.
Allergy is a disease associated with the production of antibodies from a particular class of immunoglobulin, IgE, against allergens. The development of an IgE-mediated response to common aeroallergens is also a factor which indicates predisposition towards the development of asthma. If an allergen encounters a specific IgE which is bound to an IgE Fc receptor (FcεR) on the surface of a basophil (circulating in the blood) or mast cell (dispersed throughout solid tissue), the cell becomes activated, resulting in the production and release of mediators such as histamine, serotonin, and lipid mediators.
A subject having an allergy is a subject that is currently experiencing or has previously experienced an allergic reaction in response to an allergen.
A subject at risk of developing an allergy or asthma is a subject that has been identified as having an allergy or asthma in the past but who is not currently experiencing the active disease, as well as a subject that is considered to be at risk of developing asthma or allergy because of genetic or environmental factors. A subject at risk of developing allergy or asthma can also include a subject who has any risk of exposure to an allergen or a risk of developing asthma, i.e., someone who has suffered from an asthmatic attack previously or has a predisposition to asthmatic attacks. For instance, a subject at risk may be a subject who is planning to travel to an area where a particular type of allergen or asthmatic initiator is found or it may even be any subject living in an area where an allergen has been identified. If the subject develops allergic responses to a particular antigen and the subject may be exposed to the antigen, i.e., during pollen season, then that subject is at risk of exposure to the antigen.
The generic name for molecules that cause an allergic reaction is allergen. An “allergen” as used herein is a molecule capable of provoking an immune response characterized by production of IgE. An allergen is a substance that can induce an allergic or asthmatic response in a susceptible subject. Thus, in the context of this invention, the term allergen means a specific type of antigen which can trigger an allergic response which is mediated by IgE antibody. The method and preparations of this invention extend to a broad class of such allergens and fragments of allergens or haptens acting as allergens. The list of allergens is enormous and can include pollens, insect venoms, animal dander, dust, fungal spores, and drugs (e.g., penicillin).
There are numerous species of allergens. The allergic reaction occurs when tissue-sensitizing immunoglobulin of the IgE type reacts with foreign allergen. The IgE antibody is bound to mast cells and/or basophils, and these specialized cells release chemical mediators (vasoactive amines) of the allergic reaction when stimulated to do so by allergens bridging the ends of the antibody molecule. Htamine, platelet activating factor, arachidonic acid metabolites, and serotonin are among the best known mediators of allergic reactions in man. Htamine and the other vasoactive amines are normally stored in mast cells and basophil leukocytes. The mast cells are dispersed throughout animal tissue and the basophils circulate within the vascular system. These cells manufacture and store histamine within the cell unless the specialized sequence of events involving IgE binding occurs to trigger its release.
The symptoms of the allergic reaction vary, depending on the location within the body where the IgE reacts with the antigen. If the reaction occurs along the respiratory epithelium, the symptoms are sneezing, coughing and asthmatic reactions. If the interaction occurs in the digestive tract, as in the case of food allergies, abdominal pain and diarrhea are common. Systemic reactions, for example following a bee sting, can be severe and often life-threatening.
Delayed-type hypersensitivity, also known as type IV allergy reaction, is an allergic reaction characterized by a delay period of at least 12 hours from invasion of the antigen into the allergic subject until appearance of the inflammatory or immune reaction. The T lymphocytes (sensitized T lymphocytes) of individuals in an allergic condition react with the antigen, triggering the T lymphocytes to release lymphokines (macrophage migration inhibitory factor (MIF), macrophage activating factor (MAF), mitogenic factor (MF), skin-reactive factor (SRF), chemotactic factor, neovascularization-accelerating factor, etc.), which function as inflammation mediators, and the biological activity of these lymphokines, together with the direct and indirect effects of locally appearing lymphocytes and other inflammatory immune cells, give rise to the type IV allergy reaction. Delayed allergy reactions include tuberculin type reaction, homograft rejection reaction, cell-dependent type protective reaction, contact dermatitis hypersensitivity reaction, and the like, which are known to be most strongly suppressed by steroidal agents. Consequently, steroidal agents are effective against diseases which are caused by delayed allergy reactions. Long-term use of steroidal agents at concentrations currently being used can, however, lead to the serious side-effect known as steroid dependence. The methods of the invention solve some of these problems, by providing for lower and fewer doses to be administered.
Immediate hypersensitivity (or anaphylactic response) is a form of allergic reaction which develops very quickly, i.e., within seconds or minutes of exposure of the patient to the causative allergen, and it is mediated by IgE antibodies made by B lymphocytes. In nonallergic patients, there is no IgE antibody of clinical relevance; but, in a person suffering with allergic diseases, IgE antibody mediates immediate hypersensitivity by sensitizing mast cells which are abundant in the skin, lymphoid organs, in the membranes of the eye, nose and mouth, and in the respiratory tract and intestines.
Mast cells have surface receptors for IgE, and the IgE antibodies in allergy-suffering patients become bound to them. As discussed briefly above, when the bound IgE is subsequently contacted by the appropriate allergen, the mast cell is caused to degranulate and to release various substances called bioactive mediators, such as histamine, into the surrounding tissue. It is the biologic activity of these substances which is responsible for the clinical symptoms typical of immediate hypersensitivity; namely, contraction of smooth muscle in the airways or the intestine, the dilation of small blood vessels and the increase in their permeability to water and plasma proteins, the secretion of thick sticky mucus, and in the skin, redness, swelling and the stimulation of nerve endings that results in itching or pain.
“Asthma” as used herein refers to a disorder of the respiratory system characterized by inflammation, narrowing of the airways, and increased reactivity of the airways to inhaled agents. Asthma is frequently, although not exclusively, associated with an atopic or allergic condition. Symptoms of asthma include recurrent episodes of wheezing, breathlessness, and chest tightness, and coughing, resulting from airflow obstruction. Airway inflammation associated with asthma can be detected through observation of a number of physiological changes, such as, denudation of airway epithelium, collagen deposition beneath basement membrane, edema, mast cell activation, inflammatory cell infiltration, including neutrophils, inosineophils, and lymphocytes. As a result of the airway inflammation, asthma patients often experience airway hyper-responsiveness, airflow limitation, respiratory symptoms, and disease chronicity. Airflow limitations include acute bronchoconstriction, airway edema, mucous plug formation, and airway remodeling, features which often lead to bronchial obstruction. In some cases of asthma, sub-basement membrane fibrosis may occur, leading to persistent abnormalities in lung function.
Research over the past several years has revealed that asthma likely results from complex interactions among inflammatory cells, mediators, and other cells and tissues resident in the airway. Mast cells, inosineophils, epithelial cells, macrophage, and activated T-cells all play an important role in the inflammatory process associated with asthma. Djukanovic R et al. (1990) Am Rev Respir Dis 142:434-457. It is believed that these cells can influence airway function through secretion of preformed and newly synthesized mediators which can act directly or indirectly on the local tissue. It has also been recognized that subpopulations of T-lymphocytes (Th2) play an important role in regulating allergic inflammation in the airway by releasing selective cytokines and establishing disease chronicity. Robinson D S et al. (1992) N Engl J Med 326:298-304.
Asthma is a complex disorder which arises at different stages in development and can be classified based on the degree of symptoms as acute, subacute or chronic. An acute inflammatory response is associated with an early recruitment of cells into the airway. The subacute inflammatory response involves the recruitment of cells as well as the activation of resident cells causing a more persistent pattern of inflammation. Chronic inflammatory response is characterized by a persistent level of cell damage and an ongoing repair process, which may result in permanent abnormalities in the airway.
A “subject having asthma” is a subject that has a disorder of the respiratory system characterized by inflammation, narrowing of the airways and increased reactivity of the airways to inhaled agents. Asthma is frequently, although not exclusively, associated with atopic or allergic symptoms. An “initiator” as used herein refers to a composition or environmental condition which triggers asthma. Initiators include, but are not limited to, allergens, cold temperatures, exercise, viral infections, SO2.
The compositions and methods of the invention can be used alone or in conjucnction with other agents and methods useful in the treatment of asthma. An “asthma/allergy medicament” as used herein is a composition of matter which reduces the symptoms of, prevents the development of, or inhibits an asthmatic or allergic reaction. Various types of medicaments for the treatment of asthma and allergy are described in the Guidelines For The Diagnosis and Management of Asthma, Expert Panel Report 2, NIH Publication No. 97/4051, Jul. 19, 1997, the entire contents of which are incorporated herein by reference. The summary of the medicaments as described in the NIH publication is presented below. In most embodiments the asthma/allergy medicament is useful to some degree for treating both asthma and allergy.
Medications for the treatment of asthma are generally separated into two categories, quick-relief medications and long-term control medications. Asthma patients take the long-term control medications on a daily basis to achieve and maintain control of persistent asthma. Long-term control medications include anti-inflammatory agents such as corticosteroids, chromolyn sodium and nedocromil; long-acting bronchodilators, such as long-acting β2-agonists and methylxanthines; and leukotriene modifiers. The quick-relief medications include short-acting β2 agonists, anti-cholinergics, and systemic corticosteroids. There are many side effects associated with each of these drugs and none of the drugs alone or in combination is capable of preventing or completely treating asthma.
Asthma medicaments include, but are not limited, PDE-4 inhibitors, bronchodilator/beta-2 agonists, K+ channel openers, VLA-4 antagonists, neurokin antagonists, thromboxane A2 (TXA2) synthesis inhibitors, xanthines, arachidonic acid antagonists, 5 lipoxygenase inhibitors, TXA2 receptor antagonists, TXA2 antagonists, inhibitor of 5-lipox activation proteins, and protease inhibitors.
Bronchodilator/β2 agonists are a class of compounds which cause bronchodilation or smooth muscle relaxation. Bronchodilator/β2 agonists include, but are not limited to, salmeterol, salbutamol, albuterol, terbutaline, D2522/formoterol, fenoterol, bitolterol, pirbuerol methylxanthines and orciprenaline. Long-acting β2 agonists and bronchodilators are compounds which are used for long-term prevention of symptoms in addition to the anti-inflammatory therapies. Long-acting β2agonists include, but are not limited to, salmeterol and albuterol. These compounds are usually used in combination with corticosteroids and generally are not used without any inflammatory therapy. They have been associated with side effects such as tachycardia, skeletal muscle tremor, hypokalemia, and prolongation of QTc interval in overdose.
Methylxanthines, including for instance theophylline, have been used for long-term control and prevention of symptoms. These compounds cause bronchodilation resulting from phosphodiesterase inhibition and likely adenosine antagonism. Dose-related acute toxicities are a particular problem with these types of compounds. As a result, routine serum concentration must be monitored in order to account for the toxicity and narrow therapeutic range arising from individual differences in metabolic clearance. Side effects include tachycardia, tachyarrhythmias, nausea and vomiting, central nervous system stimulation, headache, seizures, hematemesis, hyperglycemia and hypokalemia. Short-acting β2 agonists include, but are not limited to, albuterol, bitolterol, pirbuterol, and terbutaline. Some of the adverse effects associated with the administration of short-acting β2 agonists include tachycardia, skeletal muscle tremor, hypokalemia, increased lactic acid, headache, and hyperglycemia.
Conventional methods for treating or preventing allergy have involved the use of anti-histamines or desensitization therapies. Anti-histamines and other drugs which block the effects of chemical mediators of the allergic reaction help to regulate the severity of the allergic symptoms but do not prevent the allergic reaction and have no effect on subsequent allergic responses. Desensitization therapies are performed by giving small doses of an allergen, usually by injection under the skin, in order to induce an IgG-type response against the allergen. The presence of IgG antibody helps to neutralize the production of mediators resulting from the induction of IgE antibodies, it is believed. Initially, the subject is treated with a very low dose of the allergen to avoid inducing a severe reaction and the dose is slowly increased. This type of therapy is dangerous because the subject is actually administered the compounds which cause the allergic response and severe allergic reactions can result.
Allergy medicaments include, but are not limited to, anti-histamines, steroids, and prostaglandin inducers. Anti-histamines are compounds which counteract histamine released by mast cells or basophils. These compounds are well known in the art and commonly used for the treatment of allergy. Anti-histamines include, but are not limited to, astemizole, azelastine, betatastine, buclizine, ceterizine, cetirizine analogues, CS 560, desloratadine, ebastine, epinastine, fexofenadine, HSR 609, levocabastine, loratidine, mizolastine, norastemizole, terfenadine, and tranilast.
Prostaglandin inducers are compounds which induce prostaglandin activity. Prostaglandins function by regulating smooth muscle relaxation. Prostaglandin inducers include, but are not limited to, S-5751.
The asthma/allergy medicaments also include steroids and immunomodulators. The steroids include, but are not limited to, beclomethasone, fluticasone, triamcinolone, budesonide, corticosteroids and budesonide.
Corticosteroids include, but are not limited to, beclomethasome dipropionate, budesonide, flunisolide, fluticaosone propionate, and triamcinolone acetonide. Although dexamethasone is a corticosteroid having anti-inflammatory action, it is not regularly used for the treatment of asthma/allergy in an inhaled form because it is highly absorbed and it has long-term suppressive side effects at an effective dose. Dexamethasone, however, can be used according to the invention for the treating of asthma/allergy because when administered in combination with nucleic acids of the invention it can be administered at a low dose to reduce the side effects. Some of the side effects associated with corticosteroid include cough, dysphonia, oral thrush (candidiasis), and in higher doses, systemic effects, such as adrenal suppression, osteoporosis, growth suppression, skin thinning and easy bruising. Barnes & Peterson (1993) Am Rev Respir Dis 148:S1-S26; and Kamada A K et al. (1996) Am J Respir Crit Care Med 153:1739-48.
Systemic corticosteroids include, but are not limited to, methylprednisolone, prednisolone and prednisone. Cortosteroids are associated with reversible abnormalities in glucose metabolism, increased appetite, fluid retention, weight gain, mood alteration, hypertension, peptic ulcer, and aseptic necrosis of bone. These compounds are useful for short-term (3-10 days) prevention of the inflammatory reaction in inadequately controlled persistent asthma. They also function in a long-term prevention of symptoms in severe persistent asthma to suppress and control and actually reverse inflammation. Some side effects associated with longer term use include adrenal axis suppression, growth suppression, dermal thinning, hypertension, diabetes, Cushing's syndrome, cataracts, muscle weakness, and in rare instances, impaired immune function. It is recommended that these types of compounds be used at their lowest effective dose (guidelines for the diagnosis and management of asthma; expert panel report to; NIH Publication No. 97-4051; July 1997).
The immunomodulators include, but are not limited to, the group consisting of anti-inflammatory agents, leukotriene antagonists, IL-4 muteins, soluble IL-4 receptors, immunosuppressants (such as tolerizing peptide vaccine), anti-IL-4 antibodies, IL-4 antagonists, anti-IL-5 antibodies, soluble IL-13 receptor-Fc fusion proteins, anti-IL-9 antibodies, CCR3 antagonists, CCR5 antagonists, VLA-4 inhibitors, and downregulators of IgE.
Leukotriene modifiers are often used for long-term control and prevention of symptoms in mild persistent asthma. Leukotriene modifiers function as leukotriene receptor antagonists by selectively competing for LTD-4 and LTE-4 receptors. These compounds include, but are not limited to, zafirlukast tablets and zileuton tablets. Zileuton tablets function as 5-lipoxygenase inhibitors. These drugs have been associated with the elevation of liver enzymes and some cases of reversible hepatitis and hyperbilirubinemia. Leukotrienes are biochemical mediators that are released from mast cells, inosineophils, and basophils that cause contraction of airway smooth muscle and increase vascular permeability, mucous secretions and activate inflammatory cells in the airways of patients with asthma.
Other immunomodulators include neuropeptides that have been shown to have immunomodulating properties. Functional studies have shown that substance P, for instance, can influence lymphocyte function by specific receptor-mediated mechanisms. Substance P also has been shown to modulate distinct immediate hypersensitivity responses by stimulating the generation of arachidonic acid-derived mediators from mucosal mast cells. McGillies J et al. (1987) Fed Proc 46:196-9 (1987). Substance P is a neuropeptide first identified in 1931. Von Euler and Gaddum J Physiol (London) 72:74-87 (1931). Its amino acid sequence was reported by Chang et al. in 1971. Chang M M et al. (1971) Nature New Biol 232:86-87. The immunoregulatory activity of fragments of substance P has been studied by Siemion I Z et al. (1990) Molec Immunol 27:887-890 (1990).
Another class of compounds is the down-regulators of IgE. These compounds include peptides or other molecules with the ability to bind to the IgE receptor and thereby prevent binding of antigen-specific IgE. Another type of downregulator of IgE is a monoclonal antibody directed against the IgE receptor-binding region of the human IgE molecule. Thus, one type of downregulator of IgE is an anti-IgE antibody or antibody fragment. Anti-IgE is being developed by Genentech. One of skill in the art could prepare functionally active antibody fragments of binding peptides which have the same function. Other types of IgE downregulators are polypeptides capable of blocking the binding of the IgE antibody to the Fc receptors on the cell surfaces and displacing IgE from binding sites upon which IgE is already bound.
One problem associated with downregulators of IgE is that many molecules do not have a binding strength to the receptor corresponding to the very strong interaction between the native IgE molecule and its receptor. The molecules having this strength tend to bind irreversibly to the receptor. However, such substances are relatively toxic since they can bind covalently and block other structurally similar molecules in the body. Of interest in this context is that the α chain of the IgE receptor belongs to a larger gene family where, e.g., several of the different IgG Fc receptors are contained. These receptors are absolutely essential for the defense of the body against, e.g., bacterial infections. Molecules activated for covalent binding are, furthermore, often relatively unstable and therefore they probably have to be administered several times a day and then in relatively high concentrations in order to make it possible to block completely the continuously renewing pool of IgE receptors on mast cells and basophilic leukocytes.
Chromolyn sodium and nedocromil are used as long-term control medications for preventing primarily asthma symptoms arising from exercise or allergic symptoms arising from allergens. These compounds are believed to block early and late reactions to allergens by interfering with chloride channel function. They also stabilize mast cell membranes and inhibit activation and release of mediators from inosineophils and epithelial cells. A four to six week period of administration is generally required to achieve a maximum benefit.
Anticholinergics are generally used for the relief of acute bronchospasm. These compounds are believed to function by competitive inhibition of muscarinic cholinergic receptors. Anticholinergics include, but are not limited to, ipratropium bromide. These compounds reverse only cholinerigically-mediated bronchospasm and do not modify any reaction to antigen. Side effects include drying of the mouth and respiratory secretions, increased wheezing in some individuals, and blurred vision if sprayed in the eyes.
In addition to standard asthma/allergy medicaments, other methods for treating asthma/allergy have been used either alone or in combination with established medicaments. One preferred, but frequently impossible, method of relieving allergies is allergen or initiator avoidance. Another method currently used for treating allergic disease involves the injection of increasing doses of allergen to induce tolerance to the allergen and to prevent further allergic reactions.
Allergen injection therapy (allergen immunotherapy) is known to reduce the severity of allergic rhinitis. This treatment has been theorized to involve the production of a different form of antibody, a protective antibody which is termed a “blocking antibody”. Cooke R A et al. (1935) Serologic Evidence of Immunity with Coexisting Sensitization in a Type of Human Allergy, Exp Med 62:733. Other attempts to treat allergy involve modifying the allergen chemically so that its ability to cause an immune response in the patient is unchanged, while its ability to cause an allergic reaction is substantially altered. These methods, however, can take several years to be effective and are associated with the risk of side effects such as anaphylactic shock.
The compositions and methods of the invention can be used to modulate an immune response. The ability to modulate an immune response allows for the prevention and/or treatment of particular disorders that can be affected via immune system modulation.
Treatment after a disorder has started aims to reduce, ameliorate, or altogether eliminate the disorder, and/or its associated symptoms, or prevent it from becoming worse. Treatment of subjects before a disorder has started (i.e., prophylactic treatment) aims to reduce the risk of developing the disorder. As used herein, the term “prevent” refers to the prophylactic treatment of patients who are at risk of developing a disorder (resulting in a decrease in the probability that the subject will develop the disorder), and to the inhibition of further development of an already established disorder.
Different doses may be necessary for treatment of a subject, depending on activity of the compound, manner of administration, purpose of the immunization (i.e., prophylactic or therapeutic), nature and severity of the disorder, age and body weight of the subject. The administration of a given dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units. Multiple administration of doses at specific intervals of weeks or months apart is usual for boosting antigen-specific immune responses.
Combined with the teachings provided herein, by choosing among the various active compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and preferred mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is entirely effective to treat the particular subject. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular therapeutic agent being administered (e.g., in the case of an immunostimulatory nucleic acid, the type of nucleic acid, i.e., a CpG nucleic acid, the number of unmethylated CpG motifs or their location in the nucleic acid, the degree of modification of the backbone to the oligonucleotide, etc.), the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular nucleic acid and/or other therapeutic agent without necessitating undue experimentation.
Subject doses of the compounds described herein typically range from about 0.1 μg to 10,000 mg, more typically from about 1 μg/day to 8000 mg, and most typically from about 10 μg to 100 μg. Stated in terms of subject body weight, typical dosages range from about 0.1 μg to 20 mg/kg/day, more typically from about 1 to 10 mg/kg/day, and most typically from about 1 to 5 mg/kg/day.
The pharmaceutical compositions containing nucleic acids and/or other compounds can be administered by any suitable route for administering medications. A variety of administration routes are available. The particular mode selected will depend, of course, upon the particular agent or agents selected, the particular condition being treated, and the dosage required for therapeutic efficacy. The methods of this invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of an immune response without causing clinically unacceptable adverse effects. Preferred modes of administration are discussed herein. For use in therapy, an effective amount of the nucleic acid and/or other therapeutic agent can be administered to a subject by any mode that delivers the agent to the desired surface, e.g., mucosal, systemic.
Administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan. Routes of administration include but are not limited to oral, parenteral, intravenous, intramuscular, intranasal, sublingual, intratracheal, inhalation, subcutaneous, ocular, vaginal, and rectal. For the treatment or prevention of asthma or allergy, such compounds are preferably inhaled, ingested or administered by systemic routes. Systemic routes include oral and parenteral. Inhaled medications are preferred in some embodiments because of the direct delivery to the lung, the site of inflammation, primarily in asthmatic patients. Several types of devices are regularly used for administration by inhalation. These types of devices include metered dose inhalers (MDI), breath-actuated MDI, dry powder inhaler (DPI), spacer/holding chambers in combination with MDI, and nebulizers.
The therapeutic agents of the invention may be delivered to a particular tissue, cell type, or to the immune system, or both, with the aid of a vector. In its broadest sense, a “vector” is any vehicle capable of facilitating the transfer of the compositions to the target cells. The vector generally transports the immunostimulatory nucleic acid, antibody, antigen, and/or disorder-specific medicament to the target cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
In general, the vectors useful in the invention are divided into two classes: biological vectors and chemical/physical vectors. Biological vectors and chemical/physical vectors are useful in the delivery and/or uptake of therapeutic agents of the invention.
Most biological vectors are used for delivery of nucleic acids and this would be most appropriate in the delivery of therapeutic agents that are or that include immunostimulatory nucleic acids.
In addition to the biological vectors discussed herein, chemical/physical vectors may be used to deliver therapeutic agents including immunostimulatory nucleic acids, antibodies, antigens, and disorder-specific medicaments. As used herein, a “chemical/physical vector” refers to a natural or synthetic molecule, other than those derived from bacteriological or viral sources, capable of delivering the nucleic acid and/or other medicament.
A preferred chemical/physical vector of the invention is a colloidal dispersion system. Colloidal dispersion systems include lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system of the invention is a liposome. Liposomes are artificial membrane vessels which are useful as a delivery vector in vivo or in vitro. It has been shown that large unilamellar vesicles (LUVs), which range in size from 0.2-4.0 μm can encapsulate large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form. Fraley et al. (1981) Trends Biochem Sci 6:77.
Liposomes may be targeted to a particular tissue by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein. Ligands which may be useful for targeting a liposome to an immune cell include, but are not limited to: intact or fragments of molecules which interact with immune cell specific receptors and molecules, such as antibodies, which interact with the cell surface markers of immune cells. Such ligands may easily be identified by binding assays well known to those of skill in the art. In still other embodiments, the liposome may be targeted to the cancer by coupling it to a one of the immunotherapeutic antibodies discussed earlier. Additionally, the vector may be coupled to a nuclear targeting peptide, which will direct the vector to the nucleus of the host cell.
Lipid formulations for transfection are commercially available from QIAGEN, for example, as EFFECTENE™ (a non-liposomal lipid with a special DNA condensing enhancer) and SUPERFECT™ (a novel acting dendrimeric technology).
Liposomes are commercially available from Gibco BRL, for example, as LIPOFECTIN™ and LIPOFECTACE™, which are formed of cationic lipids such as N-[1-(2,3 dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB). Methods for making liposomes are well known in the art and have been described in many publications. Liposomes also have been reviewed by Gregoriadis G (1985) Trends Biotechnol 3:235-241.
In one embodiment, the vehicle is a biocompatible microparticle or implant that is suitable for implantation or administration to the mammalian recipient. Exemplary bioerodible implants that are useful in accordance with this method are described in PCT International application no. PCT/US/03307 (Publication No. WO95/24929, entitled “Polymeric Gene Delivery System”. PCT/US/0307 describes a biocompatible, preferably biodegradable polymeric matrix for containing an exogenous gene under the control of an appropriate promoter. The polymeric matrix can be used to achieve sustained release of the therapeutic agent in the subject.
The polymeric matrix preferably is in the form of a microparticle such as a microsphere (wherein the nucleic acid and/or the other therapeutic agent is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the nucleic acid and/or the other therapeutic agent is stored in the core of a polymeric shell). Other forms of the polymeric matrix for containing the therapeutic agent include films, coatings, gels, implants, and stents. The size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix is introduced. The size of the polymeric matrix further is selected according to the method of delivery which is to be used, typically injection into a tissue or administration of a suspension by aerosol into the nasal and/or pulmonary areas. Preferably when an aerosol route is used the polymeric matrix and the nucleic acid and/or the other therapeutic agent are encompassed in a surfactant vehicle.
The polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when the matrix is administered to a nasal and/or pulmonary surface that has sustained an injury. The matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time. In some preferred embodiments, the nucleic acid are administered to the subject via an implant while the other therapeutic agent is administered acutely. Biocompatible microspheres that are suitable for delivery, such as oral or mucosal delivery, are disclosed in Chickering et al. (1996) Biotech Bioeng 52:96-101 and Mathiowitz E et al. (1997) Nature 386:410-414 and PCT Pat. Application WO97/03702.
Both non-biodegradable and biodegradable polymeric matrices can be used to deliver the nucleic acid and/or the other therapeutic agent to the subject. Biodegradable matrices are preferred. Such polymers may be natural or synthetic polymers. The polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable, particularly for the nucleic acid agents. The polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multi-valent ions or other polymers.
Bioadhesive polymers of particular interest include bioerodible hydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules, (1993) 26:581-587, the teachings of which are incorporated herein. These include polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).
If the therapeutic agent is a nucleic acid, the use of compaction agents may also be desirable. Compaction agents also can be used alone, or in combination with, a biological or chemical/physical vector. A “compaction agent”, as used herein, refers to an agent, such as a histone, that neutralizes the negative charges on the nucleic acid and thereby permits compaction of the nucleic acid into a fine granule. Compaction of the nucleic acid facilitates the uptake of the nucleic acid by the target cell. The compaction agents can be used alone, i.e., to deliver a nucleic acid in a form that is more efficiently taken up by the cell or, more preferably, in combination with one or more of the above-described vectors.
Other exemplary compositions that can be used to facilitate uptake of a nucleic acid include calcium phosphate and other chemical mediators of intracellular transport, microinjection compositions, electroporation and homologous recombination compositions (e.g., for integrating a nucleic acid into a preselected location within the target cell chromosome).
The compounds may be administered alone (e.g., in saline or buffer) or using any delivery vectors known in the art. For instance the following delivery vehicles have been described: cochleates (Gould-Fogerite et al., 1994, 1996); Emulsomes (Vancott et al., 1998, Lowell et al., 1997); ISCOMs (Mowat et al., 1993, Carlsson et al., 1991, Hu et., 1998, Morein et al., 1999); liposomes (Childers et al., 1999, Michalek et al., 1989, 1992, de Haan 1995a, 1995b); live bacterial vectors (e.g., Salmonella, Escherichia coli, Bacillus calmatte-guerin, Shigella, Lactobacillus) (Hone et al., 1996, Pouwels et al., 1998, Chatfield et al., 1993, Stover et al., 1991, Nugent et al., 1998); live viral vectors (e.g., Vaccinia, adenovirus, Herpes Simplex) (Gallichan et al., 1993, 1995, Moss et al., 1996, Nugent et al., 1998, Flexner et al., 1988, Morrow et al., 1999); microspheres (Gupta et al., 1998, Jones et al., 1996, Maloy et al., 1994, Moore et al., 1995, O'Hagan et al., 1994, Eldridge et al., 1989); nucleic acid vaccines (Fynan et al., 1993, Kuklin et al., 1997, Sasaki et al., 1998, Okada et al., 1997, Ishii et al., 1997); polymers (e.g. carboxymethylcellulose, chitosan) (Hamajima et al., 1998, Jabbal-Gill et al., 1998); polymer rings (Wyatt et al., 1998); proteosomes (Vancott et al., 1998, Lowell et al., 1988, 1996, 1997); sodium fluoride (Hashi et al., 1998); transgenic plants (Tacket et al., 1998, Mason et al., 1998, Haq et al., 1995); virosomes (Gluck et al., 1992, Mengiardi et al., 1995, Cryz et al., 1998); and, virus-like particles (Jiang et al., 1999, Leibl et al., 1998).
The formulations of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.
The term pharmaceutically-acceptable carrier means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal. The term carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.
For oral administration, the compounds (i.e., nucleic acids, antigens, antibodies, and other therapeutic agents) can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any carriers.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The compounds, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long-acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer R (1990) Science 249:1527-1533, which is incorporated herein by reference.
The nucleic acids and optionally other therapeutics and/or antigens may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).
The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the compounds into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the compounds into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. Liquid dose units are vials or ampoules. Solid dose units are tablets, capsules and suppositories.
Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the compounds, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109. Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di-, and tri-glycerides; hydrogel release systems; silastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which an agent of the invention is contained in a form within a matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.
The invention also provides efficient methods of identifying immunostimulatory compounds and optimizing the compounds and agents so identified. Generally, the screening methods involve assaying for compounds which inhibit or enhance signaling through a particular TLR. The methods employ a TLR, a suitable reference ligand for the TLR, and a candidate immunostimulatory compound. The selected TLR is contacted with a suitable reference compound (TLR ligand) and a TLR-mediated reference signal is measured. The selected TLR is also contacted with a candidate immunostimulatory compound and a TLR-mediated test signal is measured. The test signal and the reference signal are then compared. A favorable candidate immunostimulatory compound may subsequently be used as a reference compound in the assay. Such methods are adaptable to automated, high throughput screening of candidate compounds. Examples of such high throughput screening methods are described in U.S. Pat. Nos. 6,103,479; 6,051,380; 6,051,373; 5,998,152; 5,876,946; 5,708,158; 5,443,791; 5,429,921; and 5,143,854.
As used herein “TLR signaling” refers to an ability of a TLR polypeptide to activate the Toll/IL-1R (TIR) signaling pathway, also referred to herein as the TLR signal transduction pathway. Changes in TLR activity can be measured by assays designed to measure expression of genes under control of κB-sensitive promoters and enhancers. Such genes can be naturally occurring genes or they can be genes artificially introduced into a cell. Naturally occurring reporter genes include the genes encoding IL-1β, IL-6, IL-8, the p40 subunit of interleukin 12 (IL-12 p40), and the costimulatory molecules CD80 and CD86. Other genes can be placed under the control of such regulatory elements and thus serve to report the level of TLR signaling.
The assay mixture comprises a candidate immunostimulatory compound. Typically, a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a different response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration of agent or at a concentration of agent below the limits of assay detection. Candidate immunostimulatory compounds may encompass numerous chemical classes, although typically they are organic compounds. In some embodiments, the candidate immunostimulatory compounds are small RNAs or small organic compounds, i.e., organic compounds having a molecular weight of more than 50 yet less than about 2500 Daltons. Polymeric candidate immunostimulatory compounds can have higher molecular weights, e.g., oligonucleotides in the range of about 2500 to about 12,500. Candidate immunostimulatory compounds also may be biomolecules such as nucleic acids, peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like. Where the candidate immunostimulatory compound is a nucleic acid, the candidate immunostimulatory compound typically is a DNA or RNA molecule, although modified nucleic acids having non-natural bonds or subunits are also contemplated.
Candidate immunostimulatory compounds may be obtained from a wide variety of sources, including libraries of natural, synthetic, or semisynthetic compounds, or any combination thereof. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily modified through conventional chemical, physical, and biochemical means. Further, known pharmacological agents may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc., to produce structural analogs of the candidate immunostimulatory compounds.
A variety of other reagents also can be included in the mixture. These include reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc., which may be used to facilitate optimal protein-protein and/or protein-nucleic acid binding. Such a reagent may also reduce non-specific or background interactions of the reaction components. Other reagents that improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, antimicrobial agents, and the like may also be used.
The order of addition of components, incubation temperature, time of incubation, and other parameters of the assay may be readily determined. Such experimentation merely involves optimization of the assay parameters, not the fundamental composition of the assay. Incubation temperatures typically are between 4° C. and 40° C., more typically about 37° C. Incubation times preferably are minimized to facilitate rapid, high throughput screening, and typically are between 1 minute and 10 hours.
After incubation, the level of TLR signaling is detected by any convenient method available to the user. For cell-free binding type assays, a separation step is often used to separate bound from unbound components. The separation step may be accomplished in a variety of ways. For example, separation can be accomplished in solution, or, conveniently, at least one of the components is immobilized on a solid substrate, from which the unbound components may be easily separated. The solid substrate can be made of a wide variety of materials and in a wide variety of shapes, e.g., microtiter plate, microbead, dipstick, resin particle, etc. The substrate preferably is chosen to maximize signal-to-noise ratios, primarily to minimize background binding, as well as for ease of separation and cost.
Separation may be effected, for example, by removing a bead or dipstick from a reservoir, emptying or diluting a reservoir such as a microtiter plate well, rinsing a bead, particle, chromatographic column or filter with a wash solution or solvent. The separation step preferably includes multiple rinses or washes. For example, when the solid substrate is a microtiter plate, the wells may be washed several times with a washing solution, which typically includes those components of the incubation mixture that do not participate in specific bindings such as salts, buffer, detergent, non-specific protein, etc. Where the solid substrate is a magnetic bead, the beads may be washed one or more times with a washing solution and isolated using a magnet.
Detection may be effected in any convenient way for cell-based assays such as measurement of an induced polypeptide within, on the surface of, or secreted by the cell. Examples of detection methods useful in cell-based assays include fluorescence-activated cell sorting (FACS) analysis, bioluminescence, fluorescence, enzyme-linked immunosorbent assay (ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR), and the like. Examples of detection methods useful in cell-free assays include bioluminescence, fluorescence, ELISA, RT-PCR, and the like.
EXAMPLES
Example 1
Responsiveness of Human PBMC to G,U-Containing Oligoribonucleotides
Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors, plated at 3×105 cells/well, stimulated in vitro with various test and control immunostimulatory agents for 16 hours, and then analyzed by enzyme-linked immunosorbent assay (ELISA) using matched antibody pairs from BD-Pharmingen for secreted cytokines IL-12 p40 and TNF-α, performed according to the manufacturer's protocol. Also included were certain negative controls, including medium alone and DOTAP (10 μg/200 μl culture well; “Liposomes”) alone. The control immunostimulatory agents included the imidazoquinolone R-848 (2 μg/ml), lipopolysaccharide (LPS; 1 μg/ml), Pam3Cys (5 μg/ml), poly IC (50 μg/ml), and CpG DNA (50 μg/ml). These are reported ligands for TLR7, TLR4, TLR2, TLR3, and TLR9, respectively. Test immunostimulatory agents included the following RNA molecules, each at 50 μg/ml, with and without DOTAP (10 μg total “with Liposomes” and “without Liposomes”, respectively): GUGUUUAC alone; GUAGGCAC alone; GUGUUUAC in combination with GUAGGCAC; GUAGGA; GAAGGCAC; CUAGGCAC; CUCGGCAC; and CCCCCCCC. These RNA oligonucleotides each contained a phosphorothioate linkage between the penultimate and 3′ terminal nucleoside.
FIG. 1 depicts the responsiveness of human PBMC to the test and control agents listed above, as measured by secreted amounts of IL-12 p40 (pg/ml). As can be seen in FIG. 1, PBMCs were responsive to R-848, LPS, Pam3Cys, and poly IC, while they were unresponsive to DOTAP alone. Significantly, human PBMC secreted large amounts of IL-12 p40 (10-20 ng/ml) in response to G,U-containing RNA oligonucleotides GUGUUUAC alone; GUAGGCAC alone; GUGUUUAC in combination with GUAGGCAC; CUAGGCAC; and CUCGGCAC, each in combination with DOTAP. Also significantly, human PBMC did not secrete significant amounts of IL-12 p40 in response to G,U-free RNA oligonucleotides GAAGGCAC and CCCCCCCC. The immunostimulatory effect of the G,U-containing RNA molecules appeared to be greatly enhanced by the inclusion of DOTAP. In this experiment, the G,U-containing 6-mer RNA GUAGGA appeared to exert little, if any immunostimulatory effect either with or without DOTAP.
FIG. 2 depicts the responsiveness of human PBMC to the test and control agents listed above, as measured by secreted amounts of TNF-α. A similar pattern of results was observed as in FIG. 1, i.e., human PBMC secreted large amounts of TNF-α (40-100 ng/ml) in response to G,U-containing RNA oligonucleotides GUGUUUAC alone; GUAGGCAC alone; GUGUUUAC in combination with GUAGGCAC; CUAGGCAC; and CUCGGCAC, each in combination with DOTAP. Also similar to the results in FIG. 1, human PBMC did not secrete significant amounts of TNF-α in response to G,U-free RNA oligonucleotides GAAGGCAC and CCCCCCCC, or in response to the G,U-containing 6-mer RNA GUAGGA. The immunostimulatory effect of the G,U-containing RNA molecules appeared to be greatly enhanced by the inclusion of DOTAP.
It will be appreciated in this example that the following partial self-complementarity basepairing is possible, where G-U wobble basepairs are shown joined with a dot and G-C and A-U basepairs are shown joined by a line:
Example 2
Dose-Response Behavior of Human PBMC to G,U-Containing Oligoribonucleotides
The experiments described in the preceding example were repeated with varied concentrations of RNA oligonucleotides in order to assess the dose-response behavior of human PBMCs to G,U-containing RNA oligonucleotides of the invention. A total of 10, 3 or 1 μg RNA was added to 10 μg DOTAP and then added to the 200 μl culture wells. After 16 hours IL-12 p40 and TNF-α ELISAs were performed as described in Example 1.
FIG. 3 depicts the dose-response of human PBMC to the various RNAs as measured by secreted amounts of IL-12 p40 (ng/ml). As can be seen from FIG. 3, human PBMC secreted increasing amounts of IL-12 p40 in response to increasing amounts of G,U-containing RNA oligomers GUGUUUAC; GUAGGCAC; CUAGGCAC; and CUCGGCAC, each in combination with DOTAP. Conversely, FIG. 3 also shows that human PBMC appeared not to secrete IL-12 p40 in response to any of the tested amounts of G,U-free RNA oligomers GAAGGCAC or CCCCCCCC.
Corresponding dose-response of human PBMC to the various RNAs was measured by secreted amounts of TNF-α. A similar pattern of results was observed as in FIG. 3, i.e., human PBMC secreted increasing amounts of TNF-α in response to increasing amounts G,U-containing RNA oligonucleotides GUGUUUAC; GUAGGCAC; CUAGGCAC; and CUCGGCAC, each in combination with DOTAP. Also similar to the results in FIG. 3, human PBMC did not appear to secrete significant amounts of TNF-α in response to any of the tested amounts of G,U-free RNA oligonucleotides GAAGGCAC and CCCCCCCC.
Example 3
Base Sequence Sensitivity of RNA Oligomers
Point mutations were made to the RNA oligonucleotide GUAGGCAC by substituting A or C at selected positions. The various oligoribonucleotides included the following: GUAGGCAC; GUAGGA; GAAGGCAC; AUAAACAC; AUAGACAC; AUAAGCAC; GUAAACAC; CUAGGCAC; CUCGGCAC; and GUGUUUAC. The oligonucleotides were titrated onto human PBMC isolated from healthy donors and plated at 3×105 cells/well. A total of 10 μg RNA was added to 10 μg DOTAP and then added to the 200 μl culture wells. Human TNF-α was measured by ELISA using matched antibody pairs from BD-Pharmingen according to the manufacturer's protocol. Results are shown in FIG. 4.
Example 4
Effect of DOTAP on Human PBMC Response to Various Stimuli
In order to characterize further the role of DOTAP in the immunostimulatory effects of the G,U-containing RNA oligomers observed in the previous examples, human PBMCs were isolated from healthy donors, plated at 3×105 cells/well, and stimulated in the presence of known TLR ligands, either with or without DOTAP (“with Liposomes” or “without Liposomes”, respectively). The known TLR ligands examined were total RNA prepared from hyphae (hyphae), total RNA prepared from yeast (yeast), total RNA prepared from promyelocytic cell line HL-60 (HL60), in vitro transcribed ribosomal RNA for E. coli Sp6, in vitro transcribed ribosomal RNA for E. coli T7, LPS, poly IC, Pam3Cys, and R-848. Medium alone and DOTAP alone were used as negative controls. The panel of RNAs from the previous examples, again at 10 μg/ml and without DOTAP, was also included.
Total RNA was isolated from the human promyelocytic cell line HL-60 using Trizol (Sigma). Prior to isolation, cells were treated for 4 hours with 500 μM hydrogen peroxide (H2O2), which induces apoptosis in this cell line (HL60 500). Untreated cells served as control (HL60 0).
Candida albicans RNA was isolated from yeast or hyphae (induced by 4 h incubation with 10% fetal calf serum). Cells from a 100 ml culture were pelleted, washed and resuspended in 10 ml of Tris/EDTA buffer (10 mM, 1 mM). RNA was isolated by extraction with hot acidic phenol according to methods described in Ausubel F M et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, New York.
The genomic fragment of E. coli 16S RNA was amplified with the primers 5′-ATTGAAGAGTTTGATCATGGCTCAGATTGAACG-3′ (SEQ ID NO:5) and 5′-TAAGGAGGTGATCCAACCGCAGGTTCC-3′ (SEQ ID NO:6) from genomic E. coli DNA and cloned into the pGEM T easy vector. In vitro transcription was performed using T7 or Sp6 RNA polymerase. Transcribed RNA was further purified by chloroform/phenol extraction, precipitated, and used at 10 μg.
Following 16 hour incubation, ELISAs were performed as before to assess secretion of IL-12 p40 and TNF-α. Representative results are shown in FIG. 5.
FIG. 5 depicts the effect of DOTAP on the amount of IL-12 p40 secreted by human PBMC following incubation with and without DOTAP. As can be seen from the figure, the following stimuli appeared to exert greater immunostimultory effect in the presence of DOTAP than in its absence: hyphae, yeast, E. coli Sp6, and E. coli T7. The following stimuli appeared to exert reduced immunostimultory effect in the presence of DOTAP than in its absence: LPS, poly IC. The following stimuli appeared to exert about the same immunostimultory effect in the presence or absence of DOTAP: HL60, Pam3Cys and R-848.
Example 5
Immunostimulatory Effect of G,U-Containing RNA Oligomers is Species- and MyD88-Dependent
The following murine cells were isolated and incubated with various RNAs and other known TLR ligands in order to assess species-, cell type-, and signaling pathway-specificity: wild type macrophages in the presence of IFN-γ; MyD88-deficient macrophages in the presence of IFN-γ; J774 (mouse macrophage cell line); and RAW 264.7 (mouse macrophage cell line, e.g., ATCC TIB-71). Murine bone macrophages were generated from wild type or MyD88-deficient C57BL/6 mice by culturing bone marrow cells with 50 ng/ml M-CSF for 5 days. Cells were seeded at 25,000 cells/well and treated with 20 ng/ml IFN-γ for 16 hours. The murine macrophage cell lines RAW and J774 were seeded at 10,000 cells/well.
The following test and control agents were examined: R-848 (2 μg/ml), ODN 1668 (CpG DNA; 5′-TCCATGACGTTCCTGATGCT-3′; SEQ ID NO:7); LPS (1 μg/ml); poly IC (50 μg/ml); Pam3Cys (5 μg/ml); Ionomycin/TPA; the following RNA molecules, each with (“+ Lipo”) and without DOTAP (10 μg/200 μl culture well): GUGUUUAC alone (RNA1); GUAGGCAC alone (RNA2); GUGUUUAC in combination with GUAGGCAC (RNA1/2); UCCGCAAUGGACGAAAGUCUGACGGA (RNA6; SEQ ID NO:8); GAGAUGGGUGCGAGAGCGUCAGUAUU (RNA9; SEQ ID NO:9); and the following DNA molecules, corresponding to RNA1, RNA2, and RNA1/2: GTGTTTAC alone (DNA1); GTAGGCAC alone (DNA2); and GTGTTTAC in combination with GTAGGCAC (DNA1/2). These RNA and DNA oligonucleotides each contained a phosphorothioate linkage between the penultimate and 3′ terminal nucleoside. RNA6 and RNA9 each contained in addition a phosphorothioate linkage between the penultimate and 5′ terminal nucleoside. RNA6 corresponds to a ribosomal RNA stem loop derived from Listeria monocytogenes. RNA9 corresponds to a stem loop derived from human immunodeficiency virus (HIV, an RNA retrovirus). The cells were cultured for 12 hours and supernatants were harvested. Murine IL-12 p40, IL-6, and TNF-α were measured by ELISA using matched antibody pairs from BD-Pharmingen according to the manufacturer's protocol. Representative results are shown in FIG. 6.
Panel A of FIG. 6 shows that wild type murine macrophages in the presence of IFN-γ secrete significant amounts of IL-12 p40 in response to R-848; ODN 1668 (CpG DNA); LPS; poly IC; Pam3Cys; and G,U-containing RNA oligomers GUGUUUAC in combination with GUAGGCAC (with DOTAP). In contrast, Panel B of FIG. 6 shows that MyD88-deficient murine macrophages in the presence of IFN-γ secrete little or no IL-12 p40 in response to any of the test and control agents examined, thus demonstrating a dependence on MyD88 for immunostimulatory response to these compounds. Such a result is consistent with participation by a TLR in the immunostimulatory response to any of these compounds, including in particular the G,U-containing RNA oligonucleotides of the invention. Panels C and D of FIG. 6 show generally similar, if somewhat attenuated, response patterns of J774 and RAW 264.7 mouse macrophage cell lines as for wild type murine macrophages in the presence of IFN-γ, as shown in Panel A. Essentially similar results were found in parallel ELISAs measuring IL-6 and TNF-α.
In additional studies involving MyD88 wild-type cells, it was observed that addition of bafilomycin largely or completely abrogated the immunostimulatory effect of the RNA oligomers. Together with the MyD88-dependence, this observation is consistent with involvement of at least one of TLR3, TLR7, TLR8, and TLR9.
Example 6
Use of Cholesteryl Ester in Place of Cationic Lipid
In order to investigate the possibility of using cholesteryl ester-modified RNA oligomer in place of RNA oligomer plus cationic lipid, RNA oligomer GUGUGUGU was prepared with (R 1058) and without (R 1006) a 3′ cholesteryl ester modification. These two RNA oligomers with and without DOTAP, were added over a range of concentrations to overnight cultures of human PBMC. Culture supernatants were harvested, and human TNF-α, IL-12 p40, and IFN-α were measured by ELISA using matched antibody pairs from BD-Pharmingen according to the manufacturer's protocol. Representative results for experiments including DOTAP are shown in Table 1.
TABLE 1
Cholesteryl Ester Modification in Place of DOTAP
TNF-α
TNF-α
IFN-α
IFN-α
+DOTAP
−DOTAP
+DOTAP
−DOTAP
EC50
max
EC50,
max
EC50
max
EC50
max
ID
μM
pg/ml
μM
pg/ml
μM
pg/ml
μM
pg/ml
R 1006
2.8
40000
7.8
2200
4.5
5000
—
—
R 1058
0.2
75000
1.0
3000
0.5
3800
0.5
1500
The results indicate that R 1058, with the cholesteryl ester modification, is more potent than R 1006, having the same base sequence but without cholesterol, both with and without DOTAP.
Example 7
Effect of Oligomer Length
RNA oligomers GUGUGUGU, GUGUGUG, GUGUGU, GUGUG, GUGU, GUG, and GU, with and without DOTAP, were added over a range of concentrations to overnight cultures of human PBMC. Culture supernatants were harvested, and human TNF-α, IL-12 p40, and IFN-α were measured by ELISA using matched antibody pairs from BD-Pharmingen according to the manufacturer's protocol. Representative results for experiments including DOTAP are shown in Table 2.
TABLE 2
Effect of RNA Oligomer Length
TNF-α
IL-12 p40
IFN-α
ID
SEQ
EC50, μM
max pg/ml
EC50, μM
max pg/ml
EC50, μM
max pg/ml
R 1006
GUGUGUGU
2.8
40000
1.6
7000
4.5
5000
R 1048
GUGUGUG
2.2
30000
2.6
10000
4.6
2700
R 1049
GUGUGU
6.7
30000
2.1
8000
4.8
3400
R 1050
GUGUG
7.6
40000
3.9
14000
6.9
400
R 1051
GUGU
—
—
>20
14000
—
—
R 1052
GUG
—
—
>20
6000
5.5
800
R 1053
GU
—
—
>20
5000
—
—
Example 8
Effect of Stabilization of Internucleoside Linkages
GUGUGUGU RNA oligomers were synthesized with specific phosphorothioate and phosphodiester linkages as shown in Table 2, where “*” represents phosphorothioate and “_” represents phosphodiester. RNA oligomers, with and without DOTAP, were added over a range of concentrations to overnight cultures of human PBMC. Culture supernatants were harvested, and human TNF-α, IL-12 p40, and IFN-α were measured by ELISA using matched antibody pairs from BD-Pharmingen according to the manufacturer's protocol. Representative results for experiments including DOTAP are shown in Table 3.
TABLE 3
UZ,4/36 Effect of Stabilization of Internucleoside Linkages
TNF-α
IFN-α
ID
SEQ
EC50, μM
max, pg/ml
EC50, μM
max, pg/ml
R 1006
G*U*G*U*G*U*G*U
2.8
40000
4.5
5000
R 1054
G*U_G*U*G*U*G*U
5.6
40000
6.7
3700
R 1055
G*U_G*U_G*U*G*U
>20
20000
—
—
R 1056
G*U_G*U_G*U_G*U
>20
12000
—
—
R 1057
G_U_G_U_G_U_G_U
—
—
0.1
6000
In like manner, an all-phosphodiester 40-mer capable of forming a stem-loop structure and having a base sequence as provided by 5′-CACACACUGCUUAAGCGCUUGCCUGCUUAAGUAGUGUGUG-3′ (R 1041; SEQ ID NO:10) was synthesized and tested in overnight culture with human PBMC. This RNA oligomer was found to be very potent in its ability to induce IFN-α, with an EC50 of <0.1 μM and a maximum of 5000 pg/ml.
Example 9
DNA:RNA Conjugates
A series of DNA:RNA conjugates, each containing the RNA sequence GUGUGUGU and a poly-dT or a poly-dG sequence, was prepared. The oligomers were as follows, where again “*” represents phosphorothioate and “_” represents phosphodiester:
(R 1060; SEQ ID NO:11)
G*U*G*U*G*U*G*U_dG_dG*dG*dG*dG*dG
(R 1061; SEQ ID NO:12)
dG*dG*dG*dG_dG_G*U*G*U*G*U*G*U
(R 1062; SEQ ID NO:13)
G*U*G*U*G*U*G*U*dT*dT*dT*dT*dT*dT
(R 1063; SEQ ID NO:14)
dT*dT*dT*dT*dT*G*U*G*U*G*U*G*U
Human PBMC were cultured overnight in the presence of added DNA:RNA conjugate, with and without DOTAP. Culture supernatants were harvested and human TNF-α, IL-6, IL-12 p40, IP-10, and IFN-α were measured by ELISA using matched antibody pairs from BD-Pharmingen according to the manufacturer's protocol. Representative results for experiments including DOTAP are shown in Table 4.
TABLE 4
Immunostimulatory DNA:RNA Conjugates
TNF-α
IL-6
IP-10
EC50,
EC50,
EC50,
ID
μM
max pg/ml
μM
max pg/ml
μM
max pg/ml
R 1060
4.9
20000
—
—
—
—
R 1061
4.3
20000
>20
10000
1.1
180
R 1062
0.3
80000
0.4
28000
0.1
400
R 1063
0.3
60000
0.8
28000
0.1
250
Example 10
Transfer RNA
Human PBMC were cultured overnight in the presence of various concentrations (1, 3, and 10 μg/ml) of tRNA obtained from wheat germ, bovine, yeast, and E. coli sources, added to the culture medium with and without DOTAP. Culture supernatants were harvested and human TNF-α and IL-12 p40 were measured by ELISA using matched antibody pairs from BD-Pharmingen according to the manufacturer's protocol. Yeast and E. coli tRNAs, and to a lesser extent bovine tRNA, induced TNF-α and IL-12 p40 when DOTAP was also present. In addition, E. coli tRNA at 3 and 10 μg/ml induced minor amounts of both cytokines even without DOTAP.
Example 11
HIV RNA
Hunan PBMC were incubated overnight with either of two key G,U-rich sequences, namely 5′-GUAGUGUGUG-3′ (SEQ ID NO:2) and 5′-GUCUGUUGUGUG-3′ (SEQ ID NO:3), corresponding to nt 99-108 and 112-123 of HIV-1 strain BH10, respectively, each with and without DOTAP. Culture supernatants were harvested, and human IL-12 p40 and TNF-α were measured by ELISA using matched antibody pairs from BD-Pharmingen according to the manufacturer's protocol. Representative results are shown in FIG. 7. The figure shows that both of these RNA molecules, at micromolar concentrations in the presence of DOTAP, induced 50-100 ng/ml of TNF and 50-200 ng/ml of IL-12 p40.
Example 12
Responsiveness of Human PBMC to Stringent Response Factor
When bacteria are starved they enter into a programmed response termed the stringent response. This involves the production of nucleic acid alarmones and ribosomal loss. Bacteria growing at high rates contain 70,000-80,000 ribosomes accounting for as much as 50% of their dry weight. As growth slows, unneeded ribosomes are hydrolyzed. It was hypothesized that rapidly growing cells in their early stationary phase contain large amounts of oligoribonucleotides that are released into the media when the cells enter a neutral pH environment.
FIG. 10 depicts the responsiveness of human PBMC to stringent response factor (SRF). SRF is produced by rapidly growing bacteria (in this case Listeria monocytogenes) in rich media until their late log phase. The bacteria were pelleted and resuspended in an equal volume of PBS for 24 h. The mixture is centrifuged to remove the bacteria. The supernatant is sterilized by passing it through a 0.2 μm filter. The sterilized solution was passed through a molecular filter with a cutoff of 10 kDa. This fraction was separated on a C18 column and the eluant was tested. At a concentration of 5 μg/ml SRF induced TNF from human PBMC. If SRF was treated with any of three RNAses the activity was destroyed. The activity was not due to substances other than RNA because the RNase-treated SRF had near background stimulatory ability. This implied activity was due to RNA.
Example 13
Responsiveness of Human PBMC to Ribonucleoside Vanadyl Complexes
During studies of SRF it was surprisingly determined that the RNAse inhibitor, ribonucleoside vanadyl complexes (RVCs), could stimulate human PBMC to produce TNF (FIG. 11) and IL-6.
FIG. 11 depicts the responsiveness of human PBMC to the ribonucleoside vanadyl complexes (RVCs). It was unexpectedly discovered during testing of RNAse inhibitors that RVCs were stimulatory for human PBMC. 2 mM RVC induced the release of substantial TNF. Also tested was the anti-viral imidazoquinoline, resiquimod (R-848) denoted as X and used at 0.1 μg/ml.
Example 14
Responsiveness of Human TLR7 and Human TLR8 to Ribonucleosides
The observations of Example 13 could be extended to 293 cells genetically reconstituted with TLR7 and TLR8 but not non-transfected 293 cells (FIG. 12). During analysis of individual ribonucleoside vanadyl complexes, it was unexpectedly determined that a mixture of the ribonucleosides A, U, C, and G or the single ribonucleoside G was effective in the absence of vanadate at stimulating PBMC to produce TNF and TLR7 or TLR8 to activate NF-kB (FIG. 12).
FIG. 12 depicts the responsiveness of human TLR7 and human TLR8 to ribonucleosides. It was determined that the response by human PBMC to RNA or RVC was mediated by TLR7 or TLR8 and further that the response could be driven by ribonucleosides only. Human 293 cells were either mock-transfected or transfected with human TLR7 or human TLR8 and monitored for responsiveness to ribonucleosides. The open reading frames of human TLR7 (hTLR7) and human TLR8 (hTLR8) were amplified by PCR from a cDNA library of human PBMC using the following primers pairs: for TLR7, 5′-CACCTCTCATGCTCTGCTCTCTTC-3′ (SEQ ID NO:15) and 5′-GCTAGACCGTTTCCTTGAACACCTG-3# (SEQ ID NO:16); and for TLR8, 5′-CTGCGCTGCTGCAAGTTACGGAATG-3′ (SEQ ID NO:17) and 5′-GCGCGAAATCATGACTTAACGTCAG-3′ (SEQ ID NO:18). The sequence information for primer selection was obtained from Genbank accession numbers AF240467 and AF245703. All full-length TLR fragments were cloned into pGEM-T Easy vector (Promega, Mannheim, Germany), excised with NotI, cloned into the expression vector pcDNA 3.1 (−) (Invitrogen, Karlsruhe, Germany) and sequenced. Sequences of the coding region of hTLR7 and hTLR8 correspond to the accession numbers AF240467 (SEQ ID NO:25) and AF245703, respectively (SEQ ID NO:29).
For monitoring transient NF-κB activation, 3×106 293 HEK cells (ATCC, VA, USA) were electroporated at 200 volt and 960 μF with 1 μg TLR expression plasmid, 20 ng NF-κB luciferase reporter-plasmid and 14 μg of pcDNA3.1 (−) plasmid as carrier in 400 μl RPMI medium supplemented with 25% fetal bovine serum (FCS). Cells were seeded at 105 cells per well and after over night culture stimulated with R-848 (denoted in FIG. 12 as X; commercially synthesized by GLSynthesis Inc., Worcester, Mass., USA), RVCs or ribonucleosides for a further 7 hours. Stimulated cells were lysed using reporter lysis buffer (Promega, Mannheim, Germany), and lysate was assayed for luciferase activity using a Berthold luminometer (Wildbad, Germany).
As depicted in FIG. 12, TLR7 transfectants responded to R-848, RVCs, a mixture of ribonucleosides (A, G, C, U at 0.5 mM) and the ribonucleoside guanosine. Likewise TLR8 showed a similar response pattern.
Example 16
Responsiveness of TLR7 and TLR8 to Mixtures of Two Ribonucleosides
FIG. 13 depicts the responsiveness of TLR7 and TLR8 to mixtures of two ribonucleosides. In an experiment conducted as in FIG. 11 it was determined that TLR 8 responded best to a combination of the ribonucleosides G and U, however, TLR7 responded best to G alone. Additionally it can be seen that a minor response was given by a combination of C and U. These data show that ribonucleosides of the proper composition serve as ligands for TLR7 and TLR8. The nonspecific stimulus of TPA served as a control only. X denotes R-848.
Example 17
Human PBMC Respond to a Mixture of the Ribonucleosides G and U
FIG. 14 depicts the response of human PBMC to a mixture of the ribonucleosides G and U. It can be appreciated that the ribonucleosides G and U act synergistically to induce TNF from human PBMC. In this example the ratio of G:U of 1:10 was optimal.
Example 18
Human PBMC Respond to G,U-Rich Oligoribonucleotides
FIG. 15 depicts how human PBMC respond to RNA G,U-rich oligonucleotides. Both RNA and DNA oligonucleotides 5′-GUUGUGGUUGUGGUUGUG-3′ (SEQ ID NOs:1 and 19) were tested at 30 μM on human PBMC and TNF was monitored. Human PBMC were responsive to G,U-rich RNA oligonucleotides and not G,U-rich DNA oligonucleotides.
Example 19
Human PBMC Respond to Oxidized RNA
FIG. 16 depicts the response of human PBMC to oxidized RNA. Ribosomal 16S RNA was isolated from E. coli and subjected to chemical oxidation. The treatments were (mod A) 0.2 mM ascorbic acid plus 0.2 mM CuCl2 for 30 min at 37° C. or (mod B) 0.2 mM ascorbic acid plus 0.02 mM CuCl2 for 30 min at 37° C. This treatment induces oxidation at the 8 position of guanosine and also induces strand breaks 3′ of the modified guanosine. It was shown that ribosomal RNA induced TNF production from human PBMC. It was also evident that oxidation of ribosomal RNA greatly potentiates the response.
Example 20
Human TLR7 Responds to Oxidized Guanosine Ribonucleoside
FIG. 17 depicts human TLR7 and TLR8 responses to the oxidized guanosine ribonucleoside. Cells mock-transfected or transfected with human TLR 7 or human TLR8, as in Example 14, were tested for responsiveness to 7-allyl-8-oxoguanosine (loxoribine) at 1 mM. It can be clearly shown that human TLR7 is responsive to 7-allyl-8-oxoguanosine. Thus it appears that a ligand for TLR 7 is oxidized nucleic acids.
Example 21
Human TLR7 Responds to Other Modified Guanosine Ribonucleoside
FIG. 18 depicts human TLR7 responses to the other modified guanosine ribonucleoside. Cells transfected with human TLR7, as in Example 14, were tested for a dose-dependent response to 7-allyl-8-oxoguanosine (loxoribine). Additionally other modified guanosines were tested. It can be clearly shown that human TLR 7 was responsive to 7-allyl-8-oxoguanosine in a dose-dependent manor. As shown above, human TLR7 was responsive to guanosine; however FIG. 18 also shows that human TLR7 responded mildly to the deoxy form of guanosine as well as to 8-bromo-guanosine.
Example 22
Distribution of Human TLRs
FIG. 19 depicts the distribution of human TLR1-TLR9. Various purified human immune cells were screened by PCR for TLR1 through 9 expression. It was shown that human lymphoid CD123+ dendritic cells (DC) were strongly positive for TLR9 and TLR7 while weaker for TLR8. The converse was shown however for myeloid CD11c+ DC. This is very relevant because the two types of DC have very different functions in the immune system. Significantly, FIG. 19 also shows that human neutrophils were strongly positive for human TLR8 while very weak for TLR9 and negative for TLR7. This is also relevant because neutrophils are very often the first cells to engage infectious pathogens and thus believed to initiate responses.
Example 23
HEK-293 cell were stably transfected with human TLR7 or human TLR8. Additionally, the cells were stably transfected with NF-κB-luciferase reporter construct. The cells were titrated with varing amounts of RNA oligonucleotides and cultured for 16 h. Luciferase activity was measured by standard methods and normalizied versus mock-stimulated transfectants. Luciferase activity measured for the mock-stimulated transfectant was set to a value of 1-fold NF-κB induction. Results are shown in FIG. 20, where old NF-κB induced by the stimulating RNA oligonucleotide is plotted versus the concentration of test ribonucleotide. Stimulation with GUGUGUGU is shown for human TLR8. Stimulation with GUAGUCAC is shown for human TLR7 and human TLR8.
Equivalents
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.
All references, patents and patent publications that are recited in this application are incorporated in their entirety herein by reference.
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11368333
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zoetis belgium
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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514/44
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Mar 31st, 2022 03:01PM
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Mar 31st, 2022 03:01PM
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Zoetis
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:zts
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Zoetis
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Feb 17th, 2015 12:00AM
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Dec 15th, 2008 12:00AM
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https://www.uspto.gov?id=US08956628-20150217
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Bacteriophage preparations and method of use thereof
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Disclosed herein are purified bacteriophage preparations that effectively lyse a plurality of C. perfringens strains. In one embodiment, a purified bacteriophage preparation includes four or more C. perfringens-specific bacteriophage, wherein each bacteriophage has lytic activity against at least five C. perfringens strains. In another embodiment, the purified bacteriophage preparation includes five or more C. perfringens-specific bacteriophage.
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8956628
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1. The A purified bacteriophage preparation comprising CPAS-7 (accession number PTA-8479), CPAS-15 (accession number PTA-8480), CPAS-16 (accession number PTA-8481) and CPLV-42 (accession number PTA-8483) wherein each bacteriophage has lytic activity against at least five C. perfringens strains which cause necrotic enteritis in poultry.
2. The purified bacteriophage preparation of claim 1, wherein the at least five C. perfringens strains comprise ATCC strain 3624 and ATCC strain 9856.
3. The purified bacteriophage preparation of claim 1 wherein the C. perfringens strains comprise at least five of ATCC strain 25768, ATCC strain 3624, ATCC strain 9856, ATCC strain 3628, ATCC strain 13124, ATCC strain PTA-8495, NRRL strain B-50143, NRRL strain B-50144, NRRL strain B-50145, or a combination thereof.
4. The purified bacteriophage preparation of claim 1 wherein the preparation has lytic activity against ATCC strain 25768, ATCC strain 3624, ATCC strain 9856, ATCC strain 3628, ATCC strain 13124, ATCC strain PTA-8495, NRRL strain B-50143, NRRL strain B-50144, NRRL strain B-50145.
5. The purified bacteriophage preparation of claim 4, wherein the bacteriophage preparation is incapable of infecting at least ten strains of E. coli, L. monocytogenes, S. enterica, and P. aeruginosa.
6. The purified bacteriophage preparation of claim 1 comprising five or more C. perfringens-specific bacteriophage, wherein each bacteriophage has lytic activity against at least five C. perfringensstrains.
7. The purified bacteriophage preparation of claim 6, comprising CPAS-12 (accession number PTA-8479), CPAS-15 (accession number PTA-8480), CPAS-16 (accession number PTA-481), CPLV-42 (accession number PTA-8483) and CPAS-7 (accession number PTA-8482 wherein each bacteriophage has lytic activity against at least five C. perfringens strains which cause necrotic enteritis in poultry.
8. The purified bacteriophage preparation of claim 7 wherein the at least five C. perfringens strains comprise ATCC strain 3624 and ATCC strain 9856.
9. The purified bacteriophage preparation of claim 7 wherein the C. perfringens strains comprise at least five of ATCC strain 25768, ATCC strain 3624, ATCC strain 9856, ATCC strain 3628, ATCC strain 13124, ATCC strain PTA-8495, NRRL strain B-50143, NRRL strain B-50144, NRRL strain B-50145, or a combination thereof.
10. The purified bacteriophage preparation of claim 7 wherein the preparation has lytic activity against ATCC strain 25768, ATCC strain 3624, ATCC strain 9856, ATCC strain 3628, ATCC strain 13124, ATCC strain PTA-8495, NRRL strain B-50143, NRRL strain B-50144, NRRL strain B-50145.
11. The purified bacteriophage preparation of claim 10, wherein the bacteriophage preparation is incapable of infecting at least ten strains of E. coli, L. monocytogenes, S. enterica, and P. aeruginosa.
12. The purified bacteriophage preparation of claim 1, wherein the bacteriophage preparation lyses greater than or equal to 85% of at least 40 screened C. perfringens strains, and wherein the bacteriophage preparation is incapable of infecting at least ten strains of E. coli, L. monocytogenes, S. enterica, and P. aeruginosa.
13. The purified bacteriophage preparation of claim 12, wherein the bacteriophage preparation lyses greater than or equal to 85% of at least 45 screened C. perfringens strains.
14. The purified bacteriophage preparation of claim 13, wherein each of the individual C. perfringens-specific bacteriophage lyses 15% to 90% of the screened C. perfringens strains.
15. The purified bacteriophage preparation of claim 1 further comprising a pharmaceutically acceptable excipient.
16. The purified bacteriophage preparation of claim 15, wherein the pharmaceutically acceptable excipient is a water-conditioning agent.
17. The purified bacteriophage preparation of claim 16, wherein the water-conditioning agent is a 50mM citrate-phosphate-thiosulfate buffer comprising about 40 mg sodium thiosulfate, 6.0 gm disodium phosphate (anhydrous), 1.1 gm citric acid (anhydrous) per liter of deionized water, pH 7.0, added at a 1:10 or greater ratio.
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17
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CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application Ser. No. 61/013,325 filed on Dec. 13, 2007, which is incorporated in its entirety by reference herein.
BACKGROUND
Antibiotic use enhances the growth of healthy domesticated poultry and livestock. Although extensive bans and restrictions have not been implemented in the United States as they have in the E.U. and other countries, pressure for antibiotic alternatives has increased due to concerns of increasing antibiotic resistance among foodborne bacteria. Banning or markedly reducing the agricultural and farm-veterinary use of antibiotics may have a profoundly negative impact on the safety of foods and on the treatment of sick flocks or herds of domesticated livestock, however. Thus, effective, safe, and environmentally friendly alternative(s) to antibiotics are needed to address these concerns and needs.
Viruses that kill bacteria were first identified in the early part of the 20th century by Frederick Twort and Felix d'Herelle who called them bacteriophages or bacteria-eaters (from the Greek phago meaning to eat or to devour). Because of their remarkable antibacterial activity, phages were used to treat diseases of economically important animals/domesticated livestock almost immediately after their discovery, and therapeutic applications for humans closely followed. However, with the advent of antibiotics, phage therapy gradually fell out-of-favor in the United States and Western Europe, and virtually no subsequent research was done on the potential therapeutic applications of phages for bacterial diseases of humans or animals. The emergence of antibiotic-resistance in bacteria, however, has rekindled interest in therapeutic bacteriophages. Phage therapy may have a positive impact on human health by improving the safety of foods in the U.S.A. and elsewhere, and by helping to reduce safely the use of antibiotics in agribusiness.
Among the bacteria that cause significant morbidity and mortality in chickens, C. perfringens is one of the most notorious pathogens. In chickens, C. perfringens infections are often manifested as necrotic enteritis that occur later in the production cycle, often following a coccidial infection or other insult to the gastrointestinal tract. It is thus desirable to develop bacteriophage preparations suitable to reduce morbidity and mortality in chickens.
SUMMARY
Disclosed herein are purified bacteriophage preparations that effectively lyse a plurality of C. perfringens strains. In one embodiment, a purified bacteriophage preparation comprises four or more C. perfringens-specific bacteriophage, wherein each bacteriophage has lytic activity against at least five C. perfringens strains. In another embodiment, the purified bacteriophage preparation comprises five or more C. perfringens-specific bacteriophage.
In another embodiment, a method of reducing chicken mortality due to C. perfringens infection comprises administering a purified bacteriophage preparation comprising four or more C. perfringens-specific bacteriophage, wherein each bacteriophage has lytic activity against at least five C. perfringens strains.
In another embodiment, a method of selecting a C. perfringens host strain suitable to propagate bacteriophage from a plurality of test strains comprises microbiologically confirming one test strain from the plurality of test strains as a C. perfringens species to produce a confirmed strain; associating the confirmed strain with a poultry disease to produce a disease-associated strain; and applying one or more additional selective criterion to the disease-associated strain selected from minimal antibiotic resistance and absence of animal-virulence markers other than those for C. perfringens to produce the C. perfringens host strain suitable to propagate bacteriophage.
In yet another embodiment, a method of producing a bacteriophage cocktail comprises mixing four or more C. perfringens-specific bacteriophage, wherein each bacteriophage has lytic activity against at least five C. perfringens strains.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a phylogenetic analysis of C. perfringens strains.
FIG. 2 is a dendrogram portraying the genetic diversity of various C. perfringens strains based on SmaI-digested PFGE patterns of C. perfringens DNA.
FIG. 3 shows phage plaques produced by representative bacteriophage infecting C. perfringens strain Cp 42.
FIG. 4: Pulsed-field gel electrophoresis analysis of undigested phage DNA isolated from each monophage. Lane M, Low Range PFG Marker (NEB); lane 1, CPAS-7; lane 2, CPAS-15; lane 3, CPAS-16; lane 4, CPLV-42; lane 5, CPTA-12; lane 6, CPAS-37.
FIG. 5 shows XmnI digested phage DNA from each monophage. Lane M, GeneRuler DNA Ladder Mix (Fermentas); lane 1, CPLV-42; lane 2, CPAS-7; lane 3, CPAS-15; lane 4, CPTA-37; lane 5, CPAS-12; lane 6, CPAS-16.
FIG. 6 shows structural protein profiles for each C. perfringens monophage. Lane 1, CPLV-42; lane 2, CPAS-12; lane 3, CPAS-7; lanes 4, CPAS-16; lane 5, CPAS-15; lane 6, CPTA-37.
FIG. 7 shows electron micrographs of C. perfringens bacteriophages.
The above-described and other features will be appreciated and understood by those skilled in the art from the following detailed description, drawings, and appended claims.
DETAILED DESCRIPTION
Disclosed herein are purified bacteriophage preparations that effectively lyse a plurality of C. perfringens strains. Lysis of particular strains is demonstrated by the drop-on-lawn method, which is standard in the art. The bacteriophage preparations are suitable to reduce morbidity and mortality in chickens.
In one embodiment, a purified bacteriophage preparation comprises four or more C. perfringens-specific bacteriophage, wherein each bacteriophage has lytic activity against at least five C. perfringens strains. In another embodiment, the purified bacteriophage preparation comprises five or more C. perfringens-specific bacteriophage.
In one specific embodiment, the bacteriophage preparation comprises CPAS-12 (accession number PTA-8479), CPAS-15 (accession number PTA-8480), CPAS-16 (accession number PTA-8481) and CPLV-42 (accession number PTA-8483). In another embodiment, the bacteriophage preparation consists essentially of CPAS-12, CPAS-15, CPAS-16 and CPLV-42. In yet another embodiment, the bacteriophage preparation consists of CPAS-12, CPAS-15, CPAS-16 and CPLV-42.
In another specific embodiment, the bacteriophage preparation comprises CPAS-7 (accession number PTA-8478), CPAS-12, CPAS-15, CPAS-16 and CPLV-42. In another embodiment, the bacteriophage preparation consists essentially of CPAS-7, CPAS-12, CPAS-15, CPAS-16 and CPLV-42. In yet another embodiment, the bacteriophage preparation consists of CPAS-7, CPAS-12, CPAS-15, CPAS-16 and CPLV-42.
In one embodiment, the C. perfringens strains are ATCC strain 25768, ATCC strain 3624, ATCC strain 9856, ATCC strain 3628, ATCC strain 13124, ATCC strain PTA-8495, NRRL strain B-50143, NRRL strain B-50144, NRRL strain B-50145, and combinations comprising one or more of the foregoing strains. In a specific embodiment, the at least five C. perfringens strains comprise ATCC strain 3624 and ATCC strain 9856.
In one embodiment, the bacteriophage preparations are characterized by their specificity and effectiveness against C. perfringens strains. In one embodiment, the purified bacteriophage preparation lyses greater than or equal to 85% of at least 40 screened C. perfringens strains, wherein the bacteriophage preparation is incapable of infecting at least 10 strains of E. coli, L. monocytogenes, S. enterica, and P. aeruginosa. In another embodiment, the purified bacteriophage preparation lyses greater than or equal to 85% of at least 45 screened C. perfringens strains. In yet another embodiment, each of the individual C. perfringens-specific bacteriophage lyses 15% to 90% of the screened C. perfringens strains.
The term “purified” in reference to a bacteriophage or bacteriophage preparation does not require absolute purity (such as a homogeneous preparation). Instead, it is an indication that the bacteriophage or bacteriophage preparation is relatively more pure than in the natural environment (compared to the natural level, this level should be at least 2-5 fold greater, e.g., in terms of mg/mL). Purification is according to any method known to those of ordinary skill in the art that will result in a preparation of bacteriophage substantially free from other nucleic acids, proteins, carbohydrates, lipids, or subcellular organelles. Individual bacteriophage may be purified to electrophoretic homogeneity. Purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
Purity of phage stocks can be deter mined by pulsed-field gel electrophoresis (PFGE) of uncut DNA. In one embodiment, approximately 100-200 ng of the phage DNA is electrophoresed in a 1% SeaKem Gold Agarose (Cambrex, Rockland, Me.) gel with 0.5× Sodium boric acid (1×SB: 10 mM sodium hydroxide pH adjusted to 8.5 with boric acid) buffer at 14° C. in a CHEF Mapper XA PFGE apparatus (Bio-Rad Laboratories, Hercules, Calif.). The run time is 12 hours with a voltage of 6 V/cm and a linearly ramped pulse time of 0.06 s to 8.53 seconds. The gels ware stained with ethidium bromide and visualized with UV light.
In one embodiment, endotoxin levels for lots of phage produced are determined by a Limulus amoebocyte lysate (LAL) assay using a chromogenic assay (QCL-1000 Kit, BioWhittaker, Walkersville, Md.) following the manufacturers' recommendations. A standard curve with E. coli endotoxin (supplied with kit) ranging from 0.1 to 1.0 endotoxin units (EU) per milliliter endotoxin is constructed for each assay by plotting the optical density at 405 nm (OD405) versus EU/ml. Phage samples are assayed diluted in sterile water (USP, Baxter, Deerfield, Ill.) and the absorbance at 405 nm was measured with a μQuant (Bio-Tek Instruments, Inc., Winooski, Vt.) microplate reader. Endotoxin concentrations for each sample are calculated by linear regression from the standard curve.a Standards and samples are analyzed in triplicate.
In one embodiment, carbohydrate content for all lots of phage produced is determined by an anthrone method. A standard curve with glucose ranging from 10 to 250 μg/ml is constructed for each assay by plotting the optical density at 625 nm (OD625) versus μg/ml of glucose concentration. Phage samples are assayed in PBS and the absorbance at 625 nm is measured with a μQuant (Bio-Tek Instruments, Inc.) microplate reader. Carbohydrate concentrations for each sample are calculated by linear regression from the standard curve. Standards and samples are analyzed in duplicate.
In one embodiment, the total protein content for lots of phage produced is determined by a bicinchoninic acid (BCA) assay using a colorimetric assay (BCA™ Protein Assay Kit, Pierce, Rockford, Ill.) following the manufacturers' recommendations. A standard curve with bovine serum albumin (BSA, supplied with kit) ranging from 20 to 2,000 μg/ml protein is constructed for each assay by plotting the optical density at 562 nm (OD562) versus μg/ml BSA. The absorbance at 562 nm is measured with a μQuant (Bio-Tek Instruments, Inc.) microplate reader. Total protein concentrations for each sample are calculated by linear regression from the standard curve. Standards and samples are analyzed in triplicate.
The term “strain” means bacteria or bacteriophage having a particular genetic content. The genetic content includes genomic content as well as recombinant vectors. Thus, for example, two otherwise identical bacterial cells would represent different strains if each contained a vector, e.g., a plasmid, with different phage open reading frame inserts.
By “treatment” or “treating” is meant administering a bacteriophage preparation for prophylactic and/or therapeutic purposes. The term “prophylactic treatment” refers to treating an animal that is not yet infected but is susceptible to or otherwise at risk of a bacterial infection. The term “therapeutic treatment” refers to administering treatment to an animal already suffering from infection.
The term “bacterial infection” means the invasion of the host organism, animal or plant, by pathogenic bacteria. This includes the excessive growth of bacteria which are normally present in or on the body of the organism, but more generally, a bacterial infection is any situation in which the presence of a bacterial population(s) is damaging to a host organism. Thus, for example, an organism suffers from a bacterial infection when excessive numbers of a bacterial population are present in or on the organism's body, or when the effects of the presence of a bacterial population(s) is damaging to the cells, tissue, or organs of the organism.
The terms “administer”, “administering”, and “administration” refer to a method of giving a dosage of a bacteriophage preparation to an organism. Suitable methods include topical, oral, intravenous, transdermal, intraperitoneal, intramuscular, or intrathecal. The preferred method of administration varies depending on various factors, e.g., the components of the bacteriophage preparation, the site of the potential or actual bacterial infection, the bacterium involved, and the infection severity.
In the context of treating a bacterial infection a “therapeutically effective amount” or “pharmaceutically effective amount” indicates an amount of a bacteriophage preparation which has a therapeutic effect. This generally refers to the inhibition, to some extent, of the normal cellular functioning of bacterial cells that render or contribute to bacterial infection.
The dose of bacteriophage preparation that is useful as a treatment is a “therapeutically effective amount”. Thus, as used herein, a therapeutically effective amount means an amount of a bacteriophage preparation that produces the desired therapeutic effect as judged by clinical trial results. This amount can be routinely determined by one skilled in the art and will vary depending on several factors, such as the particular bacterial strain involved and the particular bacteriophage preparation used.
In one embodiment, a bacteriophage preparation optionally includes one or more pharmaceutically acceptable excipients. In one embodiment, the excipient is a water-conditioning agent, for example agents suitable for water dechlorination and/or phage stabilization. Such agents are innocuous to the bacteriophage cocktail, but when added prior to or simultaneously with C. perfringens bacteriophage or bacteriophage cocktails, act to dechlorinate municipal levels of chlorine, which if untreated would kill or significantly reduce the viability of the bacteriophage or bacteriophage cocktail. Exemplary water-conditioning agents include amino acids and/or salts which help to normalize the pH and ionic balance of the bacteriophage cocktail, when added to diverse water sources used for the animal's drinking and for phage delivery. In one embodiment, the water-conditioning agent is a 50 mM citrate-phosphate-thiosulfate (CPT) buffer, comprising about 40 mg sodium thiosulfate, 6.0 gm disodium phosphate (anhydrous), 1.1 gm citric acid (anhydrous) per liter of deionized water, pH 7.0. By including water-conditioning agents in the cocktail or adding separately to the treatment water, the water-conditioning agents act to both stabilize and protect the bacteriophage cocktail in a commercial preparation suitable for routine field use.
A method of producing a bacteriophage cocktail comprises mixing four or more C. perfringens-specific bacteriophage, wherein each bacteriophage has lytic activity against at least 5 C. perfringens strains. In another embodiment, a method of producing a bacteriophage cocktail comprises mixing five or more C. perfringens-specific bacteriophage, wherein each bacteriophage has lytic activity against at least 5 C. perfringens strains
Once C. perfringens cocktail have been selected, further testing can be employed to refine the specificity of the cocktail. In one embodiment, a bacteriophage cocktail is tested against additional C. perfringens strains which have antibiotic resistance genes to test the phage cocktail against antibiotic-resistant strains and evaluate the cocktail's potential for use in the field against antibiotic-resistant Clostridia. In another embodiment, a bacteriophage cocktail is tested against C. perfringens strains derived from animal species other than chickens to further evaluate and define the host range of the strain or strains to include additional animal species (e.g., swine, cattle, turkey, sheep, exotics, dogs, cats, and the like) In another embodiment, a bacteriophage cocktail is tested against Clostridium of different species (i.e., other than C. perfringens), which will further define the range of effectiveness of the phage cocktail. In yet another embodiment, a bacteriophage cocktail is tested against additional gram-positive bacteria (e.g., both reference strains and animal-associated types, aerobic and anaerobic) to further evaluate and define host range. In another embodiment, a bacteriophage cocktail is tested against additional gram-negative and gram-variable microorganisms to further evaluate host range. In another embodiment, a bacteriophage cocktail is tested for pre-conditioning or additive formulations, using different levels of stabilizing and dechlorinating water-conditioning agents, for optimally maintaining the viability of the phage cocktail under a wide range of water types and chlorination levels as may be expected in field usage conditions.
In another embodiment, the method further comprises testing the potential of the bacteriophage cocktail for the development of intrinsic phage resistance in the target host. Testing includes challenging test C. perfringens host strains with individual and/or combined bacteriophage cocktail phage over several cycles, and ascertaining the rate of resistance development toward the individual phages as well as that of the combination(s) of phages. It is anticipated that the combination bacteriophage cocktail will have significantly less development of resistance against a given individual host strain. An optimum combination of bacteriophages may be further elucidated using known mathematical optimization techniques or software packages (Box-Hunter, Latin Squares, Taguchi, Simplex, etc.) as applied to the bacteriophage resistance data generated from such experimentation.
Advantages of bacteriophage therapy include high bactericial activity, high selectivity permitting targeting of specific pathogens while leaving desirable bacterial flora intact, specificity for prokaryotic cells, and environmental benignity. In livestock and poultry applications, bacteriophage have the advantage of specificity that should not select for phage-resistance in non-target bacterial species, the possible emergence of resistance against phages will not affect the susceptibility of the bacteria to antibiotics used to treat humans, and, unlike antibiotics, phage preparations can readily be modified in response to changes in bacterial pathogen populations or susceptibility.
The poultry and livestock industries use antibiotics for three main purposes: (i) prophylactically, to prevent disease in flocks, herds, etc., (ii) to treat sick livestock, and (iii) to improve digestion and utilization of feed, which often results in improved weight gain. Antibiotics used in the latter setting often are referred to as “growth-promoting antibiotics” or GPAs. Most GPAs are not commonly used in human medicine, and they are usually administered, in small amounts, to poultry and other livestock via food. Bacteriophages can effectively replace and/or reduce the use of antibiotics in all three of the above-mentioned settings.
Among the bacteria that cause significant morbidity and mortality in chickens, C. perfringens is one of the most notorious pathogens. In order to identify effective bacteriophage for a genetically diverse population of C. perfringens strains, isolates of C. perfringens were first identified. In order to identify effective bacteriophage, it is useful to identify C. perfringens strains that affect poultry at various locations within the United States. As shown herein, forty-one strains of C. perfringens were isolated from various sources and characterized by pulsed-field gel electrophoresis (PFGE) typing. (FIG. 1) Among the 35 strains subjected to PFGE, phylogenetic analysis showed that these strains clustered into 15 heterogenic groups. (FIG. 2) Among these 15 PFGE types, P6 is the predominant type (10 strains) followed by P4 (6 strains). Additional C. perfringens strains may be obtained from publicly available collections.
One important factor in the identification of bacteriophage is the selection of C. perfringens strains suitable for their identification. In one embodiment, a method of selecting a C. perfringens host strain suitable to propagate bacteriophage from a plurality of test strains, comprises microbiologically confirming one test strain from the plurality of test strains as a C. perfringens species to produce a confirmed strain; associating the confirmed strain with a poultry disease to produce a disease-associated strain; applying one or more additional selective criterion to the disease-associated strain selected from minimal antibiotic resistance and absence of animal-virulence markers other than those for C. perfringens to produce the C. perfringens host strain suitable to propagate bacteriophage. In one embodiment, the selection criterion is minimal antibiotic resistance and the antibiotic resistance is tetracycline, ampicillin, tylosin, erythromycin, lincomycin, chloramphenicol or other drug resistance. The selection of strains absent from antibiotic resistance minimizes the potential transduction of plasmid or chromosomal-borne antibiotic resistance genes, into the subsequent bacteriophage cocktail genomes. The advantage of this applied criterion, is to in advance, limit any potential resistance genes in a bacteriophage cocktail preparation. The selective criterion used for these phage cocktail host strains, are a unique extension of a unique library of C. perfringens strains, combined with microbiological knowledge of antibiotic resistance, along with skills in running antibiotic susceptibility tests to ascertain the resistance profiles of the submitted host strains.
Six novel bacteriophages of the Siphoviridae or Myoviridae families that infect Clostridium perfringens were isolated from environmental water or sewage sources. Phage are characterized, for example, at both the protein and nucleic acid level. The optimal host strain for propagation of each bacteriophage is identified and all phage are preferably negative for endogenous phage. In addition, each bacteriophage is characterized by PFGE, RAPD, SDS-PAGE, and other approaches. Stocks of all six monophages and their respective host strains are made for use in characterization and production of each phage.
The C. perfringens-specific monophages are capable of specifically infecting C. perfringens strains, and are not capable of infecting/growing on E. coli, L. monocytogenes, S. enterica, and P. aeruginosa. As used herein, the teen C. perfringens-specific refers to bacteriophage and bacteriophage preparations that are capable of infecting a plurality of C. perfringens strains and are incapable of infecting at least 10 strains of E. coli, L. monocytogenes, S. enterica, and P. aeruginosa.
Six bacteriophages that infect Clostridium perfringens are sequenced. (SEQ ID NOs:1-6) Five of the six phages are sequenced, and each predicted open reading frame is identified in each genome. Each of the predicted genes was annotated. None of the 17 undesirable genes (Table 5.1.1) is found in the genomes of any of the five phages for which sequences were available.
Two phage cocktails, INT-401 (CPAS-7, CPAS-12, CPAS-15, CPAS-16, and CPLV-42) and INT-402 (CPAS-12, CPAS-15, CPAS-16, CPLV-42), are prepared from five of the six monophages isolated. Both cocktails are effective in killing greater than 85% of the 46 C. perfringens strains screened. INT-401 was selected for use in proof-of-principle efficacy studies designed to determine the prevention of necrotic enteritis in C. perfringens challenged broiler chickens.
Oral Gavage of Test Article (INT-401 phage cocktail) to birds on the day of challenge (Day 14) and for the next four days significantly reduced mortality due to NE. Growth performance in this group was numerically equivalent to the non-challenged control, and appeared to be better compared to the challenged, but phage-untreated chickens. Given the fact that many chickens are naturally colonized with C. perfringens, the latter observation warrants further elucidation, to better examine the possible growth performance-enhancing benefits of the phage preparation.
Two of the three “in ovo” treatments had numerically reduced NE mortality (9.6 and 14.8%) when compared to the Challenged control (25.9%).
Oral Gavage of Test Article prior to challenge, or spray of Test Article to chicks at the hatchery, was ineffective in preventing NE mortality due to C. perfringens challenge.
The results of the studies herein suggest that C. perfringens-specific phage preparation can be effective in significantly reducing chicken mortality due to C. perfringens infections in chickens such as those causing necrotic enteritis when administered shortly after the bacterial challenge. Further dosing- and delivery-optimization studies are warranted, together with further fine-tuning of the product for the optimal efficacy.
Exemplary means of administration of the bacteriophage preparations are oral administration, intramuscular injection, subcutaneous injection, intravenous injection, intraperitoneal injection, eye drop, nasal spray, and the like. When the subject to be treated is a bird, the bird may be a hatched bird, including a newly hatched (i.e., about the first three days after hatch), adolescent, and adult birds. Birds may be administered the vaccine in ovo, as described in U.S. Pat. No. 4,458,630 to Sharma, for example, incorporated herein by reference.
In one embodiment, the bacteriophage preparation is administered in an animal feed such as poultry feed. The bacteriophage preparation is prepared in a number of ways. For instance, it can be prepared simply by mixing the different appropriate compounds to produce the bacteriophage preparation. The resulting bacteriophage preparation can then be either mixed directly with a feed, or more conventionally impregnated onto a cereal-based carrier material such as milled wheat, maize or soya flour. Such an impregnated carrier constitutes a feed additive, for example.
The bacteriophage preparation may be mixed directly with the animal feed, or alternatively mixed with one or more other feed additives such as a vitamin feed additive, a mineral feed additive or an amino acid feed additive. The resulting feed additive including several different types of components can then be mixed in an appropriate amount with the feed. It is also possible to include the bacteriophage preparation in the animal's diet by incorporating it into a second (and different) feed or drinking water which the animal also has access to. Accordingly, it is not essential that the bacteriophage preparation is incorporated into the usual cereal-based main feed of an animal.
In one embodiment, included are methods of identifying an optimized field delivery modes and conditions for phage cocktail applications. In one embodiment, an optimized administration condition is water administration, for up to 3 days at temperatures up to 50° C.
The bacteriophage preparation can be used for a wide variety of animals, but use of the bacteriophage preparation is particularly preferred in domestic animals and farm livestock. Animals which may in particular benefit from the bacteriophage preparation include poultry (such as chickens, turkeys, ducks and geese), ruminants (such as cattle, horses and sheep), swine (pigs), rodents (such as rabbits) and fish. The bacteriophage preparation is particularly useful in broiler chickens.
The invention is further illustrated by the following non-limiting examples.
EXAMPLES
Example 1
Characterization of Clostridium perfringens Isolates
Media: Brain Heart Infusion (BHI) broth or BHI agar was used to grow all isolates. All media were obtained from EMD Chemicals, Gibbstown, N.J.
Microorganisms: Forty-two C. perfringens strains were employed. One strain (Cp 20) did not grow and was excluded from further analysis. As part of the collection process, isolates were checked for purity and frozen at −80° C. in 30% glycerol. Most of the work was performed in an anaerobic chamber (Plas-Labs, Inc. Lansing, Mich.), that contained a 90% N2-5% H2-5% CO2 atmosphere.
Bacteriophage: All bacteriophages were isolated from environmental water sources.
Phage Isolation: Samples of water collected for the isolation of phage were mixed with 10×BHI broth, inoculated with a single C. perfringens strain of interest and incubated anaerobically at 37° C. overnight. The samples were centrifuged (8,000×g, 10 min) to remove the bacterial cells and sterile filtered (0.22 μm Stericup™, Millipore, Bedford, Mass.). Filtrates were serially diluted in BHI broth and titered using the soft-agar overlay method. Briefly, dilutions of each filtrate were mixed with log-phase bacterial culture, incubated at 37° C. for 10 minutes, molten soft-agar added, poured onto BHI agar plates and incubated anaerobically at 37° C. overnight. Individual plaques were picked from the overlay plates and titered a second time as an initial step in ensuring that each phage was pure.
Screening for Endogenous Phage: Clostridium perfringens strains used for propagating the phages were screened for endogenous bacteriophage by the drop on lawn method. Liquid cultures of the host strains were grown overnight, centrifuged (9,500×g, 5 minutes) to remove the bacteria and filtered through a 0.22 μm syringe filter (Millipore). The same strains were grown in BHI broth to an OD600 of 0.1-0.3. Two hundred microliters of each screening strain was mixed with molten soft-agar and poured onto a BHI agar plate. After the soft-agar hardened 10 μl of each host strain filtrate was spotted onto the plates with the screening strains. Lytic activity was observed after overnight anaerobic incubation at 37° C.
Clostridium perfringens Host Strain Typing: The 41 C. perfringens strains received were typed by PFGE using the National Molecular Subtyping Network (PulseNet) standard protocol. Clostridium perfringens strains were grown on BHI agar overnight anaerobically at 37° C. and suspended in 75 mM NaCl-25 mM EDTA (pH 8.0) (CSB) buffer to an OD610 of 1.3-1.4. The bacterial cells were embedded in 1.2% SeaKem® Gold Agarose (Cambrex, Rockland, Me.) by mixing equal volumes (0.4 mL) of the cell suspension and melted agarose made in TE buffer. Plugs were made in 1.5-mm thick molds (Bio-Rad Laboratories, Hercules, Calif.) and solidified at 4° C. The cells were lysed by incubation in lysis buffer (50 mM Tris-HCl [pH 8.0], 50 mM EDTA [pH 8.0], 1% N-laurylsarcosine, and proteinase K [1 mg/ml]) at 55° C. overnight. The plugs were washed at 54° C. with shaking three times for 15 minutes each in sterile water and then three times in TE buffer. The plugs were stored at 4° C. in TE buffer.
Plugs were equilibrated with restriction endonuclease buffer at 25° C. overnight. The plugs were digested with SmaI (New England Biolabs, Beverly, Mass.) according to the manufacturers' recommendations overnight. Restriction fragments were separated by electrophoresis through a 1% agarose gel in 0.5× Tris-borate-EDTA (10× TBE, EMD Chemicals) with 1 mM thio-urea at 14° C. in a CHEF Mapper XA PFGE apparatus (Bio-Rad Laboratories). The run time was 20 hours with a voltage of 6 V/cm and a linearly ramped pulse time of 0.4 seconds to 40 seconds. The size range analyzed was 40-1,400 kilobases.
Data Handling & Analysis: A large zone of clearing (lytic activity) produced on lawns of any of the C. perfringens strains where the culture filtrate was applied were considered positive for endogenous phage.
Host Strain Typing: Analysis of the PFGE banding patterns was done with Quantity One (ver. 4.41) and Molecular Analyst Fingerprinting (ver. 1.6) software (Bio-Rad Laboratories) to determine the genetic relatedness of the strains. The dendrogram was constructed by the UPGMA algorithm with a 4% tolerance for fragment shifts. Isolates were considered closely related if their PFGE patterns differed by less than three fragments when digested with the restriction endonuclease SmaI.
PFGE Results: Forty-one strains of C. perfringens were isolated from various sources and characterized by pulsed-field gel electrophoresis (PFGE) typing. (Table 1) Among the 35 strains (one strain did not grow and six were not type-able due to nuclease problems) subjected to PFGE, phylogenetic analysis showed that these strains clustered into 15 heterogenic groups. (FIG. 1) Among these 15 PFGE types, P6 is the predominant type (10 strains) followed by P4 (6 strains). C. perfringens strain Cp27 has ATCC accession number PTA-8495.
TABLE 1
Clostridium perfringens isolates
Intralytix
Isolation
Pathogenic
PFGE
ID
Alpharma IDa
Year
Source
Location
(Yes/No)
Comment
Type
Cp 1
7998 B
1995
Roney
Canada
Yes
P1
Cp 2
UAZ 75
—
—
—
—
P2
Cp 3
Wallers
1993
—
IL
Yes
P3
Cp 4
Pennington
1993
—
IL
Yes
P4
Cp 5
96-7413
1996
Roney
AL
Yes
NT
Cp 6
UAZ 74
—
—
—
—
P5
Cp 7
Warren
1993
—
IL
Yes
P6
Cp 8
AU1
1996
—
AL
Yes
Gangrenous Dermatitis
P7
Cp 9
95-949
1995
Fitz-Coy
East Coast
Yes
NT
Cp 10
M1
2000
Fitz-Coy
East Coast
Yes
P8
Cp 11
Harmes
1993
—
IL
Yes
P3
Cp 12
94-5223
1994
Thayer
GA
Yes
P6
Cp 13
D00-20250
2000
Fitz-Coy
MN
Yes
NT
Cp 14
UDE 95-1377
1995
Fitz-Coy
DE
Yes
P9
Cp 15
95-1046
1995
Fitz-Coy
DE
Yes
Gall Bladder
NT
Cp 16
F96-01993
1996
Fitz-Coy
CA
Yes
P6
Cp 17
UAZ 257
—
—
—
—
P10
Cp 18
94-5228
1994
Thayer
GA
Yes
P11
Cp 19
Gresbrecht A
1993
—
IL
Yes
P6
Cp 20
96-2873
1996
Roney
AL
Yes
Did not grow
*
Cp 21
URZ298
—
—
—
—
P12
Cp 22
FC1
1995
Fitz-Coy
East Coast
Yes
P13
Cp 23
Kendall
1993
—
IL
Yes
P4
Cp 24
UDE 95-1372
1995
Fitz-Coy
DE
Yes
P14
Cp 25
C97M3
1997
—
CO
Yes
P4
Cp 26
Reed
1993
—
IL
Yes
P4
Cp 27
AU2
1996
Roney
AL
Yes
Gangrenous Dermatitis
P7
Cp 28
A1A
2002
Skinner
DE
Yes
P15
Cp 29
96-7414
1996
Roney
AL
Yes
P13
Cp 30
94-5230
1994
Thayer
GA
Yes
P6
Cp 31
94-5224
1994
Thayer
GA
Yes
P6
Cp 32
FC2
1995
Fitz-Coy
East Coast
Yes
P4
Cp 33
94-5229
1994
Thayer
GA
Yes
P6
Cp 34
7998C
1995
—
Canada
Yes
P1
Cp 35
S1-1
2000
Fitz-Coy
East Coast
Yes
P6
Cp 36
94-5227
1994
Thayer
GA
Yes
P6
Cp 37
Jones
1993
—
IL
Yes
P6
Cp 38
6A
2002
Skinner
NJ
Yes
P14
Cp 39
S1-7
2000
Fitz-Coy
East Coast
Yes
NT
Cp 40
7998A
1995
Roney
Canada
Yes
P1
Cp 41
95-1000
1995
Fitz-Coy
—
—
P4
Cp 42
AU3
1996
Roney
—
—
NT
* Isolate failed to grow.
NT = Not Typed.
All isolates were from intestines unless otherwise noted. All isolates were of chicken origin.
A dendrogram portraying the genetic diversity of various C. perfringens strains based on SmaI-digested PFGE patterns of C. perfringens DNA is shown in FIG. 2. Among the strains making up the 15 PFGE types, 16 strains (about 46%) were grouped in PFGE types P6 (10 strains) and P4 (6 strains). The remaining 20 strains clustered into eight PFGE types represented by a single strain (PFGE types P2, P5, P8, P9, P10, P11, P12 and P15), four PFGE types represented by two strains each (PFGE types P3, P7, P13 and P14), and one PFGE type represented by three strains (PFGE type P1). While some of the strains within the same PFGE type were associated with the same geographic location/source/year of isolation (e.g., both strains in PFGE type P3 have come from the Illinois Disease Lab, and they both were isolated in 1995), the number of strains in the PFGE types other than P4 and P6 was too small for making generalized conclusions about their specific association with any given facility/location. Strains in the PFGE types P4 and P6 did not appear to be associated with a specific geographic location/source of isolation (e.g., strains in the PFGE type P6 were isolated from various sources in Illinois, Georgia and California). FIG. 3 shows phage plaques produced by representative bacteriophage infecting C. perfringens strain Cp 42.
In sum, four to six candidate bacteriophages lytic for C. perfringens were isolated on phylogenetically distinct strains from environmental water sources each obtained from a different poultry farm or processing plant.
Example 2
Characterization of Phages Capable of Infecting Clostridium perfringens
The methods from Example 1 were also used in Example 2 where appropriate.
Phage Sterility: Microbial contamination was determined by (1) plating 1 mL aliquots of test sample on LB agar plates and incubating replicate plates at 37° C. and 30° C. for 48 hours and (2) pre-incubating 1 mL aliquots of test sample at 37° C. for 24 hours then plating the samples on LB agar and incubating the plates for 24 hours at 37° C. One set of plates was incubated aerobically and another set anaerobically as indicated. Any bacterial growth at the indicated times denotes contamination.
Phage Purity: Purity of phage stocks was determined by pulsed-field gel electrophoresis (PFGE) of uncut DNA. Approximately 100-200 ng of the phage DNA was electrophoresed in a 1% SeaKem® Gold Agarose (Cambrex, Rockland, Me.) gel with 0.5× Sodium boric acid (1×SB: 10 mM sodium hydroxide pH adjusted to 8.5 with boric acid) buffer at 14° C. in a CHEF Mapper XA PFGE apparatus (Bio-Rad Laboratories, Hercules, Calif.). The run time was 12 hours with a voltage of 6 V/cm and a linearly ramped pulse time of 0.06 seconds to 8.53 seconds. The gels were stained with ethidium bromide and visualized with UV light.
Nucleic Acid Characterization: DNA from each batch of bacteriophage Was isolated by a standard phenol-chloroform extraction method. Proteinase K (200 μg/ml) and RNase A (1 μg/ml) were added to phage samples with a titer≧1×109 PFU/ml and incubated at 37° C. for 30 minutes followed by 56° C. for an additional 30 minutes. SDS/EDTA was add to a final concentration of 0.1% and 5 mM respectively and incubated at room temperature for 5 minutes. The samples were extracted once with buffered phenol, once with phenol-chloroform and once with chloroform. Phage DNA was ethanol precipitated and resuspended in 10 mM Tris-HCl (pH 8.0)-0.1 mM EDTA (TE) buffer.
Restriction maps of the phage genomes were made by digesting approximately 1 μg of the phage DNA with 10 units of XmnI (New England Biolabs, Beverly, Mass.) according to the manufacturers' recommendations. Restriction fragments were separated on a 1.0% agarose gel for 16 hours at 20 V in 1× Tris-acetate-EDTA (10×TAE, EMD Chemicals) buffer and bands visualized by staining with ethidium bromide.
Protein Characterization: Phage proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, phage samples with a titer≧1×108 PFU/ml were denatured in a boiling water bath for 5 minutes in NuPAGE® LDS buffer fortified with DTT (Invitrogen, Carlsbad, Calif.). Aliquots were electrophoresed in a precast NuPAGE® Novex 4 to 12% Bis-Tris continuous gradient gel (Invitrogen) at 120 V for 110 minutes. Proteins was visualized on gels by silver-staining using a SilverXPress® (Invitrogen) according to the manufacturers' recommendations.
Clostridium perfringens Phage Susceptibility: Forty-six C. perfringens strains were screened for their susceptibility to the six monophages and two cocktails by the drop on lawn method. Strains was streaked onto BHI agar plates and incubated at 37° C. anaerobically overnight. One colony of each strain was inoculated into a separate 15-ml culture tube containing BHI broth and incubated at 37° C. anaerobically until the OD600 reached 0.1-0.3. One hundred microliters of each strain was mixed with BHI soft-agar and poured onto a BHI agar plate. After the soft-agar hardened 10 μl of each phage was spotted in triplicate onto the plates inoculated with the C. perfringens strains. Lytic activity was observed after overnight anaerobic incubation at 37° C.
Preparation of Phage Manufacturing Batches: Shake flask batches of each phage were carried out in 2-L flasks containing 1.5 L of BHI broth. Clostridium perfringens strains were grown in BHI broth anaerobically overnight at 37° C., subcultured and grown to an OD600 of 0.1-0.3. Cultures were infected at an MOI previously determined to be optimal for each phage (See Table 5.1.4). Growth was monitored spectrophotometrically until lysis occurred and phage harvested by vacuum filtration (Stericup, Millipore). Batches of each phage were concentrated separately and buffer exchanged with PBS by tangential flow filtration in a Pellicon® 2 Mini Cassette using a 50 kDa filter (Millipore).
Endotoxin Levels: Endotoxin levels for all lots of phage produced were determined by a Limulus amoebocyte lysate (LAL) assay using a chromogenic assay (QCL-1000 Kit, BioWhittaker, Walkersville, Md.) following the manufacturers' recommendations. A standard curve with E. coli endotoxin (supplied with kit) ranging from 0.1 to 1.0 endotoxin units (EU) per milliliter endotoxin was constructed for each assay by plotting the optical density at 405 nm (OD405) versus EU/ml. Phage samples were assayed diluted in sterile water (USP, Baxter, Deerfield, Ill.) and the absorbance at 405 nm was measured with a μQuant (Bio-Tek Instruments, Inc., Winooski, Vt.) microplate reader.
Endotoxin concentrations for each sample were calculated by linear regression from the standard curve. Standards and samples were analyzed in triplicate.
Carbohydrate Content: Carbohydrate content for all lots of phage produced was determined by an anthrone method. A standard curve with glucose ranging from 10 to 250 μg/mL was constructed for each assay by plotting the optical density at 625 nm (OD625) versus μg/mL of glucose concentration. Phage samples were assayed in PBS and the absorbance at 625 nm was measured with a μQuant™ (Bio-Tek Instruments, Inc.) microplate reader.
Carbohydrate concentrations for each sample were calculated by linear regression from the standard curve. Standards and samples were analyzed in duplicate.
Total Protein Content: The total protein content for all lots of phage produced was determined by a bicinchoninic acid (BCA) assay using a colorimetric assay (BCA™ Protein Assay Kit, Pierce, Rockford, Ill.) following the manufacturer's recommendations. A standard curve with bovine serum albumin (BSA, supplied with kit) ranging from 20 to 2,000 μg/mL protein was constructed for each assay by plotting the optical density at 562 nm (OD562) versus μg/mL BSA. The absorbance at 562 nm was measured with a μQuant® (Bio-Tek Instruments, Inc.) microplate reader.
Total protein concentrations for each sample were calculated by linear regression from the standard curve. Standards and samples were analyzed in triplicate.
Electron Microscopy: High-titer phage lysates (≧108 PFU/ml) were centrifuged 31,000×g for 2 hours and resuspended in 100 mM ammonium acetate (pH 7.0). A drop of phage suspension was deposited on a carbon-coated Formvar copper grid of 400 mesh. The phages were negatively stained by adding a drop of potassium phosphotungstate (1-2%, pH 7) and after one minute the excess fluid was withdrawn. Pictures of the phage particles were taken with a Philips EM 300 transmission electron microscope at an acceleration voltage of 60 kV with a primary magnification of 29,700×.
Results: Six novel bacteriophages that infect Clostridium perfringens were isolated from environmental water or sewage sources. Each phage was characterized at both the protein and nucleic acid level. The optimal host strain for propagation of each bacteriophage was identified and all were negative for endogenous phage. (Table 2)
TABLE 2
Optimal C. perfringens host strains for propagating
C. perfringens-specific phages
Phage
Host
CPLV-42
Cp 27
CPAS-7
Cp 8
CPAS-12
Cp 26
CPTA-37
Cp 27
CPAS-15
Cp 8
CPAS-16
Cp 42
Stocks of all six monophages and their respective host strains were made for use in characterization and production of each phage.
PFGE analysis of uncut DNA from each of the isolated phages showed that they were pure monophages with genome sizes of 36 to 50 kb (FIG. 4). DNA from each of the monophages isolated was digestible with XmnI (FIG. 5). All six of the monophages showed different protein profiles on SDS-PAGE or RFLP profiles confirming that all six monophages are different from one another. (FIG. 6)
Electron microscopy showed the bacteriophages to be members of the Siphoviridae or Myoviridae families of icosahedral head phages (FIG. 7) with long tails and double-stranded DNA genomes.
The ability of the six C. perfringens-specific monophages to infect 46 C. perfringens strains is shown in Table 3. Phage CPLV-42, CPAS-16, CPTA-37, CPAS-7, CPAS-12, and CPAS-15 infected 20%, 87%, 13%, 39%, 74%, and 52% of the strains screened respectively. The specificity of the phages for C. perfringens was examined by screening the susceptibility of ten strains of E. coli, L. monocytogenes, S. enterica, and P. aeruginosa. None of these 40 strains were infected by any of the monophages isolated against C. perfringens (Table 4).
TABLE 3
Susceptibility of C. perfringens strains to C. perfringens-specific bacteriophages
Phage
Cocktail
Strain
CPLV-42
CPAS-16
CPAS-12
CPAS-15
CPAS-7
CPTA-37
INT-401
INT-402
ATCC 13124
+
+
+
+
+
−
+
+
Cp 1
−
+
−
−
−
−
+
+
Cp 2
−
−
+
+
+
−
+
+
Cp 3
−
+
+
−
−
−
+
+
Cp 4
−
+
+
+
−
−
+
+
Cp 5
−
+
+
+
−
−
+
+
Cp 6
−
+
+
−
−
−
+
+
Cp 7
+
+
+
+
+
−
+
+
Cp 8
+
+
−
+
+
−
+
+
Cp 9
−
+
+
+
−
−
+
+
Cp 10
−
+
−
+
−
−
+
+
Cp 11
−
+
+
−
−
−
+
+
Cp 12
−
+
+
−
−
−
+
+
Cp 13
−
+
+
+
−
−
+
+
Cp 14
−
+
−
+
+
−
+
+
Cp 15
+
+
+
−
−
−
+
+
Cp 16
−
+
+
+
+
−
+
+
Cp 17
−
+
+
+
+
−
+
+
Cp 19
−
+
+
−
−
−
+
+
Cp 21
−
+
+
−
−
−
+
+
Cp 22
+
+
+
+
−
−
+
+
Cp 23
−
+
+
+
+
−
+
+
Cp 24
−
+
+
+
−
−
+
+
Cp 25
−
+
+
+
+
−
+
+
Cp 26
−
+
+
−
−
−
+
+
Cp 27
+
+
+
−
−
+
+
+
Cp 28
−
+
−
−
−
−
+
−
Cp 29
−
+
−
−
+
−
+
−
Cp 30
−
+
+
−
−
−
+
+
Cp 31
−
−
+
−
−
−
+
−
Cp 32
+
+
−
+
+
+
+
−
Cp 33
−
+
+
+
−
−
+
−
Cp 34
−
−
−
+
+
+
+
+
Cp 35
−
+
−
+
+
−
+
+
Cp 36
−
−
+
+
−
−
+
+
Cp 37
−
+
+
−
−
−
+
+
Cp 38
−
+
+
+
+
−
+
+
Cp 39
−
+
−
−
−
−
+
+
Cp 40
−
+
−
−
−
−
+
+
Cp 41
−
−
+
−
+
+
+
+
Cp 42
+
+
+
+
+
+
+
+
Cp 43
−
+
+
−
−
−
+
+
Cp 44
−
+
+
−
+
−
+
+
Cp 45
−
+
+
−
−
−
+
+
Cp 46
−
−
−
−
−
−
−
−
Cp 47
+
+
+
+
+
+
+
+
TABLE 4
Susceptibility of other bacterial strains to C. perfringens-specific bacteriophages
Phage
Strain
CPLV-42
CPAS-16
CPAS-12
CPAS-15
CPAS-7
CPTA-37
Pseudomonas
aeruginosa
Pa 1
−
−
−
−
−
−
Pa 3
−
−
−
−
−
−
Pa 7
−
−
−
−
−
−
Pa 15
−
−
−
−
−
−
Pa 21
−
−
−
−
−
−
Pa 33
−
−
−
−
−
−
Pa 42
−
−
−
−
−
−
Pa 62
−
−
−
−
−
−
Pa 65
−
−
−
−
−
−
Pa 72
−
−
−
−
−
−
Salmonella
enterica
SE 24
−
−
−
−
−
−
SS 28
−
−
−
−
−
−
ST 31
−
−
−
−
−
−
SHE 43
−
−
−
−
−
−
SH 49
−
−
−
−
−
−
S 45
−
−
−
−
−
−
S AE 72
−
−
−
−
−
−
SK 103
−
−
−
−
−
−
SR 114
−
−
−
−
−
−
SH 162
−
−
−
−
−
−
Listeria
monocytogenes
Lm 6
−
−
−
−
−
−
Lm 10
−
−
−
−
−
−
Lm 23
−
−
−
−
−
−
Lm 31
−
−
−
−
−
−
Lm 35
−
−
−
−
−
−
Lm 49
−
−
−
−
−
−
Lm 62
−
−
−
−
−
−
Lm 67
−
−
−
−
−
−
Lm 79
−
−
−
−
−
−
Lm 86
−
−
−
−
−
−
Escherichia coli
Ec 3
−
−
−
−
−
−
Ec 26
−
−
−
−
−
−
Ec 37
−
−
−
−
−
−
Ec 41
−
−
−
−
−
−
Ec 56
−
−
−
−
−
−
Ec 60
−
−
−
−
−
−
Ec 65
−
−
−
−
−
−
Ec 68
−
−
−
−
−
−
Ec 73
−
−
−
−
−
−
Ec 77
−
−
−
−
−
−
The following strains were used to demonstrate the overall activity of the phage cocktail TNT-401, versus a standardized set of Clostridium perfringens strains available from depositories. Strains were evaluated for lysis by spotting 10 microliters containing 108 pfu/ml onto pure lawns of each test strain spread onto BHI agar, and incubating overnight at 37° C.
TABLE 5
Depository
Strain Identifier Number
INT-401 Lysis
ATCC
25768
+
ATCC
3624
+
ATCC
9856
+
ATCC
3628
+
ATCC
13124
+
ATCC
PTA-8495
+
NRRL
B-50143 (CP8)
+
NRRL
B-50144 (CP26)
+
NRRL
B-50145 (CP42)
+
6 monophages were then isolated. (Table 6) Two phage cocktails, INT-401 (CPAS-7, CPAS-12, CPAS-15, CPAS-16, and CPLV-42) and INT-402 (CPAS-12, CPAS-15, CPAS-16, CPLV-42), were prepared from five of the six monophages isolated. Table 7 gives the levels of endotoxin, total carbohydrate, and total protein in C. perfringens-specific cocktails. Both cocktails were effective in killing greater than 85% of the 46 C. perfringens strains screened. INT-401 was selected for use in proof-of-principle efficacy studies designed to determine the prevention of necrotic enteritis in C. perfringens challenged broiler chickens. This cocktail contains the five bacteriophages with the broadest host range and is likely to provide a broader (compared to INT-402) spectrum of activity in actual use against wild-type C. perfringens strains.
TABLE 6
Summary of C. perfringens monophage batch preparation
Culture Time
Titer
Phage
Host Strain
MOI
(h)
(PFU/ml)
CPLV-42
Cp 27
1
5
5 × 1010
CPAS-16
Cp 42
1
3
1 × 108
CPAS-12
Cp 26
1
4-5
1 × 108
CPTA-37
Cp 27
1
4.5
4 × 108
CPAS-7
Cp 8
1
4-5
5 × 108
CPAS-15
Cp 8
1
4-5
6 × 109
TABLE 7
Levels of endotoxin, total carbohydrate, and total protein in
C. perfringens-specific cocktails
Content
INT-401
INT-402
Endotoxin (EU/ml)
384
288
Total Carbohydrate
170
43
(μg/ml)
Total protein (μg/ml)
661
175
Example 3
Protocol for Bacteriophage Treatment as Therapy or Prevention of Necrotic Enteritis in Broiler Chickens Challenged with Clostridium Perfringens
Broiler chickens: A total of 576 male day-old broiler chickens were assigned to treatment on day 0. There were no vaccinations (Mareks or Bronchitis) or antibiotics applied to eggs or chicks at the hatchery.
Housing: The 64-pen broiler chicken research facility at Maple Leaf Agresearch was used to conduct the study. Forty-eight pens, each providing approximately 10 square feet of floor space, were assigned to treatment groups. Each pen had a concrete floor and nylon mesh partitions supported by PVC frame. Adjacent pens were separated by a solid 12-inch high plastic barrier at bird level. Each pen was permanently identified by number and contained 12 birds on day zero. The barn was heated by two natural gas heaters, which were equally spaced and positioned to warm incoming air at the south wall of the building. Air was exhausted by fans located on the north-facing wall of the building. Each pen contained one nipple-type drinker, which provided clean drinking water ad libitum. Water was de-chlorinated. Dry feed was provided ad libitum in trough-type feeders (one per pen) of 5-kg capacity. New wood shavings were used as bedding.
Management: Lighting program, barn temperature, litter type and other management practices were typical of commercial broiler chicken producers in the local geographic area and is fully documented in the raw data. Birds, which were moribund and unable to reach food or water, were culled and euthanized by carbon dioxide gas.
Bodyweight, pen number, date of death and cause of death were determined by necropsy and recorded for each bird culled or found dead during the study.
Experimental design: A randomized complete block design was used to study the effects of eight treatments. The treatments were as follows:
TABLE 8
Treatment design
Treatment
C. perfringens
code
Challenge
Test Article
1
No
No
2
Yes
No
3
Yes
Yes
4
Yes
Yes
5
Yes
Yes
6
Yes
Yes
7
Yes
Yes
8
Yes
Yes
There were 8 pens per block (8 treatments) and 6 blocks (replicates) for a total of 48 pens. (See Section 4.2, Deviation #2 for change to above treatment to block assignment).
Treatment Groups:
Treatment 1—Control-UUC (no C. perfringens challenge or bacteriophage administration)
Treatment 2—Control-IUC (C. perfringens challenge without bacteriophage cocktail)
Treatment 3—In ovo injection of phage cocktail at day 18 of incubation.
Treatment 4—Spray application of phage cocktail to chicks after hatching.
Treatment 5—In ovo injection of phage cocktail at day 18 of incubation and spray application of phage cocktail to chicks after hatching.
Treatment 6—In ovo injection of phage cocktail at day 18 of incubation, spray application of phage cocktail to chicks after hatching and oral gavage of bacteriophage cocktail on Day 7 through 13.
Treatment 7—Bacteriophage cocktail administered via oral gavage from Day 7 through Day 13 (oral gavage ceased on day of Clostridium perfringens challenge)
Treatment 8—Bacteriophage cocktail administered via oral gavage beginning on Day 14 (concurrent with Clostridium perfringens challenge) through Day 18.
Feeding program: The following feeding program was used in the study:
TABLE 9
Feeding program
Day
Feed Type
Formulation number*
0-13
Starter
282
9:00 p.m. Day13 to
None. Feed was
None
9:00 a.m. Day 14
withdrawn
14-21
Starter
282
Feed sampling: The investigator's representative was present during feed manufacture. Ten representative samples were taken from each batch of final feed, composited and divided into three samples for proximate analysis, and retainer samples, respectively.
Administration of Clostridium perfringens challenge: A Clostridium perfringens isolate originating from a field case of necrotic enteritis in Ontario was used in the study. Inoculum contained approximately 108 cfu Clostridium perfringens per mL at time of feeding. Feed was withdrawn from all birds for approximately 8 hours prior to first introduction of challenge. Inoculum was administered to birds via feed in the afternoon and night commencing Day 14 P.M. and ending Day 15 A.M. using trough-type feeders. A suitable quantity of assigned feed (approximately 0.150 kg) and an amount of inoculum equal to approximately 1.5 times the weight of feed was added to each feeder. When this procedure was complete for all pens assigned to challenge, feeders were returned to their corresponding pens. Inoculum-feed mixture remaining at the end of the half-day period was weighed and discarded.
Lesion scoring of sacrificed birds: Three birds were randomly selected from each pen on Day 16 and euthanized. These birds were scored grossly for necrotic enteritis and coccidiosis lesions:
TABLE 10
Necrotic enteritis scoring
Necrotic
enteritis score
Description
0
Normal, no evidence of gross lesions
1
Thin, friable small intestine
2
Focal necrosis and/or ulceration
3
Patchy necrosis
4
Severe extensive necrosis (typically seen in
birds which have died from NE)
Clostridium perfringens culture of small intestinal segment: A small intestinal segment was collected from 40 birds that died on or after Day 15 and had a gross diagnosis of necrotic enteritis. The segment was forwarded to the Department of Pathology at the University of Guelph for C. perfringens culture. Culture results were reported as positive or negative for C. perfringens. Samples of positive bacterial cultures were forwarded to Intralytix for testing for phage susceptibility. In addition, 144 ileum samples were collected from the birds sacrificed for C. perfringens lesion scoring on Day 16. These samples were quantitatively tested for C. perfringens at the above referenced laboratory and microbiological samples were forwarded to Intralytix for additional characterization of phage activity.
Necropsy: All birds that died or were euthanized were submitted to the study pathologist for gross necropsy to determine the cause of death.
Observations and Calculation of Variables:
1) Bodyweight and number of birds per pen on Days 0, 14, and 21.
2) Amounts of each feed consumed by each pen.
3) Bodyweight and date of death for birds which were culled or died.
4) Feed conversion ratio was calculated on a pen basis as feed consumed/[total weight of live birds+total weight of dead and culled birds+total weight of sacrificed birds] for the 0-14, 14-21 and 0-21 Day periods.
5) Average bodyweight per pen was calculated as total weight of live birds at time of weighing/number of live birds at time of weighing.
6) Daily feed intake (grams) per live bird day was calculated on a pen basis for Day 0-14, Day 14-21 and Day 0-21.
7) Apparent cause of death was recorded for all birds that died or were culled. Total mortality and mortality from necrotic enteritis will be calculated on a pen basis.
8) Evaluation of the effects of the in ovo injection treatments on percent hatch and chick health at the hatchery.
9) Necrotic enteritis lesion score of sacrificed birds (Day 16)
10) Birds were observed on a flock basis at least once daily and observations recorded.
Test substance disposition: Remaining bacteriophage cocktail test substance was destroyed by incineration and destruction is documented in the study records.
Bird disposition: Birds (treated and control) were humanely euthanized at the end of the study and disposed of via incineration and method and date of disposition was recorded in the study records. Hatchery waste and unused in ovo bacteriophage injected eggs were disposed of via incineration. Hatched chicks that had been in ovo injected or sprayed with bacteriophage but not assigned to the study, were humanely euthanized and disposed of via incineration.
Original data: Original data is submitted to the sponsor together with the final report. An exact copy of the final report and data will be maintained at Maple Leaf Agresearch for a minimum of two years.
Documentation: All raw (original) data was recorded in black ink on data sheets bearing the trial number. Corrections were made by drawing a single line through the original entry and writing the correct entry beside it together with initials of the person making the correction, the date the correction was made and the reason for the correction. Defined error codes were used to record reason for correcting a data point.
Statistical analysis: Randomized complete block design was be used. Pen location within the facility was the blocking factor. The pen was the experimental unit for statistical analysis. A one-way treatment structure was utilized with each treatment being replicated six times (once within each block, except as detailed in Deviation #2, Section 4.2). Mortality data was transformed using an arcsine transformation prior to analysis of variance. Mixed models analysis was used to analyze all data. Means were compared using an appropriate multiple range test.
Amendment #1: This amendment clarified dates, eliminated vaccine administration and any potential interference vaccine might have with the Test Article, and detailed exact doses of Test article to be administered during “in ovo” injection (0.2 mL), spraying (about 7 mL per 100 chicks) and oral dosing (0.5 mL per bird per day).
Amendment #2: This amendment redefined the dosages of “in ovo” administration (0.05 mL per egg) and spray (7 to 22 mL per 100 chicks). The upper range of 22 mL was actually used for the spray.
Deviations: Two deviations occurred and are described in the Protocol section of the Study Binder. A brief description follows:
Deviation #1: Only 12 birds were assigned to pens instead of the 15 described in the protocol. This will have an impact on the statistical power of the study, particularly the mortality data.
Deviation #2: Block 3 was assigned two treatment 2 pens and no treatment 5 pen causing an imbalance in the design. Least square means will be reported to correct for the unequal representation per treatment group. This is not expected to have a major influence on the power of the study.
Example 4
Results for Bacteriophage Treatment as Therapy or Prevention of Necrotic Enteritis in Broiler Chickens Challenged with Clostridium Perfringens
The results of this study are summarized in Tables 10 to 12. A detailed statistical analysis was performed.
Hatchery: “In ovo” injection of eggs with Test Article (0.05 mL per egg) for Treatments 3, 5 and 6 was performed at 18 days of incubation using an Embrex machine and followed the standard industry protocol with the following exceptions: Marek's vaccine and antibiotic (Excenel) were not included. This standard procedure also involved applying a small amount of chlorine solution over the injection hole just post injection. For this trial, 1259 fertile eggs were transferred without being injected and hatched at 96.6%. The 775 fertile eggs injected with Test Article hatched at 95.5%.
TABLE 11
Delivery Routes of Bacteriophage on weights of broiler
chickens challenged with necrotic enteritis
Average live weights (kg)
Treatment1
Day 0
Day 14
Day 21
Day 35
Day 42
Control
.046
.330
.750A
1.917A
2.776A
Challenged control
.046
.340
.618C
1.530C
2.342C
BMD control
.045
.340
.634C
1.795B
2.686AB
Gavaged phage
.045
.328
.641C
1.762B
2.601B
Phage in water
.046
.348
.694B
1.812AB
2.664AB
Phage in feed
.045
.333
.658BC
1.754B
2.592B
SEM2
.000
.010
.018
.045
.059
Pr > F
.5309
.7492
.0003
.0001
.0004
1LSMEANS were provided for each treatment. The treatment groups included a control, challenged control, BMD 50 g/ton as a medicated control, oral gavaged phage, phage provide via water and phage provide via feed. The bacteriophage used was Intralytix C. perfringens Phage Cocktail - 4.8 × 109 pfu/ml. On Day 14, all birds were orally inoculated with a coccidial inoculum containing approximately 5,000 oocysts of E. maxima per bird. All groups, except the control, were challenged with Clostridium perfringens on Days 18, 19, and 20. Oral Administration of phage cocktail via gavages, drinking water and feed application will occurred on days 17, 18, 19, 20, and 21.
2Standard error of the LSMEANS.
A,B,CMeans within columns with different superscripts are significantly different (P < .05).
Three Treatments (#'s 4, 5 and 6, Table 10) were also sprayed with Test Article at the hatchery after hatch. A commercial spray cabinet designed for administering coccidiosis vaccine was used to deliver the Test Article at a rate of 22 mL per box or approximately 0.22 mL per bird. These birds were held in the hatchery for an extra ½ hour to permit drying prior to transport to the research farm.
Challenged pens were provided with 1.66 kg of the Clostridia perfringens inoculum/feed mixture and all consumed at least 1.25 kg except one, a challenged control pen. This pen suffered from severe water restriction due to a technical problem and for this reason was removed from the analysis. Due to the deviation described above the challenged control (Treatment 2) was assigned one extra pen and Treatment 5 one less pen. With this slight imbalance in design, least square means are reported. There was no significant (P>0.05) difference between challenged groups in quantity of inoculum consumed.
The primary criteria for evaluating the effectiveness of Test Article and its method of administration is mortality attributable directly to Clostridia perfringens challenge. No birds died from Necrotic Enteritis (NE) in the non-challenged control (Treatment 1) and this was significantly (P<0.01) different than the challenged control with 25.9% percent of birds in a pen dying of NE. Birds treated (Treatment 8) by Oral Gavage (OG) from the day of challenge (day 14) until day 18 had the lowest mortality (5.6%) of the phage treated groups and this was not significantly (P>0.05) different from the non-challenged control (Treatment 1). Two of the “In ovo” groups (Treatments 3 and 6) had intermediate NE mortality, which was not significantly (P>0.05) different from either Control groups (Treatments 1 and 2).
Three birds per pen were sacrificed at Day 16 to detect lesions typical of NE and to determine the presence of Clostridia perfringens in either a defined segment (approximately 3 to 4 cm distal to the duodenum) if no lesions were present or a segment surrounding an identified lesion. Although not significantly (P>0.05) different from the other treatment groups, there were no “typical” NE lesions found in the non-challenged control (Treatment 1). No significant (P>0.05) difference between treatments was found for lesion scores.
Clostridia perfringens (Cp) bacterium were isolated from all groups including the non-challenged control. We do not know if the strain isolated from the non-challenged control was the same as the challenged strain. However, Treatment 1 was numerically lower for Bacterial Scores for Cultures and this was consistent with the significantly (P<0.05) lowest score (Table 1) for Smears. No other trends were evident in the Bacterial Score means for either Cultures or Smears between the other treatment groups.
TABLE 12
Delivery Routes of Bacteriophage on weight gains of broiler chickens
challenged with necrotic enteritis
Average weight gain(kg)
Days
Days
Days
Treatment1
0-14
Days 0-21
14-21
Days 0-35
0-42
Control
.284
.705A
.421A
1.871A
2.730A
Challenged control
.294
.572C
.278C
1.484C
2.296C
BMD control
.295
.589C
.294C
1.750B
2.641AB
Gavaged phage
.283
.596C
.313BC
1.716B
2.556B
Phage in water
.302
.648B
.346B
1.766AB
2.618AB
Phage in feed
.287
.612BC
.325BC
1.709B
2.547B
SEM2
.010
.018
.014
.045
.059
Pr > F
.7559
.0003
.0001
.0001
.0004
1LSMEANS were provided for each treatment. The treatment groups included a control, challenged control, BMD 50 g/ton as a medicated control, oral gavaged phage, phage provide via water and phage provide via feed. The bacteriophage used was Intralytix C. perfringens Phage Cocktail - 4.8 × 109 pfu/ml. On Day 14, all birds were orally inoculated with a coccidial inoculum containing approximately 5,000 oocysts of E. maxima per bird. All groups, except the control, were challenged with Clostridium perfringens on Days 18, 19, and 20. Oral Administration of phage cocktail via gavages, drinking water and feed application will occurred on days 17, 18, 19, 20, and 21.
2Standard error of the LSMEANS.
A,B,CMeans within columns with different superscripts are significantly different (P < 0.05).
There was a significantly (P<0.05) higher chick weight for one of the groups (Treatment 4) receiving Test Article by Spray at the hatchery. This was not significantly different than one (Treatment 5) of the other two groups receiving the Spray. This may be a result of these Treatments retaining more moisture from the Spray procedure. Pre-challenge, on Day 14, there were no significant differences (P>0.05) in body weight between the Treatments. After challenge, at Day 21, the non-challenged control (Treatment 1) was significantly (P<0.05) heavier than the birds receiving Test Article by Oral Gavage (Treatment 7) prior to challenge. No other differences in growth performance were detected.
Total mortality in the non-challenged control was high at 13.9%. As indicated in the necropsy records much of this non-NE mortality was due to internal infections, omphalitis (yolk sac infections) and sudden death. This high early chick mortality is not typical. Total mortality was significantly (P<0.05) lower for the non-challenged control (Treatment 1, 13.9%) and the birds receiving Oral Gavage from day 14 to day 18 (Treatment 8, 12.5%) than Treatment 5 (37.0%).
TABLE 13
Delivery Routes of Bacteriophage on feed conversion of broiler chickens
challenged with necrotic enteritis
Feed conversion ratio (feed to gain)2
Days
Days
Days
Treatment1
0-14
Days 0-21
14-21
Days 0-35
0-42
Control
1.703
1.532D
1.417C
1.709D
1.892D
Challenged control
1.600
1.912A
2.284A
2.483A
3.226A
BMD control
1.561
1.829AB
2.130A
2.077B
2.652B
Gavaged phage
1.662
1.760BC
1.864B
1.814CD
2.086C
Phage in water
1.562
1.676C
1.778B
1.813CD
2.066C
Phage in feed
1.680
1.777ABC
1.868B
1.841C
2.089C
SEM3
.045
.051
.081
.047
.039
Pr > F
.1359
.0002
.0001
.0001
.0001
1LSMEANS were provided for each treatment. The treatment groups included a control, challenged control, BMD 50 g/ton as a medicated control, oral gavaged phage, phage provide via water and phage provide via feed. The bacteriophage used was Intralytix C. perfringens Phage Cocktail - 4.8 × 109 pfu/ml. On Day 14, all birds were orally inoculated with a coccidial inoculum containing approximately 5,000 oocysts of E. maxima per bird. All groups, except the control, were challe Oral Administration of phage cocktail via gavages, drinking water and feed application will occurred on days 17, 18, 19, 20, and 21.
2The feed conversion ratio was adjusted for the weights of mortality and removed weights.
3Standard error of the LSMEANS.
A,B,C,DMeans within columns with different superscripts are significantly different (P < 0.05).
TABLE 14
Delivery Routes of Bacteriophage on mortality and lesion scores
of broiler chickens challenged with necrotic enteritis
Mortality (%)2
Necrotic
Totals include all causes
Necrotic
enteritis
Days
Days
Days
enteritis
lesion
Treatment1
0-21
0-35
0-42
Days 0-42
scores3
Control
2.67CD
2.67D
4.00D
0D
0B
Challenged control
41.33A
66.00A
66.67A
64.00A
.9A
BMD control
24.67B
51.33B
53.33B
50.00B
1.1A
Gavaged phage
10.00C
16.67C
18.00C
14.00C
.1B
Phage in water
.67D
67D
3.33D
0D
.1B
Phage in feed
2.00CD
3.33D
5.33D
.66D
.4B
SEM4
2.97
2.71
2.81
2.76
.2
Pr > F
.0001
.0001
.0001
.0001
.0006
1LSMEANS were provided for each treatment. The treatment groups included a control, challenged control, BMD 50 g/ton as a medicated control, oral gavaged phage, phage provide via water and phage provide via feed. The bacteriophage used was Intralytix C. perfringens Phage Cocktail - 4.8 × 109 pfu/ml. On Day 14, all birds were orally inoculated with a coccidial inoculum containing approximately 5,000 oocysts of E. maxima per bird. All groups, except the control, were challenged with Clostridium perfringens on Days 18, 19, and 20. Oral Administration of phage cocktail via gavages, drinking water and feed application will occurred on days 17, 18, 19, 20, and 21.
2Percentage data were analyzed with and without transformation (arc sin square root).
3On Day 22, scoring was based on a 0 to 3 score, with 0 being normal and 3 being the most severe.
4Standard error of the LSMEANS.
A,B,C,DMeans within columns with different superscripts are significantly different (P < .05).
The non-challenged control (Treatment 1) had the numerically highest (102 grams per bird per day) feed intake and this was significantly (P<0.05) more than Treatment 5 (84 grams per bird per day). Although the means comparison was not significant (P>0.05), Treatment 8 had the numerically best FCR (1.477) of the Phage treated groups and equal to the performance of the non-challenged control.
Conclusions
1. A successful Clostridia perfringens (Cp) challenge was achieved. The positive control had 25.9% of the birds die of Necrotic Enteritis compared to the negative control (0.0%).
2. Oral Gavage of Test Article to birds on the day of challenge (Day 14) and for the next four days significantly reduced mortality due to NE. Growth performance in this group was numerically equivalent to the Non-Challenged control.
3. Two of the three “In ovo” treatments had numerically reduced NE mortality (9.6 and 14.8%) when compared to the Challenged control (25.9%).
4. Oral Gavage of Test Article prior to challenge was ineffective in preventing NE mortality due to Cp challenge.
5. Spray of Test Article to chicks at the hatchery did not significantly (P>0.05) reduce NE mortality from Cp challenge.
6. The precision of this trial was reduced by several factors including fewer birds being assigned to pens at day old than specified in the protocol and high early non-challenge related mortality.
Example 5
Sequence Analysis of C. perfringens Bacteriophage
Media: Brain Heart Infusion (BHI) broth or BHI agar supplemented with 250 mg/L cycloserine was used to grow all C. perfringens isolates. Luria-Bertani (LB) broth and LB agar was used to grow all aerobic strains. All media were obtained from EMD Chemicals, Gibbstown, N.J.
Microorganisms: Clostridium perfringens strains were from the Intralytix, Inc. Culture Collection, Baltimore, Md. As part of the collection process, isolates were checked for purity and frozen at −80° C. in 30% glycerol. Most of the work was performed in an anaerobic chamber (Plas-labs, Lansing, Mich.), that contained a 90% N2-5% H2-5% CO2 atmosphere. Escherichia coli, Listeria monocytogenes, Salmonella enterica, and Pseudomonas aeruginosa strains were from the Intralytix, Inc. Culture Collection and all were grown aerobically.
Bacteriophage: All bacteriophages were isolated from environmental water, industrial wastewater, or sewage sources.
Phage DNA Isolation: DNA from each batch of bacteriophage was isolated by a standard phenol-chloroform extraction method. Proteinase K (200 μg/mL) and RNase A (1 μg/mL) were added to phage samples with a titer≧1×109 PFU/ml and incubated at 37° C. for 30 minutes followed by 56° C. for an additional 30 minutes. SDS/EDTA was add to a final concentration of 0.1% and 5 mM respectively and incubated at room temperature for 5 minutes. The samples were extracted once with buffered phenol, once with phenol-chloroform and once with chloroform. Phage DNA was ethanol precipitated and resuspended in 10 mM Tris-HCl (pH 8.0)-0.1 mM EDTA ('1′E) buffer.
Phage Sequencing: The DNA from each of the phages was sequenced using standard automated sequencing methods.
Sequence Analysis: To identify the predicted open reading frames (ORFs) WPA uses a combination of CRITICA (1) and GLIMMER (2). The results from these programs are combined and the optimal open reading frames are extracted from the combined data set.
Each of the two programs uses different algorithms for identifying open reading frames, and each has its benefits and drawbacks. However, by combining the output from both tools WPA is able to optimize the predicted ORFs that they can identify.
WPA uses an automated annotation system in which assignments are generated primarily by sequence similarity searches between the de novo identified ORFs and other ORFs in their databases. In this case, the BLAST algorithm was used to compare predicted protein sequences for the annotation. In addition to the publicly available databases for comparison WPA has a phage database that contains approximately 400 phage genomes which they feel represents the best phage dataset available.
Following the automated annotation and assignment phase, the assignments for each gene are manually curated by Intralytix to see if any of the 17 undesirable genes (Table 15) are present.
TABLE 15
List of undesirable genes encoded in bacteriophage genomes
Toxin and its Encoding Gene
Bacterial Pathogen
Enterotoxin A (entA)
Staphylococcus aureus
Enterotoxin A (sea, sel)
Staphylococcus
Enterotoxin A (sea)
Staphylococcus aureus
Staphylokinase (sak)
Staphylococcus aureus
Enterotoxin P (sep)
Staphylococcus aureus
Exfoliative toxin A (eta)
Staphylococcus aureus
Diphtheria toxin (tox)
Corynebacterium
diphtheriae
Shiga toxins (stx1, 2)
Escherichia coli
Cytotoxin (ctx)
Pseudomonas aeruginosa
Cholera toxin (ctxA)
Vibrio cholerae
Cholera toxin (ctxB)
Vibrio cholerae
Zonula occludens toxin (zot)
Vibrio cholerae
Neurotoxin (C1)
Clostridium botulinum
Enterohaemolysin (hly)
Escherichia coli
Streptococcal exotoxin A
Streptococcus pyogenes
(speA)
Streptococcal exotoxin C
Streptococcus pyogenes
(speC)
Streptococcal exotoxin K
Streptococcus pyogenes
(speK)
Five of the six phages were sequenced. The sequences of each of the five phage genomes were obtained and each predicted open reading frame identified in each genome (Table 16). Each of the predicted genes was annotated. None of the 17 undesirable genes (Table 15) were found in the genomes of any of the five phages for which sequences were available (Table 17).
TABLE 16
Number of predicted ORFs for each C. perfringens-specific
bacteriophage
Number of Open
Reading Frames
Phage
(ORFs)
1
67
2
77
3
69
4
47
5
67
TABLE 17
Annotations of all predicted genes for each C. perfringens-specific
bacteriophage genome
Gene ID
Annotated Function
CPAS-7
1
Presumed portal vertex protein
2
Ring-infected erythrocyte surface antigen
3
Hypothetical protein
4
Hypothetical protein
5
FKBP-type peptidyl-prolyl cis-trans isomerase (trigger factor)
6
Isocitrate dehydrogenase kinase/phosphatase
7
Phage-like element PBSX protein xkdH
8
Phase 1 flagellin
9
Type I restriction-modification system restriction
subunit (EC 3.1.21.3)
10
Sarcosine oxidase, alpha subunit
11
Hypothetical protein
12
Phage-like element PBSX protein xkdK
13
Phage-like element PBSX protein xkdM
14
Hypothetical protein
15
Hypothetical protein
16
Phage protein
17
Hypothetical protein
18
Phage-like element PBSX protein xkdQ
19
Ribosomal protein S4 and related proteins
20
Phage-like element PBSX protein xkdS
21
Hypothetical protein
22
Phage-like element PBSX protein xkdT
23
Tail fiber
24
Heat shock protein 90
25
Hypothetical protein
26
Bacteriocin uviB precursor
27
N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28)
28
ABC transporter, permease protein
29
No hits
30
Transposase
31
DNA repair protein RadA
32
Transcriptional regulator
33
Hypothetical protein
34
Hypothetical protein
35
Thymidine kinase (EC 2.7.1.21)
36
Tryptophanyl-tRNA synthetase (EC 6.1.1.2)
37
CMP-binding factor
38
3-isopropylmalate dehydratase large subunit (EC 4.2.1.33)
39
DNA ligase
40
Putative penicillin-binding protein
41
Signal transducer and activator of transcription 1
42
Chemotaxis protein CHED
43
Gramicidin S synthetase I (EC 5.1.1.11)
44
ABC transporter ATP-binding protein
45
Putative ATP-dependent DNA helicase
46
DNA polymerase I
47
Hypothetical protein
48
Hypothetical protein
49
Phenylalanyl-tRNA synthetase beta chain
50
Terminase large subunit
51
Terminase small subunit
52
Hypothetical protein
53
CobT protein
54
Putative chromosome segregation protein, SMC
ATPase superfamily
55
Deoxycytidylate deaminase (EC 3.5.4.12)
56
aceE; pyruvate dehydrogenase e1 component
oxidoreductase protein
57
Hypothetical protein
58
Hypothetical protein
59
Aspartic acid-rich protein aspolin2
60
CDEP
61
Nucleolin
62
Peptide ABC transporter, ATP-binding protein
63
GTP-binding protein SAR1
64
gp56 dCTPase
65
Hypothetical protein
66
Homeobox-leucine zipper protein
67
DNA repair protein recN
CPAS-16
1
Developmentally regulated GTP-binding protein 1
2
Presumed portal vertex protein
3
Ring-infected erythrocyte surface antigen
4
Hypothetical protein
5
Hypothetical protein
6
FKBP-type peptidyl-prolyl cis-trans isomerase (trigger factor)
7
Isocitrate dehydrogenase kinase/phosphatase
8
Phage-like element PBSX protein xkdH
9
High-affinity potassium transporter
10
Sarcosine oxidase, alpha subunit
11
Hypothetical protein
12
Phage-like element PBSX protein xkdK
13
Phage-like element PBSX protein xkdM
14
Hypothetical protein
15
Hypothetical protein
16
Phage protein
17
Hypothetical protein
18
Phage-like element PBSX protein xkdQ
19
Ribosomal protein S4 and related proteins
20
Phage-like element PBSX protein xkdS
21
Hypothetical protein
22
Phage-like element PBSX protein xkdT
23
Tail fiber
24
Heat shock protein 90
25
Hypothetical protein
26
Bacteriocin uviB precursor
27
N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28)
28
Enterotoxin
29
Membrane protein
30
No hits
31
Thymidylate synthase (EC 2.1.1.45)
32
Heat shock protein (dnaJ-2)
33
Hypothetical protein
34
DNA polymerase I
35
DNA polymerase I
36
Putative ATP-dependent DNA helicase
37
ABC transporter ATP-binding protein
38
Terminase large subunit
39
Terminase small subunit
40
CobT protein
41
Putative chromosome segregation protein, SMC
ATPase superfamily
42
Deoxycytidylate deaminase (EC 3.5.4.12)
43
aceE; pyruvate dehydrogenase e1 component
oxidoreductase protein
44
Hypothetical protein
45
Hypothetical protein
46
Genomic DNA, chromosome 3, BAC clone: F1D9
47
SWF/SNF family helicase
48
Aspartic acid-rich protein aspolin2
49
CDEP
50
Nucleolin
51
Hypothetical protein
52
Putative reductase
53
Hypothetical protein
54
Hypothetical protein
55
Hypothetical protein
56
Tail fiber
57
gp56 dCTPase
58
Acetate kinase (EC 2.7.2.1)
59
GTP-binding protein SAR1
60
Peptide ABC transporter, ATP-binding protein
61
Cytochrome b (EC 1.10.2.2)
62
Homeobox-leucine zipper protein
63
DNA repair protein recN
64
Transposase
65
DNA repair protein RadA
66
Exonuclease I
67
Transcriptional regulator
68
Hypothetical protein
69
Thymidine kinase (EC 2.7.1.21)
70
Tryptophanyl-tRNA synthetase (EC 6.1.1.2)
71
CMP-binding factor
72
3-isopropylmalate dehydratase large subunit (EC 4.2.1.33)
73
DNA ligase
74
Putative penicillin-binding protein
75
Signal transducer and activator of transcription 1
76
Chemotaxis protein CHED
77
Gramicidin S synthetase I (EC 5.1.1.11)
CPAS-15
1
Gramicidin S synthetase I (EC 5.1.1.11)
2
Chemotaxis protein CHED
3
Signal transducer and activator of transcription 1
4
Putative penicillin-binding protein
5
DNA ligase
6
3-isopropylmalate dehydratase large subunit (EC 4.2.1.33)
7
CMP-binding factor
8
Tryptophanyl-tRNA synthetase (EC 6.1.1.2)
9
Thymidine kinase (EC 2.7.1.21)
10
Hypothetical protein
11
Hypothetical protein
12
Transcriptional regulator
13
Exonuclease I
14
DNA repair protein RadA
15
Transposase
16
ABC transporter ATP-binding protein
17
Y56A3A.29a protein
18
DNA polymerase I
19
DNA polymerase I
20
Thymidylate synthase (EC 2.1.1.45)
21
Hypothetical protein
22
SWF/SNF family helicase
23
Aspartic acid-rich protein aspolin2
24
CDEP
25
Hypothetical protein
26
Terminase large subunit
27
Terminase small subunit
28
CobT protein
29
Putative chromosome segregation protein, SMC
ATPase superfamily
30
Deoxycytidylate deaminase (EC 3.5.4.12)
31
aceE; pyruvate dehydrogenase e1 component
oxidoreductase protein
32
Hypothetical protein
33
Hypothetical protein
34
DNA repair protein recN
35
Homeobox-leucine zipper protein
36
Hypothetical protein
37
No hits
38
Hypothetical protein
39
N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28)
40
Bacteriocin uviB precursor
41
Hypothetical protein
42
Heat shock protein 90
43
Tail fiber
44
PROBABLE SUCCINYL-COA SYNTHETASE
BETA CHAIN PROTEIN
45
Phage-like element PBSX protein xkdT
46
Hypothetical protein
47
Phage-like element PBSX protein xkdS
48
Ribosomal protein S4 and related proteins
49
Phage-like element PBSX protein xkdQ
50
Hypothetical protein
51
Phage protein
52
Phage protein
53
Hypothetical protein
54
Hypothetical protein
55
Phage-like element PBSX protein xkdM
56
Phage-like element PBSX protein xkdK
57
Hypothetical protein
58
Sarcosine oxidase, alpha subunit
59
High-affinity potassium transporter
60
Phage-like element PBSX protein xkdH
61
Putative nodulation protein
62
Isocitrate dehydrogenase kinase/phosphatase
63
FKBP-type peptidyl-prolyl cis-trans isomerase (trigger factor)
64
Hypothetical protein
65
Hypothetical protein
66
Ring-infected erythrocyte surface antigen
67
Presumed portal vertex protein
68
GTP-binding protein SAR1
69
Hypothetical protein
CPLV-42
1
Hypothetical protein
2
Ribosomal protein S4 and related proteins
3
Hypothetical protein
4
F14H3.11 protein
5
Myosin heavy chain, cardiac muscle beta isoform
6
Holin
7
N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28)
8
Developmentally regulated GTP-binding protein 1
9
Hypothetical protein
10
DNA internalization-related competence protein ComEC/Rec2
11
Phage-related protein
12
Antirepressor
13
Phage protein
14
Hypothetical protein
15
Excinuclease ABC subunit B
16
Putative oxidoreductase protein
17
DNA topoisomerase I (EC 5.99.1.2)
18
Imidazole glycerol phosphate synthase
subunit hisF (EC 4.1.3.—)
19
Methyl-accepting chemotaxis protein
20
Hypothetical protein
21
Aminomethyltransferase (EC 2.1.2.10)
22
Hypothetical protein
23
Hypothetical protein
24
Hypothetical protein
25
Hypothetical protein
26
Hypothetical protein
27
Hypothetical protein
28
DNA polymerase III, subunit beta
29
Terminase large subunit
30
lin2585
31
Portal protein
32
Genomic DNA, chromosome 3, BAC clone: F1D9
33
Deoxyguanosinetriphosphate triphosphohydrolase (dgtP)
34
Virulence-associated protein E
35
Hypothetical protein
36
3′-phosphoadenosine 5′-phosphosulfate
sulfotransferase (PAPS reductase)/FAD synthetase
and related enzymes, COG0175
37
Phosphoadenosine phosphosulfate reductase (EC 1.8.4.8)
38
Hypothetical protein
39
Hypothetical protein
40
WRKY transcription factor 22
41
D-threonine dehydrogenase
42
Hypothetical protein
43
Hypothetical protein
44
Transcriptional regulator
45
Single-strand binding protein
46
Hypothetical protein
47
Sensor histidine kinase
CPAS-12
1
Presumed portal vertex protein
2
Ring-infected erythrocyte surface antigen
3
Hypothetical protein
4
Hypothetical protein
5
FKBP-type peptidyl-prolyl cis-trans isomerase (trigger factor)
6
Isocitrate dehydrogenase kinase/phosphatase
7
Phage-like element PBSX protein xkdH
8
High-affinity potassium transporter
9
Sarcosine oxidase, alpha subunit
10
Hypothetical protein
11
Phage-like element PBSX protein xkdK
12
Phage-like element PBSX protein xkdM
13
Hypothetical protein
14
Hypothetical protein
15
Phage protein
16
Hypothetical protein
17
Phage-like element PBSX protein xkdQ
18
Ribosomal protein S4 and related proteins
19
Phage-like element PBSX protein xkdS
20
Hypothetical protein
21
Phage-like element PBSX protein xkdT
22
Tail fiber
23
Heat shock protein 90
24
Hypothetical protein
25
Bacteriocin uviB precursor
26
N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28)
27
ABC transporter, permease protein
28
No hits
29
Gramicidin S synthetase I (EC 5.1.1.11)
30
Chemotaxis protein CHED
31
Signal transducer and activator of transcription 1
32
Putative penicillin-binding protein
33
DNA ligase
34
3-isopropylmalate dehydratase large subunit (EC 4.2.1.33)
35
CMP-binding factor
36
Tryptophanyl-tRNA synthetase (EC 6.1.1.2)
37
Thymidine kinase (EC 2.7.1.21)
38
Nucleolin
39
Transcriptional regulator
40
DNA repair protein RadA
41
Transposase
42
Hypothetical protein
43
Thymidylate synthase (EC 2.1.1.45)
44
Heat shock protein (dnaJ-2)
45
DNA polymerase I
46
Putative ATP-dependent DNA helicase
47
ABC transporter ATP-binding protein
48
Terminase large subunit
49
Terminase small subunit
50
CobT protein
51
Putative chromosome segregation protein, SMC
ATPase superfamily
52
Deoxycytidylate deaminase (EC 3.5.4.12)
53
aceE; pyruvate dehydrogenase e1 component
oxidoreductase protein
54
Hypothetical protein
55
Hypothetical protein
56
Genomic DNA, chromosome 3, BAC clone: F1D9
57
SWF/SNF family helicase
58
Aspartic acid-rich protein aspolin2
59
CDEP
60
Nucleolin
61
Alpha/beta hydrolase fold: Esterase/lipase/thioesterase
family . . .
62
Normocyte-binding protein 1
63
Hypothetical protein
64
DNA repair protein recN
65
Homeobox-leucine zipper protein
66
Hypothetical protein
67
gp56 dCTPase
Example 5
Use of a Water-conditioning Agent in the Phage Cocktail
Phage cocktail INT-40 at a final concentration of 1×107 pfu/mL was placed into treated (containing 50 mM citrate-phosphate-thiosulfate (CPT) buffer, comprising about 40 mg sodium thiosulfate, 6.0 gm disodium phosphate (anhydrous), 1.1 gm citric acid (anhydrous) per liter of deionized water, pH 7.0 (added at a 1:10 ratio to water) and untreated (distilled water) solutions containing added bleach at the levels indicated in Table 18, and allowed to stand for one hour at room temperature. Samples were taken, and 10 microliters were spotted onto BHI agar medium containing lawns of C. perfringens ATCC 13124 and allowed to dry. Plates were incubated overnight at 37° C., and phage inactivation was scored by the absence of a lytic clearing zone visible on the bacterial lawn.
Results: The results in Table 1 demonstrated the thiosulfate-containing buffer was able to protect phage cocktail INT-401 against oxidation due to chlorine bleach exposure. This conditioning agent could therefore be applied to chlorinated water as a means to allow the phage cocktail to retain activity in a commercial poultry watering system.
TABLE 18
Water Conditioning Agent Allowing Protection of Phage Cocktail
INT-401 in the presence of Hypochlorite
Hypochlorite
Lysis Response vs. ATCC 13124
Concentration (ppm)
Conditioned Water
Unconditioned Water
0
++
++
0.5
++
+
1
++
+
2
+
−
4
+
−
6
+
−
Lysis Response:
++ Clear Lysis
+ Partial Lysis
− No Lysis
The terms “first,” “second,” and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another, and the terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. All ranges disclosed herein are inclusive and combinable.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims.
All cited patents, patent applications, and other references are incorporated herein by reference in their entirety.
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12334863
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zoetis products llc
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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424/247.1
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Mar 31st, 2022 03:01PM
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Mar 31st, 2022 03:01PM
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Zoetis
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:zts
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Zoetis
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Oct 21st, 2014 12:00AM
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Aug 25th, 2010 12:00AM
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https://www.uspto.gov?id=US08865409-20141021
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Assay methods for MDV-1
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A method for the quantification of a vaccine strain and/or a virulent strain of Marek's Disease Virus Serotype-1 (MDV-1) in a sample from a bird, comprising the steps of: (i) providing a biological sample from the bird and optionally isolating nucleic acid from the biological sample; (ii) subjecting the biological sample of (i) to real-time quantitative PCR (qPCR) comprising: (a) amplification of a region of the pp38 gene within the nucleic acid sample of (i), said region containing a consistent single nucleotide polymorphism (SNP) difference between vaccine and virulent strains of MDV-1; and (b) contacting the amplified nucleic acid of (a) with a detectable nucleic acid probe specific for the SNP of the vaccine strain of MDV-1 and/or a detectable nucleic acid probe specific for the SNP of the virulent strain of MDV-1; and (iii) Measuring changes in the detectable signal produced by the probe of (ii). Methods are also provided for the absolute quantification of vaccine and virulent viruses.
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8865409
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1. A method for the quantification of a vaccine strain, and a virulent strain of Marek's Disease Virus Serotype-1 (MDV-1) in a sample from a bird, comprising the steps of:
(i) providing a biological sample from the bird;
(ii) subjecting the biological sample of (i) to real-time quantitative PCR (qPCR) comprising:
(a) amplification of a region of the pp38 gene within the nucleic acid sample of (i), said region containing a consistent single nucleotide polymorphism (SNP) difference between vaccine and virulent strains of MDV-1, wherein the amplification step is performed with the following nucleic acid amplification primers: Forward primer: 5′ GAGCTAACCGGAGAGGGAGA 3′ set forth in SEQ ID NO:5 and Reverse primer: 5′ CGCATACCGACTTTCGTCAA 3′ set forth in SEQ ID NO:6; and
(b) contacting the amplified nucleic acid of (a) with a detectable nucleic acid probe specific for the SNP of the vaccine strain and a detectable nucleic acid probe specific for the SNP of the virulent strain of MDV-1 wherein said detectable nucleic acid probes comprise the vaccine strain-specific probe set forth in SEQ ID NO: 1 and at least one of the following virulent strain-specific probes: SEQ ID NO: 2 and SEQ ID NO: 3; and
(iii) measuring the detectable signal produced by each of the vaccine strain probe and the virulent stain probes of (ii); and
(iv) comparing the detectable signal measured in (iii) for each specific probe to determine the amount of virulent and vaccine strain present in the sample.
2. The method of claim 1 wherein the quantification of the strain(s) is relative quantification.
3. The method of claim 1 wherein the method optionally includes the step of isolating nucleic acid from the biological sample of step (i) and subjecting the isolated nucleic acid to the qPCR of step (ii).
4. The method of claim 1 wherein the quantification of the strain(s) is absolute quantification.
5. The method of claim 1 wherein the qPCRs of step (ii) further comprise a concomitant known standard qPCR utilizing a probe labeled with a different detectable signal to allow absolute quantification of the vaccine strain, the virulent strain or both the vaccine and virulent strains.
6. The method of claim 1 wherein the standard qPCR system is an Ovo reaction which uses the Ovo host gene present in chicken DNA.
7. The method of claim 1 wherein the vaccine strain is an attenuated vaccine strain.
8. The method of claim 7 wherein the vaccine strain is the CVI988 vaccine.
9. The method of claim 1 wherein the probe or probes used are homolabelled fluorogenic probes comprising two identical reporter dyes that are capable of quenching each other and becoming dequenched once the probe is cleaved.
10. The method of claim 9 where the probe or probes are AllGlo miniprobes.
11. The method of claim 1 wherein the virulent MDV strain is selected from the group consisting of RB1B MDV, 584A MDV, 595 MDV, 684A MDV, Md5 MDV, HPRS-B14 MDV, JM102 MDV, 660A MDV, 675A MDV, 549 MDV, 571 MDV and C12-130 MDV.
12. The method of claim 1 wherein the nucleic acid sample provided is derived from the following tissues: blood, spleen, liver, skin, ovaries, bursa, thymus, kidney or feather tips.
13. The method of claim 1 wherein the detectable signal comprises a fluorescing agent.
14. The method of claim 13 wherein the fluorescing agent comprises a fluorescent dye selected from the group consisting of MAR, JUP, NEP, SAT and URA.
15. The method of claim 14 wherein the detectable signal is the dye SUP.
16. A method for the identification of the cause of failure of an MDV-1 vaccine to protect against infection with a virulent strain of MDV-1, the development or the testing of an MDV-1 vaccine comprising the quantification both the virulent and vaccine strain of MDV-1 in a sample from the bird according to the method of claim 1.
17. The method of claim 6 wherein the standard qPCR Ovo reaction comprises the amplification of an Ovo gene amplicon with the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO 8 and detection of the amplicon with the Ovo specific detection probe of SEQ ID NO: 9.
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17
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This application commences the national stage under 35 U.S.C. §371 of PCT International Application No. PCT/GB2010/001602, filed on Aug. 25, 2010, which claims the priority benefit of Great Britain Application No. 0914826.3, filed on Aug. 25, 2009. The prior applications are incorporated herein by reference in their entirety.
The present invention relates to assay methods and, in particular, to methods for analysing levels of vaccine strains and levels of virulent strains of virus, particularly Marek's Disease Virus serotype-1 (MDV-1), in samples from birds.
BACKGROUND
Marek's Disease Virus (MDV) is a herpesvirus, which causes lymphoproliferative disease in chickens. Even after the introduction of vaccines against MDV, the infection still causes considerable losses in the poultry industry. MDV is divided into three serotypes, all of which establish latent infections. Serotype 1 includes oncogenic viruses, serotype 2 non-oncogenic viruses and serotype 3 includes the turkey herpesviruses (HVT) (Bülow et al (1976) Zentralblatt für Veterinarmedizin, 23B, 391-402).
Handberg et al (2001) Avian Pathology 30: 243-249 describe the use of serotype 1- and serotype 3-specific PCR for the detection of MDV in chickens. Tissue samples were taken from blood (buffy-coat cells), spleen, liver, skin, feather tips and ovaries.
CVI988 (Rispens) strain, a naturally-attenuated MDV serotype-1 (MDV-1), was introduced in the mid-1990s (Rispens et al., (1972) Avian Diseases, 16, 108-125; de Boer et al., (1986) Avian Diseases, 30, 276-283) and is to-date the most effective vaccine against MDV. However, even in light of vaccination programmes, MDV outbreaks continue to cause significant losses. These outbreaks can have several causes including: inhibition by maternal antibodies of vaccine virus replication (King et al., (1981) Avian Diseases, 25, 74-81; de Boer et al., (1986) supra); suppression of the immune response to vaccine by environmental stresses or co-infections with immunosuppressive pathogens; infection with a virulent MDV strain prior to establishment of full vaccinal immunity; infection of vaccinated and fully immunocompetent birds with a hypervirulent MDV strain (Witter et al., (2005) Avian Pathology, 34, 75-90); and, finally, administration of insufficient vaccine dose which fails to induce protective immunity.
As such it is important to be able to accurately measure MDV vaccine and challenge (virulent) virus individually, including measuring one or both, in the same individual chicken for both experimental and commercial reasons. Experimentally, it is very useful to be able to examine the interactions between vaccine and challenge virus, with a view to better understanding the mechanism of vaccinal protection and differences in efficiency between vaccines. Commercially, it is important to assist in identifying the causes of vaccine failures, such as administration of a sub-optimal vaccine dose (Landman & Verschuren, 2003), interference with vaccine virus replication by maternal antibodies (King et al., 1981; de Boer et al., 1986), and infection with hypervirulent MDV field strains (Witter et al., 2005).
A highly sensitive real-time quantitative PCR (q-PCR) assay for absolute quantitation of MDV-1 in chicken tissues has been developed (Baigent et al., 2005a) and used very successfully to investigate the kinetics of CVI988 vaccine virus replication in tissues of experimental and commercial chicks in the absence of challenge virus infection (Baigent et al., 2005b, 2006). However this method cannot distinguish between CVI988 and challenge virus. Other groups have developed real-time PCR assays to quantify MDV-2 (Renz et al., 2006) and MDV-3 (herpesvirus of turkeys) (Islam et al., 2006a). These serotype-specific assays can be used to quantify both vaccine and virulent challenge virus in individual chickens, where challenge virus (always MDV-1) is of a different serotype to vaccine virus (MDV-2 or -3) (Islam et al., 2006b).
However, the most widely used and effective MDV vaccine is a naturally-attenuated serotype-1 strain (CVI988, Rispens) (Rispens et al., 1972; de Boer et al., 1986) and, to date, real-time PCR to distinguish challenge virus from serotype-1 vaccine virus has not been possible, because sequence differences between them are very limited. A difference in the number of repeats in the 132-bp repeat region can be used to distinguish CVI988 from virulent strains using conventional PCR (Becker et al., 1992; Silva, 1992). However, the repetitive nature of this region precludes its use in quantitative PCR. Differences in the meq gene and the ICP4 gene are not consistent between CVI988 and virulent strains. A qPCR system has also been developed (Baigent, unpublished) to distinguish between BAC (Bacterial Artificial Chromosome) cloned MDV-1 and wild-type MDV-1 by targeting the BAC-specific sequence of the BAC cloned MDV-1 and US2 gene of the wild-type MDV-1, this can be used to distinguish between BAC cloned vaccine strains and wild-type virulent strains but only when such vaccine stains are BAC cloned. Importantly, such a method could not be used commercially, since no commercial MDV-1 vaccines are BAC cloned.
The pp38 gene shows a consistent single nucleotide difference between the CVI988 vaccine strain and virulent strains of MDV-1, however q-PCR primers are difficult to design which would be able to distinguish such a small difference.
DESCRIPTION OF THE INVENTION
The present invention provides methods for quantifying vaccine and/or virulent MDV-1 strains of Marek's Disease Virus Serotype-1 in birds based on a single nucleotide polymorphism (SNP) in the pp38 gene. These methods also include methods for the quantification of the relative and/or absolute amounts of vaccine and virulent strains and determination of copy number of each strain.
In a first aspect the invention provides a method for the quantification of a vaccine strain of Marek's Disease Virus Serotype-1 (MDV-1) in a sample from a bird, comprising the steps of:
(i) providing a biological sample from the bird;
(ii) subjecting the biological sample of (i) to real-time quantitative PCR (qPCR) comprising:
(a) amplification of a region of the pp38 gene within the nucleic acid sample of (i), said region containing a consistent single nucleotide polymorphism (SNP) difference between vaccine and virulent strains of MDV-1; and
(b) contacting the amplified nucleic acid of (a) with a detectable nucleic acid probe specific for the SNP of the virulent strain of MDV-1;
(iii) Measuring changes in the detectable signal produced by the probe of (ii)
In a second aspect the invention provides a method for the quantification of a virulent strain of Marek's Disease Virus Serotype-1 (MDV-1) in a sample from a bird, comprising the steps of:
(i) providing a biological sample from the bird;
(ii) subjecting the biological sample of (i) to real-time quantitative PCR (qPCR) comprising:
(a) amplification of a region of the pp38 gene within the nucleic acid sample of (i), said region containing a consistent single nucleotide polymorphism (SNP) difference between vaccine and virulent strains of MDV-1; and
(b) contacting the amplified nucleic acid of (a) with a detectable nucleic acid probe specific for the SNP of the virulent strain of MDV-1;
(iii) Measuring changes in the detectable signal produced by the probe of (ii)
In a third aspect the invention provides a method for the quantification of a vaccine strain and a virulent strain of Marek's Disease Virus Serotype-1 (MDV-1) in a sample from a bird, comprising the steps of:
(i) providing biological samples from the bird;
(ii) subjecting the biological samples of (i) to real-time quantitative PCR (qPCR) comprising:
(a) amplification of a region of the pp38 gene within the nucleic acid samples of
(i), said region containing a consistent single nucleotide polymorphism (SNP) difference between vaccine and virulent strains of MDV-1; and
(b) contacting the amplified nucleic acid of (a) with a detectable nucleic acid probe specific for the SNP of the vaccine strain and a second nucleic acid probe specific for the SNP of the virulent strain of MDV-1;
(iii) Measuring changes in the detectable signal produced by each probe of (ii); and optionally
(iv) comparing the changes in the detectable signal measured in (iii) for each specific probe.
In one embodiment of the invention the quantification of the virulent and vaccine strains may be a relative quantification.
The methods of the invention optionally may include the step of isolating nucleic acid from the biological sample of step (i) and subjecting the isolated nucleic acid to the qPCR of step (iii).
The biological sample provided from the bird may comprise bird nucleic acid or nucleic acid as isolated from the bird (i.e. may include both bird nucleic acids and nucleic acid from other sources within the bird—e.g. a virus). In one embodiment of the invention the biological sample provided is derived from the following tissues: blood, spleen, liver, skin, ovaries, bursa, thymus, kidney or feather tips of individual birds (Baigent et al., (2005a) Journal of Virological Methods, 123, 53-64; Baigent et al., (2005b) Journal of General Virology, 86, 2989-2998). Use of feather tips provides a means of sampling birds in the field and is the simplest and least invasive procedure.
The pp38 gene encodes a phosphoprotein that plays an important role in the pathogenesis of MDV. The pp38 gene has been shown to be necessary to establish cytolytic infection in B-cells, to produce an adequate level of latently infected T-cells and to maintain the transformed state in vivo. (Gimeno et al. 2005)
A single-nucleotide polymorphism (SNP) is a DNA sequence variation occurring when a single nucleotide (A, T, C, or G) in the genome (or other shared sequence) differs between members of a species, or between paired chromosomes in an individual, or in this case between vaccine and virulent strains of MDV-1. For example, two sequenced DNA fragments from different individuals, AAGCCTA to AAGCTTA, contain a difference in a single nucleotide. In this case we say that there are two alleles, the C polymorphism and the T polymorphism. Almost all common SNPs have only two alleles.
Real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (Q-PCR/qPCR) or kinetic polymerase chain reaction is a technique based on the polymerase chain reaction (PCR) and is used to both amplify and quantify target genetic material. qPCR enables both detection and quantification (as relative amount or absolute number of copies when normalized to DNA input or additional normalizing genes) of a specific sequence in a DNA sample. qPCR follows the general procedure of PCR with the extra feature that the accumulating amplified DNA is quantified in real time after every cycle of the qPCR. Such quantification can be achieved by the common methods of fluorescing agents that intercalate with the accumulating DNA or modified nucleic acid probes complimentary to the amplified DNA sequence that produce a detectable signal on binding with the accumulating DNA. Therefore qPCR is a widely used and very useful method of detecting and quantifying a particular nucleic acid sequence in a sample. Methods of qPCR are well known to those skilled in the art and are described in textbooks such as Sambrook and Russell, Molecular Cloning: a Laboratory Manual. Volumes 1, 2 and 3 (2001, 3rd Edition).
Here a method is provided for detecting vaccine and/or virulent strains of MDV-1 in a nucleic acid sample through the means of Real-Time PCR (qPCR) and using an agent such as a nucleic acid probe capable of producing a detectable signal upon binding with the nucleic acid produced during the qPCR. Such a detectable signal may conveniently be provided by labelling the probe with a fluorescing dye such as JUP which has a wavelength detectable in the VIC detection channel of the qPCR instrument.
To achieve specificity in a duplex assay (i.e. an assay with two forms of nucleic acid to be detected) specificity is usually required at three levels:
(1) PCR product specificity: In the present invention a universal primer pair is used, complimentary to a conserved region of the pp38 gene, and as a result both CVI988 and virulent strains of MDV-1 will be amplified equally and as such there is no specificity at this level.
(2) Probe specificity: In the present invention there is good probe specificity, since both probes bind minimally to the mis-matched PCR product, instead binding specifically to either the vaccine or virulent strains of MDV-1.
(3) Detector specificity: In the present invention there is no cross talking between the detection channels of the known standard reaction and the pp38 reaction and as such detector specificity is achieved.
The detectable signal produced during the qPCR may be provided by labelling the probe with any agent capable of producing a detectable signal such as a fluorescing agent such as a fluorescent dye, for example MAR, JUP, NEP, SAT, URA, FAM, HEX, TET, VIC, JOE, TAMRA, ethidium bromide, LC Green, SYBR Green I, YO-PRO-1, BEBO and SYTO9; a chromophore, such as a chromophore dye, for example Cy3, Cy5, Pyr(10)PC; or a chromophore dye pair suitable for use in FRET analysis for example the dye pair TOTO 1 and TOTO 3.
In a further embodiment of the invention the quantification of the strain(s) is absolute quantification. In one embodiment of the invention, the qPCRs of step (ii) further comprise a concomitant known standard qPCR (normalisation reaction) utilising a probe labelled with a different detectable signal to allow absolute quantification of the attenuated and/or virulent strains. The different detectable signal allows the signal produced to be detected in a separate detection channel of the qPCR instrument. In one embodiment this standard qPCR system is the Ovo reaction, which uses the Ovo host gene present in chicken DNA and conveniently may use the detectable signal FAM-BHQ1 (available from suppliers such as Eurogentec). Such a normalisation reaction allows accurate comparison of samples by taking account of the amount of DNA used in the reaction. Ovo is a gene present in chicken DNA and two copies of Ovo gene is equivalent to one cell, so the Ovo standard curve can be used to calculate the Ovo Ct value which is equivalent to 10000 cells. For the test samples, a standard curve produced using CVI988 DNA and the CVI988-specific reaction is used to calculate the copy no. (from CVI988-specific Ct value) of CVI988 in the sample, while a standard curve produced using RB-1B DNA and the virulent-specific reaction is used to calculate the copy no. (from virulent-specific Ct value) of virulent MDV in the sample. The Ovo standard curve is used to work out Ovo copy no. (from Ovo Ct value) in the sample. Ovo copy number (i.e. cell number) will be different in different samples so, in order to be able to accurately compare sample A with sample B it is calculated how many MDV copies would be in each sample if those samples had 10000 cells.
Using the signal provided by the aforementioned nucleic acid probe, the amount of DNA amplified by each cycle is quantifiable by comparison with the known standard. A cycle-threshold (Ct) value is produced as to allow the comparison of relative amounts of DNA produced for different samples. This Ct value is effectively a measure of the cycle of the qRT-PCR where the signal produced passes beyond a background threshold value.
This measurement is determined by plotting relative concentrations of DNA present during the exponential phase of the PCR reaction (measured as fluorescence) against cycle number on a logarithmic scale (so an exponentially increasing quantity will give a straight line). A threshold for detection of fluorescence above background is determined and the cycle at which the fluorescence from a sample crosses the threshold is the cycle threshold, Ct.
In other words, the Ct value is defined as the cycle number at the threshold level of log based fluorescence (i.e. the lowest fluorescence observed above background levels). This is the observed and measured value in real-time PCR experiments (qPCR).
The production of a detectable signal in a qPCR with each of the specific probes (one specific for the attenuated vaccine strain and one specific for a virulent strain of MDV-1) allows relative determination of the amount of each of the vaccine strain and the MDV-1 virulent strain in the nucleic sample provided from a specific bird. This is possible as the nucleic acid probes have a sequence complimentary to the sequence specific for either the vaccine or virulent strains of MDV-1 and accordingly will only bind to one of the specific strains. In the embodiment of the invention which includes a known standard, Ct values can be calculated as described and used in absolute quantification of the specific strain of virus produced in the qPCR reaction and as such a comparison of the amount of each strain is possible.
In one embodiment of the invention the vaccine strain of MDV-1 is an attenuated vaccine and in a further embodiment the vaccine strain is the attenuated vaccine CVI988.
Virus vaccines are dead, inactivated or attenuated forms of viruses or purified products derived from them and are given to a subject in order to induce an immune response to such a virus without the subject experiencing the usual negative effects or illness caused by the virus in its virulent form. Following such an immune response, immunity is created in the subject against exposure to the pathogen in its virulent form.
Several types of vaccine may be used and they represent different strategies used to try to reduce risk of illness, while retaining the ability to induce a beneficial immune response. Commonly used virus vaccines fall into the categories of killed vaccines, attenuated vaccines, toxoid vaccines, subunit vaccines and conjugate vaccines.
Attenuated vaccines contain live attenuated viruses—these are live viruses that can replicate. They are naturally occurring in a non-virulent form or have been cultivated under conditions that disable their virulent properties, or which use closely-related but less dangerous organisms to produce a broad immune response. They typically provoke more durable immunological responses than other forms of vaccine.
In one embodiment of the invention, the probes are complimentary to a sequence of pp38 incorporating the region of up to 30 base pairs around the SNP.
In a particular embodiment of the invention the probes may contain within a longer sequence or comprise solely of the following sequences or a fragment thereof:
Vaccine strain-specific probe:
5′ CCCACCGTGACAGCC 3′
SEQ ID NO. 1
Virulent strain-specific probe:
5′ CCCACTGTGACAGCC 3′;
SEQ ID NO. 2
or
a second virulent strain-specific probe:
5′ CTCCCACTGTGACAGCC 3
SEQ ID NO. 3
In a further aspect of the invention, a method is provided wherein the qPCR of step (ii) further comprises the use of a ‘total’ nucleic acid probe in step (ii) capable of detecting both vaccine and virulent strains. Such a total probe is useful as a control as the amount of virus detected by the total probe should be equal to the sum of virus detected by both the vaccine-specific and virulent-specific probes in their specific qPCRs.
In a particular aspect of the invention the total probe specific for both strains may contain within a longer sequence or comprise solely of the following sequence or a fragment thereof:
5′ GCTACCGCCTGAGCC 3′
SEQ ID NO. 4
The primers used within the qPCR will amplify a region incorporating the single nucleotide polymorphism and as such can be used in combination with any of the nucleic acid probes described above. In a preferred embodiment of the invention, the forward and reverse primers used in the amplification of the nucleic acid sample may contain within a longer sequence or comprise solely of the following sequences or a fragment thereof:
Forward primer:
5′ GAGCTAACCGGAGAGGGAGA 3′
SEQ ID NO. 5
Reverse primer:
5′ CGCATACCGACTTTCGTCAA 3′
SEQ ID NO. 6
The polynucleotides of the invention also provide polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides recited above. Polynucleotides according to the invention can have, e.g., at least about 65%, at least about 70%, at least about 75%, at least about 80%, 81%, 82%, 83%, 84%, more typically at least about 85%, 86%, 87%, 88%, 89%, more typically at least about 90%, 91%, 92%, 93%, 94%, and even more typically at least about 95%, 96%, 97%, 98%, 99% sequence identity to a polynucleotide recited above.
The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above. The polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA. Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for example, methods for determining hybridization conditions that can routinely isolate polynucleotides of the desired sequence identities.
Oligonucleotides as used in the present invention can be produced by methods routine to those skilled in the art and are discussed in text books such as Ch10 Molecular Cloning: A Laboratory Manual (Sambrook and Russell 2001).
As discussed, the specificity of the probes used is due to a consistent single nucleotide polymorphism (SNP) in the pp38 gene between attenuated and virulent strains of MDV-1. In an embodiment of the invention the probes used to detect this SNP are homolabelled fluorogenic probes comprising two identical reporter dyes that are capable of quenching each other and becoming dequenched once the probe is cleaved. Preferably such probes are AllGlo miniprobes (AlleLogic Biosciences Corp, Hayward, Calif.).
AllGlo miniprobes are novel homolabelled fluorogenic probes. Unlike conventional dual-labeled Taqman probes, AllGlo miniprobes are comprised of two identical reporter dyes that are capable of quenching each other and become dequenched once the probe is cleaved. Thus, probe cleavage results in the release of two signal-generating dyes per probe, increasing detection sensitivity compared with Taqman probes.
AllGlo miniprobes are significantly shorter than conventional dual-labeled probes (e.g. 15-16 nucleotides). The shortened length makes quenching more efficient and results in low fluorescent baselines. The specificity is greater than that of Taqman probes, such that an AllGlo miniprobe can detect a single point mutation. AllGlo miniprobes are preferably used with the fluorescing dyes MAR, JUP, NEP, SAT or URA available from Allelogic Biosciences.
In one embodiment of the invention, the virulent MDV-1 strain is selected from RB1B MDV, 584A MDV, 595 MDV, 684A MDV, Md5 MDV, HPRS-B14 MDV, JM102 MDV, 660A MDV, 675A MDV, 549 MDV, 571 MDV, or C12-130 MDV.
In one aspect the method of the invention may be used in the diagnosis of infection with virulent MDV-1 in birds whether or not they have been previously vaccinated with, for example, an attenuated vaccine strain. As the method has the ability to simultaneously monitor the behaviour of the vaccine and the virulent strains, it would be the diagnostic method of choice in situations such as identifying the cause of vaccine failures and in a further aspect the method may be used as a research tool in the development and/or testing of MDV-1 vaccines. The method may be particularly useful in common situations in which it is not known if a bird is infected with both vaccine and virulent strains prior to testing for either strain.
In one embodiment, the invention may be provided as a kit of parts comprising:
(i) primers that will amplify a region of the pp38 gene incorporating a consistent single nucleotide polymorphism (SNP) between vaccine and virulent strains of MDV-1; and
(ii) a labelled nucleic acid probe for the pp38 polymorphism specific to the vaccine strain of MDV-1.
In a further embodiment, the invention may be provided as a kit of parts comprising:
(i) primers that will amplify a region of the pp38 gene incorporating a consistent single nucleotide polymorphism (SNP) between vaccine and virulent strains of MDV-1; and
(ii) a labelled nucleic acid probe for the pp38 polymorphism specific to the virulent strains of MDV-1.
In a further embodiment, the invention may be provided as a kit of parts comprising:
(i) primers that will amplify a region of the pp38 gene incorporating a consistent single nucleotide polymorphism (SNP) between vaccine and virulent strains of MDV-1;
(ii) a labelled nucleic acid probe for the pp38 polymorphism specific to vaccine strain; and
(iii) a labelled nucleic acid probe for the pp38 polymorphism specific to virulent strains of MDV-1.
In further embodiments the kit of parts may optionally include standard qPCR reagents and/or a known standard qPCR utilising a probe labelled with a different detectable signal. In one embodiment the vaccine strain quantified by the kit of parts is the CVI988 vaccine.
In a further embodiment the nucleic acid probe(s) of the kit of parts may contain within a longer sequence or comprise solely of the following sequences or a fragment thereof:
Vaccine strain-specific probe:
5′ CCCACCGTGACAGCC 3′;
SEQ ID NO. 1
and/or
Virulent strain-specific probe:
5′ CCCACTGTGACAGCC 3′
SEQ ID NO. 2
The kit of parts may further comprises a total probe that binds both vaccine strains and virulent strains which in one embodiment has the following sequence:
5′ GCTACCGCCTGAGCC 3′
SEQ ID NO. 4
PREFERRED EMBODIMENTS
Examples embodying certain aspects of the invention will now be described with reference to the following figures in which:
FIG. 1 shows the binding location of primers bold underline and probes
in the pp38 gene for (a) CVI988 (vaccine strain) SEQ ID NO. 10 and (b) Md5 SEQ ID NO. 11 (representative virulent strain). The probes binding the consistent polymorphism G/A are the specific probes, the other probe is the total probe. The G/A polymorphism is not consistent in all virulent strains
FIG. 2 shows the specificity of each of the two miniprobes for (a) CVI988 (vaccine strain) and (b) RB1B (representative virulent strain) by plotting change in fluorescence against cycle number of the qPCR.
FIG. 3 shows DNA standard curves of Ct value versus copy number of infected Chick Embryo Fibroblast (CEF) cells produced for the following qPCR reactions: (a) CVI988 (vaccine strain) probe with nucleic acid from CVI988 (vaccine strain) infected CEF; (b) RB1B (representative virulent strain) probe with nucleic acid from RB1B (representative virulent strain) infected CEF; (c) dual-specificity probe with nucleic acid from CVI988 (vaccine strain) infected CEF; and (d) dual-specificity probe with nucleic acid from RB1B (representative virulent strain) infected CEF. Copy number values were produced by calibrating the fluorescence results produced with those produced from plasmid DNA of known copy number.
FIG. 4 shows the specificity of the nucleic acid probes for MDV-serotype-3 vaccine HVT, MDV-serotype-2 vaccine SB-1, 3 different sources of MDV-1 vaccine CVI988 and for MDV-1 virulent strains of varying virulence across a range of 12 different virulent stains of MDV-1. Serotypes (1, 2 or 3) and pathotypes (v, vv or vv+) are shown for each of the MDV-1 strains. (v=virulent, vv=very virulent, vv+=very virulent plus).
FIG. 5 shows the standard curve produced for the Ovo qPCR reaction using an Ovo probe labelled with FAM-BHQ1 and calibrated serial dilutions of non-infected Chick Embryo Fibroblast (CEF) DNA.
FIG. 6 shows the gene map for MDV-1 and the location of the key pp38 and US2 genes within the CVI988 vaccine and RB1B challenge virus.
FIG. 7 shows the comparison of standard curves of Ct-values versus copy number of infected cells produced in the following qPCR reactions: (a) CVI988 (vaccine strain) probe with nucleic acid from CVI988 (vaccine strain) infected CEF; (b) RB1B (representative virulent strain) probe with nucleic acid from RB1B (representative virulent strain) infected CEF compared to those produced from the equivalent reactions in the BAC/US2 qPCR method.
FIG. 8 shows the copy number produced for 40 birds using qPCR systems specific for BAC cloned vaccine virus (column 1), virulent/challenge virus (column 2) and total virus (column 3) and comparing the present qPCR invention versus BAC/US2 based qPCR for each. Birds are indicated as to whether they fall into the following four groups: 1) non-vaccinated, non-challenged; 2) vaccinated, non-challenged; 3) vaccinated, challenged; and 4) non-vaccinated, challenged.
FIG. 9 shows correlation of levels of virus measured between the qPCR of the present invention and the BAC/US2 qPCR system for (a) vaccine virus and (b) virulent virus.
FIG. 10 shows (a) correlation of levels of virus measured between total (dual-specific) qPCR and the additive result of vaccine-specific and virulent-specific for the qPCR of the present invention; (b) correlation of levels of virus measured between the additive result of vaccine-specific and virulent-specific for the qPCR of the present invention and the additive result of the BAC/US2 qPCR system; and (c) correlation between levels of virus measured between total (dual-specific) qPCR of the present invention and the additive result of the BAC/US2 qPCR system.
EXAMPLE 1
Design of qPCR Primers and Probes
The MDV pp38 gene sequence shows two single nucleotide differences between the attenuated vaccine CVI988 and Md5 (a virulent strain) (FIG. 1). The polymorphism highlighted in large bold underline (G/A) represents a consistent difference between the MDV-1 vaccine virus and the MDV-1 virulent viruses for all which we have sequence (RB1B MDV, 584A MDV, 595 MDV, 684A MDV, Md5 MDV, HPRS-B14 MDV, JM102 MDV, 660A MDV, 675A MDV, 549 MDV, 571 MDV, C12-130 MDV) and this was the polymorphism which was incorporated into the specific probes. The polymorphism highlighted in bold underline italics (G/A) is not consistently present in all strains of virulent virus. Table 1 lists the primer sequences together with the two probes (pp38Vacc_G_JUP specific for the attenuated vaccine CVI988; and pp38Vir_A_JUP(1) specific for virulent viruses) that were designed to cover the polymorphic region. A third probe pp38-total_JUP targeting a region common to pp38 of all MDV-1 strains was also designed. All three probes can be used in combination with the same primer pair (see Table 1).
Table 1 also shows a second probe (pp38Vir_A_JUP(3)) targeting specifically virulent viruses was designed so that the vaccine-specific, virulent-specific and total pp38 probes all have a similar melting temperature. The performance of pp38Vir_A_JUP(3) is very similar to that of pp38Vir_A_JUP(1) (the other virulent strain-specific probe); however, because it covers the second ‘non-consistent’ polymorphic site, it is possible that affinity for some field strains may be reduced (see FIG. 1 and Table 1). The probes are labelled with the dye JUP (AlleLogic Biosciences), which is detected in the VIC detection channel of the real-time PCR instrument.
TABLE 1
CVI988 MDV1-
Virulent MDV1-
Total
specific pp38 PCR
specific pp38 q-PCR
pp38 q-PCR
F. Primer
GAGCTAACCGGAGAG
GAGCTAACCGGAGAG
GAGCTAACCGGAG
GGAGA SEQ ID NO. 5
GGAGA SEQ ID NO. 5
AGGGAGA SEQ ID
NO. 5
R. Primer
CGCATACCGACTTTCG
CGCATACCGACTTTCG
CGCATACCGACTT
TCAA SEQ ID NO. 6
TCAA SEQ ID NO. 6
TCGTCAA SEQ ID
NO. 6
Probes
CCCACCGTGACAGCC
CCCACTGTGACAGCC
GCTACCGCCTGAG
available
pp38Vacc_G_JUP
pp38Vir_A_JUP(1)
CC SEQ ID NO. 4
Tm = 57.7 SEQ ID
Tm = 54.1 SEQ ID
pp38-total_JUP
NO. 1
NO. 2
Tm = 57.4
C CCCACTGTGACAGCC
SEQ ID NO. 3
pp38Vir_A_JUP(3) (*)
Tm = 57.1
Probe
−
−
+
strand
Amp. size
99 bp
99 bp
99 bp
Primers and probes were designed and manufactured by AlleLogic Biosciences, Hayward, CA, USA.
EXAMPLE 2
Infection of Chick Embryo Fibroblast (CEF) Cells
Chick Embryo Fibroblast (CEF) cells were infected with either CVI988 vaccine strain or RB1B virulent strain of MDV-1. Five days post infection, serial dilutions were prepared from these cells for use in singleplex qPCRs.
CEF (chick embryo fibroblast) cell monolayers were established in culture 75 cm2 flasks and infected with approximately 1000 plaque forming units (pfu) of cell-associated virus. After 5 days in culture at 38° C., cells were harvested and DNA was prepared using phenol-chloroform extraction method. Reactions are 25 ul volume and contain: Absolute Blue qPCR mastermix (ABgene), 400 nM of each primer (i.e. Ovo primers+virus specific primers), 200 nM of each probe (i.e. Ovo probe+virus-specific probe), approximately 100 ng DNA sample. Reactions are performed on an ABI7500 real-time PCR machine using the cycling parameters: 50° C. for 2 min, 95° C. for 15 min, followed by 40 cycles of 95° C. (15 sec) and 60° C. (1 min).
EXAMPLE 3
Specificity of the qPCRs
Singleplex Reactions
Reactions were set up essentially as previously described (Baigent et al., 2005a), each containing ‘ABsolute q-PCR master mix’ (ABgene, Epsom, UK), forward and reverse primer, and one of the probes. An ABI 7500FAST® q-PCR system (Applied Biosystems, Foster City, Calif., USA) was used to amplify and detect the reaction products, under the following conditions: 50° C. for 2 min, 95° C. for 15 min, followed by 40 cycles of 95° C. (15 sec) and 60° C. (1 min). Primer and probe combinations for each singleplex qPCR are shown in Table 2.
TABLE 2
CVI988 MDV1-
Virulent MDV1-
specific pp38 PCR
specific pp38 q-PCR
F.
GAGCTAACCGGAGAGGGAGA
GAGCTAACCGGAGAGGGAGA
Primer
SEQ ID NO. 5
SEQ ID NO. 5
R.
CGCATACCGACTTTCGTCAA
CGCATACCGACTTTCGTCAA
Primer
SEQ ID NO. 6
2SEQ ID NO. 5
Probe
CCCACCGTGACAGCC
CCCACTGTGACAGCC
pp38Vacc_G_JUP
pp38Vir_A_JUP(1)
Tm = 57.7
Tm = 54.1
SEQ ID NO. 1
SEQ ID NO. 2
Specificity of the probes was initially assessed by using serial 10-fold dilutions of DNA prepared from chick embryo fibroblast (CEF) cells, five days post infection with either CVI988 or RB1B (a virulent strain of MDV-1).
FIG. 2 demonstrates the change in fluorescence produced by (a) CVI988-specific probe and (b) virulent-specific probe using nucleic acid template from either CVI988 or RBIB (a virulent strain) or no template control. The data clearly shows the signal to only be produced at significant levels when the probe specific for the nucleic acid is used in the qPCR and as such specificity of the probes is clearly demonstrated.
EXAMPLE 4
Standard Curves for qPCR
DNA standards were prepared from CVI988-infected CEF cells and from RB-1B-infected CEF cells and accurately quantified by calibration against plasmid DNA of known copy number. Ten-fold serial dilutions were prepared. FIG. 3(a) shows the standard curve produced using CVI988-specific qPCR on CVI988 DNA. FIG. 3(b) shows the standard curve produced using virulent-specific qPCR on RB-1B DNA. FIG. 3(c) shows the standard curve produced using total pp38 qPCR on CVI988 DNA. FIG. 3(d) shows the standard curve produced using total pp38 qPCR on RB-1B DNA.
Each reaction was of good efficiency, and produced a good standard curve. The pp38 total probe detected CVI988 (vaccine) DNA and RB-1B (virulent) DNA and with similar efficiency as predicted. No-template-control wells were negative for all probes.
EXAMPLE 5
Specificity of the pp38 Nucleic Acid Probes for a Range of MDV-1 Strains
The specificity of pp38Vir_A_JUP (virulent-specific) probe and pp38Vacc_G_JUP (CVI988 vaccine-specific) probes was tested on a range of 12 MDV-1 strains of varying virulence, a range of 3 different sources of MDV-1 vaccine strain CVI988 and 2 non-serotype-1 vaccine strains (FIG. 4).
Thresholds (delta Rn) were set at: Ovo reaction (0.2), virulent-specific pp38 reaction (0.6), CVI988-specific pp38 reaction (0.35), total pp38 reaction (0.1). The virulent-specific probe detected all virulent strains of MDV1 (but did not detect CVI988), and the vaccine-specific probe detected only CVI988, but from all 3 sources. Neither probe detected the MDV-2 and MDV-3 vaccine strains SB-1 and HVT. The data confirm the consistent specificity of pp38Vacc_G_JUP for the MDV-1 vaccine CVI988 strain of varying sources and the specificity of pp38Vir_A_JUP for virulent MDV-1 strains of varying virulence. The data also shows specificity of both probes for MDV-1 strains over MDV-2 or MDV-3 strains.
EXAMPLE 6
Adaptation of the Assays for Absolute Quantification
In order to use the MiniProbe q-PCR reactions for absolute quantification of vaccine and challenge virus in tissue samples (expressed as virus genomes per 104 cells), it was necessary to optimise duplexing of the pp38 reactions with Ovo reaction. The Ovo probe was labelled with FAM-BHQ1 so as to be detected in a different channel to the pp38 probe fluorescence. The target amplicon of the Ovo qPCR is 71 base pairs and primers and probe sequences used are shown in table 3 below. Primers and probes were produced by Sigma Aldrich and Eurogentec.
TABLE 3
Chicken
Ovo
CACTGCCACTGGGCTCTGT
71
ovo-
forward
SEQ ID NO. 7
bp
transferrin
primer
gene
(Jeltsch et
Ovo
GCAATGGCAATAAACCTCCAA
al., 1987)
reverse
SEQ ID NO. 8
primer
Ovo
AGTCTGGAGAAGTCTGTGCAGCCTCCA
probe
SEQ ID NO. 9
(5′ FAM label, 3′ BHQ label)
Two copies of Ovo gene is equivalent to one cell, so the Ovo standard curve can be used to calculate the Ovo Ct value which is equivalent to 10000 cells. CVI988 DNA and the CVI988-specific reaction is used to calculate the copy no. (from CVI988-specific Ct value) of CVI988 in the sample, while a standard curve produced using RB-1B DNA and the virulent-specific reaction is used to calculate the copy no. (from virulent-specific Ct value) of virulent MDV in the sample. The Ovo standard curve is used to work out Ovo copy no. (from Ovo Ct value) in the sample. Ovo copy number (i.e. cell number) will be different in different samples so, in order to be able to accurately compare sample A with sample B it is calculated how many MDV copies would be in each sample if those samples had 10000 cells.
The standard curve for the Ovo reaction was prepared using calibrated serial dilutions of non-infected CEF DNA and is shown in FIG. 5.
EXAMPLE 7
Testing and Validation of Duplex qPCR on Chicken DNA Samples
Since the primer pair will amplify both CVI988 and virulent MDV-1 DNA, these two target sequences will compete for the same primers when both target sequences are present. Samples from laboratory experiments, and from the field, will contain CVI988 and virulent MDV-1 in various ratios. To confirm that competition for primers does not prevent the amplification (and thus detection) of the less abundant virus where one virus is present at high levels, and the other at only low levels, the ability of the reactions to accurately quantify both CVI988 and virulent MDV-1 in mixed samples was examined.
Chicken tissue DNA samples were taken from an experiment in which chickens had been vaccinated with a BAC-cloned CVI988 virus (pCVI988) and challenged with RB-1B. Four groups of chicks were (1) non-vaccinated, non-challenged; (2) vaccinated, non-challenged; (3) vaccinated and challenged; (4) non-vaccinated, challenged. Spleen samples were collected at various time points post vaccination and challenge. The vaccine virus was pCVI988 and the challenge (virulent) virus WT RB-1B which should allow distinction between the two viruses by both the CVI988 pp38/virulent pp38 system and by a second qPCR system which we have developed to distinguish between BAC cloned MDV (using a BAC-specific sequence) and wild-type MDV (by targeting the US2 gene). This latter qPCR system could thus be used to validate the CVI988 pp38/virulent pp38 qPCR system). Total virus was also measured using the pp38-total reaction. A plan for the validation of the qPCR system is shown in Table 4 and indicated on the viral gene maps of FIG. 6.
TABLE 4
Miniprobe pp38 qPCR
Standard qPCR
system target
system target
Vaccine MDV
CVI988-specific pp38
BAC sequence
1000pfu, 1 d, i.m.
sequence
pCVI988
Challenge MDV
Virulent ‘MDV1-specific’
US2 sequence
1000pfu, 15 d, i.p.
pp38
RB-1B (vvMDV-1)
Total MDV
Total pp38
—
Each of the five virus-specific qPCR reactions shown in Table 4 was performed in duplex with the Ovo qPCR to allow absolute quantification of virus per 10000 cells. FIGS. 7(a) and 7(b) show the standard curves used for quantifying vaccine and virulent virus using both qPCR systems.
FIG. 8 shows level of vaccine, challenge and total MDV measured in 40 birds, from four groups, 1) non-vaccinated, non-challenged; 2) vaccinated, non-challenged; 3) vaccinated, challenged; and 4) non-vaccinated, challenged. Samples having a baseline value (0.6) are negative.
The first two columns show measurement of vaccine virus by CVI-specific pp38 qPCR (col 1) and BAC-specific qPCR (col 2). In both systems, vaccine virus was detected only in the two vaccinated groups. Agreement between the copy number values obtained with the two systems was good (see also FIG. 9(a)).
Columns 3 and 4 show measurement of challenge virus by virulent-specific pp38 qPCR (col 3) and US2-specific qPCR (col 4). In both systems, challenge virus was detected only in the two challenged groups. Agreement between the copy number values obtained with the two systems was good (see also FIG. 9(b)).
Columns 5, 6, and 7 show measurement of total MDV by pp38 qPCR to measure both viruses (col 5), additive results from CVI-specific pp38 qPCR and virulent-specific pp38 qPCR (col 6), and additive results from BAC-specific qPCR and US2-specific qPCR (col 7). Agreement between the copy number values obtained with these three systems was very good (FIGS. 10(a), (b) & (c)).
These data confirm that the CVI-pp38 qPCR and the virulent MDV1-pp38 qPCR show excellent specificity, and can be used to accurately quantify CVI988 vaccine and virulent MDV-1 strains in tissue samples including those containing both viruses in biologically relevant amounts.
REFERENCES
1. Baigent, S. J., Petherbridge, L. J., Howes, K., Smith, L. P., Currie, R. J., Nair, V. K., 2005a. Absolute quantitation of Marek's disease virus genome copy number in chicken feather and lymphocyte samples using real-time PCR. J. Virol. Methods 123, 53-64.
2. Baigent, S. J., Smith, L. P., Currie, R. J., Nair, V. K., 2005b. Replication kinetics of Marek's disease vaccine virus in feathers and lymphoid tissues using PCR and virus isolation. J Gen Virol. 86, 2989-2998.
3. Baigent, S. J., Smith, L. P., Nair, V. K., Currie, R. J., 2006. Vaccinal Control of Marek's Disease: Current challenges, and future strategies to maximize protection. Vet. Immunol. Immunopathol. 112, 78-86.
4. Becker Y, Asher Y, Tabor E, Davidson I, Malkinson M, Weisman Y: Polymerase chain reaction for differentiation between pathogenic and non-pathogenic serotype 1 Marek's disease viruses (MDV) and vaccine viruses of MDV-serotypes 2 and 3. J Virol Methods 1992; 40:307-322.
5. de Boer, G. F., Groenendal, J. E., Boerrigter, H. M., Kok, G. L., Pot, J. M., 1986. Protective efficacy of Marek's disease virus (MDV) CVI-988 CEF65 clone C against challenge infection with three very virulent MDV strains. Avian Dis. 30, 276-283.
6. Islam, A., Cheetham, B. F., Mahony, T. J., Young, P. L., Walkden-Brown, S. W., 2006a. Absolute quantitation of Marek's disease virus and Herpesvirus of turkeys in chicken lymphocyte, feather tip and dust samples using real-time PCR. J. Virol. Methods 132, 127-134.
7. Gimeno I M, Witter R L, Hunt H D, Reddy S M, Lee L F, Silva R F. The pp38 gene of Marek's disease virus (MDV) is necessary for cytolytic infection of B cells and maintenance of the transformed state but not for cytolytic infection of the feather follicle epithelium and horizontal spread of MDV. J. Virol. 2005 April; 79(7):4545-9
8. Islam, A. F., Walkden-Brown, S. W., Islam, A., Underwood, G. J., Groves, P. J., 2006b. Relationship between Marek's disease virus load in peripheral blood lymphocytes at various stages of infection and clinical Marek's disease in broiler chickens. Avian Pathol. 35, 42-48.
9. King, D., Page, D., Schat, K. A., Calnek, B. W., 1981. Difference between influences of homologous and heterologous maternal antibodies on response to serotype-2 and serotype-3 Marek's disease vaccines. Avian Dis. 25, 74-81.
10. Landman, W. J., Verschuren, S. B., 2003. Titration of Marek's disease cell-associated vaccine virus (CVI 988) of reconstituted vaccine and vaccine ampoules from Dutch hatcheries. Avian Dis. 47, 1458-1465.
11. Renz, K. G., Islam, A., Cheetham, B. F., Walkden-Brown, S. W., 2006. Absolute quantification using real-time polymerase chain reaction of Marek's disease virus serotype 2 in field dust samples, feather tips and spleens. J. Virol. Methods. 135, 186-191.
12. Rispens, B. H., Van Vloten, J., Mastenbroek, N., Maas, H. J. L., Schat, K. A., 1972. Control of Marek's disease in the Netherlands. I. Isolation of an avirulent Marek's disease virus (strain CVI 988) and its use in laboratory vaccination trials. Avian Dis. 16, 108-125.
13. Silva R F: Differentiation of pathogenic and non-pathogenic serotype 1 Marek's disease viruses (MDVs) by the polymerase chain reaction amplification of the tandem direct repeats within the MDV genome. Avian Dis 1992; 36:521-528.
14. Sambrook and Russell: Molecular Cloning: A Laboratory Manual. Volume 1-3 (2001 3rd Ed)
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13391563
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zoetis w llc
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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435/ 6.12
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Mar 31st, 2022 03:01PM
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Mar 31st, 2022 03:01PM
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Zoetis
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Health Care
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Pharmaceuticals & Biotechnology
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