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nasdaq:biib
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Biogen
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Sep 13th, 2011 12:00AM
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Jul 16th, 2004 12:00AM
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https://www.uspto.gov?id=US08017733-20110913
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Polyalkylene polymer compounds and uses thereof
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The invention relates to novel polyalkylene glycol compounds and methods of using them. In particular, compounds comprising a novel polyethylene glycol conjugate are used alone, or in combination with antiviral agents to treat a viral infection, such as chronic hepatitis C.
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8017733
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1. A composition having the structure according to the formula:
wherein E is hydrogen, a straight- or branched-chain C1 to C20 alkyl group, or a detectable label;
a is an integer from 4 to 10,000;
each Z and Z′ is independently hydrogen; a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group; C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl; a substituted or unsubstituted aryl or heteroaryl group; or a substituted or unsubstituted alkaryl group wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein in the substituted groups the substitution is selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, an aromatic moiety, a heteroaromatic moiety, imino, silyl, ether, and alkylthio, provided that at least one Z or Z′ is not hydrogen;
R* is a linking moiety formed from the reaction of a moiety selected from the group consisting of aldehyde, aldehyde hydrate, and acetal with B, wherein B is a biologically-active molecule or precursor thereof that comprises interferon-beta-1a (IFN-β-1a);
each n is 0 or an integer from 1 to 5; and
p is 1, 2, or 3.
2. The composition of claim 1, wherein R* is methylene and wherein B is attached to R* by a bond between the methylene and an amine of the biologically-active molecule.
3. The composition of claim 2, wherein the amine is an amino terminus of the biologically-active molecule.
4. The composition according to claim 1 having the structure according to the formula:
wherein Z is a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group; C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl; a substituted or unsubstituted aryl or heteroaryl group; or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein in the substituted groups the substitution is selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, an aromatic moiety, a heteroaromatic moiety, imino, silyl, ether, and alkylthio.
5. The composition of claim 4, wherein Z is methyl and n is one.
6. A pharmaceutical composition comprising the composition according to claim 1 and a pharmaceutically-acceptable carrier.
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6
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FIELD OF THE INVENTION
The invention relates to novel polyalkylene glycol compounds, conjugates of the polymers and proteins, and uses thereof.
BACKGROUND OF THE INVENTION
Covalent attachment of hydrophilic polymers, such as polyalkylene glycol polymers, also known as polyalkylene oxides, to biologically-active molecules and surfaces is of interest in biotechnology and medicine.
In particular, much research has focused on the use of poly(ethylene glycol) (PEG), also known as or poly(ethylene oxide) (PEO), conjugates to enhance solubility and stability and to prolong the blood circulation half-life of molecules.
In its most common form, PEG is a linear polymer terminated at each end with hydroxyl groups:
HO—CH2CH2O—(CH2CH2O)n—CH2CH2—OH.
The above polymer, alpha-, omega-dihydroxylpoly(ethylene glycol), can also be represented as HO-PEG-OH, where it is understood that the -PEG-symbol represents the following structural unit:
—CH2CH2—(CH2CH2O)n—CH2CH2—
where n typically ranges from about 4 to about 10,000. PEG is commonly used as methoxy-PEG-OH, or mPEG, in which one terminus is the relatively inert methoxy group, while the other terminus is a hydroxyl group that is subject to ready chemical modification. Additionally, random or block copolymers of different alkylene oxides (e.g., ethylene oxide and propylene oxide) that are closely related to PEG in their chemistry can be substituted for PEG in many of its applications.
To couple PEG to a molecule of interest, it is often necessary to activate the PEG by preparing a derivative of the PEG having a reactive functional group at least at one terminus. The functional group is chosen based on the type of available reactive group on the molecule that will be coupled to the PEG.
PEG is a polymer having the properties of solubility in water and in many organic solvents, lack of toxicity, and lack of immunogenicity. One use of PEG is to covalently attach the polymer to insoluble molecules to make the resulting PEG-molecule “conjugate” soluble. For example, it has been shown that the water-insoluble drug paclitaxel, when coupled to PEG, becomes water-soluble. Greenwald, et al., J. Org. Chem., 60:331-336 (1995).
The prodrug approach, in which drugs are released by degradation of more complex molecules (prodrugs) under physiological conditions, is a powerful component of drug delivery. Prodrugs can, for example, be formed by bonding PEG to drugs via linkages which are degradable under physiological conditions. The lifetime of PEG prodrugs in vivo depends upon the type of functional group(s) forming linkages between PEG and the drug. In general, ester linkages, formed by reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on the drug hydrolyze under physiological conditions to release the drug, while amide and carbamate linkages, formed from amine groups on the drug, are stable and do not hydrolyze to release the free drug. It has been shown that hydrolytic delivery of drugs from PEG esters can be favorably controlled to a certain extent by controlling the number of linking methylene groups in a spacer between the terminal PEG oxygen and the carbonyl group of the attached carboxylic acid or carboxylic acid derivative. For example, Harris et al., in U.S. Pat. No. 5,672,662, describe PEG butanoic acid and PEG propanoic acid, and activated derivatives thereof, as alternatives to carboxymethyl PEG for compounds where less hydrolytic reactivity in the corresponding ester derivatives is desirable. See, generally, PCT publication WO 01/46291.
One factor limiting the usefulness of proteinaceous substances for medical treatment applications is that, when given parenterally, they are eliminated from the body within a short time. This elimination can occur as a result of degradation by proteases or by clearance using normal pathways for protein elimination such as by filtration in the kidneys. Oral administration of these substances is even more problematic because, in addition to proteolysis in the stomach, the high acidity of the stomach destroys these substances before they reach their intended target tissue. The problems associated with these routes of administration of proteins are well known in the pharmaceutical industry, and various strategies are being employed in attempts to solve them. A great deal of work dealing with protein stabilization has been published. Various ways of conjugating proteins with polymeric materials are known, including use of dextrans, polyvinyl pyrrolidones, glycopeptides, polyethylene glycol, and polyamino acids. The resulting conjugated polypeptides are reported to retain their biological activities and solubility in water for parenteral applications.
Of particular interest is increasing the biological activity of interferons while reducing the toxicity involved with use of these proteins for treating human patients. Interferons are a family of naturally-occurring small proteins and glycoproteins produced and secreted by most nucleated cells in response to viral infection as well as to other antigenic stimuli. Interferons render cells resistant to viral infection and exhibit a wide variety of actions on cells. They exert their cellular activities by binding to specific membrane receptors on the cell surface. Once bound to the cell membrane, interferons initiate a complex sequence of intracellular events. In vitro studies have demonstrated that these include the induction of certain enzymes; suppression of cell proliferation, immunomodulation activities such as enhancement of the phagocytic activity of macrophages; augmentation of the specific cytotoxicity of lymphocytes for target cells; and inhibition of virus replication in virus-infected cells.
Interferons have been tested in the treatment of a variety of clinical disease states. The use of human interferon beta has been established in the treatment of multiple sclerosis. Two forms of recombinant interferon beta, have recently been licensed in Europe and the U.S. for treatment of this disease: interferon-beta-1a (AVONEX®, Biogen, Inc., Cambridge, Mass. and REBIF® Serono, Geneva, Switzerland) and interferon-beta-1b (BETASERON®, Berlex, Richmond, Calif.). Interferon beta-1a is produced in mammalian cells using the natural human gene sequence and is glycosylated, whereas interferon beta-1b is produced in E. coli bacteria using a modified human gene sequence that contains a genetically engineered cysteine-to-serine substitution at amino acid position 17 and is non-glycosylated.
Non-immune interferons, which include both alpha and beta interferons, are known to suppress human immunodeficiency virus (HIV) in both acutely and chronically-infected cells. See Poli and Fauci, 1992, AIDS Research and Human Retroviruses 8(2):191-197. Due to their antiviral activity, interferons, in particular alpha interferons, have received considerable attention as therapeutic agents in the treatment of hepatitis C virus (HCV)-related disease. See Hoofnagle et al., in: Viral Hepatitis 1981 International Symposium, 1982, Philadelphia, Franklin Institute Press; Hoofnagle et al., 1986, New Eng. J. Med. 315:1575-1578; Thomson, 1987, Lancet 1:539-541 Kiyosawa et al., 1983, in: Zuckerman, ed., Viral Hepatitis and Liver Disease, Allen K. Liss, New York pp. 895-897; Hoofnagle et al., 1985, Sem. Liv. Dis., 1985, 9:259-263.
Interferon-polymer conjugates are described in, for example, U.S. Pat. No. 4,766,106, U.S. Pat. No. 4,917,888, European Patent Application No. 0 236 987, European Patent Application No. 0 510 356 and International Application Publication No. WO 95/13090.
Chronic hepatitis C is an insidious and slowly progressive disease having a significant impact on the quality of life. Despite improvement in the quality of the blood-donor pool and the recent implementation of testing of donated blood for HCV, the estimated incidence of acute infection among persons receiving transfusions is 5 to 10%. See Alter et al., in: Zuckerman, ed., Viral Hepatitis and Liver Disease, Allen K. Liss, New York. 1988, pp. 537-542. Thus, of the approximately 3 million persons who receive transfusions in the United States each year, acute hepatitis C will develop in about 150,000. While many patients who contract hepatitis C will have subclinical or mild disease, approximately 50% will progress to a chronic disease state characterized by fluctuating serum transaminase abnormalities and inflammatory lesions on liver biopsy. It is estimated that cirrhosis will develop in up to about 20% of this group. See Koretz et al., 1985, Gastroenterology 88:1251-1254.
Interferons are known to affect a variety of cellular functions, including DNA replication, and RNA and protein synthesis, in both normal and abnormal cells. Thus, cytotoxic effects of interferon are not restricted to tumor or virus-infected cells but are also manifested in normal, healthy cells. As a result, undesirable side effects may arise during interferon therapy, particularly when high doses are required. Administration of interferon can lead to myelosuppression, thereby resulting in reduced red blood cell count, and reduced white blood cell and platelet levels. Interferons commonly give rise to flu-like symptoms (e.g., fever, fatigue, headaches and chills), gastrointestinal disorders (e.g., anorexia, nausea and diarrhea), dizziness and coughing. Often, the sustained response of HCV patients to non-PEGylated interferon treatment is low and the treatment can induce severe side effects, including, but not limited to, retinopathy, thyroiditis, acute pancreatitis, and depression.
The undesirable side effects that accompany interferon therapy frequently limit the therapeutic usefulness of interferon treatment regimes. Thus, a need exists to maintain or improve the therapeutic benefits of such therapy while reducing or eliminating the undesirable side effects.
SUMMARY OF THE INVENTION
The invention relates to novel polyalkylene glycol compounds, conjugates of these compounds, and uses thereof.
In one aspect, the invention relates to an activated polyalkylene glycol polymer having the structure according to Formula I:
wherein P is a polyalkylene glycol polymer;
X and Y are independently O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′;
Q is a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl (including fused bicyclic and bridged bicyclic ring structures), a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, sulfamoyl, sulfonate, silyl, ether, and alkylthio;
each R′, Z and Z′ is independently hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, sulfamoyl, sulfonate, silyl, ether, and alkylthio;
R is a moiety suitable for forming a bond between the compound of Formula I and a biologically-active compound or precursor thereof;
m is or 1;
each n is independently 0 or an integer from 1 to 5; and
p is 1, 2, or 3.
In another aspect, the invention relates to an activated polyalkylene glycol compound (PGC) having the structure according to Formula Ia:
where P is a polyalkylene glycol polymer, m is zero or one, n is zero or an integer from one to five, and X and Y are independently O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′.
Q is a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group. If present, the substituents can be halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, sulfamoyl, sulfonate, silyl, ether, or alkylthio. Heterocyclic and carbocyclic groups include fused bicyclic and bridged bicyclic ring structures.
Each R′ and Z is independently hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group. The substituents can be halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, or alkylthio.
Compounds which include chiral carbons can be in the R configuration, the S configuration, or may be racemic.
R is a moiety suitable for forming a bond between the compound of Formula I and a biologically-active compound or precursor thereof.
In one embodiment, R is a carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, or a glyoxal moiety.
In certain embodiments, P is a polyethylene glycol having the structure of Formula II:
E-(O—CH2CH2)a—, Formula II:
where E is hydrogen or a straight- or branched-chain C1 to C20 alkyl group and a is an integer from 4 to 10,000. For example, E can be a methyl group.
In other embodiments, E can be a detectable label, such as, for example, a radioactive isotope, a fluorescent moiety, a phosphorescent moiety, a chemiluminescent moiety, or a quantum dot.
In yet other embodiments, E is a moiety suitable for forming a bond between the compound of Formula I and a biologically-active compound or precursor thereof. For example, E can be a carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, or a glyoxal moiety.
In still other embodiments, E has the structure according to Formula III or Formula IV:
where each Q, X, Y, Z, m, and n are, independently, as defined above; and each W is, independently, hydrogen or a C1 to C7 alkyl.
R″ is a moiety suitable for forming a bond between the compound of Formula III and a biologically-active compound or precursor thereof, and R′″ is a moiety suitable for forming a bond between the compound of Formula IV and a biologically-active compound or precursor thereof. For example, R″ and R′″ can be a carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, or a glyoxal moiety. R″ and R′″ can be the same or different from R.
In particular embodiments, Q is a substituted or unsubstituted alkaryl.
In another aspect, the invention relates to an activated PGC having the structure according to Formula V:
where P, X, Y, R′, Z, R, m, and n are as defined, and T1 and T2 are, independently, absent, or a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group. The substituents can be halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, or alkylthio.
L may be absent (e.g., d is zero) or there may be from one to four (e.g., n is an integer from one to four) L substituents on the aromatic ring in addition to the T1 and T2 substituents, and each L is, independently, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group. The substituents are selected from halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio.
R is a moiety suitable for forming a bond between the compound of Formula V and a biologically-active compound or precursor thereof. For example, R is chosen from carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal.
In one embodiment of the activated polyalkylene glycol polymer of Formula V, P is a polyethylene glycol having the structure of Formula II:
E-(O—CH2CH2)a—, Formula II:
where E is hydrogen or a straight- or branched-chain C1 to C20 alkyl group and a is an integer from 4 to 10,000. For example, E can be methyl. In other embodiments, E is a detectable label, such as, for example, a radioactive isotope, fluorescent moiety, phosphorescent moiety, chemiluminescent moiety, or a quantum dot.
In another aspect, P is a polyethylene glycol having the structure of Formula II:
E-(O—CH2CH2)a—, Formula II:
where E is a moiety suitable for forming a bond between the compound of Formula V and a biologically-active compound or precursor thereof and a is an integer from 4 to 10,000. For example, E is chosen from carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal moieties.
In another aspect, E has the structure according to Formula III or Formula IV:
where Q is a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl (including fused bicyclic and bridged bicyclic ring structures), a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl; the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, and the substituents can be of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, or alkylthio.
X, Y, Z, m, and n are as defined, and each W is, independently, hydrogen or a C1 to C7 alkyl; and R″ is a moiety suitable for forming a bond between the compound of Formula III and a biologically-active compound or precursor thereof, and R′″ is a moiety suitable for forming a bond between the compound of Formula IV and a biologically-active compound or precursor thereof.
In certain embodiments, R″ and R′″ can be the same as or different from R, and are chosen from carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal moieties. In one embodiment of the compound of Formula V, X and Y, if present, are oxygen.
In another aspect the invention relates to an activated PGC having the structure according to Formula VI:
where P is a polyalkylene glycol polymer, m is zero or one, n is zero or an integer from one to five, X and Y are independently O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′, and T1 and T2 are, independently, absent, or a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group.
Each R′ and Z is, independently, hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group.
d is zero or an integer from one to four, and each L is, independently, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group. The substituents are selected from halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, or alkylthio moieties.
In one embodiment, the activated PGC according to Formula VI has the structure according to Formula VII or Formula VIII:
In one embodiment of the activated polyalkylene glycol compounds of Formulae VII and VIII, P is a polyethylene glycol having the structure of Formula II:
E-(O—CH2CH2)a—, Formula II:
where E is hydrogen or a straight- or branched-chain C1 to C20 alkyl group and a is an integer from 4 to 10,000. For example, E can be methyl. In other embodiments, E is a detectable label, such as, for example, a radioactive isotope, fluorescent moiety, phosphorescent moiety, chemiluminescent moiety, or a quantum dot.
In another aspect, P is a polyethylene glycol having the structure of Formula II:
E-(O—CH2CH2)a—, Formula II:
where E is a moiety suitable for forming a bond between the compound of Formula VII or VIII and a biologically-active compound or precursor thereof and a is an integer from 4 to 10,000. For example, E is chosen from carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal moieties.
In another aspect, E has the structure according to Formula III or Formula IV:
where Q is a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl; the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, and the substituents can be of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, or alkylthio. Heterocyclic and carbocyclic groups include fused bicyclic and bridged bicyclic ring structures
X, Y, Z, m, and n are as defined, and each W is, independently, hydrogen or a C1 to C7 alkyl; and R″ is a moiety suitable for forming a bond between the compound of Formula III and a biologically-active compound or precursor thereof, and R′″ is a moiety suitable for forming a bond between the compound of Formula IV and a biologically-active compound or precursor thereof.
In certain embodiments, R″ and R′″ can be the same as or different from R, and are chosen from carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal moieties.
In one embodiment, the activated polyalkylene glycol compound of Formula VIII, the ring substituents are located in a meta arrangement. In another embodiment, the ring substituents are located in a para arrangement.
In another embodiment, the activated polyalkylene glycol compound according to Formula VI, has the structure according to Formula IX:
where P is a polyalkylene glycol polymer, each n and u are, independently, zero or an integer from one to five; and Z is hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group.
In one embodiment of the compounds of Formula IX, the ring substituents are located in a meta arrangement. In another embodiment of the compounds of Formula IX, the ring substituents are located in a para arrangement.
In another embodiment of the compounds of Formula IX, P is a polyethylene glycol having the structure of Formula II:
E-(O—CH2CH2)a—, Formula II:
where E is hydrogen, a straight- or branched-chain C1 to C20 alkyl group, a detectable label, or a moiety suitable for forming a bond between the compound of Formula IX and a biologically-active compound or precursor thereof and a is an integer from 4 to 10,000.
In another aspect, the invention involves an activated polyalkylene glycol polymer having the structure according to Formula X:
wherein P is a polyalkylene glycol polymer;
X is O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′;
R′ is hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio;
Z and Z′ are individually hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio, provided that at least one Z or Z′ is not hydrogen;
R is a moiety suitable for forming a bond between the compound of Formula X and a biologically-active compound or precursor thereof;
each n is independently 0 or an integer from 1 to 5; and
p is 1, 2, or 3.
In another aspect, the invention involves an activated polyalkylene glycol compound (PGC) having the structure according to Formula Xa:
In these compounds, P is a polyalkylene glycol polymer, such as, for example, PEG or mPEG.
X is O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′, and R′, if present, is hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio.
Z is a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio.
R is a moiety suitable for forming a bond between the compound of Formula X and a biologically-active compound or precursor thereof; and
n is 0 or an integer from 1 to 5, such that there are between zero and five methylene groups between X and the Z-containing carbon.
In one embodiment, R is chosen from the group consisting of carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal.
In another embodiment, P is a polyethylene glycol having the structure of Formula II:
E-O—CH2CH2)a—, Formula II:
wherein E is hydrogen, a straight- or branched-chain C1 to C20 alkyl group, or a detectable label; and a is an integer from 4 to 10,000. In a further embodiment, E may be methyl.
In yet another embodiment, P is a polyethylene glycol having the structure of Formula II, wherein E is a moiety suitable for forming a bond between the compound of Formula X and a biologically-active compound or precursor thereof and a is an integer from 4 to 10,000.
In an additional embodiment, E is chosen from the group consisting of carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal. Alternatively, E may have the structure according to Formula III:
wherein P is a polyalkylene glycol polymer;
X and Y are independently O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′;
Q is a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl (including fused bicyclic and bridged bicyclic ring structures), a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio;
R′ and each Z are independently as described above;
m is 0 or 1;
each W is, independently, hydrogen or a C1 to C7 alkyl;
each n is independently 0 or an integer from 1 to 5; and
R″ is a moiety suitable for forming a bond between the compound of Formula III and a biologically-active compound or precursor thereof. Heterocyclic and carbocyclic groups include fused bicyclic and bridged bicyclic ring structures.
In still a further embodiment, E has the structure according to Formula IV:
wherein each X, Z and n are, independently, as defined;
each W is, independently, hydrogen or a C1 to C7 alkyl; and
R′″ is a moiety suitable for forming a bond between the compound of Formula IV and a biologically-active compound or precursor thereof.
In an additional embodiment, R″ is chosen from the group consisting of carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal.
In a further embodiment, R′″ is chosen from the group consisting of carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal.
In another embodiment, E is a detectable label. Additionally, E may be selected from the group consisting of radioactive isotopes, fluorescent moieties, phosphorescent moieties, chemiluminescent moieties, and quantum dots.
In still another embodiment, the activated PGC according to the invention has the structure according to Formula XI:
wherein P is a polyalkylene glycol polymer; and
n and Z are as defined.
In another embodiment, the activated polyalkylene glycol has the structure according to Formula XII:
wherein n, a, and Z are as defined above. In one embodiment, Z may be methyl. In some embodiments, n is one.
In another aspect, the invention involves an activated polyalkylene glycol compound of having the structure according to Formula XIII:
where a is an integer from 4 to 10,000.
The invention is also concerned with a composition of the activated polyalkylene glycol compounds of the invention and a biologically-active compound or precursor thereof. In various embodiments, the biologically-active compound or precursor thereof is chosen from the group consisting of a peptide, peptide analog, protein, enzyme, small molecule, dye, lipid, nucleoside, oligonucleotide, oligonucleotide analog, sugar, oligosaccharide, cell, virus, liposome, microparticle, surface, and a micelle.
In another aspect, the invention provides a composition having the structure according to Formula XIV:
wherein P is a polyalkylene glycol polymer;
X and Y are independently O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′;
Q is a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio;
each R′, Z, and Z′ is independently hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio;
R* is a linking moiety;
B is a biologically-active compound or precursor thereof;
m is 0 or 1;
each n is independently 0 or an integer from 1 to 5; and
p is 1, 2, or 3.
In another aspect, the invention involves a composition having the structure according to Formula XIVa:
wherein P is a polyalkylene glycol polymer;
X and Y are independently O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′;
Q is a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl (including fused bicyclic and bridged bicyclic ring structures), a substituted or unsubstituted aryl or heteroaryl group (including fused bicyclic and bridged bicyclic ring structures), or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio;
each R′ and Z is independently hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio;
R* is a linking moiety formed from the reaction of R with a biologically-active compound or precursor thereof;
B is a biologically-active compound or precursor thereof after conjugation with R;
m is 0 or 1; and
n is 0 or an integer from 1 to 5.
In one embodiment, R* is a linking moiety formed from the reaction of R with a biologically-active compound or precursor thereof. For example, R is a moiety selected from the group consisting of carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal.
In another embodiment, P is a polyethylene glycol having the structure of Formula II:
E-(O—CH2CH2)a—, Formula II:
wherein E is hydrogen, a straight- or branched-chain C1 to C20 alkyl group, or a detectable label; and a is an integer from 4 to 10,000. In this embodiment, E may be methyl.
In a further embodiment, P is a polyethylene glycol having the structure of Formula II:
E-(O—CH2CH2)a—, Formula II:
wherein E is a moiety suitable for forming a bond between the compound of Formula XIV and a biologically-active compound or precursor thereof and a is an integer from 4 to 10,000. Here, in still a further embodiment, E may form a bond to another biologically-active compound, B. Alternatively, E may form a bond to a biologically-active compound other than B. E may also form an additional bond to the biologically-active compound, B.
In various embodiments, E may be chosen from the group consisting of carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal. In another embodiment, E may have the structure according to Formula III:
wherein each Q, X, Y, Z, m, and n are, independently, as defined, each W is, independently, hydrogen or a C1 to C7 alkyl; and R″ is a moiety suitable for forming a bond between the compound of Formula III and a biologically-active compound or precursor thereof.
In a further embodiment, E has the structure according to Formula IV:
wherein each X, Z and n are, independently, as defined, each W is, independently, hydrogen or a C1 to C7 alkyl; and
R′″ is a moiety suitable for forming a bond between the compound of Formula IV and a biologically-active compound or precursor thereof.
In various embodiments, R″ is chosen from the group consisting of carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal.
Likewise, in other embodiments, R′″ is chosen from the group consisting of carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal.
In still other embodiments, E is a detectable label. For example, E may be selected from the group consisting of radioactive isotopes, fluorescent moieties, phosphorescent moieties, chemiluminescent moieties, and quantum dots.
In various embodiments, Q is a substituted or unsubstituted alkaryl.
In another aspect, the invention involves a composition having the structure according to Formula XV:
wherein P is a polyalkylene glycol polymer; m is zero or one; d is zero or an integer from one to four; and n is zero or an integer from one to five.
X and Y are independently O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′; and T1 and T2 are, independently, absent, or a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio.
Each R′ and Z is, independently, hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio.
Each L is, independently, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio.
R* is a linking moiety formed from the reaction of R with a biologically-active compound or precursor thereof, and B is a biologically-active compound, or precursor thereof, after conjugation with R.
For example, R may be a moiety selected from the group consisting of carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal.
In another embodiment, P is a polyethylene glycol having the structure of Formula II:
E-(O—CH2CH2)a—, Formula II:
wherein E is hydrogen, a straight- or branched-chain C1 to C20 alkyl group, or a detectable label; and a is an integer from 4 to 10,000. In this embodiment, E may be methyl.
In still another aspect, P is a polyethylene glycol having the structure of Formula II:
E-(O—CH2CH2)a— Formula II:
wherein E is a moiety suitable for forming a bond between the compound of Formula XV and a biologically-active compound or precursor thereof and a is an integer from 4 to 10,000. Here, E may be selected from the group consisting of carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal. Additionally, E may have the structure according to:
wherein Q is a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, (including fused bicyclic and bridged bicyclic ring structures), a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio;
each X, Y, Z, m, and n are, independently, as defined;
each W is, independently, hydrogen or a C1 to C7 alkyl; and
R″ is a moiety suitable for forming a bond between the compound of Formula III and a biologically-active compound or precursor thereof.
In another embodiment, E can have the structure according to Formula IV:
wherein X, Z and n are as defined;
each W is, independently, hydrogen or a C1 to C7 alkyl; and
R′″ is a moiety suitable for forming a bond between the compound of Formula IV and a biologically-active compound or precursor thereof.
In still another embodiment, R″ is chosen from the group consisting of carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal.
Likewise, in other embodiments, R′″ may selected from the group consisting of carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal.
In other embodiments, E is a detectable label. For example, E may be selected from the group consisting of radioactive isotopes, fluorescent moieties, phosphorescent moieties, chemiluminescent moieties, and quantum dots.
In another aspect, the invention relates to a composition having the structure according to Formula XVI:
where m is 0 or 1, n is 0 or an integer from 1 to 5, P is a polyalkylene glycol polymer, X and Y are independently O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′, T1 and T2 are, independently, absent, or a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, and each R′ and Z is independently hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group;
d is 0 or an integer from 1 to 4, and each L is, independently, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group. The substituents are selected from halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio groups.
R* is a linking moiety formed from the reaction of R with a biologically-active compound or precursor thereof, and B is a biologically-active compound, or precursor thereof, after conjugation with R.
For example, R may be a moiety selected from the group consisting of carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal.
In one embodiment, R* is a methylene group and B is a biologically-active molecule having an amino group, where the methylene group forms a bond with the amino group on B.
In certain embodiments, the amine is the amino terminus of a peptide, an amine of an amino acid side chain of a peptide, or an amine of a glycosylation substituent of a glycosylated peptide. For example, the peptide can be an interferon, such as interferon-beta, e.g., interferon-beta-1a.
In some embodiments, the compound according to Formula XVI has a structure according to Formula XVII:
where P is a polyalkylene glycol polymer, Z is hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, n is 0 or an integer from 1 to 5.
R* is a linking moiety formed from the reaction of R with a biologically-active compound or precursor thereof, and B is a biologically-active compound, or precursor thereof, after conjugation with R.
For example, R may be a moiety selected from the group consisting of carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal.
In one embodiment, R* of Formula XVII is a methylene group and B is a biologically-active molecule having an amino group, where the methylene group forms a bond with the amino group on B.
In certain embodiments, the amine is the amino terminus of a peptide, an amine of an amino acid side chain of a peptide, or an amine of a glycosylation substituent of a glycosylated peptide. For example, the peptide can be an interferon, such as interferon-beta, e.g., interferon-beta-1a.
In other embodiments, the compound according to Formula XVI has a structure according to Formula XVIII:
where P is a polyalkylene glycol polymer, R* is a linking moiety, B is a biologically-active molecule, and n is one or two.
In one embodiment, R* of Formula XVIII is a methylene group and B is a biologically-active molecule having an amino group, where the methylene group forms a bond with the amino group on B.
In certain embodiments, the amine is the amino terminus of a peptide, an amine of an amino acid side chain of a peptide, or an amine of a glycosylation substituent of a glycosylated peptide. For example, the peptide can be an interferon, such as interferon-beta, e.g., interferon-beta-1a.
In certain embodiments of the compound according to Formula XVI, P is a polyethylene glycol having the structure of Formula II:
E-(O—CH2CH2)a—, Formula II:
wherein E is hydrogen, a straight- or branched-chain C1 to C20 alkyl (e.g., methyl) group, a detectable label, or a moiety suitable for forming a bond between the compound of Formula XVI and a biologically-active compound or precursor thereof and a is an integer from 4 to 10,000. When E is a detectable label, the label can be, for example, a radioactive isotope, fluorescent moiety, phosphorescent moiety, chemiluminescent moiety, or a quantum dot.
In another embodiment, where E is a moiety suitable for forming a bond between the compound of Formula XVI and a biologically-active compound or precursor thereof, E is chosen from carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal moieties.
In another embodiment, E has the structure according to Formula III or Formula IV:
where Q is a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl (including fused bicyclic and bridged bicyclic ring structures), a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl; the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, and the substituents can be of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, or alkylthio.
X, Y, Z, m, and n are as defined, and each W is, independently, hydrogen or a C1 to C7 alkyl; and R″ is a moiety suitable for forming a bond between the compound of Formula III and a biologically-active compound or precursor thereof, and R′″ is a moiety suitable for forming a bond between the compound of Formula IV and a biologically-active compound or precursor thereof.
In certain embodiments, R″ and R′″ can be the same as or different from R, and are chosen from carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal moieties.
In other embodiments of the compound according to Formula XVI, the compound can have the structure according to Formula XIX:
wherein P is a polyalkylene glycol polymer, each n and u are, independently, zero or an integer from one to five, Z is hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group.
R* is a linking moiety formed from the reaction of R with a biologically-active compound or precursor thereof, and B is a biologically-active compound, or precursor thereof, after conjugation with R.
In one embodiment, R* of Formula XIX is a methylene group and B is a biologically-active molecule having an amino group, where the methylene group forms a bond with the amino group on B.
In certain embodiments, the amine is the amino terminus of a peptide, an amine of an amino acid side chain of a peptide, or an amine of a glycosylation substituent of a glycosylated peptide. For example, the peptide can be an interferon, such as interferon-beta, e.g., interferon-beta-1a.
In another aspect, the invention relates to a composition according to Formula XX:
where m is 0 or 1, d is 0 or an integer from 1 to 4, a is an integer from 4 to 10,000, and n is 0 or an integer from 1 to 5.
Each X and Y is independently O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′, or NR′, T1 and T2 are, independently, absent, or a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, and each R′ and Z is independently hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group.
When present, each L is, independently, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group. The substituents are selected from halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio.
Q is a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl (including fused bicyclic and bridged bicyclic ring structures), a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group. The substituents can be halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, or alkylthio. Each W is, independently, hydrogen or a C1 to C7 alkyl.
R* and R** are, independently, linking moieties formed from the reaction of R and R″ with a biologically-active compound or precursor thereof, and B and B′ are each a biologically-active compound, or precursor thereof, after conjugation with R and R″, respectively.
In some embodiments, B and B′ are the same type of biologically-active compound. In other embodiments, B and B′ are different biologically-active compounds. In still other embodiments, B and B′ are the same biologically active molecule. In additional embodiments, R* and R** are the same. In other embodiments, R* and R** are different.
In another aspect, the invention relates to a composition according to Formula XXI:
where m is 0 or 1, d is 0 or an integer from 1 to 4, a is an integer from 4 to 10,000, and n is 0 or an integer from 1 to 5.
X and Y are independently O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′, T1 and T2 are, independently, absent, or a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, each R′ and Z is, independently, hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group.
When present, each L is, independently, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio, and each W is, independently, hydrogen or a C1 to C7 alkyl.
R* and R** are, independently, linking moieties formed from the reaction of R and R″ with a biologically-active compound or precursor thereof, and B and B′ are each a biologically-active compound, or precursor thereof, after conjugation with R and R″, respectively.
In some embodiments, B and B′ are the same type of biologically-active compound. In other embodiments, B and B′ are different biologically-active compounds. In still other embodiments, B and B′ are the same biologically active molecule. In additional embodiments, R* and R** are the same. In other embodiments, R* and R** are different.
In another aspect, the invention involves a composition having the structure according to Formula XXII:
wherein P is a polyalkylene glycol polymer;
X is O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′;
R′ is hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio;
each Z and Z′ is independently hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio, provided that at least one Z or Z′ is not hydrogen;
R* is a linking moiety;
B is a biologically-active molecule;
each n is 0 or an integer from 1 to 5; and
p is 1, 2, or 3.
In a further aspect, the invention involves a composition having the structure according to Formula XXIIa:
wherein P is a polyalkylene glycol polymer,
X is O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′; and n is 0 or an integer from 1 to 5.
R′ is hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio;
Z is a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or, a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio;
R* is a linking moiety formed from the reaction of R with a biologically-active compound or precursor thereof, and B is a biologically-active compound, or precursor thereof, after conjugation with R.
In one embodiment, R* is formed from the reaction of a moiety selected from the group consisting of carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal with a biologically-active compound or precursor thereof.
In an additional embodiment, P is a polyethylene glycol having the structure of Formula II:
E-(O—CH2CH2)a—, Formula II:
wherein E is hydrogen, a straight- or branched-chain C1 to C20 alkyl group, or a detectable label; and a is an integer from 4 to 10,000. In this embodiment, E may be methyl.
In another embodiment, P is a polyethylene glycol having the structure of Formula II:
E-(O—CH2CH2)a—, Formula II:
wherein E is a moiety suitable for forming a bond between the compound of Formula II and a biologically-active compound or precursor thereof and a is an integer from 4 to 10,000. In this embodiment, E may bind to a biologically-active compound or precursor thereof other than B. In other embodiments, E forms an additional bond to the biologically-active compound B.
In various embodiments, E may be selected from the group consisting of carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal.
In other embodiments, E has the structure according to Formula III:
wherein P is a polyalkylene glycol polymer,
each X and Y is independently O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′;
Q is a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl (including fused bicyclic and bridged bicyclic ring structures), a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio;
R′ and each Z are independently as described above;
m is 0 or 1;
each n is independently 0 or an integer from 1 to 5;
R″ is a moiety suitable for forming a bond between the compound of Formula III and a biologically-active compound or precursor thereof; and
each W is, independently, hydrogen or a C1 to C7 alkyl.
In a further embodiment, E has the structure according to Formula IV:
wherein each X, Z and n are, independently, as defined;
each W is, independently, hydrogen or a C1 to C7 alkyl; and
R′″ is a moiety suitable for forming a bond between the compound of Formula IV and a biologically-active compound or precursor thereof.
In still further embodiments, R″ is chosen from the group consisting of carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal.
In yet other embodiments, R′″ is chosen from the group consisting of carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal.
In additional embodiments, E is a detectable label. For example, E may be selected from the group consisting of radioactive isotopes, fluorescent moieties, phosphorescent moieties, chemiluminescent moieties and quantum dots.
In another embodiment, R* is methylene and B is a biologically-active molecule attached via an amine. For example, the amine is the amino terminus of a peptide. In a further embodiment, the peptide is an interferon such as interferon-beta-1a.
In another embodiment, the invention is a composition having the structure according to Formula XXIII:
wherein n, a, R* B, and Z are as defined above. In one additional embodiment, Z is methyl and n is one.
In still a further aspect, the invention involves a composition according to Formula XXIV:
wherein m is 0 or 1, a is an integer from 4 to 10,000; and each n is independently zero or an integer from 1 to 5. Each X and Y is independently O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′; each R′ and Z is, independently, hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group; and each W is, independently, hydrogen or a C1 to C7 alkyl.
Q is a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl (including fused bicyclic and bridged bicyclic ring structures), a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio.
R* and R** are, independently, linking moieties formed from the reaction of R and R″ with a biologically-active compound or precursor thereof, and B and B′ are each a biologically-active compound, or precursor thereof, after conjugation with R and R″, respectively.
In some embodiments, B and B′ are the same type of biologically-active compound. In other embodiments, B and B′ are different biologically-active compounds. In still other embodiments, B and B′ are the same biologically active molecule. In additional embodiments, R* and R** are the same. In other embodiments, R* and R** are different.
In a further aspect, the invention involves a composition according to Formula XXV:
wherein
X is O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′; a is an integer from 4 to 10,000; and each n is independently 0 or an integer from 1 to 5.
Each and Z is independently hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, and each W is, independently, hydrogen or a C1 to C7 alkyl.
R* and R** are, independently, linking moieties formed from the reaction of R and R″ with a biologically-active compound or precursor thereof, and B and B′ are each a biologically-active compound, or precursor thereof, after conjugation with R and R″, respectively.
In some embodiments, B and B′ are the same type of biologically-active compound. In other embodiments, B and B′ are different biologically-active compounds. In still other embodiments, B and B′ are the same biologically active molecule. In additional embodiments, R* and R** are the same. In other embodiments, R* and R** are different.
The invention also involves a pharmaceutical composition containing the compositions of the invention along with a pharmaceutically-acceptable carrier. In various embodiments, the pharmaceutical composition also contains an additional biologically-active agent. For example, the biologically-active agent may be selected from the group consisting of a peptide, peptide analog, protein, enzyme, small molecule, dye, lipid, nucleoside, oligonucleotide, oligonucleotide analog, sugar, oligosaccharide, cell, virus, liposome, microparticle, surface, and a micelle. In another embodiment, the biologically-active agent is an antiviral agent.
In another aspect, the invention relates to a composition comprising the product of the reaction of the compound of Formula I and a biologically-active compound or a precursor thereof (B).
In one embodiment, the composition has the stricture according to Formula XIV:
where all variables are as defined above, and R* is a linking moiety formed by the reaction of R with a reactive moiety on the biologically-active compound or precursor thereof; and B is a biologically-active compound or precursor thereof.
In another aspect, the invention relates to a composition comprising the product of the reaction of the compound of Formula V and a biologically-active compound or a precursor thereof. In one embodiment, the composition has the structure according to Formula XV:
where all variables are as defined above, R* is a linking moiety formed by the reaction of R with a reactive moiety on the biologically-active compound or precursor thereof; and B is a biologically-active compound or precursor thereof.
In yet another embodiment, the composition has the structure according to Formula XX or XXI:
where all variables are as defined above, each W is, independently, hydrogen or a C1 to C7 alkyl, R* is a linking moiety formed by the reaction of R with a reactive moiety on the biologically-active compound, B, or precursor thereof; R** is a linking moiety formed by the reaction of R″ or R′″ with a reactive moiety on the biologically-active compound, B′, or precursor thereof; and B and B′ are, independently, a biologically-active compound or precursor thereof. In some embodiments, B and B′ are the same type of biologically-active compound. In other embodiments, B and B′ are different biologically-active compounds. In still other embodiments, B and B′ are the same biologically active molecule. In additional embodiments, R* and R** are the same. In other embodiments, R* and R** are different.
In another aspect, the invention relates to a composition comprising the product of the reaction of the compound Formula VI and a biologically-active compound or a precursor thereof.
In one embodiment; the composition has the structure according to Formula XVI:
where all variables are as defined above, R* is a linking moiety formed by the reaction of R with a reactive moiety on the biologically-active compound or precursor thereof; and B is a biologically-active compound or precursor thereof.
In another aspect, the invention relates to a composition comprising the product of the reaction of the compound of Formula VII and a biologically-active compound or a precursor thereof.
In one embodiment, the composition has the structure according to Formula XVII:
where all variables are as defined above, R* is a linking moiety formed by the reaction of R with a reactive moiety on the biologically-active compound or precursor thereof; and B is a biologically-active compound or precursor thereof.
In another aspect, the invention relates to a composition comprising the product of the reaction of the compound of Formula VIII and a biologically-active compound or a precursor thereof.
In one embodiment, the composition has the structure according to Formula XVIII:
where all variables are as defined above, R* is a linking moiety formed by the reaction of R with a reactive moiety on the biologically-active compound or precursor thereof; and B is a biologically-active compound or precursor thereof.
In another aspect, the invention relates to a composition comprising the product of the reaction of the compound of Formula IX and a biologically-active compound or a precursor thereof.
In one embodiment, the composition has the structure according to Formula XIX:
where all variables are as defined above, R* is a linking moiety formed by the reaction of R with a reactive moiety on the biologically-active compound or precursor thereof; and B is a biologically-active compound or precursor thereof.
In another aspect, the invention relates to a composition comprising the product of the reaction of the compound of Formula X and a biologically-active compound or a precursor thereof.
In one embodiment, the composition has the structure according to Formula XXII:
where all variables are as defined above, R* is a linking moiety formed by the reaction of R with a reactive moiety on the biologically-active compound or precursor thereof; and B is a biologically-active compound or precursor thereof.
In another embodiment, the composition has the structure the structure according to Formula XXIV:
where all variables are as defined above, each W is, independently, hydrogen or a C1 to C7 alkyl. R* is a linking moiety formed by the reaction of R with a reactive moiety on the biologically-active compound, B, or precursor thereof, R** is a linking moiety formed by the reaction of R″ with a reactive moiety on the biologically-active compound, B′, or precursor thereof; and B and B′ are, independently, a biologically-active compound or precursor thereof. In some embodiments, B and B′ are the same type of biologically-active compound. In other embodiments, B and B′ are different biologically-active compounds. In still other embodiments, B and B′ are the same biologically active molecule. In additional embodiments, R* and R** are the same. In other embodiments, R* and R** are different.
In other embodiments, the composition has the structure according to Formula XXV:
where all variables are as defined in claims above, each W is, independently, hydrogen or a C1 to C7 alkyl.
R* and R** are, independently, linking moieties formed from the reaction of R and R″ with a biologically-active compound or precursor thereof, and B and B′ are each a biologically-active compound, or precursor thereof, after conjugation with R and R″, respectively.
In some embodiments, B and B′ are the same type of biologically-active compound. In other embodiments, B and B′ are different biologically-active compounds. In still other embodiments, B and B′ are the same biologically active molecule. In additional embodiments, R* and R** are the same. In other embodiments, R* and R** are different.
In another aspect, the invention involves a method of treating a patient with a susceptible viral infection, comprising administering to the patient an effective amount of a composition having the structure according to Formula XIV:
wherein P is a polyalkylene glycol polymer,
X and Y are independently O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′;
Q is a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl (including fused bicyclic and bridged bicyclic ring structures), a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio;
each R′, Z and Z′ is independently hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio;
R* is a linking moiety;
B is a biologically-active compound or precursor thereof;
m is 0 or 1;
each n is 0 or an integer from 1 to 5; and
p is 1, 2, or 3.
In a further aspect, the invention involves a method of treating a patient with a susceptible viral infection by administering to the patient an effective amount of a composition having the structure according to Formula XIVa:
wherein P is a polyalkylene glycol polymer, m is 0 or 1; and n is 0 or an integer from 1 to 5.
X and Y are independently O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′; and Q is a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl (including fused bicyclic and bridged bicyclic ring structures), a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio.
Each R′ and Z is independently hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio;
R* is a linking moiety formed from the reaction of R with a biologically-active compound or precursor thereof, and B is a biologically-active compound, or precursor thereof, after conjugation with R.
In one embodiment, B is a biologically-active peptide such as interferon. For example, this interferon may be interferon-beta-1a.
In further embodiments, the composition also includes a biologically-active agent selected from the group consisting of a small molecule antiviral, a nucleic acid antiviral and a peptide antiviral. For example, the antiviral agent may be selected from the group consisting of ribavirin, levovirin, 3TC, FTC, MB686, zidovudine, acyclovir, gancyclovir, viramide, VX-497, VX-950, and ISIS-14803.
In various embodiments, the viral infection in need of treatment is chronic hepatitis C.
In an additional aspect, the invention involves a method of treating a patient with a susceptible viral infection by administering to the patient an effective amount of a composition having the structure according to Formula XV:
wherein P is a polyalkylene glycol polymer; m is 0 or 1; d is 0 or an integer from 1 to 4; n is 0 or an integer from 1 to 5; and X and Y are independently O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′.
T1 and T2 are, independently, absent, or a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio.
Each R′ and Z is independently hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio.
Each L is, independently, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety heteroaromatic moiety, imino, silyl, ether, and alkylthio;
R* is a linking moiety formed from the reaction of R with a biologically-active compound or precursor thereof, and B is a biologically-active compound, or precursor thereof, after conjugation with R.
In various embodiments, B is a biologically-active peptide such as interferon. For example, in one embodiment, B is interferon-beta-1a.
In another embodiment, the composition further contains a biologically-active agent selected from the group consisting of a small molecule antiviral, a nucleic acid antiviral and a peptidic antiviral. In other embodiments, the antiviral agent may be selected from the group consisting of ribavirin, levovirin, 3TC, FTC, MB686, zidovudine, acyclovir, gancyclovir, viramide, VX497, VX-950, and ISIS-14803. In addition, the viral infection can be chronic hepatitis C.
In a further aspect, the invention involves a method of treating a patient with a susceptible viral infection by administering to the patient an effective amount of a composition having the structure according to Formula XVI:
where P is a polyalkylene glycol polymer, m is 0 or 1; d is 0 or an integer from 1 to 4; n is 0 or an integer from 1 to 5; X and Y are independently O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′; T1 and T2 are, independently, absent, or a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group; and each R′ and Z is independently hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group.
Each L is, independently, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio.
R* is a linking moiety formed from the reaction of R with a biologically-active compound or precursor thereof, and B is a biologically-active compound, or precursor thereof, after conjugation with R.
In various embodiments, B is a biologically-active peptide such as interferon. For example, B may be interferon-beta-1a.
In still further embodiments, the composition further contains a biologically-active agent selected from the group consisting of a small molecule antiviral, a nucleic acid antiviral and a peptidic antiviral. For example, the antiviral agent may be selected from the group consisting of ribavirin, levovirin, 3TC, FTC, MB686, zidovudine acyclovir, gancyclovir, viramide, VX497, VX-950, and ISIS-14803.
In another embodiment, the viral infection is chronic hepatitis C.
In a further aspect, the invention involves a method of treating a patient with a susceptible viral infection by administering to the patient an effective amount of a composition having the structure according to Formula XX:
wherein m is 0 or 1; d is 0 or an integer from 1 to 4; a is an integer from 4 to 10,000; and n is 0 or an integer from 1 to 5.
Each X and Y is independently O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′; T1 and T2 are, independently, absent, or a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group; each R′ and Z is independently hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group; and each W is, independently, hydrogen or a C1 to C7 alkyl.
Each L is, independently, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio.
Q is a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl (including fused bicyclic and bridged bicyclic ring structures), a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio.
R* and R** are, independently, linking moieties formed from the reaction of R and R″ with a biologically-active compound or precursor thereof, and B and B′ are each a biologically-active compound, or precursor thereof, after conjugation with R and R″, respectively.
In some embodiments, B and B′ are the same type of biologically-active compound. In other embodiments, B and B′ are different biologically-active compounds. In still other embodiments, B and B′ are the same biologically active molecule. In additional embodiments, R* and R** are the same. In other embodiments, R* and R** are different.
In various embodiments, B is a biologically-active peptide such as interferon. For example, in one embodiment, B is interferon-beta-1a.
In other embodiments, the composition further contains a biologically-active agent selected from the group consisting of a small molecule antiviral, a nucleic acid antiviral and a peptidic antiviral. For example, the antiviral agent may be selected from the group consisting of ribavirin, levovirin, 3TC, FTC, MB686, zidovudine, acyclovir, gancyclovir, viramide, VX-497, VX-950, and ISIS-14803.
In a further embodiment, the viral infection is chronic hepatitis C.
In a further aspect, the invention involves a method of treating a patient with a susceptible viral infection by administering to the patient an effective amount of a composition having the structure according to Formula XXI:
where m is 0 or 1; d is 0 or an integer from 1 to 4; a is an integer from 4 to 10,000; each n is 0 or an integer from 1 to 5; each X and Y is independently O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′; T1 and T2 are, independently, absent, or a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group; each R′ and Z is independently hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group; and each W is, independently, hydrogen or a C1 to C7 alkyl.
Each L is, independently, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio.
R* and R** are, independently, linking moieties formed from the reaction of R and R″ with a biologically-active compound or precursor thereof, and B and B′ are each a biologically-active compound, or precursor thereof, after conjugation with R and R″, respectively.
In some embodiments, B and B′ are the same type of biologically-active compound. In other embodiments, B and B′ are different biologically-active compounds. In still other embodiments, B and B′ are the same biologically active molecule. In additional embodiments, R* and R** are the same. In other embodiments, R* and R** are different.
In various embodiments, B is a biologically-active peptide such as an interferon. For example, B may be is interferon-beta-1a.
In another embodiment, the composition further contains a biologically-active agent selected from the group consisting of a small molecule antiviral, a nucleic acid antiviral and a peptidic antiviral. For example, the antiviral agent may be selected from the group consisting of ribavirin, levovirin, 3TC, FTC, MB686, zidovudine, acyclovir, gancyclovir, viramide, VX-497, VX-950, and ISIS-14803.
In a further embodiment, the viral infection is chronic hepatitis C.
In another aspect, the invention involves a method of treating a patient with a susceptible viral infection, comprising administering to the patient an effective amount of a composition having the structure according to Formula XXII:
wherein
P is a polyalkylene glycol polymer;
X is O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′;
R′ is hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio;
each Z and Z′ is independently hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio provided that at least one Z or Z′ is not hydrogen;
R* is a linking moiety;
B is a biologically-active molecule.
m is 0 or 1;
each n is 0 or an integer from 1 to 5; and
p is 1, 2, or 3.
In still another aspect, the invention involves a method of treating a patient with a susceptible viral infection, comprising administering to the patient an effective amount of a composition having the structure according to Formula XXIIa:
where: P is a polyalkylene glycol polymer; m is 0 or 1; n is 0 or an integer from 1 to 5; X is O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′; and R′ is hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio.
Z is a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio.
R* is a linking moiety formed from the reaction of R with a biologically-active compound or precursor thereof, and B is a biologically-active compound, or precursor thereof, after conjugation with R.
In various embodiments, B is a biologically-active peptide such as interferon. For example, B may be interferon-beta-1a.
In another embodiment, the composition further contains a biologically-active agent selected from the group consisting of a small molecule antiviral, a nucleic acid antiviral and a peptidic antiviral. For example, the antiviral agent may be selected from the group consisting of ribavirin, levovirin, 3TC, FTC, MB686, zidovudine, acyclovir, gancyclovir, viramide, VX-497, VX-950, and ISIS-14803.
In a further embodiment, the viral infection is chronic hepatitis C.
In yet another aspect, the invention involves a method of treating a patient with a susceptible viral infection by administering to the patient an effective amount of a composition having the structure according to Formula XXIV:
where: m is 0 or 1; a is an integer from 4 to 10,000; each n is independently 0 or an integer from 1 to 5; each X and Y is independently O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′; each R′ and Z is independently hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group; and each W is, independently, hydrogen or a C1 to C7 alkyl.
Q is a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl (including fused bicyclic and bridged bicyclic ring structures), a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio.
R* and R** are, independently, linking moieties formed from the reaction of R and R″ with a biologically-active compound or precursor thereof, and B and B′ are each a biologically-active compound, or precursor thereof, after conjugation with R and R″, respectively.
In some embodiments, B and B′ are the same type of biologically-active compound. In other embodiments, B and B′ are different biologically-active compounds. In still other embodiments, B and B′ are the same biologically active molecule. In additional embodiments, R* and R** are the same. In other embodiments, R* and R** are different.
In various embodiments, B is a biologically-active peptide such as interferon. For example, B may be interferon-beta-1a.
In other embodiments, the composition further contains a biologically-active agent selected from the group consisting of a small molecule antiviral, a nucleic acid antiviral and a peptidic antiviral. For example, the antiviral agent may be selected from the group consisting of ribavirin, levovirin, 3TC, FTC, MB686, zidovudine, acyclovir, gancyclovir, viramide, VX-497, VX-950, and ISIS-14803.
In still other embodiments, the viral infection is chronic hepatitis C.
In an additional aspect, the invention involves a method of treating a patient with a susceptible viral infection by administering to the patient an effective amount of a composition having the structure according to Formula XXV:
wherein each W is, independently, hydrogen or a C1 to C7 alkyl; a is an integer from 4 to 10,000; each n is independently 0 or an integer from 1 to 5; X is O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′; and each and Z is independently hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group.
R* and R** are, independently, linking moieties formed from the reaction of R and R″ with a biologically-active compound or precursor thereof, and B and B′ are each a biologically-active compound, or precursor thereof, after conjugation with R and R″, respectively.
In some embodiments, B and B′ are the same type of biologically-active compound. In other embodiments, B and B′ are different biologically-active compounds. In still other embodiments, B and B′ are the same biologically active molecule. In additional embodiments, R* and R** are the same. In other embodiments, R* and R** are different.
In various embodiments, B is a biologically-active peptide such as interferon. For example, B may be interferon-beta-1a.
In another embodiment, the composition further contains a biologically-active agent selected from the group consisting of a small molecule antiviral, a nucleic acid antiviral and a peptidic antiviral. For example, the antiviral agent may be selected from the group consisting of ribavirin, levovirin, 3TC, FTC, MB686, zidovudine, acyclovir, gancyclovir, viramide, VX-497, VX-950, and ISIS-14803.
In still other embodiments, the viral infection is chronic hepatitis C.
The present invention is also concerned with a method of treating a patient suspected of having hepatitis C infection by administering to the patient a combination of any of the compositions of the invention and an antiviral agent. In various embodiments, the composition and the antiviral agent are administered simultaneously, sequentially, or alternatively.
In one embodiment, the antiviral agent is ribavirin.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be further understood from the following description with reference to the tables, in which:
FIG. 1 is a reducing SDS-PAGE gel showing the purity of unmodified IFN-β-1a and 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a: Lane A: molecular weight markers (from top to bottom; 100 kDa, 68 kDa, 45 kDa, 27 kDa, and 18 kDa, respectively); Lane B: 4 μg of unmodified IFN-β-1a; Lane C: 4 μg of 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a.
FIG. 2 depicts traces of the size exclusion chromatography of unmodified IFN-β-1a and 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a: Panel A: molecular weight standards; Panel B: 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a; Panel C, unmodified IFN-β-1a.
FIG. 3 is a trace of the size exclusion chromatography of 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde-modified IFN-β-1a.
FIG. 4 is a reducing SDS-PAGE gel showing the purity of unmodified IFN-β-1a and 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a: Lane A: 2.5 μg of 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a; Lane B: 2.5 μg of unmodified IFN-β-1a; Lane C: molecular weight markers (from top to bottom; 100 kDa, 68 kDa, 45 kDa, 27 kDa, and 18 kDa, respectively).
FIG. 5 depicts traces of the size exclusion chromatography of 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a; Panel A: molecular weight standards; Panel B: 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a.
FIG. 6 is a reducing SDS-PAGE gel depicting the stability of 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a: Lane A: molecular weight markers (from top to bottom; 100 kDa, 68 kDa, 45 kDa, 27 kDa, 18 kDa, and 15 kDa, respectively); Lanes B, C, D, and E: 2 μg of 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a removed for assay at day 0, 2, 5, and 7, respectively.
FIGS. 7A-B show the antiviral activity of various PEGylated human IFN-β-1a samples as a function of protein concentration: FIG. 7A; unmodified IFN-β-1a (o), 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a (□), 20 kDa mPEG-O-p-methylphenyl-O-2-methylpropionaldehyde-modified IFN-β-1a (Δ), and 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde-modified IFN-β-1a (⋄). FIG. 7B; unmodified IFN-β-1a (∘), 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a (□), 20 kDa mPEG-O-p-phenylpropionaldehyde-modified IFN-β-1a (Δ), and 20 kDa mPEG-O-m-phenylacetaldehyde-modified IFN-β-1a (⋄).
FIGS. 8A-B are graphs depicting the pharmacokinetics of unmodified and various PEGylated human IFN-β-1a samples: FIG. 8A: Unmodified IFN-β-1a (upper panel) and IFN-β-1a modified with 20 kDa mPEG-O-2-methylpropionaldehyde (lower panel); FIG. 8B: IFN-β-1a modified with 20 kDa mPEG-O-p-methylphenyl-O-2-methylpropionaldehyde (upper panel) and 20 kDa mPEG-O-p-phenylacetaldehyde (lower panel).
FIGS. 9A-B are graphs depicting the pharmacokinetics of unmodified and various PEGylated human IFN-β-1a samples: FIG. 9A: Unmodified IFN-β-1a (upper panel) and IFN-β-1a modified with 20 kDa mPEG-O-p-phenylpropionaldehyde (lower panel); FIG. 9B: IFN-β-1a modified with 20 kDa mPEG-O-m-phenylacetaldehyde (upper panel) and 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde (lower panel).
FIG. 10 is a bar graph comparing a single administration of 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a, with daily administration of unmodified IFN-β-1a at reducing the number of radially-oriented neovessels in nu/nu mice carrying SK-MEL-1 human malignant melanoma cells: treatment with vehicle control once on day 1 only (bar A); treatment with 1 MU (5 μg) of unmodified IFN-β-1a daily on days 1-9 inclusive (bar B); treatment with 1 MU (10 μg) of 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a once on day 1 only (bar C); and treatment with vehicle control daily on days 1-9 inclusive (bar D).
DETAILED DESCRIPTION OF THE INVENTION
The invention is directed to compounds and methods useful in the treatment of various diseases and disorders. As explained in detail below, such diseases and disorders include, in particular, those which are susceptible to treatment with interferon therapy, including but not limited to viral infections such as hepatitis infections and autoimmune diseases such as multiple sclerosis.
The compounds of the invention include novel, activated polyalkylene glycol compounds according to Formula I:
where P is a water soluble polymer such as a polyalkylene glycol polymer. A non-limiting list of such polymers include other polyalkylene oxide homopolymers such as polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof. Other examples of suitable water-soluble and non-peptidic polymer backbones include poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxypropylmethacrylamide), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine) and copolymers, terpolymers, and mixtures thereof. In one embodiment, the polymer backbone is poly(ethylene glycol) or monomethoxy polyethylene glycol (mPEG) having an average molecular weight from about 200 Da to about 400,000 Da. It should be understood that other related polymers are also suitable for use in the practice of this invention and that the use of the term PEG or poly(ethylene glycol) is intended to be inclusive and not exclusive in this respect. The term PEG includes poly(ethylene glycol) in any of its forms, including alkoxy PEG, difunctional PEG, multi-armed PEG, forked PEG, branched PEG, pendent PEG, or PEG with degradable linkages therein.
In the class of compounds represented by Formula I, there are between zero and five methylene groups between Y and the Z-containing carbon (e.g., n is zero or an integer from one to five) and m is zero or one, e.g., Y is present or absent.
X and Y are, independently, O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′. In some embodiments, X and Y are oxygen.
Q is a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl (including fused bicyclic and bridged bicyclic ring structures), a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group. The substituents can be halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, sulfamoyl, sulfonate, silyl, ether, or alkylthio.
The Z substituent is hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group. The substituents can be halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, sulfamoyl, sulfonate, silyl, ether, or alkylthio.
When X or Y is NR′, R′ can be hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl, wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group. The substituents can be halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, sulfamoyl, sulfonate, silyl, ether, or alkylthio.
R is a reactive functional group, i.e., an activating moiety capable of reacting to form a linkage or a bond between the compound of Formula I and a biologically-active compound or precursor thereof. Thus, R represents the “activating group” of the activated polyalkylene glycol compounds (PGCs) represented by Formula I. R can be, for example, a carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, or glyoxal. In particular embodiments, R is an aldehyde hydrate.
Specific examples of R in the literature include N-succinimidyl carbonate (see e.g., U.S. Pat. Nos. 5,281,698, 5,468,478), amine (see e.g., Buckmann et al. Makromol. Chem. 182:1379 (1981), Zaplipsky et al. Eur. Polym. J. 19:1177 (1983)), hydrazide (See, e.g., Andresz et al. Makromol. Chem. 179:301 (1978)), succinimidyl propionate and succinimidyl butanoate (see, e.g. Olson et al. in Poly (ethylene glycol) Chemistry & Biological Applications, pp 170-181, Harris & Zaplipsky Eds., ACS, Washington, D.C., 1997; see also U.S. Pat. No. 5,672,662), succinimidyl succinate (See. e.g., Abuchowski et al. Cancer Biochem. Biophys. 7:175 (1984) and Joppich et al. Macrolol. Chem. 180:1381(1979), succinimidyl ester (see, e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonate (see, e.g., U.S. Pat. No. 5 5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. J. Biochem. 94:11(1979), Elling et al., Biotech. Appl. Biochem. 13:354 (1991), oxycarbonylimidazole (see, e.g. Beauchamp, et al., Anal. Biochem. 131:25 (1983). Tondelli et al. J. Controlled Release 1:251 (1985)), p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl. Biochem. Biotech., 11:141 (1985); and Sartore et al., Appl. Biochem. Biotech. 27:45 10 (1991)), aldehyde (see, e.g., Harris et al. J. Polym. Sci. Chem. Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No. 5,252,714), maleimide (see, e.g., Goodson et al. Bio/Technology 8:343 (1990), Romani et al. in Chemistry of Peptides and Proteins 2:29 (1984)), and Kogan, Synthetic Comm. 22:2417 (1992)), orthopyridyl-disulfide (see, e.g., Woghiren, et al. Bioconj. Chem. 4:314 (1993)), acrylol (see, e.g., Sawhney 15 et al., Macromolecules, 26:581 (1993)), vinylsulfone (see, e.g., U.S. Pat. No. 5,900,461). In addition, two molecules of the polymer of this invention can also be linked to the amino acid lysine to form a di-substituted lysine, which can then be further activated with N-hydroxysuccinimide to form an active N-succinimidyl moiety (see, e.g., U.S. Pat. No. 5,932,462).
The terms “functional group”, “active moiety”, “active group”, “activating group”, “activating moiety”, “reactive site”, “chemically-reactive group” and “chemically-reactive moiety” are used in the art and herein to refer to distinct, definable portions or units of a molecule. The terms are somewhat synonymous in the chemical arts and are used herein to indicate the portions of molecules having a characteristic chemical activity and which are typically reactive with other molecules. The term “active,” when used in conjunction with functional groups, is intended to include those functional groups that react readily with electrophilic or nucleophilic groups on other molecules, in contrast to those groups that require strong catalysts or highly impractical reaction conditions in order to react. For example, as would be understood in the art, the term “active ester” would include those esters that react readily with nucleophilic groups such as amines. Typically, an active ester will react with an amine in aqueous medium in a matter of minutes, whereas certain esters, such as methyl or ethyl esters, require a strong catalyst in order to react with a nucleophilic group.
In the compounds of the invention as defined above, the functional group R becomes a linking moiety, R*, after it has reacted with a biologically-active molecule to form a linkage or bond between the activated polyalkylene glycol compound (PGC) and the biologically-active compound. Thus, B is a biologically-active compound after conjugation to the PGC and R* is a moiety formed by the reaction of R on the activated PGC with one or more reactive functional groups on the biologically-active compound, B, such that a single covalent attachment results between the PGC and biologically-active compound. In a preferred embodiment, R* is a moiety formed by the reaction of R on the activated PGC with a single reactive functional group on the biologically-active compound, such that a covalent attachment results between the activated polyalkylene glycol compound (PGC) and the biologically-active compound.
The biologically-active compound or precursor thereof (B) is preferably not adversely affected by the presence of the PGC. Additionally, B either naturally has a functional group which is able to react with and form a linkage with the activated PGC, or is modified to contain such a reactive group.
As used herein, a precursor of B is an inactive or less active form of B that changes to the active or more active form, respectively, upon contact with physiological conditions, e.g., administration to a subject. Such changes can be conformational or structural changes, including, but not limited to, changing from a protected form to a non-protected form of B. As used herein, such change does not include release of the conjugated PGCs of this invention.
As would be understood in the art, the term “protected” refers to the presence of a protecting group or moiety that prevents reaction of the chemically-reactive functional group under certain reaction conditions. The protecting group will vary depending on the type of chemically-reactive group being protected. For example, if the chemically-reactive group is an amine or a hydrazide, the protecting group can be selected from the group of tert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). If the chemically-reactive group is a thiol, the protecting group can be orthopyridyldisulfide. If the chemically-reactive group is a carboxylic acid, such as butanoic or propionic acid, or a hydroxyl group, the protecting group can be benzyl or an alkyl group such as methyl or ethyl. Other protecting groups known in the art may also be used in the invention.
The terms “linking moiety”, “linkage” or “linker” are used herein to refer to moieties or bonds that are formed as the result of a chemical reaction and typically are covalent linkages. Thus, the linkage represented by bond R*—B in the above formulae results from the reaction between an activated moiety, R, on the PGC with a biologically-active compound, i.e. B′—R* is the linking moiety formed from R upon reaction with B′, and B is the biologically-active compound as conjugated to the PGC by reaction of a functional group on B′ with R.
As used herein, the term “biologically-active compound” refers to those compounds that exhibit one or more biological responses or actions when administered to a subject and contain reactive groups that contain reactive moieties that are capable of reacting with and conjugating to at least one activated PGC of the invention. The term “biologically-active molecule”, “biologically-active moiety” or “biologically-active agent” when used herein means any substance which can affect any physical or biochemical properties of any subject, including but not limited to viruses, bacteria, fungi, plants, animals, and humans. In particular, as used herein, biologically-active molecules include any substance intended for diagnosis, cure, mitigation, treatment, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental well-being of humans or animals.
Examples of biologically-active molecules include, but are not limited to, peptides, peptide analogs, proteins, enzymes, small molecules, dyes, lipids, nucleosides, oligonucleotides, analogs of oligonucleotides, sugars, oligosaccharides, cells, viruses, liposomes, microparticles, surfaces and micelles.
Classes of biologically-active agents that are suitable for use with the invention include, but are not limited to, chemokines, lymphokines, antibodies, soluble receptors, anti-tumor agents, anti-anxiety agents, hormones, growth factors, antibiotics, fungicides, fungistatic agents, anti-viral agents, steroidal agents, antimicrobial agents, germicidal agents, antipyretic agents, antidiabetic agents, bronchodilators, antidiarrheal agents, coronary dilation agents, glycosides, spasmolytics, antihypertensive agents, antidepressants, antianxiety agents, other psychotherapeutic agents, corticosteroids, analgesics, contraceptives, nonsteroidal anti-inflammatory drugs, blood glucose lowering agents, cholesterol lowering agents, anticonvulsant agents, other antiepileptic agents, immunomodulators, anticholinergics, sympatholytics, sympathomimetics, vasodilatory agents, anticoagulants, antiarrhythmics, prostaglandins having various pharmacologic activities, diuretics, sleep aids, antihistaminic agents, antineoplastic agents, oncolytic agents, antiandrogens, antimalarial agents, antileprosy agents, and various other types of drugs. See Goodman and Gilman's The Basis of Therapeutics (Ninth Edition, Pergamon Press, Inc., USA, 1996) and The Merck Index (Thirteenth Edition, Merck & Co., Inc., USA, 2001), each of which is incorporated herein by reference.
Biologically-active compounds include any compound that exhibits a biological response in its present form, or any compound that exhibits a biological response as a result of a chemical conversion of its structure from its present form. For example, biologically-active compounds will include any compound that contains a protective group that, when cleaved, results in a compound that exhibits a biological response. Such cleavage can be the result, for example, of an in vivo reaction of the compound with endogenous enzymes or a pre-administration reaction of the compound, including its reaction with the activated PGCs of this invention. As a further example, biologically-active compounds will also include any compound which undergoes a stereotransformation, in vivo or ex vivo, to form a compound that exhibits a biological response or action.
Biologically-active compounds typically contain several reactive sites at which covalent attachment of the activated PGC is feasible. For example, amine groups can undergo acylations, sulfhydryl groups can undergo addition reactions and alkylations, carbonyl and carboxyl groups can undergo acylations, and aldehyde and hydroxyl groups can undergo amination and reductive amination. One or more of these reactions can be used in the preparation of the polyalkylene glycol-modified biologically-active compounds of the invention. In addition, biologically-active compounds can be modified to form reactive moieties on the compound that facilitate such reactions and the resultant conjugation to the activated PGC.
Those of ordinary skill will recognize numerous reaction mechanisms available to facilitate conjugation of the activated PGC to a biologically-active compound. For example, when the activating moiety, R, is a hydrazide group, it can be covalently coupled to sulfhydryl, sugar, and carbonyl moieties on the biologically-active compounds (after these moieties undergo oxidation to produce aldehydes). The reaction of hydrazide activating moieties (R) with aldehydes on biologically-active compounds (B′) creates a hydrazone linkage (R*—B). When R is a maleimide group, it can be reacted with a sulfhydryl group to form a stable thioether linkage. If sulfhydryls are not present on the biologically-active compound, they may be created through disulfide reduction or through thiolation with 2-iminothiolane or SATA. When R is an imidoester it will react with primary amines on B′ to form an imidoamide linkage. Imidoester conjugation is usually performed between pH 8.5-9.0. When connecting the activated PGCs to biologically-active proteins, imidoesters provide an advantage over other R groups since they do not affect the overall charge of the protein. They carry a positive charge at physiological pH, as do the primary amines they replace. Imidoester reactions are carried out between 0° C. and room temperature (e.g., at 4° C.), or at elevated temperatures under anhydrous conditions. When R is an NHS-ester, its principal target is primary amines. Accessible α-amine groups, for example those present on the N-termini of peptides and proteins, react with NHS-esters to form a covalent amide bond.
In some embodiments, R*—B is a hydrolytically-stable linkage. A hydrolytically stable linkage means that the linkage is substantially stable in water and does not react with water at useful pHs, e.g., the linkage is stable under physiological conditions for an extended period of time, perhaps even indefinitely. In other embodiments, R*—B is a hydrolytically-unstable or degradable linkage. A hydrolytically-unstable linkage means that the linkage is degradable in water or in aqueous solutions, including for example, blood. Enzymatically-unstable or degradable linkages also means that the linkage can be degraded by one or more enzymes.
As understood in the art, polyalkylene and related polymers may include degradable linkages in the polymer backbone or in the linker group between the polymer backbone and one or more of the terminal functional groups of the PGC molecule. For example, ester linkages formed by the reaction of, e.g., PGC carboxylic acids or activated PGC carboxylic acids with alcohol groups on a biologically-active compound generally hydrolyze under physiological conditions to release the agent. Other hydrolytically-degradable linkages include carbonate linkages; imine linkages resulted from reaction of an amine and an aldehyde (See, e.g., Ouchi et al., Polymer Preprints, 38(1):582-3 (1997)); phosphate ester linkages formed by reacting an alcohol with a phosphate group; acetal linkages that are the reaction product of an aldehyde and an alcohol; orthoester linkages that are the reaction product of a formate and an alcohol; peptide linkages formed by an amine group, e.g., at an end of a the PGC, and a carboxyl group of a peptide; and oligonucleotide linkages formed by a phosphoramidite group, e.g., at the end of a polymer, and a 5′ hydroxyl group of an oligonucleotide.
The polyalkylene glycol, P, can be polyethylene glycol, having the structure of Formula II:
E-(O—CH2CH2)a—, Formula II:
wherein a is an integer from 4 to 10,000 and E is hydrogen or a straight- or branched-chain C1 to C20 alkyl group, a detectable label, or a moiety suitable for forming a bond between the compound of Formula I and a biologically-active compound or precursor thereof.
Thus, when E is a moiety suitable for forming a bond between the compound of Formula I and a biologically-active compound or precursor thereof, E can be a carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, or glyoxal. It is to be understood that E should be compatible with R so that reaction between E and R does not occur.
By “detectable label” is meant any label capable of detection. Non-limiting examples include radioactive isotopes, fluorescent moieties, phosphorescent moieties, chemiluminescent moieties, and quantum dots. Other detectable labels include biotin, cysteine, histidine, haemagglutinin, myc or flag tags.
In some embodiments, E has the structure according to Formula III or Formula IV:
Each Q, X, Y, Z, m, and n are as defined above, and each W is, independently, hydrogen or a C1 to C7 alkyl.
In this class of compounds, R″ is a moiety suitable for forming a bond between the compound of Formula III and a biologically-active compound or precursor thereof; and R′″ is a moiety suitable for forming a bond between the compound of Formula IV and a biologically-active compound or precursor thereof.
R″ and R′″ can be of carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, or glyoxal. It is to be understood that R″ and R′″ should be compatible with R so that reaction with R does not occur.
As used herein, R″ and R′″, upon conjugation to a biologically-active compound or precursor thereof, form linking moieties as defined above. Thus, R** is a linking moiety formed by the reaction of the R″ or R′″ group on the activated PGC with a reactive functional group on the biologically-active compound, such that a covalent attachment results between the PGC and the biologically-active compound. R and R″ or R′″ can be the same moiety or different moieties, and the biologically-active compound bound to each can be the same or different.
As used herein, the term “alkyl” refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g. C1-C30 for straight chain, C3-C30 for branched chain), and more preferably 20 or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6, or 7 carbons in the ring structure.
Moreover, the term “alkyl” (or “lower alkyl”) is intended to include both “unsubstituted alkyls” and “substituted alkyls”, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl may include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl, and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), —CF3, —CN and the like. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls, —CF3, —CN, and the like.
The term “aralkyl”, as used herein, refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group). Exemplary aralkyl groups include, but are not limited to, benzyl and more generally (CH2)nPh, where Ph is phenyl or substituted phenyl, and n is 1, 2, or 3.
The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond, respectively.
Unless the number of carbons is otherwise specified, “lower alkyl” as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths. Preferred alkyl groups are lower alkyls. In preferred embodiments, a substituent designated herein as alkyl is a lower alkyl.
The term “aryl” as used herein includes 5-, 6-, and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as “aryl heterocycles” or “heteroaromatics.” The aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, —CF3, —CN, or the like. The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, and/or heterocyclyls.
The terms ortho, meta and para apply to 1,2-, 1,3-, and 1,4-disubstituted benzenes, respectively. For example, the names 1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.
The terms “heterocyclyl” or “heterocyclic group” refer to 3- to 10-membered ring structures, more preferably 3- to 7-membered rings, whose ring structures include one to four heteroatoms. Heterocycles can also be polycycles. Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. The heterocyclic ring can be substituted at one or more positions with substituents as described above, such as, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, —CF3, —CN, or the like.
The term “carbocycle”, as used herein, refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon.
Heterocycles and carbocycles include fused bicyclic and bridged bicyclic ring structures.
As used herein, the term “nitro” means —NO2; the term “halogen” designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term “hydroxyl” means —OH; and the term “sulfonyl” means —SO2—.
The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula:
wherein R9, R10 and R′10 each independently represent a hydrogen, an alkyl, an alkenyl, —(CH2)m—R8, or R9 and R10 taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; R8 represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8.
The term “alkylamine” as used herein means an amine group, as defined above, having a substituted or unsubstituted alkyl attached thereto, i.e., at least one of R9 and R10 is an alkyl group.
The term “acylamino” is art-recognized and refers to a moiety that can be represented by the general formula:
wherein R9 is as defined above, and R′11 represents a hydrogen, an alkyl, an alkenyl or —(CH2)m—R8, where m and R8 are as defined above.
The term “amido” is art-recognized as an amino-substituted carbonyl and includes a moiety that can be represented by the general formula:
wherein R9, R10 are as defined above. Preferred embodiments of the amide will not include imides which may be unstable.
The term “amidine” is art-recognized as a group that can be represented by the general formula:
wherein R9, R10 are as defined above.
The term “guanidine” is art-recognized as a group that can be represented by the general formula:
wherein R9, R10 are as defined above.
The term “alkylthio” refers to an alkyl group, as defined above, having a sulfur radical attached thereto. In preferred embodiments, the “alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl, —S-alkynyl, and —S—(CH2)m—R8, wherein m and R8 are defined above. Representative alkylthio groups include methylthio, ethylthio, and the like.
The term “carbonyl” is art-recognized and includes moieties that can be represented by the general formula:
wherein X is a bond or represents an oxygen or a sulfur, and R11 represents a hydrogen, an alkyl, an alkenyl, —(CH2)m—R8 or a pharmaceutically-acceptable salt, R′11 represents a hydrogen, an alkyl, an alkenyl or —(CH2)m—R8, where m and R8 are as defined above. Where X is an oxygen and R11 or R′11 is not hydrogen, the formula represents an “ester”. Where X is an oxygen, and R11 is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when R11 is a hydrogen, the formula represents a “carboxylic acid”. Where X is an oxygen, and R′11 is hydrogen, the formula represents a “formate”. In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a “thiolcarbonyl” group. Where X is a sulfur and R11 or R′11 is not hydrogen, the formula represents a “thioester.” Where X is a sulfur and R11 is hydrogen, the formula represents a “thiocarboxylic acid.” Where X is a sulfur and R′11 is hydrogen, the formula represents a “thioformate.” On the other hand, where X is a bond, and R11 is not hydrogen, the above formula represents a “ketone” group. Where X is a bond, and R11 is hydrogen, the above formula represents an “aldehyde” group.
The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy, and the like. An “ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH2)m—R8, where m and R8 are described above.
The term “sulfonate” is art-recognized and includes a moiety that can be represented by the general formula:
in which R41 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.
The term “sulfate” is art recognized and includes a moiety that can be represented by the general formula:
in which R41 is as defined above.
The term “sulfonamido” is art recognized and includes a moiety that can be represented by the general formula:
in which R9 and R′11 are as defined above.
The term “sulfamoyl” is art recognized and includes a moiety that can be represented by the general formula:
in which R9 and R10 are as defined above.
The term “sulfonyl”, as used herein, refers to a moiety that can be represented by the general formula:
in which R44 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl.
The term “sulfoxido” as used herein, refers to a moiety that can be represented by the general formula:
in which R44 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.
It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein above. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.
A comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry; this list is typically presented in a table entitled Standard List of Abbreviations. The abbreviations contained in said list, and all abbreviations utilized by organic chemists of ordinary skill in the art are hereby incorporated by reference.
In some embodiments, the compounds of the invention have the structure according to Formula V:
X, Y, m, n, Z, and R′ are as defined above, and R is an activating moiety as defined i above, suitable for forming a bond between the compound of Formula V and a biologically-active compound or precursor. In particular embodiments, R is an aldehyde hydrate.
P is as defined above, and can be represented by Formula II
E-(O—CH2CH2)a—, Formula II:
where E is as described above, and in some embodiments, can be represented by Formula III or IV.
T1 and T2 are, independently, absent, or a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group. The substituents can be halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, or alkylthio.
When d is zero, there are no additional substituents (L) on the aromatic ring. When d is an integer from 1 to 4, the substituents (L) can be a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group. The substituents can be halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, or alkylthio.
When R is an aldehyde, the compounds fall within those represented by Formula VI:
where all other variables are as defined above.
For example, when X and Y are oxygen and R is an aldehyde, the compounds of the invention are represented by compound J.
where the T1 and T2 substituents can be in the ortho, meta, or para arrangement.
Where the T1 and T2 substituents are straight-chain alkyl groups, and d is zero, the compounds are represented by Formula IX:
where each u is independently zero or an integer from one to five and all other variables are as defined above. In particular embodiments, Z is hydrogen or methyl.
Particular classes of compounds falling within Formula IX can be represented by Formulae VII and VIII:
Some representative activated polyalkylene glycol compounds include the following, where the polyalkylene glycol polymer is PEG or mPEG:
In some embodiments, the compounds of the invention are represented by Formula X:
where, as above, n is zero or an integer from one to five, and X is O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′.
When X is NR′, R′ can be hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, or alkylthio. Z can be a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group. When present, the substituents can be halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, or alkylthio.
As defined above, R is an activating moiety suitable for forming a bond between the compound of Formula X and a biologically-active compound or precursor thereof. In some embodiments, R is an aldehyde hydrate.
P is a polyalkylene glycol polymer as defined above, and can be represented by Formula II:
E-(O—CH2CH2)a—, Formula II:
where E and a are as described above, and in some embodiments, can be represented by Formula III or IV. In some embodiments, E is methyl, and, therefore, P is mPEG.
When R is an aldehyde and X is oxygen, the compounds fall within the structure according to Formula XI:
where P, Z and n are as defined for Formula X.
When P is mPEG, the compounds are described by Formula XII:
and when n is one and Z is methyl, the compound is represented by Formula XIII:
wherein a is an integer from 4 to 10,000.
Examples of synthetic pathways for making compounds according to the invention are set forth in the Examples below.
The invention also includes compositions of the activated polyalkylene glycol compounds (PGCs) of the invention and one or more biologically-active compounds. As described above, biologically-active compounds are those compounds that exhibit a biological response or action when administered to a subject. Unconjugated biologically-active compounds may be administered to a subject in addition to the compounds of the invention. Additionally, biologically-active compounds may contain reactive groups that are capable of reacting with and conjugating to at least one activated PGC of the invention.
The invention also includes conjugates of the novel PGCs with biologically-active compounds. In one embodiment, the conjugates are formed from a compound of Formula I and a biologically-active compound (B) and are described according to Formula XIV:
As above, m is zero or one so that Y is present or absent, n is zero or an integer from one to five, and X and Y are independently O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′.
Q is a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl (including fused bicyclic and bridged bicyclic ring structures), a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group. When present, the substituents can be halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, or alkylthio.
Each R′ and Z is independently hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, wherein the substituents are selected from the group consisting of halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, and alkylthio;
R* is a linking moiety formed from the reaction of R with a corresponding functional group on the biologically-active compound, B, as described above. For example, R* is formed from the reaction of a moiety such as a carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, or glyoxal functionality with a biologically-active compound or precursor thereof.
P is a polyalkylene glycol polymer as defined above, and can be represented by Formula II:
E-(O—CH2CH2)a—, Formula II:
where E is hydrogen, a straight- or branched-chain C1 to C20 alkyl group (e.g., methyl), a detectable label, or a moiety suitable for forming a bond between the compound of Formula XIV and a biologically-active compound or precursor thereof. As above, a is an integer from 4 to 10,000.
Where E is a detectable label, the label can be, for example, a radioactive isotope, a fluorescent moiety, a phosphorescent moiety, a chemiluminescent moiety, or a quantum dot.
When E is a moiety suitable for forming a bond between the compound of Formula XIV and a biologically-active compound or precursor thereof, E can form a bond to another molecule of the biologically-active compound (B) so that the activated polyalkylene glycol compound is bound at either terminus to a molecule of the same type of biologically-active compound, to produce a dimer of the molecule.
In some embodiments, E forms a bond to a biologically-active compound other than B, creating a heterodimer of biologically-active compounds or precursors thereof.
In other embodiments, E forms an additional bond to the biologically-active compound, B, such that both E and R are bound through different functional groups of the same molecule of the biologically-active compound or precursor thereof.
When E is capable of forming a bond to a biologically-active molecule or precursor thereof, E can be the same as or different from R and is chosen from carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal moieties.
When E is capable of forming a bond to a biologically-active molecule or precursor thereof, E can have the structure according to Formula III or Formula IV:
where each Q, X, Y, Z, m, and n are, independently, as defined above, each W is, independently, hydrogen or a C1 to C7 alkyl, R″ is a moiety suitable for forming a bond between the compound of Formula III and a biologically-active compound or precursor thereof, and R′″ is a moiety suitable for forming a bond between the compound of Formula IV and a biologically-active compound or precursor thereof.
R″ and R′″ are, independently chosen from carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal moieties.
When Q in Formula XIV is a substituted or unsubstituted alkaryl, the conjugate is formed from an activated polyalkylene glycol of Formula V and a biologically-active molecule (B), and is described according to Formula XV:
where T1 and T2 are, independently, absent, or a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group. When present, the substituents can be halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, or alkylthio. In some embodiments, T1 and T2, if present, are straight- or branched-chain saturated or unsaturated or C1 to C20 alkyl or heteroalkyl group.
d is zero (e.g., there are no L substituents on the aromatic ring) or an integer from 1 to 4. Each L is, when present, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group. The substituents can be halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, or alkylthio.
All other variables are as described above, including P, which is a polyalkylene glycol polymer, and can be represented by Formula II:
E-(O—CH2CH2)a—, Formula II:
where E is hydrogen, a straight- or branched-chain C1 to C20 alkyl group (e.g., methyl), a detectable label, or a moiety suitable for forming a bond between the compound of Formula XV and a biologically-active compound or precursor thereof. As above, a is an integer from 4 to 10,000.
Where E is a detectable label, the label can be, for example, a radioactive isotope, a fluorescent moiety, a phosphorescent moiety, a chemiluminescent moiety, or a quantum dot.
When E is a moiety suitable for forming a bond between the compound of Formula XV, and a biologically-active compound, B, E can form a bond to another molecule of the biologically-active compound (B) so that the activated polyalkylene glycol compound is bound at either terminus to a molecule of the same type of biologically-active compound, to produce a dimer of the molecule.
In some embodiments, E forms a bond to a biologically-active compound other than B, creating a heterodimer of biologically-active compounds or precursors thereof.
In other embodiments, E forms an additional bond to the biologically-active compound, B, such that both E and R are bound through different functional groups of the same molecule of the biologically-active compound or precursor thereof.
When E is capable of forming a bond to a biologically-active molecule or precursor thereof, E can be the same as or different from R and is chosen from carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal moieties.
When E can form a bond with a biologically-active compound or precursor thereof, in some embodiments, E can be Formula III or Formula IV:
where each Q, X, Y, Z, m, and n are, independently, as defined above, each W is, independently, hydrogen or a C1 to C7 alkyl, R″ is a moiety suitable for forming a bond between the compound of Formula III and a biologically-active compound or precursor thereof, and R′″ is a moiety suitable for forming a bond between the compound of Formula IV and a biologically-active compound or precursor thereof.
R″ and R′″ are, independently chosen from carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal moieties.
When bound at both ends to a biologically-active compound or precursor thereof, these bifunctional molecules can be represented according to Formula XX or Formula XXI:
where each X and Y, T1 and T2, R′ and Z, L, Q, m, n, a, and n are as described above, and each W is, independently, hydrogen or a C1 to C7 alkyl. R* and R** are, independently, linking moieties formed from the reaction of R and R″ with a biologically-active compound or precursor thereof, and B and B′ are each a biologically-active compound, or precursor thereof, after conjugation with R and R″, respectively.
In some embodiments, B and B′ are the same type of biologically-active compound. In other embodiments, B and B′ are different biologically-active compounds. In still other embodiments, B and B′ are the same biologically active molecule. In additional embodiments, R* and R** are the same. In other embodiments, R* and R** are different. For example, in some embodiments, E can form a bond to another molecule of the biologically-active compound (B═B′) so that the activated PGC is bound at either terminus to a molecule of the same type of biologically-active compound, to produce a dimer of the molecule. In some embodiments, E forms a bond to a biologically-active compound other than B (B is not B′), creating a heterodimer of biologically-active compounds or precursors thereof. In other embodiments, E forms an additional bond to the biologically-active compound, B, such that both E (through R″ or R′″) and R are bound through different functional groups of the same molecule of the biologically-active compound or precursor thereof.
In some embodiments, R* or R** is methylene group and B or B′ is a biologically-active molecule containing an amino group, where the methylene group forms a bond with the amino group on B. For example, the amine can be the amino terminus of a peptide, an amine of an amino acid side chain of a peptide, or an amine of a glycosylation substituent of a glycosylated peptide. In some embodiments, the peptide is an interferon, such as interferon-beta, e.g., interferon-beta-1a. In some embodiments, this type of bond is formed by a reductive alkylation reaction.
Where the T1 and T2 substituents of Formula XV are straight-chain alkyl groups, X and Y are oxygen, and d is zero, the conjugates are represented by Formula XIX:
where each u is independently zero or an integer from one to five and all other variables are as defined above. In particular embodiments, Z is hydrogen or methyl.
Particular classes of compounds falling within Formula XV can be represented by Formulae XVII and XVIII formed from the reaction of Formulae VII and VIII, respectively, with a biologically-active compound, or precursor thereof:
where n is zero or an integer from one to five, P is a polyalkylene glycol polymer, as described above, Z is hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, R* is a linking moiety as described above, B is a biologically-active molecule. These compounds can be bifunctional or monofunctional, depending on the identity of E, as described above.
In some embodiments, R* is a methylene group and B is a biologically-active molecule containing an amino group, where the methylene group forms a bond with the amino group on B. For example, the amine scan be the amino terminus of a peptide, an amine of an amino acid side chain of a peptide, or an amine of a glycosylation substituent of a glycosylated peptide. In some embodiments, the peptide is an interferon, such as interferon-beta, e.g., interferon-beta-1a. In some embodiments, this type of bond is formed by a reductive alkylation reaction.
The conjugates of the invention can also be formed from reaction of compounds according to Formula X with a biologically-active compound or precursor thereof, to form conjugates according to Formula XXIIa:
where B is a biologically-active molecule, as described above and n is zero or an integer from one to five.
X is O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′, when X is NR′, R′ is hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group. If present, the substituents can be halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, or alkylthio.
Z is a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group. The substituents can be halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, or alkylthio.
R* is a linking moiety formed from the reaction of R with a corresponding functional group on the biologically-active compound, B, as described above. For example, R* is formed from the reaction of a moiety such as a carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, or glyoxal functionality with a biologically-active compound or precursor thereof.
In some embodiments, Z is methyl and n is one.
P is a polyalkylene glycol polymer as defined above, and can be represented by Formula II:
E-(O—CH2CH2)a—, Formula II:
where E is hydrogen, a straight- or branched-chain C1 to C20 alkyl group (e.g., methyl), a detectable label, or a moiety suitable for forming a bond between the compound of Formula XXII and a biologically-active compound or precursor thereof. As above, a is an integer from 4 to 10,000.
Where E is a detectable label, the label can be, for example, a radioactive isotope, a fluorescent moiety, a phosphorescent moiety, a chemiluminescent moiety, or a quantum dot.
When E is capable of forming a bond to a biologically-active molecule or precursor thereof, a bifunctional molecule results. E can form a bond to another molecule of the biologically-active compound (B) so that the activated polyalkylene glycol compound is bound at either terminus to a molecule of the same type of biologically-active compound, to produce a dimer of the molecule.
In some embodiments, E forms a bond to a biologically-active compound other than B, creating a heterodimer of biologically-active compounds or precursors thereof.
In other embodiments, E forms an additional bond to the biologically-active compound, B, such that both E and R are bound through different functional groups of the same molecule of the biologically-active compound or precursor thereof.
When E is capable of forming a bond to a biologically-active molecule or precursor thereof, E can be the same as or different from R and is chosen from carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal moieties.
In some embodiments, E can have the structure according to Formula III or Formula IV:
where each Q, X, Y, Z, m, and n are, independently, as defined above, each W is, independently, hydrogen or a C1 to C7 alkyl, R″ is a moiety suitable for forming a bond between the compound of Formula III and a biologically-active compound or precursor thereof, and R′″ is a moiety suitable for forming a bond between the compound of Formula IV and a biologically-active compound or precursor thereof.
R″ and R′″ are, independently chosen from carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal moieties, and can be the same or different from R.
When bound at both ends to a biologically-active compound or precursor thereof, these bifunctional molecules can be represented according to Formula XXIV or Formula XXV:
where each X and Y is independently O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, or NR′, and each R′ and Z is, independently, hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group.
Q is a C3 to C8 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl (including fused bicyclic and bridged bicyclic ring structures), a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group. If present, the substituents can be halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, or alkylthio.
Each W is, independently, hydrogen or a C1 to C7 alkyl, m is zero or one, a is an integer from 4 to 10,000, and each n is independently 0 or an integer from 1 to 5.
R* and R** are independently linking moieties as described above, B and B′ are independently biologically-active molecules and can be the same or different.
E (through R″ or R′″) can form a bond to another molecule of the biologically-active compound (B) so that the activated polyalkylene glycol compound is bound at either terminus to a molecule of the same type of biologically-active compound, to produce a dimer of the molecule.
In some embodiments, E (through R″ or R′″) forms a bond to a biologically-active compound other than B, creating a heterodimer of biologically-active compounds or precursors thereof.
In other embodiments, E (through R″ or R′″) forms an additional bond to the biologically-active compound, B, such that both E and R are bound through different functional groups of the same molecule of the biologically-active compound or precursor thereof.
R″ and R′″ can be the same as or different from R, and are chosen from carboxylic acid, ester, aldehyde, aldehyde hydrate, acetal, hydroxy, protected hydroxy, carbonate, alkenyl, acrylate, methacrylate, acrylamide, substituted or unsubstituted thiol, halogen, substituted or unsubstituted amine, protected amine, hydrazide, protected hydrazide, succinimidyl, isocyanate, isothiocyanate, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, hydroxysuccinimidyl, azole, maleimide, sulfone, allyl, vinylsulfone, tresyl, sulfo-N-succinimidyl, dione, mesyl, tosyl, and glyoxal moieties.
In some embodiments, R* or R** is a methylene group and B or B′ is a biologically-active molecule containing an amino group, where the methylene group forms a bond with the amino group on B. For example, the amine can be the amino terminus of a peptide, an amine of an amino acid side chain of a peptide, or an amine of a glycosylation substituent of a glycosylated peptide. In some embodiments, the peptide is an interferon, such as interferon-beta, e.g., interferon-beta-1a. In some embodiments, this type of bond is formed by a reductive alkylation reaction.
The conjugates of the invention can be prepared by coupling a biologically-active compound to a polyalkylene glycol compound as described in the Examples. In some embodiments, the coupling is achieved via a reductive alkylation reaction.
Biologically-active compounds of interest include any substance intended for diagnosis, cure mitigation, treatment, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental well-being of humans or animals. Examples of biologically-active molecules include, but are not limited to, peptides, peptide analogs, proteins, enzymes, small molecules, dyes, lipids, nucleosides, oligonucleotides, analogs of oligonucleotides, sugars, oligosaccharides, cells, viruses, liposomes, microparticles, surfaces and micelles. This class of compounds also include precursors of these types of molecules. Classes of biologically-active agents that are suitable for use with the invention include, but are not limited to, cytokines, chemokines, lymphokines, soluble receptors, antibodies, antibiotics, fungicides, anti-viral agents, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones, growth factors, steroidal agents, and the like.
The biologically-active compound can be a peptide, such as an interferon, including interferon-beta (e.g., interferon-beta-1a) or interferon-alpha.
Because the polymeric modification with a PGC of the invention reduces antigenic responses, a foreign peptide need not be completely autologous in order to be used as a therapeutic. For example, a peptide, such as interferon, used to prepare polymer conjugates may be prepared from a mammalian extract, such as human, ruminant, or bovine interferon, or can be synthetically or recombinantly produced.
For example, in one aspect, the invention is directed to compounds and methods for treating conditions that are susceptible of treatment with interferon alpha or beta. Administration of a polyalkylene glycol conjugated interferon beta (hereinafter “PGC IFN-beta”, “PGC IFN-β”, e.g., PEG IFN-beta”, “PEG IFN-β” “PEGylated IFN-beta”, or “PEGylated IFN-β”) provides improved therapeutic benefits, while substantially reducing or eliminating entirely the undesirable side effects normally associated with conventionally practiced interferon alpha or beta treatment regimes.
The PGC IFN-beta can be prepared by attaching a polyalkylene polymer to the terminal amino group of the IFN beta molecule. A single activated polyalkylene glycol molecule can be conjugated to the N-terminus of IFN beta via a reductive alkylation reaction.
The PGC IFN-beta conjugate can be formulated, for example, as a liquid or a lyophilized powder for injection. The objective of conjugation of IFN beta with a PGC is to improve the delivery of the protein by significantly prolonging its plasma half-life, and thereby provide protracted activity of IFN beta.
The term “interferon” or “IFN” as used herein means the family of highly homologous species-specific proteins that inhibit viral replication and cellular proliferation and modulate immune response. Human interferons are grouped into two classes; Type I, including α- and β-interferon, and Type II, which is represented by γ-interferon only. Recombinant forms of each group have been developed and are commercially available. Subtypes in each group are based on antigenic/structural characteristics.
The terms “beta interferon”, “beta-interferon”, “beta IFN”, “beta-IFN”, “β interferon”, “β-interferon”, “β IFN”, “IFN”, “interferon beta”, “interferon-beta”, “interferon β”, “interferon-β”, “IFN beta”, “IFN-beta”, “IFN β”, “IFN-β”, and “human fibroblast interferon” are used interchangeably herein to describe members of the group of interferon beta's which have distinct amino acid sequences as have been identified by isolating and sequencing DNA encoding the peptides.
Additionally, the terms “beta interferon 1a”, “beta interferon-1a” “beta-interferon 1a”, “beta-interferon-1a”, “beta IFN 1a”, “beta IFN-1a”, “beta-IFN 1a”, “beta-IFN-1a”, “β interferon 1a”, “β interferon-1a”, “β-interferon 1a”, “β-interferon-1a”, “β IFN 1a”, “β IFN-1a”, “β-IFN 1a”, “β-IFN-1a”, “interferon beta 1a, “interferon beta-1a”, “interferon-beta 1a”, “interferon-beta-1a”, “interferon β 1a”, “interferon β-1a”, “interferon-β 1a”, “interferon-β-1a”, “IFN beta 1a”, “IFN beta-1a”, “IFN-beta 1a”, “IFN-beta-1a”, “IFN β 1a”, “IFN β-1a”, “IFN-β 1a”, “IFN-β-1a” are used interchangeably herein to describe recombinantly- or synthetically-produced interferon beta that has the naturally-occurring (wild type) amino acid sequences.
The advent of recombinant DNA technology applied to interferon production has permitted several human interferons to be successfully synthesized, thereby enabling the large-scale fermentation, production, isolation, and purification of various interferons to homogeneity. Recombinantly produced interferon retains some—or most of—its in vitro and in vivo antiviral and immunomodulatory activities. It is also understood that recombinant techniques could also include a glycosylation site for addition of a carbohydrate moiety on the recombinantly-derived polypeptide.
The construction of recombinant DNA plasmids containing sequences encoding at least part of human fibroblast interferon and the expression of a polypeptide having immunological or biological activity of human fibroblast interferon is also contemplated. The construction of hybrid beta-interferon genes containing combinations of different subtype sequences can be accomplished by techniques known to those of skill in the art.
Typical suitable recombinant beta-interferons which may be used in the practice of the invention include but are not limited to interferon beta-1a such as AVONEX® available from Biogen, Inc., Cambridge, Mass., and interferon-beta-1b such as BETASERON® available from Berlex, Richmond, Calif.
There are many mechanisms by which IFN-induced gene products provide protective effects against viral infection. Such inhibitory viral effects occur at different stages of the viral life cycle. See. U.S. Pat. No. 6,030,785. For example, IFN can inhibit uncoating of viral particles, penetration, and/or fusion caused by viruses.
Conditions that can be treated in accordance with the present invention are generally those that are susceptible to treatment with interferon. For example, susceptible conditions include those, which would respond positively or favorably (as these terms are known in the medical arts) to interferon beta-based therapy. For purposes of the invention, conditions that can be treated with interferon beta therapy described herein include those conditions in which treatment with an interferon beta shows some efficacy, but in which the negative side effects of IFN-β treatment outweigh the benefits. Treatment according to the methods of the invention results in substantially reduced or eliminated side effects as compared to conventional interferon beta treatment. In addition, conditions traditionally thought to be refractory to IFN-β treatment, or those for which it is impractical to treat with a manageable dosage of IFN-β, can be treated in accordance with the methods of the present invention.
The PGC IFN-β compounds of the invention can be used alone or in combination with one or more agents useful for treatment for a particular condition. At least one pilot study of recombinant interferon beta-1a for the treatment of chronic hepatitis C has been conducted. See generally Habersetzer et al., Liver 30:437-441 (2000), incorporated herein by reference. For example, the compounds can be administered in combination with known antiviral agents for treatment of a viral infection. See Kakumu et al., Gastroenterology 105:507-12 (1993) and Pepinsky, et al., J. Pharmacology and Experimental Therapeutics, 297:1059-1066 (2001), incorporated herein by reference.
As used herein, the term “antivirals” may include, for example, small molecules, peptides, sugars, proteins, virus-derived molecules, protease inhibitors, nucleotide analogs and/or nucleoside analogs. A “small molecule” as the term is used herein refers to an organic molecule of less than about 2500 amu (atomic mass units), preferably less than about 1000 amu. Examples of suitable antiviral compounds include, but are not limited to, ribavirin, levovirin, MB6866, zidovudine 3TC, FTC, acyclovir, gancyclovir, viramide, VX497, VX-950, and ISIS-14803.
Exemplary conditions which can be treated with interferon include, but are not limited to, cell proliferation disorders, in particular multiple sclerosis, cancer (e.g., hairy cell leukemia, Kaposi's sarcoma, chronic myelogenous leukemia, multiple myeloma, basal cell carcinoma and malignant melanoma, ovarian cancer, cutaneous T cell lymphoma), and viral infections. Without limitation, treatment with interferon may be used to treat conditions which would benefit from inhibiting the replication of interferon-sensitive viruses. For example, interferon can be used alone or in combination with AZT in the treatment of human immunodeficiency virus (HIV)/AIDS or in combination with ribavirin in the treatment of HCV. Viral infections which may be treated in accordance with the invention include, but are not limited to, hepatitis A, hepatitis B, hepatitis C, other non-A/non-B hepatitis, herpes virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex, human herpes virus type 6 (HHV-6), papilloma, poxvirus, picomavirus, adenovirus, rhinovirus, human T lymphotropic virus-type 1 and 2 (HTLV-1/-2), human rotavirus, rabies, retroviruses including HIV, encephalitis, and respiratory viral infections. The methods of the invention can also be used to modify various immune responses.
A correlation between HCV genotype and response to interferon therapy has been observed. See U.S. Pat. No. 6,030,785; Enomoto et al., N. Engl. J. Med. 334:77-81 (1996); Enomoto et al., J. Clin. Invest. 96:224-30 (1995). The response rate in patients infected with HCV-1b is less than 40%. See U.S. Pat. No. 6,030,785. Similar low response rates have also been observed in patients infected with HCV-1a. See id.; Hoofnagel et al., Intervirology 37:87-100 (1994). However, the response rate in patients infected with HCV-2 is nearly 80%. See U.S. Pat. No. 6,030,785; Fried et al., Semin. Liver Dis. 15:82-91 (1995). In fact, an amino acid sequence of a discrete region of the NS5A protein of HCV genotype 1b was found to correlate with sensitivity to interferon. See U.S. Pat. No. 6,030,785, incorporated herein by reference. See also Enomoto et al. 1996; Enomoto et al. 1995. This region has been identified as the interferon sensitivity determining region (ISDR). See id.
The PGC IFN-beta conjugate is administered in a pharmacologically-effective amount to treat any of the conditions described above, and is based on the IFN beta activity of the polymeric conjugate. The term “pharmacologically-effective amount” means the amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by a researcher or clinician. It is an amount that is sufficient to significantly affect a positive clinical response while maintaining diminished levels of side effects. The amount of PGC IFN-beta which may be administered to a subject in need thereof is in the range of 0.01-100 μg/kg, or more preferably 0.01-10 μg/kg, administered in single or divided doses.
Administration of the described dosages may be every other day, but preferably occurs once a week or once every other week. Doses are administered over at least a 24 week period by injection.
Administration of the dose can be oral, topical, intravenous, subcutaneous, intramuscular, or any other acceptable systemic method. Based on the judgment of the attending clinician, the amount of drug administered and the treatment regimen used will, of course, be dependent on the age, sex and medical history of the patient being treated, the neutrophil count (e.g., the severity of the neutropenia), the severity of the specific disease condition and the tolerance of the patient to the treatment as evidenced by local toxicity and by systemic side-effects.
In practice, the conjugates of the invention are administered in amounts which will be sufficient to inhibit or prevent undesired medical conditions or disease in a subject, such as a mammal, and are used in the form most suitable for such purposes. The compositions are preferably suitable for internal use and include an effective amount of a pharmacologically-active compound of the invention, alone or in combination with other active agents, with one or more pharmaceutically-acceptable carriers. The compounds are especially useful in that they have very low, if any, toxicity.
The conjugates herein described can form the active ingredient of a pharmaceutical composition, and are typically administered in a mixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as “carrier” materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like. The compositions typically will include an effective amount of active compound or the pharmaceutically-acceptable salt thereof, and in addition, and may also include any carrier materials as are customarily used in the pharmaceutical sciences. Depending on the intended mode of administration, the compositions may be in solid, semi-solid or liquid dosage form, such as, for example, injectables, tablets, suppositories, pills, time-release capsules, powders, liquids, suspensions, or the like, preferably in unit dosages.
Conventional pharmaceutical compositions comprising a pharmacologically-effective amount of a conjugate, e.g., PGC IFN-beta, together with pharmaceutically-acceptable carriers, adjuvants, diluents, preservatives and/or solubilizers may be used in the practice of the invention. Pharmaceutical compositions of interferon include diluents of various buffers (e.g., arginine, Tris-HCl, acetate, phosphate) having a range of pH and ionic strength, carriers (e.g., human serum albumin), solubilizers (e.g., tween, polysorbate), and preservatives (e.g., benzyl alcohol). See, for example, U.S. Pat. No. 4,496,537.
Administration of the active compounds described herein can be via any of the accepted modes of administration for therapeutic agents. These methods include systemic or local administration such as oral, nasal, parenteral, transdermal, subcutaneous, or topical administration modes.
For instance, for oral administration in the form of a tablet or capsule (e.g., a gelatin capsule), the active drug component can be combined with an oral, non-toxic pharmaceutically-acceptable inert carrier such as ethanol, glycerol, water, and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents, and coloring agents can also be incorporated into the mixture. Suitable binders include starch, magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, sugars, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, its magnesium or calcium salt, and/or polyethylene glycol and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum starches, agar, alginic acid or its sodium salt, or effervescent mixtures, and the like. Diluents, include, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine.
The conjugates of the invention can also be administered in such oral dosage forms as timed-release and sustained-release tablets or capsules, pills, powders, granules, elixers, tinctures, suspensions, syrups, and emulsions.
Liquid, particularly injectable compositions can, for example, be prepared by dissolving, dispersing, etc. The active compound is dissolved in or mixed with a pharmaceutically-pure solvent such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form the injectable solution or suspension. Additionally, solid forms suitable for dissolving in liquid prior to injection can be formulated. Injectable compositions are preferably aqueous isotonic solutions or suspensions. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically-valuable substances.
The conjugates of the present invention can be administered in intravenous (e.g., bolus or infusion), intraperitoneal, subcutaneous or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions.
Parental injectable administration is generally used for subcutaneous, intramuscular or intravenous injections and infusions. For example, when a subcutaneous injection is used to deliver 0.01-100 μg/kg, or more preferably 0.01-10 μg/kg of PEGylated IFN-beta over one week, two injections of 0.005-50 μg/kg, or more preferably 0.005-5 μg/kg, respectively, may be administered at 0 and 72 hours. Additionally, one approach for parenteral administration employs the implantation of a slow-release or sustained-released system, which assures that a constant level of dosage is maintained, according to U.S. Pat. No. 3,710,795, incorporated herein by reference.
Furthermore, preferred conjugates for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen. Other preferred topical preparations include creams, ointments, lotions, aerosols, sprays and gels, wherein the amount administered would be 10-100 times the dose typically given by parenteral administration.
For solid compositions, excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like may be used. The active compound defined above, may be also formulated as suppositories using for example, polyalkylene glycols, for example, propylene glycol, as the carrier. In some embodiments, suppositories are advantageously prepared from fatty emulsions or suspensions.
The conjugates of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, containing cholesterol, stearylamine, or phosphatidylcholines. In some embodiments, a film of lipid components is hydrated with an aqueous solution of drug to a form lipid layer encapsulating the drug, as described in U.S. Pat. No. 5,262,564.
Conjugates of the present invention may also be delivered by the use of immunoglobulin fusions as individual carriers to which the compound molecules are coupled. The compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl-methacrylamide-phenol, polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues. The conjugates can also be coupled to proteins, such as, for example, receptor proteins and albumin. Furthermore, the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as for example, sodium acetate, triethanolamine oleate, etc.
The dosage regimen utilizing the conjugates is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. The activity of the compounds of the invention and sensitivity of the patient to side effects are also considered. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
Oral dosages of the present invention, when used for the indicated effects, will range between about 0.01-100 μg/kg/day orally, or more preferably 0.01-10 μg/kg/day orally. The compositions are preferably provided in the form of scored tablets containing 0.5-5000 μg, or more preferably 0.5-500 μg of active ingredient.
For any route of administration, divided or single doses may be used. For example, compounds of the present invention may be administered daily or weekly, in a single dose, or the total dosage may be administered in divided doses of two, three or four.
Any of the above pharmaceutical compositions may contain 0.1-99%, 1-70%, or, preferably, 1-50% of the active compounds of the invention as active ingredients.
As described above, the course of the disease and its response to drug treatments may be followed by clinical examination and laboratory findings. The effectiveness of the therapy of the invention is determined by the extent to which the previously described signs and symptoms of a condition, e.g., chronic hepatitis, are alleviated and the extent to which the normal side effects of interferon (i.e., flu-like symptoms such as fever, headache, chills, myalgia, fatigue, etc. and central nervous system related symptoms such as depression, paresthesia, impaired concentration, etc.) are eliminated or substantially reduced.
In some embodiments, a polyalkylated compound of the invention (e.g., a PEGylated interferon) is administered in conjunction with one or more pharmaceutical agents useful for treatment for a particular condition. For example, a polyalkylated protein can be administered in combination with a known antiviral agent or agent for treatment of a viral infection. Such antiviral compounds include, for example, ribavirin, levovirin, MB6866, and zidovudine 3TC, FTC, acyclovir, gancyclovir, viramide, VX497, VX-950, and ISIS-14803.
The conjugate and antiviral can be simultaneously administered (e.g., the agents are administered to a patient together); sequentially administered (e.g., the agents are administered to the patient one after the other); or alternatively administered (e.g., the agents are administered in a repeating series, such as agent A then agent B, then agent A, etc.).
In the practice of the invention, the preferred PGC IFN-beta (e.g., PEG IFN-beta) may be administered to patients infected with the hepatitis C virus. Use of PEG IFN-beta-1a is preferred.
Patients are selected for treatment from anti-HCV antibody-positive patients with biopsy-documented chronic active hepatitis.
In order to follow the course of HCV replication in subjects in response to drug treatment, HCV RNA may be measured in serum samples by, for example, a nested polymerase chain reaction assay that uses two sets of primers derived from the NS3 and NS4 non-structural gene regions of the HCV genome. See Farci et al., 1991, New Eng. J. Med. 325:98-104. Ulrich et al., 1990. J. Clin. Invest., 86:1609-1614.
Antiviral activity may be measured by changes in HCV-RNA titer. HCV RNA data may be analyzed by comparing titers at the end of treatment with a pre-treatment baseline measurement. Reduction in HCV RNA by week 4 provides evidence of antiviral activity of a compound. See Kleter et al., 1993, Antimicrob. Agents Chemother. 37(3):595-97; Orito et al., 1995, J. Medical Virology, 46:109-115. Changes of at least two orders of magnitude (>2 log) is interpreted as evidence of antiviral activity.
A person suffering from chronic hepatitis C infection may exhibit one or more of the following signs or symptoms: (a) elevated serum alanine aminotransferase (ALT), (b) positive test for anti-HCV antibodies, (c) presence of HCV as demonstrated by a positive test for HCV-RNA, (d) clinical stigmata of chronic liver disease, (e) hepatocellular damage. Such criteria may not only be used to diagnose hepatitis C, but can be used to evaluate a patient's response to drug treatment.
Elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are known to occur in uncontrolled hepatitis C, and a complete response to treatment is generally defined as the normalization of these serum enzymes, particularly ALT. See Davis et al., 1989, New Eng. J. Med. 321:1501-1506. ALT is an enzyme released when liver cells are destroyed and is symptomatic of HCV infection. Interferon causes synthesis of the enzyme 2′,5′-oligoadenylate synthetase (2′5′OAS), which in turn, results in the degradation of the viral mRNA. See Houglum, 1983, Clinical Pharmacology 2:20-28. Increases in serum levels of the 2′5′OAS coincide with decrease in ALT levels.
Histological examination of liver biopsy samples may be used as a second criteria for evaluation. See, e.g., Knodell et al., 1981, Hepatology 1:431-435, whose Histological Activity Index (portal inflammation, piecemeal or bridging necrosis, lobular injury, and fibrosis) provides a scoring method for disease activity.
Safety and tolerability or treatment may be determined by clinical evaluations and measure of white blood cell and neutrophil counts. This may be assessed through periodic monitoring of hematological parameters e.g., white blood cell, neutrophil, platelet, and red blood cell counts).
Various other extended- or sustained-release formulations can be prepared using conventional methods well known in the art.
Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. All patents and publications cited herein are incorporated by reference.
EXAMPLES
Example 1
Synthesis of Activated Polyalkylene Glycols
A) Alkylation of Alcohols
Activated polyalkylene glycols are synthesized by alkylating a polyalkylene glycol having a free terminal hydroxyl functionality. A generic reaction is outlined in Scheme I:
The polyalkylene glycol (P—OH) is reacted with the alkyl halide (A) to form the ether (B). Compound B is then hydroxylated to form the alcohol (C), which is oxidized to the aldehyde (D). In these compounds, n is an integer from zero to five and Z can be a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group. Z can also be a C3 to C7 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl (the alkyl is a C1 to C20 saturated or unsaturated alkyl) or heteroalkaryl group. For substituted compounds, the substituents can be halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, or alkylthio. Typically, P—OH is polyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG) having a molecular weight of 5,000 to 40,000 Daltons (Da).
For example, the synthesis of mPEG-O-2-methylpropionaldehyde is outlined in Scheme II.
mPEG-OH with a molecular weight of 20, 000 Da (mPEG-OH 20 kDa; 2.0 g, 0.1 mmol, Sunbio) was treated with NaH (12 mg, 0.5 mmol) in THF (35 mL). Fifty equivalents of 3-bromo-2-methylpropene (3.34 g, 5 mmol) and a catalytic amount of KI were then added to the mixture. The resulting mixture was heated to reflux for 16 h. Water (1 mL) was then added and the solvent was removed under vacuum. To the residue was added CH2Cl2 (25 mL) and the organic layer was separated, dried over anhydrous Na2SO4, and the volume was reduced to approximately 2 mL. This CH2Cl2 solution was added to ether (150 mL) drop-wise. The resulting white precipitate was collected, yielding 1.9 g of compound 1. 1HNMR (CDCl3, 400 MHz) showed δ 4.98 (s, 1H), 4.91 (s, 1H), 1.74 (s, 3H).
To compound 1 (1.9 g, 0.1 mmol) in THF (20 mL) and CH2Cl2 (2 mL) at 0° C., was added BH3 in THF (1.0 M, 3.5 mL). The mixture was stirred in an ice bath for 1 h. To this mixture, NaOH was added slowly (2.0 M, 2.5 mL), followed by 30% H2O2 (0.8 mL). The reaction was warmed to room temperature and stirred for 16 h. The above work-up procedure was followed (CH2Cl2, precipitated from ether) to yield 1.8 g of 2 as a white solid. 1HNMR (CDCl3, 400 MHz) showed δ 1.80 (m, 1H), 0.84 (d, 3H).
Compound 2 (250 mg) was dissolved in CH2Cl2 (2.5 mL) and Dess-Martin periodinate (DMP; 15 mg) was added with stirring for 30 min at room temperature. To the mixture was added saturated NaHCO3 and Na2S2O3 (2 mL) and the mixture was stirred at room temperature for 1 h. The above work-up procedure was followed to give 3 (mPEG-O-2-methylpropionaldehyde, 120 mg) as a white solid. 1HNMR (CDCl3, 400 MHz) showed δ 9.75 (s, 1H), 2.69 (m, 1H), 1.16 (d, 3H).
A similar procedure is followed for aromatic alcohols, as shown in Scheme III:
In general, the aromatic alcohol (E) is reacted with the alkyl halide (A) to form the mono ether (F). The remaining alcohol group of compound F is then converted to the halide (e.g., bromide) in Compound G, which is reacted with the polyalkylene glycol (P—OH) to give the ether (H). This compound is then converted to the aldehyde (J) through a hydroboration to the primary alcohol (I) followed by oxidation. In these compounds, n is an integer from zero to five, d is zero or an integer from one to four, and Z can be a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group. Z can also be a C3 to C7 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl (the alkyl is a C1 to C20 saturated or unsaturated alkyl) or heteroalkaryl group. For substituted compounds, the substituents can be halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, or alkylthio.
Additionally, T1 and T2 are, independently, absent, or a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, and can be ortho, meta, or para to each other. Each L (when present) is, independently, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C7 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl wherein the alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group. The substituents can be halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, or alkylthio.
Usually, P—OH is polyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG) having a molecular weight of 5,000 to 40,000 Da.
For example, the synthesis of mPEG-O-p-methylphenyl-O-2-methylpropionaldehyde (8) is shown in Scheme IV;
To a solution of 4-hydroxybenzylalcohol (2.4 g, 20 mmol) in THF (50 mL) and water (2.5 mL) was first added sodium hydroxide (1.5 g, 37.5 mmol) and then 3-bromo-2-methylpropene (4.1 g, 30 mmol). This reaction mixture was heated to reflux for 16 h. To the mixture was added 10% citric acid (2.5 mL) and the solvent was removed under vacuum. The residue was extracted with ethyl acetate (3×15 mL) and the combined organic layers were washed with saturated NaCl (10 mL), dried and concentrated to give compound 4. (3.3 g, 93%). 1HNMR (CDCl3, 400 MHz) showed δ 7.29 (m, 2H), 6.92 (m, 2H), 5.14 (s, 1H), 5.01 (s, 1H), 4.56 (s, 2H), 4.46 (s, 2H), 1.85 (s, 3H).
Mesyl chloride (MsCl; 2.5 g, 15.7 mmol) and triethyl amine (TEA; 2.8 mL, 20 mmol) were added to a solution of compound 4 (2.0 g, 11.2 mmol) in CH2Cl2 (25 mL) at 0° C. and the reaction was placed in the refrigerator for 16 h. A usual work-up yielded a pale yellow oil (2.5 g, 87%). 1HNMR (CDCl3, 400 MHz) showed δ 7.31 (m, 2H), 6.94 (m, 2H), 5.16 (s, 1 H), 5.01 (s, 1H), 5.03 (s, 2H), 4.59 (s, 2H), 4.44 (s, 2H), 3.67 (s, 31), 1.85 (s, 3 H). This oil (2.4 g, 9.4 mmol) was dissolved in THF (20 mL) and LiBr (2.0 g, 23.0 mmol) was added. The reaction mixture was heated to reflux for 1 h and was then cooled to room temperature. Water (2.5 mL) was added to the mixture and the solvent was removed under vacuum. The residue was extracted with ethyl acetate (3×15 mL) and the combined organic layers were washed with saturated NaCl (10 mL), dried over anhydrous Na2SO4, and concentrated to give the desired bromide 5 (2.3 g, 96%) as a pale yellow oil. 1HNMR (CDCl3, 400 MHz) showed δ 7.29 (m, 2H), 6.88 (m, 2H), 5.11 (s, 1H), 4.98 (s, 1H), 4.53 (s, 2H), 4.44 (s, 2H), 1.83 (s, 3H).
mPEG-OH 20 kDa (2.0 g, 0.1 mmol, Sunbio) was treated with NaH (12 mg, 0.5 mmol) in THF (35 mL) and compound 5 (0.55 g, 22.8 mmol) was added to the mixture with a catalytic amount of KI. The resulting mixture was heated to reflux for 16 h. Water (1.0 mL) was added to the mixture and the solvent was removed under vacuum. To the residue was added CH2Cl2 (25 mL) and the organic layer was separated, dried over anhydrous Na2SO4, and the volume was reduced to approximately 2 mL. Drop-wise addition to an ether solution (150 mL) resulted in a white precipitate which was collected to yield 6 (1.5 g) as a white powder. 1HNMR (CDCl3, 400 MHz) showed δ 7.21 (d, 2H), 6.90 (d, 2H), 5.01 (s, 1H), 4.99 (s, 1H), 4.54 (s, 2H), 4.43 (s, 2H), 1.84 (s, 3H).
To a solution of compound 6 (1.0 g, 0.05 mmol) in THF (10 mL) and CH2Cl2 (2 mL) cooled to 0° C., was added BH3/THF (1.0 M, 3.5 mL) and the reaction was stirred for 1 h. A 2.0 M NaOH solution (2.5 mL) was added slowly and followed by 30% H2O2 (0.8 mL). The reaction mixture was allowed to warm to room temperature and stirred for 16 h. The above work-up procedure was followed (CH2Cl2, precipitated from ether) to yield 7 (350 mg) as a white solid. 1HNMR (CDCl3, 400 MHz) showed δ 7.21 (d, 2H), 6.84 (d, 2H), 4.54 (s, 2 H), 2.90 (m, 2H), 1.96 (d, 3H).
Compound 7 (150 mg, 0.0075 mmol) was dissolved in CH2Cl2 (1.5 mL) and DMP (15 mg) was added while the reaction mixture was stirred at room temperature for 1.5 h. 1HNMR (CDCl3, 400 MHz) showed δ 9.76 (s, 1H), 7.21 (d, 2H), 6.78 (d, 2H), 4.44 (s, 2 H), 4.14 (m, 2H), 2.85 (m, 1H), 1.21 (d, 3H). To the mixture was added saturated NaHCO3 (0.5 mL) and Na2S2O3 (0.5 mL) and stirring continued at room temperature for 1 h. The above work-up procedure was followed (CH2Cl2 solution, precipitated from ether) to give 8 (92 mg) as a white solid.
Similarly, mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde (9) was synthesized as outlined in Scheme V.
To a solution of 3-hydroxybenzylalcohol (2.4 g, 20 mmol) in THF (50 mL) and water (2.5 mL) was first added sodium hydroxide (1.5 g, 37.5 mmol) and then 3-bromo-2-methylpropene (4.1 g, 30 mmol). This reaction mixture was heated to reflux for 16 h. To the mixture was added 10% citric acid (2.5 mL) and the solvent was removed under vacuum. The residue was extracted with ethyl acetate (3×15 mL) and the combined organic layers were washed with saturated NaCl (10 mL), dried and concentrated to give compound 10 (3.2 g, 90%). 1HNMR (CDCl3, 400 MHz) showed δ 7.26 (m, 1H), 6.94 (m, 2H), 6.86 (m, 1H), 5.11 (s, 1H), 5.01 (s, 1H), 4.61 (s, 1H), 4.44 (s, 2H), 1.82 (s, 3H).
MsCl (2.5 g, 15.7 mmol) and TEA (2.8 mL, 20 mmol) were added to a solution of compound 10 (2.0 g, 11.2 mmol) in CH2Cl2 (25 mL) at 0° C. and the reaction was placed in the refrigerator for 16 h. A usual work-up yielded a pale yellow oil (2.5 g, 87%). 1HNMR (CDCl3, 400 MHz) showed δ 7.31 (m, 1H), 7.05 (m, 2H), 6.91 (m, 1H), 5.16 (s, 1 H), 5.04 (s, 1H), 4.59 (s, 1H), 4.46 (s, 2H), 3.71 (s, 3H), 1.84 (s, 3H). This oil (2.4 g, 9.4 mmol) was dissolved in THF (20 mL) and LiBr (2.0 g, 23.0 mmol) was added. The reaction mixture was heated to reflux for 1 h and was then cooled to room temperature. To the mixture was added water (2.5 mL) and the solvent was removed under vacuum. The residue was extracted with ethyl acetate (3×15 mL) and the combined organic layers were washed with saturated NaCl (10 mL), dried over anhydrous Na2SO4, and concentrated to give the desired bromide 11 (2.2 g, 92%) as a pale yellow oil. 1HNMR (CDCl3, 400 MHz) showed δ 7.29 (m, 1H), 6.98 (m, 2H), 6.85 (m, 1H), 5.14 (s, 2H), 4.98 (s, 2H), 4.50 (s, 2H), 4.44 (s, 2H), 1.82 (d, 3H).
mPEG-OH 20 kDa (2.0 g, 0.1 mmol, Sunbio) was treated with NaH (12 mg, 0.5 mmol) in THF (35 mL) and compound 11 (0.55 g, 22.8 mmol) was added to the mixture with a catalytic amount of KI. The resulting mixture was heated to reflux for 16 h. Water (1.0 mL) was added to the mixture and the solvent was removed under vacuum. To the residue was added CH2Cl2 (25 mL) and the organic layer was separated, dried over anhydrous Na2SO4, and the volume was reduced to approximately 2 mL. Dropwise addition to an ether solution (150 mL) resulted in a white precipitate which was collected to yield 12 (1.8 g) as a white powder. 1HNMR (CDCl3, 400 MHz) showed δ 7.19 (m, 1H), 6.88 (m, 2H), 6.75 (m, 1H), 4.44 (s, 2H), 4.10 (m, 2H), 1.82 (d, 3H).
To a solution of compound 12 (1.0 g, 0.05 mmol) in THF (7.5 mL) and CH2Cl2 (2.5 mL) cooled to 0° C., was added BH3/THF (1.0 M, 3.5 mL) and the reaction was stirred for 1 h. A 2.0 M NaOH solution (3 mL) was added slowly, followed by 30% H2O2 (0.85 mL). The reaction mixture was allowed to warm to room temperature and stirred for 16 h. The above work-up procedure was followed (CH2Cl2, precipitated from ether) to yield 13 (450 mg) as a white solid. 1HNMR (CDCl3, 400 MHz) showed δ 7.15 (m, 1H), 6.84 (m, 2H), 6.69 (m, 1 H), 4.50 (s, 2H), 2.90 (m, 2H), 1.95 (d, 3H).
Compound 13 (200 mg, 0.01 mmol) was dissolved in CH2Cl2 (1.5 mL) and DMP (20 mg) was added while the reaction mixture was stirred at room temperature for 1 h. 1HNMR (CDCl3, 400 MHz) showed δ 9.74 (s, 1H), 7.17 (m, 1H), 6.86 (m, 2H), 6.74 (m, 1 H), 4.48 (s, 2H), 4.15 (m, 2H), 2.78 (m, 1H), 1.22 (d, 3H). To the mixture was added saturated NaHCO3 (0.5 mL) and Na2S2O3 (0.5 mL) and stirring continued at room temperature for 1 h. The above work-up procedure was followed (CH2Cl2 solution, precipitated from ether) to give 9 (142 mg) as a white solid.
B) Generation Via Reaction with Aromatic Alcohols
Activated polyalkylene glycols are synthesized by a Mitsunobu reaction between a polyalkylene glycol having a free terminal hydroxyl functionality and an aromatic alcohol. The reaction scheme is outlined in Scheme VI.
The polyalkylene glycol (P—OH) is reacted with an alcohol (K) to form the ether (L). In these compounds, m is zero or one, d is zero or an integer from one to four, and n is zero or an integer from one to five. Y is O, S, CO, CO2, COS, SO, SO2, CONR′, SO2NR′, and NR′. T1 and T2 are, independently, absent, or a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group.
R′ and Z are, independently, hydrogen, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group.
Each L (if present) is, independently, a straight- or branched-chain, saturated or unsaturated C1 to C20 alkyl or heteroalkyl group, C3 to C7 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted alkaryl. The alkyl is a C1 to C20 saturated or unsaturated alkyl or heteroalkaryl group, and the substituents can be halogen, hydroxyl, carbonyl, carboxylate, ester, formyl, acyl, thiocarbonyl, thioester, thioacetate, thioformate, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic moiety, heteroaromatic moiety, imino, silyl, ether, or alkylthio.
P is a polyalkylene glycol polymer. Usually, P—OH is polyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG) having a molecular weight of 5,000 to 40,000 Da.
For example, a synthesis of mPEG-O-p-phenylacetaldehyde (6) is outlined in Scheme VII.
4-hydroxyphenylacetaldehyde (15) was synthesized as described in Heterocycles, 2000, 53, 777-784. 4-Hydroxyphenethyl alcohol (Compound 14, 1.0 g, 7.3 mmol, Aldrich) was dissolved in dimethylsulfoxide (8 mL, Aldrich). With stirring, TEA (2.2 mL, 16 mmol, Aldrich) was added slowly. Pyridine-sulfur trioxide (SO3.py) complex (2.5 g, 16 mmol, Aldrich) was completely dissolved in dimethylsulfoxide (9 mL, Aldrich) and this solution was added drop-wise to the alcohol, with vigorous stirring. After stirring for 1 h at room temperature, the reaction was diluted with CH2Cl2, then washed with ice-cold water. The organic layer was dried over Na2SO4, filtered, and concentrated to dryness. Purification using silica gel chromatography with hexane-ethyl acetate as eluent (5:1, then 2:1) yielded 488 mg (49%) of 4-hydroxyphenylacetaldehyde (5).
mPEG-OH 20 kDa (101 mg, 0.005 mmol) and 4-hydroxyphenylacetaldehyde (15) (39 mg, 0.29 mmol) were azeotroped four times with toluene, then taken up in anhydrous CH2Cl2 (2 mL, Aldrich). To this solution was added triphenylphosphine (PPh3; 66 mg, 0.25 mmol, Aldrich) and then diisopropylazodicarboxylate (DIAD; 49 μL, 0.25 mmol, Aldrich) with stirring. After 3 days of stirring at room temperature, the reaction mixture was added drop-wise to vigorously-stirred diethyl ether. The resulting precipitate was isolated by filtration and washed three times with diethyl ether. The crude material was taken up in CH2Cl2 and washed with water. The organic layer was dried over Na2SO4, filtered, and concentrated to dryness. The material was taken up in minimum CH2Cl2, then precipitated by adding drop-wise to stirred diethyl ether. This material was collected by filtration, washed three times with diethyl ether and dried to give 63 mg (62%) of mPEG-O-p-phenylacetaldehyde (16).
A synthesis of mPEG-O-p-phenylpropionaldehyde (17) was prepared in a similar manner.
4-hydroxyphenylpropionaldehyde was prepared by a synthesis analogous to that for 4-hydroxyphenylacetaldehyde (Heterocycles, 2000, 53, 777-784). 3-(4-Hydroxyphenyl)-1-propanol (1.0 g, 6.6 mmol, Aldrich) was dissolved in dimethylsulfoxide (8 mL, Aldrich). TEA (2.0 mL, 14 mmol, Aldrich) was added slowly with stirring. Pyridine-sulfur trioxide (SO3.py) complex (2.3 g, 15 mmol, Aldrich) was completely dissolved in dimethylsulfoxide (9 mL, Aldrich) and this solution was added drop-wise to the alcohol, with vigorous stirring. After stirring for 1 h at room temperature, the reaction was diluted with CH2Cl2, then washed with ice-cold water. The organic layer was dried over Na2SO4, filtered, and concentrated to dryness. Purification using silica gel chromatography with hexane-ethyl acetate as eluent (5:1, then 2:1) yielded 745 mg (75%) of 4-hydroxyphenylpropionaldehyde.
mPEG-OH 20 kDa (100 mg, 0.005 mmol) and 4-hydroxyphenylpropionaldehyde (40 mg, 0.27 mmol) were azeotroped four times with toluene, then taken up in anhydrous CH2Cl2 (2 mL, Aldrich). To this solution was added triphenylphosphine (66 mg, 0.25 mmol, Aldrich) and then diisopropylazodicarboxylate (49 μL, 0.25 mmol, Aldrich) with stirring. After 3 days stirring at room temperature, the reaction mixture was added drop-wise to vigorously-stirred diethyl ether. The resulting precipitate was isolated by filtration and washed three times with diethyl ether. The crude material was taken up in CH2Cl2 and washed with water. The organic layer was dried over Na2SO4, filtered, and concentrated to dryness. The material was taken up in minimum CH2Cl2, then precipitated by adding drop-wise to stirred diethyl ether. This material was collected by filtration, washed three times with diethyl ether and dried to give 60 mg (60%) of mPEG-O-p-phenylpropionaldehyde (17).
mPEG-O-m-phenylacetaldehyde (18) was also prepared in this way.
3-hydroxyphenylacetaldehyde was prepared by a synthesis analogous to that of 4-hydroxyphenylacetaldehyde (Heterocycles, 2000, 53, 777-784). 3-Hydroxyphenethyl alcohol (1.0 g, 7.5 mmol, Aldrich) was dissolved in dimethylsulfoxide (8 mL, Aldrich). TEA (2.0 mL, 14 mmol, Aldrich) was added slowly with stirring. Pyridine-sulfur trioxide (SO3.py) complex (2.4 g, 15 mmol, Aldrich) was completely dissolved in dimethylsulfoxide (8 mL, Aldrich) and this solution was added drop-wise to the alcohol, with vigorous stirring. After stirring for 1 h at room temperature, the reaction was quenched with ice-cold water, then extracted with CH2Cl2. The organic layer was dried over Na2SO4, filtered, and concentrated to dryness. Purification using silica gel chromatography with hexane-ethyl acetate as eluent (3:1, then 1:1) yielded 225 mg (22%) of 3-hydroxyphenylacetaldehyde.
mPEG-OH 20 kDa (307 mg, 0.015 mmol) and 3-hydroxyphenylacetaldehyde (117 mg, 0.86 mmol) were azeotroped four times with toluene, then taken up in anhydrous CH2Cl2 (5 mL, Aldrich). To this solution was added triphenylphosphine (200 mg, 0.76 mmol, Aldrich) and then diisopropylazodicarboxylate (147 μL, 0.75 mmol, Aldrich) with stirring. After 3 days of stirring at room temperature, the reaction mixture was added drop-wise to vigorously-stirred diethyl ether. The resulting precipitate was isolated by filtration and washed three times with diethyl ether and dried to yield 284 mg (93%) of mPEG-O-m-phenylacetaldehyde (18).
Chiral PEG-cinnamate-N-hydroxy succinimate (NHS) compounds are generated, for example, as shown in Schemes VIII and IX:
PEG-Dihydrourocanate-NHS compounds are also generated via a Mitsunobu reaction, as shown in Scheme X:
PEG-Dihydrocinnamate-NHS compounds are also generated from an aromatic alcohol as shown in Scheme XI:
PEG-benzofurans and PEG-indoles are generated as shown in Schemes XII and XIII:
C) Generation Via Reaction of PEG-Amines
PEG amines are reacted with alkyl halides to generate PEG-amides. An example of the generation of a PEG-amide-bicyclooctane-NHS conjugate is shown in Scheme XIV:
A PEG-primary amine is conjugated with an aryl-halide to form a PEG-secondary amine conjugate, which is then reacted under Heck conditions (a stereospecific Palladium-catalyzed coupling of an alkene with an organic halide or triflate lacking sp3 hybridized β-hydrogens) with an NHS-alkene to form the desired PEG-conjugate. The synthesis of a pyrimidine-containing conjugate is shown in Scheme XV:
PEG-sulfonamide conjugates are also synthesized in this manner, as shown in Scheme XVI:
D) Compounds Generated Via Reaction with Heterocycles
PEG compounds are reacted with ring- or non-ring nitrogens in heterocycles to form reactive PEG species. Representative reactions are shown in Schemes XVII for aminopyrrolidine and XVIII for various piperazines:
Example 2
Preparation of Peptide Conjugates
The peptide conjugates according to the present invention can be prepared by reacting a protein with an activated PGC molecule. For example, interferon (IFN) can be reacted with a PEG-aldehyde in the presence of a reducing agent (e.g., sodium cyanoborohydride) via reductive alkylation to produce the PEG-protein conjugate, attached via an amine linkage. See, e.g., European Patent 0154316 B1.
Human IFN-β-1a was PEGylated with the following activated polyalkylene glycols of the invention: 20 kDa mPEG-O-2-methylpropionaldehyde, 20 kDa mPEG-O-p-methylphenyl-O-2-methylpropionaldehyde, 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde, 20 kDa mPEG-O-p-phenylacetaldehyde, 20 kDa mPEG-O-p-phenylpropionaldehyde, and 20 kDa mPEG-O-m-phenylacetaldehyde. The PEGylated proteins were purified to homogeneity from their respective reaction mixtures and subjected to a series of characterization tests to ascertain the identity, purity, and potency of the modified proteins.
A detailed description of the preparation and characterization of human IFN-β-1a modified with 20 kDa mPEG-O-2-methylpropionaldehyde, 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde, and 20 kDa mPEG-O-p-phenylacetaldehyde follows.
A) Preparation and Characterization of 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a
Human IFN-β-1a was PEGylated at its N-terminus with 20 kDa mPEG-O-2-methylpropionaldehyde. The product of the reductive alkylation chemistry used to incorporate the PEG onto the IFN-β-1a backbone resulted in the formation of an amine linkage which is extremely stable against degradation. The PEGylated IFN-β-1a was subjected to extensive characterization, including analysis by SDS-PAGE, size exclusion chromatography (SEC), peptide mapping, and assessment of activity in an in vitro antiviral assay. The purity of the product, as measured by SDS-PAGE and SEC, was greater than 90%. In the PEGylated sample there was no evidence of aggregates. Residual levels of unmodified IFN-β-1a in the product were below the limit of quantitation, but appear to represent about 1% of the product. The specific activity of the PEGylated IFN-β-1a in the antiviral activity assay was reduced approximately 2-fold compared to the unmodified IFN-β-1a (EC50=32 pg/mL for 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a versus EC50=14 pg/mL for unmodified IFN-β-1a). The PEGylated IFN-β-1a bulk was formulated at 30 μg/mL in phosphate-buffered saline (PBS) pH 7.3, containing 14 mg/mL human serum albumin (HSA), similar to the formulation used for AVONEX® (Biogen, Cambridge, Mass.) which has been subjected to extensive characterization. The material was supplied as a frozen liquid which was stored at −70° C.
The properties of 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a are summarized in Table 1:
TABLE 1
Properties of 20 kDa mPEG-O-2-methylpropionaldehyde-modified
IFN-β-1a
Pegylation efficiency
>90%
IFN-β-1a/PEG ratio
1:1
Purity
>90%
Site of attachment
N-terminus
Antiviral activity EC50
32 pg/mL
1. Preparation of 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a. 10 mL of nonformulated AVONEX® (IFN-β-1a bulk intermediate, a clinical batch of bulk drug that passed all tests for use in humans, at 250 μg/mL in 100 mM sodium phosphate pH 7.2, 200 mM NaCl) was diluted with 12 mL of 165 mM MES pH 5.0 and 50 μL of 5 N HCl. The sample was loaded onto a 300 μL SP-Sepharose FF column (Pharmacia). The column was washed with 3×300 μL of 5 mM sodium phosphate pH 5.5, 75 mM NaCl, and the protein was eluted with 5 mM sodium phosphate pH 5.5, 600 mM NaCl. Elution fractions were analyzed for their absorbance at 280 nm and the concentration of IFN-β-1a in the samples estimated using an extinction coefficient of 1.51 for a 1 mg/mL solution. The peak fractions were pooled to give an IFN-β-1a concentration of 3.66 mg/mL, which was subsequently diluted to 1.2 mg/mL with water.
To 0.8 mL of the IFN-β-1a from the diluted SP-Sepharose eluate pool, 0.5 M sodium phosphate pH 6.0 was added to 50 mM, sodium cyanoborohydride (Aldrich) was added to 5 mM, and 20 kDa mPEG-O-2-methylpropionaldehyde was added to 5 mg/mL. The sample was incubated at room temperature for 16 h in the dark. The PEGylated IFN-β-1a was purified from the reaction mixture on a 0.5 mL SP-Sepharose FF column as follows: 0.6 mL of the reaction mixture was diluted with 2.4 mL 20 mM MES pH 5.0, and loaded on to the SP-Sepharose column. The column was washed with sodium phosphate pH 5.5, 75 mM NaCl and then the PEGylated IFN-β-1a was eluted from the column with 25 mM MES pH 6.4, 400 mM NaCl. The PEGylated IFN-β-1a was further purified on a Superose 6 HR 10/30 FPLC sizing column with 5 mM sodium phosphate pH 5.5, 150 mM NaCl as the mobile phase. The sizing column (25 mL) was run at 20 mL/h and 0.5 mL fractions were collected. The elution fractions were analyzed for protein content by absorbance at 280 nm, pooled, and the protein concentration of the pool determined. The PEGylated IFN-β-1a concentration is reported in IFN equivalents as the PEG moiety does not contribute to absorbance at 280 nm. Samples of the pool were removed for analysis, and the remainder was diluted to 30 μg/mL with HSA-containing formulation buffer, aliquoted at 0.25 mL/vial, and stored at −70° C.
2. UV spectrum of purified 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a. The UV spectrum (240-340 nm) of 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a was obtained using the pre-HSA-formulated bulk sample. The PEGylated sample exhibited an absorbance maximum at 278-279 nm and an absorbance minimum at 249-250 nm, consistent with that observed for the unmodified IFN-β-1a bulk intermediate. The protein concentration of the PEGylated product was estimated from the spectrum using an extinction coefficient of ε2800.1%=1.51. The protein concentration of the PEGylated bulk was 0.23 mg/mL. No turbidity was present in the sample as evident by a lack of absorbance at 320 nm.
3. Characterization of 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a by SDS-PAGE. 4 μg of unmodified and 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a were subjected to SDS-PAGE under reducing conditions on a 10-20% gradient gel. The gel was stained with Coomassie brilliant blue R-250, and is shown in FIG. 1 (Lane A, molecular weight markers (from top to bottom; 100 kDa, 68 kDa, 45 kDa, 27 kDa, and 18 kDa, respectively); Lane B, unmodified IFN-β-1a; Lane C, 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a). SDS-PAGE analysis of 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a revealed a single major band with an apparent mass of 55 kDa, consistent with modification by a single PEG. No higher mass forms resulting from the presence of additional PEG groups were detected. In the purified, PEGylated product, unmodified IFN-β-1a was detected; however, the amount is below the limit of quantitation. The level of unmodified IFN-β-1a in the preparation is estimated to account for only about 1% of the total protein.
4. Characterization of 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a by size exclusion chromatography. Unmodified and 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a were subjected to SEC on an analytical Superose 6 HR10/30 FPLC sizing column using PBS pH 7.2 as the mobile phase. The column was run at 20 mL/h and the eluent monitored for absorbance at 280 nm, as shown in FIG. 2: Panel A: molecular weight standards (670 kDa, thyroglobulin; 158 kDa, gamma globulin; 44 kDa, ovalbumin; 17 kDa, myoglobin; 1.3 kDa, vitamin B12), Panel B: 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a; Panel C: unmodified IFN-β-1a. The 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a eluted as a single sharp peak with an apparent molecular mass of approximately 200 kDa, consistent with the large hydrodynamic volume of the PEG. No evidence of aggregates was observed. Unmodified IFN-β-1a in the preparation was detected but was below the limit of quantitation. Based on the size of the peak, the unmodified IFN-β-1a accounts for 1% or less of the product, consistent with that observed using SDS-PAGE.
5. Analysis of 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a by peptide mapping. The specificity of the PEGylation reaction was evaluated by peptide mapping. Unmodified and 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a were digested with endoproteinase Lys-C from Achromobacter (Wako Bioproducts) and the resulting cleavage products were fractionated by reverse-phase HPLC on a Vydac C4 column using a 30 min gradient from 0 to 70% acetonitrile, in 0.1% TFA. The column eluent was monitored for absorbance at 214 nm.
All of the predicted peptides from the endoproteinase Lys-C digest of IFN-β1a have been identified previously by N-terminal sequencing and mass spectrometry (Pepinsky et al., (2001) J Pharmacology and Experimental Therapeutics 297:1059), and, of these, only the peptide that contains the N-terminus of IFN-β-1a was altered by modification with 20 kDa mPEG-O-2-methylpropionaldehyde; as evident by its disappearance from the peptide map. The mapping data therefore indicate that the PEG moiety is specifically attached to this peptide. The data further indicate that the PEG modification is targeted at the N-terminus of the protein since only the N-terminal modification would result in the specific loss of this peptide.
B) Preparation and Characterization of 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde-modified IFN-β-1a
Human IFN-β-1a was PEGylated at the N-terminus with 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde. The product of the reductive alkylation chemistry that was used to incorporate the PEG onto the IFN-β-1a backbone results in the formation of an amine linkage which is extremely stable against degradation. The PEGylated IFN-β-1a was subjected to extensive characterization, including analysis by SDS-PAGE, SEC, peptide mapping, and assessment of activity in an in vitro antiviral assay. The purity of the product as measured by SDS-PAGE and SEC was greater than 95%. In the PEGylated IFN-β-1a sample there was no evidence of aggregates. Residual levels of unmodified IFN-β-1a in the product were below the limit of quantitation, but appear to represent about 1% of the product. The specific activity of the PEGylated IFN-β-1a in the antiviral activity assay was reduced approximately 2-fold compared to the unmodified IFN-β-1a (EC50=31 pg/mL for 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde-modified IFN-β-1a versus EC50=14 pg/mL for unmodified IFN-β-1a). The PEGylated IFN-β-1a bulk was formulated at 30 μg/mL in PBS pH 7.2 containing 15 mg/mL HSA, similar to the formulation used for AVONEX® which has been subjected to extensive characterization. The material was supplied as a frozen liquid which was stored at −70° C.
The properties of 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde-modified IFN-β-1a are summarized in Table 2:
TABLE 2
Properties of 20 kDa mPEG-O-m-methylphenyl-O-2-
methylpropionaldehyde-modified IFN-β-1a
PEGylation efficiency
>80%
IFN-β-1a/PEG ratio
1:1
Purity
>95%
Site of attachment
N-terminus
Antiviral activity EC50
31 pg/mL
1. Preparation of 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde-modified IFN-β-1a. 80 mL of nonformulated AVONEX® (IFN-β-1a bulk intermediate, a clinical batch of bulk drug that passed all tests for use in humans, at 254 μg/mL in 100 mM sodium phosphate pH 7.2, 200 mM NaCl) was diluted with 96 mL of 165 mM MES pH 5.0, and 400 μL of 5 N HCl. The sample was loaded onto a 1.2 mL SP-Sepharose FF column (Pharmacia). The column was washed with 6.5 mL of 5 mM sodium phosphate pH 5.5, 75 mM NaCl, and the protein was eluted with 5 mM sodium phosphate pH 5.5, 600 mM NaCl. Elution fractions were analyzed for their absorbance at 280 nm and the concentration of IFN-β-1a in the samples was estimated using an extinction coefficient of 1.51 for a 1 mg/mL solution. The peak fractions were pooled to give an IFN-β-1a concentration of 4.4 mg/mL. To 2.36 mL of the 4.4 mg/mL IFN-β-1a from the SP-Sepharose eluate pool, 0.5 M sodium phosphate pH 6.0 was added to 50 mM, sodium cyanoborohydride (Aldrich) was added to 5 mM, and 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde, was added to 10 mg/mL. The sample was incubated at room temperature for 21 h in the dark. The PEGylated IFN-β-1a was purified from the reaction mixture on a 8.0 mL SP-Sepharose FF column as follows: 9.44 mL of reaction mixture was diluted with 37.7 mL of 20 mM MES pH 5.0, and loaded onto the SP-Sepharose column. The column was washed with sodium phosphate pH 5.5, 75 mM NaCl and then the PEGylated IFN-β-1a was eluted from the column with 25 mM MES pH 6.4, 400 mM NaCl. The PEGylated IFN-β-1a was further purified on a Superose 6 HR 10/30 FPLC sizing column with 5 mM sodium phosphate pH 5.5, 150 mM NaCl as the mobile phase. The sizing column (25 mL) was run at 24 mL/h and 0.25 mL fractions were collected. The elution fractions were analyzed for protein content by SDS-PAGE, pooled, and the protein concentration of the pool determined. The PEGylated IFN-β-1a concentration is reported in IFN equivalents after adjusting for the contribution of the PEG to the absorbance at 280 nm using an extinction coefficient of 2 for a 1 mg/mL solution of the PEGylated IFN-β-1a. Samples of the pool were removed for analysis, and the remainder was diluted to 30 μg/mL with HSA-containing formulation buffer, aliquoted at 0.25 mL/vial, and stored at −70° C.
2. UV spectrum of purified 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde-modified IFN-β-1a. The UV spectrum (240-340 nm) of 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde-modified IFN-β-1a was obtained using the pre-HSA-formulated bulk sample. The PEGylated sample exhibited an absorbance maximum at 278-279 nm and an absorbance minimum at 249-250 nm, consistent with that observed for the unmodified IFN-β-1a bulk intermediate. The protein concentration of the PEGylated product was estimated from the spectrum using an extinction coefficient of ε2800.1%=2.0. The protein concentration of the PEGylated bulk was 0.42 mg/mL. No turbidity was present in the sample as evident by the lack of absorbance at 320 nm.
3. Characterization of 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde-modified IFN-β-1a by SDS-PAGE. 2.1 μg of 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde-modified IFN-β-1a was subjected to SDS-PAGE under reducing conditions on a 4-20% gradient gel. The gel was stained with Coomassie brilliant blue R-250. SDS-PAGE analysis of 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde-modified IFN-β-1a revealed a single major band with an apparent mass of 55 kDa consistent with modification by a single PEG. In the purified PEGylated product unmodified IFN-β-1a was detected; however, the amount is below the limit of quantitation. It is estimated that the level of unmodified IFN-β-1a in the preparation accounts for only about 1% of the total protein.
4. Characterization of 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde-modified IFN-β-1a by size exclusion chromatography. 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde-modified IFN-β-1a was subjected to SEC on an analytical Superose 6 HR10/30 FPLC sizing column using PBS pH 7.0 as the mobile phase. The column was run at 24 mL/h and the eluent was monitored for absorbance at 280 nm. The PEGylated IFN-β-1a eluted as a single sharp peak with no evidence of aggregates (FIG. 3).
5. Analysis of 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde-modified IFN-β-1a by peptide mapping. The specificity of the PEGylation reaction was evaluated by peptide mapping. 13.3 μg of unmodified and 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde-modified IFN-β-1a were digested with 20% (w/w) of endoproteinase Lys-C from Achromobacter (Wako Bioproducts) in PBS containing 5 mM DTT, 1 mM EDTA, at pH 7.6, at room temperature for 30 h (final volume=100 μL). 4 μL of 1 M DTT and 100 μL of 8 M urea were then added and the samples incubated for 1 h at room temperature. The peptides were separated by reverse-phase HPLC on a Vydac C18 column (214TP51) using a 70 min gradient from 0-63% acetonitrile, in 0.1% TFA, followed by a 10 min gradient from 63-80% acetonitrile, in 0.1% TFA. The column eluent was monitored for absorbance at 214 nm.
All of the predicted peptides from the endoproteinase Lys-C digest of IFN-β-1a have been identified previously by N-terminal sequencing and mass spectrometry (Pepinsky et al., (2001) J Pharmacology and Experimental Therapeutics 297:1059), and, of these, only the peptide that contains the N-terminus of IFN-β-1a was altered by modification with 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde; as evident by its disappearance from the map. The mapping data therefore indicate that the PEG moiety is specifically attached to this peptide. The data further indicate that the PEG modification is targeted at the N-terminus of the protein since only the N-terminal modification would result in the specific loss of this peptide.
C) Preparation and Characterization of 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a
Human IFN-β-1a was PEGylated at the N-terminus with 20 kDa mPEG-O-p-phenylacetaldehyde. The product of the reductive alkylation chemistry that was used to incorporate the PEG onto the IFN-β-1a backbone results in the formation of an amine linkage which is extremely stable against degradation. The PEGylated IFN-β-1a was subjected to extensive characterization, including analysis by SDS-PAGE, SEC, peptide mapping, and assessment of activity in an in vitro antiviral assay. The purity of the product as measured by SDS-PAGE and SEC was greater than 95%. In the PEGylated IFN-β-1a sample there was no evidence of aggregates. Residual levels of unmodified IFN-β-1a in the product were below the limit of quantitation, but appear to represent about 1% of the product. In a stability test, no aggregation or degradation of 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a was evident in Tris-buffer pH 7.4, following an incubation at 37° C. for up to 7 days. The specific activity of the PEGylated IFN-β-1a in the antiviral activity assay was reduced approximately 2-fold compared to the unmodified IFN-β-1a (EC50=31 pg/mL for 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a versus EC50=14 pg/mL for unmodified IFN-β-1a). The PEGylated IFN-β-1a bulk was formulated at 30 μg/mL in PBS pH 7.3 containing 14 mg/mL HSA, similar to the formulation used for AVONEX® which has been subjected to extensive characterization. The material was supplied as a frozen liquid which was stored at −70° C.
The properties of 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a are summarized in Table 3:
TABLE 3
Properties of 20 kDa mPEG-O-p-phenylacetaldehyde-modified
IFN-β-1a
Pegylation efficiency
>80%
IFN-β-1a/PEG ratio
1:1
Purity
>95%
Site of attachment
N-terminus
Antiviral activity EC50
31 pg/mL
1. Preparation of 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a. 20 mL of nonformulated AVONEX® (IFN-β-1a bulk intermediate, a clinical batch of bulk drug that passed all tests for use in humans, at 250 μg/mL in 100 mM sodium phosphate pH 7.2, 200 mM NaCl) was diluted with 24 mL of 165 mM MES pH 5.0, 100 μL of 5 N HCl, and 24 mL water. The sample was loaded onto a 600 μL SP-Sepharose FF column (Pharmacia). The column was washed with 2×900 μL of 5 mM sodium phosphate pH 5.5, 75 mM NaCl, and the protein was eluted with 5 mM sodium phosphate pH 5.5, 600 mM NaCl. Elution fractions were analyzed for their absorbance at 280 nm and the concentration of IFN-β-1a in the samples was estimated using an extinction coefficient of 1.51 for a 1 mg/mL solution. The peak fractions were pooled to give an IFN-β-1a concentration of 2.3 mg/mL. To 1.2 mL of the IFN-β-1a from the SP-Sepharose eluate pool, 0.5 M sodium phosphate pH 6.0 was added to 50 mM, sodium cyanoborohydride (Aldrich) was added to 5 mM, and 20 kDa mPEG-O-p-phenylacetaldehyde, was added to 10 mg/mL. The sample was incubated at room temperature for 18 h in the dark. The PEGylated IFN-β-1a was purified from the reaction mixture on a 0.75 mL SP-Sepharose FF column as follows: 1.5 mL of reaction mixture was diluted with 7.5 mL of 20 mM MES pH 5.0, 7.5 mL water, and 5 μL 5 N HCl, and loaded onto the SP-Sepharose column. The column was washed with sodium phosphate pH 5.5, 75 mM NaCl and then the PEGylated IFN-β-1a was eluted from the column with 20 mM MES pH 6.0, 600 mM NaCl. The PEGylated IFN-β-1a was further purified on a Superose 6 HR 10/30 FPLC sizing column with 5 mM sodium phosphate pH 5.5, 150 mM NaCl as the mobile phase. The sizing column (25 mL) was run at 20 mL/h and 0.5 mL fractions were collected. The elution fractions were analyzed for protein content by absorbance at 280 nm, pooled, and the protein concentration of the pool determined. The PEGylated IFN-β-1a concentration is reported in IFN equivalents after adjusting for the contribution of the PEG (20 kDa mPEG-O-p-phenylacetaldehyde has an extinction coefficient at 280 nm of 0.5 for a 1 mg/mL solution) to the absorbance at 280 nm using an extinction coefficient of 2 for a 1 mg/mL solution of the PEGylated IFN-β-1a. Samples of the pool were removed for analysis, and the remainder was diluted to 30 μg/mL with HSA-containing formulation buffer, aliquoted at 0.25 mL/vial, and stored at −70° C.
2. UV spectrum of purified 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a. The UV spectrum (240-340 nm) of 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a was obtained using the pre-HSA-formulated bulk sample. The PEGylated sample exhibited an absorbance maximum at 278-279 nm and an absorbance minimum at 249-250 nm, consistent with that observed for the unmodified IFN-β-1a bulk intermediate. The protein concentration of the PEGylated product was estimated from the spectrum using an extinction coefficient of ε2800.1%=2.0. The protein concentration of the PEGylated bulk was 0.10 mg/mL. No turbidity was present in the sample as evident by the lack of absorbance at 320 nm.
3. Characterization of 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a by SDS-PAGE. 2.5 μg of unmodified and 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a were subjected to SDS-PAGE under reducing conditions on a 10-20% gradient gel. The gel was stained with Coomassie brilliant blue R-250, and is shown in FIG. 4 (Lane A: 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a; Lane B: unmodified IFN-β-1a; Lane C: molecular weight markers (from top to bottom; 100 kDa, 68 kDa, 45 kDa, 27 kDa, and 18 kDa, respectively)). SDS-PAGE analysis of 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a revealed a single major band with an apparent mass of 55 kDa consistent with modification by a single PEG. No higher mass forms resulting from the presence of additional PEG groups were detected. In the purified PEGylated product unmodified IFN-β-1a was detected; however, the amount is below the limit of quantitation. It is estimated that the level of unmodified IFN-β-1a in the preparation accounts for only about 1% of the total protein.
4. Characterization of 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a by size exclusion chromatography. 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a was subjected to SEC on an analytical Superose 6 HR10/30 FPLC sizing column using PBS pH 7.2 as the mobile phase. The column was run at 20 mL/h and the eluent was monitored for absorbance at 280 nm, as shown in FIG. 5: Panel A: molecular weight standards (670 kDa, thyroglobulin; 158 kDa, gamma globulin; 44 kDa, ovalbumin; 17 kDa, myoglobin; 1.3 kDa, vitamin B12); Panel B: 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a. The PEGylated IFN-β-1a eluted as a single sharp peak with an apparent molecular mass of approximately 200 kDa consistent with the large hydrodynamic volume of the PEG. No evidence of aggregates was observed. Unmodified IFN-β-1a in the preparation was detected but was below the limit of quantitation. Based on the size of the peak, the unmodified IFN-β-1a accounts for 1% or less of the product, consistent with that observed using SDS-PAGE.
5. Analysis of 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a by peptide mapping. The specificity of the PEGylation reaction was evaluated by peptide mapping. Unmodified and 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a were digested with endoproteinase Lys-C from Achromobacter (Wako Bioproducts) and the resulting cleavage products were fractionated by reverse-phase HPLC on a Vydac C4 column using a 30 min gradient from 0 to 70% acetonitrile, in 0.1% TFA. The column eluent was monitored for absorbance at 214 nm.
All of the predicted peptides from the endoproteinase Lys-C digest of IFN-β-1a have been identified previously by N-terminal sequencing and mass spectrometry (Pepinsky et al., (2001) J Pharmacology and Experimental Therapeutics 297:1059), and, of these, only the peptide that contains the N-terminus of IFN-β-1a was altered by modification with 20 kDa mPEG-O-p-phenylacetaldehyde; as evident by its disappearance from the map. The mapping data therefore indicate that the PEG moiety is specifically attached to this peptide. The data further indicate that the PEG modification is targeted at the N-terminus of the protein since only the N-terminal modification would result in the specific loss of this peptide.
6. Stability of 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a. To test the stability of 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a, samples were diluted to 0.1 μg/mL with 100 mM Tris-HCl buffer, pH 7.4, and were then incubated at 37° C. for up to 7 days. 20 μL of sample (2 μg) was removed at days 0, 2, 5, and 7, and analyzed by SDS-PAGE under reducing conditions, as shown in FIG. 6: Lane A: molecular weight markers (from top to bottom; 100 kDa, 68 kDa, 45 kDa, 27 kDa, 18 kDa, and 15 kDa, respectively); Lanes B, C, D, and E: mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a removed at day 0, 2, 5, and 7, respectively. No evidence of aggregation or degradation of PEGylated IFN-β-1a was observed even after 7 days at 37° C.
Example 3
Specific Activity of PEGylated Human IFN-β-1a in an In Vitro Antiviral Assay
The specific antiviral activity of PEGylated IFN-β-1a samples was tested on human lung carcinoma cells (A549 cells) that had been exposed to encephalomyocarditis (EMC) virus, and using the metabolic dye 2,3-bis[2-Methoxy-4-nitro-5-sulfo-phenyl]-2H-tetrazolium-5-carboxyanilide (MTT; M-5655, Sigma, St. Louis, Mo.) as a measure of metabolically-active cells remaining after exposure to the virus. Briefly, A549 cells were pretreated for 24 h with either unmodified or PEGylated IFN-β-1a (starting at 66.7 pg/mL and diluting serially 1.5-fold to 0.8 pg/mL) prior to challenge with virus. The cells were then challenged for 2 days with EMC virus at a dilution that resulted in complete cell killing in the absence of IFN. Plates were then developed with MTT. A stock solution of MTT was prepared at 5 mg/mL in PBS and sterile-filtered, and 50 μL of this solution was diluted into cell cultures (100 μL per well). Following incubation at room temperature for 30-60 min, the MTT/media solution was discarded, cells were washed with 100 μL PBS, and finally the metabolized dye was solubilized with 100 μL 1.2 N HCl in isopropanol. Viable cells (as determined by the presence of the dye) were quantified by absorbance at 450 nm. Data were analyzed by plotting absorbance against the concentration of IFN-β-1a, and the activity of IFN-β-1a was defined as the concentration at which 50% of the cells were killed i.e., the 50% cytopathic effect (EC50) or 50% maximum OD450. The assay was performed eight times for unmodified IFN-β-1a and three to four times with the various PEGylated IFN-β-1a samples. For each assay, duplicate data points for each protein concentration were obtained. Representative plots of cell viability versus the concentration of unmodified or PEGylated IFN-β-1a are shown in FIGS. 7A and 7B. In FIG. 7A, the symbols are as follows: unmodified IFN-β-1a (∘), 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a (□), 20 kDa mPEG-O-p-methylphenyl-O-2-methylpropionaldehyde-modified IFN-Δ-1a (Δ), and 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde-modified IFN-β-1a (⋄). In FIG. 7B, the symbols are as follows: unmodified IFN-β-1a (∘), 20 kDa mPEG-O-p-phenylacetaldehyde-unmodified IFN-β-1a (□), 20 kDa mPEG-O-p-phenylpropionaldehyde-modified IFN-β-1a (Δ), and 20 kDa mPEG-O-m-phenylacetaldehyde-modified IFN-β-1a (⋄).
The EC50 values (the concentration at half-maximal viral protection) for IFN-β-1a modified with 20 kDa mPEG-O-2-methylpropionaldehyde, 20 kDa mPEG-O-p-methylphenyl-O-2-methylpropionaldehyde, 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde, 20 kDa mPEG-O-p-phenylacetaldehyde, 20 kDa mPEG-O-p-phenylpropionaldehyde, and 20 kDa mPEG-O-m-phenylacetaldehyde are shown in Table 4. All PEGylated IFNs-β-1a were modified and purified to homogeneity essentially as described for 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a, 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde-modified IFN-β-1a, and 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a as described above.
TABLE 4
Specific antiviral activity of unmodified and PEGylated IFNs-β-1a
Mean EC50
Protein
(pg/mL)
Unmodified IFN-β-1a
14 (range 12-16)
20 kDa mPEG-O-2-methylpropionaldehyde-
32 (range 26-37)
modified IFN-β-1a
20 kDa mPEG-O-p-methylphenyl-O-2-
41 (range 36-47)
methylpropionaldehyde-modified IFN-β-1a
20 kDa mPEG-O-m-methylphenyl-O-2-
31 (range 27-35)
methylpropionaldehyde-modified IFN-β-1a
20 kDa mPEG-O-p-phenylacetaldehyde-
31 (range 25-39)
modified IFN-β-1a
20 kDa mPEG-O-p-phenylpropionaldehyde-
31 (range 27-34)
modified IFN-β-1a
20 kDa mPEG-O-m-phenylacetaldehyde-
27 (range 25-29)
modified IFN-β-1a
Example 4
Pharmacokinetics of Intravenously-Administered Unmodified and PEGylated IFNs-β-1a in Rats
Canulated female Lewis rats were injected intravenously with either 80 μg/kg of unmodified IFN-β-1a or 24 μg/kg of the following PEGylated IFNs-β-1a; 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a, 20 kDa mPEG-O-p-methylphenyl-O-2-methylpropionaldehyde-modified IFN-β-1a, 20 kDa mPEG-O-p-phenylacetaldehyde-modified IFN-β-1a, 20 kDa mPEG-O-p-phenylpropionaldehyde-modified IFN-β-1a, 20 kDa mPEG-O-m-phenylacetaldehyde-modified IFN-β-1a, and 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde-modified IFN-β-1a. Both the unmodified and PEGylated proteins were formulated in the presence of 14-15 mg/mL HSA as a carrier. For the unmodified protein, blood (0.2 mL) was obtained via the canula at different time points; immediately prior to administration, and at 0.083, 0.25, 0.5, 1.25, 3, and 5 hours post-administration. For the PEGylated proteins, blood (0.2 mL) was obtained via the canula immediately prior to administration, and at 0.083, 0.25, 0.5, 1.25, 3, 24, 48, and 72 h post-administration. Whole blood was collected into serum separator tubes (Beckton Dickinson No. 365956) and incubated at room temperature for 60 min to allow for clotting. The clotted blood was centrifuged for 10 min at 4° C., and the serum removed and stored at −70° C. until the time of assay.
The serum samples were then thawed and tested in antiviral assays. The serum samples were diluted 1:50 into serum-containing medium (Dulbecco's Modified Eagles Medium containing 10% (v/v) fetal bovine serum, 100 U each of penicillin and streptomycin, and 2 mM L-glutamine) and tested in antiviral assays. Samples were titrated into designated wells of a 96 well tissue culture plate containing human lung carcinoma cells (A549, #CCL-185, ATCC, Rockville, Md.). Dilutions of a standard (66.7, 44.4, 29.6, 19.8, 13.2, 8.8, 5.9, 3.9, 2.6, 1.7, 1.2, and 0.8 pg/mL of the same form of IFN-β-1a administered to the rat) and of three serum samples were assayed on each plate. The A549 cells were pretreated with diluted serum samples for 24 h prior to challenge with encephalomyelocarditis (EMC) virus. Following a 2 day incubation with virus, viable cells were stained with a solution of MTT (at 5 mg/mL in phosphate buffer) for 1 h, washed with phosphate buffer, and solubilized with 1.2 N HCl in isopropanol. The wells were then read at 450 nm. Standard curves of the unmodified or PEGylated IFN-β-1a were generated for each plate and used to determine the amount of unmodified or PEGylated IFN-β-1a in each test sample. Pharmacokinetic parameters were then calculated using non-compartmental analysis with WinNonLin version 3.0 or 3.3 software.
FIG. 8A shows the concentration versus time plots for unmodified IFN-β-1a (upper panel) and IFN-β-1a modified with 20 kDa mPEG-O-2-methylpropionaldehyde (lower panel), and FIG. 8B shows the concentration versus time plots for IFN-β-1a modified with 20 kDa mPEG-O-p-methylphenyl-O-2-methylpropionaldehyde (upper panel) and 20 kDa mPEG-O-p-phenylacetaldehyde (lower panel). Data points are averages from measurements from 3 rats.
Table 5 shows the pharmacokinetic parameters Cmax (maximal observed concentration), ti/2 (elimination half-life) AUC (area under the curve), Vss (distribution volume at steady state), clearance rate, and MRT (mean residence time) for unmodified IFN-β-1a and these forms of PEGylated IFN-β-1a. The data shown in FIGS. 8A and 8B and in Table 5 were obtained in the same study.
FIG. 9A shows the concentration versus time plots for unmodified IFN-β-1a (upper panel) and IFN-β-1a modified with 20 kDa mPEG-O-p-phenylpropionaldehyde (lower panel). Data points are averages from measurements from 2 rats. FIG. 9B shows the concentration versus time plots for IFN-β-1a modified with 20 kDa mPEG-O-m-phenylacetaldehyde (upper panel) and 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde (lower panel). Data points are averages from measurements from 3 rats.
Table 6 shows the pharmacokinetic parameters for unmodified IFN-β-1a and these forms of PEGylated IFN-β-1a. The data shown in FIGS. 9A and 9B and in Table 6 were obtained in the same study; independent from the data shown in FIGS. 8A and 8B, and in Table 5.
As is clear from the data shown in FIGS. 8A, 8B, 9A, and 9B, and in Tables 5 and 6, PEGylation of IFN-β-1a with the PEG molecules of the invention improves the pharmacokinetic properties of IFN-β-1a. In all cases, the PEGylated proteins were cleared less rapidly than unmodified IFN-β-1a, resulting in clearance rates of 3.9-8.3 mL/h/kg as compared to 160-170 mL/h/kg for the unmodified protein. As a consequence of the reduced clearance rates, the mean residence time (MRT) increased from approximately 1 h for the unmodified protein to 4.8-7.6 h for the PEGylated proteins. Similarly, the elimination half-life (t1/2) increased from approximately 1 h for the unmodified protein to 5.2-13 h for the PEGylated proteins. The area under the curve (AUC) values were also significantly increased upon PEGylation of IFN-β-1a. For unmodified IFN-β-1a, the AUC was approximately 0.5 μg·h/mL while for the PEGylated proteins the AUC values ranged from approximately 3 to 6 μg·h/mL, despite the fact that the PEGylated proteins were dosed at a level 3.3-fold lower than the unmodified protein. For the maximal observed concentration (Cmax), the values were generally higher for unmodified IFN-β-1a than for the PEGylated proteins, reflecting the lower dose of the modified proteins administered. For the volume of distribution at steady state (Vss), the values for all the PEGylated proteins were lower than for unmodified IFN-β-1a, indicating a restriction in their ability to exit the central blood compartment.
TABLE 5
Pharmacokinetic parameters for unmodified IFN-β-1a, 20 kDa mPEG-O-2-
methylpropionaldehyde-modified IFN-β-1a, 20 kDa mPEG-O-p-methylphenyl-O-2-
methylpropionaldehyde-modified IFN-β-1a, and 20 kDa mPEG-O-p-phenylacetaldehyde-
modified IFN-β-1a following intravenous administration in ratsa
20 kDa mPEG-O-p-
20 kDa mPEG-O-2-
methylphenyl-O-2-
20 kDa mPEG-O-p-
Unmodified
methylpropionaldehyde-
methylpropionaldehyde-
phenylacetaldehyde-
Parameter
Units
IFN-β-1a
modified IFN-β-1a
modified IFN-β-1a
modified IFN-β-1a
Cmax
pg/mL
1,400,000
720,000
710,000
590,000
t1/2
h
0.98
13
11
6.8
AUC
pg · h/mL
510,000
4,800,000
4,500,000
2,900,000
Vss
mL/kg
160
39
40
53
Clearance
mL/h/kg
160
5.0
5.3
8.3
MRT
h
0.98
7.6
7.4
6.4
aThe pharmacokinetic data for the unmodified and PEGylated IFNs-β-1a shown were obtained in the same study
TABLE 6
Pharmacokinetic parameters for unmodified IFN-β-1a, 20 kDa mPEG-O-p-
phenylpropionaldehyde-modified IFN-β-1a, 20 kDa mPEG-O-m-phenylacetaldehyde-
modified IFN-β-1a, and 20 kDa mPEG-O-m-methylphenyl-O-2-methylpropionaldehyde-
modified IFN-β-1a following intravenous administration in ratsa
20 kDa mPEG-O-m-
20 kDa mPEG-O-m-
20 kDa mPEG-O-p-
phenylacetaldehyde-
methylphenyl-O-2-
Unmodified
phenylpropionaldehyde-
modified
methylpropionaldehyde-
Parameter
Units
IFN-β-1a
modified IFN-β-1a
IFN-β-1a
modified IFN-β-1a
Cmax
pg/mL
670,000
930,000
550,000
700,000
t1/2
h
0.92
5.2
7.7
7.1
AUC
pg · h/mL
470,000
4,700,000
3,800,000
6,200,000
Vss
mL/kg
140
25
46
21
Clearance
mL/h/kg
170
5.1
6.4
3.9
MRT
h
0.81
4.8
7.2
5.5
aThe pharmacokinetic data for the unmodified and PEGylated IFNs-β-1a shown were obtained in the same study.
Example 5
Comparative Pharmacokinetics and Pharmacodynamics of Unmodified and PEGylated Human IFN-β-1a in Non-Human Primates
Single and repeat dose comparative studies are conducted with unmodified and PEGylated IFN-β-1a to determine their relative stability and activity in non-human primates. In these studies, the pharmacokinetics and pharmacodynamics of the PEGylated IFN-β-1a conjugates is compared to that of unmodified IFN-β-1a and reasonable inferences can be extended to humans.
Animals and Methods
Study 1 (Repeat Dose)
This is a parallel group, repeat dose study to evaluate the comparative pharmacokinetics and pharmacodynamics of unmodified and PEGylated IFN-β-1a. Healthy primates (e.g., rhesus monkeys) are used for this study. Prior to dosing, all animals are evaluated for signs of ill health by a laboratory animal veterinarian on two occasions within 14 days prior to test article administration; one evaluation must be within 24 h prior to the first test article administration. Only healthy animals receive the test article. Evaluations include a general physical examination and pre-dose blood draws for baseline clinical pathology and baseline antibody level to IFN-β-1a. All animals are weighed and body temperatures are recorded within 24 h prior to test article administrations. Twelve subjects are enrolled and assigned to groups of three to receive 1×106 U/kg of unmodified or PEGylated IFN-β-1a, but otherwise identical IFN-β-1a. Administration is by either the subcutaneous (SC) or intravenous (IV) routes. Six male animals receive test article by the IV route (3 per treatment) and another 6 male animals receive test article by the SC route (3 per treatment). All animals must be naive to IFN-β treatment. Each animal is dosed on two occasions, the doses are separated by four weeks. The dose volume is 1.0 mL/kg. Blood is drawn for pharmacokinetic testing at 0, 0.083, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24, 48, 72, and at 96 hours following each injection. Blood samples for measurement of the IFN-induced biological response marker, serum neopterin, are drawn at 0, 24, 48, 72, 96, 168, 336, and at 504 h following administration of study drug. Evaluations during the study period include clinical observations performed 30 min and 1 h post-dose for signs of toxicity. Daily cage-side observations are performed and general appearance, signs of toxicity, discomfort, and changes in behavior are recorded. Body weights and body temperatures are recorded at regular intervals through 21 days post-dose.
Study 2 (Single Dose)
This is a parallel group, single dose study to evaluate the comparative pharmacokinetics and pharmacodynamics of unmodified and PEGylated IFN-β-1a. Healthy primates (e.g., rhesus monkeys) are used for this study. Prior to dosing, all animals are evaluated for signs of ill health by a laboratory animal veterinarian on two occasions within 14 days prior to test article administration; one evaluation must be within 24 h prior to the first test article administration. Only healthy animals receive the test article. Evaluations include a general physical examination and pre-dose blood draws for baseline clinical pathology and baseline antibody level to IFN-β-1a. All animals are weighed and body temperatures are recorded within 24 h prior to test article administrations. Twenty subjects are enrolled and assigned to one of five groups of four animals (2 male and 2 female per group) to receive either 1×106 U/kg of unmodified or PEGylated IFN-β-1a intramuscularly (IM), or 2×105 U/kg, 1×106 U/kg, or 5×106 U/kg of PEGylated IFN-β-1a intravenously (IV). All animals must be naive to IFN-β treatment. The dose volume is generally 1.0 mL/kg. Blood is drawn for pharmacokinetic testing at 0, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, and at 96 hours, and at 7, 14, 21, and at 28 days following administration of study drug. Blood samples for measurement of the IFN-induced biological response marker, 2′-5′-oligoadenylate synthase (2′-5′-OAS), are drawn at 0, 12, 24, 48, 72, and at 96 hours, and at 7, 14, 21, and at 28 days following administration of study drug. Evaluations during the study period include clinical observations performed 30 min and 1 h post-dose for signs of toxicity. Daily cage-side observations are performed and general appearance, signs of toxicity, discomfort, and changes in behavior are recorded. Body weights and body temperatures are recorded at regular intervals through 28 days post-dose.
Assay Methods
Levels of IFN-β-1a in serum are quantitated using a cytopathic effect (CPE) bioassay. The CPE assay measures levels of IFN-mediated antiviral activity. The level of antiviral activity in a sample reflects the number of molecules of active IFN contained in that sample at the time the blood is drawn. This approach has been the standard method to assess the pharmacokinetics of IFN-β. The CPE assay detects the ability of IFN-β to protect human lung carcinoma cells (A549, #CCL-185, ATCC, Rockville, Md.) from cytotoxicity due to encephalomyocarditis (EMC) virus. The cells are preincubated for 15-20 h with serum samples to allow the induction and synthesis of IFN-inducible proteins that are responsible for the antiviral response. EMC virus is then added and incubated for a further 30 h before assessment of cytotoxicity is made using a crystal violet stain. An internal IFN-β standard as well as a PEGylated IFN-β-1a internal standard is tested concurrently with samples on each assay plate. This standard is calibrated against a natural human fibroblast IFN reference standard (WHO Second International Standard for Interferon, Human Fibroblast, Gb-23-902-53). Each assay plate also includes cell growth control wells containing neither IFN-β of any kind nor EMC, and virus control wells that contain cells and EMC but no IFN-β. Control plates containing the standard and samples are also prepared to determine the effect, if any, of the samples on cell growth. These plates are stained without the addition of virus. Samples and standards are tested in duplicate on each of two replicate assay plates, yielding four data points per sample. The geometric mean concentration of the four replicates is reported. The limit of detection in this assay is 10 U/mL. Serum concentrations of neopterin are determined at the clinical pharmacology unit using commercially-available assays. Serum concentrations of 2′-5′-OAS are determined at a contract laboratory using a validated commercially-available assay.
Pharmacokinetic and Statistical Methods
Rstrip™ software (MicroMath, Inc., Salt Lake City, Utah) is used to fit data to pharmacokinetic models. Geometric mean concentrations are plotted by time for each group. Since assay results are expressed in dilutions, geometric means are considered more appropriate than arithmetic means. Serum IFN levels are adjusted for baseline values and non-detectable serum concentrations are set to 5 U/mL, which represents one-half the lower limit of detection. For IV infusion data, a two compartment IV infusion model is fit to the detectable serum concentrations for each subject, and the SC data are fit to a two compartment injection model.
The following pharmacokinetic parameters are calculated:
(i) observed peak concentration, Cmax (U/mL);
(ii) area under the curve from 0 to 48 h, AUC (U×h/mL) using the trapezoidal rule;
(iii) elimination half-life (h);
and, from IV infusion data (if IV is employed):
(iv) distribution half-life (h);
(v) clearance (mL/h/kg)
(vi) apparent volume of distribution, Vd (mL/kg).
WinNonlin (Version 1.0, Scientific Consulting Inc., Apex, N.C.) software is used to calculate the elimination half-lives after IV and SC injection. For neopterin and 2′-5′-OAS, arithmetic means by time are presented for each group, Emax, the maximum change from baseline, is calculated. Cmax, AUC, and Emax are submitted to a one-way analysis of variance to compare dosing groups. Cmax and AUC are logarithmically-transformed prior to analysis; geometric means are reported.
Example 6
Anti-Angiogenic Effects of PEGylated Human IFN-β-1a; the Ability of PEGylated IFN-β-1a to Inhibit Endothelial Cell Proliferation In Vitro
Human venous endothelial cells (Cell Systems, Cat. # 2V0-P75) and human dermal microvascular endothelial cells (Cell Systems, Cat. # 2M1-C25) are maintained in culture with CS-C Medium Kit (Cell Systems, Cat. # 4Z0-500). 24 h prior to the experiment, cells are trypsinized, and resuspended in assay medium, 90% M199 and 10% fetal bovine serum (FBS), and are adjusted to desired cell density. Cells are then plated onto gelatin-coated 24 or 96 well plates, either at 12, 500 cells/well or 2,000 cells/well, respectively. After overnight incubation, the assay medium is replaced with fresh medium containing 20 ng/mL of human recombinant basic Fibroblast Growth Factor (bFGF) (Becton Dickinson, Cat. # 40060) and various concentrations of unmodified or PEGylated IFN-β-1a of the invention or positive control (endostatin can be used as a positive control, as could an antibody to bFGF) are added. The final volume is adjusted to 0.5 mL in the 24 well plate or 0.2 mL in the 96 well plate. After 72 h, cells are trypsinized for Coulter counting, frozen for CyQuant fluorescence reading, or labeled with [3H]-thymidine. This in vitro assay tests the PEGylated human IFN-β-1a molecules of the invention for effects on endothelial cell proliferation which may be indicative of anti-angiogenic effects in vivo. See O'Reilly, et al., Cell 88: 277-285 (1997).
Example 7
In Vivo Models to Test Anti-Angiogenic and Neovascularization Effects of PEGylated Human IFN-β-1a and PEGylated Rodent IFNs-β
Unmodified IFN-β-1a and 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a were tested for their ability to inhibit the formation of radially-oriented vessels entering the periphery of SK-MEL-1 human malignant melanoma tumors in athymic nude homozygous (nu/nu) mice. SK-MEL-1 cells were grown in culture to 80% confluency, and then 2×106 cells inoculated intradermally (0.1 mL volume on day 0) into the flank in the mid-axillary line in three week old athymic nude homozygous (nu/nu) NCR mice (Taconic, Germantown, N.Y.). 24 hours later (day 1), groups of three mice each received the following subcutaneous doses of vehicle control, unmodified IFN-β-1a, or 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a:
Group A: 0.1 mL of 45.6 mg/mL human serum albumin (vehicle control) once on day 1 only
Group B: 0.1 mL of 45.6 mg/mL human serum albumin containing 1 MU (5 μg) of unmodified IFN-β-1a daily on days 1-9 inclusive
Group C: 0.1 mL of 45.6 mg/mL human serum albumin containing 1 MU units (10 μg) of 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a once on day 1 only
Group D: 0.1 mL of 45.6 mg/mL human serum albumin (vehicle control) daily on days 1-9 inclusive
Mice were sacrificed on day 10 (Avertin, 0.5 mL intraperitoneally) and the tumor inoculation site assessed for neovascularization, measured by an observer blind as to treatment group. Vessels were counted under fixed magnification under a dissecting microscope. Every radially-oriented vessel entering the periphery of the tumor was scored as a single vessel. Each group consisted of three mice.
As shown in FIG. 10, a single administration of 1 MU of 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a (group C) was as effective at reducing the number of neovessels as daily administration of 1 MU of unmodified IFN-β-1a (group B). However, the effect of the 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a is more pronounced when considering that daily administration of the vehicle alone had some inhibitory effect (compare group A, vehicle given once, with group D, vehicle given daily).
A variety of other models have also been developed which can be used to test the anti-angiogenic and anti-neovascularization effects of the PEGylated molecules of the invention. Some of these models have been described in U.S. Pat. Nos. 5,733,876 (Mar. 31, 1998: “Method of inhibiting angiogenesis”) and 5,135,919 (Aug. 4, 1992: “Method and a pharmaceutical composition for the inhibition of angiogenesis”). Other assays include the shell-less chorioallantoic membrane (CAM) assay of Taylor and Folkman; Nature 297:307 (1982) and Crum et al., Science 230:1375 (1985); the mouse dorsal air sac method anti-angiogenesis model of Folkman et al.; J. Exp. Med. 133: 275 (1971), and the rat corneal micropocket assay of Gimbrone, Jr. et al., J. Natl. Cancer Inst. 52:413 (1974) in which corneal vascularization is induced in adult male rats of the Sprague-Dawley strain (Charles River, Japan) by implanting 500 ng of bFGF (bovine, R & D Systems, Inc.), impregnated in ethylene-vinyl acetate copolymer pellets, in each cornea. In addition, a model exists in which angiogenesis is induced in NIH-Swiss or athymic nude (nu/nu) mice after implantation of MCF-7 breast carcinoma or NIH-OVCAR-3 ovarian carcinoma cells as described by Lindner and Borden; Int. J. Cancer 71:456 (1997). Additional tumor cell lines including (but not limited to) SK-MEL-1 human malignant melanoma cells may also be used to induce angiogenesis as described above. Various doses, with various dosing frequencies, and for various duration can be tested for both the unmodified and PEGylated IFN-β-1a proteins of the invention.
Other methods for testing PEGylated murine and rat IFN-β for anti-angiogenic effects in an animal model include (but are not limited to) protocols for screening new potential anticancer agents as described in the original Cancer Chemotherapy Reports, Part 3, Vol. 3, No. 2, September 1972 and the supplement In Vivo Cancer Models, 1976-1982, NIH Publication No. 84-2635, February 1984. Because of the species specificity of Type I interferons, to assess the anti-angiogenic activity of PEGylated IFN-β in rodent models, PEGylated rodent IFN-β preparations (e.g., murine and rat) are generated. Such screening methods are exemplified by a protocol to test for the anti-angiogenic effects of PEGylated murine IFN-β on subcutaneously-implanted Lewis Lung Carcinoma:
Origin of Tumor Line
This tumor line arose spontaneously in 1951 as a carcinoma of the lung in a C57BL/6 mouse.
Summary of Test Procedure
A tumor fragment is implanted subcutaneously in the axillary region of a B6D2F1 mouse. The test agent (i.e., a PEGylated interferon of the invention) is administered at various doses, subcutaneously (SC) or intraperitoneally (IP) on multiple days following tumor implantation. The parameter measured is median survival time. Results are expressed as a percentage of control survival time.
Animals
Propagation: C57BL/6 mice.
Testing: B6D2F1 mice.
Weight: Mice are within a 3 g weight range, with a minimum weight of 18 g for males and 17 g for females.
Sex: One sex is used for all test and control animals in one experiment.
Source: One source, if feasible, for all animals in one experiment.
Experiment Size
Ten animals per test group.
Tumor Transfer
Propagation:
Fragment: Prepare a 2-4 mm fragment of a SC donor tumor.
Time: Day 13-15.
Site: Implant the fragment SC in the axillary region with a puncture in the inguinal region.
Testing
Fragment: Prepare a 24 mm fragment of SC donor tumor.
Time: Day 13-15.
Site: Implant the fragment SC in the axillary region with a puncture in the inguinal region.
Testing Schedule
Day 0: Implant tumor. Run bacterial cultures. Test positive control compound in every odd-numbered experiment. Prepare materials. Record deaths daily.
Day 1: Check cultures. Discard experiment if contaminated. Randomize animals.
Treat as instructed (on day 1 and on following days).
Day 2: Recheck cultures. Discard experiment if contaminated.
Day 5: Weigh Day 2 and day of initial test agent toxicity evaluation.
Day 14: Control early-death day.
Day 48: Control no-take day.
Day 60: End and evaluate experiment. Examine lungs for tumor.
Quality Control
Schedule the positive control compound (NSC 26271; Cytoxan at a dose of 100 mg/kg/injection) in every odd-numbered experiment, the regimen for which is intraperitoneal on Day 1 only. The lower Test/Control limit for the positive control is 140%. The acceptable untreated control median survival time is 19-35.6 days.
Evaluation
The parameter measured is median survival time. Compute the mean animal body weights for Day 1 and Day 5, compute Test/Control ratio for all test groups. The mean animal body weights for staging day and final evaluation day are computed. The Test/Control ratio is computed for all test groups with >65% survivors on Day 5. A Test/Control ratio value <86% indicates toxicity. An excessive body weight change difference (test minus control) may also be used in evaluating toxicity.
Criteria for Activity
An initial Test/Control ratio greater than or equal to 140% is considered necessary to demonstrate moderate activity. A reproducible Test/Control ratio value of greater than or equal to 150% is considered significant activity.
Example 8
In Vivo Models to Test the Antiproliferative and Anti-Tumor Effects of PEGylated Human IFN-β-1a and PEGylated Rodent IFNs-β
Various in vivo models are available to test the anti-proliferative and anti-tumor effects of unmodified and PEGylated human IFNs-β-1a of the invention. In a model described by Bailon et al., Bioconjugate Chemistry 12:195 (2001), athymic nude mice (Harlan) are implanted subcutaneously with 2×106 human renal A498, human renal ACHN, or human renal G402 cells under the rear flank and 3-6 weeks allowed for tumors to develop. Unmodified or PEGylated human IFN-β-1a is then administered at various doses, with various dosing frequencies, and for various duration, and tumor volume measured and compared between treatments. In another model described by Lindner and Borden, J. Interferon Cytokine Res 17: 681 (1997), athymic nude (nu/nu) oophorectomized female BALB/c mice are implanted with 2×106 MCF-7 (plus estradiol), MDA-MB-231, MDA-MB468, or BT-20 human breast carcinoma cells, NIH-OVCAR-3 human ovarian carcinoma cells, HT-29 human colon carcinoma cells, or SK-MEL-1 or FEMX human malignant melinoma cells, into the dermis overlying the mammary glands nearest the axillae, and the size of the tumors assessed as a function of time. Unmodified or PEGylated human IFN-β-1a is then administered at various doses, with various dosing frequencies, and for various duration, and tumor volume measured and compared between treatments. Other models for testing the anti-proliferative and anti-tumor effects of PEGylated human IFN-β-1a include (but are not limited to) local and metastatic lung cancer models described by Qin et al., Molecular Therapy 4: 356 (2001), and nude mouse xenograft models of human colorectal cancer liver metastases described by Tada et al., J Clinical Investigation 108: 83 (2001).
Other methods for testing PEGylated murine and rat IFN-β for anti-proliferative and anti-tumor effects in animal models include (but are not limited to) a mouse model of malignant mesothelioma described by Odaka et al., Cancer Res 61: 6201 (2001), local and metastatic lung cancer models described by Qin et al., Molecular Therapy 4: 356 (2001), and syngeneic mouse models of colorectal cancer liver metastases described by Tada et al., J Clinical Investigation 108: 83 (2001).
Example 9
In Vivo Models to Test Anti-Viral Effects of PEGylated Murine IFN-β and PEGylated Human IFN-β-1a
An in vivo mouse model is available to test the effect of unmodified and PEGylated murine IFN-β on the levels of human Hepatitis B Virus (HBV) in HBV-transgenic SCID mice. Larkin et al., Nature Medicine 5:907 (1999). In this model, transgenic SCID mice carrying a head-to-tail dimer of the human HBV genome have detectable levels of HBV replicative forms and pre-genomic RNA in the liver, and HBV virus in the serum. Hepatocytes from the transgenic mice are also positive for the HBsAg, HBcAg, and HbxAg proteins, indicative of viral replication. An example of a protocol for comparing unmodified and PEGylated murine IFN-β in this model is given below:
30 mice (5 groups of 5 plus 5 spare) with comparable viral titer are titered at two independent time points (at least 1 week apart) to establish a baseline titer and to ensure that their titers remain constant prior to dosing with murine IFN-β. Groups of 5 mice are dosed 3 times per week (Monday, Wednesday, and Friday) subcutaneously with the following samples, as shown in Table 7.
TABLE 7
Group
Dosing sample
1
Vehicle control (1 mg/mL murine serum albumin, MSA)
2
30 U unmodified murine IFN-β in 1 mg/mL MSA
3
300 U unmodified murine IFN-β in 1 mg/mL MSA
4
3000 U unmodified murine IFN-β in 1 mg/mL MSA
5
30 U PEGylated murine IFN-β in 1 mg/mL MSA
6
300 U PEGylated murine IFN-β in 1 mg/mL MSA
7
3000 U PEGylated murine IFN-β in 1 mg/mL MSA
Viral titers are determined weekly during dosing and weekly to bi-weekly for 6 months following dosing. Plots of viral titer against time are constructed for a comparison of vehicle and IFN-β-treated animals with respect to the clearance and reestablishment of viral titer. A second study is then performed with the appropriate doses of unmodified and PEGylated murine IFN-β with 10-20 mice per group for a total of 30-60 mice (10-20 for control, 10-20 for unmodified murine IFN-β, and 10-20 for PEGylated murine IFN-β). Viral titers are assessed as above, and at sacrifice, serum is analyzed for viral titer as well as for HbsAg by SDS-PAGE and Western blotting. Livers are also removed, frozen or fixed as necessary, and stained for the presence of HbsAg, HbcAg, and HbxAg. Other appropriate histological, histochemical, or biochemical tests familiar to those in the art may also be performed on serum and tissue samples.
An in vivo mouse model is also available to test the effect of unmodified and PEGylated human IFN-β-1a on the levels of human Hepatitis C Virus, (HCV) in mice carrying chimeric human livers. Mercer et al., Nature Medicine 7:927 (2001). In this model, normal human hepatocytes are grafted into SCID mice carrying a plasminogen activator transgene (Alb-uPA) and the mice inoculated with serum from humans infected with the different genotypes of HCV. The engrafted human liver cells become infected by the virus and the virus replicates. Levels of HCV RNA in the serum can be quantified by PCR, as well as the levels of positive and negative (replicative form) RNA in the liver cells. An appropriate study protocol similar to (but not limited to) that described above for unmodified and PEGylated murine IFN-β in transgenic HBV SCID mice is performed to assess the efficacy of unmodified and PEGylated human IFN-β-1a in this model i.e. to determine the effect of treatment on HCV titer, liver histology, serum ALT levels, and the presence of HCV replicative forms in the engrafted human liver tissue. Other appropriate histological, histochemical, or biochemical tests familiar to those in the art may also be performed on serum and tissue samples.
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10892830
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biogen idec ma inc.
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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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530/351
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Apr 1st, 2022 05:10PM
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Apr 1st, 2022 05:10PM
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Biogen
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:biib
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Biogen
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Oct 2nd, 2007 12:00AM
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Apr 2nd, 2002 12:00AM
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https://www.uspto.gov?id=US07276241-20071002
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Methods of treating a tumor that expresses APRIL by administering BCMA
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A novel receptor in the TNF family is provided: APRIL-R. Chimeric molecules and antibodies to APRIL-R and methods of use thereof are also provided.
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7276241
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1. A method of treating a mammal for a tumor that expresses A Proliferation Inducing Ligand (APRIL), said method comprising administering to the mammal a therapeutically effective amount of a polypeptide capable of binding to APRIL, wherein the polypeptide comprises at least one of the following amino acid sequences:
(a) an amino acid sequence which is at least 95% identical to amino acids 8 to 41 of SEQ ID NO:8; and
(b) an amino acid sequence which is at least 95% identical to amino acids 1 to 52 of SEQ ID NO:8.
2. The method of claim 1, wherein the polypeptide comprises amino acids 8 to 41 of SEQ ID NO:8.
3. The method of claim 1, wherein the polypeptide further comprises a signal sequence of a secreted protein.
4. The method of claim 1, wherein the polypeptide further comprises an Fc domain of an immunoglobulin.
5. The method of claim 4, wherein the immunoglobulin is lgG.
6. The method of claim 5, wherein the immunoglobulin is human.
7. The method of claim 6, wherein the polypeptide comprises amino acids 24-302 of SEQ ID NO:12.
8. The method of any one of claims 1-7, wherein the mammal is a human.
9. The method of any one of claims 1-7, wherein the tumor is a carcinoma.
10. The method of claim 9, wherein the mammal is a human.
11. The method of claim 9, wherein the carcinoma is selected from the group consisting of lung carcinoma, colon carcinoma, prostate carcinoma, and breast carcinoma.
12. The method of claim 11, wherein the mammal is a human.
13. A method of inhibiting the activity of A Proliferation Inducing Ligand (APRIL) in a mammal having a tumor that expresses APRIL, said method comprising administering to the mammal an effective amount of a polypeptide capable of binding to APRIL, wherein the polypeptide comprises at least one of the following amino acid sequences:
(a) an amino acid sequence which is at least 95% identical to amino acids 8 to 41 of SEQ ID NO:8; and
(b) an amino acid sequence which is at least 95% identical to amino acids 1 to 52 of SEQ ID NO:8.
14. The method of claim 13, wherein the polypeptide comprises amino acids 8 to 41 of SEQ ID NO:8.
15. The method of claim 13, wherein the polypeptide further comprises a signal sequence of a secreted protein.
16. The method of claim 13, wherein the polypeptide further comprises an Fc domain of an immunoglobulin.
17. The method of claim 16, wherein the immunoglobulin is lgG.
18. The method of claim 17, wherein the immunoglobulin is human.
19. The method of claim 18, wherein the polypeptide comprises amino acids 24-302 of SEQ ID NO:12.
20. The method of any one of claims 13-19, wherein the mammal is a human.
21. The method of any one of claims 13-19, wherein the tumor is a carcinoma.
22. The method of claim 21, wherein the mammal is a human.
23. The method of claim 21, wherein the carcinoma is selected from the group consisting of lung carcinoma, colon carcinoma, prostate carcinoma, and breast carcinoma.
24. The method of claim 23, wherein the mammal is a human.
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24
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RELATED APPLICATIONS
This is a continuation of PCT/US00/27579, filed on Oct. 5, 2000, which claims priority from U.S. provisional application Ser. No. 60/157,933 filed on Oct. 6, 1999, U.S. provisional application Ser. No. 60/181,807 filed 11 Feb. 2000 and U.S. provisional application Ser. No. 60/215,688 filed 30 Jun. 2000.
FIELD OF THE INVENTION
The present invention relates generally to methods of treatment for cancer. The methods involve the administration of certain tumor necrosis factor (TNF) antagonists.
BACKGROUND OF THE INVENTION
Members of the tumor-necrosis factor (TNF) family of cytokines are involved in an ever expanding array of critical biological functions. Each member of the TNF family acts by binding to one or more members of a parallel family of receptor proteins. These receptors in turn signal intracellularly to induce a wide range of physiological or developmental responses. Many of the receptor signals influence cell fate, and often trigger terminal differentiation. Examples of cellular differentiation include proliferation, maturation, migration, and death.
TNF family members are Type II membrane bound proteins, having a short intracellular N-terminal domain, a transmembrane domain, and the C-terminal receptor binding domains lying outside the cell surface. In some cases the extracellular portion of the protein is cleaved off, creating a secreted form of the cytokine. While the membrane bound proteins act locally, presumably through cell contact mediated interaction with their receptors, the secreted forms have the potential to circulate or diffuse, and therefore can act at distant sites. Both membrane bound and secreted forms exist as trimers, and are thought to transduce their signal to receptors by facilitating receptor clustering.
The TNF receptor protein family is characterized by having one or more cysteine rich extracellular domains. Each cysteine rich region creates a disulfide-bonded core domain, which contributes to the three dimensional structure that forms the ligand binding pocket. The receptors are Type I membrane bound proteins, in which the extracellular domain is encoded by the N-terminus, followed by a transmembrane domain and a C-terminal intracellular domain. The intracellular domain is responsible for receptor signaling. Some receptors contain an intracellular “death domain”, which can signal cell apoptosis, and these can be strong inducers of cell death. Another class of receptors can weakly induce cell death; these appear to lack a death domain. A third class of receptors do not induce cell death. All classes of receptors can signal cell proliferation or differentiation instead of death, depending on cell type or the occurrence of other signals.
A well studied example of the pluripotent nature of TNF family activity is the nominant member, TNF. TNF can exist as a membrane bound cytokine or can be cleaved and secreted. Both forms bind to the two TNF receptors, TNF-R55 and TNF-R75. Originally described on the basis on its' ability to directly kill tumor cells, TNF also controls a wide array of immune processes, including inducing acute inflammatory reactions, as well as maintaining lymphoid tissue homeostasis. Because of the dual role this cytokine can play in various pathological settings, both agonist and antagonist reagents have been developed as modifiers of disease. For example TNF and LTα (which also signals through the TNF receptors) have been used in treatment for cancers, especially those residing in peripheral sites, such as limb sarcomas. In this setting direct signaling by the cytokine through the receptor induces tumor cell death (Aggarwal and Natarajan, 1996. Eur Cytokine Netw 7:93-124).
In immunological settings, agents that block TNF receptor signaling (e.g., anti-TNF mAb, soluble TNF-R fusion proteins) have been used to treat diseases like rheumatoid arthritis and inflammatory bowel disease. In these pathologies TNF acts to induce cell proliferation and effector function, thereby exacerbating autoimmune disease, and in this setting blocking TNF binding to its receptor(s) has therapeutic benefit (Beutler, 1999. J Rheumatol 26 Suppl 57:16-21).
A more recently discovered ligand/receptor system appears amenable to similar manipulations. Lymphotoxin beta (LTβ), a TNF family member which forms heterotrimers with LTα, bind to the LTβ-R. Some adenocarcinoma tumor cells which express LTβ-R can be killed or differentiated when treated with an agonistic anti-LTβ-R mAb (Browning et al., 1996. J Exp Med 183: 867-878). In immunological settings it has been shown that anti-LTβ mAb or soluble LTβ-R-Ig fusion protein can block the development of inflammatory bowel diseases, possibly by influencing dendritic cell and T cell interaction (Mackay et al., 1998. Gastroenterology 115:1464-1475).
The TRAIL system also has potential as a cancer therapy. TRAIL interacts with a number of membrane bound and soluble receptors. Two of these receptors, TRAIL-R1 and TRAIL R2 (also called DR4 and DR5), transmit death inducing signals to tumor cells but not to normal cells, which express additional TRAIL receptors that do not induce death. These additional receptors are thought to function as decoys. The use of soluble TRAIL to kill tumor cells relies on the selective expression of decoy receptors on normal but tumor tissue (Gura, 1997. Science 277: 768).
Tumor cells themselves often express a variety of decoy receptors that block immune recognition or effector functions. Indeed some tumors overexpress TRAIL decoy receptors, apparently to avoid TRAIL mediated death (Sheikh et al., 1999. Oncogene 18: 4153-4159). This limits the utility of TRAIL as an anti-tumor agent in some settings. Similar observations have been made about a decoy receptor for FAS-L, which is overexpressed by lung and colon cancer cells (Pitti et al., 1998. Nature 396: 699-703), and for the IL-1 receptor antagonist (Mantovani et al., 1998. Ann. N Y Acad. Sci. 840: 338-351). Decoy receptors are also employed by viral genomes to protect infected host cells from host defense mechanisms.
APRIL (A Proliferation Inducing Ligand) is a new member of the TNF family of proteins. APRIL expression and functional studies suggest that this protein is utilized by tumor cells to induce rapid proliferation. Tumor cell lines treated with soluble APRIL protein or transfected with APRIL cDNA grow rapidly in vitro. APRIL transfected cells implanted into immunodeficient mice grow rapidly as tumors. Finally, human tumor cells, but not normal tissue, express high levels of APRIL messenger RNA. These observations suggest that APRIL binds to a receptor that is also expressed by tumor cells, setting up autocrine or paracrine tumor cell activation. In addition, it is possible that APRIL acts in other disease settings, such that activating or blocking the APRIL pathway would have additional utility. For example, underexpression or overexpression of APRIL may play a role in developmental defects, since development is often characterized by the carefully controlled balance between cell proliferation and cell death. Similarly, APRIL may act in cell proliferative diseases, such as those that occur in connection with some autoimmune diseases (e.g., lupus) or in inflammatory diseases where cell populations expand rapidly (e.g., bacterial sepsis).
Based on the known utility of using agonists and antagonists of TNF and TNF receptor family members as disease modifiers, the APRIL pathway presents itself as an important target for drug development. This is particularly true for cancer therapy since tumor cells appear to produce and utilize APRIL to support their own growth, and are therefore unlikely to produce decoy receptors or other antagonists of the APRIL pathway. Thus the APRIL pathway is uniquely different from, for example, the TRAIL or FAS-L pathways, which can be thwarted by tumor decoy receptors.
Current treatments for cancer are inadequate for many tumor types, due to poor efficacy, low impact on survivorship, toxicity that causes severe side effects, or combinations thereof. Therefore there is a need to identify and develop additional methods for treating cancer growth which can provide efficacy without inducing severe side effects. Antagonists of the APRIL pathway, including anti-APRIL mAbs, anti-APRIL receptor mAbs, soluble APRIL receptor-Ig fusion proteins, natural antagonists, small molecule antagonists, and chemical, pharmaceutical, or other antagonists would thus be useful.
To this end we have identified B cell mediated protein (BCM or BCMA) as a receptor for APRIL.
SUMMARY OF THE INVENTION
Applicants have found that BCMA is a receptor for the tumor necrosis factor, APRIL. APRIL is the same molecule previously described in WO 99 12965, which is incorporated by reference herein. The APRIL receptor is referred to hereinafter as “APRIL-R”. The present invention is directed to methods of treatment and pharmaceutical preparations for use in the treatment of mammalian species having or at risk of having cancer. Such subjects include subjects already afflicted with cancer, or which have already received cancer therapy.
The methods and compositions of this invention capitalize in part upon the discovery that certain agents that are cancer therapeutic agents, defined herein as APRIL-R antagonists, including for example, anti-APRIL-R antibodies, may be used in the treatment of subjects at risk of developing cancer as defined herein or the need for cancer treatment.
The cancer therapeutic agents of the invention may be administered by any route of administration which is compatible with the selected agent, and may be formulated with any pharmaceutically acceptable carrier appropriate to the route of administration. Preferred routes of administration are parenteral and, in particular, intravenous, intraperitoneal, and intracapsular. Treatments are also preferably conducted over an extended period on an outpatient basis. Daily dosages of the cancer therapeutic agents are expected to be in the range of about 0.01-1000 μg/kg body weight, and more preferably about 10-300 μg/kg body weight, although precise dosages will vary depending upon the particular cancer therapeutic agent employed and the particular subject's medical condition and history.
The treatments of the present invention are useful in eradicating a substantially clonal population (colony) of transformed cells from the body of a mammal, or to suppress or to attenuate the growth of the colony, which is most commonly referred to as a tumor. As such they are useful in prolonging the lives, and in maintaining the quality of life, of subjects at risk of, or already afflicted with cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the nucleic acid sequence (SEQ ID NO:1) of a cDNA for murine APRIL and its derived amino acid sequence (SEQ ID NO:3) as mapped in vector pCCM213.10. Shown underlined is the myc epitope and the amino acids derived from FasL. The beginning of APRIL extracellular domain coding sequence is indicated by arrows.
FIG. 2 shows the nucleic acid sequence (SEQ ID NO:4) and its derived amino acid sequence (SEQ ID NO:6) of FLAG-human APRIL construct for expression in mammalian cells. The map indicates the signal sequence (1-15); the FLAG epitope (AA 16-23) and the beginning of human APRIL extracellular domain coding sequence (32-end).
FIG. 3A shows the nucleic acid sequence (SEQ ID NO:7) and amino acid sequence (SEQ ID NO:8) of full length human BCMA. FIG. 3B shows the nucleic acid sequence (SEQ ID NO:11) of pJST538, a plasmid encoding a human APRIL-R-hIgGFc fusion construct and its derived amino acid sequence (SEQ ID NO:12).
FIG. 4 shows binding of myc-murine APRIL to the murine B cell lymphoma line A20. 3 separate experiments show specific binding of APRIL to A20 cells compared to A) unstained cells and cells stained with R1532 only, B) cells stained with RANKL-L and R1532 and C) cells stained with APRIL and an irrelevant rabbit sera.
FIG. 5 shows binding of myc-murine APRIL to the human B cell lymphoma line RAJI. 2 separate experiments show specific binding of APRIL to RAJI cells compared to A) unstained cells and cells stained with R1532 only, and cells stained with RANK-1 and R1532 and B) cells stained with APRIL and an irrelevant rabbit sera
FIG. 6 shows that APRIL binding to A20 cells (A) and Raji cells (B) is competed using soluble BAFF protein or soluble BCMA-Ig protein.
FIG. 7 shows binding of FLAG-human APRIL to various cell lines: A) A20 cells, B) HT29 cells, C)NIH3T3 cells. Specific binding is demonstrated using biotinylated anti-FLAG mAb M2 detection compared to binding seen with an irrelevant isotype control mAb or without addition of FLAG-APRIL.
FIG. 8 shows immunoprecipitation of myc-mAPRIL using BCMA-Fc fusion protein. FIG. 8A shows specific hBCMA-Fc/myc-mAPRIL and positive control OPG-Fc/Rank-1 immunoprecipitations, compared to negative controls shown in FIG. 8B. FIGS. 8C and 8D demonstrate that the amounts of protein loaded were equivalent.
FIG. 9 shows an ELISA format experiments demonstrating that FLAG-h APRIL binds to hBCMA-fc fusion protein. Various receptor-Fc fusion proteins were coated onto the ELISA plates and bound with FLAG-tagged ligands. A)Detection of the bound ligands revealed that only APRIL and hBAFF specifically bind to hBCMA-Fc, but not hCD40-Fc. B) Dose titration showing that the ELISA signal detected after binding hAPRIL or hBAFF onto hBCMA-Fc coated plates is linearly dependent on the amount of protein added.
FIG. 10 show an immunoprecipitation of FLAG-hAPRIL and FLAG-HBAFF by hBMCA-Fc fusion protein. Upper 4 panels show the equivalence of the protein loads in each immunoprecipitation, while the lower panels show that hAPRIL and hBAFF are immunoprecipitated by hBCMA-Fc but not hTRAIN-Fc.
FIG. 11 shows BiaCore analysis of the binding of myc-APRIL (FIG. 11A), FLAG-hBAFF (FIG. 11B), and FLAG-mBAFF (FIG. 11C) to hBCMA, hLTbeta receptor, or hTNF-R80 or blank showing specific binding only to hBCMA.
FIG. 12 shows APRIL binding to BCMA transfected cells. 293EBNA cells were transfected with a plasmid that expresses full length hBCMA. Cells were harvested 48 hours later using 5 mM EDTA and stained with myc-nAPRIL. Panel A shows that the extent of staining is dose dependent. Panel B shows that the staining decreased to background level using a soluble BCMA-Ig protein.
FIG. 13 shows the growth of NIH3T3 cells implanted subcutaneously in immunodeficient (Nu/Nu) mice treated with control reagents or with BCMA-Ig fusion protein. In this model the NIH3T3 cells form a fibrosarcoma.
FIG. 14 shows the growth of the human colon carcinoma SW480 implanted subcutaneously in immunodeficient (Nu/Nu) mice treated with control reagents or with hBCMA-Ig fusion protein.
FIG. 15A shows the growth of the human colon carcinoma HT29 implanted subcutaneously in immunodeficient (Nu/Nu) mice treated with control reagents or with hBCMA-Ig fusion protein. FIG. 15B shows the growth of the human lung carcinoma A549 implanted subcutaneously in immunodeficient (Nu/Nu) mice treated with control reagents or with hBCMA-Ig fusion protein
DETAILED DESCRIPTION
Definitions
In order to more clearly and concisely point out the subject matter of the claimed invention, the following definitions are provided for specific terms used in the following written description and appended claims.
The invention will now be described with reference to the following detailed description of which the following definitions are included:
The terms “APRIL receptor” or “APRIL-R” when used herein encompass native sequence APRIL-R and APRIL-R variants. The APRIL-R may be isolated from a variety of sources, such as from murine or human tissue types or from another source, or prepared by recombinant or synthetic methods. The term APRIL-R further refers to a polypeptide which is capable of binding to the tumor necrosis family member, APRIL, or to homologs or fragments thereof. An example of an APRIL-R is BCMA.
The term “BCMA” or “BCM” refers to the novel protein for B cell maturation as described in Gras et al. (1995), International Immunology, 7: 1093-1106, “BCMAp: an integral membrane protein in the golgi apparatus of human mature B lymphocytes”; Y. Laabi et al. (1992), EMBO J., 11, 3897-3904, “A new gene BCM on Chromosome 16 is fused to the interleukin 2 gene by a t(4;16) (q26;p13) translocation in a malignant T cell lymphoma”.
A “native sequence APRIL-R” comprises a polypeptide having the same amino acid sequence as APRIL-R derived from nature. Such native sequence APRIL-R can be isolated from nature or can be produced by recombinant or synthetic means. The native sequence APRIL-R can be naturally-occurring truncated or secreted forms of the APRIL-R (e.g. soluble forms containing for instance, an extracellular domain sequence), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants of the APRIL-R. In one embodiment of the invention, the native sequence APRIL-R is a mature or full-length native sequence APRIL-R polypeptide comprising amino acids 1 to 184 of SEQ ID NO: 8 or fragment thereof.
The “APRIL-R extracellular domain” or “APRIL-R ECD” refers to a form of APRIL-R which is essentially free of transmembrane and cytoplasmic domains of APRIL-R. Ordinarily, APRIL-R extracellular domain will have less than 1% of such transmembrane and cytoplasmic domains and will preferably have less than 0.5% of such domains. Optionally, APRIL-R ECD will comprise amino acid residues 1 to 51, or 1 to 52, or 1 to 53 of SEQ ID NO: 8. In a preferred embodiment, the APRIL-ECD comprises amino acid residues 4 to 51 of SEQ ID NO: 8 or more preferably amino acid residues 8 to 41 of SEQ ID NO:8. It will be understood by the skilled artisan that the transmembrane domain identified for the APRIL-R polypeptide of the present invention is identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain. The exact boundaries of a transmembrane domain may vary but most likely by no more than about 5 amino acids at either end of the domain specifically mentioned herein.
“APRIL-R variant” means an active APRIL-R as defined below having at least about 80% amino acid sequence identity with the APRIL-R having the deduced amino acid sequence shown in SEQ ID NO:5 for a full-length native sequence APRIL-R or with a APRIL-R ECD sequence. Such APRIL-R variants include, for instance, APRIL-R polypeptides wherein one or more amino acid residues are added, or deleted, at the end or C-terminus of the sequence of SEQ ID NO:8. Ordinarily, a APRIL-R variant will have at least about 80% or 85% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, and even more preferably at least about 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO:8.
“Percent (%) amino acid sequence identity” with respect to APRIL-R sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the APRIL-R sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publically available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared.
The term “epitope tagged” when used herein refers to a chimeric polypeptide comprising APRIL-R, or a domain sequence thereof, fused to a “tag polypeptide”. The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, or which can be identified by some other agent, yet is short enough such that it does not interfere with activity of the APRIL-R. The tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least 6 amino acid residues and usually between about 8 to about 50 amino acid residues (preferably, about 10 to about 20 residues).
“Isolated” when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminate components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by us of a spinning cup sequenator, or (2) to homogeneity SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or preferably, silver stain. Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the APRIL-R's natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.
The term “antibody” is used in the broadest sense and specifically covers single APRIL-R monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies) and anti-APRIL-R antibody compositions with polyepitopic specificity. The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts.
A “purified preparation” or a “substantially pure preparation” of a polypeptide, as used herein, means a polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it naturally occurs. Preferably, the polypeptide is also separated from other substances, e.g., antibodies, matrices, etc., which are used to purify it.
The terms, “treating”, “treatment” and “therapy” as used herein refers to curative therapy, prophylactic therapy, and preventative therapy.
The terms “peptides”, “proteins”, and “polypeptides” are used interchangeably herein.
“Biologically active” as used herein, means having an in vivo or in vitro activity which may be performed directly or indirectly. Biologically active fragments of APRIL-R may have, for example, 70% amino acid homology with the active site of the receptor, more preferably at least 80%, and most preferably, at least 90% homology. Identity or homology with respect to the receptor is defined herein as the percentage of amino acid residues in the candidate sequence which are identical to the APRIL-R residues in SEQ ID NO:8.
The term “mammal” as used herein refers to any animal classified as a mammal including humans, cows, horses, dogs, mice and cats. In preferred embodiment of the invention, the mammal is a human.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature.
Reference will now be made in detail to the present preferred embodiments of the invention. This invention relates to the use of APRIL-R and APRIL-R related molecules to effect the growth and maturation of B-cells and non-B cells, specifically as they relate to tumor cells. The invention also relates to the use of APRIL-R and APRIL-R related molecules to effect responses of the immune system, as necessitated by immune-related disorders. Additionally, this invention encompasses the treatment of cancer and immune disorders through the use of a APRIL-R, or APRIL-R related gene through gene therapy methods.
The APRIL-R and homologs thereof produced by hosts transformed with the sequences of the invention, as well as native APRIL-R purified by the processes known in the art, or produced from known amino acid sequences, are useful in a variety of methods for anticancer, antitumor and immunoregulatory applications. They are also useful in therapy and methods directed to other diseases.
Another aspect of the invention relates to the use of the polypeptide encoded by the isolated nucleic acid encoding the APRIL-R in “antisense” therapy. As used herein, “antisense” therapy refers to administration or in situ generation of oligonucleotides or their derivatives which specifically hybridize under cellular conditions with the cellular mRNA and/or DNA encoding the ligand of interest, so as to inhibit expression of the encoded protein, i.e. by inhibiting transcription and/or translation. The binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix. In general, “antisense” therapy refers to a range of techniques generally employed in the art, and includes any therapy which relies on specific binding to oligonucleotide sequences.
An antisense construct of the present invention can be delivered, for example, as an expression plasmid, which, when transcribed in the cell, produces RNA which is complementary to at least a portion of the cellular mRNA which encodes Kay-ligand. Alternatively, the antisense construct can be an oligonucleotide probe which is generated ex vivo. Such oligonucleotide probes are preferably modified oligonucleotides which are resistant to endogenous nucleases, and are therefore stable in vivo. Exemplary nucleic acids molecules for use as antisense oligonucleotides are phosphoramidates, phosphothioate and methylphosphonate analogs of DNA (See, e.g., U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed, for example, by Van Der Krol et al., (1988) Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res 48: 2659-2668, specifically incorporated herein by reference.
The APRIL-R of the invention, as discussed above, is a member of the TNF receptor family. The protein, fragments or homologs thereof may have wide therapeutic and diagnostic applications.
The polypeptides of the invention specifically interact with APRIL, a polypeptide previously described in WO99/12964 incorporated by reference herein. However, the peptides and methods disclosed herein enable the identification of molecules which specifically interact with the APRIL-R or fragments thereof.
The claimed invention in certain embodiments includes methods of using peptides derived from APRIL-R which have the ability to bind to APRIL. Fragments of the APRIL-R's can be produced in several ways, e.g., recombinantly, by PCR, proteolytic digestion or by chemical synthesis. Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end or both ends of a nucleic acid which encodes the polypeptide. Expression of the mutagenized DNA produces polypeptide fragments.
Polypeptide fragments can also be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-moc or t-boc chemistry. For example, peptides and DNA sequences of the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragment, or divided into overlapping fragments of a desired length. Methods such as these are described in more detail below.
Generation of Soluble Forms of APRIL-R
Soluble forms of the APRIL-R can often signal effectively and hence can be administered as a drug which now mimics the natural membrane form. It is possible that the APRIL-R claimed herein are naturally secreted as soluble cytokines, however, if not, one can reengineer the gene to force secretion. To create a soluble secreted form of APRIL-R, one would remove at the DNA level the N-terminus transmembrane regions, and some portion of the stalk region, and replace them with a type I leader or alternatively a type II leader sequence that will allow efficient proteolytic cleavage in the chosen expression system. A skilled artisan could vary the amount of the stalk region retained in the secretion expression construct to optimize both ligand binding properties and secretion efficiency. For example, the constructs containing all possible stalk lengths, i.e. N-terminal truncations, could be prepared such that proteins starting at amino acids 1 to 52 would result. The optimal length stalk sequence would result from this type of analysis.
Generation of Antibodies Reactive with the APRIL-R
The invention also includes antibodies specifically reactive with the claimed APRIL-R or its co-receptors. Anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide. Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers, or other techniques, well known in the art.
An immunogenic portion of APRIL-R or its co-receptors can be administered in the presence of an adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.
In a preferred embodiment, the subject antibodies are immunospecific for antigenic determinants of APRIL-R or its co-receptors, e.g. antigenic determinants of a polypeptide of SEQ ID NO:8, or a closely related human or non-human mammalian homolog (e.g. 70, 80 or 90 percent homologous, more preferably at least 95 percent homologous). In yet a further preferred embodiment of the present invention, the anti-APRIL-R or anti-APRIL-co-receptor antibodies do not substantially cross react (i.e. react specifically) with a protein which is e.g., less than 80 percent homologous to SEQ ID NO:8; preferably less than 90 percent homologous with SEQ ID NO:8; and, most preferably less than 95 percent homologous with SEQ ID NO:8. By “not substantially cross react”, it is meant that the antibody has a binding affinity for a non-homologous protein which is less than 10 percent, more preferably less than 5 percent, and even more preferably less than 1 percent, of the binding affinity for a protein of SEQ ID NO.8.
The term antibody as used herein is intended to include fragments thereof which are also specifically reactive with APRIL-R, or its receptors. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab′)2 fragments can be generated by treating antibody with pepsin. The resulting F(ab′)2 fragment can be treated to reduce disulfide bridges to produce Fab′ fragments. The antibodies of the present invention are further intended to include biospecific and chimeric molecules having anti-APRIL-R or anti-APRIL-co-receptor activity. Thus, both monoclonal and polyclonal antibodies (Ab) directed against APRIL-R, and their co-receptors, and antibody fragments such as Fab′ and F(ab′)2, can be used to block the action of the APRIL-R and its respective co-receptors.
Various forms of antibodies can also be made using standard recombinant DNA techniques. (Winter and Milstein, Nature 349: 293-299 (1991) specifically incorporated by reference herein.) For example, chimeric antibodies can be constructed in which the antigen binding domain from an animal antibody is linked to a human constant domain (e.g. Cabilly et al., U.S. Pat. No. 4,816,567, incorporated herein by reference). Chimeric antibodies may reduce the observed immunogenic responses elicited by animal antibodies when used in human clinical treatments.
In addition, recombinant “humanized antibodies” which recognize APRIL-R or its co-receptors can be synthesized. Humanized antibodies are chimeras comprising mostly human IgG sequences into which the regions responsible for specific antigen-binding have been inserted. Animals are immunized with the desired antigen, the corresponding antibodies are isolated, and the portion of the variable region sequences responsible for specific antigen binding are removed. The animal-derived antigen binding regions are then cloned into the appropriate position of human antibody genes in which the antigen binding regions have been deleted. Humanized antibodies minimize the use of heterologous (i.e. inter species) sequences in human antibodies, and thus are less likely to elicit immune responses in the treated subject.
Construction of different classes of recombinant antibodies can also be accomplished by making chimeric or humanized antibodies comprising variable domains and human constant domains (CH1, CH2, CH3) isolated from different classes of immunoglobulins. For example, antibodies with increased antigen binding site valencies can be recombinantly produced by cloning the antigen binding site into vectors carrying the human: chain constant regions. (Arulanandam et al., J. Exp. Med., 177: 1439-1450 (1993), incorporated herein by reference.)
In addition, standard recombinant DNA techniques can be used to alter the binding affinities of recombinant antibodies with their antigens by altering amino acid residues in the vicinity of the antigen binding sites. The antigen binding affinity of a humanized antibody can be increased by mutagenesis based on molecular modeling. (Queen et al., Proc. Natl. Acad. Sci. 86: 10029-33 (1989)) incorporated herein by reference.
Generation of Analogs: Production of Altered DNA and Peptide Sequences
Analogs of the APRIL-R can differ from the naturally occurring APRIL-R in amino acid sequence, or in ways that do not involve sequence, or both. Non-sequence modifications include in vivo or in vitro chemical derivatization of the APRIL-R. Non-sequence modifications include, but are not limited to, changes in acetylation, methylation, phosphorylation, carboxylation or glycosylation.
Preferred analogs include APRIL-R biologically active fragments thereof, whose sequences differ from the sequence given in SEQ ID NO:8, by one or more conservative amino acid substitutions, or by one or more non-conservative amino acid substitutions, deletions or insertions which do not abolish the activity of APRIL-ligand. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g. substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and, phenylalanine, tyrosine.
Uses
The full length APRIL-R gene (SEQ ID NO:8) or portions thereof may be used as hybridization probes for a cDNA library to isolate, for instance, still other genes which have a desired sequence identity to the APRIL-R sequence disclosed in SEQ ID NO: 6. Nucleotide sequences encoding APRIL-R can also be used to construct hybridization probes for mapping the gene which encodes the APRIL-R and for the genetic analysis of individuals with genetic disorders. Screening assays can be designed to find lead compounds that mimic the biological activity of a APRIL-R. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates. Small molecules contemplated include synthetic organic or inorganic compounds. Nucleic acids which encode APRIL-R or its modified forms can also be used to generate either transgenic animals or “knock out” animals which in turn are useful in the development and screening of therapeutically useful reagents, including for example cancer reagents.
The APRIL-R and homologs thereof produced by hosts transformed with the sequences of the invention, as well as native APRIL-R purified by the processes known in the art, or produced from known amino acid sequences, are useful in a variety of methods for anticancer applications.
In one embodiment of the invention is a method of treating a mammal for a condition associated with undesired cell proliferation by administering to the mammal a therapeutically effective amount of a composition comprising an APRIL-R antagonist, wherein the APRIL-R antagonist comprises a polypeptide that antagonizes the interaction between APRIL and its cognate receptor or receptors, with a pharmaceutically acceptable recipient.
In a preferred embodiment the cognate receptor of APRIL on the surface of the cell is BCMA.
The method can be used with any APRIL-R antagonist that has a polypeptide that antagonizes the interaction between APRIL and its cognate receptor or receptors. Examples of APRIL-R antagonists include but are not limited to soluble APRIL-R polypeptide, including but not limited to soluble BCMA; soluble chimeric APRIL-R molecules, including but not limited to BCMA-IgG-Fc and anti-APRIL-R antibody homologs, including but not limited to anti-BCMA monoclonal antibody.
The method of the invention can be used with any condition associated with undesired cell proliferation. In particular the methods of the present invention can be used to treat tumor cells which express APRIL and/or APRIL-R (i.e. BCMA).
Examples of cancers whose cell proliferation is modulated by APRIL may be screened by measuring in vitro the level of APRIL and/or APRIL-R (i.e. BCMA) message expressed in tumor tissue libraries. Tumor tissue libraries in which APRIL and/or APRIL-R (i.e. BCMA) message is highly expressed would be candidates. Alternatively, one may screen for candidates searching the public and private databases (i.e. Incyte data base) with, for example, the full length human APRIL cDNA sequence. Applying these techniques, it was found, for example, that APRIL mRNA expression was detected in a large number of tumor types, including but not limited to those found in Table 1 below:
TABLE 1
Library Description
Prostate tumor line, LNCaP, CA, 50M, untreated, TIGR
T-lymphocyte tumor, lymphoma, TIGR
Ovary tumor, papillary serous cystadenoCA
Lung, mw/adenoCA, COPD, 47M
Breast tumor, adenoCA, 46F, SUB, m/BRSTNOT33
Ganglion, dorsal root, cervical, aw/lymphoma, 32M, NORM
Brain tumor, frontal, neuronal neoplasm, 32M
Prostate tumor, adenoCA, 59M, SUB, m/PROSNOST19
Colon tumor, hepatic flexure, adenoCA, 55M, SUB, m/COLATMT01
Pancreatic tumor, TIGR
Paraganglion tumor, paraganglioma, aw/renal cell CA, 46M
Breast, mw/ductal CA, 43F, m/BRSTTUT16
Kidney tumor, renal cell CA, 51F
Bladder, mw/TC CA, CA in situ, 60M, m/BLADTUT04
Uterus tumor, endometrial, F, TIGR
Prostate, BPH, mw/adenoCA, PIN, 59M
Lung, mw/adenoCA, 53M, m/LUNGTUT17
Bone tumor/line, MG-63, osteoSAR/giant cell, M/F, pool, RP
Brain, frontal cortex, aw/lung CA, 77M
Colon tumor, adenoCA, NORM, SUB, CGAP
Lung tumor, squamous cell CA, 57M
Lung, mw/adenoCA, 63M
Prostate, AH, mw/adenoCA, 50M, m/PROSTUT01
Periph blood, B-lymphocytes, CLL, pool, NORM, 3′ CGAP
Colon tumor, adenoCA, pool, NORM, 3′/5′ CGAP
Kidney, mw/renal cell CA, 8,53F, pool, NORM
Ovary, dermoid cyst, 22F
Colon tumor, adenoCA, NORM, 3′ CGAP
Colon tumor, adenoCA, 3′, CGAP
Prostate, BPH, mw/adenoCA, 70M, SUB
Ovary tumor, mets colon adenoCA, 58F
Uterus, myometrium, mw/leiomyoma, 43F
Sm intestine, ileum, mw/CUC, 25F
Lymph node, peripancreatic, aw/pancreatic adenoCA, 65M
Ovary, aw/leiomyomata, 36F, NORM
Lung, mw/spindle cell carcinoid, 62F
Lung tumor, squamous CA, 50M
Brain tumor, meningioma, 36M
Tumor, adenoCA, 65F, m/PANCNOT08
Lung, mw/endobronchial carcinoid, 33M
Adrenal gland, mw/pheochromocytoma, 43F, m/ADRETUT07
Brain tumor, frontal, meningioma, 50M
Kidney tumor, clear cell type cancer, pool, NORM, 3′ CGAP
Breast, mw/lobular CA, 67F
Lung, mw/mets osteoSAR, aw/pleura mets, 58M, NORM
Prostate tumor, adenoCA, 59M, SUB, m/PROSNOT19
Sm intestine tumor, ileum, mets endometrial adenoCA, 64F
Ovary tumor, adenoCA, 58F
Breast, NF breast disease, 46F
Brain tumor, frontal, mets hypernephroma, 58M
Kidney tumor, Wilms', pool, WM/WN
Lung, mw/mets thyroid CA, 79M, m/LUNGTUT02
Lung tumor, mets thyroid CA, 79M, m/LUNGNOT03
Parathyroid tumor, adenoma, M/F, NORM, WM
Pancreatic tumor, anaplastic CA, 45F
Ovary, mw/mucinous cystadenoCA, 43F, m/OVARTUT01
Lung tumor, squamous cell CA, pooled, NORM, CGAP
Breast tumor, adenoCA, 46F, m/BRSTNOT17
Uterus, mw/leiomyoma, aw/colon adenoCA, 45F
Lung, mw/adenoCA, aw/node, diaphragm mets, 63F
Breast tumor, adenoCA, 46F, m/BRSTNOT33
Prostate tumor, adenoCA, 66M, m/PROSNOT15, PROSDIN01
Breast tumor, adenoCA, 54F, m/BRSTNOT03
Germ cell tumor, pool, SUB, 3′ CGAP
Bone marrow, tibia, aw/mets alveolar rhabdomyoSAR, 16M
Prostate, AH, mw/adenoCA, 57M, m/PROSTUT04
Breast, PF changes, mw/adenoCA, 55F, m/BRSTTUT01
Uterus tumor, serous papillary CA, F, pooled, 3′ CGAP
Ovary tumor, mucinous cystadenoCA, 43F, m/OVARNOT03
Breast, PF changes, mw/adenoCA, intraductal CA, 43F
Breast, mw/ductal CA, CA in situ, aw/node mets, 62F
Neuroganglion tumor, ganglioneuroma, 9M
Pancreas tumor, adenoCA, 3′ CGAP
Uterus tumor, endometrial adenoCA, F, pooled, 3′ CGAP
Lung tumor, neuroendocrine carcinoid, pool, NORM, 3′ CGAP
The APRIL-R antagonists of the subject invention which are used in treating conditions associated with undesired cell proliferation, in particular tumor therapy, advantageously inhibit tumor cell growth greater than 10%, 20%, 30% or 40% and most advantageously greater than 50%. The APRIL-R antagonists are obtained through screening (see, for example, the discussion in Example 6). For example, APRIL-R antagonists can be selected on the basis of growth inhibiting activity (i.e. greater than 10%, 20%, 30%, 40% or 50%) against the human colon carcinoma HT29 or human lung carcinoma A549 (see, for example, the discussion in FIG. 15) which are derived from a colon and lung tumor respectively.
Another embodiment of the invention, provides methods of inhibiting B-cell and non-B cell growth, dendritic cell-induced B-cell growth and maturation or immunoglobulin production in an animal using APRIL-R polypeptide.
In another embodiment, the invention provides methods of using APRIL-R in the treatment of autoimmune diseases, hypertension, cardiovascular disorders, renal disorders, B-cell lympho-proliferate disorders, immunosuppressive diseases, organ transplantation, inflammation, and HIV. Also included are methods of using agents for treating, suppressing or altering an immune response involving a signaling pathway between APRIL-R and its ligand.
The present invention also provides pharmaceutical compositions comprising a APRIL-R polypeptide and a pharmaceutically acceptable excipient. Suitable carriers for a APRIL-R polypeptide, for instance, and their formulations, are described in Remington' Pharmaceutical Sciences, 16th ed., 1980, Mack Publishing Co., edited by Oslo et al. Typically an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of the carrier include buffers such as saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7.4 to about 7.8. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers, which matrices are in the form of shaped articles, e.g. liposomes, films or microparticles. It will be apparent to those of skill in the art that certain carriers may be more preferable depending upon for instance the route of administration and concentration of the a APRIL-R polypeptide being administered.
Administration may be accomplished by injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular) or by other methods such as infusion that ensure delivery to the bloodstream in an effective form.
Practice of the present invention will employ, unless indicated otherwise, conventional techniques of cell biology, cell culture, molecular biology, microbiology, recombinant DNA, protein chemistry, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning: A Laboratory Manual, 2nd edition. (Sambrook, Fritsch and Maniatis, eds.), Cold Spring Harbor Laboratory Press, 1989; DNA Cloning, Volumes I and II (D. N. Glover, ed), 1985; Oligonucleotide Synthesis, (M. J. Gait, ed.), 1984; U.S. Pat. No. 4,683,195 (Mullis et al.,); Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins, eds.), 1984; Transcription and Translation (B. D. Hames and S. J. Higgins, eds.), 1984; Culture of Animal Cells (R. I. Freshney, ed). Alan R. Liss, Inc., 1987; Immobilized Cells and Enzymes, IRL Press, 1986; A Practical Guide to Molecular Cloning (B. Perbal), 1984; Methods in Enzymology, Volumes 154 and 155 (Wu et al., eds), Academic Press, New York; Gene Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos, eds.), 1987, Cold Spring Harbor Laboratory; Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds.), Academic Press, London, 1987; Handbook of Experiment Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds.), 1986; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, 1986.
The following Examples are provided to illustrate the present invention, and should not be construed as limiting thereof.
EXAMPLES
The following methods were used in the Examples disclosed hereinafter.
Methods:
Cloning and Expression of Myc-Tagged Murine APRIL (CCM776) in Pichia pastoris.
The expression vector pCCM213.10 was constructed by taking PDR004 (H98 muAPRIL with superFAS-ligand stalk attached to N terminus along with FLAG epitope tag) and excising out the mu APRIL coding sequence from Sac I to Not1. Synthetic oligonucleotides LTB-559 and 560 form a Xho-1-Sac1 linker which contain an alpha mating factor leader sequence, myc epitope tag, as well as the KEL motif from FAS ligand. Both the muAPRIL fragment and linker were ligated into the Xho-1-Not1 sites of pccm211, a Pichia pastoris expression plasmid.
PCCM213.10 was linearized with Stu 1, electroporated into GS115 strain (his4-) and plated into minimal media containing dextrose. HIS4 transformants were analyzed for protein expression by inoculating a single representative colony in rich meida (BMGY: Buffered glycerol complex medium) and allowing it to grow to density for 48 hours at 30C. Cultures were spun, and cell pellets were resuspended (1:5) in a rich induction media containing 1.5% methanol (BMMY:Buffered methanol complex media). After two days of induction at 30C, supernatants were run out on SDS-PAGE and assessed for the presence of muAPRIL. Coomassie staining and Western blot (with the anti-myc mAb 9E10) showed that one strain, CCM776, produced adequate amounts of the glycosylated form myc-tagged-H98 muAPRIL protein.
Myc-mAPRIL Purification
Myc-mApril, a protein of 149 amino acids was expressed in pichia. This protein has an isoelectric point of 7.45. 175 ml of pichia supernatant was dialyzed and buffer exchanged to 10 mM Tris pH 6.8 overnight and then passed through a 20 ml SP column. The column was washed extensively with 10 mM Tris-HCl, pH 6.8, and eluted with 250 n mM NaCl in PBS. A second step purification was achieved using a gel filtration column (S300). Fractions containing myc-April from 20 ml SP column were concentrated by centrifugation to a volume of 7 ml. After gel filtration, we recovered 8 mg of myc-APRIL as detected by OD and coomassie gel. We also performed Western blot analysis using mouse monoclonal 9E10 antibody (anti-myc) showing that the myc tag is intact after the purification steps. N terminal sequence verified that the purified protein corresponds to myc-mApril.
FLAG-Human April Purification.
Plasmid ps429 (subsequently named p1448) was used to transiently transfect 293 T cells using lipofectamine reagent (Gibco-Brl) and serum free media. The plasmid, constructed in the mammalian expression vector PCR3 (Invitrogen) encodes the receptor-binding domain of human APRIL, with an N-terminal protein into the cell culture media. FLAG-APRIL protein was purified from serum free media using an anti-FLAG mAb M2 column and excess purified FLAG peptide, following the manufacturers' instructions (Kodak).
HBMCA-Fc Purification.
HBMCA-Fc was transiently tranfected into 293 cells. Conditioned media from 293 cells over-expressing hBCM-Fc was loaded into a protein A column. Protein was eluted using 25 mM phosphate 100 nM NaCl pH 2.8 followed by neutralization with 1/20 volume of 0.5 M NaPO4 pH 8.6. Selected fractions based in OD 280 were subject to reducing and non-reducing SDS-PAGE gels and western blots to identify the purified protein. 3 mg of protein were recovered from 500 ml of conditioned media.
Myc-mAPRIL Binds to Various Cell Lines in FACS Analysis.
450 ng/ml of purified myc-mAPRIL was bound to cell lines in 100 ul PBS/2% FBS+Fc blocking reagents (FcBlock@ 20 ug/ml (Pharmingen) and purified human IgG@ 10 ug/ml (Sandoz) on ice for 1 hour. Positive binding was revealed using specific rabbit anti-murine APRIL antisera (1:500) and donkey anti-rabbit IgG-FITC (Jackson). Cell lines A20, Raji, NIH3T3, and HT29 were maintained in media as suggested by the supplier (ATCC Bethesda, Md.). BJAB cells were cultured in HEPES-buffered RPMI supplemented with 10% FBS and L-glutamine. In competition assays 450 ng/ml myc-murine APRIL was added with 1 ug/ml of competitor protein.
Example 1
Detection of APRIL Binding to APRIL-R Using a Plate Assay
In this example, BCMA Association with April was tested.
In order to test whether BCMA associates with April we performed a co-immunoprecipitation experiment. Both soluble proteins hBCMA-Fc and myc-mApril were used in this experiment.
HBCMA-Fc and LTbR-Fc were added with different TNF ligands: myc-mApril; myc-CD40L and myc-RANKL into media containing 10% FBS for ½ hour at room temperature. Fc proteins were bound to protein A beads for 1-2 hours, washed three times with 1 ml of PBS, analyzed by immunoblotting with mouse monoclonal 9E10 (anti-myc) antibody and developed using enhanced chemiluminescence.
We detected myc-APRIL in hBCMA-Fc immunoprecipitates indicating that BCMA interacts with April in a specific way since other TNF ligands, myc-CD40L and myc-RANKL did not have the ability to bind to BCMA. Myc-April does not associate with LTbR-Fc.
The same membrane was stripped and reblotted with anti-hIG-HRP to show that the same amount of LTbR-Fc with BCMA-Fc were used in the immunoprecipitates.
Example 2
hBCMA Binds to Flag-hAPRIL
This example describes that hBCMA-FC interacts with FLAG-hAPRIL.
ELISA analysis: Coated plates with receptor-Fc fusion proteins (hBCMA-Fc-739 or hTNFR2-Fc-492) at 1 ug/ml in carbonate pH 9.6, overnight, 4C. Blocked for 2 hours at room temperature using PBS/5% non fat dry milk/05% Tween-20. 2× serial dilution of ligands were made in 100 ul of blocking buffer (TNFa-197 from 1000 ng/ml, muBAFF-657 from 1000 ng/ml, hApril-507 from 2000 ng/ml (inactive), hApril-429 from 5× concentrated media). After incubation with ligands the plate was washed in PBS) 0.5% Tween-20 and probed with 0.5 ug/ml anti-FLAG mAb M2 in dilution buffer. The antibody was then detected using anti-mouse-PO 1/2000 with enzymatic development (OPD).
Immunoprecipitation experiments: 293T cells were transfected with indicated expression plasmid (Rec-Fc or flag ligand) in 9 cm plate. Transfected cells were left for 5d in 8 ml Optimem media (Gibco-BRL). Immunoprecipitation were performed by mixing 200 ul of each receptor-conditioned media with 200 ul of each ligand-conditioned media+400 ul PBS+10 ul ProtG-Sepharose. These were rotated 1 h on a wheel, washed 4× with 1 ml PBS, then boiled in 50 ul sample buffer (+DTT). 20 ul of each immunoprecipitation was loaded per lane. Reveal blotting was done with 1 ug/ml anti-FLAG M2 mAb (Sigma, St Louis Mo.) and anti-mouse PO ( 1/2000). A reprobe blot with anti-human-PO was also checked: 100 ul conditioned media was precipitated with MeOH/CHC13/lysozyme. This mix was boiled in 50 ul sample buffer (+DTT) and 20 ul was loaded. A Reveal blot was performed with anti-FLAG mAb M2 (1 ug/ml) and anti-mouse-PO ( 1/2000).
Example 3
BiaCore Analysis
This example describes the binding of myc-mAPRIL; hKayL-440 (hBAFF); and Flag-mBAFF to hBCMA-Ig, hLT-R-Ig, or hp80 TNFR-Ig. All experiments were performed at 25C with a 10 ul/ml minute flow rate.
Each experiment was performed using HBS buffer (10MM HEPES, 150 mM NaCl, 0.005% P20 surfactant, at pH 7.4). The same solution was used both as running buffer and as sample diluent.
The CM5 chip (BIAcore, Inc.) surface was first activated with N-hydroxysuccinimide/N-ethyl-N′-(3-diethylaminopropyl)-carbodiimide hydrochloride (BIAcore). Twenty ul of hBCMA-Ig; fifteen ul of hLT-R05-Ig and 10 ul of hp80-TNFR, diluted to 30 g/ml in 10 mM acetic acid were then blocked with once with 30 ul and again with 15 ul of ethanolamine-HCL (pH 8.5). This resulted in a surface density of 1600-3700 resonance units (RU). The chip was regenerated with 20 ul of 1 mM formic acid. These rejections were repeated five times to establish a reproducible and stable baseline.
For the experiment, 100 ul of myc-mApril, hKayL-440, and FLAG-mBAFF each was diluted to 30 ug/ml in diluent buffers and was injected over the surface of the chip. Immediately after each injection, the chip was washed with 500 ul of the diluent buffer. The surface was regenerated between experiments by injecting 20 ul of 1 mM formic acid; followed with another 15 ul injection formic acid. After regeneration, the chip was equilibrated with the dilution buffer.
Example 4
Generation of Soluble Receptor Forms
To form a receptor inhibitor for use in humans, one requires the human receptor cDNA sequence of the extracellular domain. If the mouse form is known, human cDNA libraries can be easily screened using the mouse cDNA sequence and such manipulations are routinely carried out in this area. With a human cDNA sequence, one can design oligonucleotide primers to PCR amplify the extracellular domain of the receptor in the absence of the transmembrane and intracellular domains. Typically, one includes most of the amino acids between the last disulfide linked “TNF domain” and the transmembrane domain. One could vary the amount of “stalk” region included to optimize the potency of the resultant soluble receptor. This amplified piece would be engineered to include suitable restriction sites to allow cloning into various C-terminal Ig fusion chimera vectors. Alternatively, one could insert a stop signal at the 3′ end and make a soluble form of the receptor without resorting to the use of a Ig fusion chimera approach. The resultant vectors can be expressed in most systems used in biotechnology including yeast, insect cells, bacteria and mammalian cells and examples exist for all types of expression. Various human Fc domains can be attached to optimize or eliminate FcR and complement interactions as desired. Alternatively, mutated forms of these Fc domains can be used to selectively remove FcR or complement interactions or the attachment of N-linked sugars to the Fc domain which has certain advantages.
Example 5
Generation of Agonistic or Antagonistic Antibodies
The above described soluble receptor forms can be used to immunize mice and to make monoclonal antibodies by conventional methods. The resultant mAbs that are identified by ELISA methods can be further screened for agonist activity either as soluble antibodies or immobilized on plastic in various in vitro cellular assays. Often the death of the HT29 cell line is a convenient system that is sensitive to signaling through many TNF receptors. If this line does not possess the receptor of interest, that full length receptor can be stably transfected into the HT29 line to now allow the cytotoxicity assay to work. Alternatively, such cells can be used in the Cytosensor apparatus to assess whether activation of the receptor can elicit a pH change that is indicative of a signaling event. TNF family receptors signal well in such a format and this method does not require one to know the actual biological events triggered by the receptor. The agonistic mAbs would be “humanized” for clinical use. This procedure can also be used to define antagonistic mAbs. Such mAbs would be defined by the lack of agonist activity and the ability to inhibit receptor-ligand interactions as monitored by ELISA, classical binding or BIAcore techniques. Lastly, the induction of chemokine secretion by various cells in response to an agonist antibody can form a screening assay.
Example 6
Screening for Inhibitors of the Receptor-Ligand Interaction
Using the receptor-Ig fusion protein, one can screen either combinatorial libraries for molecules that can bind the receptor directly. These molecules can then be tested in an ELISA formatted assay using the receptor-Ig fusion protein and a soluble form of the ligand for the ability to inhibit the receptor-ligand interaction. This ELISA can be used directly to screen various natural product libraries etc. for inhibitory compounds. The receptor can be transfected into a cell line such as the HT29 line to form a biological assay (in this case cytotoxicity) that can then form the screening assay.
Example 7
In vivo Tumor Growth Inhibition
The effectiveness of BCMA-Ig as a tumor growth antagonist was tested using a number of different tumor cell lines grown in vivo. Athymic (Nu/Nu), immunodeficient mice were used for these studies, and tumor cells were implanted subcutaneously. For the SW480 tumor line, which grows aggressively, we implanted 8×105 cells in 100 μl pyrogen-free, sterile PBS. One control group was left untreated (n=5), while other groups were dosed with 100 μgs control-Ig (n=6) or 100 μgs BCMA-Ig (n=6) proteins. Dosing began just prior to implantation, with subsequent doses every 7 days thereafter. Tumor diameter was measured using a micrometer, and the volume is calculated using the formula vol=4/3Πr3.
SW480 colon carcinoma tumors grow very quickly using the Nu/Nu mouse model, and palpable tumors were detected within 10 days. After 24 days the average control tumor volume was 0.3 cm3, while the average volume of BCMA-Ig treated tumors was 0.19 cm3, a reduction of 46% in tumor burden. The colon carcinoma HT29 also grows aggressively in the Nu/Nu model. For these experiments 1×106 cells in 100 μl pyrogen-free, sterile PBS were implanted subcutaneously, and the dosing regimen was as described for SW480. Palpable tumors were detected after 7 days, and in the control groups most of the tumors grew very rapidly. After 42 days the average tumor volume in the control groups (untreated and control-Ig treated, n=12) was 0.485 cm3, while the average tumor size in the BCMA-Ig treated group (n=5) was 0.095 cm3, a reduction of 80% in tumor burden. After 50 days 30% of the mice in the control group were scored as terminal due to tumor sizes greater than 1.5 cm3, and the experiment was halted. In contrast to the control group 0% of the mice in the BCMA-Ig treated group were scored as terminal. These results are shown in table 2.
TABLE 2
Tumor volumes and lethality in the HT29 model
after 50 days treatment.
tumor vol
terminal
control animals (untreated and control-Ig treated)
0.22
−
0.22
−
0.35
−
0.61
−
0.73
−
1.74
+
2.53
+
1.51
+
0.90
−
0.44
−
0.32
−
1.92
+
ave:
0.96
%:
30
BCMA-Ig treated
0.11
−
0.32
−
0.13
−
0.56
−
0.33
−
ave:
0.29
%:
0
This demonstrates a 70% reduction in average tumor volume and a significant effect on mortality in the HT29 model of tumor growth using BCMA-Ig treatment.
The lung carcinoma tumor line A549 grows more slowly than the colon carcinoma lines described above. For this cell line we implanted 1×106 cells in 100 μl pyrogen-free, sterile PBS, and treated using the regimen described previously. Palpable tumors were detected approximately 20 days after implantation. 50 days after tumor implantation the average tumor volume in the control groups (untreated and control-Ig treated; n=16) was 0.2 cm3 while the average tumor volume in the BCMA-Ig treated group (n=7) was 0.1 cm3, a reduction of 50% in tumor volume. In the BCMA-Ig treated group 57% of the mice had a tumor of less than 0.1 cm3 after 50 days, while only 6% of the control treated mice retained such a small tumor burden. 60 days after tumor implantation the average tumor volume in the control group had increased to 0.3 cm3. In contrast the average tumor volume in the BCMA-Ig treated group was still less than 0.2 cm3 (0.188).
For the murine NIH3T3 line, which also grows more slowly than the colon carcinoma lines, we implanted 5×106 cells in 100 μl pyrogen-free, sterile PBS, and treated as described above. The NIH3T3 cells form a fibrosarcoma tumor when implanted subcutaneously in Nu/Nu mice. After 4 weeks palpable tumors were detected, and in the control groups (n=11) these tumors expanded in volume over the next 10 days to reach an average size of 0.136 cm3. In contrast the tumor volume in the BCMA-Ig-treated group (n=5) only reached a size of 0.03 cm3, a 78% reduction in tumor burden. At day 48 after tumor implantation the average tumor volume in the controls groups had reached 1.6 cm3, while the average tumor volume in the BCMA-Ig treated group was only 0.8 cm3, a 50% reduction in tumor volume. By day 52, 82% ( 9/11) of the animals in the control groups had been scored as terminal based on a tumor volume of greater than 1.5 cm3, leaving only 18% of the animals still alive. In contrast 40% (⅖) of the animals in the BCMA-Ig treated group had a tumor of such volume that they had to be sacrificed, leaving 60% of the animals still alive. These results are tabulated in Table 3.
TABLE 3
Survivorship data in the NIH3T3 model.
Days after implantation
% survival
38
42
48
52
control
100
90
64
18
BCMA-Ig
100
100
80
60
The results showing the growth of NIH3T3 tumors over time are illustrated in FIG. 13. The results showing the growth of SW480 tumors over time are illustrated in FIG. 14. The results showing the growth of the HT29 tumors over time, and a scattergram showing individual animals on day 42 after tumor implantation, are illustrated in FIG. 15A. The results showing the growth of A549 tumors in individual animals on days 50 and 60 after tumor implantation are shown in FIG. 15B.
The results for the tumor growth inhibition for the NIH3T3 tumor cell line are shown in FIG. 13. The results for the tumor growth inhibition for the SW480 tumor cell line are shown in FIG. 14. The results for the tumor growth inhibition for the HT29 and A549 tumor cell lines are shown in FIG. 15.
Example 8
BCMA-IgG Causes a Reduction in the Number of B Cells in Normal Mice
Eight-week-old female BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, Me.).
Mice (3/group) received i.p. either PBS, 400 μg of human BCMA-huIgG1 (hBCMA-Ig) fusion protein (supplied by Teresa Cachero, Biogen), or 400 μg of purified human IgG (HuIgG) (Sandoz, Basel, Switzerland) on days −8, −5, −1 and +2. Mice received 100 μl of 10% sheep red blood cells (SRBC) (Colorado Serum Company, Denver, Colo.) on day 0.
At the time of sacrifice blood was collected via cardiac puncture into tubes containing EDT, and red blood cells were lysed in a hypotonic buffer. Blood was also collected without EDTA for serum preparation. Single cell suspensions were prepared from spleens and mesenteric lymph nodes (MLN) and red blood cells were lysed in a hypotonic buffer. Flow cytometry was performed using PE-conjugated anti-CD45R/B220, anti-syndecan/CD138 and anti-B7.2, and FITC-conjugated anti-IgM and anti-CD45R/B220. All mAbs were purchased from Pharmingen (San Diego, Calif.). Briefly, Fc receptors were blocked with 10 μg/ml Fc Block (Pharmingen) for 15 min. on ice, followed by addition of PE- and FITC-conjugated mabs and incubated on ice for 20-30 min. Cells were washed 1× and suspended in 0.5% paraformaldehyde. Cell fluorescence data were acquired on a FACSCalibur™ flow cytometer (Becton Dickinson, San Jose, Calif.) and analyzed using CELLQuest™ software (Becton Dickinson).
After treatment with hBCMA-Ig there was approximately a 50% reduction in the number of B cells in peripheral blood and in the peripheral lymphoid organs examined. B220high IgMlow B cells accounted for 23.4% and 21.5% of cells in PBS-treated and HulgG-treated mice, respectively, whereas this population represented only 9.9% of cells in hBCMA-Ig-treated mice. Plasma cells (sndecan/CD138+) appeared to be slightly decreased as well with 5.7% and 4.8% present in the blood of PBS-treated and HuIgG-treated mice, respectively, compared with 3.9% in hBCMA-Ig-treated mice. The B7.2 molecule was upregulated on 3.1% and 4.5% of B220+cells in PBS-treated and HuIgG-treated mice, respectively, compared with 1.9% in hBCMA-Ig-treated mice.
In the spleen B220high B cells were markedly reduced in hBCMA-Ig-treated mice representing 18.8%, compared with 36.7% and 40% in PBS- and HuIgG-treated mice, respectively. This decline was observed in both IgMhigh and IgMlow subpopulations (see Table 1). There was no change observed in the newly formed B cell compartment in the spleen, B220low IgMhigh (data not shown). Plasma cells (syndecan/CD 138+) appeared to be slightly decreased as well with 3.3% and 3.4% present in the spleen of PBS-treated and HuIgG-treated mice, respectively, compared with 2.4% in hBCMA-Ig-treated mice.
The MLN exhibited a decline in B220+B cells with 14.1% present in hBCMA-Ig-treated mice compared with 26.7% and 35.8% in PBS-treated and HuIgG-treated mice, respectively. The data are summarized in Table 3.
TABLE 3
B cell populations in hBCMA-Ig, PBS and HuIgG-treated mice1.
B220high
Blood
IgMlow
Syndecan
B7.2/B220low
PBS
23.4 ± 5.7
5.7 ± 1.5
3.1 ± 0.5
HuIgG
21.5 ± 4.5
4.8 ± 0.9
4.5 ± 1.0
HBCMA-Ig
9.9 ± 1.8
3.9 ± 0.6
1.9 ± 0.5
B220high
Spleen
IgMlow
B220high IgM+
Syndecan
PBS
27.8 ± 1.6
11.9 ± 1.6
3.3 ± 0.8
HuIgG
30.5 ± 2
11.8 ± 1.0
3.4 ± 0.7
HBCMA-Ig
10.6 ± 0.2
8.4 ± 0.2
2.4 ± 0.2
MLN
B220+
PBS
26.7
HuIgG
35.8 ± 3.3
HBCMA-Ig
14.1 ± 5.9
1The mice were treated as described in the Materials and Methods section, and the data are given as percent ± standard Deviation
The decreased percentage of B7.2+B cells in the blood and plasma cells in the blood and spleens of hBCMA-Ig-treated mice after immunization with SRBCs suggests that there is inhibition of B cell activation and/or maturation, and potentially increased elimination of activated B cells. A very minor percent of antigen-specific B cells would be activated and respond to any antigen, in this case SRBC. Because the hBCMA-Ig treatment resulted in such a dramatic reduction in the percent of B cells in all tissues examined, ˜50%, the activity of hBCMA-Ig appears to also target resting, mature B cells.
It is therefore contemplated that BCMA fusion protein may be used as a therapeutic drug with clinical application in B cell-mediated diseases. Diseases would include those that are autoimmune in nature such as systemic lupus erythematosus, myasthenia gravis, autoimmune hemolytic anemia, idiopathic thrombocytopenia purpura, anti-phospholipid syndrome, Chaga's disease, Grave's disease, Wegener's Granulomatosis, Poly-arteritis Nodosa and Rapidly Progressive Glomerulonephritis. The therapeutic agent would also have application in plasma cell disorders such as multiple myeloma, Waldenstrom's macroglobulinemia, Heavey-chain disease, Primary or immunocyte-associated amyloidosis, and Monoclonal gammopathy of undetermined significance (MGUS). Oncology targets would include B cell carcinomas, leukemias, and lymphomas.
It will be apparent to those skilled in the art that various modifications and variations can be made in the polypeptides, compositions and methods of the invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided that they come within the scope of the appended claims and their equivalents.
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10115192
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biogen idec ma inc.
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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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424/185.1
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Apr 1st, 2022 05:10PM
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Apr 1st, 2022 05:10PM
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Biogen
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:biib
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Biogen
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Sep 15th, 2009 12:00AM
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May 25th, 1995 12:00AM
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https://www.uspto.gov?id=US07588755-20090915
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DNA sequences, recombinant DNA molecules and processes for producing human fibroblast interferon-like polypeptides
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DNA sequences, recombinant DNA molecules and hosts transformed with them which produce polypeptides displaying a biological or immunological activity of human fibroblast interferon, the genes coding for these polypeptides and methods of making and using these DNA sequences, molecules, hosts, genes and polypeptides. The DNA sequences are characterized in that they code for a polypeptide displaying a biological or immunological activity of human fibroblast interferon. In appropriate hosts these DNA sequences and recombinant DNA molecules permit the production and identification of genes and polypeptides displaying a biological or immunological activity of human fibroblast interferon and their use in antiviral and antitumor or anticancer agents.
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7588755
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1. A method for immunomodulation or treating a viral conditions, a viral disease, cancers or tumors comprising the step of administering to a patient in need of such treatment a therapeutically effective amount of a composition comprising:
a recombinant polypeptide produced by a non-human host transformed by a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of:
(a) DNA sequences which are capable of hybridizing to any of the DNA inserts of G-pBR322(Pst)/HFIF1, G-pBR322(Pst)/HFIF3 (DSM 1791), G-pBR322(Pst)/HFIF6 (DSM 1792), and G-pBR322(Pst)/HFIF7 (DSM 1793) under hybridizing conditions of 0.75 M NaCl at 68° C. and washing conditions of 0.3 M NaCl at 68° C., and which code for a polypeptide displaying antiviral activity, and (b) DNA sequences which are degenerate as a result of the genetic code to the DNA sequences defined in (a);
said DNA sequence being operatively linked to an expression control sequence in the recombinant DNA molecule.
2. The method according to claim 1, wherein said DNA sequence is selected from DNA sequences of the formulae:
ATGACCAACAAGTGTCTCCTCCAAATTGCTCTCCTGTTGTGCTTCTCCACTACAGCT CTTTCCATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAGTGT CAGAAGCTCCTGTGGCAATTGAATGGGAGGCTTGAATACTGCCTCAAGGACAGGAT GAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCAGCAGTTCCAGAAGGAGGACG CCGCATTGACCATCTATGAGATGCTCCAGAACATCTTTGCTATTTTCAGACAAGATT CATCTAGCACTGGCTGGAATGAGACTATTGTTGAGAACCTCCTGGCTAATGTCTATC ATCAGATAAACCATCTGAAGACAGTCCTGGAAGAAAAACTGGAGAAAGAAGATTTC ACCAGGGGAAAACTCATGAGCAGTCTGCACCTGAAAAGATATTATGGGAGGATTCT GCATTACCTGAAGGCCAAGGAGTACAGTCACTGTGCCTGGACCATAGTCAGAGTGG AAATCCTAAGGAACTTTTACTTCATTAACAGACTTACAGGTTACCTCCGAAAC, and ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAGTGTCAGAAG CTCCTGTGGCAATTGAATGGGAGGCTTGAATACTGCCTCAAGGACAGGATGAACTTT GACATCCCTGAGGAGATTAAGCAGCTGCAGCAGTTCCAGAAGGAGGACGCCGCATT GACCATCTATGAGATGCTCCAGAACATCTTTGCTATTTTCAGACAAGATTCATCTAG CACTGGCTGGAATGAGACTATTGTTGAGAACCTCCTGGCTAATGTCTATCATCAGAT AAACCATCTGAAGACAGTCCTGGAAGAAAAACTGGAGAAAGAAGATTTCACCAGGG GAAAACTCATGAGCAGTCTGCACCTGAAAAGATATTATGGGAGGATTCTGCATTACC TGAAGGCCAAGGAGTACAGTCACTGTGCCTGGACCATAGTCAGAGTGGAAATCCTA AGGAACTTTTACTTCATTAACAGACTTACAGGTTACCTCCGAAAC.
3. The method according to claim 1 wherein the polypeptide is selected from polypeptides of the formulae:
Met-Thr-Asn-Lys-Cys-Leu-Leu-Gln-Ile-Ala-Leu-Leu-Leu-Cys-Phe-Ser-Thr-Thr-Ala-Leu-Ser-Met-Ser-Tyr-Asn-Leu-Leu-Gly-Phe-Leu-Gln-Arg-Ser-Ser-Asn-Phe-Gln-Cys-Gln-Lys-Leu-Leu-Trp-Gln-Leu-Asn-Gly-Arg-Leu-Glu-Tyr-Cys-Leu-Lys-Asp-Arg-Met-Asn-Phe-Asp-Ile-Pro-Glu-Glu-Ile-Lys-Gln-Leu-Gln-Gln-Phe-Gln-Lys-Glu-Asp-Ala-Ala-Leu-Thr-Ile-Tyr-Glu-Met-Leu-Gln-Asn-Ile-Phe-Ala-Ile-Phe-Arg-Gln-Asp-Ser-Ser-Ser-Thr-Gly-Trp-Asn-Glu-Thr-Ile-Val-Glu-Asn-Leu-Leu-Ala-Asn-Val-Tyr-His-Gln-Ile-Asn-His-Leu-Lys-Thr-Val-Leu-Glu-Glu-Lys-Leu-Glu-Lys-Glu-Asp-Phe-Thr-Arg-Gly-Lys-Leu-Met-Ser-Ser-Leu-His-Leu-Lys-Arg-Tyr-Tyr-Gly-Arg-Ile-Leu-His-Tyr-Leu-Lys-Ala-Lys-Glu-Tyr-Ser-His-Cys-Ala-Trp-Thr-Ile-Val-Arg-Val-Glu-Ile-Leu-Arg-Asn-Phe-Tyr-Phe-Ile-Asn-Arg-Leu-Thr-Gly-Tyr-Leu-Arg-Asn, and Met-Ser-Tyr-Asn-Leu-Leu-Gly-Phe-Leu-Gln-Arg-Ser-Ser-Asn-Phe-Gln-Cys-Gln-Lys-Leu-Leu-Trp-Gln-Leu-Asn-Gly-Arg-Leu-Glu-Tyr-Cys-Leu-Lys-Asp-Arg-Met-Asn-Phe-Asp-Ile-Pro-Glu-Glu-Ile-Lys-Gln-Leu-Gln-Gln-Phe-Gln-Lys-Glu-Asp-Ala-Ala-Leu-Thr-Ile-Tyr-Glu-Met-Leu-Gln-Asn-Ile-Phe-Ala-Ile-Phe-Arg-Gln-Asp-Ser-Ser-Ser-Thr-Gly-Trp-Asn-Glu-Thr-Ile-Val-Glu-Asn-Leu-Leu-Ala-Asn-Val-Tyr-His-Gln-Ile-Asn-His-Leu-Lys-Thr-Val-Leu-Glu-Glu-Lys-Leu-Glu-Lys-Glu-Asp-Phe-Thr-Arg-Gly-Lys-Leu-Met-Ser-Ser-Leu-His-Leu-Lys-Arg-Tyr-Tyr-Gly-Arg-Ile-Leu-His-Tyr-Leu-Lys-Ala-Lys-Glu-Tyr-Ser-His-Cys-Ala-Trp-Thr-Ile-Val-Arg-Val-Glu-Ile-Leu-Arg-Asn-Phe-Tyr-Phe-Ile-Asn-Arg-Leu-Thr-Gly-Tyr-Leu-Arg-Asn.
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This is a division, of application Ser. No. 07/387,503, filed Jul. 28, 1989, now abandoned which is a continuation of Ser. No. 06/250,609, filed Apr. 3, 1981 now abandoned, entitled DNA SEQUENCES, RECOMBINANT DNA MOLECULES AND PROCESSES FOR PRODUCING HUMAN FIBROBLAST INTERFERON-LIKE POLYPEPTIDES.
TECHNICAL FIELD OF INVENTION
This invention relates to DNA sequences, recombinant DNA molecules and process for producing human fibroblast interferon-like polypeptides. More particularly, the invention relates to DNA sequences expressed in appropriate host organism. The recombinant DNA molecules disclosed herein are characterized by DNA sequences that code for polypeptides whose amino acid sequence and composition are substantially consistent with human fibroblast interferon and which have an immunological or biological activity of human fibroblast interferon. As will be appreciated from the disclosure to follow, the DNA sequences, recombinant DNA molecules and processes of this invention may be used in the production of polypeptides useful in antiviral and antitumor or anticancer agents and methods and in immunomodulation.
BACKGROUND ART
In this application the interferon nomenclature announced in Nature, 286, p. 2421 (Jul. 10, 1980) will be used. This nomenclature replaces that used in our earlier applications from which this application claims priority. E.g., IF is now designated IFN and fibroblast interferon is now designated IFN-β.
Two classes of interferons (“IFN”) are known to exist. Interferons of Class I are small, acid stable (glyco)-proteins that render cells resistant to viral infection (A. Isaacs and J. Lindenmann, “Virus Interference I The Interferon”, Proc. Royal Soc. Ser. B., 147, pp. 258-67 (1957) and W. E. Stewart, II, The Interferon System, Springer-Verlag (1979) (hereinafter “The Interferon System”)). Class II IFNs are acid labile. At present, they are poorly characterized. Although to some extent cell specific (The Interferon System, pp. 135-45), IFNs are not virus specific. Instead, IFNs protect cells against a wide spectrum of viruses.
Human interferon (“BuIFN”) has been classified into three groups α, β and γ. HuIFN-β or fibroblast interferon is produced upon appropriate induction in diploid fibroblast cells. It is also produced in minor amounts, together with a major amount of HuIFN-α, in lymphoblastoid cells. IFN-β made from these cells has been extensively purified and characterized (E. Knight, Jr., “Interferon: Purification And Initial Characterization From Human Diploid Cells”, Proc. Natl. Acad. Sci. USA, 73, pp. 520-23 (1976)). It is a glyco-protein of about 20,000 molecular weight (M. Wiranowska-Stewart, et al., “Contributions Of Carbohydrate Moieties To The Physical And Biological Properties Of Human Leukocyte, Lympho-blastoid And Fibroblast Interferons”, Abst. Ann. Meeting Amer. Soc. Microbiol., p. 246 (1978)). It is also heterogeneous in regard to size presumably because of the carbohydrate moities.
The amino acid composition of authentic human fibroblast interferon has also been reported (E. Knight, Jr., et al., “Human Fibroblast Interferon: Amino Acid Analysis And Amino-Terminal Amino Acid Sequence”, Science, 207, pp. 525-26 (1980)). And, elucidation of the amino acid sequence of authentic human fibroblast interferon is in progress. To date, the amino acid sequence of the NH2 terminus of the authentic mature protein has been reported for the first 13 amino acid residues: Met-Ser-Tyr-Asn-Leu-Leu-Gly-Phe-Leu-Gln-Arg-Ser-Ser . . . . (E. Knight, Jr., et al., supra).
Two distinct genes, one located on chromosome 2, the other on chromosome 5, have been reported to code for IFN-β (D. L. Slate and F. H. Ruddle, “Fibroblast Interferon In Man Is Coded By Two Loci On Separate Chromosomes”, Cell, 16, pp. 171-80 (1979)). Other studies, however, indicate that the gene for IFN-β is located on chromosome 9 (A. Medger, et al., “Involvement Of A Gene On Chromosome 9 In Human Fibroblast Interferon Production”, Nature, 280, pp. 493-95 (1979)).
Although authentic HuIFN-β is glycosylated, removal of the carbohydrate moiety (P. J. Bridgen, et al., “Human Lymphoblastoid Interferon”, J. Biol. Chem., 252, pp. 6585-87 (1977)) or synthesis of IFN-β in the presence of inhibitors which purport to preclude glyco-sylation (W. E. Stewart, II, et al., “Effect of Glyco-sylation Inhibitors On The Production And Properties Of Human Leukocyte Interferon”,Virology, 97, pp. 473-76 (1979); J. Fujisawa, et al., “Nonglycosylated Mouse L Cell Interferon Produced By The Action Of Tunicamycin”, J. Biol. Chem., 253, pp. 8677-79 (1978); E. A. Havell, et al., “Altered Molecular Species Of Human Interferon Produced In The Presence Of Inhibitors of Glycosylation”, J. Biol. Chem., 252, pp. 4425-27 (1977); The Interferon System, p. 181) yields a smaller form of IFN-β which still retains most or all of its IFN activity.
HuIFN-β, like many human proteins, may also be polymorphic. Therefore, cells of particular individuals may produce IFN-β species within the more general IFN-β class which are physiologically similar but structurally slightly different from the prototype of the class of which it is a part. Therefore, while the protein structure of the IFN-β may be generally well-defined, particular individuals may produce IFN-βs that are slight variations thereof.
IFN-β is usually not detectable in normal or healthy cells (The Interferon System, pp. 55-57). Instead, the protein is produced as a result of the cell's exposure to an IFN inducer. IFN inducers are usually viruses but may also be non-viral in character, such as natural or synthetic double-stranded RNA, intra-cellular microbes, microbial products and various chemical agents. Numerous attempts have been made to take advan-tage of these non-viral inducers to render human cells resistant to viral infection (S. Baron and F. Dianzani (eds.), Texas Reports On Biology And Medicine, 35 (“Texas Reports”), pp. 528-40 (1977)). These attempts have not been very successful. Instead, use of exogenous HuIFN-β itself is now preferred.
Interferon therapy against viruses and tumors or cancers has been conducted at varying dosage regimes and under several modes of administration (The Interferon System, pp. 305-321). For example, interferon has been effectively administered orally, by innoculation—intravenous, intramuscular, intranasal, intradermal and subcutaneous—, and in the form of eye drops, ointments and sprays. It is usually administered one to three times daily in dosages of 104 to 107 units. The extent of the therapy depends on the patient and the condition being treated. For example, virus infections are usually treated by daily or twice daily doses over several days to two weeks and tumors and cancers are usually treated by daily or multiple daily doses over several months or years. The most effective therapy for a given patient must of course be determined by the attending physician, who will consider such well known factors as the course of the disease, previous therapy, and the patient's response to interferon in selecting a mode of administra-tion and a dosage regime.
As an antiviral agent, HuIFN has been used to treat the following: respiratory infections (Texas Reports, pp. 486-96); herpes simplex keratitis (Texas Reports, pp. 497-500; R. Sundmacher, “Exogenous Interferon in Eye Diseases”, International virology IV, The Hague, Abstract nr. W2/11, p. 99 (1978)); acute hemorrhagic conjunctivitis (Texas Reports, pp. 501-10); adenovirus keratoconjunctivitis (A. Romano, et al., ISM Memo I-A8131 (October, 1979)); varicella zoster (Texas Reports, pp. 511-15); cytomegalo-virus infection (Texas Reports, pp. 523-27); and hepatitis B (Texas Reports, pp. 516-22). See also The Interferon System, pp. 307-19. However, large-scale use of IFN as an antiviral agent requires larger amounts of IFN than heretofore have been available.
IFN has other effects in addition to its anti-viral action. For example, it antagonizes the effect of colony stimulating factor, inhibits the growth of hemo-poietic colony-forming cells and interferes with the normal differentiation of granulocyte and macrophage precursors (Texas Reports, pp. 343-49). It also inhibits erythroid differentiation in DMSO-treated Friend leukemia cells (Texas Reports, pp. 420-28). It is significant that some cell lines may be considerably more sensitive to HuIFN-β than to HuIFN-α in these regards (S. Einhorn and H. Strander, “Is Interferon Tissue-Specific?—Effect Of Human Leukocyte And Fibroblast Interferons On The Growth Of Lymphoblastoid And Osteosarcoma Cell Lines”, J. Gen. Virol., 35, pp. 573-77 (1977); T. Kuwata, et al., “Comparison Of The Suppression Of Cell And Virus Growth In Transformed Human Cells By Leukocyte And Fibroblast Interferon”, J. Gen. Virol., 43, pp. 435-39 (1979)).
IFN may also play a role in regulation of the immune response. For example, depending upon the dose and time of application in relation to antigen, IFN can be both immunopotentiating and immunosuppressive in vivo and in vitro (Texas Reports, pp. 357-69). In addition, specifically sensitized lymphocytes have been observed to produce IFN after contact with antigen. Such antigen-induced IFN could therefore be a regulator of the immune response, affecting both circulating antigen levels and expression of cellular immunity (Texas Reports, pp. 370-74). IFN is also known to enhance the activity of killer lymphocytes and antibody-dependent cell-mediated cyto-toxicity (R. R. Herberman, et al., “Augmentation By Interferon Of Human Natural And Antibody-Dependent Cell-Mediated Cytotoxicity”, Nature, 277, pp. 221-23 (1979); P. Beverley and D. Knight, “Killing Comes Naturally”, Nature, 278, pp. 119-20 (1979); Texas Reports, pp. 375-80; J. R. Huddlestone, et al., “Induction And Kinetics Of Natural Killer Cells in Humans Following Interferon Therapy”, Nature, 282, pp. 417-19 (1979); S. Einhorn, et al., “Interferon And Spontaneous Cytotoxicity In Man. II. Studies In Patients Receiving Exogenous Leukocyte Interferon”, Acta Med. Scand., 204, pp. 477-83 (1978)). Both may be directly or indirectly involved in the immunological attack on tumor cells.
Therefore, in addition to its use as an antiviral agent, HuIFN has potential application in antitumor and, anticancer therapy (The Interferon System, pp. 319-21 and 394-99). It is now known that IFNs affect the growth of many classes of tumors in many animals (The Interferon System, pp. 292-304). They, like other anti-tumor agents, seem most effective when directed against small tumors. The antitumor effects of animal IFN are dependent on dosage and time but have been demonstrated at concentrations below toxic levels. Accordingly, numerous investigations and clinical trials have been and continue to be conducted into the antitumor and anticancer properties of HuIFNs. These include treatment of several malignant diseases such as osteosarcoma, acute myeloid leukemia, multiple myeloma and Hodgkin's disease (Texas Reports, pp. 429-35). In addition, HuIFN-β has recently been shown to cause local tumor regression when injected into subcutaneous tumoral nodules in melanoma and breast carcinoma-affected patients (T. Nemoto, et al., “Human Interferons And Intralesional Therapy Of Melanoma And Breast Carcinoma”, Amer. Assoc. For Cancer Research, Abs nr. 993, p. 246 (1979)). Although the results of these clinical tests are encouraging, the antitumor and anticancer applications of IFN-β have been severely hampered by lack of an adequate supply of purified IFN-β.
Significantly some cell lines which resist the anticellular effects of IFN-α remain sensitive to IFN-β. This differential effect suggests that IFN-β may be usefully employed against certain classes of resistant tumor cells which appear under selective pressure in patients treated with high doses of IFN-α (T. Kuwata, et al., supra; A. A. Creasy, et al., “The Role of G0-G1 Arrest In The Inhibition Of Tumor Cell Growth By Interferon”, Abstracts, Conference On Regulatory Functions Of Interferons, N.Y. Acad. Sci., nr. 17 (Oct. 23-26, 1979)).
At the biochemical level IFNs induce the formation of at least 3 proteins, a protein kinase (B. Lebleu, et al., “Interferon, Double-Stranded RNA And Protein Phosphorylation”, Proc. Natl. Acad. Sci. USA, 73, pp. 3107-11 (1976); A. G. Hovanessian and I. M. Kerr, “The (2′-5′) Oligoadenylate (ppp A2′-5A2′-5′A) Synthetase And Protein Kinase(s) From Interferon-Treated Cells”, Eur. J. Biochem., 93, pp. 515-26 (1979)), a (2′-5′)oligo(A) polymerase (A. G. Hovanessian, et al., “Synthesis Of Low-Molecular Weight Inhibitor Of Protein Synthesis With Enzyme From Interferon-Treated Cells”, Nature, 268, pp. 537-39 (1977); A. G. Hovanessian and I. M. Kerr, Eur. J. Biochem, supra) and a phosphodiesterase (A. Schmidt, et al., “An Interferon-Induced Phosphodiesterase Degrading (2′-5′)oligoisoadenylate And The C-C-A Terminus Of tRNA”, Proc. Natl. Acad. Sci. USA, 76, pp. 4788-92 (1979)).
Both IFN-β and IFN-α appear to trigger similar enzymatic pathways (C. Baglioni, “Interferon-Induced Enzymatic Activities And Their Role In The Antiviral State”, Cell, 17, pp. 255-64 (1979)) and both may share a common active core because they both recognize a chromosome 21-coded cell receptor (M. Wiranowska-Stewart, “The Role Of Human Chromosome 21 In Sensitivity To Interferons”, J. Gen. Virol., 37, pp. 629-34 (1977)). The appearance of one or more of these enzymes in cells treated with IFN should allow a further characterization of proteins with IFN-like activity.
Today, HuIFN-β is produced by human cell lines grown in tissue culture. It is a low yield, expensive process. One large producer makes only 40−50×108 units of crude IFN-β per year (V. G. Edy, et al., “Human Interferon: Large Scale Production In Embryo Fibroblast Cultures”, in Human Interferon (W. R. Stinebring and P. J. Chapple, eds.), Plenum Publishing Corp., pp. 55-60 (1978)). On purification by adsorption to controlled pore glass beads, IFN-β of specific activity of about 106 units/mg may be recovered in 50% yield from the crude cell extracts (A. Billiau, et al., “Human Fibroblast Interferon For Clinical Trials: Production, Partial Purification And Characterization”, Antimicrobial Agents And Chemotherapy, pp. 49-55 (1979)). Further purification to a specific activity of about 109 units/mg is accomplished by zinc chelate affinity chromatography in about 100% yield (A. Billiau, et al., “Production, Purification And Properties Of Human Fibroblast Interferon”, Abstracts, Conference On Regulatory Functions Of Interferons, N.Y. Acad. Sci., nr 29 (Oct. 23-26, 1979)). Because the specific activity of HuIFN-β is so high, the amount of IFN-β required for commercial applications is low. For example, 100 g of pure IFN-β would provide between 3 and 30 million doses.
Recent advances in molecular biology have made it possible to introduce the DNA coding for specific non-bacterial eukaryotic proteins into bacterial cells. In general, with DNA other than that prepared via chemical synthesis, the construction of such recombinant DNA molecules comprises the steps of producing a single-stranded DNA copy (cDNA) of a purified messenger RNA (mRNA) template for the desired protein; converting the cDNA to double-stranded DNA; linking the DNA to an appropriate site in an appropriate cloning vehicle to form a recombinant DNA molecule and transforming an appropriate host with that recombinant DNA molecule. Such transformation may permit the host to produce the desired protein. Several non-bacterial genes and proteins have been obtained in E. coli using recombinant DNA technology. These include, for example, IFN-α (S. Nagata, et al., “Synthesis In E. coli Of A Polypeptide With Human Leukocyte Interferon Activity”, Nature, 284, pp. 316-20 (1980)). In addition, recombinant DNA technology has been employed to produce a plasmid said to contain a gene sequence coding for IFN-β (T. Taniguchi, et al., “Construction And Identification Of A Bacterial Plasmid Containing The Human Fibroblast Interferon Gene Sequence”, Proc. Japan Acad. Ser. B, 55, pp. 464-69 (1979)).
However, in neither of the foregoing has the actual gene sequence of IFN-β been described and in neither has that sequence been compared to the initial amino acid sequence or amino acid composition of authentic IFN-β. The former work is directed only to IFN-α, a distinct chemical, biological and immunological Class I interferon from IFN-β (cf. supra). The latter report is based solely on hybridization data. These data alone do not enable one to determine if the selected clone contains the complete or actual gene sequence coding for IFN-β or if the cloned gene sequence will be able to express IFN-β in bacteria. Hybridization only establishes that a particular DNA insert is to some extent homologous with and complementary to a mRNA component of the poly(A) RNA that induces interferon activity when injected into oocytes. Moreover, the extent of any homology is dependent on the hybridization conditions chosen for the screening process. Therefore, hybridization to a mRNA component of poly(A) RNA alone does not demonstrate that the selected DNA sequence is a sequence which codes for HuIFN-β or a polypeptide which displays the immunological or biological activity of HuIFN-β or that such sequence will be useful in producing such polypeptides in appropriate hosts.
At a seminar in Zurich on Feb. 25, 1980, Taniguchi stated that he had determined the nucleotide sequence for one of his hybridizing clones. He also stated that the first 13 amino acids coded for by that sequence were identical to that determined by Knight, et al., supra, for authentic. HuIFN-β. Taniguchi did not disclose the full nucleotide sequence for his clone or compare its amino acid composition with that determined for authentic HUIFN-β. Taniguichi has since reported the full nucleotide sequence for his hybridizing clone (T. Taniguichi et al., Gene, 10, pp. 11-15 (1980)). The sequence differs by one nucleotide from that described and claimed in British patent application 8011306, filed Apr. 3, 1980, an application to which the present application claims priority. The amino acid sequence reported by Taniguichi is identical to the amino acid sequence described and claimed in the foregoing application 8011306. Taniguichi had also not reported the expression in an appropriate host of polypeptides which display an immunological or biological activity of HuIFN-β at the time of the filing of British patent application 80.18701, filed Jun. 6, 1980, an application to which this application claims priority. It is this expression in a host of polypeptide(s) displaying an immunological or biological activity of HuIFN-β and the methods, polypeptides, genes and recombinant DNA molecules thereof, which characterize this invention.
Nor is this invention addressed as is the apparent suggestion of Research Disclosure No. 18309, pp. 361-62 (1979) to prepare pure or substantially pure IFN-α mRNA before attempting to clone the IFN gene or to produce fibroblast interferon-like polypeptides in bacterial hosts.
Finally, it should be recognized that the selection of a DNA sequence or the construction of a recombinant DNA molecule which hybridizes to a mRNA from polyA RNA, that mRNA producing HuIFN activity in oocytes, is not sufficient to demonstrate that the DNA sequence or the hybrid insert of the recombinant DNA molecule corresponds to HuIFN. Instead, in the absence of a comparison of the amino acid sequence coded for by a particular DNA sequence and the amino acid sequence of the authentic protein, only the production of a polypeptide that displays an immunological or biological activity of HuIFN can actually demonstrate that the selected DNA sequence or constructed recombinant DNA molecule corresponds to HuIFN. More importantly, it is only after such HuIFN activity is shown that the DNA sequence, recombinant DNA molecule or sequences related to them may be usefully employed to select other sequences corresponding to HuIFN in accordance with this invention or to produce recombinant DNA molecules that may express products having an immunological or biological activity of HuIFN-β.
It will therefore be appreciated from the foregoing that the problem of producing HuIFN-β with the use of recombinant DNA technology is much different than any of the above described processes. Here, a particular DNA sequence of unknown structure—that coding for the expression of HuIFN-β in an appropriate host—must be found in and separated from a highly complex mixture of DNA sequences in order for it to be used in the production of HuIFN-β. Furthermore, this location and separation problem is exacerbated by the predicted exceedingly low concentration of the desired DNA sequence in the complex mixture and the lack of an effective means for rapidly analyzing the many DNA sequences of the mixture to select and separate the desired sequence.
DISCLOSURE OF THE INVENTION
The present invention solves the problems referred to by locating and separating DNA sequences that code for the expression of HuIFN-β in an appropriate host thereby providing DNA sequences, recombinant DNA molecule and methods by means of which a host is transferred to produce a polypeptide displaying an immunological or biological activity of human fibroblast interferon.
By virtue of this invention, it is possible to obtain polypeptides displaying an immunological or biological activity of HuIFN-β for use in antiviral, antitumor or anticancer agents and methods. This invention allows the production of these polypeptides in amounts and by methods hitherto not available.
As will be appreciated from the disclosure to follow, the DNA sequences and recombinant DNA molecules of the invention are capable of directing the production, in an appropriate host, of polypeptides displaying an immunological or biological activity of HuIFN-β. Replication of these DNA sequences and recombinant DNA molecules in an appropriate host also permits the production in large quantities of genes coding for these polypeptides. The molecular structure and properties of these polypeptides and genes may be readily determined. The polypeptides and genes are useful, either as produced in the host or after appropriate derivatization or modification, in compositions and methods for detecting and improving the production of these products themselves and for use in antiviral and antitumor or anticancer agents and methods and immunomodulation.
This process is therefore distinguishable from the prior processes, above mentioned, in that this process, contrary to the noted prior processes, involves the preparation and selection of DNA sequences and recombinant DNA molecules which contain appropriate DNA sequences which code for at least one polypeptide displaying an immunological or biological activity of HuIFN-β.
It will be appreciated from the foregoing that a basic aspect of this invention is the provision of a DNA sequence which is characterized in that it codes for a polypeptide displaying an immunological or biological activity of HuIFN-β and is selected from the group consisting of the DNA inserts of G-pHFIF-1, G-pHFIF-3, G-pHFIF-6, G-pHFIF-7, G-pPla-HFIF-67-12, G-pPla-HFIF-67-12Δ19, G-pP1a-HFIF-67-8, G-pP1a-HFIF-67-12Δ279T, G-pP1a-HFIF-67-12Δ218M1, G-pP1a-HFIF-67-12ΔM1, G-pP1a-HFIF-67-12Δ19BX-2, DNA sequences which hybridize to any of the foregoing DNA inserts, DNA sequences, from whatever source obtained, including natural, synthetic or semi-synthetic sources, related by mutation, including single or multiple, base substitutions, deletions, insertions and inversions to any of the foregoing DNA sequences or inserts, and DNA sequences comprising sequences of codons which on expression code for a polypeptide displaying similar immunological or biological activity to a polypeptide coded for on expression of the codons of any of the foregoing DNA sequences. The sequences of this invention are further characterized in that they permit the production of HuIFN-β and HuIFN-β-like polypeptides in hosts.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic outline of one embodiment of a process of this invention for preparing a mixture of recombinant DNA molecules, some of which are characterized by inserted DNA sequences that code for the polypeptides of this invention.
FIG. 2 is a schematic outline of the initial clone screening process of this invention.
FIG. 3 is a schematic outline of one embodiment of a clone screening process using DNA sequences prepared in accordance with the invention.
FIG. 4 displays the composite nucleotide sequence of the coding strand of HuIFN-β DNA. The sequence is numbered from the beginning of the insert well into the untranslated area of the insert. Nucleotides 65-127 represent a signal sequence and nucleotides 128-625 represent the “mature” fibroblast interferon. The amino acid sequences of the signal polypeptide are depicted above their respective nucleotide sequences; the amino acids of the signal polypeptide being numbered from −21 to −1 and the amino acids of mature interferon being numbered from 1 to 166. Review of the restriction and fragment analysis data of the HuIFN-β DNA present in the cultures deposited in connection with Great Britain in patent application 80.11306; filed Apr. 3, 1980, has resulted in two nucleotides being changed in FIG. 4 as compared to FIG. 4 of that British patent application. These changes are in the untranslated sequence preceding the proposed signal sequence of HuIFN-β DNA. These changes do not effect the sequence of HuIFN-β DNA or the amino acid sequence of its translation product and do not alter the sequence's use as an hydridization probe to screen clones for HuIFN-β related DNA inserts.
FIG. 5 displays the orientation and restriction maps of several plasmids in accordance with this invention.
FIG. 6 is a comparison of the amino acid composition of human fibroblast interferon as determined in accordance with this invention and that determined from authentic fibroblast interferon.
FIG. 7 displays a restriction map of the HuIFN-β gene of this invention and the sequencing strategy used in sequencing pHFIF3, pHFIF6, and pHFIF7.
FIG. 8 is a schematic outline of the construction of recombinant DNA molecule pPLa-HFIF-67-1 of this invention.
FIG. 9 is a schematic outline of the construction of recombinant DNA molecule pPLa-HFIF-67-12 and pPLa-HFIF-67-12Δ19 of this invention.
FIG. 10 is a schematic outline of the construction of recombinant DNA molecule pPLc-HFIF-67-8 of this invention.
FIG. 11 is a schematic outline of the orientation and partial restriction map of pPLa-HFIF-67-12 of this invention.
FIG. 12 is a schematic outline of the orientation and partial restriction map of pPLa-HFIF-67-12Δ19 of this invention.
FIG. 13 is a schematic outline of the orientation and partial restriction map of pPLc-HFIF-67-8 of this invention.
BEST MODE OF CARRYING OUT THE INVENTION
In order that the invention herein described may be more fully understood, the following detailed description is set forth.
In the description the following terms are employed:
Nucleotide—A monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon (1′ carbon of the pentose) and that combination of base and sugar is called a nucleoside. The base characterizes the nucleotide. The four DNA bases are adenine (“A”), guanine (“C”), cytosine (“C”), and thymine (“T”). The four bases are A, G, C and uracil (“U”).
DNA Sequence—A linear array of nucleotides connected one to the other by phosphodiester bonds between the 3′ and 5′ carbons of adjacent pentoses.
Codon—A DNA sequence of three nucleotides (a triplet) which encodes through mRNA an amino acid, a translation start signal or a translation termination signal. For example, the nucleotide triplets TTA, TTG, CTT, CTC, CTA and CTG encode for the amino acid leucine (“Leu”), TAG, TAA and TGA are translation stop signals and ATG is a translation start signal.
Reading Frame—The grouping of codons during translation of mRNA into amino acid sequences. During translation the proper reading frame must be maintained. For example, the DNA sequence GCTGGTTGTAAG may be expressed in three reading frames or phases, each of which affords a different amino acid sequence:
GCT GGT TGT AAG—Ala-Gly-Cys-Lys
G CTG GTT GTA AG—Leu-Val-Val
GC TGG TTG TAA G—Trp-Leu-(STOP)
Polypeptide—A linear array of amino acids connected one to the other by peptide bonds between the α-amino and carboxy groups of adjacent amino acids.
Genome—The entire DNA of a cell or a virus. It includes inter alia the structural genes coding for the polypeptides of the substance, as well as operator, promoter and ribosome binding and interaction sequences, including sequences such as the Shine-Dalgarno sequences.
Structural Gene—A DNA sequence which encodes through its template or messenger RNA (“mRNA”) a sequence of amino acids characteristic of a specific polypeptide.
Transcription—The process of producing mRNA from a structural gene.
Translation—The process of producing a polypeptide from mRNA.
Expression—The process undergone by a structural gene to produce a polypeptide. It is a combination of transcription and translation.
Plasmid—A nonchromosomal double-stranded DNA sequence comprising an intact “replicon” such that the plasmid is replicated in a host cell. When the plasmid is placed within a unicellular organism, the characteristics of that organism may be changed or transformed as a result of the DNA of the plasmid. For example, a plasmid carrying the gene for tetracycline resistance (TetR) transforms a cell previously sensitive to tetracycline into one which is resistant to it. A cell transformed by a plasmid is called a “transformant”.
Phage or Bacteriophage—Bacterial virus many of which consist of DNA sequences encapsidated in a protein envelope or coat (“capsid”).
Cloning Vehicle—A plasmid, phage DNA or other DNA sequence which is able to replicate in a host cell, characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without attendant loss of an essential biological function of the DNA, e.g., replication, production of coat proteins or loss of promoter or binding sites, and which contain a marker suitable for use in the identification of transformed cells, e.g., tetracycline resistance or ampicillin resistance. A cloning vehicle is often called a vector.
Cloning—The process of obtaining a population of organisms or DNA sequences derived from one such organism or sequence by asexual reproduction.
Recombinant DNA Molecule or Hybrid DNA—A molecule consisting of segments of DNA from different genomes which have been joined end-to-end outside of living cells and have the capacity to infect some host cell and be maintained therein.
Expression Control Sequence—A sequence of nucleotides that controls and regulates expression of structural genes when operatively linked to those genes. They include the lac system, major operator and promoter regions of phage λ, the control region of fd coat protein and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells and their viruses.
Referring now to FIG. 1, we have shown therein a schematic outline of one embodiment of a process for preparing a mixture of recombinant DNA molecules, some of which include inserted DNA sequences that characterize this invention.
Preparation of Poly(a)RNA Containing Human Fibroblast Interferon mRNA (IFN-β mRNA)
The RNA used in this invention was extracted from human VGS cells, a diploid fibroblast cell line which can be propagated in monolayer cultures at 37° C. IFN-β is produced in these cells on induction with poly(I,C) in the presence of cycloheximide.
For a typical RNA isolation, each of 20 roller bottles of diploid VGS cells in confluent monolayer was “primed” overnight with 100 units/ml IFN-β and the cultures induced for 1 h with 100 μg/ml poly(I,C) and 50 μg/ml cycloheximide, incubated with cycloheximide (50 μg/ml) for 4 h, harvested by scraping into phosphate-buffered saline and spun down. The cells were lysed for 15 min at 0° C. to remove the intact nuclei containing the DNA and to isolate the cytoplasmic RNA by suspending them in hypotonic buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl and 1.5 mM MgCl2) and adding NP40 to 1%. Nuclei were removed by pelleting in a Sorvall SS-34 rotor for 5 min at 3000 rpm. Sodium dodecyl sulphate (“SDS”) and EDTA were added to the supernatant to 1% and 10 mM, respectively, and the mixture extracted 5 times with 2× vol of 1:1 redistilled phenol and chloroform-isoamyl alcohol (25:1), the aqueous phases containing the RNA being separated by centrifugation in a Sorvall SS-34 rotor at 8000 rpm for 10 min after each extraction. The RNA was precipitated from the aqueous phase by addition of 1/10 vol of 2 M sodium acetate (pH 5.1) and 2.5 vol ethanol. Usually, 60 to 90 μg of total cytoplasmic RNA were obtained per roller bottle.
Other procedures to extract the cytoplasmic RNA have also been used. For example; the cells were totally lysed after homogenization in 0.2 M Tris-HCl (pH 9.0), 50 mM NaCl, 20 mM EDTA and 0.5% SDS and extracted with phenol-chloroform as above (F. H. Reynolds, et al., “Interferon Activity Produced By Translation Of Human Interferon Messenger RNA In Cell-Free Ribosomal Systems And In Xenopus Oocytes”, Proc. Natl. Acad. Sci. USA, 72, pp. 4881-87 (1975)) or the washed cells were suspended in 400 μl 0.1 M NaCl, 0.01 M Tris-HCl (pH 7.5), and 0.001 M EDTA (“NTE buffer”) and 2.5 ml 4 M guanidinium-isothiocyanate and 1 M β-mercaptoethanol in 20 mM sodium acetate (pH 5.0) were added and the cells homogenized. The lysate was layered on a 1.3-ml 5.7 M CsCl cushion in a Beckman SW-60 Ti nitrocellulose tube, spun for 17 h at 39000 rpm to pellet the RNA and separate it from DNA, proteins and lipids and the RNA extracted once with phenol-chloroform (J. Morser, et al., “Characterization Of Interferon Messenger RNA From Human Lymphoblastoid Cells”, J. Gen. Virol., 44, pp. 231-34 (1979)).
The total RNA was assayed for the presence of IFN-β mRNA by injection into the cytoplasm of Xenopus laevis oocytes and determination of the IFN-β activity induced therein (Reynold, et al., supra). The assay was conducted by dissolving the RNA in water and injecting about 50 nl into each oocyte. The oocytes were incubated overnight at room temperature in Barth medium (J. Gurdon, J. Embryol. Exper. Morphol., 20, pp. 401-14 (1968)), homogenized in part of the medium, the debris removed by centrifugation, and the IFN-β activity of the supernatant determined. Detection of IFN-β activity was by reduction of virus-induced cytopathic effect (W. E. Stewart and S. E. Sulkin, “Interferon Production In Hampsters Experimentally Infected With Rabies Virus”, Proc. Soc. Exp. Biol. Med., 123, pp. 650-53 (1966)). The challenge virus was vesicular stomatitis virus (Indiana strain) and the cells were human diploid fibroblasts trisomic for chromosome 21 to afford higher IFN-β sensitivity. IFN-β activity is expressed relative to the IFN reference standard 69/19.
Poly(A) RNA containing IFN-β mRNA was isolated from the cytoplasmic RNA by adsorption to oligo(dT)-cellulose (type 7; P-L Biochemicals) in 0.4 M NaCl, 10 mM Tris-HCl (pH 7.8), 10 mM EDTA and 0.2% SDS for 10 min at room temperature. RNA aggregation was minimized by heating the RNA for 2 min at 70° C. prior to adsorption. After washing the cellulose with the above-mentioned buffer, the poly(A) RNA fraction was eluted with 10 mM Tris-HCl (pH 7.8), 1 mM EDTA and 0.2% SDS. It usually comprised 4-5% of the total RNA, as measured by optical density at 260 nm.
A further purification to enrich the poly(A)RNA in IFN-β mRNA was effected by formamide-sucrose gradients (T. Pawson, et al., “The Size of Rous Sarcoma Virus mRNAs Active In Cell-Free Translation”, Nature, 268, pp. 416-20 (1977)). These gradients gave much higher resolution than non-denaturing sucrose gradients. Usually about 80 μg poly(A) RNA was dissolved in 50% formamide, 100 mM LiCl, 5 mM EDTA, 0.2% SDS and 10 mM Tris-HCl (pH 7.4), heated at 37° C. for 2 min to prevent aggregation and loaded on a 5-20% sucrose gradient in a Beckman SW-60 Ti polyallomer tube. After centrifugation at 20° C. for 4½ h at 60000 rpm in the Beckman SW-60 Ti rotor with total 14C-labeled eukaryotic RNA serving as size markers, the gradient was fractionated and the optical density of the fractions determined. All RNA fractions were precipitated twice with 0.5 M NaCl and 2.5 vol ethanol and assayed for interferon mRNA activity as described above. These purification processes result in about a 40-fold enrichment in the IFN-β mRNA content of the poly(a) RNA.
Although the RNA from VGS cells appeared to contain only one IFN-β-related mRNA fraction, RNA from other cell lines appears to contain at least another, and perhaps more, IFN-β-related mRNA fractions. This latter mRNA does not hybridize to the former mRNA but does code for a protein that displays IFN-β activity and is inactivated by antisera to authentic IFN-β. The cloning and expression of such mRNA and other mRNA's which are related to it by hybridization are also part of this invention because the processes hereinafter described are applicable thereto.
Alternatively, the oligo(dT)-adsorbed mRNA (60 μg) was fractionated by electrophoresis in a 4% polyacrylamide gel in 7 M urea, 0.1% SDS, 50 mM Tris-borate (pH 8.3), and 1 mM EDTA, the mRNA being dissolved in this buffer and heated 1 min at 55° C. before application to the gel. After electrophoresis, sections of 2 mm width were cut from the gel and the RNA eluted from each homogenized gel section, further freed from impurities by adsorption to oligo(dT)-cellulose and assayed for IFN-β mRNA as before.
At this point it should be recognized that even the poly(A) RNA product obtained from the formamide-sucrose gradients and the polyacrylamide gel fractionation contains a very large number of different mRNA's. Except for the mRNA specific for IFN-β, the other mRNAs are undesirable contaminants (FIG. 1). Unfortunately, these contaminant RNAs behave similarly to HuIFN-β mRNA throughout the remainder of the cloning process of this invention. Therefore, their presence in the poly(A) RNA will result in the ultimate preparation of a large number of unwanted bacterial clones which contain genes that may code for polypeptides other than IFN-β. This contamination presents complex screening problems in the isolation of the desired IFN-β hybrid clones. In the case of IFN-β, the screening problem is further exacerbated by the lack of a sufficiently purified sample of HuIFN-β mRNA or DNA or portion thereof to act as a screening probe for the identification of the desired clones. Therefore, the screening process for the IFN-β clones is very time-consuming and difficult. Further, because only a very small percentage of IFN-β clones themselves are expected to express IFN-β in a biologically or immunologically active form, the isolation of an active clone is a “needle in a haystack” screening process.
Advantageously, we may use recombinant DNA technology to provide a purified sample of HuIFN-β mRNA or cDNA or a portion thereof. This purified mRNA or cDNA can then be used to screen rapidly very large numbers of bacterial clones and thereby markedly increase the probability of isolating a clone which expresses IFN-β in an active form.
Synthesis of Double-Stranded cDNA Containing IFN-β cDNA
Poly(A) RNA enriched in IFN-β mRNA was used as a template to prepare complementary DNA (“cDNA”), essentially as described by R. Devos, et al., “Construction And Characterization Of A Plasmid Containing A Nearly Full-Size DNA Copy Of Bacteriophage MS2 RNA”, J. Mol. Biol., 128, pp. 595-619 (1979) for the construction of a plasmid containing a DNA copy of bacteriophage MS2 RNA.
Single-stranded cDNA was prepared from the poly(A) RNA by RNA-dependent DNA polymerase (25 units) from avian myeloblastosis virus (“AMV”) reverse transcriptase (a gift from Dr. J. Beard, Life Sciences, Gulfport, Fla.), initiated by a (dT)10 primer (6 μg, Miles) hybridized to the poly(A) tail of the RNA, in 50 μl 50 mM Tris-HCl (pH 8.3), 10 mM MgCl2, 30 mM β-mercaptoethanol, 4 mM Na4P2O7, 2.5 μg/μl inactivated bovine serum albumin, dTTP, dATP, dCTP and dGTP, each at 0.5 mM and α-32P-DATP (20 μCi, Amersham). After 30 min at 41° C., the reaction was terminated by the addition of EDTA to 10 mM, the reaction mixture extracted with equal vol of phenol:chloroform:isoamyl alcohol (25:24:1) and the aqueous phase layered on a Sephadex G50 column and eluted in TE buffer (10 mM Tris-HCl (pH 7.5) 1 mM EDTA). The void fractions displaying radioactivity were precipitated by the addition of 10 μg E. coli transfer RNA, potassium acetate (pH 5.1) to 0.2 M and 2.5 vol ethanol.
The cDNA population synthesized above is in fact a complex mixture of cDNAs originating from the different mRNAs which were present in the enriched poly(A) mRNA (FIG. 1). In addition, because of premature termination by AMV reverse transcriptase, many of the cDNAs are incomplete copies of the various mRNAs in the poly(A) RNA (not shown in FIG. 1).
Before rendering the cDNA double-stranded, it is removed from its association to the complementary template RNA by precipitation with ethanol and incubation in TE buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA) with ribonuclease T1 (10 units, Sankyo Co., Ltd) and pancreatic ribonuclease A (10 μg, Sigma) to 10 μl for 30 min at 37° C. (the ribonucleases being free of single-strand-specific endo- and exo-deoxyribonucleases). The removal of the template strand by ribonuclease instead of with alkali avoids possible cDNA mutation by alkali-catalyzed deamination.
The cDNA strand may be rendered double-stranded by DNA polymerase I (A. Efstratiadis, et al., “Enzymatic In Vitro Synthesis Of Globin Genes”, Cell, 7, pp. 279-88 (1976)). The 10 μl ribonuclease/cDNA mixture from above was diluted to 20 μl with MgCl2 to 10 mM, DTT to 10 mM, potassium phosphate (pH 6.9) to 100 mM, dATP, dCTP, dTTP, and dGTP each to 0.3 mM, α-32P-DATP (20 μCi, Amersham) and DNA polymerase I (40 units, Biolabs). After 6 h at 15° C., EDTA to 10 mM and SDS to 0.1% were added and the double-stranded cDNA isolated by extraction (phenol:chloroform:isoamyl alcohol), chromatography (Sephadex G50) and precipitation of void fractions as before.
To open the single-stranded hairpin loop which remains on the double stranded cDNA structure, the precipitated cDNA was dissolved in 100 μl 0.2 M NaCl, 50 mM sodium acetate (pH 4.5), 10 mM zinc acetate and 2 μg heat-denatured calf thymus DNA and reacted with S1 nuclease (5 units, Sigma) for 30 min at 37° C. Addition of EDTA to 10 mM, extraction with phenol:chloroform:isoamyl alcohol and precipitation of the aqueous phase by the addition of 10 μg E. coli transfer RNA as carrier, 0.2 M sodium acetate (pH 5.1) and 2.5 vol ethanol yielded a blunt-ended double stranded cDNA mixture. This mixture is heterogeneous both as a consequence of the heterogeneity of the poly(A) RNA used as a template to prepare it (FIG. 1) and of the premature termination of the cDNA transcripts by the AMV reverse transcriptase (not shown in FIG. 1).
To lessen the effect of the latter heterogeneity, the double stranded cDNA was sized by electrophoresis on a 4% polyacrylamide gel in 50 mM Tris-borate buffer (pH 8.3) and 1 mM EDTA, 5′-32P-labelled restriction fragments (φX174 (RF)-DNA) serving as size markers. DNA bands of appropriate size (e.g., size classes 800-900 bp, 700-800 bp, 650-700 bp and 550-650 bp) were selected. Because the double-stranded cDNA prepared from the polyacrylamide gel electrophoresed poly(A) RNA displayed a prominent band about 850 bp, this band was considered to represent the full-length DNA. The bands were eluted by crushing the gel in 0.5 M ammonium acetate and 0.1% SDS and stirring overnight. After the debris had been removed by centrifugation, the DNA was adsorbed to hydroxylapatite powder, loaded on a Sephadex G50 column in 5 mM sodium phosphate (pH 7.5), washed extensively with buffer, eluted with 0.45 M sodium phosphate (pH 7.5) and immediately desalted by the sieving effect of the Sephadex G50 matrix. The fractions containing the eluted DNA, as monitored by the 32P-radioactivity, were precipitated by the addition of 10 μg E. coli transfer RNA, sodium acetate to 0.2 M and 2.5 vol ethanol.
The efficiency of the cDNA preparation, described above, is exemplified by a typical experiment where about 2 μg poly(A) RNA after formamide-sucrose gradient yielded about 16 ng double-stranded cDNA having a size range of 800 to 900 bp.
Again, it must be recognized that this double-stranded cDNA is a mixture of a large number of cDNAs, only a very few of which are IFN-β cDNA (FIG. 1).
Cloning of Double-Stranded DNA
A wide variety of host/cloning vehicle combinations may be employed in cloning the double-stranded cDNA prepared in accordance with this invention. For example, useful cloning vehicles may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as various known derivatives of SV40 and known bacterial plasmids, e.g., plasmids from E. coli including col E1, pCR1, pBR322, pMB9 and their derivatives, wider host range plasmids, e.g., RP4, phage DNAs, e.g., the numerous derivatives of phage λ, e.g., NM 989, and other DNA phages, e.g., M13 and Filamenteous single stranded DNA phages and vectors derived from combinations of plasmids and phage DNAs such as plasmids which have been modified to employ phage DNA or other expression control sequences or yeast plasmids such as the 2μ plasmid or derivatives thereof. Useful hosts may include bacterial hosts such as E. coli HB101, E. coli X1776, E. coli X2282, E. coli MRCI and strains of Pseudomonas, Bacillus subtilis, Bacillus stearothermophilus and other bacilli, yeasts and other fungi, animal or plant hosts such as animal (including human) or plant cells in culture or other hosts. Of course, not all host/vector combinations may be equally efficient. The particular selection of host/cloning vehicle combination may be made by those of skill in the art after due consideration of the principles set forth without departing from the scope of this invention.
Furthermore, within each specific cloning vehicle, various sites may be selected for insertion of the double-stranded DNA. These sites are usually designated by the restriction endonuclease which cuts them. For example, in pBR322 the PstI site is located in the gene for β-lactamase, between the nucleotide triplets that code for amino acids 181 and 182 of that protein. This site was initially employed by S. Nagata et al., supra, in their synthesis of polypeptides displaying an immunological or biological activity of IFN-α. One of the two HindIII endonuclease recognition sites is between the triplets coding for amino acids 101 and 102 and one of the several Taq sites at the triplet coding for amino acid 45 of β-lactamase in pBR322. In similar fashion, the EcoRI site and the PvuII site in this plasmid lie outside of any coding region, the EcoRI site being located between the genes coding for resistance to tetracycline and ampicillin, respectively. This site was employed by T. Taniguchi et al., supra, in their recombinant synthetic scheme. These sites are well recognized by those of skill in the art. It is, of course, to be understood that a cloning vehicle useful in this invention need not have a restriction endonuclease site for insertion of the chosen DNA fragment. Instead, the vehicle could be joined to the fragment by alternative means.
The vector or cloning vehicle and in particular the site chosen therein for attachment of a selected DNA fragment to form a recombinant DNA molecule is determined by a variety of factors, e.g., number of sites susceptible to a particular restriction enzyme, size of the protein to be expressed, susceptibility of the desired protein to proteolytic degradation by host cell enzymes, contamination or binding of the protein to be expressed by host cell proteins difficult to remove during purification, expression characteristics, such as the location of start and stop codons relative to the vector sequences, and other factors recognized by those of skill in the art. The choice of a vector and an insertion site for a particular gene is determined by a balance of these factors, not all selections being equally effective for a given case.
Although several methods are known in the art for inserting foreign DNA into a cloning vehicle or vector to form a recombinant DNA molecule, the method preferred for initial cloning in accordance with this invention is digesting the plasmid (in particular pBR322) with that restriction enzyme specific to the site chosen for the insertion (in particular PstI) and adding dA tails to the 3′ termini by terminal transferase. In similar fashion, the double-stranded cDNA is elongated by the addition of dT tails to the 3′ termini to allow joining to the tailed plasmid. The tailed plasmid and cDNA are then annealed to insert the cDNA in the appropriate site of the plasmid and to circularize the hybrid DNA, the complementary character of the tails permitting their cohesion (FIG. 1). The resulting recombinant DNA, molecule now carries an inserted gene at the chosen PstI restriction site (FIG. 1). This method of dA-dT tailing for insertion is described by D. A. Jackson, et al., “Biochemical Methods For Inserting New Genetic Information Into DNA Of Simian Virus 40: Circular SV40 DNA Molecules Containing Lambda Phage Genes And The Galactose Operon Of Escherichia coli”, Proc. Natl. Acad. Sci. USA, 69, pp. 2904-909 (1972) and R. Devos, et al., supra. It results in about 3 times as many recombinant DNA plasmids as dG-dC tailing.
Of course, other known methods of inserting DNA sequences into cloning vehicles to form recombinant DNA molecules are equally useful in this invention. These include, for example, dG-dC tailing, direct ligation, synthetic linkers, exonuclease and polymerase-linked repair reactions followed by ligation, or extension of the DNA strand with DNA polymerase and an appropriate single-stranded template followed by ligation.
It should, of course, be understood that the nucleotide sequences or cDNA fragments inserted at the selected site of the cloning vehicle may include nucleotides which are not part of the actual structural gene for the desired polypeptide or may include only a fragment of the complete structural gene for the desired protein. It is only required that whatever DNA sequence is inserted, a transformed host will produce a polypeptide having a biological or immunological activity of HuIFN-β or that the DNA sequence itself is of use as a hybridization probe to select clones which contain DNA sequences useful in the production of polypeptides having an immunological or biological activity of HuIFN-β.
The cloning vehicle or vector containing the foreign gene is employed to transform a host so as to permit that host to express polypeptides displaying an immunological or biological activity of HuIFN-β for which the hybrid gene codes. The selection of an appropriate host is also controlled by a number of factors recognized by the art. These include, for example, compatibility with the chosen vector, toxicity of proteins encoded by the hybrid plasmid, ease of recovery of the desired protein, expression characteristics, bio-safety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for expression of a particular recombinant DNA molecule.
In the present synthesis, the preferred initial cloning vehicle is the bacterial plasmid pBR322 and the preferred initial restriction endonuclease site therein is the PstI site (FIG. 1). The plasmid is a small (molecular weight approx. 2.6 megadaltons) plasmid carrying resistance genes to the antibiotics ampicillin (Amp) and tetracycline (Tet). The plasmid has been fully characterized (F. Bolivar, et al., “Construction And Characterization Of New Cloning Vehicles II. A Multi-Purpose Cloning System”, Gene, pp. 95-113 (1977); J. G. Sutcliffe, “pBR322 Restriction Map Derived From The DNA Sequence: Accurate DNA Size Markers Up To 4361 Nucleotide Pairs Long”, Nucleic Acids Research, 5, pp. 2721-28 (1978); J. G. Sutcliffe, “Complete Nucleotide Sequence Of The Escherichia coli Plasmid pBR322”, Cold Spring Harbor Symposium, 43, I, pp. 77-90 (1978)). Insertion of the DNA product in this site provides a large number of bacterial clones each of which contains one of the DNA genes or fragments thereof present in the cDNA product previously prepared. Again, only a very few of these clones will contain the gene for IFN-β or fragments thereof (FIG. 1) and none of them may permit the expression of polypeptides displaying an immunological or biological activity of IFN-β. The preferred initial host in accordance with this invention is E. coli HB101.
1. Preparation of PstI-Cleaved, da-Elongated pBR322
Plasmid pBR322 was digested completely at 37° C. with PstI endonuclease (New England Biolabs) in 10 mM Tris-HCl (pH 7.6), 7 mM MgCl2, 7 mM 2-mercaptoethanol. The mixture was extracted with 1 vol phenol and 10 vol ether and precipitated with 2.5 vol ethanol:0.2 M sodium acetate solution.
Addition of homopolymeric dA tails (FIG. 1) by terminal deoxynucleotidyl transferase (TdT) (purified according to L. Chang and F. J. Bollum, “Deoxynucleotide-Polymerizing Enzymes Of Calf Thymus Gland”, J. Biol. Chem., 246, pp. 909-16 (1971)) was done in a 50-μl reaction volume containing 0.14 M potassium cacodylate, 30 mM Tris-HCl (pH 6.8), 1 mM COSO4, 0.2 μg/μl heat-inactivated bovine serum albumin, 0.8 mM DTT, 0.2 mM DATP and some α-32 P-DATP. Incubation was at 37° C. for 5 min before EDTA was added to 10 mM and SDS to 0.1% and the mixture extracted with phenol and chromatographed on Sephadex G50 in TE buffer. The void fractions, containing the linearized and elongated pBR322, were further purified by adsorption in 10 mM Tris-HCl (pH 7.8), 1 mM EDTA and 0.4 M NaCl to oligo(dT) cellulose. After extensive washing, the desired fractions were eluted with 10 mM Tris-HCl (pH 7.8) and 1 mM EDTA.
2. Preparation of dT-Elongated DNA
Double-stranded DNA was elongated with dTMP residues in similar fashion to that described above for dA tailing of pBR322, except that dTTP and some 3H-dTTP replaced the dATP and α-32P-ATP. Purification on oligo(dT) cellulose was, of course, omitted. As before, the dT-elongated DNA is a mixture of different species, only a very few of which are HuIFN-β-related (FIG. 1).
3. Preparation of Ca++-Treated E. coli HB101
Ca++-treated E. coli HB101 was prepared by the method of E. M. Lederberg and S. N. Cohen, “Transformation Of Salmonella Typhimurium By Plasmid Deoxyribonucleic Acid”, J. Bacteriol., 119, pp. 1072-74 (1974) by inoculating the E. coli HB101 (a gift from H. Boyer) into 5 ml LB medium (10 parts bactotryptone, 5 parts yeast extract and 5 parts NaCl per liter) and cultures grown overnight at 37° C. The fresh cultures were diluted 1/100 in 20 ml LB medium and grown to a density of about 2×108 bacteria per ml, quickly chilled in ice and pelleted at 6000 rpm for 5 min in a Sorvall SS34 rotor at 4° C. The cells, kept at 0-4° C., were washed with 20 ml 100 mM CaCl2. After 20 min in ice, the cells were repelleted and resuspended in 2 ml 100 mM CaCl2 and maintained at 0° C. for 15 min. Aliquots (200 μl), supplemented with glycerol to 11%, could be stored for several months at −80° C. without loss of activity (D. A. Morrison, “Transformation In Escherichia coli: Cryogenic Preservation Of Competent Cells”, J. Bacteriol., 132, pp. 349-51 (1977)).
4. Annealing of dA-Elongated pBR322 and dT-Elongated DNA
The vector's and DNA insert's complementary dA- and dT-tails permit annealing to form the initially desired hybrid plasmid or recombinant DNA molecule. For this purpose, the da-tailed PstI-cleaved pBR322 vector and the mixture of sized dT-tailed cDNAs were dissolved in TSE buffer (10 mM Tris-HCl (pH 7.6), 1 mM EDTA, 100 mM NaCl) to 1.5 μg/ml plasmid and to a molar ratio of plasmid to DNA insert of 1.5 to 2.0. After heating to 65° C. for 10 min, the mixture was cooled slowly to room temperature over 4 h.
The product is, of course, a large mixture of different recombinant DNA molecules and some cloning vehicles without inserted DNA sequences. However, each recombinant DNA molecule contains a cDNA segment at the PstI site. Each such cDNA segment may comprise a gene or a fragment thereof. Only a very few of the cDNA fragments code for HuIFN-β or a portion thereof (FIG. 1). The vast majority code for one of the other proteins or portions thereof whose mRNAs were part of the poly(A) RNA used in the process of this invention (FIG. 1). It should also be understood that none of the clones of the above-prepared library may permit the expression of polypeptides displaying an immunological or biological activity of IFN-β.
5. Transfection of E. coli HB101 with the Annealed Hybrid Plasmids
P3 containment facilities were used as necessary for the transfection process and all subsequent steps in which the resulting transformed bacteria were handled. Aliquots (90 μl or less) of the above mixture were cooled to 0° C. and 1 M CaCl2 added to 0.1 M. Aliquots (100 μl or less) of this solution were added to 200 μl CA++-treated E. coli HB101 in ice and after standing at 0° C. for 30 min, the cells were heat-shocked for 5 min at 37° C. and cooled again at 0° C. for 15 min. After addition of 2 ml LB-medium, the cells were incubated at 37° C. in a shaking water bath for 30 to 45 min and the bacterial suspension plated out onto 1.2% agar plates, containing LB medium supplemented with 10 μg/ml tetracycline.
Since plasmid pBR322 includes the gene for tetracycline resistance, E. coli hosts which have been transformed with a plasmid having that gene intact will grow in cultures containing that antibiotic to the exclusion of those bacteria not so transformed. Therefore, growth in tetracycline-containing culture permits selection of hosts transformed with a recombinant DNA molecule or recyclized vector.
After 24 h at 37° C., individual colonies were picked and suspended in 100 μl LB medium (supplemented as above) in the wells of microtiter plates (Dynatech). After incubation at 37° C. overnight, 11 μl dimethylsulfoxide were mixed into each well and the trays sealed with adhesive tape. The plates were stored at −20° C. and a library of 17,000 individual clones of transformed E. coli HB101 was prepared. This library was derived from 270 fmoles (128 ng) dT-tailed cDNA inserts, which in turn were synthesized from 4.4 μg gradient purified poly(A) RNA. About 98% of the clones of this library were sensitive to carbenicillin (a more stable ampicillin derivative). Therefore, about 98% of the library contained a plasmid having an insert in the PstI-site of the β-lactamase gene of pBR322 and only about 2% contained a recircularized vector without insert.
These 17,000 clones contain a variety of recombinant DNA molecules representing complete or partial copies of the mixture of mRNAs in the poly(A) RNA preparation from HuIFN-β-producing cells (FIG. 2). The majority of these will contain only a single recombinant DNA molecule. Only a very few of these recombinant DNA molecules are related to HuIFN-β. Accordingly, the clones must be screened to separate the HuIFN-β-related clones from the others.
Screening for a Clone Containing HuIFN-β cDNA
There are several approaches to screen for bacterial clones containing HuIFN-PcDNA. These include, for example, RNA selection hybridization (Alwine, et al., infra), differential hybridization (T. P. St. John and R. W. Davis, “Isolation Of Galactose-Inducible DNA Sequences From Saccharomyces Cerevisiae By Differential Plaque Filter Hybridization”, Cell, 16, pp. 443-452 (1979)); hybridization with a synthetic probe (B. Noyes, et al., “Detection And Partial Sequence Analysis Of Gastrin mRNA By Using An Oligodeoxynucleotide Probe”, Proc. Natl. Acad. Sci. USA, 76, pp. 1770-74 (1979)) or screening for clones that produce the desired protein by immunological (L. VIIIa-Komaroff, et al., “A Bacterial Clone Synthesizing Proinsulin”, Proc. Natl. Acad. Sci. USA, 75, pp. 3727-31 (1978)) or biological (A. C. Y. Chang, et al., “Phenotypic Expression In E. coli Of A DNA Sequence Coding For Mouse Dihydrofolate Reductase”, Nature, 275, pp. 617-24 (1978)) assays. We have chosen RNA selection hybridization as being the most convenient and promising method for primary screening.
A. RNA Selection Hybridization Assay
1. Overview of the Initial Assay
Referring now to FIG. 2, the recombinant DNA molecules were isolated from individual cultures of about 46 clones sensitive to carbenicillin and resistant to tetracycline from the above library of clones (two mixtures of 2 clones shown in FIG. 2) (Step A). The recombinant DNA molecules were cleaved and hybridized to total RNA containing HuIFN-β mRNA prepared as before (Step B). All recombinant DNA molecule-total RNA hybrids were separated from the non-hybridized total RNA (Step C). The hybridized total RNA was recovered from the hybrids and purified (Step D). The recovered RNA was assayed for HuIFN-β mRNA activity as above (Step E). If, and only if, the mixture of recombinant DNA molecules contains a recombinant DNA molecule having an inserted nucleotide sequence capable of hybridizing to the HuIFN-β mRNA in the total RNA, under stringent hybridization conditions, will the mRNA released from that hybrid cause the formation of HuIFN-β in oocytes, because mRNA released from any other recombinant DNA molecule-total RNA hybrid will not be IFN-β-related. If a group of 46 clones gave a positive response, the clones were regrouped into 6 subgroups (4 subgroups of 8 and 2 subgroups of 7) and each subgroup assayed as before. This process was continued until a single clone responding to this assay was identified.
There is no assurance that the recombinant DNA molecules and bacterial cultures transformed therewith, which are thus identified, contain the complete IFN-β cDNA sequence or even that the DNA sequence actually codes for IFN-β or will permit the clone to express polypeptides displaying an immunological or biological activity of IFN-β. However, the recombinant DNA molecules will certainly contain extensive nucleotide sequences complementary to the IFN-β mRNA coding sequence. Therefore, the recombinant DNA molecule may at least be used as a source of a probe to screen rapidly other recombinant DNA molecules and clones transformed with them to identify further sets of clones which may contain an authentic or complete IFN-β nucleotide coding sequence. These clones may then be analyzed for possible expression of polypeptides displaying a biological or immunological activity of IFN-β. And, the nucleotide sequence of the inserted DNA fragment of these hybrid plasmids and its amino acid translation product may be determined and correlated, if possible, to the amino acid composition and initial sequence reported for authentic IFN-β (supra).
2. Execution of the Initial Assay
Step A—Preparation of The Recombinant DNA Molecule Mixture
Replicas of a microtiter plate containing 96 clones from the above library of clones were made on LB-agar plates, one containing 10 μg/ml tetracycline and the other supplemented with 100 μg/ml carbenicillin. In this manner, two sets of about 45-46 clones, resistant to tetracycline and sensitive to carbenicillin, were picked and grown separately overnight at 37° C. in 100 ml LB medium, containing 10 μg/ml tetracycline. These cultures were pooled, spun down in a Sorvall GS-3 rotor at 8000 rpm for 10 min, washed twice with TES buffer (50 mM Tris-HCl (pH 8), 5 mM EDTA, 5 mM NaCl) and resuspended in 40 ml TES per 1 of initial culture volume. The cells were lysed with lysozyme-Triton X-100 (M. Kahn, et al., “Plasmid Cloning Vehicles Derived From Plasmids Col El, F, R6K And RK2” in Methods In Enzymology, 68: Recombinant DNA (R. Wu, ed.) (1980) (in press)). Forty ml of the TES suspended cells were combined with 20 ml 10% sucrose in 50 mM Tris-HCl (pH 8) and lysozyme to 1.3 mg/ml and allowed to stand at room temperature for 20 min. To this suspension were added 1 ml 0.5 M EDTA-NaOH (pH 8), 8 ml 0.2% Triton X-100, 25 mM EDTA, 50 mM Tris-HCl (pH 8) and the lysis completed at room temperature for 30 min. Cellular debris and most of the chromosomal DNA were removed by pelleting in a Beckman SW27 rotor at 24000 rpm for 45 min. The supernatant was cooled in ice, combined with 1/3 vol 40% polyethylene glycol 6000-2 M NaCl and allowed to stand overnight at 0° C. The resulting precipitate was collected in a Sorvall HB4 rotor at 5000 rpm for 10 min at 4° C. and dissolved in TES buffer. The solution, with 0.2 vol 10 mg/ml ethidium bromide (Serva) and CsCl to 1 g/ml, was centrifuged in a Beckmann R60 Ti-rotor at 40000 rpm for at least 48 h, one polyallomer tube usually being sufficient for the lysate from 1-2 l of original culture volume. Two DNA bands could be visualized in the tube under UV-illumination. The band of highest density corresponds to plasmid form I DNA, the second band corresponds to form-II and form III plasmid DNAs and some chromosomal DNA. The first band was collected from the tube, ethidium bromide removed by six isoamyl alcohol extractions, and the aqueous phase diluted with 3 vol water-supplemented with up to 0.2 M sodium acetate (pH 5.1) before DNA precipitation with 2.5 vol ethanol. The DNA was redissolved, extracted with phenol and again precipitated with ethanol. The quality of the DNA was monitored by electrophoresis on a 1% agarose gel in 40 mM Tris-HOAc (pH 7.8), 20 mM sodium acetate, 2 mM EDTA, followed by ethidium bromide staining. If the DNA was contaminated with too much RNA, it was further purified by neutral sucrose-gradient centrifugation: 300 μg DNA in 10 mM Tris-HCl (pH 7.6) and 1 mM EDTA were loaded on a 36-ml 5-20% sucrose gradient in 10 mM Tris-HCl (pH 7.6), 1 mM EDTA, 1 M NaCl, centrifuged in polyallomer tubes for 16 h at 24000 rpm in a Beckmann SW27 rotor at 18° C. and the DNA containing fractions (OD260) pooled and precipitated with sodium acetate-ethanol.
Step B—Hybridization of the DNA with Total RNA
About 150 μg DNA, thus prepared, were combined with some uniformly labelled 32P-marker DNA and 2 μg pSTNV-1 DNA (a recombinant plasmid containing a full, size cDNA copy of satellite tobacco necrosis virus (“STNV”)-RNA; J. Van Emmelo, et al., “Construction And Characterization Of A Plasmid Containing A Nearly Full-Size DNA Copy Of Satellite Tobacco Necrosis Virus RNA”, J. Mol. Biol., (in press) as internal control, sheared by sonication in an MSE sonicator and precipitated with sodium acetate-ethanol.
A diazobenzyloxymethyl (DBM)-cellulose solid matrix (Cf., J. C. Alwine, et al., “Method For Detection Of Specific RNAs In Agarose Gels By Transfer To Diazobenzyl Oxymethyl Paper And Hybridizing With DNA Probes”, Proc. Natl. Acad. Sci. USA, 74, pp. 5350-54 (1977)) was prepared according to the method of J. C. Alwine, et al., “Detection Of Specific RNAs Or Specific Fragments Of DNA Fractionation In Gels And Transfer To Diazobenzyloxymethyl Paper”, Methods-Enzymology, 68:Recombinant DNA-(R. Wu, ed.) (1980). For a paper matrix, a sheet of Whatman 540 paper was evenly soaked in a solution containing 2-3 mg 1-(m-nitrobenzyloxy)methylpyridinium chloride (NBPC/BDH and 0.7 ml sodium acetate trihydrate in 28.5 μl water per cm2, incubated at 60° C. until dry and for further 10 min, and baked at 130-135° C. for 30-40 min. After washing several times with water (about 20 min), 3 times with acetone (about 20 min), and drying it was stored. The paper was incubated at 60° C. for 30 min in 0.4 ml 20% sodium dithionite-water per cm2 with occasional shaking. The paper was again washed four times with water, once with 30% acetic acid for 5 min and four times with water, transferred to 0.3 ml per cm2 ice-cold 1.2 M HCl to which 10 mg/ml fresh NaNO2 had been added immediately before use for 30 min at 0° C., and washed twice quickly with ice-cold water and once with 80% dimethyl sulfoxide (spectrophotometric grade, Merck)-20% 25 mM sodium phosphate (pH 6.0). For a powder matrix essentially the same procedure was followed using microgranular cellulose powder (Whatman CC31), the quantities being expressed against the corresponding weight of the cellulose matrix.
Initially, we used a powder matrix because the capacity for binding was higher, so relatively smaller volumes for hybridization, washes and elution could be used. Subsequently, we used a paper matrix for individual clone screening. Use of paper permits efficient elution with water which proved superior for the later assay of IFN-βmRNA.
The DNA prepared above was dissolved in 25 mM sodium phosphate (pH 6.0) heated for 1 min, chilled and four vol DMSO added. Coupling to the matrix (50 mg (powder) or a paper disc (10 mm dia.)) usually proceeded over a weekend at 4° C. with continuous mixing. The volume of the DNA was kept rather small to allow close contact with the matrix and thereby enhance efficient coupling of the DNA to the matrix. After coupling, the matrix was washed four times with water and four times with 0.4 N NaOH at 37° C. for 10 min each, again four times with water at room temperature and finally twice with hybridization buffer (50% formamide (deionized, Baker), 40 mM piperazine-N,N′-bis(2-ethane sulfonic acid) (pH 6.4) (“PIPES, Sigma), 1 mM EDTA, 0.6 M NaCl and 0.1% SDS) at 4° C. Coupling efficiencies were measured by 32P-radioactivity.
Twenty μg total RNA, prepared as before, and 50 ng STNV-RNA were dissolved in 250 μl (50 μl for paper matrix) hybridization buffer and added to the DNA coupled matrix. The matrix was heated to 70° C. for 2 min and held at 37° C. overnight with gentle mixing.
Step C—Separation of Hybridized Total RNA-DNA from Non-Hybridized Total RNA
After centrifugation of the powder matrix, the unhybridized RNAs were removed and the matrix washed seven times with a total 2 ml 50% formamide, 10 mM PIPES (pH 6.4), 1 mM EDTA, 0.3 M NaCl and 0.1% SDS, the lower salt content of these washes destabilizing non-specific RNA-DNA binding. Each wash was followed by centrifugation and resuspension of the matrix in the buffer. For subsequent assay, the first wash was pooled with the unhybridized RNA (“Fraction 1”) and washes 2-4 (“Fraction 2”) and washes 5-7 (“Fraction 3”) were pooled. In hybridizations to a paper matrix, a similar procedure was utilized except that the total wash volume was limited 1 ml.
Step D—Purification Of Hybridized Total RNA
The hybridized total RNA-DNA was eluted from a powder matrix with 3 elutions of a total 900 μl 99% formamide, 0.2% SDS at 70° C. for 2 min and chilled in ice. The total hybridization procedure and elution with formamide were essentially as described by A. G. Smith (personal communication). The hybridized total RNA-DNA was eluted from a paper matrix by first washing with 100 μl of ice cold water and following that with two water elutions (total 300 μl) at 80° C. for 2 min. For subsequent assay these elutions and the 100 μl wash were pooled (“Fraction 4”).
To one-half of each of the 4 fractions, 0.1 μg calf liver tRNA or ribosomal RNA were added (Fractions 1A, 2A, 3A and 4A) and to the other half 8 μg eukaryotic poly(A) RNA or ribosomal RNA were added (Fractions 1B, 2B, 3B, 4B). The fractions were purified by precipitation by the addition of 0.5 M NaCl and 2.5 vol ethanol to removal traces of formamide and other impurities.
Step E—Determination of IFN-β mRNA Activity
Fractions 1A, 2A, 3A and 4A were translated in 25 μl nuclease-treated rabbit reticulocyte lysate (prepared according to the procedure of R. B. Pelham and R. J. Jackson, “An Efficient mRNA-Dependent Translation System For Reticulocyte Lysates”, Eur. J. Biochem., 7, pp. 247-56 (1976)) by the procedure of B. LeBleu, et al., “Translation Of Mouse Interferon mRNA In Xenopus Laevis Oocytes And In Rabbit Reticulocyte Lysates”, Biochem. Biophys. Res. Commun., 82, pp. 665-673 (1978) except that 250 mM spermidine-HCl, 1 mM fructose-1,6-diphosphate were added in the presence of 35S-methionine (0.5 mCi/ml, Amersham). After incubation, 25 μl reticulocyte lysate, from above, were combined with 1 μl 10% deoxycholate-10% Triton X100 and 2 μl antiserum-PBS (1:9) and heated at 37° C. for 1 h. Twenty μl Staphylococcus aureus Cowan I (freshly washed, S. W. Kessler, et al., “Rapid Isolation Of Antigens From Cells With A Staphylococcal Protein A-Antibody Adsorbent: Parameters Of The Interaction Of Antibody-Antigen Complexes With Protein A”, J. Immunology, 115, pp. 1617-1624 (1975)) in 10% 100 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.05% NP40 were added and the mixture maintained at 20° C. for 30 min and centrifuged in an Eppendorf 5412 centrifuge for 2 min. The pellet was washed and centrifuged twice with PBS and the final pellet dissolved in sample buffer and electrophoresed on a 13% polyacrylamide gel as described by U. K. Laemmli, et al., “Cleavage Of Structural Proteins During The Assembly Of The Head Of Bacteriophage T4”, Nature, 227, pp. 680-85 (1970), and autoradiographed. Comparison of the STNV-RNA translation products in Fractions 1A and 4A provide an indication of the efficiency of hybridization and RNA degradation in the process.
Fractions 1B, 2B, 3B and 4B were dissolved in 2 μl water and assayed in oocytes for IFN-β mRNA content as described above.
3. Subsequent Assay—Hybridization to Nitrocellulose Sheets
Some subsequent assays of individual clones were done on nitrocellulose sheets (M. Cochet, et al., “Cloning Of An Almost Full-Length Chicken Conalbumin Double-Stranded cDNA”, Nucleic Acids Research, 6, pp. 2435-2452 (1979)). The DNA was dissolved in 2M NaCl and 0.2 M NaOH, heated at 100° C. for 1 min, chilled, and spotted on detergent free Millipore filters (pore size 0.45 um; 7 mm dia.). The filters were baked for 2 h at 80° C., washed in 0.3 M NaCl, 2 mM EDTA, 0.1% SDS, 10 mM Tris-HCl (pH 7.5) and dried at room temperature. The RNA was hybridized for 3 h at 47° C. in 30% formamide, 0.5 M NaCl, 0.4% SDS, 2 mM EDTA, 50 mM PIPES (pH 7.5). Hybridization was stopped by dilution with 10 vol 0.1 M NaCl and the filters were washed several times in 15 ml 0.3 M NaCl, 0.1% SDS, 2 mM EDTA, 10 mM Tris-HCl (pH 7.5) by shaking at 45° C. and several times in the same solution without SDS at 4° C. Elution of the hybridized RNA-DNA was effected in 30 μl 5 mM potassium chloride at 100° C. for 1 min.
4. Results of the RNA Selection Hybridization Assay
Sixteen groups of about 46 clones were screened (Groups A-P). In six of the groups, Fraction 1B contained the only IFN-β mRNA activity, in eight of the groups no IFN-β mRNA was detected and in two groups (Groups C and O) IFN-β mRNA was observed in Fraction 4B. The group C and O assays are reported in the following format: logarithm of IFN-β units (calibrated against reference standard 69/19), detected in the assay of Fraction 1B (non-hybridized) and in the assay of Fraction 4B (hybridized). The limit of detection was 0.1.
Group
Fraction 1B
Fraction 4B
C
1.0
0
0.5
0.5
0
0.2
0
0
0
0.2
0.5
Group 0 was subdivided into 6 subgroups (Subgroups 01 to 06; four of eight clones and two of seven clones) and hybridized and assayed as before, except that a 400 ml culture per clone was used. The subgroups gave the following results, presented in the same format as above. Hybridization was carried out on DMB-cellulose powder except as otherwise indicated.
Subgroup
Fraction 1B
Fraction 4B
01
0
1.2
0
1.5
0
0.5
0
0.5
0.2
0.5
0
1.2*
02
0.7
0
03
0.7
0
0.5
0
04
0
0
*DBM cellulose paper method.
Subgroup
Fraction 1B
Fraction 4B
05
0.5
0
06
0
0
Subgroup 01 was subdivided into its individual clones (designated clones 01/1-01/8) and hybridized and assayed as before, except that a 700 ml culture per clone was used. The hybridization was again carried out on DBM-cellulose powder except as otherwise indicated
Clone
Fraction 1B
Fraction 4B
01/1
0.2
0
0.7
0
0.7
0*
1.0
0**
01/2
1.2
0
0.2
0*
0.7
0**
01/3
1.2
0
1.0
0.2*
1.2
1.0(?)*
1.2
0**
01/4
1.2
0
1.2
0
1.0
0*
1.2
0**
01/5
0.7
0
0.7
≦0.2*
1.0
0
01/6
0.7
0
1.0
≦0.2*
0.5
0**
*DBM cellulose paper method.
**Nitrocellulose sheets.
Clone
Fraction 1B
Fraction 4B
01/7
0.5
0
1.2
0*
<0.2
0.5**
01/8
0
1.7*
<0.2
1.2*
0
0.7**
0
1.0**
*DBM cellulose paper method
**Nitrocellulose sheets
Therefore, clone 01/8 contains a recombinant DNA molecule capable of hybridizing IFN-β mRNA from total RNA containing IFN-β mRNA.
Non-specific RNA-DNA binding is highly unlikely, because a comparison of Fractions 1A and 4A revealed substantially no non-specific binding of STNV DNA in these same experiments. E.g., as monitored by translation in a rabbit reticulocyte lysate in the presence of 35S-methionine, followed by gel electrophoresis, as described above. Clone 01/8 was designated E. coli HB101(G-pBR322(Pst)/HFIF1 (“G-HB101-pHFIF1”), its recombinant DNA molecule G-pBR322(Pst)HFIF1 (“pHFIF1”) and its hybrid insert “PHFIF1 fragment”. This nomenclature indicates that the clone and recombinant DNA molecule originated in Gent (“G”) and comprises plasmid pBR322 containing, at the PstI site HuIFN-β cDNA (“HFIF”), the particular molecule being the first located (“1”).
Identification of Clones Containing Recombinant DNA-Molecules Cross-Hybridizing to pHFIF1
pHFIF1, isolated above, was used to screen the library of clones, prepared previously, for bacterial clones containing recombinant DNA molecules having related hybrid DNA inserts, by colony hybridization (M. Grunstein and D. S. Hogness, “A Method For The Isolation Of Cloned DNA's That Contain A Specific Gene”, Proc. Natl. Acad. Sci. USA, 72, pp. 3961-3965 (1975)). This method allows rapid identification of related clones by hybridization of a radioactive probe made from pHFIF1 to the DNA of lysed bacterial colonies fixed in nitrocellulose filters.
The library of clones stored in microtiter plates, as described above, was replicated on similar size nitrocellulose sheets (0.45 μm pore diameter, Schleicher and Schuel or Millipore), which had been previously boiled to remove-detergent, and the sheets placed on LB-agar plates, containing tetracycline (10 μg/ml). Bacterial colonies were grown overnight at 37° C. Lysis and fixation of the bacteria on the nitrocellulose sheets took place by washing consecutively in 0.5 N NaOH (twice for 7 min), 1 M Tris-HCl (pH 7.5) (7 min), 0.5 M Tris-HCl (pH 7.5) and 1.5 M NaCl (7 min), 2×SSC (0.15 M NaCl. 0.015 M sodium citrate (pH 7.2) (7 min)). After thorough rinsing with ethanol and air drying, the sheets were baked at 80° C. for 2 h in vacuo and stored at room temperature.
A Hinf I restriction fragment specific for the pHFIF1 fragment (infra) served as the probe for colony hybridization, described infra. This fragment (˜170 base pairs) was purified by electrophoresis of the Hinf digestion products of pHFIF1 in a 6% polyacrylamide gel. After staining the DNA bands with ethidiumbromide, the specific fragment was eluted, reelectrophoresed and 32P-labelled by “nick translation” (P. W. J. Rigby et al., “Labeling Deoxyribonucleic Acid To High Specific Activity In Vitro By Nick Translation With DNA Polymerase I”, J. Mol. Biol., 113, pp. 237-251 (1977)) by incubation in 50μ 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 20 mM β-mercaptoethanol, containing 2.5 μl each of dCTP, dTTP and dGTP at 400 μM, 100 pmoles α-ATP (Amersham, 2000 Ci/mmole) and 2.5 units of DNA-polymerase I (Boehringer) at 14° C. for 45 min. The unreacted deoxynucleoside triphosphates were removed by gel filtration over Sephadex G-50 in TE buffer. The highly 32P-labelled DNA was precipitated with 0.1 vol of 2 M sodium acetate (pH 5.1) and 2.5 vol of ethanol at 20° C.
Hybridization of the above probe to the filter impregnated DNA was carried out essentially as described by D. Hanaban and M. Meselson (personal communication): The filters, prepared above, were preincubated for 2 h at 68° C. in 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin, 0.15 M NaCl, 0.03 M Tris-HCl (pH 8), 1 mM EDTA, and rinsed with 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 0.75 M NaCl, 0.15 M Tris-HCl (pH 8), 5 mM TDTA and 0.5% SDS. The hybridization proceeded overnight at 68° C. in a solution identical to the rinsing solution above using the 32P-labelled probe which had been denatured at 100° C. for 5 min prior to use. The hybridized filters were washed twice with 0.3 M NaCl, 0.06 M Tris-HCl (pH 8), 2 mM EDTA for 2 h at 68° C. before air drying and auto-radiography.
About 1350 clones, originating from the 800-900 DNA size class, were screened. Thirteen colonies, including pHFIF1, gave a positive result. These clones were designated G-HB101-pHFIF1 to 13 and their recombinant DNA molecules pHFIF1 to 13. One of the clones, pHFIF2, was hybridized with poly(A) mRNA containing IFN-β mRNA and assayed using DBM-cellulose paper (supra). Because the total IFN-RNA activity was detected in the hybridized fraction and the unhybridized RNA did not contain any detectable activity, it is clear that clones identified by colony hybridization to a part of the pHFIF1 fragment also hybridized to IFN-β mRNA.
It is, of course, evident that this method of clone screening using the HuIFN-β DNA insert of pHFIF1 or another DNA insert of a clone identified using the DNA insert of pHFIF1, as described above, may be employed equally well on other clones containing DNA sequences arising from recombinant DNA technology, synthesis, natural sources or a combination thereof or clones containing DNA sequences related to any of the above DNA sequences by mutation, including single or multiple, base substitutions, insertions, inversions, or deletions. Therefore, such DNA sequences and their identification also fall within this invention. It is also to be understood that DNA sequences, which are not screened by the above DNA sequences, yet which as a result of their arrangement of nucleotides code for those polypeptides coded for by the above DNA sequences also fall within this invention.
Characterization of the IFN-β-Related Recombinant Plasmids
The thirteen clones (pHFIF1-13) which were detected by colony hybridization were further characterized. A physical map of the inserts of these clones was constructed and the orientation of the inserts in the various clones was determined.
The physical maps of the plasmids were constructed by digestion with various restriction enzymes (New England Biolabs) in 10 mM Tris-HCl (pH 7.6), 7 mM MgCl2 and 7 mM β-mercaptoethanol at 37° C. by well-known procedures. The products of digestion were electrophoresed in 2.2% agarose or 6% polyacrylamide gels in 40 mM Tris-HOAc (pH 7.8), 20 mM EDTA. They were analyzed after visualization by staining with ethidiumbromide and compared with the detailed physical map of pBR322 (J. G. Sutcliffe, supra). Restriction maps of the different plasmids were constructed on the basis of these digestion patterns. These were refined by sequencing the DNA inserts in various of the plasmids, substantially by the procedure of A. M. Maxam and W. Gilbert, “A New Method For Sequencing DNA”, Proc. Natl. Acad. Sci. USA, 74, pp. 560-564 (1977).
Plasmid DNA was prepared from various of the pHFIF1-13 in accordance with this invention by the method of Kahn et al. (supra), employed previously herein to isolate the DNA from the sets of clones for screening. The isolated form I DNA was purified by neutral sucrose-gradient centrifugation as before and restricted by various restriction enzymes, essentially as recommended by the supplier (New England Biolabs).
Restricted DNA was dephosphorylated for 30 min at 65° C. in the presence of 4 units bacterial alkaline phosphatase and 0.1% SDS. Following two phenol extractions and ethanol precipitation, the DNA was 5′-terminally labelled with γ-32P-ATP (˜3000 Ci/mmole) and polynucleotide kinase (P-L Biochemicals, Inc.).
For sequencing, labelled fragments were handled in two ways. Some were purified on a polyacrylamide gel prior to cleavage with a second restriction enzyme. Others were immediately cleaved with a second restriction enzyme. In both cases the desired fragments were separated on a polyacrylamide gel in Tris-borate-EDTA buffer. FIG. 7 displays the various restriction fragments (the circles indicating the label and the arrow the direction of sequencing) and the sequencing strategy employed using pHFIF1, pHFIF3, pHFIF6 and pHFIF7.
The fragments were degraded according to the method of A. M. Maxam and W. Gilbert (supra). The products were fractionated on polyacrylamide gels of various concentrations and lengths in 50 mM Tris-borate, 1 mM EDTA (pH 8.3) at 900 V to 2000 V.
Each stretch of cDNA insert was sequenced from both strands and each restriction site which served as labelled terminus was sequenced using a fragment spanning it. The composite nucleotide sequence thus obtained for the coding strand of IFN-β DNA or gene and its corresponding amino acid sequence is depicted in FIG. 4. Because none of plasmids pHFIF1-13 contained the complete gene for HuIFN-β, FIG. 4 results from a combination of the data from at least two such plasmids. In this regard, FIG. 5 displays the relationship of inserts pHFIF1, pHFIF3, pHFIF6 and pHFIF7, the solid arrows or chevrons indicating the orientation of the various parts of the inserts.
Referring now to FIG. 4, the heteropolymeric part of the insert is flanked on one end by a segment rich in T's and by a string of A's (probably reflecting the polyA terminus of the mRNA). For reference the insert is numbered from first nucleotide of the composite insert to a nucleotide well into the untranslated section of the insert. An ATG initiation triplet at position 65-67 and a TGA termination triplet at position 626-628 define a reading frame uninterrupted by nonsense codons. Any other translatable sequence, i.e., in different reading frames, flanked by an ATG or a GTG and a termination signal is too short to code for a polypeptide of the expected size of IFN-β. Therefore, the region between nucleotides 65 and 625 most likely includes the nucleotide sequence for the composite DNA sequence that codes for IFN-β in accordance with this invention.
This sequence does not exclude the possibility that modifications to the gene such as mutations, including single or multiple, base substitutions, deletions, insertions, or inversions may not have already occurred in the gene or may not be employed subsequently to modify its properties or the properties of the polypeptides translated therefrom. Nor does it exclude any polymorphism which may result in physiologically similar but structurally slightly different genes or polypeptides than that reported in FIG. 4 (supra, p. 3). For example, another clone identified in accordance with this invention has a “T” instead of a “C” at nucleotide 90 of the nucleotide sequence coding for IFN-β. This change in the third nucleotide of the codon does not change the amino acid coded therefrom. The amino acid sequence coded for by the DNA sequence of FIG. 4 is identical to the amino acid sequence reported by Taniguichi et al., Gene, 10, pp. 11-15(1980)
It should of course be understood that cloned cDNA from polyA RNA by the usual procedures (A. Efstratiadis et al, supra) may lack 5′-terminal nucleotides and may even contain artifactual sequences (R. I. Richards et al., “Molecular Cloning And Sequence Analysis Of Adult Chicken β-Globin cDNA”, Nucleic Acids Research, 7, pp. 1137-46 (1979)). Therefore, it is not certain that the ATG located at nucleotides 65-67 is in fact the first ATG of authentic IFN-β coding sequence. However, for the purposes of the following description, it is assumed that the ATG at nucleotides 65-67 is the first ATG of authentic IFN-β mRNA.
By comparing the polypeptide coded by this region of the insert with that sequence of 13 amino-terminal amino acids of authentic human fibroblast interferon—MetSerTyrAsnLeuLeuGlyPheLeuGlnArgSerSer—determined by Knight et al. (supra), it appears that the chosen reading frame is correct and that nucleotides. 65-127 may code for a signal peptide which precedes the nucleotide sequence coding for the “mature” polypeptide.
In addition, in eukaryotic mRNAs the first AUG triplet from the 5′ terminus is usually the initiation site for protein synthesis (M. Kozak, “How Do Eukaryotic Ribosomes Select Initiation Regions In Messenger RNA?”, Cell, 15, pp. 1109-25 (1978)). Here, the codon in the composite fragment corresponding to the first amino acid of fibroblast interferon is 22 codons from the first ATG. This again suggests that the DNA sequence coding for fibroblast interferon may be preceded by a sequence determining a signal polypeptide of 21 amino acids. The presumptive signal sequence contains a series of hydrophobic amino acids. Such accumulation of hydrophobic residues is, of course, characteristic of signal sequences (c.f., B. D. Davis and P. C. Tai, “The Mechanism Of Protein Secretion Across Membranes”, Nature, 283, pp. 433-38 (1980)).
The nucleotide sequence apparently corresponding to “mature” HuIFN-β comprises 498 nucleotides, which code for 166 amino-acids. Assuming that there is no carboxyterminal processing, the molecular weight of the interferon polypeptide is 20085. The base composition of the coding sequence is 45% G+C. The codon usage within the interferon coding sequences is in reasonable agreement with that compiled for mammalian mRNAs in general (R. Grantham et al., “Coding Catalog Usage And The Genome Hypothesis”, Nucleic Acids Research, 8, pp. 49-62 (1980)). Any deviations observed may be ascribed to the small numbers involved.
The structure of the polypeptide depicted in FIG. 4 for the composite fragment, of course, does not take into account any modifications to the polypeptide caused by its interaction with in vivo enzymes, e.g., glycosylation. Therefore, it must be understood that the amino acid sequence depicted in FIG. 4 may not be identical with HuIFN-β produced in vivo.
The comparison of the first 13 amino acids of authentic fibroblast interferon (Knight et al., supra) and the sequence deduced from the composite gene of FIG. 4 shows no differences. The amino acid compositions determined directly for authentic fibroblast interferon on the one hand and that deduced from the sequence of the composite gene of this invention on the other also show substantial similarities. FIG. 6 displays a comparison of these compositions.
Although none of the recombinant DNA molecules initially prepared in accordance with this invention contain the complete DNA sequence for fibroblast interferon, they do provide a useful probe to screen collections of DNA sequences for those sequences which are related to HuIFN-β. Furthermore, a combination of portions of the inserts of these recombinant DNA molecules to afford the complete IFN-β coding sequence is, as is demonstrated below, within the skill of the art. For example, by reference to FIG. 8 it can readily be seen that the PstI-BglII fragment of pHFIF6 may be joined with the PstI-HaeII fragment of pHFIF7 or the EcoRI-PstI fragment of pHFIF6 may be joined with the PstI-HaeII fragment of pHFIF7 or the BqlII-PstI fragment of pHFIF6 may be joined with the PstI-BglII fragment of clone 7 to form a composite HuIFN-β coding sequence. The joining of these fragments may, of course, be done before or after insertion of the cloned fragment into a desired plasmid.
Preparation of Plasmids Containing the Complete DNA Sequence Coding for HuIFN-β for the Purpose of Expressing Polypeptides Displaying HuIFN-β Activity
Bacteriophage λ contains two strong promoters, PL and PR, whose activity is under the control of a repressor protein, the product of the phage gene cI. In the presence of repressor, transcription from these promoters is fully repressed. Removal of repressor turns on strong transcription from PL and PR (for review, see H. Szybalski and W. Szybalski “A Comprehensive Molecular Map Of Bacteriophage λ”, Gene, 7, 217-270 (1979)).
Derivatives of the multicopy plasmid pBR322 (F. Bolivar et al. “Construction And Characterization Of New Cloning Vehicles. II. A Multiple Cloning System”, Gene, 2, 95-113 (1977)) were constructed to incorporate the PL promoter. These plasmids are described in Great Britain patent application 80.28983, filed Sep. 8, 1980 and incorporated herein by reference.
A. Structure of Plasmids Containing the PL Promoter Plasmid pPLa2311
Plasmid pPLa2311 (shown in FIG. 8) consists of three HaeII fragments. The largest fragment, about 1940 base pairs, contains the PLOL region from bacteriophage λ and the β-lactamase gene region from pBR322 (J. Sutcliffe, “Complete Nucleotide Sequence Of The Escherichia coli Plasmid pBR322”, Cold Spring Harbor Symposium, 49, 77-90, (1978)). Adjacent to this fragment is a 370-base pair HaeII fragment derived from plasmid Col E1. The origin of replication spans the junction between these two fragments (A. Oka et al. “Nucleotide Sequence Of Small ColE1 Derivatives. Structure Of The Regions Essential For Autonomous Replication And Colicin E1 Immunity”, Mol. Gen. Genet., 172, 151-159 (1979)). The third HaeII fragment, about 1600 base pairs in length, codes for resistance to kanamycin. This fragment was originally derived from plasmid PCR1 (C. Covey et al. “A Method For The Detection Of Restriction Sites In Bacterial Plasmid DNA”, Mol. Gen. Genet., 145, 155-158 (1976)). The direction of transcription from the PL promoter runs in the same sense as the β-lactamase gene. Plasmid pPLa2311 confers resistance to 100 μg/ml carbenicillin and 50 μg/ml kanamycin.
Plasmid G-pPLa8
Plasmid G-pPLa8 (shown in FIG. 9) was derived from pPLa2311 by converting the PstI site in the P-lactamase gene to a BamHI site. This was accomplished by S1 nuclease treatment of PstI-opened pPLa2311 followed by blunt-end ligation to a BamHI linker fragment (obtained from Collaborative Research Inc., Waltham, Mass.) and recircularization of the molecule after BamHI cleavage. Plasmid pPLa8 no longer specifies for resistance to carbenicillin, but it still confers resistance to kanamycin.
Plasmid G-pPLc24
Plasmid G-pPLc24 (shown in FIG. 10) contains the β-lactamase gene and the origin of replication from pBR322. A 290 base pair HaeII-EcoRI fragment contains the PLOL region from bacteriophage A. The direction of transcription from the PL promoter is towards the EcoRI site. A 431 base pair EcoRI-BamHI fragment codes for the ribosome binding site and the first 98 amino acid residues of the bacteriophage MS2 replicase gene, obtained from plasmid pMS2-7 (R. Devos et al. “Construction And Characterization Of A Plasmid Containing A Nearly Full-size DNA Copy Of Bacteriophage MS2 RNA”, J. Mol. Biol., 128, 595-619 (1979)). Translation of the MS2 replicase protein fragment runs colinear with the transcription from the PL promoter.
B. Temperature-dependent Switch-On of PL Promoter Activity
Transcription from the PL promoter—present on plasmids pPLa2311, pPLa8 and pPLc24—is repressed by maintaining the plasmids in an E. coli strain that synthesizes the repressor protein. Due to its autoregulating mode of synthesis (M. Ptashne et al. “Autoregulation And Function Of A Repressor In Bacteriophage λ”, Science, 194, 156-161 (1976)), one copy of the cI gene on the chromosome of a lysogenic strain is able to repress fully the PL promoter present on a multicopy plasmid.
The strains employed in this invention were E. coli K12ΔHI (K12 M72 lacamΔtrpEA2 SmR (λcI857 Nam7Nam53ΔHI bio); U. Bernard et al. “Construction Of Plasmid Cloning Vehicles That Promote Gene Expression From The Bacteriophage λ PL Promoter”, Gene, 5, 59-76 (1979)) and E. coli M5219 (K12 M72 lacamtrpamSmR (λcI857ΔHI bio252); H. Greer, “The kil Gene Of Bacteriophage λ”, Virology, 66, 589-604 (1975)). Both strains harbor a defective, non-excisable λ prophage carrying a mutant cI gene. The mutant gene codes for a temperature-sensitive repressor, thus allowing turn on of transcription from the PL promoter by shifting the temperature—at 28° C. the repressor is active and represses transcription from the PL promoter but at 42° C. the repressor is inactivated and transcription from the PL promoter is switched on.
The ΔHI deletion of the prophage removes part of the cro gene and all other genes further to the right of cro (M. Castellazzi et al. “Isolation And Characterization Of Deletions In Bacteriophage λ Residing As Prophage I E. coli K12”, Mol. Gen. Genet., 117, 211-218 (1972)). The deletion of the cro gene is advantageous because accumulation of the cro protein is known to repress transcription from the PL promoter (A. Johnson et al. “Mechanism Of Action Of The cro Protein Of Bacteriophage λ”, Proc. Natl. Acad. Sci. U.S.A., 75, 1783-1787 (1978)). Strain M5219 in addition contains the bio252 deletion which removes all genes to the left cIII, including kil.
Upon temperature induction strain M5219 expresses a functional N-gene product. Strain K12ΔHI on the other hand has two amber mutations in N rendering it functionally N-negative. The product of the N gene is known to act as an anti-terminator in bacteriophage λ (J. W. Roberts, “Transcription Termination And Late Control In Phage A”, Proc. Natl. Acad. Sci. U.S.A., 72, 3300-3304 (1975)). The anti-termination effect was equally observed with terminator sequences not naturally present on phage λ DNA (e.g., the natural stop at the end of the trp operon), provided the RNA transcript starts at the PL promoter. Furthermore, polarity effects, introduced by the presence of a nonsense codon in the PL transcript, were relieved under the action of the N-gene protein (for review see N. Franklin and C. Yanofsky, “The N Protein Of A: Evidence bearing On Transcription Termination, Polarity And The Alteration Of E. coli RNA Polymerase” in RNA Polymerase (Cold Spring Harbor Laboratory, 1976) pp. 693-706).
Therefore, having the aforementioned plasmids in a thermo-inducible bacterial cI background allows experimental switching on or off of the activity of PL promoter. And, the choice of K12ΔH1 or M5219 allows transcription to proceed either in the absence or presence of the N-gene product. The latter could be advantageous, as described above, in instances where DNA regions are to be transcribed that contain transcription terminator-like sequences or slow-down sequences for the RNA polymerase.
C. Construction of Clones Which have a DNA Sequence Coding for HuIFN-β Inserted Into a Plasmid Containing the PL Promoter
In the following description, isolation of plasmid DNA, restriction analysis of DNA and ligation of DNA fragments were performed as described above for the cloning of double-stranded DNA. The transformation step was also as described above except that, when strains K12ΔHI or M5219 were used as the host, heat shock was done at 34° C. for 5 min and the transformed cells were incubated at 28° C.
1. Construction of Plasmid G-pPLa-HFIF-67-1
The rationale for this construction was the observation that combination of appropriate restriction fragments from clones G-pBR322(Pst)/HFIF6 and G-pBR322(Pst)/HFIF7 allows the reconstruction of a complete, continuous coding sequence of IFN-β. The flow of the derived fragments through the several construction steps is shown schematically in FIG. 8. Plasmid G-pBR322(Pst)/HFIF6 was cleaved with EcoRI and PstI and ligated to plasmid G-pBR322(Pst)/HFIF7 which had been cleaved with PstI and PvuI. Following ligation the mixture was digested with EcoRI and HaeII. A 4-fold molar excess of this mixture was then ligated to plasmid G-pPLa2311 which had been digested with HaeII and EcoRI. Transformants were obtained in strain C600rKmK+(λ) (which was used because of its relatively high transformation capability and because it contains a wild-type cI gene) by selection for kanamycin resistance. Of 15 transformants screened, two had lost resistance to carbenicillin. Restriction analysis of the DNA isolated from the plasmids of these transformants revealed that one had the desired structure of G-pPLa-HFIF-67-1 depicted in FIG. 8. This plasmid contained a unique EcoRI site and a unique PstI site. Combined EcoRI-PstI digestion produced two fragments—the smaller of which comigrated with a fragment obtained after EcoRI-PstI cleavage of G-pBR322(Pst)/HFIF6 BglII digestion cleaved out a small fragment of about 650 base pairs. The size of the latter fragment is consistent with the expected size after joining the proximal BglII-PstI fragment of clone G-pBR322(Pst)/HFIF6 to the distal PstI-BglII part of G-pBR322(Pst)/HFIF7 HincII digestion produced three fragments as expected from the presence of the HincII sites in the PL region, the amino-terminal part of the β-lactamase gene and the untranslated 5′ end of the DNA sequence of HuIFN-β. This plasmid was designated G-pPLa-HFIF-67-1.
Based on the aforementioned characterization by restriction enzyme analysis, plasmid G-pPLa-HFIF-67-1 should contain the complete coding sequence of for HuIFN-β. The direction of desired transcription runs colinearly with that from the PL promoter. In between the PL and the HiIFN-β coding sequence gene the plasmid still retains the poly(A·T) an inverted 3′ end fragment as in G-pBR322(Pst)/HFIF6.
2. Construction of Plasmid G-pPLa-HFIF-67-12
The next step in the constructions was aimed at removing from G-pPLa-HFIF-67-1 the poly(A·T) tail and part of the inverted 3′ end fragment (see FIG. 9). G-pPLa-EFIF-67-1 DNA was cleaved with BglII and HpaII. Since the HuIFN-β coding sequence contains no HpaII site this treatment results in the BqlII fragment containing the entire coding sequence for IFN-β and at the same time inactivates the remaining part of the vector. The resultant BglII fragment was ligated to plasmid G-pPLa8 which had been digested with BamHI. The enzymes BglII and BamHI make identical staggered ends such that BglII ends can be ligated to an opened BamHI site and vice versa. Such a reconstructed site is no longer a substrate for BglII or BamHI but is recognized by the enzyme Sau3AI (MboI) (V. Pirotta, “Two Restriction Endonucleases From Bacillus globigii”, Nucleic Acids Res., 3, 1747-1760 (1976)). Following ligation the mixture was again cleaved with BamHI to eliminate those G-pPLa8 molecules that had simply recircularized. Transformants were again obtained in C600rKmK+(λ) selecting for kanamycin resistance.
The transformants were screened by size determination of uncleaved DNA on agarose gel, as described above, for characterization of the IFN-β-related recombinant plasmids. Clones which proved slightly larger than the G-pPLa8 parent were further subjected to restriction analysis with either PstI or HincII. One clone was found which contained a single PstI site and three HincII sites. One fragment of this clone comigrated with a HincII fragment from pPLa8 derived from the PL to the β-lactamase region. Another small fragment of the clone measured about 400 base pairs—consistent with insertion of the BglII fragment into G-pPLa8 in the sense orientation with respect to the PL promoter. This plasmid was designated G-pPLa-HFIF-67-12. The steps used in the construction of this plasmid are shown schematically in FIG. 9. A more detailed map of this plasmid is shown in FIG. 11. The size of the plasmid (˜4450 base pairs) was estimated by the size of its constituent fragments, which in turn had been estimated by their relative mobility upon electrophoresis in agarose gels.
E. coli K12ΔHI and M5219 were then transformed with the characterized plasmid G-pPLa-HFIF-67-12.
Inspection of the determined nucleotide sequence around the BqlII/BamHI junction in G-pPLa-HFIF-67-12 revealed an interesting feature. The polypeptide initiated at the AUG of the β-lactamase coding sequence of that plasmid terminates at a double amber codon located within the untranslated 5′-end of the HuIFN-β coding sequence. These termination codons are located 23 nucleotides before the initiating AUG of the HuIFN-β signal peptide, i.e.:
Junction
181*BamHI/BglII
CCC.CGG.AUC.UUC.AGU.UUC.GGA.GGC.AAC.CUU.UCG.AAG.CCU. Pro-Arg-Ile-Phe-Ser-Phe-Gly-Gly-Asn-Leu-Ser-Lys-Pro-
UUG.CUC.UGG.CAC.AAC.AGG.UAG.UAG GCGACACUGUUCGUGUUGUCAAC Leu-Leu-Trp-His-Asn-Arg am am
AUG-(HuIFN-β signal peptide coding sequence)-AUG-(mature HuIFN-β coding sequence)
The boxed figure refers to the number of the amino acid residue in the β-lactamase protein of pBR322 (J. Sutcliffe, supra). The asterisk (*) indicates that the CCU codon present at this position on pBR322 was changed to CCC as a consequence of the conversion of the PstI site in pPLa2311 to a BamHI site in pPLa8 (see above).
Therefore, this construction opens the possibility of reinitiation at the AUG of the HuIFN-β signal peptide and therefore the possible expression of IFN-α fused to its signal peptide, but not fused to a part of β-lactamase. Such internal reinitiation following premature termination has been observed in the repressor gene of the E. coli lactose operon (T. Platt et al. “Translational Restarts: AUG Reinitiation Of A lac Repressor Fragment”, Proc. Natl. Acad. Sci. U.S.A., 69, 897-901 (1972)). This construction might enable the excretion of mature IFN-β by correct bacterially recognition of the HuIFN-β signal sequence.
3. Construction of Plasmid G-pPLa-HFIF-67-12Δ19
From the known sequence of pBR322 and the HuIFN-β coding sequence it can be deduced that deletion from G-pPLa-HFIF-67-12 of the small HincII fragment (from within p-lactamase up to 3 nucleotides in front of the HuIFN-β signal peptide initiating AUG) results in a continuous translational reading frame starting at the AUG of β-lactamase and terminating after the HuIFN-β coding sequence. This construction is therefore predicted to code for a polypeptide consisting of 82 amino acid residues from the β-lactamase coding sequence, one amino acid coded at the fused HincII site, the HuIFN-β signal peptide and mature HUIFN-β, i.e.:
82
GUU.AAC.AUG-(HuIFN-β signal peptide coding sequence)-AUG-Val Asn-Met
(mature HuIFN-β coding sequence)
The boxed figure refers to the number of the amino acid residue in the β-lactamase protein of pBR322 (J. Sutcliffe, supra). Therefore, this construction may afford the expression of a fused polypeptide consisting of a portion of β-lactamase fused through one amino acid to the HuIFN-β signal peptide which itself is fused to mature HuIFN-β. Such fusion protein may be excreted from the cell.
G-pPLa-HFIF-67-12 was partially digested with HincII. Following ligation at a DNA concentration of about 0.01 μg/ml, the DNA was cleaved with XorII, an isoschizomer of PvuI producing 3′ protruding ends (R. Wang et al., Biochim. Biophys. Acta, in press), and religated at low DNA concentration. Parent G-pPLa-HFIF-67-12 contains two XorII sites: one site inactivates the kanamycin gene and the other one is located in the HincII fragment to be deleted from the plasmid. The purpose of the XorII digestion-religation step is to eliminate parent DNA molecules not cleaved by the HincII enzyme. Such molecules possess two XorII sites and under conditions used for ligation, two fragments are highly unlikely to be rejoined. Transformants were obtained in C600rK−mK+(λ), selecting for kanamycin, and screened by restriction analysis for the presence of a single PvuI site. Further analysis of the clones was performed using HincII digestion. One clone missing the smallest HincII fragment, but otherwise identical to G-pPLa-HFIF-67-12 was designated G-pPLa-HFIF-67-12Δ19. The steps used in the construction of this plasmid are shown schematically in FIG. 9. A more detailed map of this plasmid is shown in FIG. 12. The size of the plasmid (˜4050 base pairs) was estimated by totaling the size of its constituent fragments, which in turn have been estimated by their relative mobility upon electrophoresis in agarose gels. E. coli K12ΔHI and M5219 were then transformed with the characterized plasmid G-pPLa-HFIF-67-12Δ19.
4. Construction of Plasmid G-pPLc-HFIF-67-8
Plasmid G-pPLc24 offers another possibility for insertion of HuIFN-β sequences in such a way that another fusion polypeptide can potentially be synthesized. Insertion of the BglII fragment from G-pPLa-HFIF-67-1 in the BamHI site of G-pPLc24 results in a continuous reading frame coding for 98 amino acid residues from the MS2 replicase gene (W. Fiers et al. “Complete Nucleotide Sequence Of Bacteriophage MS2 RNA: Primary And Secondary Structure Of The Replicase Gene”, Nature, 260, 500-507 (1976)), 27 amino acids coded by sequences between the BglII site and the initiating AUG of the signal sequence of HuIFN-β, followed by the HuIFN-β signal peptide and mature HuIFN-β, i.e.:
98
UGG GAU.CUU.CAG.UUU.CGG.AGG.CAA.CCU.UUC.GAA.GCC.UUU.GCU. Trp-Asp-Leu-Gln-Phe-Arg-Arg-Gln-Pro-Phe-Glu-Ala-Phe-Ala-
CUG.GCA.CAA.CAG.GUA.GUA.GGC.GAC.ACU.GUU.CGU.GUU.GUC.AAC. Leu-Ala-Gln-Gln-Val-Val-Gly-Asp-Thr-Val-Arg-Val-Val-Asn-
AUG-(HuIFN-β signal peptide coding sequence)-AUG-(mature Met
HuIFN-β coding sequence)
The boxed figure refers to the number of the amino acid residue in the MS2 replicase gene protein (R. Devos et al., supra; W. Fiers et al., supra). Therefore, this construction may afford the expression of a fused polypeptide consisting of a portion of MS2 replicase, fused through 27 amino acids to the HuIFN-β signal peptide which itself is fused to mature HuIFN-β.
G-pPLa-HFIF-67-1 DNA was digested with BglII and ligated with BamHI-cleaved pPLc24 DNA. The ligation mixture was recut with BamHI to eliminate parental pPLc24 molecules and transformed into C600rK−mK+(λ) selecting for resistance to carbenicillin. Transformants were analyzed by restriction with HincII. From the known positions of restriction sites on pPLc24 one can predict that insertion of the BglII-IFN-β fragment in the sense orientation with respect to PL should produce an extra HincII fragment of about 650 base pairs. A representative clone exhibiting this configuration was designated pPLc-HFIF-67-8. The steps used in the construction of this plasmid are shown schematically in FIG. 10. A more detailed map of this plasmid is shown in FIG. 13. The size of the plasmid (˜3850 base pairs) was estimated by totaling the size of its constituent fragments, which in turn had been estimated by their relative mobility upon electrophoresis in agarose gels. E. coli K12ΔHI and M5219 were transformed with the characterized plasmid G-pPLc-HFIF-67-8.
5. Construction of Plasmid G-pPla-HFIF-67-12Δ279T
Plasmid pKT279 (a gift of K. Talmadge; pKT279 is a derivative of pBR322 having a portion of the gene for β-lactamase deleted and having a PstI site constructed at amino acid 2 of β-lactamase) was digested with PstI and the 3′ terminal extension removed and the fragment blunt-ended by treatment with E. coli DNA polymerase I (Klenow-fragment) in the presence of deoxy-nucleoside-triphosphates. The PstI linearized and 3′ blunt-ended DNA fragment of pKT279 was then digested with EcoRI to produce a fragment that inter alia codes for the signal sequence of β-lactamase and the first 4 amino acids of the mature protein.
This small fragment was then used to replace the HpaI-EcoRI fragment of pPLa-HFIF-67-12Δ19 (the HpaI site resulting as a consequence of the above-described deletion from G-pPLa-HFIF-67-12) by ligation of the HpaI-EcoRI restricted pPLa-HFIF-67-12Δ19 with that fragment in the presence of T4 DNA ligase.
The predicted sequence at the PstI (blunt-ended)-HpaI junction is:
(β-lactamase signal peptide coding sequence)-CAC.CGC.AAC. His-Arg-Asn-
AUG-(HuIFN-β signal peptide coding sequence)-AUG-(mature Met
HuIFN-β coding sequence)
Consequently, the construction results in IFN-β preceded by two signal sequences in tandem—a bacterial signal sequence (β-lactamase) and the IFN-β signal sequence—connected by several amino acids. Therefore, this construction may afford the expression of mature HuIFN-β fused to two signal peptides or if the tandem combination of a bacterial signal sequence and HuIFN-β's signal sequence is recognized by the bacteria and correctly cleaved, the construction may afford the expression of mature HuIFN-β and its excretion from the cell.
E. coli M5219 was transformed with pPLa-HFIF-67-12Δ279T.
6. Construction of Plasmid G-pPLa-HFIF-67-12Δ218M1
Plasmid pKT218 (a gift of K. Talmadge; pKT218 is a derivative of pBR322 having a portion of the gene for β-lactamase and its signal sequence deleted and having a PstI site constructed at amino acid 4 of the signal sequence of β-lactamase) was digested with EcoRI and AluI to produce a fragment coding inter alia for the initial part of the β-lactamase signal peptide. This fragment was ligated in the presence of T4 DNA ligase with a fragment prepared from pPLa-HFIF-67-12Δ19 by AluI digestion in the presence of actinomycin D (0.05 mM) (to restrict the plasmid at the AluI site in the IFN-β signal peptide) and restriction with EcoRI.
The resulting plasmid, designated pPLa-HFIF-67-12Δ218M1, contained the initial part of the gene coding for the β-lactamase signal peptide, a part of the gene coding the HuIFN-β signal peptide and the gene coding for mature HuIFN-β. The predicted sequence of the pertinent region of the plasmid is:
4
CAA.GCU.CUU.UCC.AUG-(mature HuIFN-β coding sequence) Gln-Ala-Leu-Ser-Met-
The boxed figure refers to the number of the amino acid residue in the β-lactamase signal peptide of pKT218. Therefore, this construction may permit the expression of mature HuIFN-β fused to portion of a bacterial signal sequence and a portion of its own signal sequence. Again, if the bacterial host recognizes and correctly cleaves the hybrid signal sequence, mature HuIFN-β could be expressed from this plasmid and excreted from the cell.
E. coli M5219 was transformed with pPLa-HFIF-67-12Δ218M1.
7. Construction of Plasmid G-PPLa-HFIF-67-12ΔM1
Plasmid pPLa-HFIF-67-12Δ19 was linearized as before with AluI in the presence of actinomycin D (0.05 mM) to generate a cut at the AluI site in the signal peptide of HuIFN-β. After digestion with HpaI, the DNA was recircularized in the presence of T4 DNA ligase. The resulting plasmid, designated pPLa-HFIF-67-12ΔM1, had only a small part of the IFN-β signal sequence preceding the DNA sequence coding for mature IFN-β.
The predicted sequence of the junction is:
82
GUU CUC.UUU.CCA.UGA.
Val-Leu-Phe-pro-STOP
A UG-(mature HuIFN-β coding sequence)
The vertical boxed figure refers to the number of the amino acid residue in β-lactamase. The horizontal boxed figure refers to the sequence in a second reading frame. Therefore, the translation of the β-lactamase coding sequence and the remaining portion of the coding sequence for the signal peptide of IFN-β is arrested at the UGA-stop codon. However, the start-codon (AUG) for mature IFN-β is present at the same place, although in a different reading frame. Therefore, reinitiation of translation may take place at that point to produce mature HuIFN-β.
E. coli M5219 was transformed with pPLa-HFIF-67-12ΔM1.
8. Construction of Plasmid G-pPLa-HFIF-67-12Δ19 BX-2
Plasmid pPLa-HFIF-67-12Δ19 was linearized with HpaI and treated with exonuclease BAL 31 to remove base pairs sequentially from the end of the linearized DNA fragment (H. Gray et al., “Extracellular Nucleases Of Pseudomonas Bal 31” I. Characterization Of Single Strand-Specific Deoxyribendonuclease”, Nucleic Acids Res., 2, pp. 1459-92 (1975)). By varying the time and condition of the exonuclease treatment a series of DNA fragments having various numbers of nucleotides from the coding sequence for the signal peptide of HuIFN-β, if any, preceding the AUG start codon of mature HuIFN-β are constructed. These fragments may then be manipulated to construct ribosomal binding sites at varying distances from that start codon and to afford the desired secondary structure near that codon to enhance expression of mature HuIFN-β.
The exonuclease-treated fragments were blunt ended with E. coli DNA polymerase I (Klenow fragment) in the presence of dATP and dGTP to fill in any 5′ protruding ends. Subsequently, a double-stranded XhoI linker, having the sequence 5′-CCTCGAGG-3′ (Collaborative Research), was ligated onto the blunted-ended DNA fragments. These fragments were then extended with a double-stranded EcoRI linker, having the sequence 5′-CCGAATTCGG-3′ (Collaborative Research). After EcoRI digestion, the fragments having “sticky” EcoRI ends were recircularized with E. coli DNA ligase. The use of this ligase, instead of T4 DNA ligase, avoids the recircularization of blunt-ended fragments.
One plasmid was selected and designated pPLa-HFIF-67-12Δ19 BX-2. It has an XhoI site about 25 base pairs in front of the AUG start codon of mature HuIFN-β. The XhoI site is also preceded by a EcoRI site generated by the ligation of the EcoRI linker of the HpaI fragment to the EcoRI site just preceding the PL promoter in piLa-HFIF-67-12Δ19. Therefore, the β-lacta-mase coding sequence has been deleted. Furthermore, because at least part of the HuIFN-β signal sequence has been removed, only expression of mature HuIFN-β is possible.
E. coli K12ΔHI was transformed with pPLa-HFIF-67-12Δ19 BX-2.
Isolation and Characterization of HuIFN-β Made by Bacteria
A. Preparation of Bacterial Extracts
1. Induction Procedure
An aliquot from stock cultures (frozen at −80° C. in 50% glycerol-50% LB medium), including stock cultures of strains K12ΔH1 and M5219 transformed with the plasmids containing the IFN-β fragments, described above, was inoculated into fresh LB medium with the desired antibiotic and grown to saturation at 28° C. Two 500 ml batches of LB medium without antibiotic were inoculated with 1 ml each of saturated cells and grown with vigorous shaking to 28° C. to a cell density of 2×108/ml. One batch was shifted to 42° C. and continued to be shaken. Depending on the plasmid used, the culture was harvested at various times after the shift to 42° C. The control culture remaining at 28° C. was harvested at the same time as the 42° C. culture. Cells were collected by centrifugation in the GSA rotor (Sorvall) at 8000 rpm for 10 minutes. The pellets were washed in 20 ml of 50 mM Tris HCl (pH 7.4), 30 mM NaCl and repelleted in the SS34 rotor (Sorvall) for 10 minutes at 10,000 rpm. The pellet was quickly frozen in dry ice-methanol and stored at −80° C. When it was desired to shock osmotically the harvested cells the freezing step was omitted.
Two different procedures for lysis and extraction of the bacteria have been used.
2. Extraction Procedures
Lysis A
Cells were resuspended in a final volume of 4 ml of the above described buffer and lysozyme (Sigma) was added to 1 mg/ml. The incubation was for 30 min at 0° C. The suspension underwent two freeze-thaw cycles by sequential dipping in an ethanol-CO2 mixture (−80° C.) and a 37° C. water bath. The S-100 fraction was prepared by ultracentrifugation the lysed bacteria (4 ml) in a Beckman SW60 Tirotor for 1 hr at 60,000 rpm and 4° C., after which the supernatant was further used.
Lysis B
Lysis B was performed, as described above (lysis A), except that the solution of 50 mM Tris-HCl (pH 8.0)-30 mM NaCl was replaced by 50 mM HEPES (Sigma)-NaOH (pH 7.0), 30 mM NaCl, 3 mM β-mercaptoethanol and 3% newborn calf serum (Gibco).
Osmotic Shock
Immediately after harvesting and washing, the cell-pellet was resuspended in 20% sucrose, 100 mM EDTA, 100 mM Tris HCl (pH 7.4) at a maximal cell density of 1×1010/ml. The suspension was kept on ice for 10 minutes and then centrifuged for 10 minutes at 10,000 rpm in the Sorvall SS34 rotor. The sucrose solution was carefully drained from the tube and the pellet was resuspended in an equal volume of water (cell density of 1×1010/ml). The resuspended cells remained on ice for 10 min and were then again subjected to a centrifugation at 10,000 rpm for 10 minutes in the SS34 rotor (Sorvall). The supernatant was made 3% in fetal calf serum, 50 mM in HEPES buffer (pH 7), 30 mM in NaCl and 3 mM in β-mer-captoethanol. This supernatant is referred to as “osmotic shock supernatant”. It was stored at 0° C.
3. Ammonium Sulfate Precipitation
1 ml of an (NH4)2SO4 solution, saturated at room temperature, was added to 0.5 ml of control solution or an S-100 extract. This mixture was kept on ice for at least 30 min, after which the precipitate was pelleted in an Eppendorf centrifuge for 10 min at room temperature. The pellet was redissolved in PBS (phosphate buffered saline).
B. Interferon Titrations
1. Direct Anti-viral Assay
HuIFN-β was assayed in microtiter trays (Sterilin) by a CPE (cytophatic effect)-inhibition technique in human fibroblasts trisomic for chromosome 21. The cells were seeded one day before use, incubated with serial dilutions (log10=0.5) of the sample for 24 h and challenged with vesicular stomatitis virus (Indiana strain) 10−3 dilutions of a stock containing 106.9 mouse C-929 plaque forming units/ml. The CPE was recorded at 24 h after VSV challenge and the interferon endpoint was defined as the sample dilution causing 50% reduction of viral CPE. All assays included an internal standard of HuIFN-β which was itself calibrated against the NIH human fibroblast reference G023-902-527.
The cell line trisomic for chromosome 21 (henceforth referred to as T21) was derived from a skin biopsy of a female patient with Down's syndrome. Its karyotype has been established and showed diploidy for all chromosomes except for chromosome 21 (trisomic). The sensitivity of this cell line to interferon appears to be comparable to the sensitivity of cell lines trisomic for chromosome 21 described by E. De Clercq et al., “Non-antiviral Activities of Interferon Are Not Controlled By Chormosome 21”, Nature, 256, pp. 132-134 (1975) and E. De Clercq et al., “Chromosome 21 Does Not Code For An Interferon Receptor”, Nature, 264, 249-251 (1976).
In other assays the cell line E1SM (A. Billiau et al., “Human Fibroblast Interferon For Clinical Trials: Production, Partial Purification And Characterization”, Anti-microbial Agents And Chemotherapy, 16, 49-55 (1979)) has been used. This cell line is a diploid fibroblast disomic for chromosome 21 and derived from a two-month-old human fetus. Compared to the T21 cell line, E1SM is less sensitive to HuIFN-β by a factor of 10.
2. 2,5-A Synthetase Assay
Another method for detecting the presence of interferon is by the use of a 2,5-A synthetase assay. It has been shown that interferon induces this enzyme, which converts ATP into trimers (and to a lesser extent dimers, tetramers and multimers) of 2,5-A (A. Kimchi et al., “Kinetics Of The Induction Of Three Translation-Regulatory Enzymes By Interferon”, Proc. Natl. Acad. Sci. U.S.A., 76, 3208-3212 (1979).
Confluent 25 cm2 flasks containing cultures of E1SM cells (A. Billiau et al., supra) were treated for 20 h with a 1:6 dilution of bacterial extracts or control interferon in MEM-10% fetal calf serum. The cultures were detached with trypsin (0.25%), EDTA (0.17%) and extensively washed with 140 mM NaCl in 35 mM-Tris buffer (pH 7.5). All subsequent operations were carried out at 4° C. Cells were homogenized in 1.5-2.0 vol of 20 mM HEPES buffer (pH 7.4) containing 10 mM KCl, 1.5 mM magnesium acetate and 0.5 mM dithiothreitol (“lysis buffer I”) in a Dounce glass homogenizer. The homogenate was centrifuged for 20 min at 10,000×g and the supernatant (S10) stored in liquid nitrogen when not used immediately.
Confluent 96-well microtiter plates (105 cells in 0.2 ml per 0.28 cm2 well) were treated with interferon or the respective bacterial extracts as above. After 20 h treatment, plates were cooled on ice and washed three times with 140 mM NaCl in 35 mM Tris buffer (pH 7.5). The cultures were then lysed by adding to each well 5 μl of a solution containing 0.5% Nonidet P40 and 1 mM phenylmethane sulfonyl fluoride (PMSF) in lysis buffer I. After shaking vigorously for 20 min on ice, the cell lysates were collected and centrifuged for 20 min at 10,000×g as above.
3.5 μl of lysate, prepared as indicated above, (lysis A or lysis B) were incubated for 2 h at 31° C. in 6 μl of an incubation mixture containing 100 mM potassium acetate, 25 mM magnesium acetate, 10 mM HEPES/KOR (pH 7.4), 5 mM ATP, 4 mM fructose 1,6 bis-phosphate, 1 mM dithiothreitol and 20 μg/ml poly(I)-poly(C) and 2 μCi of lyophilized (α-32P)-ATP (400 Ci/mmol, from the Radio-chemical Centre, Amersham, U.K.). After stopping the reaction by heating for 3 min at 95° C. and clarification for 2 min at 9,000×g, the samples were treated with 150 U/ml of alkaline phosphatase from calf intestine (Boehringer, Mannheim, cat. nr. 405612) for 1 h at 37° C., clarified again and spotted (1 μl per sample) on thin-layer plates of polyethyleneimine-cellulose (Polygram, cel 300 PEI 20×20 cm from Macherey-Nagel Co., Duren Germany). The plates were washed two times in 2 l of distilled water and dried under vacuum before chromatography in 1 M acetic acid for 2-3 h. After drying they were submitted to autoradiography for 1-24 h.
C. Detection of HuIFN-β Activity in Bacterial Extracts
1. Control Experiments
Two main problems were encountered in the performance of the above-described assays. Both are important in the interpretation of the assay data. Bacterial extracts (including control extracts) resulting from lysis by the above described procedures seemed to include a non-interferon related factor which displayed anti-viral activity in the assay. It is unclear whether the factor itself was an anti-viral agent, or whether the factor induced an anti-viral substance, e.g., interferon, under the conditions of the assay. The presence of the factor was detected repeatedly in S100 extracts. The activity of the factor was, perhaps because of cell density, often higher in control extracts from E. coli HB101 than in similar control extracts of the K12ΔHI or M5219 host bacteria, where the activity of the factor was always less than about 0.7 log10/ml. For some reason, the anti-viral activity of the factor was reduced or sometimes even eliminated totally by precipitation with (NH4)2SO4 under conditions which also precipitated interferon in control experiments.
Due to the anti-viral activity attributable to this contaminating factor, it is difficult to draw conclusions about the presence of trace amounts of interferon in bacterial extracts. However, it was possible to discriminate between the anti-viral activity of the factor and the activity of authentic interferon by the use of the diploid fibroblasts E1SM. These cells are less sensitive to HuIFN-β than the usual cells trisomic for chromosome 21. But, the cells are much more sensitive to the factor, than they are to bona fide interferon. For example, using pMS2-7 (R. Devos et al. “Construction And Characterization Of A Plasmid Containing A Nearly Full-size DNA Copy Of Bacteriophage MS2 RNA”, J. Mol. Biol., 128, 595-619 (1979)) in E. coli HB101 (H. Boyer and D. Rouland-Dussoix, “A Complementation Analysis Of Restriction And Modification Of DNA In Escherichia coli”, J. Mol. Biol., 41, 459-472 (1969)) or K12ΔHI-G-pPLa2311 as control lysates, data demonstrating this relative effect are shown in the following table, with anti-viral activity measured as log10 units/ml.
T21
E1SM
HB101-pMS2-7
(lysis A)
0.7
HB101-pMS2-7
(lysis B, but no
<0.2
1.2
β-mercaptoethanol
and no calf serum)
HB101-pMS2-7
(lysis B)
not done
0.7
HB101-pMS2-7
(lysis B)
0.2
1.0
HB101-pMS2-7
(lysis B)
0.7
2.5
K12ΔHI-G-pPLa2311
(lysis B)
0.2
4.0
K12ΔHI-G-pPLa2311
(42° C.; osmotic
0.5
>1.7
shockate)
Furthermore, the presence of authentic HuIFN-β is reflected by a different ratio of values on T21:E1SM and a high value on T21 as compared to that caused by the presence of the factor. This is shown in the following data:
T21
E1SM
osmotic
K12ΔHI-G-pPLa2311 (42° C.)
0.5
2.5
shock supernatant
K12ΔHI-G-pPLa2311 (42° C.) +
1.5
2.5
HuIFN-β (authentic)
lysis B
HB101-pMS2-7
0.2
2.5
after
HB101-pMS2-7 + HuIFN-β
2.7
2.5
(NH4)2SO4
(authentic)
precipitation
(added before lysis)
Therefore, a comparison of the activities T21:E1SM and a measurement of the absolute activity on T21 cells permits the use of the anti-viral assays, described above, to detect unambiguously the presence of HuIFN-β in a bacterial extract. Furthermore, it should be noted that for extracts of cultures of E. coli (either K12ΔHI or M5219) transformed with some plasmids of this invention, e.g., G-pPLa-HFIF-67-12, G-pPLa-HFIF-67-12Δ19, G-pPLc-HFIF-67-8, G-pPLa-HFIF-67-12Δ279T, G-pPLa-HFIF-67-12Δ218MI, G-pPLa-HFIF-67-12ΔMI or G-pPLa-HFIF-67-12Δ19 BX-2, such interference by the unknown factor in the anti-viral assays was less severe. In these assays, the non-highly concentrated extracts (for example, cells from 150-ml cultures at 6×108 cells/ml were lysed and extracted in 4 ml) displayed a low or undetectable level of anti-viral activity attributable to the unknown factor.
The presence of this contaminating factor has also been shown to be detectable in the 2,5-A synthetase activity assay. Here, however, the factor can be eliminated completely by precipitation with (NH4)2SO4. Therefore, the actual presence of HuIFN-β in a bacterial extract, as distinguished from the presence of the contaminating factor, can also be detected unambiguously in this assay.
However, extracts from E. coli HB101/G-pBR322 (Pst)/HFIF6, which has an incomplete colinear coding sequence (only the last few base pairs are missing) and is thus unable to express a mature polypeptide, has repeatedly yielded a positive 2,5-A synthetase activity, but so far no discernable anti-viral activity. This demonstrates that the 2,5-A assay cannot be regarded as the only criterion for the presence of a complete bacteria-made interferon. It also demonstrates that less than the complete mature interferon may have useful activity.
2,5-A synthetase activity is measured by 32P incorporation into the 2,5-A trimer as shown by autoradiography. Results (repeated 3 times) are shown in the following table, with increasing positive values reflecting increased incorporation of 32P.
extract a/pHFIF/6→+++; after (NH4)2SO4 precipitation→++
extract b/pMS2-7→+++; after (NH4)2SO4 precipitation→−
extract b/pMS2-7→+++; after (NH4)2SO4 precipitation→++
plus HuIFN-β
A second important problem in these assays is the low recovery of HuIFN-β secreted by human fibroblasts during and after different experimental steps. A comparison of the recoveries of leukocyte interferon and fibroblast interferon added to an S-100 extract demonstrates that HuIFN-β is recovered with only 10% efficiency, in contrast to HuIFN-α's 100% recovery (anti-viral values are given as log10 units/ml; assayed on T21 cells).
IFN-α diluted in S-100-extract of HB101-pMS2-7 (lysis A) 2.5
IFN-α diluted in E-MEM plus 3% calf serum 2.7
IFN-β diluted in S-100-extract of HB101-pMS2-7 (lysis A) 0.7
IFN-β diluted in E-MEM-plus 3% calf serum 1.7
Other experiments where IFN-β was added to the cell pellet before lysis and extraction (even with calf serum added to 3% as a stabilizer) showed that only 10-30% IFN-β was recovered.
log10 units/ml
HEPES
T21
E1SM
HB101-pMS2-7
(lysis B, but no
pH 8
0.7(10%)
1.7
plus IFN-β
β-mercaptoethanol
pH 7
1.0(20%)
1.7
or calf serum)
pH 6
0.7(10%)
1.7
IFN-β
(same treatment as
pH 6
1.7(50%)
1.5
(no bacteria)
in lysis B)
Further experiments were carried out to test the stability and recovery of IFN-β activity. Precipitation with (NH4)2SO4, as described above, either in the presence or absence of bacterial extracts, often caused a reduction of the titer in the anti-viral assay:
log10 units/ml
Precipitation with (NH4)2SO4
before
after
IFN-β
1.0
0.5
IFN-β
2.7
2.5
HB101-pMS2-7 + IFN-β (lysis B)
1.5
1.2
K12ΔHI-G-pPLa2311 (28° C.) + IFN-β (lysis B)
1.7
1.5
K12ΔHI-G-pPLa2311 (28° C.) + IFN-β (lysis B)
2.2
3.0
Dialysis of IFN-β (overnight at 4° C. against PBS) either in the presence or in the absence of bacterial extracts, also usually resulted in a decreased recovery of IFN-β activity:
log10 units/ml
Dialysis
before
after
IFN-β in PBS
1.0
0.5
IFN-β in PBS
2.7
2.5
K12ΔHI-G-pPLa8 (28° C.) + IFN-β (lysis B)
1.2
<0.2
K12ΔHI-G-pPLa8 + IFN-β (lysis B)
3.0
1.7
K12ΔHI-G-pPLa8 + IFN-β (lysis B)
2.5
1.0
K12ΔHI-G-pPLa8 + IFN-β (lysis B)
1.5
0.5
Since IFN-β is a-Type I interferon its activity should be acid-stable. This was tested by dialyzing IFN-β samples in the presence or absence of bacterial extracts, overnight in 5 mM glycine-HCl (pH 2.2) at 4° C. This treatment caused the formation of a precipitate, which was pelleted in an Eppendorf centrifuge at 12,000×g for 2 min. The supernatant was then tested for anti-viral activity. Although some of the anti-viral activity remained following this treatment, there was a substantial loss in the amount of interferon recovered.
log10 units/ml
Dialysis
before
after
HB101-pMS2-7 (lysis A) + IFN-β
0.7
0.5
K12ΔHI-G-pPLa2311 (28° C.) osmotic
1.2
1.2
shockate + IFN-β
M5219-G-pPLa8 (42° C.) (lysis B) + IFN-β
1.2
0.7
M5219-G-pPLa8 (28° C.) (lysis B) + IFN-β
3.0
2.0
The reductions in HuIFN-β activity observed with these different treatments to the above described control extracts must be interpreted cautiously. The lower anti-viral titers do not necessarily mean that interferon is being degraded. The lower titers may be due to non-specific sticking of the HuIFN-β to dialysis membranes or to components in the bacterial extracts, e.g. membrane components. For example, it is well established that IFN-β is a hydrophobic protein (its hydrophobicity is also substantiated by its amino acid sequence) which can adhere non-specifically to tube walls or other surfaces. Furthermore, bacterial IFN-β, lacking glycosylation, may be even more hydrophobic. Therefore, conclusions on the recovery of the glycosylated IFN-β secreted by human cells may not necessarily be extrapolated to IFN-β of bacterial origin.
2. Demonstration of IFN-β Activity
a. Anti-viral Activity
Bacterial extracts of E. coli M5219 or K12ΔHI, containing the plasmids G-pPLa-HFIF-67-12, G-pPLa-HFIF-67-12Δ19, G-pPLc-HFIF-67-8, G-pPLa-HFIF-67-12Δ279T, G-pPLa-HFIF-67-12Δ218MI, G-pPLa-HFIF-67-12ΔMI, or G-pPLa-HFIF-67-12Δ19 BX-2 were analyzed for IFN-β anti-viral activity. The procedures for induction and preparation of the S-100 extracts and the osmotic shock supernatants were substantially as described above. 150 ml of bacterial culture (3−6×108 cells/ml) were used per experiment. All biological titers are given in log10 units/ml.
G-DPLa-HFIF-67-12
G-pPLa-HFIF-67-12 was employed to transform E. coli M5219 and E. coli K12ΔHI and S-100 extracts were prepared by lysis B. All samples were precipitated with (NH4)2SO4 before testing for antiviral activity.
T21
E1 SM
K12ΔHI-G-pPLa-HFIF-67-12 (28° C.)
<0.2
<1.0
K12ΔHI-G-pPLa-HFIF-67-12
0.2/0.5
<1.0/<1.0
(42° C., 90 min)
M5219-G-pPLa-HFIF-67-12 (28° C.)
<0.2
<1.0
M5219-G-pPLa-HFIF-67-12
0.7/0.7
<1.0/<1.2
(42° C., 90 min)
The second figure in the above table is the titer determined on reassay of the same sample. A control experiment where authentic IFN-β was added to E. coli HB101-pMS2-7 before lysis of the cells indicated an IFN-β recovery of 30% in the assay. Therefore, it is plain that upon induction IFN-β anti-viral activity is detected in the bacterial lysate. The titers, while below the detection level of E1SM cells, show clearly that the IFN-β activity is not due to a contaminating bacterial activity. Such a contaminating bacterial activity would give values of at least 2.0 on E1SM to correspond to the values of 0.5 or 0.7 on T21 cells (see control experiments above).
G-pPLa-HFIF-67-12Δ19
Plasmid G-pPLa-HFIF-67-12Δ19 was used to transform E. coli M5219 and S-100 extracts were prepared by lysis B. All samples were precipitated with (NH4)2SO4, as described above, and assayed for anti-viral activity. Again, the presence of HuIFN-β anti-viral activity in the extracts is plain. The value between brackets indicates the detection level, due to some toxicity of the particular samples for the human cells in tissue culture.
i) M5219-G-pPLa-HFIF-67-12Δ19 (28° C.)
ii) M5219-G-pPLa-HFIF-67-12Δ19 (42° C., 90 min, final cell density=3×108/ml)
on T21
on E1 SM
i)
<0.5
2.2 (<2.0)
ii)
2.2 (<0.5)
2.2 (<2.0)
A control experiment where authentic IFN-β was added to HB101-pMS2-7 before lysis of the cells displayed a 30% recovery. Here, the high values on T21 cells and the ratio of activity on T21 over that on E1SM indicate that there was no significant contaminating bacterial activity (as discussed above) in the temperature induced samples.
G-pPLc-HFIF-67-8
Plasmid G-pPLc-HFIF-67-8 was used to transform E. coli M5219 and S-100 extracts were prepared by lysis B. All samples were precipitated with (NH4)2SO4 and assayed for anti-viral activity.
i) M5219-G-pPLc-HFIF-67-8 (28° C.)
ii) M5219-G-pPLc-HFIF-67-8 (42° C., 180 min, final cell density=6×108/ml)
on T21
on E1 SM
i)
<0.5
2.2 (<2.0)
ii)
2.2 (<0.5)
2.2 (<2.0)
The value in the brackets indicates the detection level, due to toxicity. A control experiment where authentic IFN-β was added to HB101-pMS2-7 before lysis of the cells displayed a 30% recovery. Again, it is plain that the bacterial extract displayed HuIFN-β anti-viral activity.
In another experiment the osmotic shock supernatant of these cells was assayed for IFN-β antiviral activity:
i) control: M5219-G-pPLa-HFIF-67-12Δ19 (28° C.)
ii) M5219-G-pPLc-HFIF-67-8 (28° C.)
iii) M5219-G-pPLc-HFIF-67-8 (42° C., 180 min, cell density=6×108/ml).
The assays were performed on T21 cells, both before and after (NH4)2SO4 precipitation. The value between brackets indicates the limit of detection.
before precipitation
after precipitation
i)
<0.2
<0.2
ii)
<0.2
<0.2
iii)
1.5 (<0.2)
0.7 (<0.2)
The recovery of IFN-β was about 10% in control experiments. The control lysates did not show detectable activity on E1SM. The values obtained with the osmotic shock supernatants make plain that the temperature-induced M5219-G-pPLc-HFIF-67-8 extract has an anti-viral activity not present in the non-induced samples. Sample (iii) after precipitation with (NH4)2SO4, having a titer of 0.7 log10 units per ml, was dialysed to pH 2.2, as described above, and showed no substantial decrease of activity. This acid-stability is a particular property of type I interferons, e.g. IFN-β.
G-PPLa-HFIF-67-12Δ279T
Plasmid G-pPLa-HFIF-67-12Δ279T was used to transform E. coli M5219 and S-100 extracts were prepared by lysis B. Samples were precipitated with (NH4)2SO4 before assay by CPE on T21 cells. The extracts of cells induced at 42° C. displayed an anti-viral titer of 1.5-1.7 log10 u/ml of extract.
G-pPLa-HFIF-67-12Δ218MI
Plasmid G-pPLa-HFIF-67-12Δ218MI was used to transform E. coli M5219 and S-100 extracts were prepared by lysis B. Samples were precipitated with (NH4)2SO4 before assay by CPE on T21 cells. The extracts of cells induced at 42° C. displayed an anti-viral titer of 1.5 log10 u/ml of extract.
G-pPLa-HFIF-67-12ΔMI
Plasmid G-pPLa-HFIF-67-12ΔMI was used to transform E. coli M5219 and S-100 extracts were prepared by lysis B. Samples were precipitated with (NH4)2SO4 before assay by CPE on T21 cells. The extracts of cells induced at 42° C. displayed an anti-viral titer of 2.0 log10 u/ml of extract.
G-pPLa-HFIF-67-12Δ19 BX-2
Plasmid G-pPLa-HFIF-67-12Δ19 BX-2 was used to transform E. coli K12ΔHI and S-100 extracts were prepared by lysis B. Samples were precipitated with (NH4)2SO4 before assay by CPE on T21 and FS-4 cells. The extracts of cells induced at 42° C. displayed an anti-viral titer of 1.7-2.0 log10 u/ml of extract.
b. Antibody Neutralization of HuIFN-β Anti-Viral Activity
Further evidence substantiating bacterial expression of IFN-β is given by antibody neutralization experiments. The anti-interferon antiserum was produced in goats, immunized with 107 units of authentic IFN-β (secreted by human fibroblast cells), and purified on controlled pore glass beads (A Billiau et al., supra). After bacterial extracts were assayed as above for antiviral activity, serial dilutions of the antiserum were added to similar samples, the mixtures incubated for 1 h at 37° C., applied to human diploid fibroblasts T21 and assayed for anti-viral activity as described before. The degree of neutralization by IFN-β antiserum ranges from +++ (complete neutralization to—(no neutralization). The value between brackets indicates the approximate antiserum dilution for 50% neutralization.
1) M5219-G-pPLc-HFIF-67-8 (42° C., 180 min; which gave 2.2 log10 antiviral units/ml on T21 cells).
2) M5219-G-pPLa-8 (42° C., 180 min) to which IFN-β (from human fibroblasts) was added before lysis (which gave 1.7 log10 antiviral units on T21 cells).
dilution of antiserum
(1)
(2)
10−3
+++
+++
10−4
+
+++
10−5
± (10−4.5)
+++
10−6
−
± (10−6)
10−7
−
−
Similar results were obtained with extracts from M5219-pPLa-HFIF-67-12Δ19 (42° C.). The differences in neutralization titer between the bacterial IFN-β of this invention and authentic IFN-β may be due to differences in antigen-icity or in the specific IFN activity of these bacterial proteins relative to authentic IFN-β caused by lack of glycosylation in the bacterial proteins.
c. Stability of HuIFN-β Anti-Viral Activity
(1) Heat Treatment
IFN-β has, in contrast to IFN-α, the very unusual property that its anti-viral activity is recovered after boiling in 1% SDS, 1% β-mercaptoethanol, 5 M urea (Stewart, W. E. II et al., Distinct Molecular Species of Human Interferon, Requirements For Stabilization And Reactivation Of Human Leucocyte And Fibroblast Interferon, J. Gen. Virol., 26, 327-331, (1975)), although a 100% recovery usually is not obtained. For this assay the bacterial cells of a 150 ml culture were resuspended in the buffer for lysis B and an equal volume of 2% SDS, 2% β-mercaptoethanol and 10 M urea added, the mixture boiled for 2 min, and S-100 fractions prepared.
i) control: M5219-G-pPLa-HFIF-67-12Δ19 (28° C.)
ii) control: 3 log10 units of HuIFN-β diluted in lysis B buffer
iii) M5219-G-pPLc-HFIF-67-8 (42° C., 180 min, cell density=6×108/ml).
The assays were performed on T21-cells. The value in the brackets indicates the limit of detection, due to intrinsic toxicity.
Before boiling
After boiling
i)
<1.5
<1.5
ii)
2.2 (<1.5)
2.0 (<0.5)
iii)
3.0 (<2.0)
2.2 (<1.5)
The control experiment showed a recovery of about 10% of the IFN-β activity. There was no detectable value in E1SM in parallel control lysates. These data make plain that although only about 10% of added IFN-β is recovered in the control experiment, that IFN-β anti-viral activity was present in the extract from the temperature induced M5219-G-pPL-c-HFIF-67-8 culture even after this severe treatment. In fact, a higher antiviral activity was found after this treatment as compared to the lysis B procedure, indicating possible adherence of IFN-β to cell components in the latter procedure.
(2) Dialysis
The HuIFN-β anti-viral activity is also nondialysable. For example, after dialysis against PBS for 16 h at neutral pH and 4° C. the anti-viral activity (log10 u/ml) of the bacterial extracts was maintained, albeit at a reduced titer:
i) M5219-pPLc-HFIF-67-8 (42° C.)
ii) M5219-pPLa-HFIF-67-12Δ19 (42° C.)
iii) IFN-β in M5219-pPLa-8 (42° C.)
Before dialysis
After dialysis
i)
2.3
2.3
i)
3
2.3
i)
1.5
1.3
ii)
2.3
1.3
ii)
2.3
2
ii)
2.3
1
The observed decrease in activity after dialysis may be due to non-specific sticking of IFN-β to dialysis membranes, etc.
(3) Precipitation with (NH4)2SO4
The anti-viral activity (log10 u/ml) of the bacterial extracts of this invention was maintained after precipitation with 67% saturated ammonium sulphate (2 vol (NH4)2SO4 solution to 1 vol extract), a concentration known to precipitate HuIFN-β. After 30 min on ice, the pellet was centrifuged at 12000×g for 10 min and redissolved in PBS for assay:
i)-5219-pPLc-HFIF-67-8 (42° C.)
ii) M5219-pPLa-HFIF-67-12Δ19 (42° C.)
iii) IFN-β in M5219-pPLa-8 (42° C.)
before precipitation
after precipitation
i)
2
2
i)
2
2.3
ii)
2
2
iii)
1.3
1.3
iii)
1.5
1.3
(4) pH 2 Treatment
The anti-viral activity (log10 u/ml) of the bacterial extracts of this invention were also stable to acid. The extracts were either dialyzed for 15 h against 50 ml glycine-HCl (pH 2.2), followed by dialysis against PBS for 3 h or acidified with HCl, followed by neutralization with NaOH. After removal of the precipitate the assay was conducted:
i) M5219-pPLc-HFIF-67-8 (42° C.)
ii) M5219-pPLa-HFIF-67-12Δ19 (42° C.)
iii) IFN-β in M5219-pPLa-8 (42° C.)
before acid
after acid
i)
2
1.3
i)
0.7
0.7
ii)
2
1
iii)
3
2
d. 2,5-A Synthetase Activity
The osmotic shockates of M5219-G-pPLc-HFIF-67-8 (described above) were assayed for the presence of 2,5-A synthetase, as described above, with microtiter plates, except that Hela cells were used instead of E1SM cells. The following results were obtained:
i) M5219-G-pPLc-HFIF-67-8 (28° C.) (see above)
ii) M5219-G-pPLc-HFIF-67-8 (42° C.) (see above)
The values, reflecting the 2,5-A synthetase activity, indicate the 32P-radioactivity incorporated in the trimer form of 2,5-A.
(after substraction
(measured counts)
of endogenous background)
1)
non treated cells
3342
cmp
0
cmp
2)
bacterial extract
1972
cmp
−1370
cmp
(i): dilution 1/6
3)
bacterial extract
6960
cmp
3618
cmp
(ii): dilution 1/6
4)
bacterial extract
7037
cmp
3695
cmp
(i) + IFN-β to
1.5 log10 units/ml
5)
see 3 but incubated
3950
cmp
608
cmp
with anti-IFN-β
antiserum
6)
see 4 but incubated
2960
cmp
−382
cmp
with anti-IFN-β
antiserum
7)
control IFN-β
4463
cmp
1120
cmp
0.5 log10 units/ml
8)
control IFN-β
7680
cmp
4338
cmp
1 log10 units/ml
9)
control IFN-β
13615
cmp
10273
cmp
1.5 log10 units/ml
10)
control IFN-β
25040
cmp
21698
cmp
2 log10 units/ml
The results of the 2,5-A synthetase activity assay demonstrate that the osmotic shockate supernatant of the temperature induced M5219-G-pPLc-HFIF-67-8, which has anti-viral activity (see above), is also inducing 2,5-A synthetase activity while the non-induced bacterial strain is not. This parallels the results of the anti-viral activity assay.
The degree of stimulation of 2,5-A synthetase is equal to the activity of IFN-β added to the control lysate (compare samples (3) and (4)). Use of a concentration curve developed from samples (7) to (10)) shows that, taking into account the dilution, an activity of log10 1.7 units/ml can be estimated in both samples (3) and (4), which is compatible with the values in the direct antiviral assay, i.e. 1.5 log10 units for both samples. This series of experiments also demonstrates that the induction of 2,5-A synthetase can be neutralized by anti-IFN-β antiserum, as is the case in the antiviral assay.
e. Anti-viral Activity on Other Cell Lines
The extracts (i) and (ii) (M5219-G-pPLc-HFIF-67-8, above) were also tested for antiviral activity on different cell lines of feline, mouse, monkey or rabbit origin. They did not show any detectable antiviral activity on these cells; neither did authentic IFN-β, made by human cells. Also no activity was found on a feline lung cell line which was sensitive to human leucocyte interferon. These results provide further substantiation that the IFN-β produced by the bacteria exhibits properties essentially identical to those of IFN-β secreted by induced human fibroblast cells.
f. Sensitivity to Protease
The sensitivity of IFN-β from the bacterial hosts of this invention was tested by treatment of diluted bacterial extracts with increasing amount of trypsin for 1 h at 37° C. The anti-viral activity of the IFN-β was abolished by the trypsin at a similar concentration to that which abolished the activity of authentic IFN-β added to an inactive control lysate.
Trypsin
Endpoint
(ms/ml)
M5219-pPLa-HFIF-67-12Δ19 (42° C.) (1000 u/ml)
0.03
M5219-pPLc-HFIF-67-8 (42° C.) (1000 u/ml)
0.03
IFN-β in M5219-pPLa-8 (42° C.) (1000 u/ml)
0.03
M5219-pPLc-HFIF-67-8 (42° C.) (30 u/ml)
0.03
IFN-β in M5219-pPLa-8 (42° C.) (30 u/ml)
0.03
3. Identification of the Active IFN-β Product
Various experiments have demonstrated that pre-HuIFN-β is not active and is not processed by bacterial cells or under assay conditions to an active product. Therefore, the IFN-β activity detected in the various bacterial extracts, described above, is probably due to processing of the expected fused proteins (e.g., HuIFN-β fused to β-lactamase, MS2 or bacterial signal sequences) by the bacteria or under the conditions of the assay to an active product.
It is not certain that the active product in such extracts is mature HuIFN-β (mature HuIFN-β is, of course, the product of G-pPLa-HFIF-67-12ΔM1 and G-pPLa-HFIF-67-12Δ19 BX-2). However, fractionation of the bacterial extracts obtained, for example, from induced M5219-pPLa-HFIF-67-12Δ19 or from induced M5219-pPLc-HFIF-67-8, by polyacrylamide gel electrophoresis under denaturing conditions revealed the presence of two active products. The first of those products had an approximate size of 15000-18000 daltons and could correspond to mature IFN-β. The second product, which had a higher molecular weight, may be a fusion product or an incompletely processed product which has IFN-β activity or may be a product that is processed to mature IFN-β under the conditions of the assay. Amino acid sequencing of the various expression products, using well known techniques, will enable a determination of what protein products, if any, in addition to mature HuIFN-β, display the activity of HuIFN-β.
Improving the Yield and Activity of Polypeptides Displaying HuIFN-β Activity Produced in Accordance with this Invention
The level of production of a protein is governed by three major factors: the number of copies of its gene within the cell, the efficiency with which those gene copies are transcribed and the efficiency with which they are translated. Efficiency of transcription and translation (which together comprise expression) is in turn dependent upon nucleotide sequences, normally situated ahead of the desired coding sequence. These nucleotide sequences or expression control sequences define, inter alia, the location at which RNA polymerase interacts to initiate transcription (the promoter sequence) and at which ribosomes bind and interact with the mRNA (the product of transcription) to initiate translation. Not all such expression control sequences function with equal efficiency. It is thus of advantage to separate the specific coding sequences for the desired protein from their adjacent nucleotide sequences and to fuse them instead to other known expression control sequences so as to favor higher levels of expression. This having been achieved, the newly engineered DNA fragment may be inserted into a higher copy number plasmid or a bacteriophage derivative in order to increase the number of gene copies within the cell and thereby further improve the yield of expressed protein.
Several expression control sequences may be employed as described above. These include the operator, promoter and ribosome binding and interaction sequences (including sequences such as the Shine-Dalgarno sequences) of the lactose operon of E. coli (“the lac system”), the corresponding sequences of the tryptophan synthetase system of E. coli (“the trp system”), the major operator and promoter regions of phage λ (OLPL as described above and ORPR), a control region of Filamentous single-stranded DNA phages, or other sequences which control the expression of genes of prokaryotic or eukaryotic cells and their viruses. Therefore, to improve the production of a particular polypeptide in an appropriate host, the gene coding for that polypeptide may be prepared as before and removed from a recombinant DNA molecule closer to its former expression control sequence or under the control of one of the above expression control sequences. Such methods are known in the art.
Other methods to improve the efficiency of translation involve insertion of chemically or enzymatically prepared oligonucleotides in front of the initiating codon. By this procedure a more optimal primary and secondary structure of the messenger RNA can be obtained. More specifically, the sequence can be so designed that the initiating AUG codon occurs in a readily accessible position (i.e., not masked by secondary structure) either at the top of a hairpin or in other single-stranded regions. Also the position and sequence of the aforementioned Shine-Dalgarno segment can likewise be optimized. The importance of the general structure (folding) of the messenger RNA has been documented (D. Iserentant and W. Fiers “Secondary Structure Of mRNA And Efficiency Of Translation Initiation”, Gene, 9, 1-12 (1980).
Further increases in the cellular yield of the desired products depend upon an increase in the number of genes that can be utilized in the cell. This may be achieved by insertion of the HuIFN-β gene (with or without its transcription and translation control elements) in an even higher copy number plasmid or in a temperature-controlled copy number plasmid (i.e., a plasmid which carries a mutation such that the copy number of the plasmid-increases after shifting up the temperature; B. Uhlin et al. “Plasmids With Temperature-dependent Copy Number For Amplification Of Cloned Genes And Their Products”, Gene, 6, 91-106 (1979)). Alternatively, an increase in gene dosage can be achieved for example by insertion of recombinant DNA molecules engineered in the way described previously into the temperate bacteriophage λ, most simply by digestion of the plasmid with a restriction enzyme, to give a linear molecule which is then mixed with a restricted phage λ cloning vehicle (e.g., of the type described by N. E. Murray et al., “Lambdoid Phages That Simplify The Recovery of In Vitro Recombinants”, Mol. Gen. Genet., 150, 53-61 (1977) and N. E. Murray et al., “Molecular Cloning Of The DNA Ligase Gene From Bacteriophage T4”, J. Mol. Biol., 132, 493-505 (1979) and the recombinant DNA molecule produced by incubation with DNA ligase. The desired recombinant phage is then selected as before and used to lysogenize a host strain of E. coli.
Particularly useful A cloning vehicles contain a temperature-sensitive mutation in the repression gene cI and suppressible mutations in gene S, the product of which is necessary for lysis of the host cell, and gene E, the product which is the major capsid protein of the virus. With this system the lysogenic cells are grown at a relatively low temperature (e.g., 28°-32° C.) and then heated to a higher temperature (e.g., 40°-45° C.) to induce excision of the prophage. Prolonged growth at higher temperature leads to high levels of production of the protein, which is retained within the cells, since these are not lysed by phage gene products in the normal way, and since the phage gene insert is not encapsidated it remains available for further transcription. Artificial lysis of the cells then releases the desired product in high yield. As in this application we have also used the λ repressor system to control expression, it may be necessary to control the excision of the prophage and hence the gene copy number by a heteroimmune control region, e.g., derived from the lambdoid phage 21.
It should be understood that polypeptides displaying IFN-β activity (prepared in accordance with this invention) may be prepared in the form of a fused protein (e.g., linked to a prokaryotic N-terminal segment directing excretion), or in the form of prointerferon (e.g., starting with the interferon signal sequence which could be cleaved off upon excretion) or as mature interferon (the latter is feasible because mature fibro-blast interferon starts with methionine, an amino acid used for initiation of translation). The yield of these different forms of polypeptide may be improved by any or a combination of the procedures discussed above. Also different codons for some or all of the codons used in the present DNA sequences could be substituted. These substituted codons may code for amino acids identical to those coded for by the codons replaced but result in higher yield of the polypeptide. Alternatively, the replacement of one or a combination of codons leading to amino acid replacement or to a longer or shorter HuIFN-β-related polypeptide may alter its properties in a useful way (e.g., increase the stability, increase the solubility, increase the antiviral activity, increase t 2,5-A synthetase activity or increase the host specificity range).
One example of such improvement was obtained by inserting a DNA fragment of this invention including the DNA sequence coding for pre-IFN-β into a cloning vehicle containing the late promoter and splicing sequences of SV40 under the control of that promoter. Such construction in monkey cells yielded about 104 units/ml of processed IFN-β. Similar constructions in other cloning vectors and eukaryotic cells are also envisioned herein.
Finally, the activity of the polypeptides produced by the recombinant DNA molecules of this invention may be improved by fragmenting, modifying or derivatizing the DNA sequences or polypeptides of this invention by well-known means, without departing from the scope of this invention.
Identification of a Chromosomal Gene Coding for HuIFN-β
A collection of hybrid phage derived from fragments of fetal human chromosomal DNA which had been generated by partial cleavage with HaelII and AluI, and joined with EcoRI linkers to λ Charon 4A arms has been prepared by R. M. Lawn et al., Cell, 15, pp. 1157-74 (1978). This gene bank was screened by an “in situ” procedure (W. D. Benton and R. W. Davis, Science, 196, pp. 180-82 (1977); T. Maniatis et al., Cell, 15, pp. 687-701 (1978)); using as a probe the 32P-labelled IFN-β cDNA insert excised by TaaI-BglII restriction from pHFIF/21.* One hybridization-positive phage clone was isolated from 600,000 plaques by repeated plaque purification (T. Maniatis et al., supra). This plaque was designated λCH4A-gHFIF/1. Restriction analysis of this plaque demonstrated that it contains about 16.3 Kb of human DNA. * Plasmid pHFIF/21 was identified by the screening processes of this invention. The Taql-BglII fragment of that plasmid contains nearly the total 5′-untranslated region and the total coding region of IFN-β.
EcoRI digestion of λCH4A-gHFIF/1 generated, in addition to the two Charon 4A phage arms, eight insert fragments—4.6, 3.5, 2.4, 1.9, 1.3, 1.2, 0.8 and 0.6 Kb in length. After Southern blotting, only the 1.9 Kb fragment hybridized to the TaqI-BglII fragment of pHFIF/21.
The 1.9 Kb fragment was recloned directly into the EcoRI site of pBR325 (a derivative of pBR322 which also carries a chloramphenicol resistance marker containing a single EcoRI site). After ligation of 0.6 μg EcoRI-digested λCH4A-gHFIF/1 DNA to 100 ng pBR325 and transformation into E. coli HB101, several clones were selected. Only those clones containing the 1.9 Kb fragment hybridized to the IFN-β cDNA probe. This clone was designated p[325]-HFIF4.
Comparison of the restriction fragment derived from pHFIF/21 and p[325]-gHFIF/4 demonstrated that there are no intervening sequences in the chromosomal clone and that the DNA information carried by that clone is identical to that of pHFIF/21.
The identification and isolation of the chromosomal DNA coding for HuIFN-β enables the transformation of appropriate hosts with that DNA and the expression of HuIFN-β from it. Such expression is advantageous because the various signals associated with chromosomal DNA sequences will be present in such clones. These signals will then be available to trigger higher yields on expression and perhaps post-expression processing of the polypeptide coded for by the coding region of the chromosomal DNA.
Micro-organisms and recombinant DNA molecules prepared by the processes described herein are exemplified by cultures deposited in the culture collection Deutsche Sammlung von Mikroorganism in Gottingen, West Germany on. Apr. 2, 1980, and identified as HFIF-A to C:
A: E. coli HB101 (G-pBR322(Pst)/HFIF3)
B: E. coli HB101 (G-pBR322(Pst)/HFIF6)
C: E. coli HB101 (G-pBR322(Pst)/HFIF7)
These cultures were assigned accession numbers DSM 1791-1793, respectively. They are also exemplified by cultures deposited in the culture collection Deutsche Sammlung von Mikroorganism in Gottingen, West Germany on Jun. 5, 1980, and identified as HFIF-D to G:
D: E. coli M5219 (G-pPLa-HFIF-67-12)
E: E. coli K12ΔHI (G-pPLa-HFIF-67-12)
F: E. coli M5219 (G-pPLa-HFIF-67-12Δ19)
G: E. coli M5219 (G-pPLc-HFIF-67-8)
These cultures were assigned accession numbers DSM 1851-1854, respectively. And, by cultures deposited in the America Type Culture Collection, Rockville, Md. on Feb. 26, 1981, and identified as HFIF H and I:
H: E. coli M5219 (pPLa-HFIF-67-12ΔMI)
I: E. coli HB101 (p[325]-gHFIF/4)
These cultures were assigned accession numbers ATCC 31824 and 31825, respectively.
While we have herein before presented a number of embodiments of this invention, it is apparent that our basic construction can be altered to provide other embodiments which utilize the processes and compositions of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the claims appended hereto rather than the specific embodiments which have been presented herein before by way of example.
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08449930
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biogen idec ma inc.
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USA
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B1
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Utility Patent Grant (no pre-grant publication) issued on or after January 2, 2001.
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Open
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424/ 85.4
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Apr 1st, 2022 05:10PM
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Apr 1st, 2022 05:10PM
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Biogen
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:biib
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Biogen
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Sep 27th, 2011 12:00AM
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Mar 2nd, 2010 12:00AM
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https://www.uspto.gov?id=US08026072-20110927
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Method of identifying compounds that bind BAFF-R
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Disclosed are nucleic acids encoding BAFF-R polypeptides, as well as antibodies to BAFF-R polypeptides and pharmaceutical compositions including the same. Methods of treating tumorigenic and autoimmune conditions using the nucleic acids, polypeptides, antibodies and pharmaceutical compositions of this invention are also provided.
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8026072
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1. A method for identifying a compound that binds to BAFF-R comprising:
(a) contacting BAFF-R with a compound;
(b) determining whether the compound binds to BAFF-R.
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1
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This application is a divisional of U.S. application Ser. No. 11/426,286, filed Jun. 23, 2006 now U.S. Pat. No. 7,709,220, which is a divisional of U.S. application Ser. No. 10/380,703, filed Mar. 17, 2003 now U.S. Pat. No. 7,112,421, which is a National Stage Entry of PCT/US01/28006, filed Sep. 6, 2001, which claims the benefit of U.S. Provisional Application No. 60/312,185, filed Aug. 14, 2001 and U.S. Provisional Application No. 60/268,499, filed Feb. 13, 2001 and U.S. Provisional Application No. 60/234,140, filed Sep. 21, 2000; and U.S. Provisional Application No. 60/233,152, filed Sep. 18, 2000, which are herein incorporated by reference in their entirety.
FIELD OF THE INVENTION
The present invention provides a novel receptor protein. The invention generally relates to nucleic acids and polypeptides. The invention relates more particularly to nucleic acids encoding polypeptides related to a receptor to BAFF, a B-cell activating factor belonging to the Tumor Necrosis Factor (“TNF”) family, which is associated with the expression of B-cells and immunoglobulins. This receptor can be employed in the treatment of cancers, lymphomas, autoimmune diseases or inherited genetic disorders involving B-cells.
BACKGROUND OF THE INVENTION
The present invention relates to a novel receptor in the TNF family. A novel receptor has been identified as the BAFF receptor (“BAFF-R”).
The TNF family consists of pairs of ligands and their specific receptors referred to as TNF family ligands and TNF family receptors (Bazzoni and Beutler (1996) N. Engl. J. Med. 334(26):1717-1725. The family is involved in the regulation of the immune system and possibly other non-immunological systems. The regulation is often at a “master switch” level such that TNF family signaling can result in a large number of subsequent events best typified by TNF. TNF can initiate the general protective inflammatory response of an organism to foreign invasion that involves the altered display of adhesion molecules involved in cell trafficking chemokine production to drive specific cells into specific compartments, and the priming of various effector cells. As such, the regulation of these pathways has clinical potential.
Induction of various cellular responses mediated by such TNF family cytokines is believed to be initiated by their binding to specific cell receptors. At least two distinct TNF receptors of approximately 55 kDa (TNFR1) and 75 kDa (TNFR2) have been identified (Hohman et al. (1989) J. Biol. Chem. 264:14927-14934; and Brockhaus et al. (1990) Proc. Natl. Acad. Sci. USA 87:3127-3131). Extensive polymorphisms have been associated with both TNF receptor genes. Both TNFRs share the typical structure of cell surface receptors including extracellular, transmembrane and intracellular domains. The extracellular portion of type 1 and type 2 TNFRs contains a repetitive amino acid sequence pattern of four cysteine rich domains (CDRs). A similar repetitive pattern of CDRs exist in several other cell surface proteins, including p75 nerve growth factor receptor, the B-cell antigen CD40 amongst others.
The receptors are powerful tools to elucidate biological pathways because of their easy conversion to immunoglobulin fusion proteins. These dimeric soluble receptor forms are good inhibitors of events mediated by either secreted or surface bound ligands. By binding to these ligands they prevent the ligand from interacting with cell associated receptors that can signal. Not only are these receptor-Fc fusion proteins useful in an experimental sense, but they have been successfully used clinically in the case of TNF-R-Fc to treat inflammatory bowel disease, rheumatoid arthritis and the acute clinical syndrome accompanying OKT3 administration (Eason et al. (1996) Transplantation 61(2):224-228; Feldmann et al. (1996) Int. Arch. Allergy Immunol. 111(4):362-365; and van Dullemen et al. (1995) Gastroenterol. 109(1):129-135). One can envision that manipulation of the many events mediated by signaling through the TNF family of receptors will have wide application in the treatment of immune based diseases and also the wide range of human diseases that have pathological sequelae due to immune system involvement. A soluble form of a recently described receptor, osteoprotegerin, can block the loss of bone mass and, therefore, the events controlled by TNF family receptor signaling are not necessarily limited to immune system regulation (Simonet et al. (1997) Cell 89(2):309-319). Antibodies to the receptor can block ligand binding and hence can also have clinical application. Such antibodies are often very long-lived and may have advantages over soluble receptor-Fc fusion proteins which have shorter blood half-lives.
While inhibition of the receptor mediated pathway represents the most exploited therapeutic application of these receptors, originally it was the activation of the TNF receptors that showed clinical promise (Aggarwal and Natarajan (1996) Eur Cytokine Netw. 7(2):93-124). Activation of the TNF receptors can initiate cell death in the target cell and hence the application to tumors was and still is attractive (Eggermont et al. (1996) Ann. Surg. 224(6):756-765). The receptor can be activated either by administration of the ligand, i.e. the natural pathway or some antibodies that can crosslink the receptor are also potent agonists. Antibodies would have an advantage in oncology since they can persist in the blood for long periods whereas the ligands generally have short lifespans in the blood. As many of these receptors may be expressed more selectively in tumors or they may only signal cell-death or differentiation in tumors, agonist antibodies could be good weapons in the treatment of cancer. Likewise, many positive immunological events are mediated via the TNF family receptors, e.g. host inflammatory reactions, antibody production etc. and therefore agonistic antibodies could have beneficial effects in other, non-oncological applications.
Paradoxically, the inhibition of a pathway may have clinical benefit in the treatment of tumors. For example the Fas ligand is expressed by some tumors and this expression can lead to the death of Fas positive lymphocytes thus facilitating the ability of the tumor to evade the immune system in this case, inhibition of the Fas system could then allow the immune system to react to the tumor in other ways now that access is possible (Green and Ware (1997) Proc. Natl. Acad. Sci. USA 94(12):5986-90).
The TNF family ligand BAFF, also known as TALL-1, THANK, BLyS and zTNF4 (Schneider et al. (1999) J. Exp. Med. 189(11):1747-1756; Shu et al. (1999) J. Leukoc. Biol. 65(5):680-683; Mukhopadhyay et al. (1999) J. Biol. Chem. 274(23):15978-15981; Moore et al. (1999) Science 285(5425):260-263; Gross et al. (2000) Nature 404(6780:995-999) enhances B cell survival in vitro (Batten et al. (2000) J. Exp. Med. 192(10):1453-1466) and has emerged as a key regulator of peripheral B cell populations in vivo. Mice over-expressing BAFF display mature B cell hyperplasia and symptoms of systemic lupus erythaematosus (SLE) (Mackay et al. (1999) J. Exp. Med. 190(11):1697-1710). As well, some SLE patients have significantly increased levels of BAFF in their serum (Zhang et al. (2001) J. Immunol. 166(1):6-10). It has therefore been proposed that abnormally high levels of this ligand may contribute to the pathogenesis of autoimmune diseases by enhancing the survival of autoreactive B cells (Batten et al. (2000) J. Exp. Med. 192(10):1453-1466).
BAFF, a type 13 membrane protein, is produced by cells of myeloid origin (Schneider et al. (1999) J. Exp. Med. 189(11):1747-1756; Moore et al. (1999) Science 285(5425):260-263) and is expressed either on the cell surface or in a soluble form (Schneider et al. (1999) J. Exp. Med. 189(11):1747-1756). Two TNF receptor family members, BCMA and TACI have previously been shown to interact with BAFF (Gross et al. (2000) Nature 404:995-999; Thompson et al. (2000) J. Exp. Med. 192(1):129-135; Xia et al. (2000) J. Exp. Med. 192:137-143; Marsters et al. (2000) Curr. Biol. 10(13):785-788; Shu et al. (2000) J. Leukoc. Biol. 65(5):680-683; Wu et al. (2000) J. Biol. Chem. 275:35478-35485).
SUMMARY OF THE INVENTION
The present invention is based, in part, upon the discovery of “BAFF-R,” a BAFF receptor protein, polynucleotide sequences and the BAFF-R polypeptides encoded by these nucleic acid sequences.
In one aspect, the invention provides an isolated nucleic acid which encodes a BAFF-R polypeptide, or a fragment or derivative thereof. The nucleic acid can include, e.g., nucleic acid sequence encoding a polypeptide at least 50% identical, or at least 90% identical, to a polypeptide comprising the amino acid sequence of FIG. 2D (SEQ ID NO:5).
The invention also provides a substantially pure nucleic acid molecule comprising a sequence that hybridizes under stringent conditions to a hybridization probe, the nucleic acid sequence of the probe consisting of the coding sequence of FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4) or the complement of said coding sequence.
In some embodiments, the nucleic acid sequence encodes a polypeptide having the sequence of FIG. 2D (SEQ ID NO:5) with at least one conservative amino acid substitution.
In some embodiments, the nucleic acid sequence encodes a polypeptide that binds BAFF.
The nucleic acid can include, e.g., a nucleic acid which includes the nucleotide sequence shown in FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4) and FIG. 3 (SEQ ID NO:6).
The nucleic acid can be, e.g., a genomic DNA fragment, or it can be a cDNA molecule. Also included in the invention is a vector containing one or more of the nucleic acids described herein, and a cell containing the vectors or nucleic acids described herein.
In another aspect, the invention provides a substantially pure nucleic acid molecule encoding a fusion protein comprising at least two segments, wherein one of the segments comprises a polypeptide or fragment thereof as described in the amino acid sequences set forth in the above embodiments of the invention. The invention also provides a fusion protein comprising at least two or three segments, wherein the first segment comprises a heterologous signal polypeptide, the second comprises a polypeptide or fragment thereof as described in the BAFF-R amino acid sequences set forth in the above embodiments of the invention and the third segment comprises an immunoglobulin polypeptide. Alternatively, the first segment comprises an immunoglobulin polypeptide fragment containing a signal sequence and the second segment comprises the BAFF-R polypeptide fragment.
In other aspects, the invention provides a substantially pure binding agent that specifically binds to the polypeptide of the above-stated embodiments of the invention.
The present invention is also directed to host cells transformed with a recombinant expression vector comprising any of the nucleic acid molecules described above.
In another aspect, the invention includes a pharmaceutical composition that includes a BAFF-R nucleic acid and a pharmaceutically acceptable carrier or diluent.
In a further aspect, the invention includes a substantially purified BAFF-R polypeptide, e.g., any of the polypeptides encoded by a BAFF-R nucleic acid.
The invention also includes a pharmaceutical composition that includes a BAFF-R polypeptide and a pharmaceutically acceptable carrier or diluent.
In a still further aspect, the invention provides an antibody that binds specifically to a BAFF-R polypeptide. The antibody can be, e.g., a monoclonal or polyclonal antibody. The invention also includes a pharmaceutical composition including BAFF-R antibody and a pharmaceutically acceptable carrier or diluent. The present invention is also directed to isolated antibodies that bind to an epitope on a polypeptide encoded by any of the nucleic acid molecules described above.
The present invention is further directed to kits comprising antibodies that bind to a polypeptide encoded by any of the nucleic acid molecules described above and a negative control antibody.
The invention further provides a method for producing a BAFF-R polypeptide. The method includes providing a cell containing a BAFF-R nucleic acid, e.g., a vector that includes a BAFF-R nucleic acid, and culturing the cell under conditions sufficient to express the BAFF-R polypeptide encoded by the nucleic acid. The expressed BAFF-R polypeptide is then recovered from the cell. Preferably, the cell produces little or no endogenous BAFF-R polypeptide. The cell can be, e.g., a prokaryotic cell or eukaryotic cell.
The present invention provides a method of inducing an immune response in a mammal against a polypeptide encoded by any of the nucleic acid molecules disclosed above by administering to the mammal an amount of the polypeptide sufficient to induce the immune response.
The present invention is also directed to methods of identifying a compound that binds to BAFF-R polypeptide by contacting the BAFF-R polypeptide with a compound and determining whether the compound binds to the BAFF-R polypeptide.
The present invention is also directed to methods of identifying a compound that binds a nucleic acid molecule encoding BAFF-R polypeptide by contacting BAFF-R nucleic acid with a compound and determining whether the compound binds the nucleic acid molecule.
The invention further provides methods of identifying a compound that modulates the activity of a BAFF-R polypeptide by contacting BAFF-R polypeptide with a compound and determining whether the BAFF-R polypeptide activity is modified.
The present invention is also directed to compounds that modulate BAFF-R polypeptide activity identified by contacting a BAFF-R polypeptide with the compound and determining whether the compound modifies activity of the BAFF-R polypeptide, binds to the BAFF-R polypeptide, or binds to a nucleic acid molecule encoding a BAFF-R polypeptide.
In another aspect, the invention provides a method of diagnosing a B-cell mediated condition, e.g., an autoimmune disorder or cancer, in a subject. The method includes providing a protein sample from the subject and measuring the amount of BAFF-R polypeptide in the subject sample. The amount of BAFF-R in the subject sample is then compared to the amount of BAFF-R polypeptide in a control protein sample. An alteration in the amount of BAFF-R polypeptide in the subject protein sample relative to the amount of BAFF-R polypeptide in the control protein sample indicates the subject has a B-cell mediated condition. A control sample is preferably taken from a matched individual, i.e., an individual of similar age, sex, or other general condition but who is not suspected of having the condition. Alternatively, the control sample may be taken from the subject at a time when the subject is not suspected of having the disorder. In some embodiments, the BAFF-R polypeptide is detected using a BAFF-R antibody.
In a further aspect, the invention includes a method of diagnosing a B-cell mediated condition, e.g., autoimmune disorder in a subject. The method includes providing a nucleic acid sample, e.g., RNA or DNA, or both, from the subject and measuring the amount of the BAFF-R nucleic acid in the subject nucleic acid sample. The amount of BAFF-R nucleic acid sample in the subject nucleic acid is then compared to the amount of BAFF-R nucleic acid in a control sample. An alteration in the amount of BAFF-R nucleic acid in the sample relative to the amount of BAFF-R in the control sample indicates the subject has an autoimmune condition.
In a further aspect, the invention includes a method of diagnosing a tumorigenic or autoimmune condition in a subject. The method includes providing a nucleic acid sample from the subject and identifying at least a portion of the nucleotide sequence of a BAFF-R nucleic acid in the subject nucleic acid sample. The BAFF-R nucleotide sequence of the subject sample is then compared to a BAFF-R nucleotide sequence of a control sample. An alteration in the BAFF-R nucleotide sequence in the sample relative to the BAFF-R nucleotide sequence in said control sample indicates the subject has such a condition.
In a still further aspect, the invention provides method of treating or preventing or delaying a B-cell mediated condition. The method includes administering to a subject in which such treatment or prevention or delay is desired a BAFF-R nucleic acid, a BAFF-R polypeptide, or an anti-BAFF-R antibody in an amount sufficient to treat, prevent, or delay a tumorigenic or immunoregulatory condition in the subject.
The conditions diagnosed, treated, prevented or delayed using the BAFF-R nucleic acid molecules, polypeptides or antibodies can be a cancer or an immunoregulatory disorder. Diseases include those that are autoimmune in nature such as systemic lupus erythematosus, rheumatoid arthritis, myasthenia gravis, autoimmune hemolytic anemia, idiopathic thrombocytopenia purpura, anti-phospholipid syndrome, Chagas' disease, Grave's disease, Wegener's granulomatosis, poly-arteritis nodosa and rapidly progressive glomerulonephritis. The therapeutic agent also has application in plasma cell disorders such as multiple myeloma, Waldenstrom's macroglobulinemia, heavy-chain disease, primary or immunocyte-associated amyloidosis, and monoclonal ganunopathy of undetermined significance (MGUS). Oncology targets include B cell carcinomas, leukemias, and lymphomas.
Compositions and methods of treatment using the nucleic acids, polypeptides and antibodies of the present invention can be used with any condition associated with undesired cell proliferation. In particular, the present invention can be used to treat tumor-cells which express BAFF and/or BAFF-R.
Compositions of the invention comprising BAFF-R agonists (such as antibodies that bind to BAFF-R and mimic BAFF) also may be used to treat immune deficiencies marked by low amounts of B cells, for example. Such disorders may be caused by radiation and/or chemotherapy, for example.
In another aspect of the invention a method for decreasing aggregation of a recombinantly expressed protein is provided. The method comprises comparison of homologs of a protein or fusion protein thereof to determine conserved domains and non-identical amino acids within conserved regions. Generally, at least one non-polar amino acid is changed to an uncharged polar amino acid or to a proline, alanine or serine.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claim.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows the DNA sequence of the BJAB cDNA (SEQ ID NO:1) cloned in pJST576.
FIG. 1B shows the complete DNA sequence of the cDNA of the IMAGE clone 2000271 (EST AI250289) (SEQ ID NO:2).
FIG. 2A shows the nucleotide sequence of JST576 with an intron removed as predicted by the GENESCAN program (SEQ ID NO:3).
FIG. 2B shows a 1% agarose gel of PCR products obtained for BAFF-R using either first strand cDNA generated from BJAB or IM-9 RNA or on JST576 cDNA. Lane 1. Lambda DNA HindIII digest Lane 2. BJAB oligo dT primed BAF-225/BAF-191. Lane 3. BJAB oligo dT primed BAF-226/BAF-191. Lane 4. BJAB random primed BAF-225/BAF-191. Lane 5. BJAB random primed BAF-226/BAF-191. Lane 6. IM-9 oligo dT primed BAF-225/BAF-191. Lane 7. IM-9 oligo dT primed BAF-226/BAF-191. Lane 8. IM-9 random primed BAF-225/BAF-191. Lane 9. IM-9 random primed BAF-226/BAF-191. Lane 10. JST576 cDNA BAF-225/BAF-191. Lane 11. JST576 cDNA BAF-226/BAF-191. Lane 12. No template BAF-225/BAF-191. Lane 13. No template BAF-226/BAF-191.
FIG. 2C shows the mature JST576(BAFF-R) sequence (SEQ ID NO:4) (also GenBank Accession No. AF373846) determined by sequencing bulk PCR product flanking the predicted intron from BJAB first strand cDNA.
FIG. 2D shows the amino acid sequence of BAFF-R (JST576) (SEQ ID NO:5). The A (Ala) residue in bold indicates the sequence resulting from the use of the alternative splice acceptor site. The predicted transmembrane domain is boxed and the putative stop transfer signal is underlined.
FIG. 3 depicts the spliced version of JST576 (SEQ ID NO:6) containing 5′ UTR sequence obtained by RT-PCR from human spleen first strand cDNA, and the deduced amino acid sequence (SEQ ID NO:7). This sequence contains an upstream stop codon in frame with the ATG.
FIG. 4A shows the sequence of the murine BAFF-R cDNA (SEQ ID NO:8) (also GenBank Accession No. AF373847).
FIG. 4B shows the amino acid sequence of murine BAFF-R (SEQ ID NO:9). The Cys residues are bold and underlined and the predicted transmembrane region is boxed.
FIG. 4C shows the homology between human (SEQ ID NO:10) and murine (SEQ ID NO:9) BAFF-R protein sequences.
FIG. 5 depicts human BAFF binding to JST576 transfected cells. 293EBNA cells were co-transfected with pJST576 or CA336 (huTACI) and a GFP reporter construct. Cells assayed for BAFF binding with 1 ug/ml biotinylated myc-huBAFF followed by SAV-PE.
FIG. 6 shows human and murine BAFF binding to JST576 transfected cells. 293EBNA cells were co-transfected with pJST576 and a GFP reporter construct. Cells assayed 24 hr later for BAFF binding with 5 ug/ml flag-huBAFF or flag-muBAFF followed anti-flag monoclonal antibody M2 and donkey anti-mouse IgG-PE.
FIG. 7 shows that APRIL does not bind to JST576 transfected cells. 293EBNA cells were co-transfected with pJST576 or CA336 (huTACI) and a GFP reporter construct. Cells were assayed for APRIL binding with 1 ug/m myc-muAPRIL followed by anti-muAPRIL rat-IgG2b, biotinylated anti-rat FcG2b, and SAV-PE.
FIG. 8 shows that BAFF precipitates a protein from JST576 transfected-cells. 293EBNA cells were transfected with either BAFF-R (pJST576), vector only (CH269) or huTACI (CA336) and pulsed with 35S cysteine and methionine. Extracts were immunoprecipitated with flag-huBAFF and run on a reducing SDS-PAGE gel. Molecular weight markers are indicated at left.
FIG. 9 depicts the nucleic acid sequence (SEQ ID NO:11) and its derived amino acid sequence (SEQ ID NO:12) of a gene encoding a human BAFF-R:Fc: nucleic acid residues 1-63 encode the murine IgG-kappa signal sequence; nucleic acid residues 64-66 were used to introduce a restriction enzyme site, nucleic acid residues 67-276 encode part of the BAFF-R extracellular domain, nucleic acid residues 277-279 were used to introduce a restriction enzyme site, and nucleic acid residues 280-960 encode the Fc region of human IgG1.
FIG. 10 depicts the results of Northern blot analysis using the EcoNI fragment of JST56 as a probe. All exposures are 4 days. 10A: Clontech human Immune II blot; 10B: Clontech human 12 lane multi-tissue blot; 10C: Clontech human multi-tissue II blot.
FIG. 11 shows the result of Northern blot analysis of 20 μg of total RNA isolated from various cell lines. The blot was probed with an EcoNI restriction fragment of JST576 and exposed for 4 days. The ability of the cell lines to bind BAFF, as determined by FACS analysis, is indicated below the lane.
FIG. 12 shows the results of immunoprecipitation results using BAFF-R:Fc. Human BAFF is immunoprecipitated with BAFF-R:Fc or BCMA:Fc, but not Fn14-Fc. Control BAFF protein is shown in lane 1.
FIG. 13 shows that human BAFF-R:Fc blocks human BAFF binding to BJAB cells. The results of FACS analysis are shown in FIG. 13A. Curve E represents biotinylated BAFF binding to BJAB cells in the absence of BAFF-R:Fc. Curves B-D represent the ability of BAFF to bind to BJAB cells in the presence of 5 ug/ml, 1 ug/ml or 0.2 ug/ml, respectively. Curve A is the second step only curve. FIG. 13B illustrates the ability of various concentrations of BAFF-R:Fc (squares) compared to TACI:Fc (triangles) or a nonspecific fusion protein, LT_R:Fc (circles), to block the binding of BAFF to the receptor expressing BJAB cells.
FIG. 14 shows the ability of BAFF-R:Fc to block BAFF-induced co-stimulation of splenic B cells. A graph of [3H] thymidine incorporation (cpm) versus increasing amounts of hBAFF (ng/ml) is shown.
FIG. 15 shows that BAFF-R:Fc1 treatment results in a loss of peripheral B cells in normal mice.
FIG. 16 shows that treatment of mice with human and mouse BAFF-R:Fc reduces the number of splenic B220+ B cells.
FIG. 17 shows that administration of BAFF-R:Fc to mice reduces the percentage of lymph node B220+ B cells.
FIG. 18 shows that administration of BAFF-R:Fc to mice reduces peripheral blood B220+ B cells.
FIG. 19A shows FACS data from supernatants of four clones that produce antibodies that bind BAFF-R. Also shown is control supernatant which does not contain antibodies that binds BAFF-R.
FIG. 19B shows a histogram showing that two clones that block BAFF binding to BAFF-R. (a) shows the no BAFF control; (b) shows the blocking ability of the antibody from clone 2; (c) shows the blocking ability of the antibody from clone 9; and (d) shows the curve from a control antibody that does not bind BAFF-R.
FIG. 20 shows an alignment of the amino acid sequences of human BAFF-R:Fc (hBAFF-R) and mouse BAFF-R:Fc (mBAFF-R) extracellular domains and the percentage of aggregation observed upon expression of the Fc fusion proteins containing the indicated sequences. Numbered JST clones represent the amino acid sequences showing mutations (shown in underline) in the parent sequences and the resulting aggregation of expressed protein. Shown are the partial sequences for human (amino acids 2-71 of SEQ ID NO:1O; SEQ ID NO:13) and mouse (amino acids 2-71 of SEQ ID NO:9; SEQ ID NO:14) BAFF-R; and the corresponding portions for the following clones: JST659 (SEQ ID NO:15), JST66O (SEQ ID NO:16, JST661 (SEQ ID NO:17), JST662 (SEQ ID NO:18), JST663 (SEQ ID NO:19), JST673 (SEQ ID NO:20), JST674 (SEQ ID NO:21), JST675 (SEQ ID NO:22), JST672 (SEQ ID NO:23), JST676 (SEQ ID NO: 24), JST671 (SEQ ID NO:25), JST677 (SEQ ID NO:26), JST678 (SEQ ID NO:27), JST664 (SEQ ID NO:28), JST668 (SEQ ID NO:29), JST665 (SEQ ID NO:30), JST666 (SEQ ID NO: 31), and JST667 (SEQ ID NO:32).
FIG. 21 shows an autoradiograph of proteins immunoprecipitated using lysates prepared from BAFF-R-i.c.d. (BAFF-R intracellular domain) (lane 1), or control vector- (lane 2) transfected cells. Approximately 6×106 293E cells were transfected with a construct encoding BAFFR-i.c.d. or mock plasmid. After 48 hours, the cells were metabolically labeled with 35S for 24 hours, lysed with lysis buffer, precleared, and immunoprecipitated with an antimyc mAb, 9E10. The immunoprecipitates were separated by 10-20% SDS PAGE under reducing condition.
DETAILED DESCRIPTION OF THE INVENTION
The reference works, patents, patent applications, and scientific literature, including accession numbers to GenBank database sequences, that are referred to herein establish the knowledge of those with skill in the art and are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Likewise, any conflict between an art-understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter.
Standard reference works setting forth the general principles of recombinant DNA technology known to those of skill in the art include Ausubel et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York (1998); Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2D ED., Cold Spring Harbor Laboratory Press, Plainview, N.Y. (1989); Kaufman et al., Eds., HANDBOOK OF MOLECULAR AND CELLULAR METHODS IN BIOLOGY AND MEDICINE, CRC Press, Boca Raton (1995); McPherson, Ed., DIRECTED MUTAGENESIS: A PRACTICAL APPROACH, IRL Press, Oxford (1991).
The present invention discloses BAFF-R nucleic acids, isolated nucleic acids that encode BAFF-R polypeptide or a portion thereof, BAFF-R polypeptides, vectors containing these nucleic acids, host cells transformed with the BAFF-R nucleic acids, anti-BAFF-R antibodies, and pharmaceutical compositions. Also disclosed are methods of making BAFF-R polypeptides, as well as methods of screening, diagnosing, treating conditions using these compounds, and methods of screening compounds that modulate BAFF-R polypeptide activity.
The BAFF-R nucleic acids and polypeptides, as well as BAFF-R antibodies, as well as pharmaceutical compositions discussed herein, are useful, inter alia, in treating cancer and/or immunoregulatory conditions. These disorders include, e.g., B cell-mediated diseases that are autoimmune in nature such as systemic lupus erythematosus, rheumatoid arthritis myasthenia gravis, autoimmune hemolytic anemia, idiopathic thrombocytopenia purpura, anti-phospholipid syndrome, Chagas' disease, Grave's disease, Wegener's granulomatosis, poly-arteritis nodosa and rapidly progressive glomerulonephritis. This therapeutic agent also has application in plasma cell disorders such as multiple myeloma, Waldenstrom's macroglobulinemia, heavy-chain disease, primary or immunocyte-associated amyloidosis, and monoclonal gammopathy of undetermined significance (MGUS). Oncology targets include B cell carcinomas, leukemias, and lymphomas.
BAFF-R Nucleic Acids
One aspect of the invention pertains to isolated nucleic acid molecules that encode BAFF-R proteins or biologically active portions thereof. Also included are nucleic acid fragments sufficient for use as hybridization probes to identify BAFF-R-encoding nucleic acids (e.g., BAFF-R mRNA) and fragments for use as polymerase chain reaction (PCR) primers for the amplification or mutation of BAFF-R nucleic acid molecules. As used herein, the term “nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
“Probes” refer to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt) or as many as about, e.g., 6,000 nt, depending on use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
An “isolated” nucleic acid molecule is one that is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Examples of isolated nucleic acid molecules include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated BAFF-R nucleic acid molecule can contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4) and FIG. 3 (SEQ ID NO:6), or a complement of any of these nucleotide sequences, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequences of FIGS. 1A, B, 2A, C and 3 as a hybridization probe, BAFF-R nucleic acid sequences can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., Eds., MOLECULAR CLONING: A LABORATORY MANUAL 2ND ED., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausubel, et al., Eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993).
A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to BAFF-R nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
As used herein, the term “oligonucleotide” refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise portions of a nucleic acid sequence having at least about 10 nt and as many as 50 nt, preferably about 15 nt to 30 nt. They may be chemically synthesized and may be used as probes.
In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3 (SEQ ID NO:6). In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3 (SEQ ID NO:6), or a portion of this nucleotide sequence. A nucleic acid molecule that is complementary to the nucleotide sequence shown in FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3 (SEQ ID NO:6) is one that is sufficiently complementary to the nucleotide sequence shown in FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ NO:4), and FIG. 3 (SEQ ID NO:6) that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown in FIG. 1A (SEQ ID NO:1, FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4) and FIG. 3 (SEQ ID NO:6) thereby forming a stable duplex.
As used herein, the term “complementary” refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and the term “binding” means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, Van der Waals, hydrophobic interactions, etc. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
Moreover, the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3 (SEQ ID NO:6), e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically active portion of BAFF-R. Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type.
Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below. Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 45%, 50%, 70%, 80%, 95%, 98%, or even 99% identity (with a preferred identity of 80-99%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993, and below. An exemplary program is the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison, Wis.) using the default settings, which uses the algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482-489, which is incorporated herein by reference in its entirety).
A “homologous nucleic acid sequence” or “homologous amino acid sequence,” or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences encode those sequences coding for isoforms of BAFF-R polypeptide. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. In the present invention, homologous nucleotide sequences include nucleotide sequences encoding for a BAFF-R polypeptide of species other than humans, including, but not limited to, mammals, and thus can include, e.g., mouse, rat, rabbit, dog, cat cow, horse, and other organisms. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however, include the nucleotide sequence encoding human BAFF-R protein. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in FIG. 2D (SEQ ID NO:5) as well as a polypeptide having BAFF-R activity. A homologous amino acid sequence does not encode the amino acid sequence of a human BAFF-R polypeptide.
The nucleotide sequence determined from the cloning of the human BAFF-R gene allows for the generation of probes and primers designed for use in identifying and/or cloning BAFF-R homologues in other cell types, e.g., from other tissues, as well as BAFF-R homologues from other mammals. The probe/primer typically comprises a substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence of any of FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4) and FIG. 3 (SEQ ID NO:6) or an anti-sense strand nucleotide sequence of any of FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and 3 (SEQ ID NO:6) or of a naturally occurring mutant of any of FIG. 1A (SEQ ID NO:1), Fig. B (SEQ NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3 (SEQ ID NO:6).
Probes based on the human BAFF-R nucleotide sequence can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a BAFF-R protein, such as by measuring a level of a BAFF-R-encoding nucleic acid in a sample of cells from a subject e.g., detecting BAFF-R mRNA levels or determining whether a genomic BAFF-R gene has been mutated or deleted.
“A polypeptide having a biologically active portion of BAFF-R” refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. A nucleic acid fragment encoding a “biologically active portion of BAFF-R” can be prepared by isolating a portion of any of FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3 (SEQ ID NO:6) that encodes a polypeptide having a BAFF-R biological activity (biological activities of the BAFF-R proteins are described below), expressing the encoded portion of BAFF-R protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of BAFF-R. For example, a nucleic acid fragment encoding a biologically active portion of BAFF-R can optionally include a BAFF binding domain. In another embodiment, a nucleic acid fragment encoding a biologically active portion of BAFF-R includes one or more regions.
BAFF-R Variants
The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and 3 (SEQ ID NO:6) due to degeneracy of the genetic code. These nucleic acids thus encode the same BAFF-R protein as that encoded by the nucleotide sequence shown in FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3 (SEQ ID NO:6). In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in FIG. 2D (SEQ ID NO:5).
In addition to the human BAFF-R nucleotide sequence shown in any of FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3 (SEQ ID NO:6), it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of BAFF-R may exist within a population (e.g., the human population). Such genetic polymorphism in the BAFF-R gene may exist among individuals within a population due to natural allelic variation. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a BAFF-R protein, preferably a mammalian BAFF-R protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the BAFF-R gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in BAFF-R that are the result of natural allelic variation and that do not alter the functional activity of BAFF-R are intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding BAFF-R proteins from other species, and thus that have a nucleotide sequence that differs from the human sequences of FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3 (SEQ ID NO:6) are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the BAFF-R cDNAs of the invention can be isolated based on their homology to the human BAFF-R nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization-conditions. For example, a soluble human BAFF-R cDNA can be isolated based on its homology to human membrane-bound BAFF-R. Likewise, a membrane-bound human BAFF-R cDNA can be isolated based on its homology to soluble human BAFF-R.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of any of FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3 (SEQ ID NO:6). In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250 or 500 nucleotides in length. In another embodiment, an isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
Homologs (i.e., nucleic acids encoding BAFF-R proteins derived from species other than human) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular human sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.
As used herein, the phrase “stringent hybridization conditions” refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes, primers or oligonucleotides 10 nt to 50 nt) and at least about 60° C. for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
Stringent conditions are known to those skilled in the art and can be found in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions is hybridization in a high salt buffer comprising 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65° C. This hybridization is followed by one or more washes in 0.2×SSC, 0.01% BSA at 50° C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6 corresponds to a naturally occurring nucleic acid molecule. As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of any of FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and 3 (SEQ ID NO:6) or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55° C., followed by one or more washes in 1×SSC, 0.1% SDS at 37° C. Other conditions of moderate stringency that may be used are well known in the art. See, e.g., 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.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of any of FIG. 1A (SEQ ID NO:1), FIG. 2B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3 (SEQ ID NO:6) or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40° C., followed by one or more washes in 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50° C. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel et at, Eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, 1993; and Kriegler, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY, 1990; Shilo and Weinberg (1981) Proc. Natl. Acad. Sci. USA 78:6789-6792.
Conservative Mutations
In addition to naturally-occurring allelic variants of the BAFF-R sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequence of FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), FIG. 3 (SEQ ID NO:6) thereby leading to changes in the amino acid sequence of the encoded BAFF-R protein, without altering the functional ability of the BAFF-R protein. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in the sequence of any of FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3 (SEQ ID NO:6). A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of BAFF-R without altering the biological activity, whereas an “essential” amino acid residue is required for biological activity. For example, amino acid residues that are conserved among the BAFF-R proteins of the present invention, are predicted to be particularly unamenable to alteration.
In addition, amino acid residues that are conserved among family members of the BAFF-R proteins of the present invention, are also predicted to be particularly unamenable to alteration. For example, BAFF-R proteins of the present invention can contain at least one domain that is a typically conserved region in TNF family members. As such, these conserved domains are not likely to be amenable to mutation. Other amino acid residues, however, (e.g., those that are not conserved or only semi-conserved among members of the BAFF-R proteins) may not be essential for activity and thus are likely to be amenable to alteration.
Another aspect of the invention pertains to nucleic acid molecules encoding BAFF-R proteins that contain changes in amino acid residues that are not essential for activity. Such BAFF-R proteins differ in amino acid sequence from FIG. 2D (SEQ ID NO:5), yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of FIG. 2D (SEQ ID NO:5). Preferably, the protein encoded by the nucleic acid molecule is at least about 60% homologous to FIG. 2D (SEQ ID NO:5), more preferably at least about 70%, 80%, 90%, 95%, 98%, and most preferably at least about 99% homologous to FIG. 2D (SEQ ID NO:5).
An isolated nucleic acid molecule encoding a BAFF-R protein homologous to the protein of FIG. 2D can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3 (SEQ ID NO:6) such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
Mutations can be introduced into FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), or FIG. 3 (SEQ ID NO:6), for example, by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in BAFF-R is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a BAFF-R coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for BAFF-R biological activity to identify mutants that retain activity. Following mutagenesis of any of FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3 (SEQ ID NO:6), the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.
In one embodiment, a mutant BAFF-R protein can be assayed for (1) the ability to form protein:protein interactions with other BAFF-R proteins, other cell-surface proteins, or biologically active portions thereof, (2) complex formation between a mutant BAFF-R protein and a BAFF-R ligand; (3) the ability of a mutant BAFF-R protein to bind to an intracellular target protein or biologically active portion thereof; (e.g., avidin proteins); (4) the ability to bind BAFF; or (5) the ability to specifically bind a BAFF-R protein antibody.
The invention provides specific mutants encoding a BAFF-R:Fc polypeptidedesigned to alleviate aggregation of expressed protein while maintaining BAFF binding activity. Such mutants, include, for example, clones encoding the amino acid sequences of JST661 (SEQ ID NO:17), JST662 (SEQ ID NO:18), JST663 (SEQ ID NO:19), JST673 (SEQ ID NO:20), JST674 (SEQ ID NO:21), JST675 (SEQ ID NO:22), JST672 (SEQ ID NO:23), JST676 (SEQ ID NO:24), JST671 (SEQ ID NO:25), JST677 (SEQ ID NO:26); and JST678 (SEQ ID NO:27). Other embodiments include mutants encoding a BAFF-R or BAFF-R:Fc polypeptide that has similar aggregation characteristics to native human BAFF-R or BAFF-R:Fc polypeptide, but also bind BAFF, including, for example, sequences comprising the amino acid sequences of JST659 (SEQ ID NO:15), JST660 (SEQ ID NO:16), JST664 (SEQ ID NO:28), JST668 (SEQ ID NO:29), JST665 (SEQ ID NO:30), JST666 (SEQ ID NO:31), and JST667 (SEQ ID NO:32). Other embodiments include mutants encoding a BAFF-R or BAFF-R:Fc polypeptide wherein conserved amino acids between human and mouse BAFF-R are changed to other conserved amino acids and wherein the binding activity of BAFF-R or BAFF-R:Fc polypeptide to BAFF is retained. In other embodiments, the mutants encode a BAFF-R or BAFF-R:Fc polypeptide having amino acids that are not conserved between human and mouse BAFF-R which have been changed to other amino acids. Preferably, at least one nonpolar amino acid is changed to a proline residue or an uncharged polar amino acid.
Antisense
Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of FIG. 2A, C, 3 or, or fragments, analogs or derivatives thereof. An “antisense” nucleic acid comprises a nucleotide sequence that is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire BAFF-R coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a BAFF-R protein of any of FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), FIG. 3 (SEQ ID NO:6) or antisense nucleic acids complementary to a BAFF-R nucleic acid sequence of any of FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), FIG. 3 (SEQ ID NO:6) are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence encoding BAFF-R. The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the protein coding region of human BAFF-R corresponds to nucleotides 13 to 568 of FIG. 2A (SEQ ID NO:3), or nucleotides 13 to 565 of FIG. 2C (SEQ ID NO:4) or nucleotides 298 to 849 of FIG. 3 (SEQ ID NO:6)). In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding BAFF-R. The term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions).
Given the coding strand sequences encoding BAFF-R disclosed herein, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of BAFF-R mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of BAFF-R mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of BAFF-R mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a BAFF-R protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gaultier et al. (1987) Nucl. Acids Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2′-O-methylribonucleotide (Inoue et al. (1987) Nucl. Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).
Ribozymes and PNA Moieties
In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave BAFF-R mRNA transcripts to thereby inhibit translation of BAFF-R mRNA. A ribozyme having specificity for a BAFF-R-encoding nucleic acid can be designed based upon the nucleotide sequence of a BAFF-R DNA disclosed herein (i.e., SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6). For example, a derivative of a Tetrahymena L-19 IVS RNA can be-constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a BAFF-R-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, BAFF-R mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.
Alternatively, BAFF-R gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the BAFF-R (e.g., the BAFF-R promoter and/or enhancers) to form triple helical structures that prevent transcription of the BAFF-R gene in target cells. See generally, Helene (1991) Anticancer Drug Des. 6: 569-84; Helene et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14:807-15.
In various embodiments, the nucleic acids of BAFF-R can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorg. Med. Chem. 4:5-23). As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996) Bioorg. Med. Chem. 4:5-23; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93:14670-675.
PNAs of BAFF-R can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of BAFF-R can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S1 nucleases (Hyrup B. (1996) Bioorg. Med. Chem. 4:5-23); or as probes or primers for DNA sequence and hybridization (Hyrup et al. (1996), Bioorg. Med. Chem. 4:5-23; Perry-O'Keefe (1996) Proc. Natl. Acad. Sci. USA 93:14670-675).
In another embodiment, PNAs of BAFF-R can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of BAFF-R can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) Bioorg. Med. Chem. 4:5-23). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) Bioorg. Med. Chem. 4:5-23; and Finn et al. (1996) Nucl. Acids Res. 24:3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5′ end of DNA (Mag et al. (1989) Nucl. Acids Res. 17:5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn et al. (1996) above). Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment. See, Petersen et al. (1975) Bioorg. Med. Chem. Lett. 5:1119-11124.
In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. W0 88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W0 89/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agent (see, e.g., Krol et al., (1988) BioTechniques 6:958-976) or intercalating agents (see, e.g., ion, (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.
BAFF-R Polypeptides
One aspect of the invention pertains to isolated BAFF-R proteins, and biologically active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-BAFF-R antibodies. In one embodiment, native BAFF-R proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, BAFF-R proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a BAFF-R protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
An “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the BAFF-R protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of BAFF-R protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language “substantially free of cellular material” includes preparations of BAFF-R protein having less than about 30% (by dry weight) of non-BAFF-R protein (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-BAFF-R protein, still more preferably less than about 10% of non-BAFF-R protein, and most preferably less than about 5% non-BAFF-R protein. When the BAFF-R protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
The language “substantially free of chemical precursors or other chemicals” includes preparations of BAFF-R protein in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of BAFF-R protein having less than about 30% (by dry weight) of chemical precursors or non-BAFF-R chemicals, more preferably less than about 20% chemical precursors or non-BAFF-R chemicals, still more preferably less than about 10% chemical precursors or non-BAFF-R chemicals, and most preferably less than about 5% chemical precursors or non-BAFF-R chemicals.
Biologically active portions of a BAFF-R protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the BAFF-R protein, e.g., the amino acid sequence shown in SEQ ID NO:5 that include fewer amino acids than the full length BAFF-R proteins, and exhibit at least one activity of a BAFF-R protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the BAFF-R protein. A biologically active portion of a BAFF-R protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
A biologically active portion of a BAFF-R protein of the present invention may contain at least one of the above-identified domains conserved between the BAFF-R proteins. An alternative biologically active portion of a BAFF-R protein may contain at least two of the above-identified domains. Another biologically active portion of a BAFF-R protein may contain at least three of the above-identified domains. Yet another biologically active portion of a BAFF-R protein of the present invention may contain at least four of the above-identified domains.
Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native BAFF-R protein.
In an embodiment, the BAFF-R protein has an amino acid sequence shown in FIG. 2D (SEQ ID NO:5). In other embodiments, the BAFF-R protein is substantially homologous to FIG. 2D (SEQ ID NO:5) and retains the functional activity of the protein of FIG. 2D (SEQ ID NO:5), yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail below. Accordingly, in another embodiment, the BAFF-R protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of FIG. 2D (SEQ ID NO:5) and retains the functional activity of the BAFF-R proteins of FIG. 2D (SEQ ID NO:5).
In some embodiments, the invention includes specific mutants of BAFF-R:Fc polypeptide designed to alleviate aggregation of expressed protein while maintaining BAFF binding activity. Such mutants, include, for example, clones encoding the amino acid sequences of JST661 (SEQ ID NO:17), JST662 (SEQ ID NO:18), JST663 (SEQ ID NO:19), JST673 (SEQ ID NO:20), JST674 (SEQ ID NO:21), JST675(SEQ ID NO:22), JST672 (SEQ ID NO:23), JST676 (SEQ ID NO:24), JST671 (SEQ ID NO:25), JST677 (SEQ ID NO:26), and JST678 (SEQ ID NO:27). Other embodiments include mutants encoding a BAFF-R or BAFF-R:Fc polypeptide that has similar aggregation characteristics to native human BAFF-R R or BAFF-R:Fc polypeptide, but also bind BAFF, including, for example, sequences comprising the amino acid sequences of JST659 (SEQ ID NO:15), JST660 (SEQ ID NO:16), JST664 (SEQ ID NO:28), JST668 (SEQ ID NO:29), JST665 (SEQ ID NO:30), JST666 (SEQ ID NO:31), and JST667 (SEQ ID NO:32). Other embodiments include mutants encoding a BAFF-R or BAFF-R:Fc polypeptide wherein conserved amino acids between human and mouse BAFF-R are changed to other conserved amino acids and wherein the binding activity of BAFF-R or BAFF-R:Fc polypeptide to BAFF is retained. In other embodiments, the mutants encode a BAFF-R or BAFF-R:Fc polypeptide having amino acids that are not conserved between human and mouse BAFF-R which have been changed to other amino acids. Preferably, nonpolar amino acids are mutated to proline or uncharged polar amino acids.
Determining Homology Between Two or More Sequences
To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid “homology” is equivalent to amino acid or nucleic acid “identity”).
The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence shown in FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), FIG. 3 (SEQ ID NO:6).
The term “sequence identity” refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term “substantial identity” as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
Chimeric and Fusion Proteins
The invention also provides BAFF-R chimeric or fusion proteins. As used herein, a BAFF-R “chimeric protein” or “fusion protein” comprises a BAFF-R polypeptide operatively linked to a non-BAFF-R polypeptide. A “BAFF-R polypeptide” refers to a polypeptide having an amino acid sequence corresponding to BAFF-R, whereas a “non-BAFF-R polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the BAFF-R protein, e.g., a protein that is different from the BAFF-R protein and that is derived from the same or a different organism. Within a BAFF-R fusion protein the BAFF-R polypeptide can correspond to all or a portion of a BAFF-R protein. In one embodiment, a BAFF-R fusion protein comprises at least one biologically active portion of a BAFF-R protein. In another embodiment, a BAFF-R fusion protein comprises at least two biologically active portions of a BAFF-R protein. In yet another embodiment a BAFF-R fusion protein comprises at least three biologically active portions of a BAFF-R protein. Within the fusion protein, the term “operatively linked” is intended to indicate that the BAFF-R polypeptide and the non-BAFF-R polypeptide are fused in-frame to each other. The non-BAFF-R polypeptide can be fused to the N-terminus or C-terminus of the BAFF-R polypeptide. The non-BAFF-R polypeptide may be, for example, the Fc portion of an antibody. This may be operatively joined to either the N-terminus or the C-terminus of the BAFF-R polypeptide. Fc-target protein fusions have been described in Lo et al. (1998) Protein Enginering 11:495-500, and U.S. Pat. Nos. 5,541,087 and 5,726,044. The disclosures of which are herein incorporated by reference.
For example, in one embodiment a BAFF-R fusion protein comprises a BAFF-R domain operably linked to the extracellular domain of a second protein. Such fusion proteins can be further utilized in screening assays for compounds which modulate BAFF-R activity (such assays are described in detail below).
In yet another embodiment, the fusion protein is a GST-BAFF-R fusion protein in which the BAFF-R sequences are fused to the C-terminus of the GST (i.e., glutathione S-transferase) sequences. Such fusion proteins can facilitate the purification of recombinant BAFF-R.
In another embodiment, the fusion protein is a BAFF-R protein containing a heterologous signal sequence at its N-terminus. For example, since BAFF-R does not contain its own signal sequence, a heterologous signal sequence must be fused to the 5′ end of the BAFF-R coding sequence for efficient secretion of the BAFF-R fusion protein. Expression and/or secretion of BAFF-R can be increased through use of different heterologous signal sequences.
In yet another embodiment, the fusion protein is a BAFF-R-immunoglobulin fusion protein in which the BAFF-R sequences comprising one or more domains are fused to sequences derived from a member of the immunoglobulin protein family. The BAFF-R-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a BAFF-R ligand and a BAFF-R protein on the surface of a cell, to thereby suppress BAFF-R-mediated signal transduction in vivo. The BAFF-R-immunoglobulin fusion proteins can be used to affect the bioavailability of a BAFF-R cognate ligand. Inhibition of the BAFF-R ligand/BAFF-R interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g. promoting or inhibiting) cell survival. Moreover, the BAFF-R-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-BAFF-R antibodies in a subject, to purify BAFF-R ligands, and in screening assays to identify molecules that inhibit the interaction of BAFF-R with a BAFF-R ligand.
A BAFF-R chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al. Eds. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A BAFF-R-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the BAFF-R protein.
In a preferred embodiment, the BAFF-R fusion is provided by the nucleic acid (SEQ ID NO:11) and amino acid (SEQ ID NO:12) sequences of FIG. 9.
BAFF-R Agonists and Antagonists
The present invention also pertains to variants of the BAFF-R proteins that function as either BAFF-R agonists (mimetics) or as BAFF-R antagonists. Variants of the BAFF-R protein can be generated by mutagenesis, e.g., discrete point mutation or truncation of the BAFF-R protein. An agonist of the BAFF-R protein can retain substantially the same, or a subset of, the biological activities of the naturally occurring form of the BAFF-R protein. An antagonist of the BAFF-R protein can inhibit one or more of the activities of the naturally occurring form of the BAFF-R protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the BAFF-R protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the BAFF-R proteins.
Variants of the BAFF-R protein that function as either BAFF-R agonists (mimeties) or as BAFF-R antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the BAFF-R protein for BAFF-R protein agonist or antagonist activity. In one embodiment, a variegated library of BAFF-R variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of BAFF-R variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential BAFF-R sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of BAFF-R sequences therein. There are a variety of methods which can be used to produce libraries of potential BAFF-R variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential BAFF-R sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Ann. Rev. Biochem. 53:323; Itakura et al. (1977) Science 198:1056-1063; Ike et al. (1983) Nucl. Acids Res. 11:477-488.
Polypeptide Libraries
In addition, libraries of fragments of the BAFF-R protein coding sequence can be used to generate a variegated population of BAFF-R fragments for screening and subsequent selection of variants of a BAFF-R protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a BAFF-R coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the BAFF-R protein.
Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of BAFF-R proteins. The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recrusive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify BAFF-R variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6:327-331).
Anti-BAFF-R Antibodies
An isolated BAFF-R protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind BAFF-R using standard techniques for polyclonal and monoclonal antibody preparation. The full-length BAFF-R protein can be used or, alternatively, the invention provides antigenic peptide fragments of BAFF-R for use as immunogens. The antigenic peptide of BAFF-R comprises at least 8 amino acid residues of the amino acid sequence shown in FIG. 2D (SEQ ID NO:5) and encompasses an epitope of BAFF-R such that an antibody raised against the peptide forms a specific immune complex with BAFF-R. Preferably, the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of BAFF-R that are located on the surface of the protein, e.g., hydrophilic regions.
As disclosed herein, BAFF-R protein sequence of FIG. 2D (SEQ ID NO:5), or derivatives, fragments, analogs or homologs thereof, may be utilized as immunogens in the generation of antibodies that immunospecifically-bind these protein components. The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen, such as BAFF-R. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab and F(ab′)2 fragments, and an Fab expression library. In a specific embodiment, antibodies to human BAFF-R proteins are disclosed. Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies to a BAFF-R protein sequence of FIG. 2D (SEQ ID NO:5) or derivative, fragment, analog or homolog thereof. Some of these proteins are discussed below.
For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by injection with the native protein, or a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation can contain, for example, recombinantly expressed BAFF-R protein or a chemically synthesized BAFF-R polypeptide. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerin (BCG) and Corynebacterium parvum, or similar immunostimulatory agents. If desired, the antibody molecules directed against BAFF-R can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction.
The term “monoclonal antibody” or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of BAFF-R. A monoclonal antibody composition thus typically displays a single binding affinity for a particular BAFF-R protein with which it immunoreacts. For preparation of monoclonal antibodies directed towards a particular BAFF-R protein, or derivatives, fragments, analogs or homologs thereof, any technique that provides for the production of antibody molecules by continuous cell line culture may be utilized. Such techniques include, but are not limited to, the hybridoma technique (see Kohler & Milstein, (1975) Nature 256:495-497); the trioma technique; the human B-cell hybridoma technique (see Kozbor et al. (1983) Immunol. Today 4:72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al. in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., 1985, pp. 77-96). Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole et al. in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., 1985 pp. 77-96).
According to the invention, techniques can be adapted for the production of single-chain antibodies specific to a BAFF-R protein (see e.g., U.S. Pat. No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse et al. (1989) Science 246:1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a BAFF-R protein or derivatives, fragments, analogs or homologs thereof. Non-human antibodies can be “humanized” by techniques well known in the art. See e.g., U.S. Pat. No. 5,225,539. Antibody fragments that contain the idiotypes to a BAFF-R protein may be produced by techniques known in the art including, but not limited to: (i) an F(ab′)2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab′)2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) Fv fragments.
Additionally, recombinant anti-BAFF-R antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT International Application No. PCT/US86/02269; European Patent Application No. 184,187; European Patent Application No. 171,496; European Patent Application No. 173,494; PCT International Publication No. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Application No. 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060.
In one embodiment, methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art. In a specific embodiment, selection of antibodies that are specific to a particular domain of a BAFF-R protein is facilitated by generation of hybridomas that bind to the fragment of a BAFF-R protein possessing such a domain. Antibodies that are specific for one or more domains within a BAFF-R protein, e.g., domains spanning the above-identified conserved regions of BAFF-R family proteins, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
Anti-BAFF-R antibodies may be used in methods known within the art relating to the localization and/or quantitation of a BAFF-R protein (e.g., for use in measuring levels of the BAFF-R protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies for BAFF-R proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antibody derived binding domain, are utilized as pharmacologically-active compounds (hereinafter “Therapeutics”).
An anti-BAFF-R antibody (e.g., monoclonal antibody) can be used to isolate BAFF-R by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-BAFF-R antibody can facilitate the purification of natural BAFF-R from cells and of recombinantly produced BAFF-R expressed in host cells. Moreover, an anti-BAFF-R antibody can be used to detect BAFF-R protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the BAFF-R protein. Anti-BAFF-R antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, B-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; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 123I, 131I, 35S or 3H.
BAFF-R Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding BAFF-R protein, or derivatives, fragments, analogs or homologs thereof. As used herein, the term “vector” refers 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 can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can 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) are 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 “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” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention 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 recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel “Gene Expression Technology” METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif., 1990. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., BAFF-R proteins, mutant forms of BAFF-R, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for expression of BAFF-R in prokaryotic or eukaryotic cells. For example, BAFF-R can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, “Gene Expression Technology” METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif., 1990. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (1) to increase expression of recombinant protein; (2) to increase the solubility of the recombinant protein; and (3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., “Gene Expression Technology” METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif., 1990, pp. 60-89).
One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacterium with an impaired capacity to proteolytically cleave the recombinant protein. See, Gottesman, “Gene Expression Technology” METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif., 1990, pp. 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the BAFF-R expression vector is a yeast expression vector. Examples of vectors for expression in yeast (e.g., Saccharomyces cerivisae) include pYepSec1 (Baldari, et al., (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz., (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and for P. pastoris include pPIC family of vectors (Invitrogen Corp, San Diego, Calif.).
Alternatively, BAFF-R can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith et al. (1983) Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840-842) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells. See, e.g., Chapters 16 and 17 of Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2ND ED., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter, U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine box promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to BAFF-R mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub et al. (1986) “Antisense RNA as a molecular tool for genetic analysis,” Reviews—Trends in Genetics, 1(1).
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential 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 as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, BAFF-R protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL 2ND ED., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding BAFF-R or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) BAFF-R protein. Accordingly, the invention further provides methods for producing BAFF-R protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding BAFF-R has been introduced) in a suitable medium such that BAFF-R protein is produced. In another embodiment, the method further comprises isolating BAFF-R from the medium or the host cell.
Transgenic Animals
The host cells of the invention can also be used to produce nonhuman transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which BAFF-R-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous BAFF-R sequences have been introduced into their genome or homologous recombinant animals in which endogenous BAFF-R sequences have been altered. Such animals are useful for studying the function and/or activity of BAFF-R and for identifying and/or evaluating modulators of BAFF-R activity. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a “homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous BAFF-R gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
A transgenic animal of the invention can be created by introducing BAFF-R-encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal The human BAFF-R DNA sequence of FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), FIG. 3 (SEQ ID NO:6) can be introduced as a transgene into the genome of a non-human animal. Alternatively, a nonhuman homologue of the human BAFF-R gene, such as a mouse BAFF-R gene (FIG. 4A) (SEQ ID NO:8), can be isolated based on hybridization to the human BAFF-R cDNA (described further above) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to the BAFF-R transgene to direct expression of BAFF-R protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866; 4,870,009; and 4,873,191; and Hogan in MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the BAFF-R transgene in its genome and/or expression of BAFF-R mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding BAFF-R can further be bred to other transgenic animals carrying other transgenes.
To create a homologous recombinant animal, a vector is prepared which contains at least a portion of a BAFF-R gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the BAFF-R gene. The BAFF-R gene can be a human gene (e.g., FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), FIG. 3 (SEQ ID NO:6)), but more preferably, is a non-human homologue of a human BAFF-R gene. For example, a mouse homologue (FIG. 4a) of human BAFF-R gene of FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), FIG. 3 (SEQ ID NO:6) can be used to construct a homologous recombination vector suitable for altering an endogenous BAFF-R gene in the mouse genome. In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous BAFF-R gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a “knock out” vector).
Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous BAFF-R gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous BAFF-R protein). In the homologous recombination vector, the altered portion of the BAFF-R gene is flanked at its 5′ and 3′ ends by additional nucleic acid of the BAFF-R gene to allow for homologous recombination to occur between the exogenous BAFF-R gene carried by the vector and an endogenous BAFF-R gene in an embryonic stem cell. The additional flanking BAFF-R nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5′ and 3′ ends) are included in the vector. See e.g., Thomas al. (1987) Cell 51:503 for a description of homologous recombination vectors. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced BAFF-R gene has homologously recombined with the endogenous BAFF-R gene are selected (see e.g., Li et al. (1992) Cell 69:915).
The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras. See e.g., Bradley, in TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, Ed. IRL, Oxford, 1987, pp. 113-152. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley (1991) Curr. Opin. Biotechnol. 2:823-829; PCT International Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.
In another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad. Sci. USA 89:6232-6236. Another example of a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other-containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also'e produced according to the methods described in Wilmut et al. (1997) Nature 385:810-813. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G0 phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
Pharmaceutical Compositions
The BAFF-R nucleic acid molecules, BAFF-R proteins, and anti-BAFF-R antibodies (also referred to herein as “active compounds”) of the invention, and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and'5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity 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. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a BAFF-R protein or anti-BAFF-R antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are 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 powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject 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 invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved.
The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by any of a number of routes, e.g., as described in U.S. Pat. No. 5,703,055. Delivery can thus also include, e.g., intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
Uses and Methods of the Invention
The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: (a) screening assays; (b) detection assays (e.g., chromosomal mapping, tissue typing, forensic biology), (c) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenomics); and (d) methods of treatment (e.g., therapeutic and prophylactic). As described herein, in one embodiment, a BAFF-R protein of the invention has the ability to bind BAFF.
The isolated nucleic acid molecules of the invention can be used to express BAFF-R protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect BAFF-R mRNA (e.g., in a biological sample) or a genetic lesion in a BAFF-R gene, and to modulate BAFF and/or BAFF-R activity, as described further below. In addition, the BAFF-R proteins can be used to screen drugs or compounds that modulate the BAFF-R activity or expression as well as to treat disorders characterized by insufficient or excessive production of BAFF and/or BAFF-R protein, or production of BAFF-R protein forms that have decreased or aberrant activity compared to BAFF-R wild type protein. In addition, the anti-BAFF-R antibodies of the invention can be used to detect and isolate BAFF-R proteins and modulate BAFF and/or BAFF-R activity.
This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
Screening Assays
The invention provides a method (also referred to herein as a “screening assay”) for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to BAFF-R proteins or have a stimulatory or inhibitory effect on, for example, BAFF-R expression or BAFF-R activity.
In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a BAFF-R protein or polypeptide or biologically active portion thereof. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the “one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145-167).
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909-6013; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422-11426; Zuckermann et al. (1994) J. Med. Chem. 37:2678-2685; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233-1251.
Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), on chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382; Felici (1991) J. Mol. Biol. 222:301-310; Ladner U.S. Pat. No. 5,223,409).
In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of BAFF-R protein, or a biologically active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to a BAFF-R protein determined. The cell, for example, can of mammalian origin or a yeast cell. Determining the ability of the test compound to bind to the BAFF-R protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the BAFF-R protein or biologically active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation-counting. Alternatively, test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In one embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of BAFF-R protein, or a biologically active portion thereof, on the cell surface with a known compound which binds BAFF-R to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a BAFF-R protein, wherein determining the ability of the test compound to interact with a BAFF-R protein comprises determining the ability of the test compound to preferentially bind to BAFF-R or a biologically active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of BAFF-R protein, or a biologically active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the BAFF-R protein or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of BAFF-R or a biologically active portion thereof can be accomplished, for example, by determining the ability of the BAFF-R protein to bind to or interact with a BAFF-R target molecule. As used herein, a “target molecule” is a molecule with which a BAFF-R protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses a BAFF-R protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. A BAFF-R target molecule can be a non-BAFF-R molecule or a BAFF-R protein or polypeptide of the present invention. In one embodiment, a BAFF-R target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g., a signal generated by binding of a compound to a membrane-bound BAFF-R molecule) through the cell membrane and into the cell. The target, for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with BAFF-R.
Determining the ability of the BAFF-R protein to bind to or interact with a BAFF-R target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of the BAFF-R protein to bind to or interact with a BAFF-R target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising a BAFF-R-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.
In yet another embodiment, an assay of the present invention is a cell-free assay comprising contacting a BAFF-R protein or biologically active portion thereof with a test compound and determining the ability of the test compound to bind to the BAFF-R protein or biologically active portion thereof. Binding of the test compound to the BAFF-R protein can be determined either directly or indirectly as described above. In one embodiment, the assay comprises contacting the BAFF-R protein or biologically active portion thereof with a known compound which binds BAFF-R to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a BAFF-R protein, wherein determining the ability of the test compound to interact with a BAFF-R protein comprises determining the ability of the test compound to preferentially bind to BAFF-R or biologically active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-free assay comprising contacting BAFF-R protein or biologically active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the BAFF-R protein or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of BAFF-R can be accomplished, for example, by determining the ability of the BAFF-R protein to bind to a BAFF-R target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of BAFF-R can be accomplished by determining the ability of the BAFF-R protein further modulate a BAFF-R target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as previously described.
In yet another embodiment, the cell-free assay comprises contacting the BAFF-R protein or biologically active portion thereof with a known compound which binds BAFF-R to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a BAFF-R protein, wherein determining the ability of the test compound to interact with a BAFF-R protein comprises determining the ability of the BAFF-R protein to preferentially bind to or modulate the activity of a BAFF-R target molecule.
The cell-free assays of the present invention are amenable to use of both the soluble form or the membrane-bound form of BAFF-R. In the case of cell-free assays comprising the membrane-bound form of BAFF-R, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of BAFF-R is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n, 3-(3-cholamidopropyl)dimethylamminiol-1-propane sulfonate (CHAPS), 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO), or N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate.
In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize either BAFF-R or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to BAFF-R, or interaction of BAFF-R with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, GST-BAFF-R fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or BAFF-R protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of BAFF-R binding or activity determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either BAFF-R or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated BAFF-R or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with BAFF-R or target molecules, but which do not interfere with binding of the BAFF-R protein to its target molecule, can be derivatized to the wells of the plate, and unbound target or BAFF-R trapped in the wells by antibody-conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the BAFF-R or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the BAFF-R or target molecule.
In another embodiment, modulators of BAFF-R expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of BAFF-R mRNA or protein in the cell is determined. The level of expression of BAFF-R mRNA or protein in the presence of the candidate compound is compared to the level of expression of BAFF-R. mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of BAFF-R expression based on this comparison. For example, when expression of BAFF-R mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of BAFF-R mRNA or protein expression. Alternatively, when expression of BAFF-R mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of BAFF-R mRNA or protein expression. The level of BAFF-R mRNA or protein expression in the cells can be determined by methods described herein for detecting BAFF-R mRNA or protein.
In yet another aspect of the invention, the BAFF-R proteins can be used as “bait proteins” in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO 94/10300), to identify other proteins that bind to or interact with BAFF-R (“BAFF-R-binding proteins” or “BAFF-R-bp”) and modulate BAFF-R activity. Such BAFF-R-binding proteins are also likely to be involved in the propagation of signals by the BAFF-R proteins as, for example, upstream or downstream elements of the BAFF-R pathway.
The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for BAFF-R is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming a BAFF-R-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with BAFF-R.
This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
Detection Assays
Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.
Chromosome Mapping
Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is tailed chromosome mapping. Accordingly, portions or fragments of the BAFF-R, sequences, described herein, can be used to map the location of the BAFF-R genes, respectively, on a chromosome. The mapping of the BAFF-R sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
Briefly, BAFF-R genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 by in length) from the BAFF-R sequences. Computer analysis of the BAFF-R, sequences can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual chromosomes of a given species. Only those hybrids containing the species-specific gene corresponding to the BAFF-R sequences will yield an amplified fragment.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the BAFF-R sequences to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes.
Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical like colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases, will suffice to get good results at a reasonable amount of time. For a review of this technique, see Venna et al. HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUE, Pergamon Press, N.Y., 1988.
Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in McKusick, MENDELIAN INHERITANCE IN MAN, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, for example, Egeland et al. (1987) Nature, 325:783-787.
Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the BAFF-R gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
Tissue Typing
The BAFF-R sequences of the present invention can also be used to identify individuals from minute biological samples. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. The sequences of the present invention are useful as additional DNA markers for RFLP (“restriction fragment length polymorphisms,” described in U.S. Pat. No. 5,272,057).
Furthermore, the sequences of the present invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the BAFF-R sequences described herein can be used to prepare two PCR primers from the 5′ and 3′ ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue. The BAFF-R sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Much of the allelic variation is due to single nucleotide polymorphisms (SNPs), which include restriction fragment length polymorphisms (RFLPs).
Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2B (SEQ ID NO:4), FIG. 3 (SEQ ID NO:6) can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2B (SEQ ID NO:4), FIG. 3 (SEQ ID NO:6) are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
Predictive Medicine
The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining BAFF-R protein and/or nucleic acid expression as well as BAFF-R activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant BAFF-R expression or activity. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with BAFF-R protein, nucleic acid expression or activity. For example, mutations in a BAFF-R gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with BAFF-R protein, nucleic acid expression or activity.
Another aspect of the invention provides methods for determining BAFF-R protein, nucleic acid expression or BAFF-R activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as “pharmacogenomics”). Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent.)
Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of BAFF-R in clinical trials.
These and other agents are described in further detail in the following sections.
Diagnostic Assays
An exemplary method for detecting the presence or absence of BAFF-R in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting BAFF-R protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes BAFF-R protein such that the presence of BAFF-R is detected in the biological sample. An agent for detecting BAFF-R mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to BAFF-R mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length BAFF-R nucleic acid, such as the nucleic acids of any of FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2B (SEQ ID NO:4), FIG. 3 (SEQ ill NO:6) or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to BAFF-R mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.
An agent for detecting BAFF-R protein is an antibody capable of binding to BAFF-R protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. The term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect BAFF-R mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of BAFF-R mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of BAFF-R protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitation and immunofluorescence. In vitro techniques for detection of BAFF-R genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of BAFF-R protein include introducing into a subject a labeled anti-BAFF-R antibody. For example, the antibody can,e labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting BAFF-R protein, mRNA, or genomic DNA, such that the presence of BAFF-R protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of BAFF-R protein, mRNA or genomic DNA in the control sample with the presence of BAFF-R protein, mRNA or genomic DNA in the test sample.
The invention also encompasses kits for detecting the presence of BAFF-R in a biological sample. For example, the kit can comprise: a labeled compound or agent capable of detecting BAFF-R protein or mRNA in a biological sample; means for determining the amount of BAFF-R in the sample; and means for comparing the amount of BAFF-R in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect BAFF-R protein or nucleic acid.
Prognostic Assays
The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant BAFF-R expression or activity. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with BAFF-R protein, nucleic acid expression or activity in, e.g., autoimmune conditions such as autoimmune hemolytic anemia and systemic lupus erythematosus. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder. Thus, the present invention provides a method for identifying a disease or disorder associated with aberrant BAFF-R expression or activity in which a test sample is obtained from a subject and BAFF-R protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of BAFF-R protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant BAFF-R expression or activity. As used herein, a “test sample” refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant BAFF-R expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder. Thus, the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant BAFF-R expression or activity in which a test sample is obtained and BAFF-R protein or nucleic acid is detected (e.g., wherein the presence of BAFF-R protein or nucleic kid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant BAFF-R expression or activity.)
The methods of the invention can also be used to detect genetic lesions in a BAFF-R gene, thereby determining if a subject with the lesioned gene is at risk for, or suffers from, a tumorigenic or autoimmune disorder. In various embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding a BAFF-R-protein, or the mis-expression of the BAFF-R gene. For example, such genetic lesions can be detected by ascertaining the existence of at least one of (1) a deletion of one or more nucleotides from a BAFF-R gene; (2) an addition of one or more nucleotides to a BAFF-R gene; (3) a substitution of one or more nucleotides of a BAFF-R gene, (4) a chromosomal rearrangement of a BAFF-R gene; (5) an alteration in the level of a messenger RNA transcript of a BAFF-R gene, (6) aberrant modification of a BAFF-R gene, such as of the methylation pattern of the genomic DNA, (7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a BAFF-R gene, (8) a non-wild type level of a BAFF-R-protein, (9) allelic loss of a BAFF-R gene, and (10) inappropriate post-translational modification of a BAFF-R-protein. As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in a BAFF-R gene. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
In certain embodiments, detection of the lesion involves the use of a probe/pruner in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PLR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91:360-364), the latter of which can be particularly useful for detecting point mutations in the BAFF-R-gene (see Abravaya et al. (1995) Nucl. Acids Res. 23:675-682). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to a BAFF-R gene under conditions such that hybridization and amplification of the BAFF-R gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
Alternative amplification methods include: self sustained sequence replication (Guatelli et al., (1990) Proc. Natl. Acad Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) BioTechnology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
In an alternative embodiment, mutations in a BAFF-R gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Pat. No. 5,493,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
In other embodiments, genetic mutations in BAFF-R can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin et al. (1996) Human Mutation 7:244-255; Kozal et al. (1996) Nature Med. 2:753-759). For example, genetic mutations in BAFF-R can be identified in two-dimensional arrays containing light-generated DNA probes as described in Cronin et al. (1996) Human Mutation 7:244-255. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the BAFF-R gene and detect mutations by comparing the sequence of the sample BAFF-R with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert (1977) Proc. Natl. Acad. Sci. USA 74:560 or Sanger (1977) Proc. Natl. Acad. Sci. USA 74:5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve et al. (1995) Biotechnigues 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publ. No. WO 94/16101; Cohen et al. (1996) Adv Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).
Other methods for detecting mutations in the BAFF-R gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242). In general, the art technique of “mismatch cleavage” starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type BAFF-R sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent that cleaves single-stranded regions of the duplex such as which will exist due to base pair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295. In an embodiment, the control DNA or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called “DNA mismatch repair” enzymes) in defined systems for detecting and mapping point mutations in BAFF-R cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662). According to an exemplary embodiment, a probe based on a BAFF-R sequence, e.g., a wild-type BAFF-R sequence, is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Pat. No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in BAFF-R genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144; Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control BAFF-R nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In one embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).
In yet another embodiment the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 by of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys. Chem. 265:12753).
Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci. USA 86:6230). Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucl. Acids Res. 17:2437-2448) or at the extreme 3′ end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11:238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell. Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3′ end of the 5′ sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a BAFF-R gene.
Furthermore, any cell type or tissue, in which BAFF-R is expressed may be utilized in the prognostic assays described herein. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
Pharmacogenomics
Agents, or modulators that have a stimulatory or inhibitory effect on BAFF-R activity (e.g., BAFF-R gene expression), as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (e.g., cancer-related or autoimmune disorders). In conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) of the individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of BAFF-R protein, expression of BAFF-R nucleic acid, or mutation content of BAFF-R genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See e.g., Eichelbaum (1996) Clin. Exp. Pharmacol. Physiol. 23:983-985 and Linder (1997) Clin. Chem. 43:254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
Thus, the activity of BAFF-R protein, expression of BAFF-R nucleic acid, or mutation content of BAFF-R genes in an individual can be determined to thereby-select appropriate agent(s) for therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a BAFF-R modulator, such as a modulator identified by one of the exemplary screening assays described herein.
Monitoring Clinical Efficacy
Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of BAFF-R (e.g., the ability to modulate aberrant cell proliferation and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase BAFF-R gene expression, protein levels, or upregulate BAFF-R activity, can be monitored in clinical trials of subjects exhibiting decreased BAFF-R gene expression, protein levels, or downregulated BAFF-R activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease BAFF-R gene expression, protein levels, or downregulate BAFF-R activity, can be monitored in clinical trials of subjects exhibiting increased BAFF-R gene expression, protein levels, or upregulated BAFF-R activity. In such clinical trials, the expression or activity of BAFF-R and, preferably, other genes that have been implicated in, for example, a disorder, can be used as a “read out” or markers of the immune responsiveness of a particular cell.
For example, genes, including BAFF-R, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) that modulates BAFF-R activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of BAFF-R and other genes implicated in the disorder. The levels of gene expression (i.e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of BAFF-R or other genes. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent. In one embodiment, the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a BAFF-R protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the BAFF-R protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the BAFF-R protein, mRNA, or genomic DNA in the pre-administration sample with the BAFF-R protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of BAFF-R to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of BAFF-R to lower levels than detected, i.e., to decrease the effectiveness of the agent.
Methods of Treatment
The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant BAFF-R expression or activity.
Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with therapeutics that antagonize (i.e., reduce or inhibit) activity. Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, (i) a BAFF-R polypeptide, or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to a BAFF-R peptide; (iii) nucleic acids encoding a BAFF-R peptide; (iv) administration of antisense nucleic acid and nucleic acids that are “dysfunctional” (i.e., due to a heterologous insertion within the coding sequences of coding sequences to a BAFF-R peptide) are utilized to “knockout” endogenous function of a BAFF-R peptide by homologous recombination (see, e.g., Capecchi (1989) Science 244: 1288-1292); or (v) modulators (i.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide of the invention) that alter the interaction between a BAFF-R peptide and its binding partner.
Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that increase (i.e., are agonists to) activity. Therapeutics that upregulate activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, a BAFF-R peptide, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability.
Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vim) for RNA or peptide levels, structure and/or activity of the expressed peptides for mRNAs of a BAFF-R peptide). Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.).
In one aspect, the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant BAFF-R expression or activity, by administering to the subject an agent that modulates BAFF-R expression or at least one BAFF-R activity. Subjects at risk for a disease that is caused or contributed to by aberrant BAFF-R expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the BAFF-R aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of BAFF-R aberrancy, for example, a BAFF-R agonist or BAFF-R antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.
Another aspect of the invention pertains to methods of modulating BAFF-R expression or activity for therapeutic purposes. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of BAFF-R protein activity associated with the cell. An agent that modulates BAFF-R protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a BAFF-R protein, a peptide, a BAFF-R peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more BAFF-R protein activity. Examples of such stimulatory agents include active BAFF-R protein and a nucleic acid molecule encoding BAFF-R that has been introduced into the cell. In another embodiment, the agent inhibits one or more BAFF-R protein activity. Examples of such inhibitory agents include antisense BAFF-R nucleic acid molecules and anti-BAFF-R antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a BAFF-R protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) BAFF-R expression or activity. In another embodiment, the method involves administering a BAFF-R protein or nucleic acid molecule as therapy to compensate for reduced or aberrant BAFF-R expression or activity.
In one embodiment, the invention provides methods of using BAFF-R. Included in such methods are methods of inhibiting B cell growth, dendritic cell-induced B cell growth and maturation or immunoglobulin production in an animal using BAFF-R polypeptide comprising at least a BAFF binding portion of BAFF-R. Other embodiments include methods of stimulating B-cell growth, dendritic cell-induced B-cell growth and maturation or immunoglobulin production in an animal using BAFF-R polypeptide (such as by transfecting cells which are deficient in BAFF-R with vectors to allow efficient expression of BAFF-R, or by administering antibodies that bind BAFF-R and mimic BAFF).
In another embodiment, the invention provides methods of using BAFF-R in the treatment of autoimmune diseases, hypertension, cardiovascular disorders, renal disorders, B-cell lympho-proliferate disorders, immunosuppressive diseases, organ transplantation, and HIV. Also included are methods of using agents for treating, suppressing or altering an immune response involving a signaling pathway between BAFF-R and its ligand, and methods of inhibiting inflammation by administering an antibody specific for a BAFF-R or an epitope thereof.
The methods of the present invention are preferably carried out by administering a therapeutically effective amount of a BAFF-R polypeptide, a chimeric molecule comprising a BAFF-R polypeptide fused to a heterologous amino acid sequence, or an anti-BAFF-R antibody homolog.
In one embodiment, the invention provides pharmaceutical compositions comprising a BAFF-R polypeptide and a pharmaceutically acceptable excipient.
In another embodiment, the invention provides chimeric molecules comprising BAFF-R polypeptide fused to a heterologous polypeptide or amino acid sequence. An example of such a chimeric molecule comprises a BAFF-R fused to a Fc region of an immunoglobulin or an epitope tag sequence.
In another embodiment, the invention provides an antibody that specifically binds to a BAFF-R polypeptide. Optionally, the antibody is a monoclonal antibody.
In one embodiment of the invention is a method of treating a mammal for a condition associated with undesired cell proliferation by administering to the mammal a therapeutically effective amount of a composition comprising an BAFF-R antagonist, wherein the BAFF-R antagonist comprises a polypeptide that antagonizes the interaction between BAFF-R and its cognate receptor or receptors, with a pharmaceutically acceptable recipient.
In a preferred embodiment the cognate receptor of BAFF on the surface of the cell is BAFF-R.
The method can be used with any BAFF-R antagonist that has a polypeptide that antagonizes the interaction between BAFF and its cognate receptor or receptors. Examples of BAFF-R antagonists include but are not limited to soluble BAFF-R polypeptide, soluble chimeric BAFF-R molecules, including but not limited to BAFF-R-IgG-Fc and anti-BAFF-R antibody homologs.
The method of the invention can be used with any condition associated with undesired cell proliferation. In particular the methods of the present invention can be used to treat tumor cells which express BAFF and/or BAFF-R.
Examples of cancers whose cell proliferation is modulated by BAFF may be screened by measuring in vitro the level of BAFF and for BAFF-R message expressed in tumor tissue libraries. Tumor tissue libraries in which BAFF and/or BAFF-R message is highly expressed would be candidates. Alternatively, one may screen for candidates searching the public and private databases (i.e., Incyte database) with, for example, the full length human BAFF cDNA sequence.
The BAFF-R antagonists of the subject invention which are used in treating conditions associated with undesired cell proliferation, in particular tumor therapy, advantageously inhibit tumor cell growth greater than 10%, 20%, 30% or 40% and most advantageously greater than 50%. The BAFF-R antagonists are obtained through screening. For example, BAFF-R antagonists can be selected on the basis of growth inhibiting activity (i.e., greater than 10%, 20%, 30%, 40% or 50%) against the human colon carcinoma HT29 or human lung carcinoma A549 which are derived from a colon and lung tumor respectively.
Another embodiment of the invention, provides methods of inhibiting B-cell and non-B cell growth, dendritic cell-induced B-cell growth and maturation or immunoglobulin production in an animal using BAFF-R polypeptides such as those described above.
The method of inhibiting B-cell and non-B cell growth, dendritic cell-induced B-cell growth and maturation or immunoglobulin production may also include administration of an anti-BAFF-R antibody (polyclonal or monoclonal) that binds to BAFF-R and inhibits the binding of BAFF to BAFF-R. Administration of the antibody thereby inhibits B-cell and non-B cell growth, dendritic cell-induced B-cell growth and maturation or immunoglobulin production. The amount of antibody that may be suitable for use may be extrapolated from the in vivo data provided herein. Various methods are known in the art to extrapolate dosages from animal experiments, including for example, extrapolation based on body weight or surface area
In some embodiments of the invention the BAFF-R:Fc polypeptides or anti-BAFF-R antibodies are administered in an amount of about 1 to 20 mg/kg/dose. Doses may be given twice weekly, once weekly, one every two weeks or once monthly, as needed. A physician will be able to determine the proper dose by determining efficacy balanced against reducing any untoward effects of the therapy.
In another embodiment, the invention provides methods of using BAFF-R or anti-BAFF-R antibodies in the treatment of autoimmune diseases, hypertension, cardiovascular disorders, renal disorders, B-cell lympho-proliferate disorders, immunosuppressive diseases, organ transplantation, inflammation, and HIV. Also included are methods of using agents for treating, suppressing or altering an immune response involving a signaling pathway between BAFF-R and its ligand.
Methods of Inhibiting Aggregation of Expressed Protein, Including BAFF-R and BAFF-R:Fc
The invention also provides a method for inhibiting or decreasing aggregation of expressed protein, particularly human BAFF-R or huBAFF-R:Fc, which tends to aggregate during expression, frustrating purification at high yields. In the method of the invention the amino acid sequence of a protein that tends to aggregate when expressed in a recombinant system is compared to the amino acid sequence of a homolog of the protein that exhibits less aggregation activity. The two homologs will have conserved domains and non-conserved amino acids there between and perhaps interspersed therein. In general, at least one of the non-conserved amino acids amino acids of the aggregating protein may be substituted for the amino acid in the homolog to alleviate aggregation. In some embodiments, nonpolar amino acids are substituted. Nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine. In some embodiments nonpolar amino acids substitute for other nonpolar amino acids. Preferred nonpolar amino acids to inhibit or decrease aggregation are proline and alanine. In other embodiments, an uncharged polar amino acid is substituted for a nonpolar amino acid. Uncharged polar amino acids include asparagine, glutamine, serine, threonine and tyrosine.
In the method of the invention, substitutions are made that preferably allow the protein to retain biological activity. In general, non-conserved amino acids are amenable to substitution without appreciably affecting biological activity.
In a specific example of the method of the invention, human BAFF-R protein may have amino acid substitutions introduced at positions V20, P21, A22 and L27 of SEQ ID NO:5 (or V41, P42, A43, and L48 of SEQ ID NO:10) and various combinations thereof, which greatly alleviates aggregation of the protein. Similar strategies may be used for other proteins that tend to aggregate when expressed in recombinant systems. While not wishing to be bound by any particular theory of operation, it is believed that the substitution of uncharged polar amino acids for nonpolar amino acids imparts solubility to the protein and discourages aggregation of nonpolar regions between the proteins.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
Examples
Example 1
This example describes the molecular cloning of BAFF-R, a novel receptor for BAFF.
Materials and Methods
An oligo-dT primed cDNA library was made from BJAB cells, a human B cell line that binds human BAFF, and directionally cloned into the expression vector CH269. CH269 is a derivative of pCEP4 (Invitrogen) that contains the CMV promoter to drive expression of cloned DNA and also contains the oriP of EBV. This allows multicopy autonomous replication of these plasmids in cells that are stably transformed with EBNA-1, such as 293EBNA. The BJAB cDNA library was transfected into E. coli DH10B cells and seeded in a 96 well format as pools of approximately 2500 independent clones per well. DNA was prepared from these pools using the Qiagen BioRobot 9600. The DNA pools were transfected using Lipofectamine (Life Technologies) into 293EBNA cells seeded into fibronectin coated 6 well dishes. At 48 hours post-transfection the medium was removed and the cells washed with plate assay wash buffer (20 mM HEPES, 0.5 mg/ml bovine serum albumin, 0.1% NaN3). Cell monolayers were overlaid with 100 mg/ml biotinylated human recombinant soluble myc-BAFF (myc-huBAFF) in binding buffer (PBS, 2% fetal bovine serum, 0.1% NaN3) and incubated at room temperature for 1 hour. The myc-huBAFF (amino acids 136-285) used in the assay was expressed in Pichia pastoris and purified by anion exchange chromatography followed by gel filtration.
The BAFF solution was removed and the cells were washed and fixed by incubation with 1.8% formaldehyde-0.2% glutaraldehyde in PBS for 5 minutes. Cells were again washed and then incubated for 30 minutes with streptavidin conjugated with alkaline phosphatase (SAV-AP) (Jackson ImmunoResearch) at a 1:3000 dilution from stock in binding buffer. Cells were washed and stained with fast red/napthol phosphate (Pierce). Cells binding the biotin-BAFF/SAV-AP complex were identified by the presence of a red precipitate after inspection under low power microscopy. Secondary screening entailed plating out the DH10B glycerol stocks of the BAFF binding pools for single colonies, inoculating to culture in pools of 100, and repeating the BAFF binding assay as described above. Secondary screen positive pools were similarly broken down to individual clones and assayed for BAFF binding upon transfection to 293EBNA as described above. The DNA sequence of the independent BAFF binding clones was determined.
Results
One of the BAFF binding clones was pJST576. It has in insert size of 1201 base pairs (bp) not including the poly-A tail. The sequence of the insert of pJST576 is shown in FIG. 1A (SEQ ID NO:1). BLAST analysis of this clone showed homology in the Genbank database to the chromosome 22 BAC clone HS250D10 (accession number Z99716). The entire pJST576 sequence is found within this BAC. Homology was also found to the 3′ end of a human EST, AI250289 (IMAGE clone 2000271). The EST was generated from a human follicular lymphoma library. The EST AI250289 was obtained from Incyte and the sequence of the insert was determined (FIG. 1B) (SEQ ID NO:2). This sequence added 15 by of 5′ sequence to the pJST576 sequence, which is contiguous with the genomic sequence and 23 bp, which was not. The remainder of the EST sequence has perfect homology to pJST576. An open reading frame could not be identified in these clones.
Example 2
In this example, we determine that the JST576 cDNA contains an intron and then establish an open reading frame.
Methods
The GENSCAN (Burge, C. & Karlin, S. J. (1997) Mol. Biol. 268:78-94) exon prediction program was run on the JST576 cDNA sequence. The results of this program predicted that an intron was present in the cDNA. In order to determine if the prediction was correct, PCR analysis was performed on first strand cDNA from 2 cell lines expressing JST576. RNA was purified from approximately 107 BJAB or IM-9 cells using the RNeasy kit (Qiagen) following the manufacturers suggested protocol. RNA was quantitated and 5 μg was used for first strand cDNA reactions using the Superscript preamplification kit (Life Technologies). Both oligo dT and random hexamers were used to generate the first strand product. Synthesis of the first strand was done following the recommended protocol. Three (1 of each reaction, 10 ng of JST576 or no DNA was then used as a template for PCR using oligonucleotides flanking the predicted intron. The oligonucleotides used in the reaction are the 5′ oligos BAF-225 [5′-GGCCGAGTGCTTCGACCTGCT-3′] (SEQ ID NO:33) or BAF-226 [5′-GGTCCGCCACTGCGTGGCCTG-3′] (SEQ ID NO:34) and the 3′ oligo BAF-191 [5′-CACCAAGACGGCCGGCCCTGA-3′] (SEQ ID NO:35). Each reaction contained 1× Pfu buffer (Stratagene), 200 (M dNTPs, 10% DMSO, 150 ng of each oligo, and 1.25 units of Turbo Pfu polymerase (Stratagene). The reactions were run for 35 cycles at 94° C. for 30 sec., 60° C. for 1 min. and 72° C. for 1.5 min. Ten μl of each reaction was run on a 1% agarose gel. The remaining products from the BJAB and IM-9 BAF-225/191 reactions were purified using the High Pure PCR product purification kit (Roche Molecular Biochemicals) and the bulk product was subjected to DNA sequencing. In addition, PCR products using the primers BAF-225 and BAF-191 were generated from resting B cell cDNA, subcloned and individual clones were sequenced. Here 5 μl of resting B cell cDNA (Clontech) was used in a PCR reaction with the BAF-225 and BAF-191 primers as detailed above. The PCR product was then purified using the High Pure PCR product purification kit and concentrated. In order to subclone the PCR fragment, the ends of the fragment were phosphorylated and made blunt using the Sure Clone ligation kit (Amersham Pharmacia Biotech) as recommended. The resulting product was cloned into the EcoRV site of pBluescriptII (Stratagene) and transformed into E. coli. Individual colonies were grown up, the plasmid DNA miniprepped. Six independent isolates were sequenced.
Results
The mature nucleotide and amino acid sequence of JST576 predicted by the GENSCAN program is shown in FIG. 2A (SEQ ID NO:3). PCR products from BJAB and IM-9 reactions spanning the predicted intron are shown in FIG. 2B and confirm the existence of an intron in the JST576 cDNA clone. The predicted size of the PCR product from the JST576 cDNA is approximately 788 by for BAF-225/BAF-191 and 767 by for BAF-226/BAF-191. The PCR products obtained from the JST576 template are approximately this size (lanes 10 and 11). The PCR products obtained using BAF-225/BAF-191 on either oligo dT primed BJAB or IM-9 first strand cDNA (lanes 2 and 6) are the same size and significantly shorter than the product from the JST576 cDNA. The predicted size of this fragment without the predicted intron is 484 bp. The size of the PCR products is consistent with this size. The same results were obtained if BJAB or IM-9 RNA was primed with random hexamers (Innes 4 and 8). The reactions using BAF-226/BAF-191 did not work on the first strand cDNA templates. Therefore, it appears that the intron predicted by the GENSCAN program does exist in the JST576 cDNA. The sequence of the spliced product from BJAB and IM-9 RNA was confirmed by sequencing the bulk PCR product and is reflected in the sequence shown in FIG. 2C (SEQ ID NO:4). The sequence is identical to the sequence shown in FIG. 2A (SEQ ID NO:3), except for the absence of the alanine codon (GCA) at nucleotide 149 (shown in small letters). The results of sequencing 6 independent clones from the RT-PCR reaction on resting B cell cDNA indicates that both splice acceptor sites are utilized. The preferred acceptor site appears to be the product resulting in one alanine residue (5/6 clones). However, the sequence predicted by GENSCAN (SEQ ID NO:3), which contains two alanines, was observed in 1/6 clones. Therefore an open reading frame for human JST576 has been established and a single amino acid splice variant has been determined. The open reading frame predicts a protein of 184 amino acids shown in FIG. 2D (SEQ ID NO:5). The analine (A) residue in bold represents the splice variant. This protein is referred to as BAFF-R. The deduced amino acid sequence of BAFF-R includes a hydrophobic region from residues 72-100 (Hopp-Woods algorithm) and a potential transmembrane segment from residues 84-102 as analyzed by the TMPred algorithm. This region is followed by a highly charged stretch of amino acids that may function as a stop transfer signal. BAFF-R lacks an N-terminal signal sequence and is a type III membrane protein similar to the other BAFF binding proteins BCMA (Laabi et al. (1992) EMBO J. 11:3897-3904) and TACI (von Bulow and Bram, (1997) Science 278:138-141). The N-terminus is predicted to be the extracellular domain of BAFF-R and contains a 4 cysteine motif at residues 19-35 unlike any other member of the TNF receptor family. The C-terminus of BAFF-R is predicted to be the intracellular domain.
Example 3
Here we determine the DNA sequence upstream of the proposed initiating methionine for human BAFF-R including an in-frame stop codon.
Methods
A primer BAF-254 (5′GGGCGCCTACAATCTCAGCTA 3′) (SEQ ID NO:36) was made to the genomic sequence present in the BAC HS250d10 (Genbank accession number Z99716), upstream of the proposed ATG and used in a PCR reaction with the oligo BAF-236 (5′ GGCGGACCAGCAGGTCGAAGCACTC 3′) (SEQ ID NO:37). The template in the reaction was first strand cDNA made from human spleen RNA (Clontech) using the PCR preamplification kit as described by the manufacturer (Life Technologies). The PCR reaction contained 3 μl of the first strand reaction, 1× Pfu buffer (Stratagene), 10% DMSO, 0.2 mM dNTPs, 150 ng each primer and 1.25 units Pfu Turbo polymerase (Stratagene). The PCR product was purified using the High Pure PCR Product Purification kit following the manufacturer's directions (Roche Molecular Biochemicals). The ends of the PCR product were made blunt and phosphorylated using the Sure Clone ligation kit (Amersham Pharmacia Biotech), cloned into the EcoRV site of pBSK2 (Stratagene) and transformed into DH5 Colonies resulting from the ligation were miniprepped using the Wizard system (Promega) and then sequenced using an ABI machine.
Results
The sequence of the PCR product confirms that the mRNA contains sequences directly upstream of the ATG that is contained in the genomic sequence. This sequence is underlined in the sequence shown in FIG. 3. The presence of an in-frame upstream stop codon and the absence of another methionine indicate that the methionine found in the JST576 cDNA is the correct initiating methionine.
Example 4
This example describes the cloning of the murine BAFF-R cDNA.
Methods
Approximately one million phage plaques were screened from the murine A20 cell line cDNA library purchased from Stratagene (La Jolla, Calif.) as detailed by the manufacturer. The JST576 human BAFF-R cDNA was digested with EcoNI and run on a 1% low melt gel. The 425 by fragment containing was cut out of the gel and weighed. Three times the volume of water was added and the gel fragment was boiled for 5 min. The fragment was labeled with 50 μCi 32P-dCTP (Amersham) in a reaction containing 50 mM Tris pH8, 5 mM MgCl2, 10 μM β-mercaptoethanol, 200 mM HEPES pH 6.5, 20 (M dNTPs (except dCTP), 0.27 units of pd(N)6 hexanucleotides (Amersham Pharmacia Biotech) and 1 unit of Klenow enzyme (USE) overnight at room temperature. About one million counts per ml of probe was incubated with the filters in plaque screening buffer (50 mM Tris, 1% SDS, 1M NaCl, 0.1% Sodium Pyrophosphate, 0.2% PVP, 0.2% Ficoll, 0.2% BSA) overnight at 65° C. The filters were washed in 2×SSC and 0.1% SDS at 50° C. for 1.5 hrs (3×2 liters) and then exposed to x-ray film for 2 days. Approximately 36 positive plaques were identified. Of these 6 were plaque purified. The phagemids were released using the in vivo excision protocol detailed by Stratagene. The resulting colonies were grown up and the DNA was then miniprepped (Qiagen). The cDNA clones were sequenced.
Results
The murine BAFF-R consensus nucleotide sequence is presented as FIG. 4A (SEQ ID NO:8) and the amino acid sequence is presented in FIG. 4B (SEQ ID NO:9). Three of the clones contained a 10 amino acid deletion from amino acid 119 to 129 in the intracellular domain of murine BAFF-R. The alignment of the human and murine BAFF-R sequences illustrates that the 4 cysteine residues in the extracellular domain are conserved, that the position of the initiating methionine is similar and that the C-terminal region of the proteins is highly conserved (FIG. 4C), with the last 24 residues being identical. The sequences have approximately 56% identity overall.
Example 5
In this example, the ability of human recombinant soluble BAFF to bind to cells co-transfected with pJST576 and a GFP reporter plasmid is described.
Materials and Methods
The reporter plasmid encodes a membrane anchored GFP molecule and allows identification of transfected cells from non-transfected cells. 293EBNA cells were co-transfected with the reporter plasmid and pJST576 using Lipofectamine 2000 (Life Technologies). At 18-20 hr post-transfection, cells were detached from the plates with 5 mM EDTA in PBS and counted. The cells were washed twice with FACS buffer (PBS containing 10% fetal bovine serum, 0.1% NaN3) and 2.5×105 cells were incubated for 1 hour on ice with biotinylated myc-huBAFF diluted into FACS buffer over a concentration range of 8 ng/ml to 5 ug/ml. The cells were washed with FACS buffer and incubated for 30 minutes with streptavidin conjugated with phycoerythrin (SAV-PE) (Jackson ImmunoResearch) at a 1:100 dilution from stock. The cells were again washed with FACS buffer and resuspended in 1% paraformaldehyde in FACS buffer. The cells were analyzed by FACS for GFP and PE fluorescence and the data was formatted in a four quadrant dot plot. The dots in the two rightward quadrants represent cells expressing the transfection reporter GFP. The dots in the two upper quadrants represent cells having bound biotinylated myc-huBAFF with this binding revealed by SAV-PE. The cells in the upper right quadrant are transfected cells that bind biotinylated myc-huBAFF.
Results
Unstained cells and cells stained only with SAV-PE show approximately 50% are GFP positive and have been co-transfected with the reporter plasmid (FIG. 5). When cells co-transfected with the GFP reporter and pJST576 are stained with 1 ug/ml biotinylated myc-huBAFF nearly all the cells in the lower right quadrant shift up, indicating BAFF binding. A similar result is seen if a plasmid expressing huTACI is co-transfected in place of pJST576. TACI is known to bind BAFF. The cells were stained with five fold dilutions of biotinylated myc-huBAFF from 5 ug/ml to 8 ng/ml and as the concentration of biotinylated myc-huBAFF decreased the intensity of the shift decreased.
Example 6
In this example, the ability of human recombinant soluble BAFF or murine recombinant soluble BAFF to bind to cells co-transfected with pJST576 and a GFP reporter plasmid is described.
Materials and Methods
Co-transfections to 293EBNA were as described in Example 5. At 18-20 hr post-transfection, cells were detached, counted, and stained for FACS analysis similar to Example 5 with the following modifications. The cells were incubated for 1 hour on ice with 5 ug/ml of either murine or human recombinant soluble flag-BAFF, followed after washing by incubation for 30 minutes with 5 ug/ml of the anti-flag monoclonal antibody M2 (Sigma Aldrich), and then revealed by incubating the washed cells for 30 minutes with PE conjugated donkey anti-mouse IgG (Jackson ImmunoResearch) at a 1:100 dilution from stock. The cells were again washed, fixed with paraformaldehyde, and analyzed by FACS for GFP and PE positive cells.
Results
Approximately 50% of the cells are GFP positive and have therefore been co-transfected with the reporter plasmid (FIG. 6). When cells co-transfected with the GFP reporter and pJST576 are stained with 5 ug/ml of either human or murine recombinant soluble flag-BAFF, nearly all the cells in the lower right quadrant shift up. This indicates that both murine and human BAFF bind to cells transfected pJST576.
Example 7
In this example, the inability of murine recombinant soluble APRIL to bind to cells co-transfected with p3ST576 and a GFP reporter plasmid is described.
Materials and Methods
Co-transfections to 293EBNA were as described in Example 5. At 18-20 hr post-transfection, cells were detached, counted, and stained for FACS analysis similar to Example 5 with the following modifications. The cells were incubated for 1 hour on ice with 1 ug/ml of murine recombinant soluble myc-APRIL, followed after washing by incubation for 30 minutes with 5 ug/ml of anti-murine APRIL monoclonal antibody, followed by a 30 minute incubation of the washed cells with 5 ug/ml biotinylated anti-rat IgG2b (Pharmingen), and finally revealed by incubating the washed cells for 30 minutes with SAV-PE. The cells were again washed, fixed with paraformaldehyde, and analyzed by FACS for GFP and PE positive cells.
Results
Approximately 50% of the cells are GFP positive and have therefore been co-transfected with the reporter plasmid (FIG. 7). When cells co-transfected with the GFP reporter and pJST576 are stained with 1 ug/ml of murine myc-APRIL, none of the cells in the lower right quadrant shift up. This is in contrast to cells co-transfected with a plasmid expressing human TACT instead of pJST576. In these transfected cells, nearly all were positive for murine myc-APRIL binding. It has been previously shown that both BAFF and APRIL bind to both TACT and BCMA. Therefore the fact that APRIL does not bind to BAFF-R as expressed on pJST576 transfected cells indicates a specificity of BAFF-R for BAFF.
Example 8
This example describes the ability of BAFF-R as expressed from pJST576 to be co-immunoprecipitated by recombinant soluble human flag-BAFF.
Materials and Methods
293EBNA cells were transfected by Lipofectamine 2000 with pJST576, a vector only control, or a plasmid expressing huTACI as a positive control for BAFF binding. After 20 hours incubation the transfection medium was aspirated, the cells washed with PBS, and the media replaced with 35S labeling media (9 parts DMEM lacking methionine and cysteine to 1 part complete DMEM, supplemented with 10% dialyzed fetal bovine serum, 4 mM glutamine, and 100 μCi/ml 35S methionine and cysteine (Translabel, ICN Radiochemicals). Cells were incubated in this medium for six hours after which the media was removed. Cells were washed with PBS and then solubilized with 250 μl Extraction Buffer (1% Brij 98, 150 mM NaCl, 50 mM Tris pH7.5). Co-immunoprecipitations were performed by incubating 75 μl of the 35S labelled cell extracts with 5 μg recombinant soluble human flag-BAFF in 1 ml DMEM-10% fetal bovine serum-0.1% NaN3 overnight at 4° C. The anti-flag monoclonal antibody M2, 10 μg, and protein A-Sepharose were added and incubations continued for 2 hours. The Sepharose beads were collected by centrifugation, washed with FACS buffer, and resupended in SDS loading buffer with beta-mercaptoethanol as a reducing agent Samples were boiled 5 minutes, centrifuged briefly to pellet the Sepharose beads, and an aliquot run on SDS-PAGE. The gel was incubated with Enlightning (New England Nuclear), dried down, and exposed to film at −80° C.
Results
This co-immunoprecipitation binds flag-BAFF to the protein A Sepharose beads through the anti-flag antibody, M2. It will also bring down any proteins in the cell extract that bind to flag-BAFF, and these radiolabelled proteins will be detected by autoradiography. As 293EBNA cells do not bind BAFF, the empty vector control shows the background inherent in the procedure (FIG. 8). When extracts from cells transfected for TACI are co-immunoprecipitated with flag-BAFF, a band with an apparent molecular weight of approximately 34 kDa is observed. This is the approximate predicted molecular weight for full length human TACI (31.2 kDa), a protein known to bind BAFF. When extracts from cells transfected with p3ST576 are co-immunoprecipitated with flag-BAFF, a band with an apparent molecular weight of approximately 12 kDa is observed. The predicted molecular weight for BAFF-R expressed from pJST576 is 18.9 kDa. The disparity between predicted and observed molecular weights could be due to anomalous electrophoretic mobility due to the charge or conformation of BAFF-R. Another possibility is that 12 kDa is a proteolytic fragment of BAFF-R.
Example 9
This example describes the generation of soluble forms of BAFF-R. Oligonucleotide primers complementary to pJST576 can be designed to PCR amplify the BAFF-R extracellular domain in the absence of transmembrane and intracellular domains. Typically, one includes most of the stalk, or amino acid region between the ligand binding domain and the transmembrane domain. One could vary amount of stalk region included to optimize the potency of the resultant soluble receptor. This amplified fragment would be engineered with suitable restriction sites to allow cloning to various heterlogous leader sequences on the 5′ end of the fragment and to various Ig fusion chimera fusion vectors at the 3′ end. Alternatively, one can insert a stop signal at the 3′ end of the BAFF-R extracellular domain to and make a soluble form of the receptor or use another C-terminal fusion partner without resorting to the use of an instead of using the Ig fusion chimera approach. Also, one could create an N-terminal fusion protein consisting of a fusion partner containing a signal sequence followed by the N-terminal extracellular domain of BAFF-R. The resultant vectors can be expressed in most systems used in biotechnology including yeast, insect cells, bacteria, and mammalian cells and examples exist for all types of expression. Various human Fc domains can be attached to optimize or eliminate FcR and complement interactions as desired. Alternatively, mutated forms of these Fc domains can be used to selectively remove FcR or complement interactions or the attachment of N-linked sugars to the Fc domain which has certain advantages. An example of a BAFF-R:Fc fusion molecule is shown in FIG. 9. This molecule contains the type I leader sequence from a murine Ig-k gene linked by an Aat2 restriction site to the BAFF-R extracellular domain (amino acid residues 2-71 as shown in FIG. 2D) which is in turn linked by a SaII restriction site to the Fc domain of human IgG1.
Example 10
In this example we show the expression profile of BAFF-R in human tissues and cell lines using Northern blot analysis.
Materials and Methods
Various B and non-B cell lines were grown under the appropriate conditions. RNA was prepared from approximately 107 cells using the RNeasy kit (Qiagen). The RNAs were quantified and 20 μg of each sample was run on a 1.2% formaldehyde gel as described by Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 1989. The gel was blotted to a nylon membrane (BMB) and then ultraviolet (UV) cross-linked. Several human Northern blots (12 lane multi-tissue, human II and Immune system II) were purchased from Clontech. The filters was were prehybridized at 65° C. in ExpressHyb (Clontech) buffer for 30 min. and then hybridized with a randomly primed 32P labeled EcoNi fragment from the 3′ end of JST576 for about 3 hrs. The filters were washed at room temperature in 2×SSC/0.05% SDS for 45 min. and then at 50° C. in 0.1×SSC/0.1% SDS for 45 min The filter was exposed to X-ray film for 4 days using 2 intensifying screens. In addition, several human Northern blots (12 lane multi-tissue, human II and Immune system II) were purchased from Clontech, hybridized to the JST576 probe and treated as above.
Results
The mRNA for BAFF-R appears to predominately expressed in the immune system organs at this level of detection. The highest level is in the spleen and lymph nodes, but mRNA was also apparent in PBLs, thymus, small intestine and colon (FIGS. 10A, B and C). The approximate size of the message is 4.5 kb; there appears to be two mRNA populations in the samples where the gene is not highly expressed. Two mRNAs may exist in the spleen and lymph nodes as well. This may indicate that BAFF-R has alternative polyA addition sites or that the RNA undergoes alternative splicing. When a number of cell lines were examined for the presence of BAFF-R mRNA, the same 4.5 kb mRNA is detected. Only B cell lines express BAFF-R mRNA (FIG. 11). No mRNA is detected in the U266, RPM18226 and Daudi cell lines or in the non-B cell lines examined.
Example 11
In this example we show that JST576 expression is restricted to the cell lines that bind BAFF.
Materials and Methods
Cell lines were purchased from ATCC and grown under the indicated conditions. Various B and non-B cell lines were grown under the appropriate conditions. RNA was prepared from approximately 107 cells using the RNeasy kit (Qiagen). The RNAs were quantitated and 20 μg of each sample was run on a 12% formaldehyde gel as described by Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL, 1989. The gel was blotted to a nylon membrane (BMB) and then UV cross-linked. The filter was hybridized with a JST576 labeled fragment and then washed as in Example 10. The cells were checked for their ability to bind BAFF using FACS analysis. Approximately 2.5-5×105 cells were collected, and washed. FLAG-tagged BAFF, diluted in PBS+5% FCS and 0.05% sodium azide (FACS buffer), was incubated with the cells over the concentration range (S-0.125 μg/ml) for 30 min. on ice. The cells were washed with FACS buffer and incubated, for 30 min. on ice, with the anti-FLAG monoclonal antibody M2 (Sigma) at 5 μg/ml. Again the cells were washed with FACS buffer and then incubated with a 1:5000 dilution of goat anti-mouse IgG PE conjugated antibody (Jackson Immuno Research) for 30 min. on ice. The cells were washed as above and then analyzed on a FACSCalibur flow sorter (Becton-Dickinson) using CellQuest software.
Results
The results of the BAFF binding experiments are shown in Table 1. The cell lines that bind BAFF are Ramos, Namalwa, IM-9, NC-37, Raji, BJAB and SKW6.4. The level of binding is indicated by the number of + signs. The cell lines that do not bind BAFF are U266, RPMI 8226, Daudi, U937, Jurkat, HT29, A549, SW480 and ME260. The ability of the cell lines to bind BAFF is correlated to the presence of BAFF-R mRNA shown in FIG. 11.
TABLE 1
Cell Line
Type
BAFF Binding
BJAB
Burkitt lymphoma
+++
IM-9
Lymphoblast IgG
+++
NC-37
Lymphoblast EBV+
++
Ramos
Burkitt lymphoma EBV−
++
Raji
Burkitt lymphoma
++
SKW6.4
Lymphoblast IgM
++
Namalwa
Burkitt lymphoma
+
Daudi
Burkitt lymphoma EBV+
−
U266
Plasmacytoma
−
RPMI 8226
Plasmacytoma
−
U937
Monocyte
−
Jurkat
T Cell leukemia
−
HT29
Colorectal
−
adenocarcinoma
A549
Lung carcinoma
−
SW480
Colorectal
−
adenocarcinoma
ME260
Melanoma
−
Example 12
This example describes the ability of a huBAFF-R:huIgG1 fusion protein that is expressed and secreted into the conditioned media by transiently transfected 293EBNA cells to co-immunoprecipitate recombinant soluble biotinylated myc-huBAFF.
Materials and Methods
293EBNA cells were transfected by Lipofectamine2000 (LifeTechnologies) with either pJST618 which expresses huBAFF-R (aa2-71):Fc, a plasmid expressing huBCMA:huIgG1 as a positive control for BAFF binding, or a plasmid expressing huFN14:huIgG1 as a negative control for BAFF binding. After 24 hours incubation the conditioned media was harvested.
SDS-PAGE was run by mixing an equal volume of 2×SDS running buffer, with or without reducing agent, with the conditioned media and boiling for 5 minutes. The samples were then run on a 4-20% SDS polyacrylamide gel. Known quantities of purified hBCMA:Fc were run in adjacent lanes to estimate amount of hIgG1 fusion protein in the conditioned media. Samples were transferred to membranes (Immobillon P, Millipore) by western blot in 0.01M CAPS pH11-10% MeOH buffer. Membranes were blocked with 5% non-fat dry milk (NFDM) in TBST, probed with 1:3000 dilution of goat anti-human IgG-HRP (Jackson ImmunoResearch) for 1 hour, washed in TBST and exposed to film. Co-immunoprecipitations were performed by incubating 200 μl of the conditioned media with 200 ng recombinant soluble human flag-BAFF in 1 ml DMEM-10% fetal bovine serum-0.1% NaN3 overnight at 4° C. Protein A-Sepharose was added and incubations continued for 2 hours. The Sepharose beads were collected by centrifugation, washed with FACS TBST buffer, and resupended in SDS loading buffer with beta mercaptoethanol as a reducing agent. Samples were boiled 5 minutes, centrifuged briefly to pellet the Sepharose beads, and an aliquot rum on SDS-PAGE. FLAG-huBAFF, 50 ng, was run as a positive control. Samples were transferred to PVDF membranes (Immobillon P, Millipore) by western blot in 0.01M CAPS pH 11/10% MeOH buffer. Membranes were blocked with 5% NFDM-TBST, probed with 1 μg/ml anti-FLAG M2-HRP for 1 hour, washed in TBST and exposed to film.
Results
Co-immunoprecipitation brings down the various receptor:Fc fusions through the fusion partner interacting with protein A Sepharose. It will also bring down any proteins interacting with the R:IgG1 fusions, such as the flag-BAFF. Conditioned media from cells expressing hBCMA:Fc are able to co-immunoprecipitate flag-BAFF, as expected, as a band that co-migrates with flag-BAFF is observed on the western blot (FIG. 12). Conditioned media from cells expressing hFN14:Fc does not co-immunoprecipitate flag-BAFF. The conditioned media from cells expressing BAFF-R:Fc are able to co-immunoprecipitate flag-BAFF. A band that co-migrates with flag-BAFF is observed on the western blot and is of similar intensity to that co-immunoprecipitated by huBCMA:huIgG1.
Example 13
This example illustrates the ability of a BAFF-R:Fc fusion protein, in this case huBAFF-R (aa2-71):huIgG1, to block the binding of huBAFF to BJAB cells.
Materials and Methods
The huBAFF-R (2-71)-huIgG1 fusion discussed in example 9 was generated and called pJST618. This construct was transiently transfected into 293EBNA cells and the conditioned media was harvested. The fusion protein was purified by acid elution from proteinA Sepharose followed by and gel filtration chromatography. Biotinylated myc-huBAFF, 200 ng/ml, was preincubated with either 50 ul FACS buffer or with five fold serial dilutions, ranging from 5 μg/ml to 200 ng/ml, of purified huBAFF-R:Fc for 30 minutes on ice. BJAB cells (2.5×105 cells) were then incubated with these solutions on ice for one hour. Cells were washed with FACS buffer and stained with SAV-PE. The cells were analyzed by FACS for PE fluorescence and the data was formatted as overlayed histograms. Alternatively, 200 ng/ml biotinylated-BAFF was pre-incubated with two-fold serial dilutions of either hBAFF-R:Fc, hTACI:Fc, or hLTBR:Fc. Cells were stained for biotinylated BAFF binding as above.
Results
FIG. 13A shows the overlay of the histograms plotted for huBAFF binding to BJAB in the presence of various concentrations of huBAFF-R:Fc. The black line labelled “A” represents background binding of SAV-PE and the red line marked “E” represents cells stained with biotinylated myc-huBAFF without pre-incubation with BAFF-R:Fc. Pre-incubation of biotinylated myc-huBAFF with 5 μg/ml of huBAFF-R:Fc results in a shift in the histogram nearly to background levels (curve B). Pre-incubation with either 1 μg/ml (curve C) or 200 ng/ml (curve D) huBAFF-R-huIgG1 resulted in an approximate four-fold decrease in biotinylated myc-huBAFF binding.
FIG. 13B shows that both BAFF-R:Fc and TACI:Fc are able to block BAFF binding to BJAB cells. Pre-incubation with LTBR:Fc has no BAFF blocking effect.
Example 14
This example describes the ability of a BAFF-R:IgG1 fusion protein to block BAFF-induced B cell proliferation.
Material and Methods
For the in vitro proliferation assay, mouse B cells were isolated from spleens of C57Bl6 mice (8 weeks old) using a B cell recovery column (column (Cellect™ Mouse B Cell Recovery Column: Cedarlane Laboratories Limited, Ontario, Canada.). Purified B cells were analyzed by FACS and >90% were found positive for B220 staining. B cells were incubated in 96-well plates (105 cells/well in 50 ml RPMI supplemented with 10% FBS) for 72 hours in the presence or absence of 2 mg/ml of goat anti-human m chain antibody (Sigma Chemical Co.); control hIgG (10 mg/ml) huBAFF-R:Fc (10 mg/ml). The samples were done plated in triplicate and with the indicated concentrations of myc-hBAFF. Cells were pulsed for an additional 18 hours with [3H]thymidine (1 μCi/well) and harvested. [3H]Thymidine incorporation was monitored by liquid scintillation counting. Human BAFF-R:Fc fusion protein, produced as in example 13, was used in this assay., as discussed in example 9, was generated from the supernatant of pJST618 transfected 293EBNA cells. The supernatant was harvested, loaded onto a Protein A column, eluted with acid, neutralized and then subjected to gel filtration chromatography in order to obtain aggregate-free huBAFF-R:Fc protein. The BAFF used in the assay was expressed in Pichia pastoris and purified by anion exchange chromatography followed by gel filtration.
Results
FIG. 14 shows that BAFF can costimulate B cell growth in the presence of anti-m antibodies (squares) and hIgG (triangles). BAFF alone (diamonds) is not able to induce B cell proliferation. Incubation with 10 mg/ml of huBAFF-R:Fc (stars) results in a complete inhibition of BAFF-induced B cell proliferation.
Materials and Methods
Mice
Six-week old female BALB/c mice were obtained from The Jackson Laboratory (Bar Harbor, Me.) and maintained under barrier conditions in the Biogen Animal Facility.
Reagents and Treatment Regimen
Receptor fusion proteins contain the human IgG1 Fc region. Mice (5/group) received 200 μg of fusion proteins (mouse BAFF-R:Fc or human BAFF-R:Fc) 2×/week for 4 weeks, ip (intraperitoneally). Control mice received polyclonal human IgG(Panglobulin™) (HIgG), 200 μg 2×/week for 4 weeks. Three days after the last dose, blood was collected via the orbital sinus, then mice were euthanized and spleens, lymph nodes, and bone marrow were collected for analysis.
Flow Cytometric Analysis
At the time of sacrifice spleen weights were recorded. Single cell suspensions were prepared from spleen and blood after lysing red blood cells in a hypotonic solution. Single cell suspensions were also prepared from inguinal lymph nodes and bone marrow. Flow cytometry was performed using mAbs directed against B220, IgM, IgD and CD21. Splenic B cell subpopulations were defined as follicular (B220+, IgMlow, CD21low), marginal zone (B220+, IgMhi CT21hi) and newly formed (B220+, IgMhi CD21−). Briefly, ˜1.5×106 cells were incubated with 10 μg/ml of Fc Block (Pharmingen) for 10 min on ice to block Fc receptors, followed by addition of fluorescently tagged mAbs and incubated on ice for 30 min. Cells were washed 1× and resuspended in 0.5% paraformaldehyde. Cell fluorescence data were acquired on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, Calif.) and analyzed using CellQuest software (Becton Dickinson).
Results
After a 4-week treatment course with mouse or human BAFF-R:Fc there was a marked reduction in the weight of spleens from mice treated with mouse and human BAFF-R:Fc (FIG. 15), as compared to control Human IgG-treated mice. The apparent decline in splenic cellularity was found to result from a reduction in the number of splenic B cells. The mean number of total B220+ splenic B cells in mouse and human BAFF-R:Fc-treated mice, 1.8×106 and 2.6×106 cells, respectively, was significantly reduced when compared to the number of B cells in control HIgG-treated animals, which had a mean of 19.8×106 cells (FIG. 16). Examination of different subpopulations of splenic B cells, follicular, marginal zone and newly formed, indicated that the number of B cells in each subset was reduced in the BAFF-R::Fc-treated mice (Table 2), although follicular and marginal zone B cells had the greatest reduction.
TABLE 2
BAFF-R::Fc Treatment Results in a Reduction
in Splenic B Cell Subpopulations
Splenic B cell subpopulations (106 cells ± SD)
Follicular
Marginal Zone
Newly formed
Human IgG
14.5 ± 2.4
1.1 ± 0.3
1.5 ± 0.2
mBAFF-R:Fc
0.7 ± 0.1
0.06 ± 0.02
0.4 ± 0.1
hBAFF-R:Fc
1.4 ± 0.5
0.05 ± 0.02
0.5 ± 0.2
Mice received 200 μg of HIgG, mBAFF-R:Fc or hBAFF-R:Fc on days 1, 4, 8, 11, 15, 18, 22 and 25. Mice were euthanized on day 28 and spleens were harvested for analysis of B cell subsets.
Examination of the percent of B220+ B cells contained in inguinal lymph nodes (LN) showed that the mean B cell populations were markedly reduced in mouse and human BAFF-R::Fc-treated mice, 12.3%±1.4 and 18.6%±1.3, respectively, when compared to control HIgG-treated mice which had a mean of 30.8%±4.1 B cells (FIG. 17). Similar results were obtained when peripheral blood B cells were examined. 42.5%±2.9 of the lymphocytes from human IgG-treated mice were B cells, whereas only 21.2%±6.1 and 8.3%±4:5 of lymphocytes were B cells from mouse and human BAFF-R::Fc-treated mice, respectively (FIG. 18).
Although newly formed (immature) B cell and mature B cell populations were reduced in BAFF-R::Fc-treated mice, B cell precursors in the bone marrow remained unaffected (data not shown).
Discussion
These results suggest that in vivo blockade of BAFF with a soluble BAFF-R receptor fusion protein leads to the inhibition of B cell survival and/or maturation.
These results also suggest the potential use of a BAFF-R fusion protein as a therapeutic drug with clinical applications in B cell-mediated diseases. Diseases would include those that are autoimmune in nature such as systemic lupus erythematosus, myasthenia gravis, autoimmune hemolytic anemia, idiopathic thrombocytopenia purpura, anti-phospholipid syndrome, Chagas' disease, Grave's disease, Wegener's granulomatosis, poly-arteritis nodosa and rapidly progressive glomerulonephritis. This therapeutic agent would also have application in plasma cell disorders such as multiple myeloma, Waldenstrom's macroglobulinemia, heavy-chain disease, primary or immunocyte-associated amyloidosis, and monoclonal gammopathy of undetermined significance (MGUS). Oncology targets would include B cell carcinomas, leukemias, and lymphomas.
Example 16
In this example, the characterization of an initial panel of mouse monoclonal antibodies raised against the extracellular domain of BAFF-R is described. All antibodies recognize the extracellular domain of BAFF-R, and a subset of these antibodies have antagonist properties in that they prevent subsequent binding of BAFF to BAFF-R.
Materials and Methods:
RBF mice were immunized and boosted with huBAFF-R:Fc. Splenocytes from the immune mouse were fused with mouse myeloma strain FL653, a derivative of strain P3-X63-Ag8.653 to generate hybridomas by standard technologies.
Conditioned media from hybridoma clones secreting antibodies against the extracellular domain of huBAFFR were assayed by FACS. FACS binding assays were performed 293EBNA cells co-transfected with plasmids expressing full length huBAFF-R or muBAFF-R and GFP as in Example 5. Hybridoma conditioned media was diluted 1:10 in FACS buffer and incubated with the transfected cells 30 min on ice. Cells were washed with FACS buffer and binding was revealed by incubation with a 1:100 dilution of anti-mouse IgG (H+L) (Jackson ImmunoResearch) for 30 rain on ice. The cells were again washed with FACS buffer and resuspended in 1% paraformaldehyde in FACS buffer. The cells were analyzed by FACS for GFP and PE fluorescence and the data was formatted in a four quadrant dot plot as described in Example 5. BAFF blocking assays were performed by incubating 10 ug/ml proteinA purified anti-BAFF-R mAb or control antibody (MOP C21) with BJAB cells for 30 min on ice. After washing, cells were incubated with 250 ng/ml biotinylatedhuBAFF for 30 min on ice. Cells were again washed and BAFF binding was revealed by incubation with SAV-PE. The cells were analyzed by FACS for PE fluorescence and data was plotted as overlayed histograms.
Results:
The supernatants from ten clones were observed to bind huBAFF-R transfected cells. The dot plots of the FACS data of four of the ten anti-BAFF-R supernatants are shown in FIG. 19A. Transfection efficiency was approximately 50%, with nearly all transfected cells shifting to the upper right quadrant after staining with supernatants. None of these ten supernatants bound to 293EBNA cells transfected with muBAFFR (data not shown). Conditioned media from the clones that were positive for binding to BAFF-R were tested for their ability to block the interaction of BAFF with the BAFF-R expressed on the surface of BJAB cells. BJAB cells express BAFFR on their surface, and express no detectable amounts of BCMA or TACI (Thompson et al. (2001) Science August 16). Two of the ten hybridomas, clones 2 and 9, produced mAbs that were able to block the interaction of BAFF-R with BAFF. (Clone 2 was deposited with the ATCC on Sep. 6, 2001 as “anti-BAFF-R clone #2.1” (IgG1-kappa isotype) and has been assigned ATCC No. PTA-3689; Clone 9 was deposited with the ATCC on Sep. 6, 2001 as “anti-BAFF-R clone #9.1” (IgG1-kappa isotype) and has been assigned ATCC No. PTA-3688). The overlays of the histograms in FIG. 19B show that preincubation of 10 μg/ml of either mAb clone 2 (curve (b)) or 9 (curve (c)) shifts the BAFF binding curve greater than ten-fold to the left, nearly to the signal of the no BAFF control (curve (a)). The rightmost histogram (curve (d)) indicates the shift when control mAb MOP C21, anti-BAFF-R non-blocking mAbs, or no protein were incubated with the cells prior to BAFF binding.
Example 17
This example describes the construction, sequence and protein characterization of amino acid substitutions in hBAFF-R(2-71)-Fc that result in increased solubility of the recombinantly expressed molecule.
Materials and Methods:
Double stranded oligonucleotide cassettes with cohesive ends were used to introduce substitutions at targeted residues by ligation into the same sites in the hBAFF-R(2-71):IgG1 gene.
Expression plasmids were transfected into 293EBNA cells using Lipofectamine 2000 as in Example 5. Aggregation was determined by mining non-reducing SDS-PAGE of 20 hr post-transfection conditioned media, followed by western transfer, and detection with HRP conjugated anti-human IgG (1:100, Jackson ImmunoResearch) and ECL detection as in Example 12.
Immunoprecipitation experiments were performed utilizing 100 μl of 20 hr post-transfection conditioned media in 1 ml of DMEM/10% FBS/0.2% NaA3 with 200 ng flag-huBAFF. Samples were rocked for 30 min at 4° C., 30 ul protein A-Sepharose was added per tube and rocking continued for another 30 minutes. Sepharose beads were spun down and washed three times with 1 ml cold PBS. Beads were resuspended in 2×SDS reducing buffer and loaded onto 4-20% acrylamide gels. After western transfer as previously described, the ability to immunoprecipitate flag-BAFF was revealed by incubation of the filters with 1 μg/ml BRP conjugated anti-flag M2 (Sigma) followed by ECL detection.
Results:
While the human BAFF-R:Fc is highly aggregated, the murine BAFF-R:Fc is only slightly (<10%) aggregated. Deletion analysis has shown that the entire C-terminal BAFF-R moiety can be deleted from A71 to V36 (last Cys of Cysteine Rich Domain (CRD) is C35) with no decrease in aggregate formation. This would implicate the N-terminal and CRD regions of hBAFFR as being required for aggregate formation.
Initially, several murine-human BAFF-R:Fc chimeras were generated in which various amounts of N-terminal human BAFF-R sequence were replaced with the homologous murine sequence and analyzed for the effect on protein aggregation. The amino acid sequence for these and subsequent substitutions into hBAFF-R:Fc are shown in FIG. 20. This figure shows the BAFF-R moiety of both the “wild type” human (FIG. 9) and murine BAFF-R:Fc, with the numbering corresponding to the amino acid residues from the full length human (FIG. 2d) (SEQ ID NO:5) or murine BAFF-R (FIG. 4b) (SEQ ID NO:9). FIG. 20 also shows the hBAFFR-R:Fc clones with substitutions, with the substituted residues indicated in bolded, red, underlined type. The chimeras containing less than the first 21 murine residues (Q21) before switching over to human appear to aggregate similar to wild type hBAFF-R:Fc; however, those that contain at least the first 39 murine residues aggregate in a markedly reduced manner, similar to mBAFF-R. Of the additional nine residues different between these two chimeric BAFF-R:Fc constructs, four of them differ between mouse and human. This would implicate at least one of the human residues between C19 and L27, a region internal to the CRD, as being required for aggregation.
Constructs replacing the human residues with those corresponding to murine at only these 4 sites or a subset thereof were made by standard techniques. When only the 4 residues V20N P21Q A22T L27P were substituted into the human BAFFR moiety, this modified BAFF-R:Fc was not aggregated. hBAFF-R(V20N P21Q A22T L27P):Fc were still able to interact with BAFF as analyzed by immunoprecipitation. The V20N L27P substitution also reduced aggregation of hBAFF-R:Fc from approximately 90% to about 10%. Intermediate levels of aggregation were observed with P21Q L27P (40%), L27P (60%), V20N L27A (60%) and V20N L27S (60%). None of the following substitutions diminished protein aggregation: V20N P21Q A22T; V20N A22T; V20N P21Q; V2ON; and P21Q.
Example 18
This example describes p21-Arc is a protein associated with BAFF-R. The method used to determine such an interaction was immunoprecipitation.
Methods
A construct containing the cDNA encodes the intracellular domain of BAFF-R (BAFF-R-i.c.d.) with a myc tagged fused at the N-terminus was made and subcloned into CH269 plasmid at NheI (5′) and XhoI (3′) sites. The 293E cells were transfected with this construct and were lysed 72 hours after with lysis buffer containing in 150 mM NaCl, 50 mM Tris-HCl, pH7.5, 1 mM Na3VO4, 50 mM NaF and 1% Brij 97. The cell lysates were cleared with a table top centrifuge at 10,000 g for 5 minutes and were immunoprecipitated with an anti-myc monoclonal antibody, 9E10. The immunoprecipitates were separated by a 10-20% SDS-PAGE under reducing conditions and were trans-blotted onto a PVDF membrane. The blotted proteins were visualized with 0.2% Ponceau S solution and the areas corresponding to proteins specifically associated with BAFF-R were excised and subjected to N-terminal amino acid sequence analysis. An ambiguous search in the non-redundant protein database using the PATTERN SEARCH algorithm was performed for the obtained N-terminal sequence data.
Results
One of the proteins specifically associated with the myc-tagged BAFFR cytoplasmic domain has an apparent molecular weight of 21 kDa. This protein was unambiguously identified as the p21-Arc (Actin related protein complex). P21-Arc is a component of a seven subunits protein called Arp2/3 complex which was shown to be involved in the actin polymerization (Welch et al. (1997) J. Cell Biol. 138:357). Recently, an actin-binding protein, filamin, was reported to be associated with the tumor necrosis factor receptor-associated factor 2 (TRAF2) (Leonardi et al. (2000) J. Biol. Chem. 275:271). Thus, the identification of p21-Arc in the co-immunoprecipitates of BAFFR cytoplasmic domain suggests p21-Arc is either directly associated the BAFFR or indirectly associated with BAFFR via its association with TRAF2 and/or other TRAF protein which, in turn, associates with the BAFFR.
From the foregoing detailed description of the specific embodiments of the invention, it should be apparent that unique have been described. Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims which follow. In particular, it is contemplated by the inventor that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims.
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12715987
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biogen idec ma inc.
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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/ 7.1
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Apr 1st, 2022 05:10PM
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Apr 1st, 2022 05:10PM
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Biogen
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:biib
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Biogen
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May 27th, 2014 12:00AM
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Oct 30th, 2009 12:00AM
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https://www.uspto.gov?id=US08734795-20140527
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Light targeting molecules and uses thereof
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LIGHT-targeting molecules (e.g., LIGHT fusion molecules), anti-HER2 antibody molecules, compositions, e.g., pharmaceutical compositions thereof, are disclosed. Methods of using these molecules to treat, prevent and/or diagnose hyperproliferative, e.g., neoplastic, diseases or conditions, including, but not limited to, cancer and metastasis are also provided.
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8734795
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1. A LIGHT-targeting molecule, comprising an extracellular domain of a human LIGHT protein or fragment thereof, and a targeting antibody molecule that binds to HER2, wherein the targeting antibody molecule comprises a heavy chain variable (VH) amino acid sequence comprising the amino acid sequence of CDR1 (SEQ ID NO:47), CDR2 (SEQ ID NO:48) and CDR3 (SEQ ID NO:49) and a light chain variable (VL) amino acid sequence comprising the amino acid sequence of CDR1 (SEQ ID NO:74), CDR2 (SEQ ID NO:75) and CDR3 (SEQ ID NO:76), and wherein the targeting antibody molecule is linked to the extracellular domain of the human LIGHT protein or fragment thereof.
2. The LIGHT-targeting molecule of claim 1, wherein the VH is linked to a heavy chain constant region (CH), which is linked, with or without a linking group (L), to the extracellular domain of the human LIGHT protein or fragment thereof.
3. The LIGHT-targeting molecule of claim 1, which comprises a dimer or a trimer of the extracellular domain of the human LIGHT protein or fragment thereof and the targeting antibody molecule.
4. The LIGHT-targeting molecule of claim 3, wherein the extracellular domain of the LIGHT protein or fragment thereof has one or more LIGHT-associated activities chosen from: (i) binding to one or more LIGHT-receptors; (ii) inducing expression of one or more of chemokines or cytokines, chemokine or cytokine receptors, adhesion molecules, or co-stimulatory molecules; (iii) activating T cells; (iv) recruiting T cells into a tumor, cell or tissue; (v) activating or enhancing tumor-reactive T cell proliferation; (vi) creating a lymphoid-like microenvironment at a tumor cell or tissue; (vii) inducing apoptosis of a tumor cell or tissue; or (viii) stimulating an immune response in a subject.
5. The LIGHT-targeting molecule of claim 4, wherein the extracellular domain of the human LIGHT protein or fragment thereof comprises amino acids 93to 240 of SEQ ID NO:1 (human LIGHT isoform 1), or an amino sequence at least 90% identical thereto.
6. The LIGHT-targeting molecule of claim 1, which comprises a linker comprising one, two, three, four or five (G4S)4 (SEQ ID NO:134) repeats.
7. The LIGHT-targeting molecule of claim 6, comprising a heavy chain having the amino acid sequence of SEQ ID NO:4 and a light chain having the amino acid sequence of SEQ ID NO:109, or an amino acid sequence at least 90% identical to SEQ ID NO:4 or SEQ ID NO:109.
8. The LIGHT-targeting molecule of claim 1, wherein the VH amino acid sequence comprises the amino acid sequence of SEQ ID NO:11, or an amino acid sequence at least 90% identical thereto.
9. The LIGHT-targeting molecule of claim 1, wherein the VL amino acid sequence comprises the amino acid sequence of SEQ ID NO:13, or an amino acid sequence at least 90% identical thereto.
10. The LIGHT-targeting molecule of claim 1, wherein the VH amino acid sequence comprises the amino acid sequence of SEQ ID NO:11, or an amino acid sequence at least 90% identical thereto, and wherein the VL amino acid sequence comprises the amino acid sequence of SEQ ID NO:13, or an amino acid sequence at least 90% identical thereto.
11. The LIGHT-targeting molecule of claim 1, wherein the VH amino acid sequence comprises the amino acid sequence of SEQ ID NO:11.
12. The LIGHT-targeting molecule of claim 1, wherein the VL amino acid sequence comprises the amino acid sequence of SEQ ID NO:13.
13. The LIGHT-targeting molecule of claim 1, wherein the VH amino acid sequence comprises the amino acid sequence of SEQ ID NO:11, and wherein the VL amino acid sequence comprises the amino acid sequence of SEQ ID NO:13.
14. The LIGHT-targeting molecule of claim 1, wherein the extracellular domain of the LIGHT protein comprises amino acids 93 to 240 of SEQ ID NO:1.
15. A pharmaceutical composition comprising the LIGHT-targeting molecule of claim 7 and a pharmaceutically acceptable carrier.
16. A LIGHT-targeting molecule, comprising an extracellular domain of a human LIGHT protein, and a Fab fragment that binds to HER2, wherein the Fab fragment comprises:
a heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:11; and
a light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:13;
and wherein the VH domain is linked to a heavy chain constant region 1 (CH1) which is linked to the extracellular domain of a human LIGHT protein comprising amino acids 93 to 240 of SEQ ID NO:1.
17. The LIGHT-targeting molecule of claim 16, further comprising a linking group which links the CH1 to the extracellular domain of a human LIGHT protein.
18. The LIGHT-targeting molecule of claim 17, wherein the linking group comprises one, two, three, four or five (G45)4 (SEQ ID NO:134) repeats.
19. The LIGHT-targeting molecule of claim 17, wherein the linking group comprises amino acids 61 to 92 of SEQ ID NO:1.
20. A LIGHT-targeting molecule, comprising an extracellular domain of a human LIGHT protein, and a Fab fragment that binds HER2, wherein the Fab fragment comprises:
the amino acid sequence of SEQ ID NO:4; and
the amino acid sequence of SEQ ID NO:109;
and wherein the Fab fragment is linked with a linking group to the extracellular domain of a human LIGHT protein comprising amino acids 93 to 240 of SEQ ID NO:1.
21. The LIGHT-targeting molecule of claim 20, wherein the linking group comprises one, two, three, four or five (G45)4 (SEQ ID NO:134) repeats.
22. The LIGHT-targeting molecule of claim 20, wherein the linking group comprises amino acids 61 to 92 of SEQ ID NO:1.
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Patent Application Ser. No. 61/110,359, filed on Oct. 31, 2008, under 35 U.S.C. §119, the contents of which are hereby incorporated by reference in their entirety. This application also incorporates by reference the International Application filed with the U.S. Receiving Office on Oct. 30, 2009, entitled “LIGHT Targeting Molecules and Uses Thereof” and bearing PCT/US09/62870.
SEQUENCE LISTING
This application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 30, 2009, is named 983815—1_B2047—706010_seqTxt.txt, and is 130,331 bytes in size.
BACKGROUND
LIGHT, also known as TNFSF14 or CD258, is a member of the TNF superfamily (TNFSF) of ligands. Its name is derived from lymphotoxin-like, exhibits inducible expression and competes with HSV glycoprotein D for herpes virus entry mediator (HVEM), a receptor expressed by T lymphocytes. LIGHT is expressed on the surface of T cells upon activation in a tightly regulated manner (Castellano et al. (2002) J. Biol. Chem. 277 42841-51). LIGHT mediates its biological effects by binding one of three TNF superfamily receptors, including the lymphotoxin β receptor (LTβR) (Crowe et al. (1994) Science 264 707-10, Browning et al. (1997) J Immunol 159: 3288-98), the herpes virus entry mediator (HVEM) (Montgomery et al. (1996) Cell 87(3): 427-36), and decoy receptor 3 (DcR3) (Yu et al. (1999) J. Biol. Chem. 274 13733-6). Upon interaction with its receptors, LIGHT exhibits a number of immunostimulatory activities, including regulation of chemokine expression and cell adhesion molecules (Wang, J. et al. (2002) Eur. J. Immunol. 32:1969-1979). For example, LIGHT and LTα1β2 cooperate in lymphoid organogenesis and the development of lymphoid structures (Scheu, S. et al. (2002) J. Exp. Med. 195: 1613-1624; Wang, J. et al. (2002) supra). Signaling of LTβR via a LIGHT transgene has been shown to be sufficient to induce up-regulation of expression of chemokines and adhesion molecules (Wang, J. et al. (2004) J. Clin. Invest. 113: 826-835). LIGHT has also been shown to mediate CD28-independent co-stimulatory activity for T cell priming and expansion, which can lead to enhanced T cell immunity against tumors and/or increased autoimmunity (Tamada, K. et al. (2000) Nat. Med. 6:283-289; Ware, C. F. (2005) Annu. Rev. Immunol. 23:787-819; Wang, J. et al. (2005) J. Immunol. 174:8173-8182).
Given the broad range of immunostimulatory activities associated with LIGHT, the need still exists for identifying novel targeting agents for harnessing these LIGHT-associated activities for the treatment of various hyperproliferative diseases, for example, neoplastic diseases including cancer and metastasis.
SUMMARY
The present invention is based, at least in part, on the generation of LIGHT-targeting molecules (e.g., LIGHT proteins or nucleic acids encoding LIGHT proteins) that are selectively delivered to a hyperproliferative, e.g., cancerous, cell or tissue, thereby eliciting one or more anti-tumor responses, including, but not limited to, tumor cell killing and/or anti-tumor immunity. In one exemplary embodiment, the LIGHT-targeting molecule includes at least one fusion protein of a mammalian (e.g., human) LIGHT protein, or a functional variant or a fragment thereof, and an antibody molecule that binds to HER2 (referred to herein as “LIGHT-anti-HER2 fusion”). In other embodiments, novel antibody molecules against HER2 are disclosed. Thus, the present invention provides, at least in part, LIGHT-targeting molecules (e.g., LIGHT fusion molecules), anti-HER2 antibody molecules, compositions, e.g., pharmaceutical compositions thereof, as well as methods of using these molecules to treat, prevent and/or diagnose hyperproliferative, e.g., neoplastic, diseases or conditions, including, but not limited to, cancer and metastasis.
Accordingly, in one aspect, the invention features a LIGHT targeting molecule that includes at least one LIGHT moiety (e.g., a LIGHT protein, or a functional variant or a fragment thereof (e.g., the extracellular domain of LIGHT or a portion thereof), and at least one targeting moiety (e.g., a binding agent, such as an antibody molecule) that interacts, e.g., binds to, a target protein on a hyperproliferative cell (e.g., a cell surface protein expressed on a cancer or tumor cell or tissue), thereby targeting, delivering, or otherwise bringing, the LIGHT moiety to the hyperproliferative cell or tissue. In embodiments, the LIGHT molecule is linked (e.g., by chemical coupling, genetic or polypeptide fusion, non-covalent association or otherwise) to the targeting moiety. For example, the LIGHT molecule can be fused, with or without a linking group (e.g., a peptidic linking group), to the targeting moiety as a genetic or a polypeptide fusion. In other embodiments, the LIGHT molecule is covalently attached to the antibody molecule via a reactive group with or without a linking group (e.g., a non-proteinaceous biocompatible polymer). The LIGHT targeting molecule can be a monomer, dimer, trimer, tetramer, pentamer, sixmer or more of at least one LIGHT moiety and at least one targeting moiety. In embodiments, the LIGHT targeting molecules comprises, or consists essentially of, one, two, three, four, five, six, seven or eight contiguous or non-contiguous polypeptide chains. In other embodiments, the LIGHT targeting molecule comprises, or consists essentially of, one, two, three, four, five or six monomeric or dimeric subunits, each subunit comprising at least one LIGHT moiety and at least one targeting moiety. For example, the LIGHT targeting molecule may include at least one, two, three, four, five or six LIGHT fusion molecules, each fusion molecule comprising at least one LIGHT moiety and at least one targeting moiety. In some embodiments, the LIGHT fusion molecule can be a single chain polypeptide (e.g., a LIGHT moiety fused to a single chain or a single domain antibody), or at least two, three, four, five, six or more non-contiguous polypeptides forming, e.g., a dimer, trimer, tetramer, pentamer, sixmer or higher complex of non-contiguous polypeptides (e.g., a LIGHT moiety fused to one chain of an antibody molecule, e.g., a two-chain antibody or antigen-binding fragment thereof (e.g., a Fab fragment as depicted in FIG. 1 and FIG. 24). In other embodiment, the LIGHT targeting molecule comprises, or consists essentially of, two or three, LIGHT fusion molecules, each one comprising, or consisting essentially of, one LIGHT moiety (e.g., a LIGHT moiety as described herein) and one targeting moiety (e.g., a targeting moiety as described herein, e.g., a Fab antibody fragment). In another embodiment, the LIGHT targeting molecule has a trimeric or a dimeric configuration as shown in FIG. 1 or FIG. 24, respectively.
In embodiments, the LIGHT moiety of the LIGHT targeting molecule comprises, or consists essentially of, at least one LIGHT protein, or a functional variant or a fragment thereof. The LIGHT protein can be a soluble form of mammalian (e.g., human) LIGHT, e.g., a soluble form of an extracellular domain of mammalian (e.g., human) LIGHT (e.g., a full extracellular domain of LIGHT or a portion thereof). In one embodiment, the LIGHT protein, variant or fragment thereof, has one or more LIGHT-associated activities, including, but not limited to: (i) binding to one or more LIGHT-receptors (e.g., lymphotoxin β receptor (LTβR), the herpes virus entry mediator (HVEM), and/or decoy receptor 3 (DcR3)); (ii) inducing expression of one or more of chemokines or cytokines (e.g., CXCL10 (IP-10), CCL21, CXCL9, IL-5, IL-8 and/or TNF), chemokine or cytokine receptors (e.g., IL-10RA), adhesion molecules, and/or co-stimulatory molecules; (iii) activating T cells, e.g., lymphocytes (e.g., cytotoxic T lymphocytes), CD4- or CD8-expressing T cells, and/or regulatory T cells; (iv) recruiting T cells into a hyperproliferative, e.g., tumor, cell or tissue; (v) activating and/or enhancing tumor-reactive T cell proliferation; (vi) creating a lymphoid-like microenvironment, e.g., at a hyperproliferative, e.g., a tumor cell or tissue; (vii) inducing apoptosis of a hyperproliferative (e.g., tumor) cell or tissue; and/or (viii) stimulating an immune response in a subject, e.g., stimulating a subject's immune system against a hyperproliferative, e.g., a tumor or a cancerous, cell or tissue.
Exemplary LIGHT proteins of the LIGHT moiety include, or consist essentially of, the amino acid sequence from: (i) about amino acids 93 to 240 of SEQ ID NO:1 (corresponding to a portion of the extracellular domain of human LIGHT isoform 1); about amino acids 253 to 400 of SEQ ID NO:2 (corresponding to the portion of the LIGHT extracellular domain fused to the heavy chain fragment of a Fab antibody molecule of the anti-HER2 antibody molecule 71F10 via a delta 4 linker, referred to herein as “pBIIB71F10-130”); about amino acids 258 to 405 of SEQ ID NO:3 (corresponding to the portion of the LIGHT extracellular domain fused to the heavy chain fragment of a Fab antibody molecule of the anti-HER2 antibody molecule 71F10 via a G4S delta 4 linker, referred to herein as “pBIIB71F10-131”); about amino acids 245 to 392 of SEQ ID NO:4 (corresponding to the portion of the LIGHT extracellular domain fused to the heavy chain fragment of a Fab antibody molecule of the anti-HER2 antibody molecule 71F10 via a (G4S)4 linker, referred to herein as “pBIIB71F10-132”), or an amino acid sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto); or (ii) an amino acid sequence encoded by a nucleotide sequence chosen from one or more of: about nucleotides 277 to 720 of SEQ ID NO:5 (nucleotide sequence corresponding to a portion of the extracellular domain of human LIGHT isoform 1); about nucleotides 757 to 1200 of SEQ ID NO:6 (nucleotide sequence corresponding to the portion of the LIGHT extracellular domain fused to the heavy chain fragment of the 71F10 Fab antibody molecule via the delta 4 linker (pBIIB71F10-130); about nucleotides 772 to 1215 of SEQ ID NO:7 (nucleotide sequence corresponding to the portion of the LIGHT extracellular domain fused to the heavy chain fragment of the 71F10 Fab antibody molecule via the G4S delta 4 linker (pBIIB71F10-131); or about nucleotides 733 to 1176 of SEQ ID NO:8 (nucleotide sequence corresponding to the portion of the LIGHT extracellular domain fused to fused to the heavy chain fragment of the 71F10 Fab antibody molecule via the (G4S)4 linker (pBIIB71F10-132), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto).
The LIGHT moiety may, optionally, include, or consist essentially of, one or more amino acid residues (e.g., at least 10 to 35, 15 to 30, or about 20 to 26 amino acid residues) from the extracellular domain of LIGHT or a mutated form thereof, e.g., from about amino acids 61 to 92 of SEQ ID NO:1, corresponding to human LIGHT isoform 1; about amino acids 225 to 252 of SEQ ID NO:2, corresponding to 71F10 Fab-hLIGHT fusion via the delta 4 linker (pBIIB71F10-130); about amino acids 230 to 257 of SEQ ID NO:3, corresponding to 71F10 Fab-hLIGHT fusion via the G4S delta 4 linker (pBIIB71F10-131); or an amino acid sequence substantially identical thereto; or an amino acid sequence encoded by the nucleotide sequence from about nucleotides 181 to 276 of SEQ ID NO:5, corresponding to the nucleotide sequence encoding human LIGHT isoform 1; about nucleotides 673 to 756 of SEQ ID NO:6, corresponding to the nucleotide sequence encoding 71F10 Fab-hLIGHT fusion via the delta 4 linker (pBIIB71F10-130); about nucleotides 688 to 771 of SEQ ID NO:7, corresponding to the nucleotide sequence encoding 71F10 Fab-hLIGHT fusion via the G4S delta 4 linker (pBIIB 71F10-131), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto).
Variants of the LIGHT protein, or soluble fragments thereof (e.g., LIGHT extracellular domain or a portion thereof), altered to increase one or more properties of LIGHT, e.g., protein stability, immune enhancing function, can be used. For example, the LIGHT protein can be modified to have one or more protelolytic sites substantially inactivated (e.g., by deletion, mutation and/or otherwise inactivating, e.g., by amino acid insertion, of a proteolytic site). In one embodiment, amino acids EQLI (SEQ ID NO:9) comprising a proteolytic site at position 82 to 83 of the human LIGHT sequence (human LIGHT isoform 1, SEQ ID NO:1), or amino acids EKLI (SEQ ID NO:10) from positions 79-82 of the mouse LIGHT sequence are removed. In other embodiments, the LIGHT protein is from non-human origin, e.g., murine LIGHT, can be used. The amino acid and corresponding nucleotide sequences for full length mouse LIGHT are shown in SEQ ID NOs:113 and 114, respectively.
In other embodiments, the targeting moiety delivers, directs or brings, the LIGHT moiety to a desired site, e.g., a hyperproliferative, e.g., cancerous, cell or tissue, such that the LIGHT moiety induces one or more LIGHT-associated activities (e.g., one or more of the LIGHT-associated activities as described herein) against the desired site (e.g., the hyperproliferative, e.g., cancerous, cell or tissue). In certain embodiments, the targeting moiety may have an anti-tumor or cancer cell effect substantially independent from the LIGHT moiety (e.g., by inhibiting one or more activities of a cell surface protein or receptor involved in tumor growth, proliferation and/or survival, including but not limited to, receptor phosphorylation, receptor oligomerization, and/or preventing or retarding tumor cell growth or metastasis).
Without being bound by theory, Applicants believe that the targeted delivery to a tumor of LIGHT via the LIGHT targeting molecules of the invention can inhibit, block or otherwise reduce hyperproliferative and/or tumor growth through at least one or more of the following activities: (i) activation of lymphotoxin β receptor (LTβR) (e.g., triggering one or more of tumor cell cytotoxicity through LTβR signaling and/or recruitment of cytotoxic T lymphocytes into tumors); (ii) activation of HVEM; (iii) inducing expression of one or more of chemokines or cytokines (e.g., CXCL10 (IP-10), CCL21, CXCL9, IL-5, IL-8 and/or TNF), chemokine or cytokine receptors (e.g., IL-10RA), adhesion molecules, and/or co-stimulatory molecules; (iv) activating T cells, e.g., lymphocytes (e.g., cytotoxic T lymphocytes), CD4- or CD8-expressing T cells, and/or regulatory T cells; and/or (v) directing anti-tumor activity, for example, via the targeting moiety (e.g., a Fab antibody molecule) or the LIGHT moiety, thereby inducing targeted tumor cell death by mounting an effective T cell response against hyperproliferative cells or tumors, including primary tumors and metastasis. In some embodiments, the LIGHT targeting moiety causes a reduction, inhibition, or otherwise blockade of growth factor signaling (e.g., reduction of one or more signaling pathways, such a phosphorylation, receptor dimerization). A schematic of one proposed mechanism of action for the LIGHT targeting molecule is shown herein as FIG. 3.
Exemplary hyperproliferative, e.g., cancerous, cells or tissues, that can be targeted with the targeting moiety, include, but are not limited to, cancers or solid tumors of the breast, lung, stomach, ovaries, prostate, pancreas, colon, colorectum, renal, bladder, liver, head, neck, brain, as well as soft-tissue malignancies, including lymphoid malignacies, leukemia and myeloma. The targeting moiety can bind to one or more target molecules, e.g., soluble or cell surface proteins expressed on one or more of the hyperproliferative cells or tissues described herein. For example, the targeting moiety can bind to one or more of a growth factor receptor (e.g., HER2/neu, HER3, HER4, epidermal growth factor receptor (EGFR), insulin growth factor receptor (IGFR), Met, Ron, Cripto); a cancer-related integrin or integrin receptor (e.g., αvβ6, α6β4, laminin receptor (LAMR); and/or CD23, CD20, CD16, EpCAM, Tweak receptor (FN14) carcinoembryonic antigen (CEA), prostate specific membrane antigen (PSMA), TAG-72, and/or VEGF, among others. Additional examples of target molecules recognized by the targeting moieties are described herein.
In certain embodiments, the targeting moiety is an antibody molecule or a receptor ligand (e.g., a growth factor or a hormone). In embodiments where the targeting moiety is an antibody molecule, the antibody molecule can be a monoclonal or single specificity antibody, or an antigen-binding fragment thereof (e.g., a Fab, a F(ab′)2, an Fv, a single chain Fv fragment, a single domain antibody or a variant thereof (e.g., a heavy or light chain variable domain monomer or dimer, e.g., VH, VHH)); a single chain Fc fragment, a diabody (dAb), a camelid antibody; one, two, or all three complementarity determining regions (CDRs) grafted onto a repertoire of VH or VL domains, or other scaffolds (such as, e.g., a fibronectin domain or scaffold, T cell receptor, an Affibody molecule (e.g., an Affibody protein Z scaffold or other molecules as described, e.g., in Lee et al. (2008) Clin Cancer Res 14(12):3840-3849; Ahlgren et al. (2009) J. Nucl. Med. 50:781-789), Lipocalin, ankyrin repeats, LDL receptor domain, RNA aptamer, PDZ domain and microbody) (or a combination of one or more of the aforesaid antibody molecules). The antibody molecule can interact with, e.g., bind to, the desired cell surface protein (e.g., a cell surface protein as described herein). For example, the antibody molecule may include a combination of a single chain (e.g., a single chain Fc) and a Fab or a scFv. In other embodiments, the antibody molecule can be a multispecific (e.g., bivalent or bispecific) antibody or fragment thereof. In some embodiments, the antibody molecule binds to a single epitope on the cell surface protein. In other embodiments, the antibody molecule is a multi-specific antibody and binds to two or more epitopes on one or more cell surface proteins (e.g., one or more cell surface proteins as described herein). Typically, the antibody molecule is a human, a humanized, a chimeric, a camelid, a shark, or an in vitro generated antibody (or a functional fragment thereof, e.g., an antigen binding fragment as described herein). In certain embodiments, the antibody molecule binds to the cell surface protein with an affinity characterized by a dissociation constant (Kd) at least of 1×10−7 M, 1×10−8 M, 1×10−9 M, 1×10−10 M, 1×10−11 M, 1×10−12M, 1×10−13 M.
The antibody molecule can be full-length (e.g., can include at least one, and typically two, complete heavy chains, and at least one, and typically two, complete light chains) or can include an antigen-binding fragment (e.g., a Fab, F(ab′)2, Fv, a single chain Fv fragment, or other antigen binding fragment as described herein). In yet other embodiments, the antibody molecule has a heavy chain constant region (or a portion thereof, e.g., a CH1 region) chosen from, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly, chosen from, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4 of, e.g., a human, antibody. In another embodiment, the antibody molecule has a light chain constant region chosen from, e.g., the (e.g., human) light chain constant regions of kappa or lambda, or a portion thereof. The constant region can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues (—S—S— bonds), effector cell function, and/or complement function).
Exemplary LIGHT targeting molecules are shown as FIGS. 1-3 (schematic forms), 6, and 24 (dimeric form) or as SEQ ID NOs:2-4, 6-8, 101-104, 109-110, 162-163, 167-168, 173-174 and 178-179 (including nucleotide and amino acid sequences). Examples of these molecules include, but are not limited to, a fusion or a conjugate of a LIGHT moiety and a Fab fragment in monomeric, dimeric or trimeric form. In one embodiment, the N-terminal end of the LIGHT moiety (e.g., a human LIGHT fragment as described herein (e.g., about amino acids 93 to 240 of SEQ ID NO:1 (corresponding to a portion of the extracellular domain of human LIGHT isoform 1)) is covalently linked, e.g., as a polypeptide fusion, via a linking group to the C-terminal region of the Fab heavy chain constant region (e.g., a portion of, or the full, CH1 hinge region of IgG1) fused to the Fab heavy chain variable domain, while the heavy and light chains of the Fab associate with each other (FIG. 1 or FIG. 24). In one embodiment, the portion of the Fab CH1 region used in the fusion contributes to the assembly of three LIGHT-Fab fusions as a trimer depicted in FIG. 1, and exemplified by LIGHT fusions pBIIB71F10-130, pBIIB71F10-131, pBIIB71F10-132, pBIIBCD23-204 and pBIIBC06-117 (see Examples 6, 7 and 27 herein). In other embodiments, the full CH1 region of the IgG1 (e.g., about amino acids 225 to 232 of SEQ ID NO:172) is used in the fusion, which contributes to the assembly of two LIGHT-Fab fusions as a a dimer stabilized by the formation of one or more disulfide bonds as depicted in FIG. 24 (Example 28).
In certain embodiments, the LIGHT targeting molecules can include at least two non-contiguous polypeptide having the following configuration: a first polypeptide having a light chain variable domain (VL) fused to a light chain constant region (CL), for example, VL-CL; and a second polypeptide having a heavy chain variable domain (VH) fused to a portion or full heavy chain constant region (CH, particularly, CH1), which is fused, with or without a linking group (L), to the N-terminal end of the LIGHT moiety (e.g., a human LIGHT fragment as described herein) for example, VH-CH-(optionally)-L-LIGHT moiety. The LIGHT-Fab fusions can associate as dimers or as trimeric complexes (e.g., a trimeric complex of about 220 kD) or as dimeric complexes (e.g., a dimeric complex of about 150 kD). In other embodiments, a LIGHT-full antibody fusion or conjugate can be used, e.g., a fusion or conjugate wherein an N- or C-terminal region of the LIGHT moiety is covalently linked, e.g., as a polypeptide fusion, to the C-terminal end of each of the heavy chains of the full antibody (e.g., forming a LIGHT dimeric complex of about 200 kDa) (FIG. 2). In yet other embodiments, a single chain Fc fused to a C-terminal end of the VH region of a Fab is covalently linked, e.g., as a polypeptide fusion, to the LIGHT moiety in monomeric, dimeric or trimeric form (FIG. 2). In yet other embodiments, the LIGHT moiety s covalently linked, e.g., as a polypeptide fusion, to a single chain Fv in monomeric, dimeric or trimeric form (FIG. 2). In another embodiment, one or more Affibody domains are covalently linked to the LIGHT moiety (FIG. 2). In yet another embodiment, a LIGHT moiety fused to an immunoglobulin Fc region is covalently linked, e.g., as a polypeptide fusion, to a Fab in a monomeric, a dimeric or a trimeric form (FIG. 2 or FIG. 24).
In certain embodiments, the LIGHT moiety and the targeting moiety are functionally linked (e.g., by chemical coupling, genetic or polypeptide fusion, non-covalent association or otherwise). For example, the LIGHT molecule can be fused, with or without a linking group (e.g., a peptide linking group), to the targeting moiety as a genetic or a polypeptide fusion. In other embodiments, the LIGHT molecule is covalently attached to antibody molecule via a reactive group, optionally, via a biocompatible, non-proteinaceous polymer. The linking group can be any linking group apparent to those of skill in the art. For instance, the linking group can be a biocompatible polymer with a length of 1 to 100 atoms. In one embodiment, the linking group includes or consists of polyglycine, polyserine, polylysine, polyglutamate, polyisoleucine, or polyarginine residues, or a combination thereof. For example, the polyglycine or polyserine linkers can include at least five, ten, fifteen or twenty glycine and serine residues in the following configuration, (Gly)4-Ser (SEQ ID NO: 145), in one, two, three, four, five or more repeats, e.g., three or four repeats of (Gly)4-Ser (SEQ ID NO: 134). In other embodiments, linking group may include one or more amino acid residues (e.g., at least 10 to 35, 15 to 30, or about 20 to 26 amino acid residues) from the extracellular domain of LIGHT or a mutated form thereof, e.g., from about amino acids 61 to 92 of SEQ ID NO:1 of human LIGHT isoform 1, about amino acids 225 to 252 of SEQ ID NO:2 of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130), about amino acids 230 to 257 of SEQ ID NO:3 of 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131), or an amino acid sequence substantially identical thereto; or an amino acid sequence encoded by the nucleotide sequence from about nucleotides 181 to 276 of SEQ ID NO:5 of human LIGHT isoform 1, about nucleotides 673 to 756 of SEQ ID NO:6 of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130), about nucleotides 688 to 771 of SEQ ID NO:7 of 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). Alternatively, the linking group may include a combination of one or more (Gly)4-Ser (SEQ ID NO: 146) repeats and one or more amino acid residues (e.g., at least 10 to 35, 15 to 30, or about 20 to 26 amino acid residues) from the extracellular domain of LIGHT or a mutated form thereof, e.g., from about amino acids 61 to 92 of SEQ ID NO:1 of human LIGHT isoform 1, about amino acids 225 to 252 of SEQ ID NO:2 of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130), about amino acids 230 to 257 of SEQ ID NO:3 of 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131), or an amino acid sequence substantially identical thereto; or an amino acid sequence encoded by the nucleotide sequence from about nucleotides 181 to 276 of SEQ ID NO:5 of human LIGHT isoform 1, about nucleotides 673 to 756 of SEQ ID NO:6 of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130), about nucleotides 688 to 771 of SEQ ID NO:7 of 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). Exemplary linkers that can be used in the LIGHT fusions are shown as SEQ ID NO:132 (delta 4), SEQ ID NO:133 (G4S delta 4) and SEQ ID NO:134 (G4S), or amino acid sequences substantially identical thereto.
In other embodiments, the LIGHT moiety and the targeting moiety are chemically coupled, e.g., by the covalent attachment of one reactive group, e.g., a succinimidyl or maleimide containing group, to a defined amino acid of the LIGHT or the targeting moiety. In those embodiments where the LIGHT moiety and the targeting moiety are chemically coupled, the reactive group may optionally be coupled to a biocompatible polymer, e.g., a polymer having monomers chosen from one or more of AEA ((2-amino) ethoxy acetic acid), AEEA ([2-(2-amino)ethoxy)]ethoxy acetic acid) and OA (8-amino octanoic acid, also called 8-amino caprylic acid, of formula NH2—(CH2)7—COOH), or a combination thereof. The linking group can include any combinations of the aforesaid biocompatible polymers.
In one exemplary embodiment, the LIGHT targeting molecule comprises, or consists essentially of, at least one fusion molecule of a mammalian (e.g., human) LIGHT protein, or a functional variant or a fragment thereof, and an antibody molecule that binds to HER2 (referred to herein as “LIGHT-anti-HER2 fusion”). In certain embodiments, the LIGHT-anti-HER2 fusion comprises at least three fusion molecules in a trimer of about 200 to 250 kDa, typically about 220 KDa as shown in FIGS. 1 and 3 (e.g., having the amino acid sequence of SEQ ID NOs:2-4, or an amino acid sequence substantially identical thereto); or at least two fusion molecules in a dimer of about 100 to 200 kDa, typically about 150 kDa as shown in FIGS. 2 and 24 (having the amino acid sequence of SEQ ID NO:178, or an amino acid sequence substantially identical thereto). The light chain associated to the LIGHT-anti-HER2 heavy chains described herein can have the amino acid sequence shown in SEQ ID NO:109, or an amino acid sequence substantially identical thereto (or encoded by a nucleotide sequence shown in SEQ ID NO:110 or a nucleotide sequence substantially identical thereto).
Without being bound by theory, the LIGHT-anti-HER2 fusions are believed to trigger dual anti-cancer effects by inducing tumor cell killing mediated by the anti-HER2 antibody molecule, as well as stimulating localized LIGHT-mediated anti-tumor immunity. In certain embodiments, the LIGHT-anti-HER2 fusions can have one or more of the following activities: (i) bind to HER2 with an affinity characterized by a dissociation constant (Kd) of at least 1×10−7 M, 1×10−8 M, 1×10−9 M, 1×10−10 M, 1×10−11M, 1×10−12 M, 1×10−13 M; (ii) bind substantially selectively to HER2, e.g., without significant cross reactivity with other HER-family members (iii) bind to a linear or a conformation epitope on HER2 chosen from epitope of domain 1 (D1) (corresponding to about amino acids 1 to 196 of human HER2 shown in FIG. 4), epitope of domain 2 (D2) (corresponding to about amino acids 197 to 318 of human HER2 shown in FIG. 4), epitope of domain 3 (D3) (corresponding to about amino acids 319 to 508 of human HER2 shown in FIG. 4), or epitope of domain 4 (D4) (corresponding to about amino acids 508 to 630 of human HER2 shown in FIG. 4), or a combination thereof, e.g., epitope D1-2 (corresponding to about amino acids 1 to 318 of human HER2 shown in FIG. 4) or epitope D1-3 (corresponding to about amino acids 1 to 508 of human HER2 shown in FIG. 4); (iv) inhibit, block or reduce HER2 signaling (e.g., inhibit, block or reduce phosphorylation of one or more of HER2, AKT and/or MAP kinase; or inhibit, block or reduce homodimerization of HER2 or heterodimerization of HER2 and HER3, and/or HER2 with EGFR; (v) inhibit activity and/or induce cell killing of a HER2 expressing cell in vitro (e.g., MCF7 and SKBR-3 cell) and in vivo; (vi) trigger an anti-tumor immune response in vivo, e.g., in an animal model (such as a mouse tumor model carrying breast tumor cells, or a HER2-dependent colorectal and gastric xenograft tumor model), or in a human subject; and/or (vii) inducing a prolonged reduction of tumor growth or metastasis, e.g., after prolonged monotherapy or combination therapy, or after tumor relapse is detected following another chemotherapeutic therapy (e.g., standard chemotherapy or anti-HER2 antibody therapy).
In one embodiment, the LIGHT-anti-HER2 fusion comprises, or consists essentially of, at least one mammalian (e.g., human) LIGHT protein, or a variant or a fragment thereof (e.g., a LIGHT protein as described herein) and an anti-HER2 specific antibody molecule or a fragment thereof (e.g., an antibody molecule as described herein).
As described herein, the invention additionally features an antibody molecule (e.g., isolated or purified protein or polypeptide) that selectively binds to HER2 (e.g., an anti-HER2 antibody as described herein). The anti-HER2 antibodies described herein can be present in a LIGHT-targeting molecule of the invention, or can be present as single or combined entities distinct from the targeting molecules described herein.
In certain embodiments, the anti-HER2 antibody molecules present in the LIGHT-targeting molecules or as single or combined entities include one or more of the following features:
In embodiments, the anti-HER2 antibody molecule is an antibody molecule or a Fab fragment from an antibody selected from the group consisting of BIIB71F10 (comprising, or consisting of, the amino acid sequence of SEQ ID NOs:11 and 13, VH and VL, respectively, or the VH and VL amino acid of the ATCC Patent Deposit Designation PTA-10355 corresponding to the CHO cell deposit of 71F10 Fab LIGHT, or encoded by the nucleotide sequence of SEQ ID NOs: 12 and 14, VH and VL, respectively, or the nucleotide sequence of the ATCC Patent Deposit Designation PTA-10355 encoding the VH and VL amino acid of the 71F10 Fab LIGHT); BIIB69A09 (comprising, or consisting of, the amino acid sequence of SEQ ID NOs:15 and 17, VH and VL, respectively, or encoded by the nucleotide sequence of SEQ ID NOs:16 and 18, VH and VL, respectively); BIIB67F10 (comprising, or consisting of, the amino acid sequence of SEQ ID NOs:19 and 21, VH and VL, respectively, or encoded by the nucleotide sequence of SEQ ID NOs:20 and 22, VH and VL, respectively); BIIB67F11 (comprising, or consisting of, the amino acid sequence of SEQ ID NOs:23 and 25, VH and VL, respectively, or the VH and VL amino acid of the ATCC Patent Deposit Designation PTA-10357 corresponding to the CHO cell deposit of 67F11 Fab LIGHT, or encoded by the nucleotide sequence of SEQ ID NOs: 24 and 26, VH and VL, respectively, or the nucleotide sequence of the ATCC Patent Deposit Designation PTA-10357 encoding the VH and VL amino acid of the 67F11 Fab LIGHT); BIIB66A12 (comprising, or consisting of, the amino acid sequence of SEQ ID NOs:27 and 29, VH and VL, respectively; or encoded by the nucleotide sequence of SEQ ID NOs: 28 and 30, VH and VL, respectively); BIIB66C01 (comprising, or consisting of, the amino acid sequence of SEQ ID NOs:31 and 33, VH and VL, respectively or encoded by the nucleotide sequence of SEQ ID NOs: 32 and 34, VH and VL, respectively); BIIB65C10 (comprising, or consisting of, the amino acid sequence of SEQ ID NOs:35 and 37, VH and VL, respectively, or the VH and VL amino acid of the ATCC Patent Deposit Designation PTA-10358 corresponding to the CHO deposit of 65C10 Fab LIGHT, or encoded by the nucleotide sequence of SEQ ID NOs:36 and 38, VH and VL, respectively, or the nucleotide sequence of the ATCC Patent Deposit Designation PTA-10358 encoding the VH and VL amino acid of the 65C10 Fab LIGHT); BIIB65H09 (comprising, or consisting of, the amino acid sequence of SEQ ID NOs:39 and 41, VH and VL, respectively), or the VH and VL amino acid of the ATCC Patent Deposit Designation PTA-10356 corresponding to the CHO cell deposit of 65H09Fab LIGHT, or encoded by the nucleotide sequence of SEQ ID NOs: 40 and 42, VH and VL, respectively, or the nucleotide sequence of the ATCC Patent Deposit Designation PTA-10356 encoding the VH and VL amino acid of the 65H09Fab LIGHT); and BIIB65B03 (comprising, or consisting of, the amino acid sequence of SEQ ID NOs:43 and 45, VH and VL, respectively, or encoded by the nucleotide sequence of SEQ ID NOs: 44 and 46, VH and VL, respectively) (also referred to herein as “71F10,” “69A09,” “67F10,” “67F11,” “66A12,” “66C01,” “65C10,” “65H09” and “65B03”); or amino or nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). Amino acid and corresponding nucleotide sequences of VH and VL domains of each of these Fabs are shown as SEQ ID NOs:11-46. For simplicity purposes, the nucleotide and amino acid sequences disclosed herein are collectively referred to herein by their corresponding SEQ ID NOs, e.g., SEQ ID NOs:39-42, when referring to the amino acid sequence of SEQ ID NOs:39 and 41, VH and VL, respectively, and/or the corresponding ATCC deposit; or encoded by the nucleotide sequence of SEQ ID NOs: 40 and 42, VH and VL, respectively, and/or the corresponding ATCC deposit.
In other embodiments, the anti-HER2 antibody molecule has a functional activity comparable to an antibody molecule or a Fab fragment from an antibody selected from the group consisting of BIIB71F10 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:11-14), BIIB69A09 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:15-18); BIIB67F10 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:19-22); BIIB67F11 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:23-26); BIIB66A12 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:27-30); BIIB66C01 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:31-34); BIIB65C10 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:35-38); BIIB65H09 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:39-42) and BIIB65B03 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:43-46), or an amino or nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto).
In other embodiments, the anti-HER2 antibody molecule can cross-react with HER2 from one or more species chosen from human, mouse, rat, and/or cyno origin. The anti-HER2 antibody molecule can bind to HER2 with an EC50 in the range of about 1 to 120 nM, about 1 to 100 nM, about 1 to 80 nM, about 1 to 70 nM, about 1 to 60 nM, about 1 to 40 nM, about 1 to 30 nM, about 1 to 20 nM, about 1 to 15 nM, about 1 to 12 nM, about 1 to 5 nM, about 1 to 2 nM, or about 1 to 1 nM. In other embodiments, the anti-HER2 antibody molecule inhibits or reduces one or more HER2-associated biological activities with an IC50 of about 50 nM to 5 pM, typically about 100 to 250 pM or less, e.g., better inhibition. For example, the anti-HER2 antibody molecule can have one or more of the following activities: (i) inhibit, block or reduce HER2 signaling with an IC50 of about 50 nM to 5 pM, typically about 100 to 250 pM or less, e.g., better inhibition (e.g., inhibit, block or reduce phosphorylation of one or more of HER2, AKT or MAP kinase; or inhibit, block or reduce homodimerization of HER2 or heterodimerization of HER2 and HER3, or HER2 with EGFR); (ii) internalize with a slow kinetics estimated to be less than or equal to the rate of internalization for control anti-HER2 antibody, which is 8e−6s−1 in SKBR-3 cells and 2.1e−5s−1 in BT-474 cells; and/or (iii) inhibit activity and/or induce cell killing of a HER2 expressing cell in vitro (e.g., MCF7 and SKBR-3 cell) and in vivo. In one embodiment, the anti-HER2 antibody molecule associates with HER2 with kinetics in the range of 104 to 107 M−1s−1, typically 105 to 106M−1s−1. In another embodiment, the anti-HER2 antibody molecule binds to human HER2 with a kD of 0.1-100 nM. In yet another embodiment, the anti-HER2 antibody molecule has dissociation kinetics in the range of 10−2 to 10−6 s−1, typically 10−2 to 10−5 s−1. In one embodiment, the anti-HER2 antibody molecule binds to HER2, e.g., human HER2, with an affinity and/or kinetics similar (e.g., within a factor 20, 10, or 5) to a monoclonal antibody selected from the group consisting of BIIB71F10 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:11-14, or ATCC Patent Deposit PTA-10355); BIIB69A09 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:15-18); BIIB67F10 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:19-22); BIIB67F11 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:23-26, or ATCC Patent Deposit PTA-10357); BIIB66A12 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:27-30); BIIB66C01 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:31-34); BIIB65C10 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:35-38, or ATCC Patent Deposit PTA-10358); BIIB65H09 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:39-42, or ATCC Patent Deposit PTA-10356) and BIIB65B03 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:43-46). The affinity and binding kinetics of the anti-HER2 antibody molecule can be tested using, e.g., biosensor technology (BIACORE™).
In still another embodiment, the anti-HER2 antibody molecule specifically binds to an epitope, e.g., a linear or a conformational epitope, of HER2, e.g., mammalian, e.g., human HER2. In embodiments, the anti-HER2 antibody molecule competes for binding (e.g., binds to the same or similar, e.g., partially overlapping epitope) as an antibody selected from the group consisting of BIIB71F10 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:11-14, or ATCC Patent Deposit PTA-10355); BIIB69A09 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:15-18); BIIB67F10 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:19-22); BIIB67F11 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:23-26, or ATCC Patent Deposit PTA-10357); BIIB66A12 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:27-30); BIIB66C01 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:31-34); BIIB65C10 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:35-38, or ATCC Patent Deposit PTA-10358); BIIB65H09 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:39-42, or ATCC Patent Deposit PTA-10356) and BIIB65B03 (comprising, or consisting essentially of, the amino or nucleotide sequence as described herein as SEQ ID NOs:43-46). In embodiments, the anti-HER2 antibody molecule binds to a linear or a conformation epitope on HER2 chosen from epitope D1 (corresponding to about amino acids 1 to 196 of human HER2 shown in FIG. 4), epitope D2 (corresponding to about amino acids 197 to 318 of human HER2 shown in FIG. 4), epitope D3 (corresponding to about amino acids 319 to 508 of human HER2 shown in FIG. 4), or epitope D4 (corresponding to about amino acids 508 to 630 of human HER2 shown in FIG. 4), or a combination thereof, e.g., epitope D1-2 (corresponding to about amino acids 1 to 318 of human HER2 shown in FIG. 4) or epitope D1-3 (corresponding to about amino acids 1 to 508 of human HER2 shown in FIG. 4).
In one embodiment, the anti-HER2 antibody molecule includes one, two, three, four, five or all six CDR's from an antibody selected from the group consisting of BIIB71F10 (SEQ ID NOs:11 (VH), 13 (VL), or ATCC Patent Deposit PTA-10355), BIIB69A09 (SEQ ID NOs:15, 17), BIIB67F10 (SEQ ID NOs:19, 21), BIIB67F11 (SEQ ID NOs:23, 25, or ATCC Patent Deposit PTA-10357), BIIB66A12 (SEQ ID NOs:27, 29), BIIB66C01 (SEQ ID NOs:31, 33), BIIB65C10 (SEQ ID NOs:35, 37, or ATCC Patent Deposit PTA-10358), BIIB65H09 (SEQ ID NOs:39, 41, or ATCC Patent Deposit PTA-10356) and BIIB65B03 (SEQ ID NOs:43, 45), or closely related CDRs, e.g., CDRs which are identical or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions (e.g., conservative substitutions), deletions, or insertions). Optionally, the antibody molecule may include any CDR described herein.
In embodiments, the heavy chain immunoglobulin variable domain of the anti-HER2 antibody molecule comprises a heavy chain CDR1, CDR2, and/or CDR3, or having a CDR that differs by fewer than 3, 2, or 1 amino acid substitutions (e.g., conservative substitutions) from a heavy chain CDR1, CDR2, and/or CDR3 of monoclonal antibody selected from the group consisting of BIIB71F10 (SEQ ID NO:47 (CDR1), SEQ ID NO:48 (CDR2), and/or SEQ ID NO:49 (CDR3), or a CDR from ATCC Patent Deposit PTA-10355); BIIB69A09 (SEQ ID NO:50 (CDR1), SEQ ID NO:51 (CDR2), and/or SEQ ID NO:52 (CDR3)); BIIB67F10 (SEQ ID NO:53 (CDR1), SEQ ID NO:54 (CDR2), and/or SEQ ID NO:55 (CDR3)); BIIB67F11 (SEQ ID NO:56 (CDR1), SEQ ID NO:57 (CDR2), and/or SEQ ID NO:58 (CDR3), or a CDR from ATCC Patent Deposit PTA-10357); BIIB66A12 (SEQ ID NO:59 (CDR1), SEQ ID NO:60 (CDR2) and/or SEQ ID NO:61 (CDR3)); BIIB66C01 (SEQ ID NO:62 (CDR1), SEQ ID NO:63 (CDR2), and/or SEQ ID NO:64 (CDR3)); BIIB65C10 (SEQ ID NO:65 (CDR1), SEQ ID NO:66 (CDR2), and/or SEQ ID NO:67 (CDR3), or a CDR from ATCC Patent Deposit PTA-10358); BIIB65H09 (SEQ ID NO:68 (CDR1), SEQ ID NO:69 (CDR2), and/or SEQ ID NO:70 (CDR3), or a CDR from ATCC Patent Deposit PTA-10356); and BIIB65B03 (SEQ ID NO:71 (CDR1), SEQ ID NO:72 (CDR2), and/or SEQ ID NO:73 (CDR3)).
In other embodiments, the light chain immunoglobulin variable domain of the anti-HER2 antibody molecule comprises a light chain CDR1, CDR2, and/or CDR3, or having a CDR that differs by fewer than 3, 2, or 1 amino acid substitutions (e.g., conservative substitutions) from a light chain CDR1, CDR2, and/or CDR3 of monoclonal antibody selected from the group consisting of BIIB71F10 (SEQ ID NO:74 (CDR1), SEQ ID NO:75 (CDR2), and/or SEQ ID NO:76 (CDR3), or a CDR from ATCC Patent Deposit PTA-10355); BIIB69A09 (SEQ ID NO:77 (CDR1), SEQ ID NO:78 (CDR2), and/or SEQ ID NO:79 (CDR3)); BIIB67F10 (SEQ ID NO:80 (CDR1), SEQ ID NO:81 (CDR2), and/or SEQ ID NO:82 (CDR3)); BIIB67F11 (SEQ ID NO:83 (CDR1), SEQ ID NO:84 (CDR2), and/or SEQ ID NO:85 (CDR3), or a CDR from ATCC Patent Deposit PTA-10357); BIIB66A12 (SEQ ID NO:86 (CDR1), SEQ ID NO:87 (CDR2), and/or SEQ ID NO:88 (CDR3)); BIIB66C01 (SEQ ID NO:89 (CDR1), SEQ ID NO:90 (CDR2), and/or SEQ ID NO:91 (CDR3)); BIIB65C10 (SEQ ID NO:92 (CDR1), SEQ ID NO:93 (CDR2), and/or SEQ ID NO:94 (CDR3), or a CDR from ATCC Patent Deposit PTA-10358); BIIB65H09 (SEQ ID NO:95 (CDR1), SEQ ID NO:96 (CDR2), and/or SEQ ID NO:97 (CDR3), or a CDR from ATCC Patent Deposit PTA-10356) and BIIB65B03 (SEQ ID NO:98 (CDR1), SEQ ID NO:99 (CDR2), and/or SEQ ID NO:100 (CDR3)).
In certain embodiments, the amino acid sequence of the heavy chan variable domain of BIIB71F10 includes the amino acid sequence shown as SEQ ID NO:11, or is encoded by a nucleotide sequence shown as SEQ ID NO:12 or SEQ ID NO:156. The amino acid sequence of the light chan variable domain of BIIB71F10 includes the amino acid sequence shown as SEQ ID NO:13, or is encoded by a nucleotide sequence shown as SEQ ID NO:14. In other embodiments, the heavy chain and light chain variable domains of BIIB71F10 include the amino acid sequence, or is encoded by the nucleotide sequence, of ATCC Patent Deposit PTA-10355. In certain embodiments, the heavy chain variable domain of the anti-HER2 antibody molecule includes one or more of: SYGMV (SEQ ID NO:47), in CDR1, SISSSGGLTWYADSVKG (SEQ ID NO:48), in CDR2, and/or PPGIAVARDY (SEQ ID NO:49), in CDR3. In other embodiments, the light chain variable domain of the anti-HER2 antibody molecule includes one or more of: RASQGISNYLA (SEQ ID NO:74), in CDR1, AASTLQS (SEQ ID NO:75), in CDR2, and/or QKYNSALLT (SEQ ID NO:76), in CDR3, or has at least one, two or three CDRs from the heavy chain and/or light chain variable domain of ATCC Patent Deposit PTA-10355.
In certain embodiments, the amino acid sequence of the heavy chan variable domain of BIIB65H09 includes the amino acid sequence shown as SEQ ID NO:39, or is encoded by a nucleotide sequence shown as SEQ ID NO:40. The amino acid sequence of the light chan variable domain of BIIB65H09 includes the amino acid sequence shown as SEQ ID NO:41, or is encoded by a nucleotide sequence shown as SEQ ID NO:42. In other embodiments, the heavy chain and light chain variable domains of BIIB65H09 include the amino acid sequence, or is encoded by the nucleotide sequence, of ATCC Patent Deposit PTA-10356. In certain embodiments, the heavy chain variable domain of the anti-HER2 antibody molecule includes one or more of: WYSMW (SEQ ID NO:68), in CDR1, SIVSSGGQTRYADSVKG (SEQ ID NO:69), in CDR2, and/or VKGYYYYIDV (SEQ ID NO:70), in CDR3. In other embodiments, the light chain variable domain of the anti-HER2 antibody molecule includes one or more of: RASQSVDSSYLS (SEQ ID NO:95), in CDR1, GASTRAT (SEQ ID NO:96), in CDR2, and/or QQHGYSSRT (SEQ ID NO:97), in CDR3, or has at least one, two or three CDRs from the heavy chain and/or light chain variable domain of ATCC Patent Deposit PTA-10356.
In certain embodiments, the amino acid sequence of the heavy chan variable domain of BIIB67F11 includes the amino acid sequence shown as SEQ ID NO:23, or is encoded by a nucleotide sequence shown as SEQ ID NO:24. The amino acid sequence of the light chan variable domain of BIIB67F11 includes the amino acid sequence shown as SEQ ID NO:25, or is encoded by a nucleotide sequence shown as SEQ ID NO:26. In other embodiments, the heavy chain and light chain variable domains of BIIB67F11 include the amino acid sequence, or is encoded by the nucleotide sequence, of ATCC Patent Deposit PTA-10357. In certain embodiments, the heavy chain variable domain of the anti-HER2 antibody molecule includes one or more of: NYYMM (SEQ ID NO:56), in CDR1, VIGSSGGMTNYADSVKG (SEQ ID NO:57), in CDR2, and/or GYPTGYYDSSGWVYSYYGIDV (SEQ ID NO:58), in CDR3. In other embodiments, the light chain variable domain of the anti-HER2 antibody molecule includes one or more of: QASQDTDNRLH (SEQ ID NO:83), in CDR1, DAVNLKR (SEQ ID NO:84), in CDR2, and/or QHSDGLSLA (SEQ ID NO:85), in CDR3, or has at least one, two or three CDRs from the heavy chain and/or light chain variable domain of ATCC Patent Deposit PTA-10357.
In certain embodiments, the amino acid sequence of the heavy chan variable domain of BIIB65C10 includes the amino acid sequence shown as SEQ ID NO:35, or is encoded by a nucleotide sequence shown as SEQ ID NO:36. The amino acid sequence of the light chan variable domain of BIIB65C10 includes the amino acid sequence shown as SEQ ID NO:37, or is encoded by a nucleotide sequence shown as SEQ ID NO:38. In other embodiments, the heavy chain and light chain variable domains of BIIB65C10 include the amino acid sequence, or is encoded by the nucleotide sequence, of ATCC Patent Deposit PTA-10358. In certain embodiments, the heavy chain variable domain of the anti-HER2 antibody molecule includes one or more of: YYPMM (SEQ ID NO:65), in CDR1, SIWPSGGFTKYADSVKG (SEQ ID NO:66), in CDR2, and/or VSSSSWYGYLY (SEQ ID NO:67), in CDR3. In other embodiments, the light chain variable domain of the anti-HER2 antibody molecule includes one or more of: SGSSSNIGRNTVN (SEQ ID NO:92), in CDR1, SNNQRPS (SEQ ID NO:93), in CDR2, and/or AAWDDSLNAWV (SEQ ID NO:94), in CDR3, or has at least one, two or three CDRs from the heavy chain and/or light chain variable domain of ATCC Patent Deposit PTA-10358.
In certain embodiments, the amino acid sequence of the heavy chan variable domain of BIIB-65B03 includes the amino acid sequence shown as SEQ ID NO:43, or is encoded by a nucleotide sequence shown as SEQ ID NO:44. The amino acid sequence of the light chan variable domain of BIIB65B03 includes the amino acid sequence shown as SEQ ID NO:45, or is encoded by a nucleotide sequence shown as SEQ ID NO:46. In certain embodiments, the heavy chain variable domain of the anti-HER2 antibody molecule includes one or more of: WYRMN (SEQ ID NO:71), in CDR1, SIYSSGGPTNYADSVKG (SEQ ID NO:72), in CDR2, and/or EKPDYYDSSGYLDY (SEQ ID NO:73), in CDR3. In other embodiments, the light chain variable domain of the anti-HER2 antibody molecule includes one or more of: RASQSVSSSYLA (SEQ ID NO:98), in CDR1, GASSRAT (SEQ ID NO:99), in CDR2, and/or HQYGRPPV (SEQ ID NO:100), in CDR3.
In certain embodiments, the amino acid sequence of the heavy chan variable domain of BIIB66A12 includes the amino acid sequence shown as SEQ ID NO:27, or is encoded by a nucleotide sequence shown as SEQ ID NO:28. The amino acid sequence of the light chan variable domain of BIIB66A12 includes the amino acid sequence shown as SEQ ID NO:29, or is encoded by a nucleotide sequence shown as SEQ ID NO:30. In certain embodiments, the heavy chain variable domain of the anti-HER2 antibody molecule includes one or more of: MYSMQ (SEQ ID NO:59), in CDR1, VIGSSGGQTGYADSVK G (SEQ ID NO:60), in CDR2, and/or VRDYYGSGSYYLDP (SEQ ID NO:61), in CDR3. In other embodiments, the light chain variable domain of the anti-HER2 antibody molecule includes one or more of: RASQSISSYLN (SEQ ID NO:86), in CDR1, AASSLQS (SEQ ID NO:87), in CDR2, and/or QQSYSTSWT (SEQ ID NO:88), in CDR3.
In certain embodiments, the amino acid sequence of the heavy chan variable domain of BIIB66C01 includes the amino acid sequence shown as SEQ ID NO:31, or is encoded by a nucleotide sequence shown as SEQ ID NO:32. The amino acid sequence of the light chan variable domain of BIIB66C01 includes the amino acid sequence shown as SEQ ID NO:33, or is encoded by a nucleotide sequence shown as SEQ ID NO:34. In certain embodiments, the heavy chain variable domain of the anti-HER2 antibody molecule includes one or more of: WYSMS (SEQ ID NO:62), in CDR1, SISSSGGPTHYADSVKG (SEQ ID NO:63), in CDR2, and/or DSSYSGTS (SEQ ID NO:64), in CDR3. In other embodiments, the light chain variable domain of the anti-HER2 antibody molecule includes one or more of: SGSSSNIGSEYVY (SEQ ID NO:89), in CDR1, RNDQRPS (SEQ ID NO:90), in CDR2, and/or TTWDDSLSGPV (SEQ ID NO:91), in CDR3.
In certain embodiments, the amino acid sequence of the heavy chan variable domain of BIIB67F10 includes the amino acid sequence shown as SEQ ID NO:19, or is encoded by a nucleotide sequence shown as SEQ ID NO:20. The amino acid sequence of the light chan variable domain of BIIB67F10 includes the amino acid sequence shown as SEQ ID NO:21, or is encoded by a nucleotide sequence shown as SEQ ID NO:22. In certain embodiments, the heavy chain variable domain of the anti-HER2 antibody molecule includes one or more of: PYMMV (SEQ ID NO:53), in CDR1, WISPSGGYTFYADSVKG (SEQ ID NO:54), in CDR2, and/or GTYPLTY (SEQ ID NO:55), in CDR3. In other embodiments, the light chain variable domain of the anti-HER2 antibody molecule includes one or more of: SGDKLGDKYVS (SEQ ID NO:80), in CDR1, QDSKWPS (SEQ ID NO:81), in CDR2, and/or QVWDISHVV (SEQ ID NO:82), in CDR3.
In certain embodiments, the amino acid sequence of the heavy chan variable domain of BIIB69A09 includes the amino acid sequence shown as SEQ ID NO:15, or is encoded by a nucleotide sequence shown as SEQ ID NO:16. The amino acid sequence of the light chan variable domain of BIIB69A09 includes the amino acid sequence shown as SEQ ID NO:17, or is encoded by a nucleotide sequence shown as SEQ ID NO:18. In certain embodiments, the heavy chain variable domain of the anti-HER2 antibody molecule includes one or more of: RYNMW (SEQ ID NO:50), in CDR1, VIRSSGGYTGYADSVKG (SEQ ID NO:51), in CDR2, and/or WNSGYSYWDYYYGMDV (SEQ ID NO:52), in CDR3. In other embodiments, the light chain variable domain of the anti-HER2 antibody molecule includes one or more of: RASQSISSYLN (SEQ ID NO:77), in CDR1, AASSLQS (SEQ ID NO:78), in CDR2, and/or QQFNTYPIT (SEQ ID NO:79), in CDR3.
In other embodiments, the antibody molecule of the fusion includes one or more CDRs including an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NOs:50-73 and 77-100.
In yet another embodiment, the anti-HER2 antibody molecule includes at least one, two, or three Chothia hypervariable loops from a heavy chain variable region of an antibody chosen from, e.g., BIIB71F10 (SEQ ID NOs:11-12, or a Chothia hypervariable loop from ATCC Patent Deposit PTA-10355), BIIB69A09 (SEQ ID NOs:15-16), BIIB67F10 (SEQ ID NOs:19-20), BIIB67F11 (SEQ ID NOs:23-24, or a Chothia hypervariable loop from ATCC Patent Deposit PTA-10357), BIIB66A12 (SEQ ID NOs:27-28), BIIB66C01 (SEQ ID NOs:31-32), BIIB65C10 (SEQ ID NOs:35-36, or a Chothia hypervariable loop from ATCC Patent Deposit PTA-10358), BIIB65H09 (SEQ ID NOs:39-40, or a Chothia hypervariable loop from ATCC Patent Deposit PTA-10356) or BIIB65B03 (SEQ ID NOs:43-44). In yet another embodiment, the antibody molecule includes at least one, two, or three hypervariable loops from a light chain variable region of an antibody chosen from, e.g., BIIB71F10 (SEQ ID NOs:13-14, or a Chothia hypervariable loop from ATCC Patent Deposit PTA-10355), BIIB69A09 (SEQ ID NOs:17-18), BIIB67F10 (SEQ ID NOs:21-22), BIIB67F11 (SEQ ID NOs:25-26, or a Chothia hypervariable loop from ATCC Patent Deposit PTA-10357), BIIB66A12 (SEQ ID NOs:29-30), BIIB66C01 (SEQ ID NOs:33-34), BIIB65C10 (SEQ ID NOs:37-38, or a Chothia hypervariable loop from ATCC Patent Deposit PTA-10358), BIIB65H09 (SEQ ID NOs:41-42, or a Chothia hypervariable loop from ATCC Patent Deposit PTA-10356) or BIIB65B03 (SEQ ID NOs:45-46). In yet another embodiment, the antibody or fragment thereof includes at least one, two, three, four, five, or six hypervariable loops from the heavy and light chain variable regions of an antibody chosen from, e.g., BIIB71F10 (SEQ ID NOs:11-14, or a hypervariable loop from ATCC Patent Deposit PTA-10355), BIIB69A09 (SEQ ID NOs:15-18); BIIB67F10 (SEQ ID NOs:19-22), BIIB67F11 (SEQ ID NOs:23-26, or a hypervariable loop from ATCC Patent Deposit PTA-10357), BIIB66A12 (SEQ ID NOs:27-30), BIIB66C01 (SEQ ID NOs:31-34), BIIB65C10 (SEQ ID NOs:35-38, or a hypervariable loop from ATCC Patent Deposit PTA-10358), BIIB65H09 (SEQ ID NOs:39-42, or a hypervariable loop from ATCC Patent Deposit PTA-10356) or BIIB65B03 (SEQ ID NOs:43-46), or closely related hypervariable loops, e.g., hypervariable loops which are identical or which have at least one amino acid alteration, but not more than two, three or four alterations, from the sequences disclosed herein. Optionally, the protein may include any hypervariable loop described herein.
In still another example, the anti-HER2 antibody molecule includes at least one, two, or three hypervariable loops that have the same canonical structures as the corresponding hypervariable loop of BIIB71F10 (SEQ ID NOs:11-14, or a hypervariable loop from ATCC Patent Deposit PTA-10355), BIIB69A09 (SEQ ID NOs:15-18), BIIB67F10 (SEQ ID NOs:19-22), BIIB67F11 (SEQ ID NOs:23-26, or a hypervariable loop from ATCC Patent Deposit PTA-10357), BIIB66A12 (SEQ ID NOs:27-30), BIIB66C01 (SEQ ID NOs:31-34), BIIB65C10 (SEQ ID NOs:35-38, or a hypervariable loop from ATCC Patent Deposit PTA-10358), BIIB65H09 (SEQ ID NOs:39-42, or a hypervariable loop from ATCC Patent Deposit PTA-10356) or BIIB65B03 (SEQ ID NOs:43-46), e.g., the same or similar canonical structures as at least loop 1 and/or loop 2 of the heavy and/or light chain variable domains of BIIB71F10 (SEQ ID NOs:11-14, or a hypervariable loop from ATCC Patent Deposit PTA-10355), BIIB69A09 (SEQ ID NOs:15-18), BIIB67F10 (SEQ ID NOs:19-22), BIIB67F11 (SEQ ID NOs:23-26, or a hypervariable loop from ATCC Patent Deposit PTA-10357), BIIB66A12 (SEQ ID NOs:27-30), BIIB66C01 (SEQ ID NOs:31-34), BIIB65C10 (SEQ ID NOs:35-38, or a hypervariable loop from ATCC Patent Deposit PTA-10358), BIIB65H09 (SEQ ID NOs:39-42, or a hypervariable loop from ATCC Patent Deposit PTA-10356) or BIIB65B03 (SEQ ID NOs:43-46). See, e.g., Chothia et al. (1992) J. Mol. Biol. 227:799-817; Tomlinson et al. (1992) J. Mol. Biol. 227:776-798 for descriptions of hypervariable loop canonical structures. These structures can be determined by inspection of the tables described in these references.
In one embodiment, the heavy chain framework of the anti-HER2 antibody molecule (e.g., FR1, FR2, FR3, individually, or a sequence encompassing FR1, FR2, and FR3, but excluding CDRs) includes an amino acid sequence, which is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher identical to the heavy chain framework of BIIB71F10 (SEQ ID NO:11 or ATCC Patent Deposit PTA-10355), BIIB69A09 (SEQ ID NO:15); BIIB67F10 (SEQ ID NO:19); BIIB67F11 (SEQ ID NO:23 or ATCC Patent Deposit PTA-10357), BIIB66A12 (SEQ ID NO:27), BIIB66C01 (SEQ ID NO:31), BIIB65C10 (SEQ ID NO:35 or ATCC Patent Deposit PTA-10358), BIIB65H09 (SEQ ID NO:39 or ATCC Patent Deposit PTA-10356) or BIIB65B03 (SEQ ID NO:43). In embodiments, the heavy chain framework of the anti-HER2 antibody molecule has an amino acid sequence substantially homologous to human V segment sequence HV3-23 (SEQ ID NO:107) (see, e.g., Chothia et al. (1992) J. Mol. Biol. 227:799-817; Tomlinson et al. (1992) J. Mol. Biol. 227:776-798).
In another embodiment, the light chain framework of the anti-HER2 antibody molecule (e.g., FR1, FR2, FR3, individually, or a sequence encompassing FR1, FR2, and FR3, but excluding CDRs) includes, or consists essentially of, an amino acid sequence, which is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher identical to the light chain framework of BIIB71F10 (SEQ ID NO:13 or ATCC Patent Deposit PTA-10355), BIIB69A09 (SEQ ID NO:17); BIIB67F10 (SEQ ID NO:21); BIIB67F11 (SEQ ID NO:25 or ATCC Patent Deposit PTA-10357), BIIB66A12 (SEQ ID NO:29), BIIB66C01 (SEQ ID NO:33), BIIB65C10 (SEQ ID NO:37 or ATCC Patent Deposit PTA-10358), BIIB65H09 (SEQ ID NO:41 or ATCC Patent Deposit PTA-10356) or BIIB65B03 (SEQ ID NO:45). In embodiments, the heavy chain framework of the anti-HER2 antibody molecule has an amino acid sequence substantially homologous to human a VLκ I subgroup germline sequence, e.g., a VLκ consensus sequence.
In certain embodiments, the heavy chain immunoglobulin variable domain of the anti-HER2 antibody molecule includes, or consists essentially of, an amino acid sequence encoded by a nucleotide sequence that hybridizes under high stringency conditions to the complement of the nucleotide sequence encoding a heavy chain variable domain of BIIB71F10 (SEQ ID NO:12; SEQ ID NO:156, or ATCC Patent Deposit PTA-10355), BIIB69A09 (SEQ ID NO:16), BIIB67F10 (SEQ ID NO:20), BIIB67F11 (SEQ ID NO:24, or ATCC Patent Deposit PTA-10357), BIIB66A12 (SEQ ID NO:28), BIIB66C01 (SEQ ID NO:32), BIIB65C10 (SEQ ID NO:36, or ATCC Patent Deposit PTA-10358), BIIB65H09 (SEQ ID NO:40, or ATCC Patent Deposit PTA-10356) or BIIB65B03 (SEQ ID NO:44); or includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher identical to the amino acid sequence of the heavy chain variable domain of BIIB71F10 (SEQ ID NO:11 or ATCC Patent Deposit PTA-10355), BIIB69A09 (SEQ ID NO:15), BIIB67F10 (SEQ ID NO:19), BIIB67F11 (SEQ ID NO:23 or ATCC Patent Deposit PTA-10357), BIIB66A12 (SEQ ID NO:27), BIIB66C01 (SEQ ID NO:31), BIIB65C10 (SEQ ID NO:35 or ATCC Patent Deposit PTA-10358), BIIB65H09 (SEQ ID NO:39 or ATCC Patent Deposit PTA-10356) or BIIB65B03 (SEQ ID NO:43).
In other embodiments, the light chain immunoglobulin variable domain of the anti-HER2 antibody molecule includes, or consists essentially of, an amino acid sequence encoded by a nucleotide sequence that hybridizes under high stringency conditions to the complement of the nucleotide sequence encoding a light chain variable domain of BIIB71F10 (SEQ ID NO:14 or ATCC Patent Deposit PTA-10355), BIIB69A09 (SEQ ID NO:18), BIIB67F10 (SEQ ID NO:22), BIIB67F11 (SEQ ID NO:26 or ATCC Patent Deposit PTA-10357), BIIB66A12 (SEQ ID NO:30), BIIB66C01 (SEQ ID NO:34), BIIB65C10 (SEQ ID NO:38 or ATCC Patent Deposit PTA-10358), BIIB65H09 (SEQ ID NO:42 or ATCC Patent Deposit PTA-10356) or BIIB65B03 (SEQ ID NO:46); or includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher identical to a light chain variable domain of BIIB71F10 (SEQ ID NO:13 or ATCC Patent Deposit PTA-10355), BIIB69A09 (SEQ ID NO:17), BIIB67F10 (SEQ ID NO:21), BIIB67F11 (SEQ ID NO:25 or ATCC Patent Deposit PTA-10357), BIIB66A12 (SEQ ID NO:29), BIIB66C01 (SEQ ID NO:33), BIIB65C10 (SEQ ID NO:37 or ATCC Patent Deposit PTA-10358), BIIB65H09 (SEQ ID NO:41 or ATCC Patent Deposit PTA-10356) or BIIB65B03 (SEQ ID NO:45).
In certain embodiments, the LIGHT/HER2 fusions include, or consist essentially of, the amino acid sequence shown in any of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130) (SEQ ID NO:2), 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131) (SEQ ID NO:3), 71F10 Fab-hLIGHT fusion heavy chain with the (G4S) 4 linker (pBIIB71F10-132) (SEQ ID NO:4), or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto); an amino acid sequence encoded by the nucleotide sequence shown in any of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130) (SEQ ID NO:6), 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131) (SEQ ID NO:7), 71F10 Fab-hLIGHT fusion heavy chain with the (G4S)4 linker (pBIIB71F10-132) (SEQ ID NO:8), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). In certain embodiments, the LIGHT/HER2 fusions may also include, or consist essentially of, a second chain (fused or in association with the aforesaid chains) comprising or consisting essentially of the amino acid sequence shown in SEQ ID NO:109, or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto); an amino acid sequence encoded by the nucleotide sequence shown in any of SEQ ID NO:110, or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). In other embodiments, the LIGHT/HER2 fusions may also include, or consist essentially of, a second chain (fused or in association with the aforesaid chains) comprising or consisting essentially of the amino acid sequence of ATCC Patent Deposit PTA-10355, PTA-10356, PTA-10357 or PTA-10358, or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto); an amino acid sequence encoded by the nucleotide sequence of ATCC Patent Deposit PTA-10355, PTA-10356, PTA-10357 or PTA-10358, or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto).
In another exemplary embodiment, the LIGHT targeting molecule comprises at least one fusion molecule of a mammalian (e.g., human) LIGHT protein, or a functional variant or a fragment thereof, and an antibody molecule that binds to CD23 (referred to herein as “LIGHT-anti-CD23 Fab fusion”). In one embodiment, the LIGHT-anti-CD23 fusion comprises, or consists essentially of the amino acid sequence shown in any of anti-CD23 Fab-hLIGHT fusion heavy chain with the (G4S)3 or (G4S)4 linker (pBIIB CD23-204) (SEQ ID NO:101 or 174, respectively), or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto); an amino acid sequence encoded by the nucleotide sequence shown in any of anti-CD23 Fab-hLIGHT fusion heavy chain with the (G4S)3 or (G4S)4 linker (pBIIB CD23-204) (SEQ ID NO:102 or 173, respectively), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). In certain embodiments, the LIGHT/CD23 fusions may also include, or consist essentially of, a second chain (fused or in association with the aforesaid chains) comprising or consisting essentially of the amino acid sequence shown in SEQ ID NO:103, or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto); an amino acid sequence encoded by the nucleotide sequence shown in any of anti-CD23 Fab-hLIGHT fusion light chain (SEQ ID NO:104), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto).
In yet another exemplary embodiment, the LIGHT targeting molecule comprises at least one fusion molecule of a mammalian (e.g., human) LIGHT protein, or a functional variant or a fragment thereof, and an antibody molecule that binds to insulin growth factor receptor (referred to herein as “LIGHT-anti-IGFR Fab fusion”). In one embodiment, the LIGHT-anti-IGFR Fab fusion comprises, or consists essentially of the amino acid sequence shown in any of anti-IGFR Fab-hLIGHT fusion heavy chain with the (G4S)4 linker (BIIB C06-117) (SEQ ID NO:163), or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto); an amino acid sequence encoded by the nucleotide sequence shown in any of anti-IGFR Fab-hLIGHT fusion heavy chain with the (G4S)4 linker (BIIB C06-117) (SEQ ID NO:162), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). In certain embodiments, the LIGHT/IGFR fusions may also include, or consist essentially of, a second chain (fused or in association with the aforesaid chains) comprising or consisting essentially of the amino acid sequence shown in SEQ ID NO:168, or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto); an amino acid sequence encoded by the nucleotide sequence shown in any of anti-IGFR Fab-hLIGHT fusion light chain (SEQ ID NO:167), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto).
In another aspect, the invention features an antibody molecule (e.g., isolated or purified protein or polypeptide) that selectively binds to HER2 (e.g., an anti-HER2 antibody as described herein). The antibody molecule can be a monoclonal or single specificity antibody, or an antigen-binding fragment thereof (e.g., an Fab; a F(ab′)2; an Fv; a single chain Fv fragment; a single domain antibody or a variant thereof (e.g., a heavy or light chain variable domain monomer or dimer, e.g., VH, VHH)); a single chain Fc fragment; a diabody (dAb); a camelid antibody; or one, two, or all three complementarity determining regions (CDRs) grafted onto a repertoire of VH or VL domains, or other scaffolds (such as, e.g., a fibronectin domain, T cell receptor, Affibody molecule as described herein (e.g., an Affibody protein Z scaffold), fibronectin scaffold, Lipocalin, ankyrin repeats, LDL receptor domain, RNA aptamer, PDZ domain and microbody) (or a combination of one or more of the aforesaid antibody molecules). The antibody molecule can interact with, e.g., bind to, HER2, e.g., mammalian (e.g., human) HER2. For example, the antibody molecule may include a combination of a single chain (e.g., a single chain Fc) and a Fab or a scFv. In other embodiments, the antibody molecule can be a multispecific (e.g., bivalent or bispecific) antibody or fragment thereof. In some embodiments, the antibody molecule binds to a single epitope on HER2. In other embodiments, the antibody molecule is a multi-specific antibody and binds to two or more epitopes on one or more cell surface proteins (e.g., HER2 and one or more cell surface proteins as described herein). Typically, the antibody molecule is a human, humanized, chimeric, camelid, or in vitro generated antibody (or functional fragment thereof, e.g., an antibody fragment as described herein). In embodiments, the anti-HER2 antibody is generated by in vitro selection in phage. In certain embodiments, the antibody molecule binds to the cell surface protein with an affinity characterized by a dissociation constant (Kd) at least of 1×10−7 M, 1×10−8 M, 1×10−9 M, 1×10−10 M, 1×10−11 M, 1×10−12M, 1×10−13M.
The antibody molecule can be full-length (e.g., can include at least one, and typically two, complete heavy chains, and at least one, and typically two, complete light chains) or can include an antigen-binding fragment (e.g., a Fab, F(ab′)2, Fv or a single chain Fv fragment). In yet other embodiments, the antibody molecule has a heavy chain constant region chosen from, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly, chosen from, e.g., the (e.g., human) heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4. In another embodiment, the antibody molecule has a light chain constant region chosen from, e.g., the (e.g., human) light chain constant regions of kappa or lambda. The constant region can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues (—S—S— bonds), effector cell function, and/or complement function).
In certain embodiments, the anti-HER2 antibody molecules can have one or more of the activities described herein for an anti-HER2 antibody molecule.
In one embodiment, the anti-HER2 antibody molecule is an antibody molecule or a Fab fragment from, or has a functional activity comparable to, an antibody or Fab fragment selected from the group consisting of BIIB71F10, BIIB69A09, BIIB67F10, BIIB67F11, BIIB66A12, BIIB66C01, BIIB65C10, BIIB65H09 and BIIB65B03, as described herein.
In other embodiments, the anti-HER2 antibody molecule can cross-react with HER2 from one or more species chosen from human, mouse, rat, or cyno origin, and/or have one or more binding properties, e.g., affinity and/or kinetics, as described herein.
In still another embodiment, the anti-HER2 antibody molecule specifically binds to an epitope, e.g., a linear or a conformational epitope, of HER2, e.g., mammalian, e.g., human HER2, e.g., a HER2 epitope as described herein.
In one embodiment, the anti-HER2 antibody molecule includes one, two, three, four, five or all six CDR's from an antibody selected from the group consisting of BIIB71F10, BIIB69A09, BIIB67F10, BIIB67F11, BIIB66A12, BIIB66C01, BIIB65C10, BIIB65H09 and BIIB65B03, as described herein, or closely related CDRs, e.g., CDRs which are identical or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions (e.g., conservative substitutions), deletions, or insertions). Optionally, the antibody molecule may include any CDR described herein.
The amino acid sequence of the heavy chan variable domain of BIIB71F10 has the amino acid sequence shown as SEQ ID NO:11, or is encoded by a nucleotide sequence shown as SEQ ID NO:12 or SEQ ID NO:156. The amino acid sequence of the light chan variable domain of BIIB71F10 has the amino acid sequence shown as SEQ ID NO:13, or is encoded by a nucleotide sequence shown as SEQ ID NO:14. The heavy chain and light chain variable domains of BIIB71F10 include the amino acid sequence, or is encoded by the nucleotide sequence, of ATCC Patent Deposit PTA-10355.
The amino acid sequence of the heavy chan variable domain of BIIB65H09 includes the amino acid sequence shown as SEQ ID NO:39, or is encoded by a nucleotide sequence shown as SEQ ID NO:40. The amino acid sequence of the light chan variable domain of BIIB65H09 includes the amino acid sequence shown as SEQ ID NO:41, or is encoded by a nucleotide sequence shown as SEQ ID NO:42. The heavy chain and light chain variable domains of BIIB65H09 include the amino acid sequence, or is encoded by the nucleotide sequence, of ATCC Patent Deposit PTA-10356.
The amino acid sequence of the heavy chan variable domain of BIIB67F11 includes the amino acid sequence shown as SEQ ID NO:23, or is encoded by a nucleotide sequence shown as SEQ ID NO:24. The amino acid sequence of the light chan variable domain of BIIB67F11 includes the amino acid sequence shown as SEQ ID NO:25, or is encoded by a nucleotide sequence shown as SEQ ID NO:26. The heavy chain and light chain variable domains of BIIB67F11 include the amino acid sequence, or is encoded by the nucleotide sequence, of ATCC Patent Deposit PTA-10357.
The amino acid sequence of the heavy chan variable domain of BIIB65C10 includes the amino acid sequence shown as SEQ ID NO:35, or is encoded by a nucleotide sequence shown as SEQ ID NO:36. The amino acid sequence of the light chan variable domain of BIIB65C10 includes the amino acid sequence shown as SEQ ID NO:37, or is encoded by a nucleotide sequence shown as SEQ ID NO:38. The heavy chain and light chain variable domains of BIIB65C10 include the amino acid sequence, or is encoded by the nucleotide sequence, of ATCC Patent Deposit PTA-10358.
The amino acid sequence of the heavy chan variable domain of BIIB65B03 includes the amino acid sequence shown as SEQ ID NO:43, or is encoded by a nucleotide sequence shown as SEQ ID NO:44. The amino acid sequence of the light chan variable domain of BIIB65B03 includes the amino acid sequence shown as SEQ ID NO:45, or is encoded by a nucleotide sequence shown as SEQ ID NO:46.
The amino acid sequence of the heavy chan variable domain of BIIB66A12 includes the amino acid sequence shown as SEQ ID NO:27, or is encoded by a nucleotide sequence shown as SEQ ID NO:28. The amino acid sequence of the light chan variable domain of BIIB66A12 includes the amino acid sequence shown as SEQ ID NO:29, or is encoded by a nucleotide sequence shown as SEQ ID NO:30.
The amino acid sequence of the heavy chan variable domain of BIIB66C01 includes the amino acid sequence shown as SEQ ID NO:31, or is encoded by a nucleotide sequence shown as SEQ ID NO:32. The amino acid sequence of the light chan variable domain of BIIB66C01 includes the amino acid sequence shown as SEQ ID NO:33, or is encoded by a nucleotide sequence shown as SEQ ID NO:34.
The amino acid sequence of the heavy chan variable domain of BIIB67F10 includes the amino acid sequence shown as SEQ ID NO:19, or is encoded by a nucleotide sequence shown as SEQ ID NO:20. The amino acid sequence of the light chan variable domain of BIIB67F10 includes the amino acid sequence shown as SEQ ID NO:21, or is encoded by a nucleotide sequence shown as SEQ ID NO:22.
The amino acid sequence of the heavy chan variable domain of BIIB69A09 includes the amino acid sequence shown as SEQ ID NO:15, or is encoded by a nucleotide sequence shown as SEQ ID NO:16. The amino acid sequence of the light chan variable domain of BIIB69A09 includes the amino acid sequence shown as SEQ ID NO:17, or is encoded by a nucleotide sequence shown as SEQ ID NO:18.
In yet another embodiment, the anti-HER2 antibody molecule includes at least one, two, or three Chothia hypervariable loops from a heavy chain variable region of an antibody chosen from, e.g., BIIB71F10, BIIB69A09, BIIB67F10, BIIB67F11, BIIB66A12, BIIB66C01, BIIB65C10, BIIB65H09 or BIIB65B03, PTA-10355, PTA-10356, PTA-10357, or PTA-10358, as described herein. In yet another embodiment, the antibody or fragment thereof includes at least one, two, or three hypervariable loops from a light chain variable region of an antibody chosen from, e.g., BIIB71F10, BIIB69A09, BIIB67F10, BIIB67F11, BIIB66A12, BIIB66C01, BIIB65C10, BIIB65H09 or BIIB65B03, or PTA-10355, PTA-10356, PTA-10357, or PTA-10358, as described herein. In yet another embodiment, the antibody or fragment thereof includes at least one, two, three, four, five, or six hypervariable loops from the heavy and light chain variable regions of an antibody chosen from, e.g., BIIB71F10, BIIB69A09, BIIB67F10, BIIB67F11, BIIB66A12, BIIB66C01, BIIB65C10, BIIB65H09 and BIIB65B03, or PTA-10355, PTA-10356, PTA-10357, or PTA-10358, as described herein.
In still another example, the anti-HER2 antibody molecule includes at least one, two, or three hypervariable loops that have the same canonical structures as the corresponding hypervariable loop of BIIB71F10, BIIB69A09, BIIB67F10, BIIB67F11, BIIB66A12, BIIB66C01, BIIB65C10, BIIB65H09 and BIIB65B03, or PTA-10355, PTA-10356, PTA-10357, or PTA-10358, as described herein, e.g., the same or similar canonical structures as at least loop 1 and/or loop 2 of the heavy and/or light chain variable domains of BIIB71F10, BIIB69A09, BIIB67F10, BIIB67F11, BIIB66A12, BIIB66C01, BIIB65C10, BIIB65H09 and BIIB65B03, or PTA-10355, PTA-10356, PTA-10357, or PTA-10358, as described herein.
In one embodiment, the heavy chain framework of the anti-HER2 antibody molecule (e.g., FR1, FR2, FR3, individually, or a sequence encompassing FR1, FR2, and FR3, but excluding CDRs) includes an amino acid sequence, which is at least 85%, 90%, 95%, 97%, 98%, 99% or higher identical to the heavy chain framework of BIIB71F10 (SEQ ID NO:11), BIIB69A09 (SEQ ID NO:15); BIIB67F10 (SEQ ID NO:19); BIIB67F11 (SEQ ID NO:23), BIIB66A12 (SEQ ID NO:27), BIIB66C01 (SEQ ID NO:31), BIIB65C10 (SEQ ID NO:35), BIIB65H09 (SEQ ID NO:39) or BIIB65B03 (SEQ ID NO:43), or PTA-10355, PTA-10356, PTA-10357, or PTA-10358, or a heavy chain as described herein. In embodiments, the heavy chain framework of the anti-HER2 antibody molecule has an amino acid sequence substantially homologous to human V segment sequence HV3-23 (SEQ ID NO:107).
In another embodiment, the light chain framework of the anti-HER2 antibody molecule (e.g., FR1, FR2, FR3, individually, or a sequence encompassing FR1, FR2, and FR3, but excluding CDRs) includes an amino acid sequence, which is at least 85%, 90%, 95%, 97%, 98%, 99% or higher identical to the light chain framework of BIIB71F10 (SEQ ID NO:13), BIIB69A09 (SEQ ID NO:17); BIIB67F10 (SEQ ID NO:21); BIIB67F11 (SEQ ID NO:25), BIIB66A12 (SEQ ID NO:29), BIIB66C01 (SEQ ID NO:33), BIIB65C10 (SEQ ID NO:37), BIIB65H09 (SEQ ID NO:41) or BIIB65B03 (SEQ ID NO:45), PTA-10355, PTA-10356, PTA-10357, or PTA-10358, or a light chain framework as described herein. In embodiments, the heavy chain framework of the anti-HER2 antibody molecule has an amino acid sequence substantially homologous to human a VLκ I subgroup germline sequence, e.g., a VLκ consensus sequence.
In certain embodiments, the heavy chain immunoglobulin variable domain of the anti-HER2 antibody molecule includes, or consists essentially of, an amino acid sequence encoded by a nucleotide sequence that hybridizes under high stringency conditions to the complement of the nucleotide sequence encoding a heavy chain variable domain of BIIB71F10 (SEQ ID NO:12; SEQ ID NO:156), BIIB69A09 (SEQ ID NO:16); BIIB67F10 (SEQ ID NO:20); BIIB67F11 (SEQ ID NO:24), BIIB66A12 (SEQ ID NO:28), BIIB66C01 (SEQ ID NO:32), BIIB65C10 (SEQ ID NO:36), BIIB65H09 (SEQ ID NO:40) or BIIB65B03 (SEQ ID NO:44), or PTA-10355, PTA-10356, PTA-10357, or PTA-10358; or includes an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98%, 99% or higher identical to the amino acid sequence of the heavy chain variable domain of BIIB71F10 (SEQ ID NO:11), BIIB69A09 (SEQ ID NO:15); BIIB67F10 (SEQ ID NO:19); BIIB67F11 (SEQ ID NO:23), BIIB66A12 (SEQ ID NO:27), BIIB66C01 (SEQ ID NO:31), BIIB65C10 (SEQ ID NO:35), BIIB65H09 (SEQ ID NO:39) or BIIB65B03 (SEQ ID NO:43), or PTA-10355, PTA-10356, PTA-10357, or PTA-10358.
In other embodiments, the light chain immunoglobulin variable domain of the anti-HER2 antibody molecule includes, or consists essentially of, an amino acid sequence encoded by a nucleotide sequence that hybridizes under high stringency conditions to the complement of the nucleotide sequence encoding a light chain variable domain of BIIB71F10 (SEQ ID NO:14), BIIB69A09 (SEQ ID NO:18); BIIB67F10 (SEQ ID NO:22); BIIB67F11 (SEQ ID NO:26), BIIB66A12 (SEQ ID NO:30), BIIB66C01 (SEQ ID NO:34), BIIB65C10 (SEQ ID NO:38), BIIB65H09 (SEQ ID NO:42) or BIIB65B03 (SEQ ID NO:46), or PTA-10355, PTA-10356, PTA-10357, or PTA-10358; or includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher identical to a light chain variable domain of BIIB71F10 (SEQ ID NO:13), BIIB69A09 (SEQ ID NO:17); BIIB67F10 (SEQ ID NO:21); BIIB67F11 (SEQ ID NO:25), BIIB66A12 (SEQ ID NO:29), BIIB66C01 (SEQ ID NO:33), BIIB65C10 (SEQ ID NO:37), BIIB65H09 (SEQ ID NO:41) or BIIB65B03 (SEQ ID NO:45), or PTA-10355, PTA-10356, PTA-10357, or PTA-10358.
In some embodiments, the LIGHT targeting molecules and/or antibody molecules described herein are conjugated to an agent selected from the group consisting of cytotoxic agent, a therapeutic agent, cytostatic agent, a biological toxin, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, polyethylene glycol (PEG), and a combination of two or more of any said agents. In further embodiments, the cytotoxic agent is selected from the group consisting of a radionuclide, a biotoxin, an enzymatically active toxin, a cytostatic or cytotoxic therapeutic agent, a prodrugs, an immunologically active ligand, a biological response modifier, or a combination of two or more of any said cytotoxic agents. In further embodiments, the detectable label is selected from the group consisting of an enzyme, a fluorescent label, a chemiluminescent label, a bioluminescent label, a radioactive label, or a combination of two or more of any said detectable labels.
In another aspect, the invention features nucleic acid molecules (e.g., isolated or purified nucleic acids) comprising, or consisting essentially of, a nucleotide sequence encoding the LIGHT targeting molecules and/or the anti-HER2 antibody molecules described herein. In certain embodiments, nucleic acids comprise, or consist essentially of, a nucleotide sequence encoding a LIGHT-moiety (e.g., a nucleotide sequence encoding a LIGHT protein, or a functional fragment or variant thereof) and a nucleotide sequence encoding a targeting moiety functionally linked (e.g., by genetic fusion, non-covalent association or otherwise).
Exemplary nucleic acid molecules of the invention comprise, or consist essentially of, nucleotide sequences encoding LIGHT proteins of the LIGHT moiety include, or consist essentially of, the amino acid sequence from: about amino acids 93 to 240 of human LIGHT isoform 1 (corresponding to a portion of the human LIGHT extracellular domain shown as SEQ ID NO:1), about amino acids 253 to 400 of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130) (corresponding to the portion of the LIGHT extracellular domain fused to anti-HER2 antibody molecule shown as SEQ ID NO:2), about amino acids 258 to 405 of 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131) (corresponding to the portion of the LIGHT extracellular domain fused to anti-HER2 antibody molecule shown as SEQ ID NO:3), about amino acids 245 to 392 of 71F10 Fab-hLIGHT fusion heavy chain with the (G4S)4 linker (pBIIB71F10-132) (corresponding to the portion of the LIGHT extracellular domain fused to anti-HER2 antibody molecule shown as SEQ ID NO:4), or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto); In other embodiments, the nucleic acids comprise, or consist essentially of, the nucleotide sequence from: about nucleotides 277 to 720 of, human LIGHT isoform 1 (nucleotide sequence corresponding to a portion of the human LIGHT extracellular domain shown as SEQ ID NO:5), about nucleotides 757 to 1200 of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130) (nucleotide sequence corresponding to the portion of the LIGHT extracellular domain fused to anti-HER2 antibody molecule shown as SEQ ID NO:6), about nucleotides 772 to 1215 of 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131) (nucleotide sequence corresponding to the portion of the LIGHT extracellular domain fused to anti-HER2 antibody molecule shown as SEQ ID NO:7), about nucleotides 733 to 1176 of 71F10 Fab-hLIGHT fusion heavy chain with the (G4S)4 linker (pBIIB71F10-132) (nucleotide sequence corresponding to the portion of the LIGHT extracellular domain fused to anti-HER2 antibody molecule shown as SEQ ID NO:8), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). The LIGHT moiety may, optionally, include, or consist essentially of, one or more amino acid residues (e.g., at least 10 to 35, 15 to 30, or about 20 to 26 amino acid residues) from the extracellular domain of LIGHT or a mutated form thereof, e.g., from about amino acids 61 to 92 of human LIGHT isoform 1 (SEQ ID NO:1), about amino acids 225 to 252 of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130) (SEQ ID NO:2), about amino acids 230 to 257 of 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131) (SEQ ID NO:3), or an amino acid sequence substantially identical thereto; or an amino acid sequence encoded by the nucleotide sequence from about nucleotides 181 to 276 of human LIGHT isoform 1 (SEQ ID NO:5), about nucleotides 673 to 756 of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130) (SEQ ID NO:6), about nucleotides 688 to 771 of 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131) (SEQ ID NO:7), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). Variants of the LIGHT protein, or soluble fragments thereof, altered to increase one or more properties of LIGHT, e.g., protein stability, immune enhancing function, may used. For example, the LIGHT protein can be modified to have one or more protelolytic sites inactivated (e.g., by deletion, mutation or insertion, of a proteolytic site). In one embodiment, amino acids EQLI (SEQ ID NO:9) comprising a proteolytic site at position 82 to 83 of the human LIGHT sequence (human LIGHT isoform 1, SEQ ID NO:1), or amino acids EKLI (SEQ ID NO:10) from positions 79-82 of the mouse LIGHT sequence are removed. In other embodiments, the LIGHT protein is from non-human origin, e.g., murine LIGHT, can be used. The amino acid and corresponding nucleotide sequences for full length mouse LIGHT are shown in SEQ ID NOs:113 and 114, respectively.
The nucleotide sequence encoding the LIGHT molecule can be genetically fused, with or without a nucleotide sequence encoding a linking group, to a nucleotide sequence encoding the targeting moiety as a genetic fusion. In other embodiments, the nucleotide sequences encoding the LIGHT molecule and the targeting moiety can be individually expressed, and covalently attached to each other via a reactive group, optionally, via a biocompatible polymer (e.g., as described herein). Nucleic acids encoding the linking groups can include at least five, ten, fifteen or twenty glycine and serine residues in the following configuration, (Gly)4-Ser (SEQ ID NO:145), in one, two, three, four, five or more repeats, e.g., four repeats of (Gly)4-Ser (SEQ ID NO: 134). In other embodiments, linking group may include one or more amino acid residues (e.g., at least 10 to 35, 15 to 30, or about 20 to 26 amino acid residues) from the extracellular domain of LIGHT or a mutated form thereof, e.g., from about amino acids 61 to 92 of human LIGHT isoform 1 (SEQ ID NO:1), about amino acids 225 to 252 of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130) (SEQ ID NO:2), about amino acids 230 to 257 of 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131) (SEQ ID NO:3), or an amino acid sequence substantially identical thereto; or an amino acid sequence encoded by the nucleotide sequence from about nucleotides 181 to 276 of human LIGHT isoform 1 (SEQ ID NO:5), about nucleotides 673 to 756 of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130) (SEQ ID NO:6), about nucleotides 688 to 771 of 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131) (SEQ ID NO:7), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). Alternatively, the linking group encoded by the nucleic acids may include a combination of one or more (Gly)4-Ser (SEQ ID NO: 146) repeats and one or more amino acid residues (e.g., at least 10 to 35, 15 to 30, or about 20 to 26 amino acid residues) from the extracellular domain of LIGHT or a mutated form thereof, e.g., from about amino acids 61 to 92 of human LIGHT isoform 1 (SEQ ID NO:1), about amino acids 225 to 252 of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130) (SEQ ID NO:2), about amino acids 230 to 257 of 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131) (SEQ ID NO:3), or an amino acid sequence substantially identical thereto; or an amino acid sequence encoded by the nucleotide sequence from about nucleotides 181 to 276 of human LIGHT isoform 1 (SEQ ID NO:5), about nucleotides 673 to 756 of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130) (SEQ ID NO:6), about nucleotides 688 to 771 of 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131) (SEQ ID NO:7), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto).
In one exemplary embodiment, the nucleic acid molecules encode a LIGHT targeting molecule that comprises, or consists essentially of, at least one fusion molecule of a mammalian (e.g., human) LIGHT protein, or a functional variant or a fragment thereof, and an antibody molecule that binds to HER2 (referred to herein as “LIGHT-anti-HER2 fusion”). In one embodiment, the nucleic acids encoding LIGHT-anti-HER2 fusion comprises, or consists essentially of, at least one mammalian (e.g., human) LIGHT protein, or a variant or a fragment thereof (e.g., a LIGHT protein as described herein) and an anti-HER2 specific antibody molecule or a fragment thereof (e.g., an antibody molecule as described herein).
In embodiments, the nucleic acid encoding the anti-HER2 antibody molecule is an antibody molecule or a Fab fragment from an antibody selected from the group consisting of BIIB71F10 (SEQ ID NOs:11-14), BIIB69A09 (SEQ ID NOs:15-18); BIIB67F10 (SEQ ID NOs:19-22); BIIB67F11 (SEQ ID NOs:23-26), BIIB66A12 (SEQ ID NOs:27-30), BIIB66C01 (SEQ ID NOs:31-34), BIIB65C10 (SEQ ID NOs:35-38), BIIB65H09 (SEQ ID NOs:39-42) and BIIB65B03 (SEQ ID NOs:43-46), or a nucleic acid of PTA-10355, PTA-10356, PTA-10357, or PTA-10358, as described herein. In other embodiments, the nucleic acid encoding the anti-HER2 antibody molecule has a functional activity comparable to an antibody molecule or a Fab fragment from an antibody selected from the group consisting of BIIB71F10 (SEQ ID NOs:11-14), BIIB69A09 (SEQ ID NOs:15-18); BIIB67F10 (SEQ ID NOs:19-22); BIIB67F11 (SEQ ID NOs:23-26), BIIB66A12 (SEQ ID NOs:27-30), BIIB66C01 (SEQ ID NOs:31-34), BIIB65C10 (SEQ ID NOs:35-38), BIIB65H09 (SEQ ID NOs:39-42) and BIIB65B03 (SEQ ID NOs:43-46), or a nucleic acid of PTA-10355, PTA-10356, PTA-10357, or PTA-10358, as described herein.
In one embodiment, the nucleic acid molecule encoding the antibody molecule of the fusion, or the anti-HER2 antibody molecule, includes one, two, three, four, five or all six CDR's from an antibody selected from the group consisting of BIIB71F10 (SEQ ID NOs:11-14), BIIB69A09 (SEQ ID NOs:15-18); BIIB67F10 (SEQ ID NOs:19-22); BIIB67F11 (SEQ ID NOs:23-26), BIIB66A12 (SEQ ID NOs:27-30), BIIB66C01 (SEQ ID NOs:31-34), BIIB65C10 (SEQ ID NOs:35-38), BIIB65H09 (SEQ ID NOs:39-42) and BIIB65B03 (SEQ ID NOs:43-46), or a nucleic acid of PTA-10355, PTA-10356, PTA-10357, or PTA-10358, or closely related CDRs, e.g., CDRs which are identical or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions (e.g., conservative substitutions), deletions, or insertions). Optionally, the nucleic acid encodes an antibody molecule that may include any CDR described herein. In embodiments, nucleic acid encodes a heavy chain immunoglobulin variable domain that includes a heavy chain CDR1, CDR2, and/or CDR3, or having a CDR that differs by fewer than 3 amino acid substitutions (e.g., conservative substitutions) from a heavy chain CDR1, CDR2, and/or CDR3 of monoclonal antibody selected from the group consisting of BIIB71F10 (SEQ ID NO:47 (CDR1), SEQ ID NO:48 (CDR2), and/or SEQ ID NO:49 (CDR3)); BIIB69A09 (SEQ ID NO:50 (CDR1), SEQ ID NO:51 (CDR2), and/or SEQ ID NO:52 (CDR3)); BIIB67F10 (SEQ ID NO:53 (CDR1), SEQ ID NO:54 (CDR2), and/or SEQ ID NO:55 (CDR3)); BIIB67F11 (SEQ ID NO:56 (CDR1), SEQ ID NO:57 (CDR2), and/or SEQ ID NO:58 (CDR3)); BIIB66A12 (SEQ ID NO:59 (CDR1), SEQ ID NO:60 (CDR2), and/or SEQ ID NO:61 (CDR3)); BIIB66C01 (SEQ ID NO:62 (CDR1), SEQ ID NO:63 (CDR2), and/or SEQ ID NO:64 (CDR3)); BIIB65C10 (SEQ ID NO:65 (CDR1), SEQ ID NO:66 (CDR2), and/or SEQ ID NO:67 (CDR3)); BIIB65H09 (SEQ ID NO:68 (CDR1), SEQ ID NO:69 (CDR2), and/or SEQ ID NO:70 (CDR3)) and BIIB65B03 (SEQ ID NO:71 (CDR1), SEQ ID NO:72 (CDR2), and/or SEQ ID NO:73 (CDR3)), or a CDR from PTA-10355, PTA-10356, PTA-10357, or PTA-10358, as described herein. In other embodiments, the nucleic acid encoding the light chain immunoglobulin variable domain comprises a light chain CDR1, CDR2, and/or CDR3, or having a CDR that differs by fewer than 3 amino acid substitutions (e.g., conservative substitutions) from a light chain CDR1, CDR2, and/or CDR3 of monoclonal antibody antibody selected from the group consisting of BIIB71F10 (SEQ ID NO:74 (CDR1), SEQ ID NO:75 (CDR2), and/or SEQ ID NO:76 (CDR3)); BIIB69A09 (SEQ ID NO:77 (CDR1), SEQ ID NO:78 (CDR2), and/or SEQ ID NO:79 (CDR3)); BIIB67F10 (SEQ ID NO:80 (CDR1), SEQ ID NO:81 (CDR2), and/or SEQ ID NO:82 (CDR3)); BIIB67F11 (SEQ ID NO:83 (CDR1), SEQ ID NO:84 (CDR2), and/or SEQ ID NO:85 (CDR3)); BIIB66A12 (SEQ ID NO:86 (CDR1), SEQ ID NO:87 (CDR2), and/or SEQ ID NO:88 (CDR3)); BIIB66C01 (SEQ ID NO:89 (CDR1), SEQ ID NO:90 (CDR2), and/or SEQ ID NO:91 (CDR3)); BIIB65C10 (SEQ ID NO:92 (CDR1), SEQ ID NO:93 (CDR2), and/or SEQ ID NO:94 (CDR3)); BIIB65H09 (SEQ ID NO:95 (CDR1), SEQ ID NO:96 (CDR2), and/or SEQ ID NO:97 (CDR3)) and BIIB65B03 (SEQ ID NO:98 (CDR1), SEQ ID NO:99 (CDR2), and/or SEQ ID NO:100 (CDR3)), or a CDR from PTA-10355, PTA-10356, PTA-10357, or PTA-10358, as described herein. The nucleotide sequence encoding the heavy chain and light chain variable domain of the BIIB71F10, BIIB69A09, BIIB67F10, BIIB67F11, BIIB66A12, BIIB66C01, BIIB65C10, BIIB65H09 or BIIB65B03 antibody molecules are described herein.
In yet another embodiment, the nucleic acid molecule encodes an antibody molecule of the fusion, or the anti-HER2 antibody molecule, that includes at least one, two, or three Chothia hypervariable loops from a heavy chain variable region of an antibody chosen from, e.g., BIIB71F10 (SEQ ID NOs:11-12), BIIB69A09 (SEQ ID NOs:15-16); BIIB67F10 (SEQ ID NOs:19-20); BIIB67F11 (SEQ ID NOs:23-24), BIIB66A12 (SEQ ID NOs:27-28), BIIB66C01 (SEQ ID NOs:31-32), BIIB65C10 (SEQ ID NOs:34-35), BIIB65H09 (SEQ ID NOs:39-40) or BIIB65B03 (SEQ ID NOs:43-44), or a nucleic acid of PTA-10355, PTA-10356, PTA-10357, or PTA-10358, as described herein. In yet another embodiment, the nucleic acid encodes an antibody molecule of the fusion, or the anti-HER2 antibody molecule, that includes at least one, two, or three hypervariable loops from a light chain variable region of an antibody chosen from, e.g., BIIB71F10 (SEQ ID NOs:13-14), BIIB69A09 (SEQ ID NOs:17-18); BIIB67F10 (SEQ ID NOs:21-22); BIIB67F11 (SEQ ID NOs:25-26), BIIB66A12 (SEQ ID NOs:29-30), BIIB66C01 (SEQ ID NOs:33-34), BIIB65C10 (SEQ ID NOs:37-38), BIIB65H09 (SEQ ID NOs:41-42) or BIIB65B03 (SEQ ID NOs:45-46), or a nucleic acid of PTA-10355, PTA-10356, PTA-10357, or PTA-10358, as described herein. In yet another embodiment, the nucleic acid encodes an antibody molecule of the fusion, or the anti-HER2 antibody molecule, that includes at least one, two, three, four, five, or six hypervariable loops from the heavy and light chain variable regions of an antibody chosen from, e.g., BIIB71F10 (SEQ ID NOs:11-14), BIIB69A09 (SEQ ID NOs:15-18); BIIB67F10 (SEQ ID NOs:19-22); BIIB67F11 (SEQ ID NOs:23-26), BIIB66A12 (SEQ ID NOs:27-30), BIIB66C01 (SEQ ID NOs:31-34), BIIB65C10 (SEQ ID NOs:35-38), BIIB65H09 (SEQ ID NOs:39-42) or BIIB65B03 (SEQ ID NOs:43-46), or a nucleic acid of PTA-10355, PTA-10356, PTA-10357, or PTA-10358, as described herein.
In one embodiment, the nucleic acid molecule encodes an antibody molecule of the fusion, or the anti-HER2 antibody molecule, that includes all six hypervariable loops from BIIB71F10 (SEQ ID NOs:11-14), BIIB69A09 (SEQ ID NOs:15-18); BIIB67F10 (SEQ ID NOs:19-22); BIIB67F11 (SEQ ID NOs:23-26), BIIB66A12 (SEQ ID NOs:27-30), BIIB66C01 (SEQ ID NOs:31-34), BIIB65C10 (SEQ ID NOs:35-38), BIIB65H09 (SEQ ID NOs:39-42) or BIIB65B03 (SEQ ID NOs:43-46), or PTA-10355, PTA-10356, PTA-10357, or PTA-10358, or closely related hypervariable loops, e.g., hypervariable loops which are identical or which have at least one amino acid alteration, but not more than two, three or four alterations, from the sequences disclosed herein. Optionally, the nucleic acid may encode a protein including any hypervariable loop described herein.
In still another example, the nucleic acid molecule encodes an antibody molecule of the fusion, or the anti-HER2 antibody molecule, that includes at least one, two, or three hypervariable loops that have the same canonical structures as the corresponding hypervariable loop of BIIB71F10 (SEQ ID NOs:11-14), BIIB69A09 (SEQ ID NOs:15-18); BIIB67F10 (SEQ ID NOs:19-22); BIIB67F11 (SEQ ID NOs:23-26), BIIB66A12 (SEQ ID NOs:27-30), BIIB66C01 (SEQ ID NOs:31-34), BIIB65C10 (SEQ ID NOs:35-38), BIIB65H09 (SEQ ID NOs:39-42) or BIIB65B03 (SEQ ID NOs:43-46), or PTA-10355, PTA-10356, PTA-10357, or PTA-10358, e.g., the same or similar canonical structures as at least loop 1 and/or loop 2 of the heavy and/or light chain variable domains of BIIB71F10 (SEQ ID NOs:11-14), BIIB69A09 (SEQ ID NOs:15-18); BIIB67F10 (SEQ ID NOs:19-22); BIIB67F11 (SEQ ID NOs:23-26), BIIB66A12 (SEQ ID NOs:27-30), BIIB66C01 (SEQ ID NOs:31-34), BIIB65C10 (SEQ ID NOs:35-38), BIIB65H09 (SEQ ID NOs:39-42) or BIIB65B03 (SEQ ID NOs:43-46), or PTA-10355, PTA-10356, PTA-10357, or PTA-10358.
In one embodiment, the nucleic acid molecule encodes an antibody molecule of the fusion, or the anti-HER2 antibody molecule, that includes a heavy chain framework (e.g., FR1, FR2, FR3, individually, or a sequence encompassing FR1, FR2, and FR3, but excluding CDRs) that includes an amino acid sequence, which is at least 85%, 90%, 95%, 97%, 98%, 99% or higher identical to the heavy chain framework of BIIB71F10 (SEQ ID NO:11), BIIB69A09 (SEQ ID NO:15); BIIB67F10 (SEQ ID NO:19); BIIB67F11 (SEQ ID NO:23), BIIB66A12 (SEQ ID NO:27), BIIB66C01 (SEQ ID NO:31), BIIB65C10 (SEQ ID NO:35), BIIB65H09 (SEQ ID NO:39) or BIIB65B03 (SEQ ID NO:43), or PTA-10355, PTA-10356, PTA-10357, or PTA-10358. In embodiments, the heavy chain framework of the anti-HER2 antibody molecule has an amino acid sequence substantially homologous to human V segment sequence HV3-23 (SEQ ID NO:107).
In another embodiment, the nucleic acid molecule encodes a light chain framework of the antibody molecule (e.g., FR1, FR2, FR3, individually, or a sequence encompassing FR1, FR2, and FR3, but excluding CDRs) that includes, or consists essentially of, an amino acid sequence, which is at least 85%, 90%, 95%, 97%, 98%, 99% or higher identical to the light chain framework of BIIB71F10 (SEQ ID NO:13), BIIB69A09 (SEQ ID NO:17); BIIB67F10 (SEQ ID NO:21); BIIB67F11 (SEQ ID NO:25), BIIB66A12 (SEQ ID NO:29), BIIB66C01 (SEQ ID NO:33), BIIB65C10 (SEQ ID NO:37), BIIB65H09 (SEQ ID NO:41) or BIIB65B03 (SEQ ID NO:45), or PTA-10355, PTA-10356, PTA-10357, or PTA-10358. In embodiments, the heavy chain framework of the anti-HER2 antibody molecule has an amino acid sequence substantially homologous to human a VLκ I subgroup germline sequence, e.g., a VLκ consensus sequence.
In certain embodiments, the nucleic acid molecule encodes an antibody molecule of the fusion, or the anti-HER2 antibody molecule, that includes, or consists essentially of, a nucleotide sequence that hybridizes under high stringency conditions to the complement of the nucleotide sequence encoding a heavy chain variable domain of BIIB71F10 (SEQ ID NO:12; SEQ ID NO:156), BIIB69A09 (SEQ ID NO:16); BIIB67F10 (SEQ ID NO:20); BIIB67F11 (SEQ ID NO:24), BIIB66A12 (SEQ ID NO:28), BIIB66C01 (SEQ ID NO:32), BIIB65C10 (SEQ ID NO:36), BIIB65H09 (SEQ ID NO:40) or BIIB65B03 (SEQ ID NO:44), or PTA-10355, PTA-10356, PTA-10357, or PTA-10358; or includes an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98%, 99% or higher identical to the amino acid sequence of the heavy chain variable domain of BIIB71F10 (SEQ ID NO:11), BIIB69A09 (SEQ ID NO:15); BIIB67F10 (SEQ ID NO:19); BIIB67F11 (SEQ ID NO:23), BIIB66A12 (SEQ ID NO:27), BIIB66C01 (SEQ ID NO:31), BIIB65C10 (SEQ ID NO:35), BIIB65H09 (SEQ ID NO:39) or BIIB65B03 (SEQ ID NO:43), or PTA-10355, PTA-10356, PTA-10357, or PTA-10358.
In other embodiments, the nucleic acid molecule encodes an antibody molecule of the fusion, or the anti-HER2 antibody molecule, that includes, or consists essentially of, a nucleotide sequence that hybridizes under high stringency conditions to the complement of the nucleotide sequence encoding a light chain variable domain of BIIB71F10 (SEQ ID NO:14), BIIB69A09 (SEQ ID NO:18); BIIB67F10 (SEQ ID NO:22); BIIB67F11 (SEQ ID NO:26), BIIB66A12 (SEQ ID NO:30), BIIB66C01 (SEQ ID NO:34), BIIB65C10 (SEQ ID NO:38), BIIB65H09 (SEQ ID NO:42) or BIIB65B03 (SEQ ID NO:46), or PTA-10355, PTA-10356, PTA-10357, or PTA-10358; or includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher identical identical to a light chain variable domain of BIIB71F10 (SEQ ID NO:13), BIIB69A09 (SEQ ID NO:17); BIIB67F10 (SEQ ID NO:21); BIIB67F11 (SEQ ID NO:25), BIIB66A12 (SEQ ID NO:29), BIIB66C01 (SEQ ID NO:33), BIIB65C10 (SEQ ID NO:37), BIIB65H09 (SEQ ID NO:41) or BIIB65B03 (SEQ ID NO:45), or PTA-10355, PTA-10356, PTA-10357, or PTA-10358.
Exemplary nucleic acid molecules encode LIGHT/HER2 fusions that include, or consist essentially of, the amino acid sequence shown in any of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130) (SEQ ID NO:2), 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131) (SEQ ID NO:3), 71F10 Fab-hLIGHT fusion heavy chain with the (G4S)4 linker (pBIIB71F10-132) (SEQ ID NO:4), or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto); an amino acid sequence encoded by the nucleotide sequence shown in any of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130) (SEQ ID NO:6), 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131) (SEQ ID NO:7), 71F10 Fab-hLIGHT fusion heavy chain with the (G4S)4 linker (pBIIB71F10-132) (SEQ ID NO:8), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). In certain embodiments, the nucleic acid molecules encoding the LIGHT/HER2 fusions may also include, or consist essentially of, a second chain (genetically fused or in association with the aforesaid chains) comprising or consisting essentially of the amino acid sequence shown in human LIGHT isoform 1 (SEQ ID NO:1), or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). In embodiments, the nucleic acid molecules comprise, or consist essentially of, the nucleotide sequence shown in any of human LIGHT isoform 1 (SEQ ID NO:5), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto).
In another exemplary embodiment, the nucleic acid molecules encode a LIGHT targeting molecule that comprises at least one fusion molecule of a mammalian (e.g., human) LIGHT protein, or a functional variant or a fragment thereof, and an antibody molecule that binds to CD23 (referred to herein as “LIGHT-anti-CD23 fusion”). In one embodiment, the nucleic acid molecules encoding the LIGHT-anti-CD23 fusion comprises, or consists essentially of the amino acid sequence shown in any of anti-CD23 Fab-hLIGHT fusion heavy chain with the (G4S)3 or (G4S)4 linker (pBIIB CD23-204) (SEQ ID NO:101 or 174, respectively), or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). In embodiments, the nucleic acid molecules comprise, or consist essentially of, the nucleotide sequence shown in any of anti-CD23 Fab-hLIGHT fusion heavy chain the (G4S)3 or (G4S)4 linker (pBIIB CD23-204) (SEQ ID NO:102 or 173, respectively), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). In certain embodiments, the nucleic acid molecules encoding the LIGHT/CD23 fusions may also include, or consist essentially of, a second chain (fused or in association with the aforesaid chains) comprising or consisting essentially of the amino acid sequence shown in anti-CD23 Fab-hLIGHT fusion light chain (SEQ ID NO:103), or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). In embodiments, the nucleic acid molecules comprise, or consist essentially of, the nucleotide sequence shown in any of anti-CD23 Fab-hLIGHT fusion light chain (SEQ ID NO:104), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto).
In yet another embodiment, the nucleic acid molecules comprises a nucleotide sequence encoding a LIGHT targeting molecule that comprises at least one fusion molecule of a mammalian (e.g., human) LIGHT protein, or a functional variant or a fragment thereof, and an antibody molecule that binds to IGFR. In one embodiment, the nucleic acid encodes a LIGHT-anti-IGFR Fab fusion that comprises, or consists essentially of the amino acid sequence shown in any of anti-IGFR Fab-hLIGHT fusion heavy chain with the (G4S)4 linker (BIIB C06-117) (SEQ ID NO:163), or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto); the nucleotide sequence encodes the anti-IGFR Fab-hLIGHT fusion heavy chain with the (G4S)4 linker (BIIB C06-117) (SEQ ID NO:162), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). In certain embodiments, the nucleic acid molecues can also include a nucleotide sequence encoding a second chain (fused or in association with the aforesaid chains) comprising or consisting essentially of the amino acid sequence shown in SEQ ID NO:168, or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto); a nucleotide sequence encoding the anti-IGFR Fab-hLIGHT fusion light chain (comprising or consisting of SEQ ID NO:167), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto).
In yet another aspect, the invention provides a host cell comprising one or more nucleic acid molecules encoding one or more of the LIGHT targeting molecules and/or the anti-HER2 antibody molecules disclosed herein.
In other embodiments, the invention provides vectors comprising the nucleic acid molecules described herein. In further embodiments, the nucleic acid molecules are operably associated with a promoter. In additional embodiments, the invention provides host cells comprising such vectors. In further embodiments, the invention provides vectors where the polynucleotide is operably associated with a promoter.
In additional embodiments, the invention provides a method of, or process for, producing the LIGHT targeting molecules and/or the anti-HER2 antibody molecules disclosed herein. The method includes: culturing a host cell containing a vector comprising the nucleic acid molecules described herein, and recovering said LIGHT targeting molecules and/or the anti-HER2 antibody molecules. In further embodiments, the invention provides an isolated polypeptide produced by the method.
In some embodiments, the invention provides isolated polypeptides encoded by the nucleic acid molecules described herein.
Compositions, e.g., pharmaceutical compositions, that include the LIGHT targeting molecules or the anti-HER2 antibody molecules, and a pharmaceutically-acceptable carrier, are also disclosed. It is noted that the compositions, e.g., pharmaceutical compositions, may additionally include a second therapeutic agent, e.g., a second therapeutic agent as described herein (e.g., an anti-neoplastic agent). Exemplary anti-neoplastic agents, e.g., a cytotoxic agent or a cytostatic agent, that can be used in combination with the molecules of the invention include, but are not limited to, taxoids (e.g., docetaxel, paclitaxel), doxorubicin, cyclophosphamide, gencitabine and vinorelbine.
Packaged pharmaceutical compositions that include the LIGHT targeting molecules or the anti-HER2 antibody molecules, for use in treating a hyperproliferative, e.g., neoplastic, disorder or condition described herein are also encompassed by the present invention. Optionally, the packaged pharmaceutical composition is labeled and/or contains some instructions for use in treating a hyperproliferative, e.g., neoplastic, disorder or condition described herein.
In some embodiments, the invention provides a composition comprising an isolated LIGHT targeting molecule, or an antibody heavy chain variable region encoding nucleic acid molecule and/or an isolated light chain variable region encoding nucleic acid molecule, wherein the heavy chain encoding polynucleotide and the light chain encoding polynucleotide, respectively, comprise nucleic acid molecules encoding amino acid sequences identical or substantially identical, e.g., at least 85%, 90%, 95% identical to an antibody amino acid sequences disclosed herein.
In other embodiments, the LIGHT targeting molecules, antibody molecules, compositions, nucleic acid molecules encoding one of the chains of the LIGHT targeting molecule, or the antibody molecule (e.g., a heavy or light chain variable region), further comprise a nucleic acid encoding a signal peptide fused to the nucleic acid molecule encoding the LIGHT targeting molecule, or the antibody molecule.
In some embodiments, the LIGHT targeting molecules, antibody molecules, compositions, nucleic acid molecules encoding one of the chains of the LIGHT targeting molecule, or the antibody molecule (e.g., a heavy or light chain variable region), further comprise a heavy chain constant region CH1 domain fused to the VH or VL polypeptide, further comprises a heavy chain constant region CH2 domain fused to the VH polypeptide, further comprises a heavy chain constant region CH3 domain fused to the VH polypeptide, or further comprises a heavy chain hinge region fused to the VH polypeptide. In further embodiments, the heavy chain constant region is human IgG1. In certain other embodiments, the IgG1 is mutagenized according to the Kabat numbering system.
In some embodiments, the LIGHT targeting molecules, antibody molecules, compositions, the VL encoding polynucleotide further comprises a light chain constant region domain fused to the VL polypeptide. In further embodiments, the light chain constant region is human VLκ.
In some embodiments, the LIGHT targeting molecules, antibody molecules, compositions, the framework regions of the VH and VL polypeptides are human, except for five or fewer amino acid substitutions.
In some embodiments, the VH encoding polynucleotide is contained on a first vector and the VL encoding polynucleotide is contained on a second vector. In further embodiments, the VH encoding polynucleotide is operably associated with a first promoter and the VL encoding polynucleotide is operably associated with a second promoter. In certain other embodiments, the first and second promoters are copies of the same promoter. In further embodiments, the first and second promoters are non-identical.
In various embodiments of the above-described compositions, the first vector and the second vector are contained in a single host cell, or in a separate host cells.
In another aspect, the invention features a method of selectively delivering a LIGHT-targeting molecule (e.g., a LIGHT protein, variant or fragment thereof as described herein), to a hyperproliferative cell or tissue, e.g., a neoplastic cell or tissue as described herein, thereby killing, ablating, or otherwise selectively reducing the activity of the hyperproliferative cell or tissue. The method includes contacting the hyperproliferative cell with a LIGHT-targeting molecule, e.g., a molecule as described herein. The contacting step can be performed in the presence of one or more immune cells having a LIGHT receptor, e.g., a LIGHT receptor as described herein. The method can be performed in vitro, e.g., in cultured cells, or ex-vivo, e.g., as part of a therapeutic or prophylactic protocol, in a subject (e.g., a subject having a hyperproliferative disorder or condition as described herein, or an animal model as described herein (e.g., a mouse tumor model carrying breast tumor cells, or a HER2-dependent colorectal and gastric xenograft tumor model)).
In another aspect, the invention features a method of treating or preventing (e.g., curing, suppressing, ameliorating, delaying or preventing the onset of, or preventing recurrence or relapse of) a hyperproliferative, e.g., a cancerous, condition and/or disorder. The method includes administering to a subject, e.g., a subject in need of treatment, a LIGHT-targeting molecule, or anti-HER2 antibody molecule, as described herein.
In certain embodiments, the method prevents, reduces or ameliorates the recurrence or relapse of a tumor or metastasis. The method includes administering a LIGHT-targeting molecule, or anti-HER2 antibody molecule, as described herein, to a subject, e.g., a patient that is partially or completely refractory to a standard mode of therapy (e.g., chemotherapy, antibody-based and/or surgery). For example, the patient suffers from a HER2-expressing cancer (e.g., a breast, gastric or lung cancer) and has demonstrated disease progession after surgery, chemotherapy and/or antibody therapy (e.g., trastuzumab therapy). In other embodiments, the patient is a colon cancer patient that has demonstrated disease progession after surgery, chemotherapy and/or antibody therapy (e.g., VEGF or EGFR antibody therapy). In certain embodiments, the LIGHT-targeting molecule, or anti-HER2 antibody molecule, is administered to a patient who has been treated with another mode of therapy (e.g., a standard mode of therapy) for about 10 days, one to six months, six months to a year, one to two years, and so on. In certain embodiments, the subject has developed partial or complete resistance to a first-line of therapy.
Certain embodiments relating to the in vitro or in vivo methods described herein are as follows:
In further embodiments, the hyperproliferative disorder or condition is chosen from one or more of a cancer, a neoplasm, a tumor, a malignancy, or a metastasis thereof, or a recurrent malignancy (e.g., a subject that is partially or completely refractory to a first-line of treatment).
For in vivo methods, the LIGHT-targeting molecule, or anti-HER2 antibody molecule, alone or in combination with another agent (e.g., a chemotherapeutic agent as described herein), can be administered to a subject, e.g., a mammal, suffering from a hyperproliferative condition and/or disorder, in an amount sufficient to elicit at least one LIGHT-associated biological activity, in the subject.
In some embodiments, the amount or dosage of the LIGHT-targeting molecule, or anti-HER2 antibody molecule, administered can be determined, e.g., prior to administration to the subject, by testing in vitro or ex vivo the amount of the LIGHT-targeting molecule, or anti-HER2 antibody molecule, required to decrease or inhibit one or more of hyperproliferative activities, disorders or conditions described herein. The in vivo method can, optionally, include the step(s) of identifying (e.g., evaluating, diagnosing, screening, and/or selecting) a subject at risk of having, or having, one or more symptoms associated with the disorder or condition.
In various embodiments, the targeting moiety of the LIGHT targeting molecule, or the antibody molecule, specifically binds to a cell surface protein expressed on the surface of the hyperproliferative, e.g., neoplastic, cell or tissue. The targeting moiety can bind to one or more target molecules, e.g., soluble or cell surface proteins expressed on one or more of the hyperproliferative cells or tissues described herein. For example, the targeting moiety can bind to one or more of a growth factor receptor (e.g., HER-2/neu, HER3, HER4, epidermal growth factor receptor (EGFR), insulin growth factor receptor (IGFR), Met, Ron, Cripto); a cancer-related integrin or integrin receptor (e.g., αvβ6, α6β4, laminin receptor (LAMR); and/or other antigens, such as CD23, CD20, CD16, EpCAM, Tweak receptor (FN14), PSMA, and/or VEGF, among others). Additional examples of target molecules recognized by the targeting moieties are described herein.
In further embodiments, the binding of the LIGHT targeting molecule, or the antibody molecule, to the hyperproliferative, e.g., neoplastic, cell or tissue may result in one or more of: (i) binding to one or more LIGHT-receptors (e.g., lymphotoxin β receptor (LTβR), the herpes virus entry mediator (HVEM), and/or decoy receptor 3 (DcR3)); (ii) inducing expression of one or more of chemokines or cytokines (e.g., CXCL10 (IP-10), CCL21, CXCL9, IL-5, IL-8 or TNF), chemokine or cytokine receptors (e.g., IL-10RA) adhesion molecules, and/or co-stimulatory molecules; (iii) activating T cells, e.g., lymphocytes (e.g., cytotoxic T lymphocytes), CD4- or CD8-expressing T cells, and/or regulatory T cells; (iv) recruiting T cells into a hyperproliferative, e.g., tumor, site or cell; (v) activating and/or enhancing tumor-reactive T cell proliferation; (vi) creating a lymphoid-like microenvironment, e.g., at a hyperproliferative, e.g., a tumor cell or tissue; (vi) inducing apoptosis of a hyperproliferative (e.g., tumor) cell or tissue; and/or (vii) stimulating an immune response in a subject, e.g., stimulating a subject's immune system against a hyperproliferative, e.g., a tumor or a cancerous, cell or tissue.
In embodiments where the LIGHT targeting molecule, or the antibody molecule, binds to HER2, the binding of the LIGHT targeting molecule, or the antibody molecule, to the hyperproliferative, e.g., neoplastic, cell or tissue may result in one or more of: (i) binding to HER2 with an affinity of about affinity characterized by a dissociation constant (Kd) at least of 1×10−7 M, 1×10−8 M, 1×10 M, 1×10−10 M, 1×10−11 M, 1×10−12 M, 1×10−13 M; (ii) binding substantially selective to HER2, without significant cross reactivity with other HER-family members (iii) binding to a linear or a conformational epitope on HER2 chosen from D1 epitope (corresponding to about amino acids 1 to 196 of human HER2 shown in FIG. 4), D2 epitope (corresponding to about amino acids 197 to 318 of human HER2 shown in FIG. 4), D3 epitope (corresponding to about amino acids 319 to 508 of human HER2 shown in FIG. 4), or D4 epitope (corresponding to about amino acids 508 to 630 of human HER2 shown in FIG. 4), or a combination thereof; (iii) inhibiting, blocking or reducing HER2 signaling (e.g., inhibit, block or reduce phosphorylation of one or more of HER2, AKT and/or MAP kinase; or inhibit, block or reduce homodimerization of HER2 or heterodimerization of HER2 and HER3, and/or HER2 with EGFR; (iv) inhibiting activity and/or inducing cell killing of a HER2 expressing cell in vitro (e.g., MCF7 and SKBR-3 cell) and in vivo (e.g., in an animal model (such as a mouse tumor model carrying breast tumor cells, or a HER2-dependent colorectal and gastric xenograft tumor model)), or in a human subject; (v) triggering an anti-tumor immune response in vivo, and/or (vi) inducing a prolonged reduction of tumor growth or metastasis, e.g., after prolonged monotherapy or combination therapy, or after tumor relapse is detected following another chemotherapeutic therapy (e.g., standard chemotherapy or anti-HER2 antibody therapy).
In various embodiments of the above-described methods, the LIGHT-targeting molecule, or the antibody molecule inhibits tumor cell migration. In further embodiments, the tumor cell proliferation is inhibited through the prevention or retardation of tumor spread to adjacent tissues.
In various embodiments, the hyperproliferative disease or disorder is a neoplasm located in the: prostate, colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, adrenal gland, parathyroid gland, pituitary gland, testicles, ovary, thymus, thyroid, eye, head, neck, central nervous system, peripheral nervous system, lymphatic system, pelvis, skin, soft tissue, spleen, thoracic region, or urogenital tract.
Exemplary hyperproliferative, e.g., cancerous or neoplastic, cells or tissues, that can be targeted with the targeting moiety, include, but are not limited to, cancers or solid tumors of the breast, lung, stomach, ovaries, prostate, pancreas, colon, colorectum, renal, bladder, liver, head, neck, brain, as well as soft-tissue malignancies, including lymphoid malignacies, leukemia and myeloma. Additional disorders that can be treated include, but are not limited to, epithelial squamous cell cancer, melanoma, brain cancer, cervical cancer, renal cancer, testicular cancer, and thyroid cancer. In embodiments, the cancer is a HER2-expressing tumor or metastatic cancer (e.g., a HER2-expressing cancer of the breast, lung or stomach).
In various embodiments, the subject is a mammal (e.g., an animal model or a human). In further embodiments, the subject is a human, e.g., a patient with one or more of the cancers described herein. In one embodiment, the subject is a patient undergoing a standard mode of therapy, e.g., a HER2-positive patient undergoing chemotherapy and/or treatment with trastuzumab, and the LIGHT-targeting molecules and/or an anti-HER2 antibody molecule are administered as a second-line of therapy. In other embodiments, the patient is a naïve patient, e.g., the LIGHT-targeting molecules and/or an anti-HER2 antibody molecule are administered as a first-line of therapy. In other embodiment, the patient is partially or completely refractory to a standard mode of therapy. For example, the patient is a breast cancer patient that has demonstrated disease progession after chemotherapy and/or trastuzumab therapy.
In other embodiments, the targeting moiety of the LIGHT targeting molecule, or the antibody molecule, is administered, alone or combination with a second agent, as a first-line of therapy to a naïve subject, e.g., a naïve patient having a HER2-expressing breast cancer. In other embodiments, the targeting moiety of the LIGHT targeting molecule, or the antibody molecule, is administered, alone or combination with a second agent, as a second-line of therapy. In other embodiment, the targeting moiety of the LIGHT targeting molecule, or the antibody molecule, is administered to a patient that is partially or completely refractory to a standard mode of therapy. For example, the patient is a breast cancer patient that has demonstrated disease progession after chemotherapy and/or trastuzumab therapy.
In another aspect, the invention features methods for detecting and/or diagnosing, a hyperproliferative disorder or condition using a LIGHT-targeting molecules and/or an anti-HER2 antibody molecule (e.g., a binding agent as described herein). The methods include: detecting the presence of a protein target, e.g., HER2, in a sample (e.g., a purified sample or in vivo). The method comprises (i) contacting the sample with the LIGHT-targeting molecule and/or the anti-HER2 antibody molecule; and (ii) detecting formation of a complex between the LIGHT-targeting molecule and/or the anti-HER2 antibody molecule and the sample, wherein formation of the complex in the sample is indicative of the presence of the protein target in the sample. In certain embodiments, the presence of the protein target is elevated or reduced in relation to a reference value, e.g., a control sample. A change, e.g., increase or decrease, in relation to the reference value is indicative of a hyperproliferative disorder or condition. In embodiments where the protein target is HER2, an elevation in the sample relative to a reference value is indicative of the hyperproliferative disorder or condition.
The LIGHT-targeting molecules and/or the anti-HER2 antibody molecules are also collectively referred to herein as “binding agents” or “binding molecules.”
All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 depicts a schematic representation of LIGHT-Fab design.
FIG. 2 depicts a schematic representation of LIGHT fusion protein design alternatives.
FIG. 3 depicts a schematic representation of antibody-directed tumor targeting.
FIG. 4 depicts the amino acid sequences of human (SEQ ID NO:105) and rhesus (SEQ ID NO:106) ErbB2 extracellular domain with cysteine pairing.
FIG. 5 depicts the domain mapping of human anti-HER2Fabs.
FIG. 6 depicts a summary of 71F10 Fab-hLIGHT constructs used for expression. FIG. 6 discloses SEQ ID NOS 132-134, respectively, in order of appearance. A schematic representation of the 71F10 Fab-hLIGHT fusions with different linker sequences is also shown in FIG. 6.
FIG. 7 depicts the binding of 71F10 Fab-hLIGHT to human HER2-Fc measured by quantitation ELISA. FIG. 7 discloses SEQ ID NOS 147, 134 and 134, respectively, in order of appearance.
FIG. 8 depicts the binding of 71F10 Fab-hLIGHT to murine HER2-Fc measured by quantitation ELISA.
FIG. 9 depicts the demonstration of 71F10 Fab-hLIGHT trimer with expected Molecular Weight. SEC/LS analysis of 71F10 Fab-hLIGHT fusion proteins.
FIG. 10 depicts the binding of purified 71F10 Fab-hLIGHT to human (A) and murine (B) LTβR-Ig.
FIG. 11 depicts the binding of purified 71F10 Fab-hLIGHT to human (A) and murine (B) HVEM-Ig.
FIG. 12 depicts the binding of 71F10 Fab-hLIGHT to SKBR3 cells.
FIG. 13 depicts the block of binding of 71F10-LIGHT to CHO/hHER2 cells by either LTβR-Ig or HER2-Fc
FIG. 14 depicts the detection of functional hLIGHT sites using LTβR-Ig and HVEM-Ig fusions.
FIG. 15 depicts the enhancement of T cell proliferation by 71F10 Fab-hLIGHT.
FIG. 16 depicts the growth inhibition of SKBR-3 cells by recombinant hLIGHT.
FIG. 17 depicts the “U shaped” growth inhibition curve (SKBR3 cells) shown by 71F10 Fab-hLIGHT fusion proteins.
FIG. 18 depicts the LIGHT activity-dependent growth inhibition of SKBR-3 cells by 71F10 Fab-hLIGHT fusion proteins.
FIG. 19 depicts the growth inhibition of BT-474 cells by 71F10-hLIGHT.
FIG. 20 depicts the stimulation of IP-10, IL-8 secretion in HT29 cells by 71F10 Fab-hLIGHT fusion proteins.
FIG. 21 depicts the suppression of HT29 tumor growth by 71F10 Fab-hLIGHT fusion proteins. FIG. 21 shows that LIGHT targeting is required for its maximal activity.
FIG. 22 depicts the potent anti-tumor activity of 71F10 Fab-hLIGHT in N87 xenograft tumor model. FIG. 22 shows 71F10 Fab-hLIGHT can overcome tumor resistance to anti-Her2 therapies.
FIG. 23A depicts the binding of anti-IGFR C06 Fab-hLIGHT fusion protein to IGFR-Ig as measured by ELISA.
FIG. 23B depicts the binding of anti-IGFR C06 Fab-hLIGHT fusion protein to LTβR-Ig as measured by ELISA.
FIG. 24 depicts a schematic representation of dimeric form of 71F10 Fab-hLIGHT fusion protein. Amino acid residues 215-225 and 233-257 of SEQ ID NO:179 (top) and amino acid residues 215-257 of SEQ ID NO:179 (bottom) are depicted.
FIG. 25A depicts the binding of the dimeric form of 71F10 Fab-hLIGHT to HER2-Fc as shown by ELISA.
FIG. 25B depicts the simultaneous binding of the dimeric form of 71F10 Fab-LIGHT to hLTβR and HER2 as shown by ELISA.
Like reference symbols in the various drawings indicate like elements.
BRIEF DESCRIPTION OF TABLES
TABLE 1 depicts a summary of the binding activity of Fabs to HER2 measured by flow cytometry.
TABLE 2 depicts a summary of the binding activity of LIGHT fusion proteins.
TABLE 3 depicts examples of pro-inflammatory genes in HT29 cells that are effected by treatment with BIIB71F10-130 as measured by quantitative reverse transcriptase PCR.
DETAILED DESCRIPTION
The present invention is based, at least in part, on the generation of LIGHT-targeting molecules that are selectively delivered to a hyperproliferative, e.g., cancerous, cell or tissue, thereby eliciting an anti-tumor response, including tumor cell killing and/or anti-tumor immunity. In certain embodiments, the LIGHT-targeting molecules include at least one LIGHT fusion molecule that comprises a LIGHT moiety (e.g., a LIGHT protein, or a functional variant or a fragment thereof), and a targeting moiety (e.g., a binding agent, such as an antibody molecule) that interacts, e.g., binds to, a cancer protein (e.g., a cell surface protein expressed on a cancer cell or tumor), thereby delivering the LIGHT moiety in close proximity to the hyperproliferative cell or tissue. Without being bound by theory and as depicted in FIG. 3, it is believed that the LIGHT targeting molecules of the invention exert one or more of the following activities upon binding to one of its receptors, lymphotoxin β receptor (LTβR) and the herpes virus entry mediator (HVEM), exerting one or more of the following LIGHT-associated activities: i) inducing expression of one or more of chemokines or cytokines (e.g., CXCL10 (IP-10), CCL21, CXCL9, IL-5, IL-8 or TNF), chemokine or cytokine receptors (e.g., IL-10RA) adhesion molecules, and/or co-stimulatory molecules; (ii) activating T cells, e.g., lymphocytes (e.g., cytotoxic T lymphocytes), CD4- or CD8-expressing T cells, and/or regulatory T cells; (iii) recruiting T cells into a hyperproliferative, e.g., tumor, site or cell; (iv) activating and/or enhancing tumor-reactive T cell proliferation; (v) creating a lymphoid-like microenvironment, e.g., at a hyperproliferative, e.g., a tumor cell or tissue; (vi) inducing apoptosis of a hyperproliferative (e.g., tumor) cell or tissue; and/or (vii) stimulating an immune response in a subject, e.g., stimulating a subject's immune system against a hyperproliferative, e.g., a tumor or a cancerous, cell or tissue.
In one exemplary embodiment, the LIGHT targeting molecules comprises at least one fusion molecule of a mammalian (e.g., human) LIGHT protein, or a functional variant or a fragment thereof, and an antibody molecule that binds to HER2 (referred to herein as “LIGHT-anti-HER2 fusion”). Without being bound by theory, the LIGHT-anti-HER2 fusions are believed to trigger dual anti-cancer effects by inducing tumor cell killing mediated by the anti-HER2 antibody molecule, as well as stimulating localized LIGHT-mediated anti-tumor immunity. Thus, the present invention provides, in part, LIGHT-targeting molecules (e.g., LIGHT fusion molecules), antibody molecules against HER2, and methods for treating various hyperproliferative, e.g., neoplastic diseases, including cancer and metastasis using the same.
In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.
As used herein, the articles “a” and “an” refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.
The term “or” is used herein to mean, and is used interchangeably with, the term “and/or”, unless context clearly indicates otherwise.
The terms “proteins” and “polypeptides” are used interchangeably herein.
“About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values.
The term “LIGHT protein” and similar terms (“polypeptides,” “peptides” and “proteins” are used interchangeably herein) refer to a member of the TNF superfamily from any species (typically of mammalian, e.g., murine, or human or non-human primate origin), as well as functional variants thereof (including mutants, fragments and peptidomimetic forms) that retain a LIGHT-associated activity (e.g., which is capable of interacting with, e.g., binding to, a LIGHT receptor (typically of mammalian, e.g., murine or human LIGHT receptor chosen from lymphotoxin β receptor (LTβR) (Crowe et al. (1994) Science 264 707-10, Browning et al. (1997) J Immunol 159: 3288-98); the herpes virus entry mediator (HVEM) (Montgomery et al. (1996) Cell 87(3): 427-36), and/or decoy receptor 3 (DcR3) (Yu et al. (1999) J. Biol. Chem. 274 13733-6). Also encompassed are soluble forms of LIGHT, e.g., soluble forms encompassing the extracellular domain of LIGHT or functional variants thereof. Typically, LIGHT has a biological activity as described herein and one of the following features: (i) an amino acid sequence of a naturally occurring mammalian LIGHT polypeptide or a fragment thereof (e.g., a mature LIGHT), e.g., an amino acid sequence of human LIGHT isoform 1 (SEQ ID NO:1) or human LIGHT isoform 2 (SEQ ID NO:111) or mouse LIGHT (SEQ ID NO:113) or a fragment thereof (e.g., about amino acids 93 to 240 of human LIGHT isoform 1 (corresponding to a portion of the human LIGHT extracellular domain shown as SEQ ID NO:1), about amino acids 253 to 400 of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130) (corresponding to the portion of the LIGHT extracellular domain fused to anti-HER2 antibody molecule shown as SEQ ID NO:2), about amino acids 258 to 405 of 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131) (corresponding to the portion of the LIGHT extracellular domain fused to anti-HER2 antibody molecule shown as SEQ ID NO:3), about amino acids 245 to 392 of 71F10 Fab-hLIGHT fusion heavy chain with the (G4S)4 linker (pBIIB71F10-132) (corresponding to the portion of the LIGHT extracellular domain fused to anti-HER2 antibody molecule shown as SEQ ID NO:4), or (ii) an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto); (iii) an amino acid sequence encoded by the nucleotide sequence from: about nucleotides 277 to 720 of, human LIGHT isoform 1 (nucleotide sequence corresponding to a portion of the human LIGHT extracellular domain shown as SEQ ID NO:5), human LIGHT isoform 2 (SEQ ID NO:112), mouse LIGHT (SEQ ID NO:114), about nucleotides 757 to 1200 of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130) (nucleotide sequence corresponding to the portion of the LIGHT extracellular domain fused to anti-HER2 antibody molecule shown as SEQ ID NO:6), about nucleotides 772 to 1215 of 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131) (nucleotide sequence corresponding to the portion of the LIGHT extracellular domain fused to anti-HER2 antibody molecule shown as SEQ ID NO:7), about nucleotides 733 to 1176 of 71F10 Fab-hLIGHT fusion heavy chain with the (G4S)4 linker (pBIIB71F10-132) (nucleotide sequence corresponding to the portion of the LIGHT extracellular domain fused to anti-HER2 antibody molecule shown as SEQ ID NO:8), or (iv) an amino acid sequence encoded by a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto); or (v) an amino acid sequence encoded by a nucleotide sequence degenerate to a naturally occurring LIGHT nucleotide sequence or a fragment thereof, e.g., an amino acid sequence of human LIGHT isoform 1 (SEQ ID NO:1) or a fragment thereof; or (vi) a nucleotide sequence that hybridizes to one of the foregoing nucleotide sequence sequences under stringent conditions, e.g., highly stringent conditions.
The phrase “a biological activity of” a LIGHT refers to one or more of the biological activities associated with LIGHT, including but not limited to: (i) binding to one or more LIGHT-receptors (e.g., lymphotoxin β receptor (LTβR), the herpes virus entry mediator (HVEM), and/or decoy receptor 3 (DcR3)); (ii) inducing expression of one or more of chemokines or cytokines (e.g., CXCL10 (IP-10), CCL21, CXCL9, IL-5, IL-8 or TNF), chemokine or cytokine receptors (e.g., IL-10RA) adhesion molecules, and/or co-stimulatory molecules; (iii) activating T cells, e.g., lymphocytes (e.g., cytotoxic T lymphocytes), CD4- or CD8-expressing T cells, and/or regulatory T cells; (iv) recruiting T cells into a hyperproliferative, e.g., tumor, site or cell; (v) activating and/or enhancing tumor-reactive T cell proliferation; (vi) creating a lymphoid-like microenvironment, e.g., at a hyperproliferative, e.g., a tumor cell or tissue; (vii) inducing apoptosis of a hyperproliferative (e.g., tumor) cell or tissue; and/or (viii) stimulating an immune response in a subject, e.g., stimulating a subject's immune system against a hyperproliferative, e.g., a tumor or a cancerous, cell or tissue.
The methods and compositions of the present invention encompass LIGHT targeting molecules, antibody molecules and nucleic acids having the sequences specified, or sequences substantially identical or similar thereto, e.g., sequences at least 85%, 90%, 95% identical or higher to the sequence specified. In the context of an amino acid sequence, the term “substantially identical” is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence.
In the context of nucleotide sequence, the term “substantially identical” is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences having at least about 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence
Also included as polypeptides of the present invention are fragments, derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof. The terms “fragment,” “variant,” “derivative” and “analog” when referring to LIGHT proteins, LIGHT targeting molecules, antibody molecules of the present invention include any polypeptides which retain at least some of the functional properties of the corresponding native antibody or polypeptide. Fragments of polypeptides of the present invention include proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein. Variants of the polypeptides of the present invention include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants may occur naturally or be non-naturally occurring. Non-naturally occurring variants may be produced using art-known mutagenesis techniques. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives of the fragments of the present invention are polypeptides which have been altered so as to exhibit additional features not found on the native polypeptide. Examples include fusion proteins. As used herein a “derivative” of a polypeptide refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Also included as “derivatives” are those polypeptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids.
The term “functional variant” refers polypeptides that have a substantially identical amino acid sequence to the naturally-occurring sequence, or are encoded by a substantially identical nucleotide sequence, and are capable of having one or more activities of the naturally-occurring sequence.
Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes can be at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).
The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid (SEQ ID NO:1) molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to a protein (SEQ ID NO:1) protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
As used herein, the term “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions); 2) medium stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified.
It is understood that the LIGHT targeting molecules of the present invention may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on their functions. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
Various aspects of the invention are described in further detail below.
I. LIGHT Targeting Molecules
In one aspect, the invention features a LIGHT targeting molecule that includes at least one LIGHT moiety (e.g., a LIGHT protein, or a functional variant or a fragment thereof as described herein), and at least one targeting moiety (e.g., a binding agent, such as an antibody molecule) that interacts, e.g., binds to, a surface protein on a hyperproliferative cell (e.g., a cell surface protein expressed on a cancer or tumor cell or tissue), thereby delivering the LIGHT moiety to the hyperproliferative cell or tissue. In embodiments, the LIGHT molecule is functionally linked (e.g., by chemical coupling, genetic or polypeptide fusion, non-covalent association or otherwise) to the targeting moiety. For example, the LIGHT molecule can be fused, with or without a linking group, to the targeting moiety as a genetic or a polypeptide fusion. In other embodiments, the LIGHT molecule is covalently attached to the antibody molecule via a reactive group with or without a linking group (e.g., a biocompatible polymer). The LIGHT targeting molecule can be a monomer, dimer, trimer, tetramer, pentamer, or more of at least one LIGHT moiety and at least one targeting moiety. For example, the LIGHT targeting molecule may comprise at least one, two, three, four or five LIGHT fusion molecules, each one comprising at least one LIGHT moiety and at least one targeting moiety. In one embodiment, the LIGHT targeting molecule comprises, or consists essentially of, three LIGHT fusion molecules, each one comprising, or consisting essentially of, one LIGHT moiety (e.g., a LIGHT moiety as described herein) and one targeting moiety (e.g., a targeting moiety as described herein).
As used herein, a “fusion protein” refers to a protein containing two or more operably associated, e.g., linked, moieties, e.g., protein moieties. Typically, the moieties are covalently associated. The moieties can be directly associated, or connected via a spacer or linker (e.g., a linking group as described herein).
In other embodiments, the targeting moiety directs the LIGHT targeting moiety to a desired site, e.g., a hyperproliferative, e.g., cancerous, cell or tissue, such that the LIGHT moiety induces one or more LIGHT-associated activities (e.g., one or more of the LIGHT-associated activities as described herein) against the desired site (e.g., the hyperproliferative, e.g., cancerous, cell or tissue). Exemplary hyperproliferative, e.g., cancerous, cells or tissues, that can be targeted with the targeting moiety, include, but are not limited to, cancers or solid tumors of the breast, lung, stomach, ovaries, prostate, pancreas, colon, colorectum, renal, bladder, liver, head, neck, brain, as well as soft-tissue malignancies, including lymphoid malignacies, leukemia and myeloma. The targeting moiety can bind to one or more cell surface proteins expressed on one or more of the hyperproliferative cells or tissues described herein. For example, the targeting moiety, e.g., an antibody molecule as described herein, can bind to one or more of a growth factor receptor (e.g., HER-2/neu, HER3, HER4, epidermal growth factor receptor (EGFR), insulin growth factor receptor (IGFR), Met, Ron, Cripto); a cancer-related integrin or integrin receptor (e.g., αvβ6, α6β4, laminin receptor (LAMR); and/or CD23, CD20, CD16, EpCAM and/or Tweak receptor (FN14). Each one of the selected targets is described in more detail herein.
Epidermal growth factor receptor: The nucleotide acid sequences of human EGFR genomic DNA and mRNA are disclosed e.g., in Ullrich A et al., Nature 309:418-425 (1984) (isoform 1); Ilekis J. V., et al. (1995) Mol. Reprod. Dev. 41:149-156 (isoform 2); Reiter J. L. and Maihle N. J. Nucleic Acids Res. 24:4050-4056 (1996) (isoform 2); Ilekis J. V. et al., Gynecol. Oncol. 65:36-41 (1997) (isoform 2); and Reiter J. L., et al., Genomics 71:1-20 (2001) (isoforms 3 and 4). The protein sequences of human EGFR and its phosphorylation and ubiquitination sites are disclosed e.g., in Heisermann G. J. and Gill G. N. J. Biol. Chem. 263:13152-13158 (1988); Zhang Z. and Henzel W. J. Protein Sci. 13:2819-2824 (2004); Russo M. W. et al., J. Biol. Chem. 260:5205-5208 (1985); Huang F. et al, Mol. Cell. 21:737-748 (2006); and Abe Y. et al., J. Biol. Chem. 273:11150-11157 (1998). The nucleotide acid and protein sequences of mouse EGFR mRNA are disclosed in e.g., Avivi A. et al., Oncogene 7:1957-1962 (1992); Paria B. C. et al., Proc. Natl. Acad. Sci. U.S.A. 90:55-59 (1993); Luetteke N. C. et al., Genes Dev. 8:399-413 (1994); and Avivi A. et al., Oncogene 6:673-676 (1991). Human EGFR is ubiquitously expressed. Isoform 2 is also expressed in ovarian cancers. Mutations in this gene are associated with lung cancer. EGFR phosphorylates MUC1 in breast cancer cells and increases the interaction of MUC1 with C-SRC and CTNNB1/beta-catenin.
Insulin-like growth factor 1: The nucleotide acid sequences of human IGF1R genomic DNA and mRNA are disclosed e.g., in Ullrich A. et al., EMBO J. 5:2503-2512 (1986); Abbot A. M. et al., J. Biol. Chem. 267:10759-10763 (1992); The MGC Project Team, Genome Res. 14:2121-2127 (2004); Cooke D. W. et al., Biochem. Biophys. Res. Commun. 177:1113-1120 (1991); and Lee S.-T., et al., Oncogene 8:3403-3410 (1993). The protein sequences of human IGF1R are disclosed e.g., Ullrich A. et al., EMBO J. 5:2503-2512 (1986). The nucleotide acid and protein sequences of mouse IGF1R mRNA are disclosed in e.g., Wada J. et al., Proc. Natl. Acad. Sci. U.S.A. 90:10360-10364 (1993); and Wilks A. F. et al., Gene 85:67-74 (1989). Human IGF1R is expressed in various tissues. Defects in IGF1R may be a cause in some cases of resistance to insulin-like growth factor 1 (IGF1 resistance). IGF1 resistance is a growth deficiency disorder characterized by intrauterine growth retardation and poor postnatal growth accompanied with increased plasma IGF1.
HER3: The nucleotide acid and protein sequences of human HER3 are disclosed e.g., in Kraus M. H. et al., Proc. Natl. Acad. Sci. U.S.A. 86:9193-9197 (1989) (isoform 1); Plowman G. D. et al., Proc. Natl. Acad. Sci. U.S.A. 87:4905-4909 (1990) (isoform 1); Katoh M. et al., Biochem. Biophys. Res. Commun. 192:1189-1197 (1993) (isoform 2); and The MGC Project Team, Genome Res. 14:2121-2127 (2004) (isoform 1). The nucleotide acid and protein sequences of mouse HER3 are disclosed e.g., in The MGC Project Team, Genome Res. 14:2121-2127 (2004); and Moscoso L. M. et al., Dev. Biol. 172:158-169 (1995). Human HER3 is expressed in epithelial tissues and brain. It is overexpressed in a subset of human mammary tumors. Defects in HER3 are the cause of lethal congenital contracture syndrome type 2 (LCCS2); also called Israeli Bedouin multiple contracture syndrome type A. LCCS2 is an autosomal recessive neurogenic form of a neonatally lethal arthrogryposis that is associated with atrophy of the anterior horn of the spinal cord.
HER4: The nucleotide acid and protein sequences of human HER4 are disclosed e.g., in Plowman G. D., et al., Proc. Natl. Acad. Sci. U.S.A. 90:1746-1750 (1993) (isoform JM-A); Elenius K. et al., J. Biol. Chem. 272:26761-26768 (1997) (isoforms JM-A and JM-B); and The MGC Project Team, Genome Res. 14:2121-2127 (2004) (isoform JM-A). The nucleotide acid and protein sequences of mouse HER4 are disclosed e.g., in Carninci P. et al., Science 309:1559-1563 (2005); Elenius K. et al., J. Biol. Chem. 272:26761-26768 (1997); Moscoso L. M. et al., Dev. Biol. 172:158-169 (1995). Human HER4 is expressed at highest levels in brain, heart, kidney, in addition to skeletal muscle, parathyroid, cerebellum, pituitary, spleen, testis and breast; and lower levels in thymus, lung, salivary gland, and pancreas. Mutations in this gene have been associated with cancer.
MET: Met proto-oncogene (hepatocyte growth factor receptor) (MET) is also known in the art as HGFR, AUTS9, RCCP2 and c-Met. The nucleotide acid and protein sequences of human MET are disclosed e.g., in Park M. et al., Proc. Natl. Acad. Sci. U.S.A. 84:6379-6383 (1987) (isoform 2); Hillier L. W. et al., Nature 424:157-164 (2003); Chan A. M.-L. et al., Oncogene 1:229-233 (1987). Lee S.-T. et al., Oncogene 8:3403-3410 (1993); and Dean M. et al., Nature 318:385-388 (1985). The nucleotide acid and protein sequences of mouse MET are disclosed e.g., in Chan A. M.-L. et al., Oncogene 2:593-599 (1988); Wilks A. F. Gene 85:67-74 (1989); and Weidner K. M. et al., J. Cell Biol. 121:145-154 (1993). Activation of MET after rearrangement with the TPR gene produces an oncogenic protein. Defects in MET may be associated with gastric cancer. Defects in MET are a cause of hepatocellular carcinoma (HCC) and a cause of hereditary papillary renal carcinoma (HPRC) also known as papillary renal cell carcinoma 2 (RCCP2). HPRC is a form of inherited kidney cancer characterized by a predisposition to develop multiple, bilateral papillary renal tumors. The pattern of inheritance is consistent with autosomal dominant transmission with reduced penetrance. Genetic variations in MET may be associated with susceptibility to autism type 1B (AUTS1B).
RON: The nucleotide acid and protein sequences of human RON are disclosed e.g., in Ronsin C. et al., Oncogene 8:1195-1202 (1993); and Collesi C. et al., Mol. Cell. Biol. 16:5518-5526 (1996). The nucleotide acid and protein sequences of mouse RON are disclosed e.g., in Iwama A. et al., Blood 83:3160-3169 (1994); Waltz S. E. et al., Oncogene 16:27-42 (1998); and Persons D. A. et al., Nat. Genet. 23:159-165 (1999). RON is expressed in keratinocytes and lung. It confers susceptibility to friend virus induced erythroleukemia in mice.
Cripto: The nucleotide acid and protein sequences of human Cripto are disclosed e.g., in Ciccodicola A. et al., EMBO J. 8:1987-1991 (1989); Dono R. et al., Am. J. Hum. Genet. 49:555-565 (1991); Zhang Z. and Henzel W. J. Protein Sci. 13:2819-2824 (2004); Foley S. F. et al., Eur. J. Biochem. 270:3610-3618 (2003). The nucleotide acid and protein sequences of mouse Cripto are disclosed e.g., in. Dono R. et al., Development 118:1157-1168 (1993); and Liguori G. et al., Mamm. Genome 7:344-348 (1996). Cripto is preferentially expressed in gastric and colorectal carcinomas than in their normal counterparts. In mice, it is expressed at low level in specific organs of the adult animal such as spleen, heart, lung and brain. Examples of antibody molecules that bind to Cripto are described in e.g., U.S. Patent Appl. Publ. No.: 200810166341A1.
VEGFR: The nucleotide acid and protein sequences of human VEGFR are disclosed e.g., in Shibuya M. et al., Oncogene 5:519-524 (1990) (isoform Flt1); Kendall R. L. and Thomas K. A. Proc. Natl. Acad. Sci. U.S.A. 90:10705-10709 (1993) (isoform SFLT1); The MGC Project Team, Genome Res. 14:2121-2127 (2004) (isoform sFlt1); Matsushime H. et al., Jpn. J. Cancer Res. 78:655-661 (1987) (isoform Flt1); and Ito N. et al., J. Biol. Chem. 273:23410-23418 (1998). The nucleotide acid and protein sequences of mouse VEGFR are disclosed e.g., in Finnerty H. et al., Oncogene 8:2293-2298 (1993); Choi K. et al., Oncogene 9:1261-1266 (1994); and Kondo K. et al., Gene 208:297-305 (1998). VEGFR is mostly expressed in normal lung, but also in placenta, liver, kidney, heart and brain tissues. It is specifically expressed in most of the vascular endothelial cells, and also expressed in peripheral blood monocytes. It is not expressed in tumor cell lines. Isoform sFlt1 is strongly expressed in placenta.
Integrin αvβ6: Integrins are cell surface receptors that interact with the extracellular matrix (ECM) and mediate various intracellular signals. There are many types of integrin, and many cells have multiple types on their surface. Integrins are obligate heterodimers containing two distinct chains, called the α (alpha) and β (beta) subunits. In mammals, 19 α and 8 β subunits have been characterized. The nucleotide and protein sequences of human integrin αv are disclosed e.g., in Suzuki S. et al., J. Biol. Chem. 262:14080-14085 (1987); Sims M. A., Cytogenet. Cell Genet. 89:268-271 (2000); Hillier L. W. et al., Nature 434:724-731 (2005); The MGC Project Team, Genome Res. 14:2121-2127 (2004); Donahue J. P. et al., Biochim. Biophys. Acta 1219:228-232 (1994); Suzuki S. et al., Proc. Natl. Acad. Sci. U.S.A. 83:8614-8618 (1986); Cheresh D. A. et al., Cell 57:59-69 (1989). The nucleotide and protein sequences of mouse integrin av are disclosed e.g., in Almeida J. Submitted (APR-2008) to the EMBL/GenBank/DDBJ databases. The nucleotide and protein sequences of human integrin β6 are disclosed e.g., in Sheppard D. et al., J. Biol. Chem. 265:11502-11507 (1990); Hillier L. W., Nature 434:724-731 (2005); The MGC Project Team, Genome Res. 14:2121-2127 (2004); Jiang W.-M. et al., Int. Immunol. 4:1031-1040 (1992). The nucleotide and protein sequences of mouse integrin β6 are disclosed e.g., in Arend L. J. et al., J. Am. Soc. Nephrol. 11:2297-2305 (2000); Carninci P. et al., Science 309:1559-1563 (2005); The MGC Project Team, Genome Res. 14:2121-2127 (2004).
Integrin αvβ6 is mostly distributed in proliferating epithelia, e.g. lung and liver. Functions of integrin αvβ6 are disclosed in e.g., Jovanović J. et al., Biochem Soc Trans. 36(Pt 2):257-262 (2008); Wipff P. J. and Hinz B. Eur J Cell Biol. 87:601-615 (2008); Bates R. C. Future Oncol. 1:821-8 (2005); Thomas G. J. et al., J Oral Pathol Med. 35:1-10 (2006); Sheppard D. Cancer Metastasis Rev. 24:395-402 (2005); Bates R. C. and Mercurio A. M. Cancer Biol Ther. 4:365-370 (2005); Keski-Oja J. et al., Trends Cell Biol. 14:657-659 (2004); Sheppard D. Curr Opin Cell Biol. 16:552-557 (2004); Wada J. et al., Nephrol Dial Transplant. 17:75-77 (2002); Thomas G. J. and Speight P. M. Crit. Rev Oral Biol Med. 12:479-498 (2001); and Imhof B. A. et al., Curr Top Microbiol Immunol. 213:195-203 (1996).
Integrin α6β4: The nucleotide and protein sequences of human integrin α6 are disclosed e.g., in Hogervorst F. et al., Eur. J. Biochem. 199:425-433 (1991); Starr L. et al., BioTechniques 13:612-618 (1992); Tamura R. N. et al., Proc. Natl. Acad. Sci. U.S.A. 88:10183-10187 (1991); Ziober B. L. et al., J. Biol. Chem. 268:26773-26783 (1993); Shaw L. M. et al., J. Biol. Chem. 268:11401-11408 (1993); and Delwel G. O. et al., Cell Adhes. Commun. 3:143-161 (1995). The nucleotide and protein sequences of mouse integrin α6 are disclosed e.g., in Carninci P. et al., Science 309:1559-1563 (2005). The nucleotide and protein sequences of human integrin β4 are disclosed e.g., in Suzuki S, and Naitoh Y. EMBO J. 9:757-763 (1990); Hogervorst F. et al., EMBO J. 9:765-770 (1990); and Tamura R. N. et al., J. Cell Biol. 111:1593-1604 (1990). The nucleotide and protein sequences of mouse integrin β4 are disclosed e.g., in Brown J. Submitted (APR-2008) to the EMBL/GenBank/DDBJ database.
Integrin α6β4 is mostly expressed in epithelial tissues and endothelial and Schwann cells. Expression of α6β4 is increased in many epithelial tumors and it activates several key signaling molecules in carcinoma cells, including activating the phosphatidylinositol 3-kinase/Akt pathway (Bon G. et al., Breast Cancer Res. 9:203 (2007); Wilhelmsen K, A. et al., Mol Cell Biol. 26:2877-86 (2006)).
LAMR: Ribosomal protein SA (LAMR) It is also known in the art as RPSA, LRP, p40, 67LR, 37LRP, LAMBR and LAMR1. Laminins, a family of extracellular matrix glycoproteins, are the major noncollagenous constituent of basement membranes. The nucleotide and protein sequences of human LAMR are disclosed e.g., in Yow H. et al., Proc. Natl. Acad. Sci. U.S.A. 85:6394-6398 (1988); van den Ouweland A. M. W. et al., Nucleic Acids Res. 17:3829-3843 (1989); Satoh K. et al., Cancer Lett. 62:199-203 (1992); Jackers P. et al., Oncogene 13:495-503 (1996); The MGC Project Team, Genome Res. 14:2121-2127 (2004); Siyanova E. Y. et al., Dokl. Biochem. 313:227-231 (1990); Vladimirov S, N. et al., Eur. J. Biochem. 239:144-149 (1996); Selvamurugan N. and Eliceiri G. L. et al., Genomics 30:400-401 (1995); and Wewer U. M. et al., Proc. Natl. Acad. Sci. U.S.A. 83:7137-7141 (1986). The nucleotide and protein sequences of mouse LAMR are disclosed e.g., Rao C. N. et al., Biochemistry 28:7476-7486 (1989); Makrides S. et al., Nucleic Acids Res. 16:2349-2349 (1988); Coggin J. H. Jr. et al., Anticancer Res. 19:5535-5542 (1999); Carninci P. et al., Science 309:1559-1563 (2005); and The MGC Project Team, Genome Res. 14:2121-2127 (2004). It has been observed that the level of the laminin receptor transcript is higher in colon carcinoma tissue and lung cancer cell line than their normal counterparts. Also, there is a correlation between the upregulation of this polypeptide in cancer cells and their invasive and metastatic phenotype.
CD23: Fc fragment of IgE, low affinity II, receptor for (CD23) It is also known in the art as FCER2, FCE2, CD23A, IGEBF and CLEC4J. The human leukocyte differentiation antigen CD23 (FCE2) is a key molecule for B-cell activation and growth. It is the low-affinity receptor for IgE. The truncated molecule can be secreted, then functioning as a potent mitogenic growth factor. The nucleotide acid and protein sequences of human CD23 are disclosed e.g., in Ikuta K. et al., Proc. Natl. Acad. Sci. U.S.A. 84:819-823 (1987); Kikutani H. et al., Cell 47:657-665 (1986); Luedin C. et al., EMBO J. 6:109-114 (1987); The MGC Project Team, Genome Res. 14:2121-2127 (2004); Rose K. et al., Biochem. J. 286:819-824 (1992); and Yokota A. et al., Cell 55:611-618 (1988). The nucleotide acid and protein sequences of mouse CD23 are disclosed e.g., in Bettler B. et al., Proc. Natl. Acad. Sci. U.S.A. 86:7566-7570 (1989); Gollnick S. O. et al., J. Immunol. 144:1974-1982 (1990); and Kondo H. et al., Int. Arch. Allergy Immunol. 105:38-48 (1994). Anti-CD23 antibodies that can be used as targeting moieties are described, e.g., in U.S. Pat. Nos. 7,332,163 and 7,223,392.
CD20: Membrane-spanning 4-domains, subfamily A, member 1 (CD20) is also known in the art as MS4A1, B1, S7, Bp35, MS4A2, LEU-16 and MGC3969. CD20 encodes a member of the membrane-spanning 4A gene family. The nucleotide acid and protein sequences of human CD20 are disclosed e.g., in Stamenkovic I. and Seed B. J. Exp. Med. 167:1975-1980 (1988); Tedder T. F. et al., Proc. Natl. Acad. Sci. U.S.A. 85:208-212 (1988); Einfeld D. A. et al., EMBO J. 7:711-717 (1988); Tedder T. F. et al., J. Immunol. 142:2560-2568 (1989); Taylor T. D. et al., Nature 440:497-500 (2006); and The MGC Project Team, Genome Res. 14:2121-2127 (2004). The nucleotide acid and protein sequences of mouse CD20 are disclosed e.g., in Tedder T. F. et al., J. Immunol. 141:4388-4394 (1988); Carninci P. et al., Science 309:1559-1563 (2005); and The MGC Project Team, Genome Res. 14:2121-2127 (2004). CD20 is expressed on B-cells. This gene encodes a B-lymphocyte surface molecule which plays a role in the development and differentiation of B-cells into plasma cells.
CD16: Fc fragment of IgG, low affinity IIIa or IIIb, receptor (CD16) It is also know in the art as FCGR3A, FCGR3B, FCG3, CD16A, FCGR3, IGFR3, FCR-10, FCRIII, FCGRIII and FCRIIIA The nucleotide acid and protein sequences of human CD16a and CD16b are disclosed e.g., in The MGC Project Team, Genome Res. 14:2121-2127 (2004); and Scallon B. J. et al., Proc. Natl. Acad. Sci. U.S.A. 86:5079-5083 (1989). The nucleotide acid and protein sequences of human CD16b are disclosed e.g., in Ravetch J. V. and Perussia B. J. Exp. Med. 170:481-497 (1989); Simmons D. and Seed B. Nature 333:568-570 (1988); Simmons D. and Seed B. Nature 340:662-662 (1989); Peltz G. A. et al., Proc. Natl. Acad. Sci. U.S.A. 86:1013-1017 (1989); Scallon B. J. et al., Proc. Natl. Acad. Sci. U.S.A. 86:5079-5083 (1989); Bertrand G. et al., Tissue Antigens 64:119-131 (2004); and Gessner J. E. et al., J. Biol. Chem. 270:1350-1361 (1995). The nucleotide acid and protein sequences of mouse CD16 are disclosed e.g., Bonnerot C. et al., Mol. Immunol. 29: 353-361 (1992); and Kulczycki A. Jr. et al., Proc. Natl. Acad. Sci. U.S.A. 87: 2856-2860 (1990). The receptor encoded by FCGR3A is expressed on natural killer (NK) cells as an integral membrane glycoprotein anchored through a transmembrane peptide, whereas FCGR3B is expressed on polymorphonuclear neutrophils (PMN) where the receptor is anchored through a phosphatidylinositol (PI) linkage. Mutations in this gene have been linked to susceptibility to recurrent viral infections, susceptibility to systemic lupus erythematosus, and alloimmune neonatal neutropenia. The more active FCGR3B*01 allele has been associated with severe renal disease in certain systemic vasculitides.
EpCAM: Tumor-associated calcium signal transducer 1 (EpCAM) It is also known in the art as TACSTD1, EGP, KSA, M4S1, MK-1, CD326, EGP40, MIC18, TROP1, Ep-CAM, hEGP-2, C017-1A and GA733-2. This 9-exon gene encodes a carcinoma-associated antigen and is a member of a family that includes at least two type I membrane proteins. The nucleotide acid and protein sequences of human EpCAM are disclosed e.g., in Stranad J. et al., Cancer Res. 49:314-317 (1989); Simon B. et al., Proc. Natl. Acad. Sci. U.S.A. 87:2755-2759 (1990); Perez M. S, and Walker L. E. J. Immunol. 142:3662-3667 (1989); Szala S. et al., Proc. Natl. Acad. Sci. U.S.A. 87:3542-3546 (1990); The MGC Project Team, Genome Res. 14:2121-2127 (2004); Linnenbach A. J. et al., Proc. Natl. Acad. Sci. U.S.A. 86:27-31 (1989); and Chong J. M. and Speicher D. W. J. Biol. Chem. 276:5804-5813 (2001). The nucleotide acid and protein sequences of mouse EpCAM are disclosed e.g., in The MGC Project Team, Genome Res. 14:2121-2127 (2004); and Carninci P. et al., Science 309:1559-1563 (2005). This antigen is expressed on most normal epithelial cells and gastrointestinal carcinomas and functions as a homotypic calcium-independent cell adhesion molecule.
FN14: Tumor necrosis factor receptor superfamily, member 12A (FN14) It is also known in the art as TNFRSF12A, CD266 and TWEAKR. It is a receptor for TNFSF12/TWEAK. The nucleotide and protein sequences of human FN14 are disclosed e.g., in Feng S.-L. Y. et al., Am. J. Pathol. 156:1253-1261 (2000); and The MGC Project Team, Genome Res. 14:2121-2127 (2004). The nucleotide and protein sequences of mouse FN14 are disclosed e.g., in Meighan-Mantha R. L. et al., J. Biol. Chem. 274:33166-33176 (1999); Carninci P., Science 309:1559-1563 (2005); and The MGC Project Team, Genome Res. 14:2121-2127 (2004). The unprocessed precursor of human FN14 is about 129 amino acids in length and about 13911 Da in molecular weight. The unprocessed precursor of mouse FN14 is about 129 amino acids in length and about 13641 Da in molecular weight. Human FN14 is highly expressed in heart, placenta and kidney; and moderately expression in lung, skeletal muscle and pancreas. Mouse FN14 is highly expressed in fetal heart, intestine, kidney, liver, lung and skin, and in adult heart and ovary; and moderately expression in adult kidney, lung and skin.
The targeting moiety, e.g., an antibody molecule as described herein, can also bind to one or more of the following: a tyrosine-protein kinase receptor (e.g., TYRO3 (tyrosine-protein kinase receptor TYRO3, also known as tyrosine-protein kinase RSE, SKY, DTK, or byk, Mark M. R. et al., J. Biol. Chem. 269:10720-10728 (1994)); AXL (also know as tyrosine-protein kinase receptor UFO; O′Bryan J. P. et al., Mol. Cell. Biol. 11:5016-5031 (1991)); DDR1 (epithelial discoidin domain-containing receptor 1, also known as tyrosine kinase DDR, discoidin receptor tyrosine kinase, tyrosine-protein kinase CAK, cell adhesion kinase, TRK E, protein-tyrosine kinase RTK 6, HGK2, CD167 antigen-like family member A, mammary carcinoma kinase 10 (MCK-10), or CD167a; Perez J. L. et al., Oncogene 9:211-219 (1994)); DDR2 (discoidin domain-containing receptor 2, also known as receptor protein-tyrosine kinase TKT, tyrosine-protein kinase TYRO10, neurotrophic tyrosine kinase, receptor-related 3, CD167 antigen-like family member B, or CD167b; Ichikawa O. et al., EMBO J. 26:4168-4176 (2007)); ALK (ALK tyrosine kinase receptor, also known as anaplastic lymphoma kinase or CD246; Simonitsch I. et al., FASEB J. 15:1416-1418 (2001)); CSF1R (macrophage colony-stimulating factor 1 receptor, also known as Fms proto-oncogene, c-fms, or CD115; Hampe A. et al., Oncogene Res. 4:9-17 (1989))); a growth factor receptor (e.g., FGFR1 (basic fibroblast growth factor receptor 1, also known as bFGF-R, Fms-like tyrosine kinase 2, c-fgr, or CD331; Dionne C. A. et al., EMBO J. 9:2685-2692 (1990)); FGFR2 (fibroblast growth factor receptor 2, also known as keratinocyte growth factor receptor 2 or CD332; Hattori Y. et al., Proc. Natl. Acad. Sci. U.S.A. 87:5983-5987 (1990))); a growth factor (e.g., PDGF1 (also known as platelet-derived growth factor subunit A, platelet-derived growth factor A chain, or platelet-derived growth factor alpha polypeptide; Bonthron D. T. et. al., Proc. Natl. Acad. Sci. U.S.A. 85:1492-1496 (1988)); PDGF2 (also known as platelet-derived growth factor subunit B, platelet-derived growth factor B chain, platelet-derived growth factor beta polypeptide, or c-sis; Josephs S. F. et al., Science 225:636-639 (1984))); an apoptosis protein (e.g., a Netrin, e.g., Netrin-1 (Meyerhardt J. A. et al., Cell Growth Differ. 10:35-42 (1999), or Netrin-4 (also known as Beta-netrin or Hepar-derived netrin-like protein; Koch M. et al., J. Cell Biol. 151:221-234 (2000))); a tyrosine kinase (e.g., MER (Proto-oncogene tyrosine-protein kinase MER, also known as C-mer or Receptor tyrosine kinase MerTK; Graham D. K. et al., Cell Growth Differ. 5:647-657 (1994))); a hormone receptor (e.g., PRL-R (Prolactin receptor; Boutin J.-M. et al., Mol. Endocrinol. 3:1455-1461 (1989)); GH receptor (growth hormone receptor, also known as somatotropin receptor, GH-binding protein, GHBP, or Serum-binding protein; Leung D. W. et al., Nature 330:537-543 (1987))); a signal transduction protein (e.g., ephrin, e.g., ephrin A (e.g., ephrin A1, ephrin A2, ephrin A3, ephrin A4, ephrin A5; Holzman L. B. et al., Mol. Cell. Biol. 10 (11): 5830-5838 (1990)) and ephrin B (e.g., ephrin B1, ephrin B2, ephrin B3; Fletcher F. A. et al. Genomics 25 (1): 334-335 (1995)); PD-L1 (programmed cell death ligand 1, also known as B7-H1 or CD274; Dong H. et al., Nat. Med. 5:1365-1369 (1999)); neuropilin (e.g., NRP1 (Neuropilin-1, also known as Vascular endothelial cell growth factor 165 receptor or CD304; He Z. and Tessier-Lavigne M. Cell 90:739-751 (1997)); NRP2 (Neuropilin-2, also known as vascular endothelial cell growth factor 165 receptor 2; Chen H. et al., Neuron 19:547-559 (1997)); or Semaphorin (SEMA, e.g., SEMA3, SEMA4, SEMAS, SEMA6, or SEMA7; Flannery E. and Duman-Scheel M. Curr Drug Targets. 10:611-619 (2009))); a cell adhesion molecule (e.g., Mesothelin (MSLN, also known as Pre-pro-megakaryocyte-potentiating factor, CAK1 antigen, or Megakaryocyte-potentiating factor (MPF); Chang K. and Pastan I. Proc. Natl. Acad. Sci. U.S.A. 93:136-140 (1996)); Nectin, (e.g, Nectin 1, Nectin 2, Nectin 3, Nectin 4, Nectin-like protein 1, Nectin-like protein 2, Nectin-like protein 3, or Nectin-like protein 4; Takai Y. et al., Nat. Rev. Mol. Cell Biol. 9:603-615 (2008)); CEA (Carcinoembryonic antigen-related cell adhesion molecule, e.g., CEAS; Schrewe H. et al., Mol. Cell. Biol. 10:2738-2748 (1990); CEACAM6 (Carcinoembryonic antigen-related cell adhesion molecule 6, also known as Normal cross-reacting antigen, Non-specific crossreacting antigen, or CD66c; Barnett T. et al., Genomics 3:59-66 (1988))); a chemokine receptor (e.g., CCR4 (C-C chemokine receptor type 4, also known as C-C CKR-4, CC-CKR-4, K5-5, or CD194; Power C. A. et al., J. Biol. Chem. 270:19495-19500 (1995)); CXCR7 (C-X-C chemokine receptor type 7, also known as CXC-R7, G-protein coupled receptor RDC1 homolog, RDC-1, Chemokine orphan receptor 1, or G-protein coupled receptor 159; Sreedharan S. P. et al., Proc. Natl. Acad. Sci. U.S.A. 88:4986-4990 (1991))); a G-protein coupled receptor (e.g., GPR49 (Leucine-rich repeat-containing G-protein coupled receptor 5, also known as Orphan G-protein coupled receptor HG38, G-protein coupled receptor 49, or G-protein coupled receptor 67; McDonald T. et al., Biochem. Biophys. Res. Commun. 247:266-270 (1998)); SIT receptor (Sphingosine 1-phosphate receptor, also known as Endothelial differentiation G-protein coupled receptor; e.g., S1P1, S1P2, S1P3, S1P4, S1P5; Hla T. and Maciag T. J. Biol. Chem. 265:9308-9313 (1990))); an angiogenesis factor receptor (e.g., TIE2 (Angiopoietin-1 receptor, TEK, Tunica interna endothelial cell kinase, p140 TEK, or CD202b; Ziegler S. F. et al., Oncogene 8:663-670 (1993))); a membrane-bound mucin (e.g., MUC1 (also known as PEM, PEMT, Episialin, EMA, H23AG, PUM, or CD227; Lan M. S. et al., J. Biol. Chem. 265:15294-15299 (1990)), MUC2 (also known as Intestinal mucin-2; Gum J. R. Jr. et. al., J. Biol. Chem. 269:2440-2446 (1994)), MUC3 (also known as Intestinal mucin-3; Hillier L. W. et al., Nature 424:157-164 (2003)), MUC4 (also known as Pancreatic adenocarcinoma mucin, Testis mucin, ASGP, or Tracheobronchial mucin; Moniaux N. et al., Eur. J. Biochem. 267:4536-4544 (2000)), MUC5AC (also known as TBM, Major airway glycoprotein, Gastric mucin, or LeB; Escande F. et al., Biochem. J. 358:763-772 (2001)), and MUC 16 (also known as CA-125; O'Brien T. J. et al., Tumor Biol. 23:154-169 (2002))); a tumor marker (e.g., Endosialin (also known as Tumor endothelial marker 1 or CD248; a C-type lectin-like protein; St Croix B. et al., Science 289:1197-1202 (2000)); PSMA (Prostate specific membrane antigen, also known as PSA, Kallikrein-3, Semenogelase, Seminin, or P-30 antigen; Lundwall A. and Lilja H. FEBS Lett. 214:317-322 (1987)); TAG-72 (Tumor associated glycoprotein 72; a protein/sugar complex found on the surface of many cancer cells, including breast, colon, and pancreatic cells; Alles A. J. et al., Ann Surg. 219(2): 131-134 (1994)); KIM-1 (Kidney injury molecule-1, also known as T-cell immunoglobulin and mucin-containing molecule (Tim-1) or Hepatitis A virus cellular receptor 1 (HAVCR1); Feigelstock D. et al., J. Virol. 72:6621-6628 (1998))); a cell surface marker on melanoma (e.g., MART-1 (Melanoma antigen recognized by T-cells 1, also known Melan-A protein, Antigen SK29-AA, or Antigen LB39-AA; Kawakami Y. et al., Proc. Natl. Acad. Sci. U.S.A. 91:3515-3519 (1994)); gp100 (Melanocyte lineage-specific antigen GP100, also known as Melanocyte protein Pmel 17, Silver locus protein homolog, ME20-M, ME20-S, or 95 kDa melanocyte-specific secreted glycoprotein; Adema G. J. et al., J. Biol. Chem. 269:20126-20133 (1994)); TRP-1 (Tyrosinase-related protein 1, also known as DHICA oxidase, Catalase B, Glycoprotein 75, or Melanoma antigen gp75; Cohen T. et al., Nucleic Acids Res. 18:2807-2807 (1990)); TRP-2 (Tyrosinase-related protein 2, also known as DCT, DT, or L-dopachrome Delta-isomerase; Yokoyama K. et al., Biochim. Biophys. Acta 1217:317-321 (1994))); or a heat shock protein (e.g., GRP78 (78 kDa glucose-regulated protein, also known as Heat shock 70 kDa protein 5, BiP, or Endoplasmic reticulum lumenal Ca(2+)-binding protein grp78; Corrigall V. M. et al., J. Immunol. 166:1492-1498 (2001))).
The fusion proteins may additionally include a linker sequence joining the first moiety, e.g., the LIGHT moiety, to the second moiety, e.g., the targeting moiety. The linking group can be any linking group apparent to those of skill in the art. For example, the fusion protein can include a peptide linker, e.g., a peptide linker of about 5 to 50, more preferably, 10 to 35, or 15 to 33 amino acids in length; the peptide linker is about 20, 28 or 33 amino acids in length. Each of the amino acids in the peptide linker is selected from the group consisting of Gly, Ser, Asn, Thr and Ala; the peptide linker includes a Gly-Ser element. In other embodiments, the fusion protein includes a peptide linker and the peptide linker includes a sequence having the formula (Gly-Gly-Gly-Gly-Ser)y wherein y is 1, 2, 3, 4, 5, 6, 7, or 8 (SEQ ID NO:149). In one embodiment, the linking group includes or consists of polyglycine, polyserine, polylysine, polyglutamate, polyisoleucine, or polyarginine residues, or a combination thereof. For example, the polyglycine or polyserine linkers can include at least five, ten, fifteen or twenty glycine and serine residues in the following configuration, (Gly)4-Ser (SEQ ID NO: 145), in one, two, three, four, five or more repeats, e.g., four repeats of (Gly)4-Ser (SEQ ID NO: 134). In other embodiments, linking group may include one or more amino acid residues (e.g., at least 10 to 35, 15 to 30, or about 20 to 26 amino acid residues) from the extracellular domain of LIGHT or a mutated form thereof, e.g., from about amino acids 61 to 92 of human LIGHT isoform 1 (SEQ ID NO:1), about amino acids 225 to 252 of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130) (SEQ ID NO:2), about amino acids 230 to 257 of 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131) (SEQ ID NO:3), or an amino acid sequence substantially identical thereto; or an amino acid sequence encoded by the nucleotide sequence from about nucleotides 181 to 276 of human LIGHT isoform 1 (SEQ ID NO:5), about nucleotides 673 to 756 of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130) (SEQ ID NO:6), about nucleotides 688 to 771 of 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131) (SEQ ID NO:7), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). Alternatively, the linking group may include a combination of one or more (Gly)4-Ser (SEQ ID NO: 146) repeats and one or more amino acid residues (e.g., at least 10 to 35, 15 to 30, or about 20 to 26 amino acid residues) from the extracellular domain of LIGHT or a mutated form thereof, e.g., from about amino acids 61 to 92 of human LIGHT isoform 1 (SEQ ID NO:1), about amino acids 225 to 252 of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130) (SEQ ID NO:2), about amino acids 230 to 257 of 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131) (SEQ ID NO:3), or an amino acid sequence substantially identical thereto; or an amino acid sequence encoded by the nucleotide sequence from about nucleotides 181 to 276 of human LIGHT isoform 1 (SEQ ID NO:5), about nucleotides 673 to 756 of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130) (SEQ ID NO:6), about nucleotides 688 to 771 of 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131) (SEQ ID NO:7), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto).
The amino acid and nucleotide sequences of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130) are shown as SEQ ID NOs:2 and 6, respectively. The amino acid and nucleotide sequences corresponding to heavy chain of 71F10 Fab are shown starting from the N-terminus; followed by amino acids corresponding to the linking group (about amino acids 225 to 252); followed by the amino acids corresponding to human LIGHT extracellular domain (amino acids 253 to 400). The physical and chemical parameters of 71F10 Fab-Δ4huLIGHT are as follows: Extinction coefficient=1.62; pI=8.7; MW=66 kDa (x3=198 kDa); AA number=614).
The amino acid and nucleotide sequences of 71F10 Fab-hLIGHT fusion heavy chain with the G4S (SEQ ID NO:147) delta 4 linker (pBIIB71F10-131) are shown as SEQ ID NOs:3 and 7, respectively. The amino acid and nucleotide sequences corresponding to heavy chain of 71F10 Fab are shown starting from the N-terminus; followed by amino acids corresponding to the linking group (amino acids 225 to 257); followed by the amino acids corresponding to human LIGHT extracellular domain (amino acids 258 to 405). The physical and chemical parameters of 71F10 Fab-G4S-Δ4huLIGHT are as follows: Extinction coefficient=1.61; pI=8.7; MW=66 kDa (x3=198 kDa); AA number=619).
The amino acid and nucleotide sequences of 71F10 Fab-hLIGHT fusion heavy chain with the (G4S)4 (SEQ ID NO:134) linker (pBIIB71F10-132) are shown as SEQ ID NOs:4 and 8, respectively. The amino acid and nucleotide sequences corresponding to heavy chain of 71F10 Fab are shown starting from the N-terminus; followed by amino acids corresponding to the linking group (amino acids 225 to 244); followed by the amino acids corresponding to human LIGHT extracellular domain (amino acids 245 to 392). The physical and chemical parameters of 71F10 Fab-(G4S)4-Δ4huLIGHT are as follows: Extinction coefficient=1.5; pI=8.7; MW=64 kDa (x3=198 kDa); AA number=606).
In other embodiments, additional amino acid sequences can be added to the N- or C-terminus of the fusion protein to facilitate expression, detection and/or isolation or purification. For example, fusion protein may be linked to one or more additional moieties, e.g., GST, His6 tag (SEQ ID NO:150), FLAG tag. For example, the fusion protein may additionally be linked to a GST fusion protein in which the fusion protein sequences are fused to the C-terminus of the GST (i.e., glutathione S-transferase) sequences. Such fusion proteins can facilitate the purification of the fusion proteins.
In another embodiment, the fusion protein includes a heterologous signal sequence (i.e., a polypeptide sequence that is not present in a polypeptide encoded by a LIGHT nucleic acid) at its N-terminus. For example, the native LIGHT signal sequence can be removed and replaced with a signal sequence from another protein. In certain host cells (e.g., mammalian host cells), expression and/or secretion of fusion protein can be increased through use of a heterologous signal sequence. A fusion protein of the invention can be produced by standard recombinant DNA techniques (see, for example, Ausubel et al. (eds.) Current Protocols in Molecular Biology, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that encode a fusion moiety (e.g., an Fc region of an immunoglobulin heavy chain). A nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the immunoglobulin protein.
In some embodiments, fusion polypeptides exist as oligomers, such as dimers or trimers of a single contiguous polypeptides, or two or more non-contiguous polypeptides.
In other embodiments, the LIGHT or the targeting moiety is provided as a variant polypeptide having a mutation in the naturally-occurring sequence (wild type) that results in one or more higher affinity binding, increased stability, e.g., more resistant to proteolysis (relative to the non-mutated sequence), among others.
In other embodiments, additional amino acid sequences can be added to the N- or C-terminus of the fusion protein to facilitate expression, steric flexibility, detection and/or isolation or purification. The second polypeptide is preferably soluble. In some embodiments, the second polypeptide enhances the half-life, (e.g., the serum half-life) of the linked polypeptide. In some embodiments, the second polypeptide includes a sequence that facilitates association of the fusion polypeptide with a second polypeptide. In embodiments, the second polypeptide includes at least a region of an immunoglobulin polypeptide. Immunoglobulin fusion polypeptides are known in the art and are described in, e.g., U.S. Pat. Nos. 5,516,964; 5,225,538; 5,428,130; 5,514,582; 5,714,147; and 5,455,165.
It will be understood that the antibody molecules and soluble LIGHT or fusion proteins described herein can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities, such as an antibody (e.g., a bispecific or a multispecific antibody), toxins, radioisotopes, cytotoxic or cytostatic agents, among others.
Exemplary LIGHT targeting molecules include a LIGHT/HER2 fusion, e.g., a LIGHT/HER2 fusion as described herein. LIGHT/HER2 fusions include, or consist essentially of, the amino acid sequence shown in any of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130) (SEQ ID NO: 2), 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131) (SEQ ID NO:3), 71F10 Fab-hLIGHT fusion heavy chain with the (G4S)4 linker (pBIIB71F10-132) (SEQ ID NO:4), or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto); an amino acid sequence encoded by the nucleotide sequence shown in any of 71F10 Fab-hLIGHT fusion heavy chain with the delta 4 linker (pBIIB71F10-130) (SEQ ID NO:6), 71F10 Fab-hLIGHT fusion heavy chain with the G4S delta 4 linker (pBIIB71F10-131) (SEQ ID NO:7), 71F10 Fab-hLIGHT fusion heavy chain with the (G4S)4 linker (pBIIB71F10-132) (SEQ ID NO:8), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). In certain embodiments, the LIGHT/HER2 fusions may also include, or consist essentially of, a second chain (fused or in association with the aforesaid chains) comprising or consisting essentially of the amino acid sequence shown as SEQ ID NO:109, or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto); an amino acid sequence encoded by the nucleotide sequence shown in any of SEQ ID NO:110, or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto).
In another exemplary embodiment, the LIGHT targeting molecule comprises at least one fusion molecule of a mammalian (e.g., human) LIGHT protein, or a functional variant or a fragment thereof, and an antibody molecule that binds to CD23 (referred to herein as “LIGHT-anti-CD23 fusion”). In one embodiment, the LIGHT-anti-CD23 fusion comprises, or consists essentially of the amino acid sequence shown in any of anti-CD23 Fab-hLIGHT fusion heavy chain with the (G3S)3 or (G4S)4 linker (pBIIB CD23-204) (SEQ ID NO:101 or 174), or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto); an amino acid sequence encoded by the nucleotide sequence shown in any of anti-CD23 Fab-hLIGHT fusion heavy chain with the (G3S)3 or (G4S)4 linker (pBIIB CD23-204) (SEQ ID NO:102 or 173), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). In certain embodiments, the LIGHT/CD23 fusions may also include, or consist essentially of, a second chain (fused or in association with the aforesaid chains) comprising or consisting essentially of the amino acid sequence shown in anti-CD23 Fab-hLIGHT fusion light chain (SEQ ID NO:103), or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto); an amino acid sequence encoded by the nucleotide sequence shown in any of anti-CD23 Fab-hLIGHT fusion light chain (SEQ ID NO:104), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto).
In yet another exemplary embodiment, the LIGHT targeting molecule comprises at least one fusion molecule of a mammalian (e.g., human) LIGHT protein, or a functional variant or a fragment thereof, and an antibody molecule that binds to insulin growth factor receptor (referred to herein as “LIGHT-anti-IGFR Fab fusion”). In one embodiment, the LIGHT-anti-IGFR Fab fusion comprises, or consists essentially of the amino acid sequence shown in any of anti-IGFR Fab-hLIGHT fusion heavy chain with the (G4S)4 linker (BIIB C06-117) (SEQ ID NO:163), or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto); an amino acid sequence encoded by the nucleotide sequence shown in any of anti-IGFR Fab-hLIGHT fusion heavy chain with the (G4S)4 linker (BIIB C06-117) (SEQ ID NO:162), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). In certain embodiments, the LIGHT/IGFR fusions may also include, or consist essentially of, a second chain (fused or in association with the aforesaid chains) comprising or consisting essentially of the amino acid sequence shown in SEQ ID NO:168, or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto); an amino acid sequence encoded by the nucleotide sequence shown in any of anti-IGFR Fab-hLIGHT fusion light chain (SEQ ID NO:167), or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto).
Antibody Molecules
In certain embodiments, the targeting moiety is an antibody molecule against a selected hyperproliferative cell surface protein, e.g., a hyperproliferative, e.g., cancerous, cell or tissue, such that the LIGHT moiety induces one or more LIGHT-associated activities (e.g., one or more of the LIGHT-associated activities as described herein) against the desired site (e.g., the hyperproliferative, e.g., cancerous, cell or tissue). In other embodiments, novel antibody molecules against HER2 are disclosed. Exemplary hyperproliferative, e.g., cancerous, cells or tissues, that can be targeted with the targeting moiety, include, but are not limited to, cancers or solid tumors of the breast, lung, stomach, ovaries, prostate, pancreas, colon, colorectum, renal, bladder, liver, head, neck, brain, as well as soft-tissue malignancies, including lymphoid malignacies, leukemia and myeloma. The targeting moiety can bind to one or more cell surface proteins expressed on one or more of the hyperproliferative cells or tissues described herein. For example, the targeting moiety, e.g., an antibody molecule as described herein, can bind to one or more of a growth factor receptor (e.g., HER-2/neu, HER3, HER4, epidermal growth factor receptor (EGFR), insulin growth factor receptor (IGFR), Met, Ron, Cripto); a cancer-related integrin or integrin receptor (e.g., αvβ6, α6β4, laminin receptor (LAMR); and/or CD23, CD20, CD16, EpCAM and/or Tweak receptor (FN14). Each one of the selected targets is described in more detail herein.
In one embodiment, the antibody molecule binds to HER2 polypeptide (e.g., to a linear or a conformation epitope on HER2 chosen from epitope D1, epitope D2, epitope D3, or epitope D4, or a combination thereof, e.g., epitope D1-D2 or epitope D1-D3. In other specific embodiments, the antibody molecule binds to CD23 or IGFR.
In one embodiment, the antibody molecule binds to HER2 and is an antibody molecule or a Fab fragment from an antibody selected from the group consisting of BIIB71F10 (SEQ ID NOs:11-14), BIIB69A09 (SEQ ID NOs:15-18); BIIB67F10 (SEQ ID NOs:19-22); BIIB67F11 (SEQ ID NOs:23-26), BIIB66A12 (SEQ ID NOs:27-30), BIIB66C01 (SEQ ID NOs:31-33), BIIB65C10 (SEQ ID NOs:34-38), BIIB65H09 (SEQ ID NOs:39-42) and BIIB65B03 (SEQ ID NOs:43-46) (also referred to herein as 71F10, 69A09; 67F10; 67F11, 67F12, 66A12, 66C01, 65C10, 65H09 and 65B03), or the antibody molecule expressed by PTA-10355, PTA-10356, PTA-10357, or PTA-10358. In other embodiments, the anti-HER2 antibody molecule has a functional activity comparable to an antibody molecule or a Fab fragment from an antibody selected from the group consisting of BIIB71F10, BIIB69A09; BIIB67F10; BIIB67F11, BIIB66A12, BIIB66C01, BIIB65C10, BIIB65H09 and BIIB65B03, or the antibody molecule expressed by PTA-10355, PTA-10356, PTA-10357, or PTA-10358. The anti-HER2 antibody molecule can cross-react with HER2 from one or more species chosen from human, mouse, rat, or cyno origin.
The anti-HER2 antibody molecule can bind to HER2 with an EC50 in the range of about 1 to 120 nM, about 1 to 100 nM, 1 to 80 nM, about 1 to 70 nM, about 1 to 60 nM, about 1 to 40 nM, about 1 to 30 nM, about 1 to 20 nM, about 1 to 15 nM, about 1 to 12 nM, about 1 to 5 nM, about 1 to 2 nM, or about 1 to 1 nM. In other embodiments, the anti-HER2 antibody molecule inhibits or reduces one or more HER2-associated biological activities with an IC50 of about 50 nM to 5 pM, typically about 100 to 250 pM or less, e.g., better inhibition. For example, the anti-HER2 antibody molecule inhibit, block or reduce HER2 signaling with an IC50 of about 50 nM to 5 pM, typically about 100 to 250 pM or less, e.g., better inhibition (e.g., inhibit, block or reduce phosphorylation of one or more of HER2, AKT or MAP kinase; or inhibit, block or reduce homodimerization of HER2 or heterodimerization of HER2 and HER3, or HER2 with EGFR; internalize with a slow kinetics estimated to be less than or equal to the rate of internalization for control anti-HER2 antibody, which is 8e−6s−1 in SKBR-3 cells and 2.1e−5s−1 in BT-474 cells; inhibit activity and/or induce cell killing of a HER2 expressing cell in vitro (e.g., MCF7 and SKBR-3 cell) and in vivo. In one embodiment, the anti-HER2 antibody molecule associates with HER2 with kinetics in the range of 104 to 107 M−1s−1, typically 105 to 106 M−1s−1. In one embodiment, the anti-HER2 antibody molecule binds to human HER2 with a kD of 0.1-100 nM. In yet another embodiment, the anti-HER2 antibody molecule has dissociation kinetics in the range of 10−2 to 10−6 s−1, typically 10−2 to 105 s−1. In one embodiment, the anti-HER2 antibody molecule binds to HER2, e.g., human HER2, with an affinity and/or kinetics similar (e.g., within a factor 20, 10, or 5) to a monoclonal antibody selected from the group consisting of BIIB71F10, BIIB69A09; BIIB67F10; BIIB67F11, BIIB66A12, BIIB66C01, BIIB65C10, BIIB65H09 and BIIB65B03, or the antibody molecule expressed by PTA-10355, PTA-10356, PTA-10357, or PTA-10358. The affinity and binding kinetics of the anti-HER2 antibody molecule can be tested using, e.g., biosensor technology (BIACORE™).
As used herein, the term “antibody molecule” refers to a protein comprising at least one immunoglobulin variable domain sequence. The term antibody molecule includes, for example, full-length, mature antibodies and antigen-binding fragments of an antibody. For example, an antibody molecule can include a heavy (H) chain variable domain sequence (abbreviated herein as VH), and a light (L) chain variable domain sequence (abbreviated herein as VL). In another example, an antibody molecule includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequence, thereby forming two antigen binding sites, such as Fab, Fab′, F(ab′)2, Fc, Fd, Fd′, Fv, single chain antibodies (scFv for example), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies. These functional antibody fragments retain the ability to selectively bind with their respective antigen or receptor. Antibodies and antibody fragments can be from any class of antibodies including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass (e.g., IgG1, IgG2, IgG3, and IgG4) of antibodies. The antibodies of the present invention can be monoclonal or polyclonal. The antibody can also be a human, humanized, CDR-grafted, or in vitro generated antibody. The antibody can have a heavy chain constant region chosen from, e.g., IgG1, IgG2, IgG3, or IgG4. The antibody can also have a light chain chosen from, e.g., kappa or lambda.
Examples of antigen-binding fragments 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 diabody (dAb) fragment, which consists of a VH domain; (vi) a camelid or camelized variable domain; (vii) a single chain Fv (scFv), see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); (viii) a single domain antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
In embodiments, the antibody molecule is a monoclonal or single specificity antibody, or an antigen-binding fragment thereof (e.g., an Fab, F(ab′)2, Fv, a single chain Fv fragment, or a camelid variant) that binds to a hyperproliferative cell surface protein, e.g., a mammalian (e.g., human, hyperproliferative cell surface protein (or a functional variant thereof)). In embodiments, the antibody molecule binds to one or more epitopes located on the extracellular domain of the hyperproliferative cell surface protein (e.g., a hyperproliferative cell surface protein as described herein). Typically, the antibody molecule is a human, humanized, chimeric, camelid, or in vitro generated antibody to a human hyperproliferative cell surface protein (or functional fragment thereof). Typically, the antibody inhibits, reduces or neutralizes one or more activities of hyperproliferative cell surface protein (e.g., one or more biological activities of HER2 as described herein).
Antibodies of the present invention can also be single domain antibodies. Single domain antibodies can include antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and bovine. According to another aspect of the invention, a single domain antibody is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed in WO 9404678, for example. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; such VHHs are within the scope of the invention.
Antibodies of the present invention can also be Affibody molecule scaffolds, e.g., as described in Lee et al. (2008) Clin Cancer Res 14(12):3840-3849; Ahlgren et al. (2009) J. Nucl. Med. 50:781-789).
As used herein, an “immunoglobulin variable domain sequence” refers to an amino acid sequence which can form the structure of an immunoglobulin variable domain. For example, the sequence may include all or part of the amino acid sequence of a naturally-occurring variable domain. For example, the sequence may or may not include one, two, or more N- or C-terminal amino acids, or may include other alterations that are compatible with formation of the protein structure.
The term “antigen-binding site” refers to the part of an antibody molecule that comprises determinants that form an interface that binds to the antigen, e.g., HER2, or an epitope thereof. With respect to proteins (or protein mimetics), the antigen-binding site typically includes one or more loops (of at least four amino acids or amino acid mimics) that form an interface that binds to the antigen, e.g., HER2, or an epitope thereof. Typically, the antigen-binding site of an antibody molecule includes at least one or two CDRs, or more typically at least three, four, five or six CDRs.
The terms “monoclonal antibody” or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. A monoclonal antibody can be made by hybridoma technology or by methods that do not use hybridoma technology (e.g., recombinant methods).
As indicated above, the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH domain, or subset of the complementarity determining regions (CDRs), of an antibody combine to form the variable region that defines a three dimensional antigen binding site. This quaternary antibody structure forms the antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three CDRs on each of the VH and VL chains. In some instances, e.g., certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, a complete immunoglobulin molecule may consist of heavy chains only, with no light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993).
In naturally occurring antibodies, the six “complementarity determining regions” or “CDRs” present in each antigen binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding domain as the antibody assumes its three dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen binding domains, referred to as “framework” regions, show less inter-molecular variability. The framework regions largely adopt a β-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the β-sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. The amino acids comprising the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined (see, “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which are incorporated herein by reference in their entireties).
Antibodies or antigen-binding fragments, variants, or derivatives thereof of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab′ and F(ab′)2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies disclosed herein). ScFv molecules are known in the art and are described, e.g., in U.S. Pat. No. 5,892,019. Immunoglobulin or antibody molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
Antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Additionally included in the invention are antigen-binding fragments comprising any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. Antibodies or immunospecific fragments thereof of the present invention may be from any animal origin including birds and mammals. The antibodies can be human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region may be condricthoid in origin (e.g., from sharks). As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.
As used herein, the term “heavy chain portion” includes amino acid sequences derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain portion comprises at least one of: a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. In certain antibody molecules disclosed herein, the heavy chain portions of one polypeptide chain of a multimer are identical to those on a second polypeptide chain of the multimer. Alternatively, heavy chain portion-containing monomers of the invention are not identical. For example, each monomer may comprise a different target binding site, forming, for example, a bispecific antibody. The heavy chain portions of a binding polypeptide for use in the diagnostic and treatment methods disclosed herein may be derived from different immunoglobulin molecules. For example, a heavy chain portion of a polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, a heavy chain portion can comprise a hinge region derived, in part, from an IgG1 molecule and, in part, from an IgG3 molecule. In another example, a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgG1 molecule and, in part, from an IgG4 molecule.
As used herein, the term “light chain portion” includes amino acid sequences derived from an immunoglobulin light chain. Preferably, the light chain portion comprises at least one of a VL or CL domain.
Antibody molecules disclosed herein may be described or specified in terms of the epitope(s) or portion(s) of an antigen, e.g., a target polypeptide (e.g., HER2, CD23) that they recognize or specifically bind. The portion of a target polypeptide which specifically interacts with the antigen binding domain of an antibody is an “epitope,” or an “antigenic determinant.” A target polypeptide may comprise a single epitope, but typically comprises at least two epitopes, and can include any number of epitopes, depending on the size, conformation, and type of antigen. Furthermore, it should be noted that an “epitope” on a target polypeptide may be or include non-polypeptide elements, e.g., an “epitope may include a carbohydrate side chain. The minimum size of a peptide or polypeptide epitope for an antibody is thought to be about four to five amino acids. Peptide or polypeptide epitopes preferably contain at least seven, more preferably at least nine and most preferably between at least about 15 to about 30 amino acids. Since a CDR can recognize an antigenic peptide or polypeptide in its tertiary form, the amino acids comprising an epitope need not be contiguous, and in some cases, may not even be on the same peptide chain. In the present invention, peptide or polypeptide epitope recognized by antibodies of the present invention contains a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or between about 15 to about 30 contiguous or non-contiguous amino acids.
By “specifically binds,” it is generally meant that an antibody binds to an epitope via its antigen binding domain, and that the binding entails some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen binding domain more readily than it would bind to a random, unrelated epitope. The term “specificity” is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope.
By “preferentially binds,” it is meant that the antibody specifically binds to an epitope more readily than it would bind to a related, similar, homologous, or analogous epitope. Thus, an antibody which “preferentially binds” to a given epitope would more likely bind to that epitope than to a related epitope, even though such an antibody may cross-react with the related epitope.
By way of non-limiting example, an antibody may be considered to bind a first epitope preferentially if it binds said first epitope with a dissociation constant (KD) that is less than the antibody's KD for the second epitope. In another non-limiting example, an antibody may be considered to bind a first antigen preferentially if it binds the first epitope with an affinity that is at least one order of magnitude less than the antibody's KD for the second epitope. In another non-limiting example, an antibody may be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least two orders of magnitude less than the antibody's KD for the second epitope.
In another non-limiting example, an antibody molecule may be considered to bind a first epitope preferentially if it binds the first epitope with an off rate (k(off)) that is less than the antibody's k(off) for the second epitope. In another non-limiting example, an antibody may be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least one order of magnitude less than the antibody's k(off) for the second epitope. In another non-limiting example, an antibody may be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least two orders of magnitude less than the antibody's k(off) for the second epitope.
An antibody molecule disclosed herein may be said to bind a target polypeptide disclosed herein or a fragment or variant thereof with an off rate (k(off)) of less than or equal to 5×10−2 sec−1, 10−2 sec−1, 5×10−3 sec−1 or 10−3 sec−1. More preferably, an antibody of the invention may be said to bind a target polypeptide disclosed herein or a fragment or variant thereof with an off rate (k(off)) less than or equal to 5×10−4 sec−1, 10−4 sec−1, 5×10−5 sec−1, or 10 sec−15×10−6 sec−1, 10−6 sec1, 5×10−7 sec−1 or 10−7 sec−1.
An antibody molecule disclosed herein may be said to bind a target polypeptide disclosed herein or a fragment or variant thereof with an on rate (k(on)) of greater than or equal to 103M−1 sec−1, 5×103 M−1 sec−1, 104M−1 sec−1 or 5×104 M−1 sec−1. An antibody molecule of the invention may be said to bind a target polypeptide disclosed herein or a fragment or variant thereof with an on rate (k(on)) greater than or equal to 105M−1 sec−1, 5×105 M−1 sec−1, 106M−1 sec−1, or 5×106M−1 sec−1 or 107 M−1 sec−1.
An antibody molecule is said to competitively inhibit binding of a reference antibody to a given epitope if it preferentially binds to that epitope to the extent that it blocks, to some degree, binding of the reference antibody to the epitope. Competitive inhibition may be determined by any method known in the art, for example, competition ELISA assays. An antibody may be said to competitively inhibit binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.
As used herein, the term “affinity” refers to a measure of the strength of the binding of an individual epitope with the CDR of an immunoglobulin molecule. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) at pages 27-28. As used herein, the term “avidity” refers to the overall stability of the complex between a population of immunoglobulins and an antigen, that is, the functional combining strength of an immunoglobulin mixture with the antigen. See, e.g., Harlow at pages 29-34. Avidity is related to both the affinity of individual immunoglobulin molecules in the population with specific epitopes, and also the valencies of the immunoglobulins and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure, such as a polymer, would be one of high avidity.
Antibody molecules of the invention may also be described or specified in terms of their cross-reactivity. As used herein, the term “cross-reactivity” refers to the ability of an antibody, specific for one antigen, to react with a second antigen; a measure of relatedness between two different antigenic substances. Thus, an antibody is cross reactive if it binds to an epitope other than the one that induced its formation. The cross reactive epitope generally contains many of the same complementary structural features as the inducing epitope, and in some cases, may actually fit better than the original. For example, certain antibodies have some degree of cross-reactivity, in that they bind related, but non-identical epitopes, e.g., epitopes with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a reference epitope. An antibody may be said to have little or no cross-reactivity if it does not bind epitopes with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a reference epitope. An antibody may be deemed “highly specific” for a certain epitope, if it does not bind any other analog, ortholog, or homolog of that epitope.
Antibody molecules of the invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. Typical binding affinities include those with a dissociation constant or Kd less than 5×10−2 M, 10−2 M, 5×10−3M, 10−3M, 5×10−4M, 10−4M, 5×10−5M, 10−5M, 5×10−6M, 10−6M, 5×10−7M, 10−7M, 5×10−8M, 10−8 M, 5×10−9M, 10−9M, 5×10−10M, M−10 M, 5×10−11M, 10−11M, 5×10−12M, 10−12M, 5×10−13M, 10−13 M, 5×10−14 M, 10−14 M, 5×10−15 M, or 10−15 M.
Antibody molecules of the invention may be “multispecific,” e.g., bispecific, trispecific or of greater multispecificity, meaning that it recognizes and binds to two or more different epitopes present on one or more different antigens (e.g., proteins) at the same time. Thus, whether an antibody molecule is “monospecific” or “multispecific,” e.g., “bispecific,” refers to the number of different epitopes with which a binding polypeptide reacts. Multispecific antibodies may be specific for different epitopes of a target polypeptide described herein or may be specific for a target polypeptide as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material.
As used herein the term “valency” refers to the number of potential binding domains, e.g., antigen binding domains, present in an antibody, binding polypeptide or antibody. Each binding domain specifically binds one epitope. When an antibody, binding polypeptide or antibody comprises more than one binding domain, each binding domain may specifically bind the same epitope, for an antibody with two binding domains, termed “bivalent monospecific,” or to different epitopes, for an antibody with two binding domains, termed “bivalent bispecific.” An antibody may also be bispecific and bivalent for each specificity (termed “bispecific tetravalent antibodies”). In another embodiment, tetravalent minibodies or domain deleted antibodies can be made.
Bispecific bivalent antibodies, and methods of making them, are described, for instance in U.S. Pat. Nos. 5,731,168; 5,807,706; 5,821,333; and U.S. Appl. Publ. Nos. 2003/020734 and 2002/0155537, the disclosures of all of which are incorporated by reference herein. Bispecific tetravalent antibodies, and methods of making them are described, for instance, in WO 02/096948 and WO 00/44788, the disclosures of both of which are incorporated by reference herein. See generally, PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
The antibody molecule can be a polyclonal or a monoclonal antibody. In other embodiments, the antibody can be recombinantly produced, e.g., produced by phage display or by combinatorial methods. Phage display and combinatorial methods for generating antibodies are known in the art (as described in, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al. International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the contents of all of which are incorporated by reference herein).
In one embodiment, the antibody molecule is a fully human antibody (e.g., an antibody made in a mouse which has been genetically engineered to produce an antibody from a human immunoglobulin sequence), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Method of producing rodent antibodies are known in the art. Human monoclonal antibodies can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al. International Application WO 92/03918; Kay et al. International Application 92/03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet. 7:13-21; Morrison, S. L. et al. 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326).
An antibody molecule can be one in which the variable region, or a portion thereof, e.g., the CDRs, are generated in a non-human organism, e.g., a rat or mouse. Chimeric, CDR-grafted, and humanized antibodies are within the invention. Antibodies generated in a non-human organism, e.g., a rat or mouse, and then modified, e.g., in the variable framework or constant region, to decrease antigenicity in a human are within the invention. Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).
A humanized or CDR-grafted antibody will have at least one or two but generally all three recipient CDRs (of heavy and or light immuoglobulin chains) replaced with a donor CDR. The antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody. Preferably, the donor will be a rodent antibody, e.g., a rat or mouse antibody, and the recipient will be a human framework or a human consensus framework. Typically, the immunoglobulin providing the CDRs is called the “donor” and the immunoglobulin providing the framework is called the “acceptor.” In one embodiment, the donor immunoglobulin is a non-human (e.g., rodent). The acceptor framework is a naturally-occurring (e.g., a human) framework or a consensus framework, or a sequence about 85% or higher, preferably 90%, 95%, 99% or higher identical thereto.
As used herein, the term “consensus sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins, 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 can be included in the consensus sequence. A “consensus framework” refers to the framework region in the consensus immunoglobulin sequence.
An antibody molecule can be humanized by methods known in the art. Humanized antibodies can be generated by replacing sequences of the Fv variable region which are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762. Humanized or CDR-grafted antibodies can be produced by CDR-grafting or CDR substitution, wherein one, two, or all CDRs of an immunoglobulin chain can be replaced. See e.g., U.S. Pat. No. 5,225,539; Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988 Science 239:1534; Beidler et al. 1988 J. Immunol. 141:4053-4060; Winter U.S. Pat. No. 5,225,539, the contents of all of which are hereby expressly incorporated by reference. Winter describes a CDR-grafting method which may be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638A, filed on Mar. 26, 1987; Winter U.S. Pat. No. 5,225,539), the contents of which is expressly incorporated by reference.
Also within the scope of the invention are humanized antibodies in which specific amino acids have been substituted, deleted or added. Preferred humanized antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen. For example, a humanized antibody will have framework residues identical to the donor framework residue or to another amino acid other than the recipient framework residue. To generate such antibodies, a selected, small number of acceptor framework residues of the humanized immunoglobulin chain can be replaced by the corresponding donor amino acids. Preferred locations of the substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR (see e.g., U.S. Pat. No. 5,585,089). Criteria for selecting amino acids from the donor are described in U.S. Pat. No. 5,585,089, e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the contents of which are hereby incorporated by reference. Other techniques for humanizing antibodies are described in Padlan et al. EP 519596 A1, published on Dec. 23, 1992.
In one embodiment, an antibody can be made by immunizing with purified target cell antigen, or a fragment thereof, e.g., a fragment described herein, membrane associated antigen, tissue, e.g., crude tissue preparations, whole cells, preferably living cells, lysed cells, or cell fractions, e.g., membrane fractions.
The antibody molecule can be a single chain antibody. A single-chain antibody (scFv) may be engineered (see, for example, Colcher, D. et al. (1999) Ann N Y Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). The single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target.
In yet other embodiments, the antibody molecule has a heavy chain constant region chosen from, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly, chosen from, e.g., the (e.g., human) heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4. In another embodiment, the antibody molecule has a light chain constant region chosen from, e.g., the (e.g., human) light chain constant regions of kappa or lambda. The constant region can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, and/or complement function). In one embodiment the antibody has: effector function; and can fix complement. In other embodiments the antibody does not; recruit effector cells; or fix complement. In another embodiment, the antibody has reduced or no ability to bind an Fc receptor. For example, it is a isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region.
An antibody molecule can be used to isolate target proteins by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, an antibody molecule can be used to detect a target protein (e.g., in a cellular lysate or cell supernatant). Antibody molecules can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (e.g., physically linking) the antibody to a detectable substance (e.g., antibody labeling). Examples of detectable substances include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, chemiluminescent 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; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.
One of the ways in which an antibody molecule can be detectably labeled is by linking the same to an enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A., “The Enzyme Linked Immunosorbent Assay (ELISA)” Microbiological Associates Quarterly Publication, Walkersville, Md., Diagnostic Horizons 2:1-7 (1978)); Voller et al., J. Clin. Pathol. 31:507-520 (1978); Butler, J. E., Meth. Enzymol. 73:482-523 (1981); Maggio, E. (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla., (1980); Ishikawa, E. et al., (eds.), Enzyme Immunoassay, Kgaku Shoin, Tokyo (1981). Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibody molecule, it is possible to detect the antibody through the use of a radioimmunoas say (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, (March, 1986)).
Techniques for conjugating various moieties to an antibody molecules are known, see, e.g., Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. 62:119-58 (1982).
In particular, binding molecules, e.g., binding polypeptides (LIGHT targeting molecules and/or anti-HER2 antibody molecules) for use in the diagnostic and treatment methods disclosed herein may be conjugated to cytotoxins (such as radioisotopes, cytotoxic drugs, or toxins) therapeutic agents, cytostatic agents, biological toxins, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, immunologically active ligands (e.g., lymphokines or other antibodies wherein the resulting molecule binds to both the neoplastic cell and an effector cell such as a T cell), or PEG. In another embodiment, a binding molecule, e.g., a binding polypeptide, for use in the diagnostic and treatment methods disclosed herein can be conjugated to a molecule that decreases vascularization of tumors. In other embodiments, the disclosed compositions may comprise binding molecules, e.g., binding polypeptides, coupled to drugs or prodrugs. Still other embodiments of the present invention comprise the use of binding molecules, e.g., binding polypeptides, conjugated to specific biotoxins or their cytotoxic fragments such as ricin, gelonin, pseudomonas exotoxin or diphtheria toxin. The selection of which conjugated or unconjugated binding molecule to use will depend on the type and stage of cancer, use of adjunct treatment (e.g., chemotherapy or external radiation) and patient condition. It will be appreciated that one skilled in the art could readily make such a selection in view of the teachings herein.
It will be appreciated that anti-tumor antibodies labeled with isotopes have been used successfully to destroy cells in solid tumors as well as lymphomas/leukemias in animal models, and in some cases in humans. Exemplary radioisotopes include: 90Y, 125I, 131I, 123I, 111In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re and 188Re.
It will also be appreciated that, in accordance with the teachings herein, binding molecules may be conjugated to different radiolabels for diagnostic and therapeutic purposes. To this end the aforementioned U.S. Pat. Nos. 6,682,134, 6,399,061, and 5,843,439 disclose radiolabeled therapeutic conjugates for diagnostic “imaging” of tumors before administration of therapeutic antibody.
Additional preferred agents for conjugation to antibody molecules are cytotoxic drugs, particularly those which are used for cancer therapy. As used herein, “a cytotoxin or cytotoxic agent” means any agent that is detrimental to the growth and proliferation of cells and may act to reduce, inhibit or destroy a cell or malignancy. Exemplary cytotoxins include, but are not limited to, radionuclides, biotoxins, enzymatically active toxins, cytostatic or cytotoxic therapeutic agents, prodrugs, immunologically active ligands and biological response modifiers such as cytokines. Any cytotoxin that acts to retard or slow the growth of immunoreactive cells or malignant cells is within the scope of the present invention. Exemplary cytotoxins include, in general, cytostatic agents, alkylating agents, anti-metabolites, anti-proliferative agents, tubulin binding agents, hormones and hormone antagonists, and the like. Other classes of cytotoxic agents include, for example, the maytansinoid family of drugs, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, diynenes, and the podophyllotoxins.
In certain embodiments, a moiety that enhances the stability or efficacy of an antibody molecule can be conjugated. For example, in one embodiment, PEG can be conjugated to the binding molecules of the invention to increase their half-life in vivo. Leong, S. R., et al., Cytokine 16:106 (2001); Adv. in Drug Deliv. Rev. 54:531 (2002); or Weir et al., Biochem. Soc. Transactions 30:512 (2002).
Nucleic Acids Encoding LIGHT Targeting and Antibody Molecules
The present invention also provides for nucleic acid molecules encoding LIGHT targeting and antibody molecules of the invention.
In one embodiment, the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH), where at least one of the CDRs of the heavy chain variable region or at least two of the VH-CDRs of the heavy chain variable region are at least 80%, 85%, 90% or 95% identical to reference heavy chain VH-CDR1, VH-CDR2, or VH-CDR3 amino acid sequences from monoclonal HER2 antibodies disclosed herein. Alternatively, the VH-CDR1, VH-CDR2, and VH-CDR3 regions of the VH are at least 80%, 85%, 90% or 95% identical to reference heavy chain VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences from monoclonal HER2 antibodies disclosed herein. Thus, according to this embodiment a heavy chain variable region of the invention has VH-CDR1, VH-CDR2, or VH-CDR3 polypeptide sequences related to the polypeptide sequences shown in SEQ ID NOs:47-70.
In certain embodiments, the nucleic acid molecule encodes an antibody molecule of the fusion, or the anti-HER2 antibody molecule, that includes, or consists essentially of, a nucleotide sequence that hybridizes under high stringency conditions to the complement of the nucleotide sequence encoding a heavy chain variable domain of BIIB71F10 (SEQ ID NO:12; SEQ ID NO:156), BIIB69A09 (SEQ ID NO:16); BIIB67F10 (SEQ ID NO:20); BIIB67F11 (SEQ ID NO:24), BIIB66A12 (SEQ ID NO:28), BIIB66C01 (SEQ ID NO:32), BIIB65C10 (SEQ ID NO:36), BIIB65H09 (SEQ ID NO:40) or BIIB65B03 (SEQ ID NO:44), or the heavy chain variable domain of the antibody molecule expressed by PTA-10355, PTA-10356, PTA-10357, or PTA-10358; or includes an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98%, 99% or higher identical identical to the amino acid sequence of the heavy chain variable domain of BIIB71F10 (SEQ ID NO:11), BIIB69A09 (SEQ ID NO:15); BIIB67F10 (SEQ ID NO:19); BIIB67F11 (SEQ ID NO:23), BIIB66A12 (SEQ ID NO:27), BIIB66C01 (SEQ ID NO:31), BIIB65C10 (SEQ ID NO:35), BIIB65H09 (SEQ ID NO:39) or BIIB65B03 (SEQ ID NO:43), or the heavy chain variable domain of the antibody molecule expressed by PTA-10355, PTA-10356, PTA-10357, or PTA-10358.
In other embodiments, the nucleic acid molecule encodes an antibody molecule of the fusion, or the anti-HER2 antibody molecule, that includes, or consists essentially of, a nucleotide sequence that hybridizes under high stringency conditions to the complement of the nucleotide sequence encoding a light chain variable domain of BIIB71F10 (SEQ ID NO:14), BIIB69A09 (SEQ ID NO:18); BIIB67F10 (SEQ ID NO:22); BIIB67F11 (SEQ ID NO:26), BIIB66A12 (SEQ ID NO:30), BIIB66C01 (SEQ ID NO:34), BIIB65C10 (SEQ ID NO:38), BIIB65H09 (SEQ ID NO:42) or BIIB65B03 (SEQ ID NO:46), or the light chain variable domain of the antibody molecule expressed by PTA-10355, PTA-10356, PTA-10357, or PTA-10358; or includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher identical to a light chain variable domain of BIIB71F10 (SEQ ID NO:13), BIIB69A09 (SEQ ID NO:17); BIIB67F10 (SEQ ID NO:21); BIIB67F11 (SEQ ID NO:25), BIIB66A12 (SEQ ID NO:29), BIIB66C01 (SEQ ID NO:33), BIIB65C10 (SEQ ID NO:37), BIIB65H09 (SEQ ID NO:41) or BIIB65B03 (SEQ ID NO:45), or the light chain variable domain of the antibody molecule expressed by PTA-10355, PTA-10356, PTA-10357, or PTA-10358.
Exemplary nucleic acid molecules encode LIGHT/HER2 fusions that include, or consist essentially of, the amino acid sequence shown in any of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto); an amino acid sequence encoded by the nucleotide sequence shown in any of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). In certain embodiments, the nucleic acid molecules encoding the LIGHT/HER2 fusions may also include, or consist essentially of, a second chain (genetically fused or in association with the aforesaid chains) comprising or consisting essentially of the amino acid sequence shown in SEQ ID NO:1, or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). In embodiments, the nucleic acid molecules comprise, or consist essentially of, the nucleotide sequence shown in any of SEQ ID NO:5, or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto).
In another exemplary embodiment, the nucleic acid molecules encode a LIGHT targeting molecule that comprises at least one fusion molecule of a mammalian (e.g., human) LIGHT protein, or a functional variant or a fragment thereof, and an antibody molecule that binds to CD23 or IGFR. In one embodiment, the nucleic acid molecules encoding the LIGHT-anti-CD23 or the LIGHT-anti-IGFR fusion comprises, or consists essentially of the amino acid sequence shown in any of SEQ ID NO:101, 174, 163, or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). In embodiments, the nucleic acid molecules comprise, or consist essentially of, the nucleotide sequence shown in any of SEQ ID NO:102, 173, 162, or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). In certain embodiments, the nucleic acid molecules encoding the LIGHT/CD23 fusions may also include, or consist essentially of, a second chain (fused or in association with the aforesaid chains) comprising or consisting essentially of the amino acid sequence shown in SEQ ID NO:103 or 168, or an amino sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto). In embodiments, the nucleic acid molecules comprise, or consist essentially of, the nucleotide sequence shown in any of SEQ ID NO:104 or 167, or a nucleotide sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95% or higher identical thereto).
Recombinant Expression Vectors, Host Cells and Genetically Engineered Cells
The invention also includes a nucleic acid which encodes the targeting molecules and/or antibody molecules described herein. Also included are vectors which include the nucleic acid and cells transformed with the nucleic acid, particularly cells which are useful for producing an antibody, e.g., mammalian cells, e.g. CHO or lymphatic cells. The invention also includes cell lines (e.g., recombinant host cells, hybridomas), which make an antibody molecule as described herein, and method of using said cells to make antibody molecules.
In another aspect, the invention includes, vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide described herein. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid or viral vector. The vector can be capable of autonomous replication or it can integrate into a host DNA. Viral vectors include, e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses.
A vector can include a nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. The term “regulatory sequence” includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by nucleic acids as described herein.
The recombinant expression vectors of the invention can be designed for expression of proteins in prokaryotic or eukaryotic cells. For example, polypeptides of the invention can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
Purified recombinant proteins can be activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for proteins. In a preferred embodiment, a fusion protein expressed in a retroviral expression vector of the present invention can be used to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six weeks).
To maximize recombinant protein expression in E. coli is to express the protein in a host bacterium with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
The expression vector can be a yeast expression vector, a vector for expression in insect cells, e.g., a baculovirus expression vector or a vector suitable for expression in mammalian cells. When used in mammalian cells, the expression vector's control functions can be provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
In another embodiment, the promoter is an inducible promoter, e.g., a promoter regulated by a steroid hormone, by a polypeptide hormone (e.g., by means of a signal transduction pathway), or by a heterologous polypeptide (e.g., the tetracycline-inducible systems, “Tet-On” and “Tet-Off”; see, e.g., Clontech Inc., CA, Gossen and Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547, and Paillard (1989) Human Gene Therapy 9:983). In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example, the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. Regulatory sequences (e.g., viral promoters and/or enhancers) operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the constitutive, tissue specific or cell type specific expression of antisense RNA in a variety of cell types. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus.
Another aspect the invention provides a host cell which includes a nucleic acid molecule described herein, e.g., a nucleic acid molecule within a recombinant expression vector or a nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome. The terms “host cell” and “recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential 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 as used herein. A host cell can be any prokaryotic or eukaryotic cell. For example, a protein can be expressed in bacterial cells (such as E. coli), insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells e.g., COS-7 cells, CV-1 origin SV40 cells; Gluzman (1981) Cell 23:175-182). Other suitable host cells are known to those skilled in the art.
A host cell of the invention can be used to produce (i.e., express) a protein. Accordingly, the invention further provides methods for producing a protein using the host cells of the invention. In one embodiment, the method includes culturing the host cell of the invention (into which a recombinant expression vector encoding a protein has been introduced) in a suitable medium such that a protein is produced. In another embodiment, the method further includes isolating a protein from the medium or the host cell.
Inhibition of Hyperproliferative Activity
The invention provides methods of treating or preventing (e.g., curing, suppressing, ameliorating, delaying or preventing the onset of, or preventing recurrence or relapse of) a hyperproliferative, e.g., neoplastic condition and/or disorder, in a subject. The method includes administering to the subject a LIGHT targeting molecule or an anti-HER2 antibody molecule as described herein, in an amount sufficient to inhibit or reduce one or more biological activities in the hyperproliferative, e.g., neoplastic cell or tissue, thereby treating or preventing the disorder or condition.
In certain embodiments, the method prevents, reduces or ameliorates the recurrence or relapse of a tumor or metastasis. The method includes administering a LIGHT-targeting molecule, or anti-HER2 antibody molecule, as described herein, to a subject, e.g., a patient that is partially or completely refractory to a standard mode of therapy (e.g., chemotherapy, antibody-based and/or surgery). For example, the patient suffers from a HER2-expressing cancer (e.g., a breast, gastric or lung cancer) and has demonstrated disease progession after surgery, chemotherapy and/or antibody therapy (e.g., trastuzumab therapy). In this regard, the majority of the patients with metastatic breast cancer who initially respond to trastuzimab demonstrate disease progression within one year of treatment initiation (Nahta, R. et al. (2006) Nature Clinical Practice Vol. 3 (5):269-280). The LIGHT-anti-HER2 molecules of the invention can be used to treat the trastuzumab-refractory patient population. The LIGHT-anti-HER2 molecules of the invention have been shown to excert a prolonged inhibition of anti-tumor activity (beyond the inhibition detected with anti-HER2 antibodies) (FIG. 22), thus, expanding the therapeutic and prophylactic uses of these molecules.
In other embodiments, the patient is a colon cancer patient that has demonstrated disease progession after surgery, chemotherapy and/or antibody therapy (e.g., VEGF or EGFR antibody therapy). In certain embodiments, the LIGHT-targeting molecule, or anti-HER2 antibody molecule, is administered to a patient who has been treated with another mode of therapy (e.g., a standard mode of therapy) for about 10 days, one to six months, six months to a year, one to two years, and so on. In certain embodiments, the subject has developed partial or complete resistance to a first-line of therapy.
In some embodiments, the amount or dosage of the LIGHT-targeting molecule, or anti-HER2 antibody molecule, administered can be determined, e.g., prior to administration to the subject, by testing in vitro or ex vivo the amount of the LIGHT-targeting molecule, or anti-HER2 antibody molecule, required to decrease or inhibit one or more of hyperproliferative activities, disorders or conditions described herein. The in vivo method can, optionally, include the step(s) of identifying (e.g., evaluating, diagnosing, screening, and/or selecting) a subject at risk of having, or having, one or more symptoms associated with the disorder or condition.
In various embodiments of the above-described methods, the antibody or fragment thereof inhibits tumor cell migration. In further embodiments, the tumor cell proliferation is inhibited through the prevention or retardation of tumor spread to adjacent tissues.
In further embodiments, the hyperproliferative disorder or condition is chosen from one or more of a cancer, a neoplasm, a tumor, a malignancy, or a metastasis thereof, or a recurrent malignancy (e.g., a subject that is partially or completely refractory to a first-line of treatment).
In embodiments, the targeting moiety of the LIGHT targeting molecule, or the antibody molecule, is administered, alone or combination with a second agent, as a first-line of therapy to a naïve subject, e.g., a naïve patient having a HER2-expressing breast cancer. In other embodiments, the targeting moiety of the LIGHT targeting molecule, or the antibody molecule, is administered, alone or combination with a second agent, as a second-line of therapy. In other embodiment, the targeting moiety of the LIGHT targeting molecule, or the antibody molecule, is administered to a patient that is partially or completely refractory to a standard mode of therapy. For example, the patient is a breast cancer patient that has demonstrated disease progession after chemotherapy and/or trastuzumab therapy.
As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
By “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.
As used herein, phrases such as “a subject that would benefit from administration of a binding molecule” and “an animal in need of treatment” includes subjects, such as mammalian subjects, that would benefit from administration of a binding molecule used, e.g., for detection of an antigen recognized by a binding molecule (e.g., for a diagnostic procedure) and/or from treatment, i.e., palliation or prevention of a disease such as cancer, with a binding molecule which specifically binds a given target protein. As described in more detail herein, the binding molecule can be used in unconjugated form or can be conjugated, e.g., to a drug, prodrug, or an isotope.
In various embodiments, the subject is a mammal (e.g., an animal model or a human). In further embodiments, the subject is a human, e.g., a patient with one or more of the cancers or neoplastic conditions described herein. In one embodiment, the subject is a patient undergoing a standard mode of therapy, e.g., a HER2-positive patient undergoing chemotherapy and/or treatment with trastuzumab, and the LIGHT-targeting molecules and/or an anti-HER2 antibody molecule are administered as a second-line of therapy. In other embodiments, the patient is a naïve patient, e.g., the LIGHT-targeting molecules and/or an anti-HER2 antibody molecule are administered as a first-line of therapy. In other embodiment, the patient is partially or completely refractory to a standard mode of therapy. For example, the patient is a breast cancer patient that has demonstrated disease progession after chemotherapy and/or trastuzumab therapy.
By “hyperproliferative disease or disorder” is meant all neoplastic cell growth and proliferation, whether malignant or benign, including all transformed cells and tissues and all cancerous cells and tissues. Hyperproliferative diseases or disorders include, but are not limited to, precancerous lesions, abnormal cell growths, benign tumors, malignant tumors, and “cancer.” In certain embodiments of the present invention, the hyperproliferative disease or disorder, e.g., the precancerous lesion, abnormal cell growth, benign tumor, malignant tumor, or “cancer” comprises cells which express, over-express, or abnormally express a target cell antigen.
Additional examples of hyperproliferative diseases, disorders, and/or conditions include, but are not limited to neoplasms, whether benign or malignant, located in the: prostate, colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital tract. Such neoplasms, in certain embodiments, express, over-express, or abnormally express a target cell antigen.
Other hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above. In certain embodiments of the present invention the diseases involve cells which express, over-express, or abnormally express a target cell antigen.
As used herein, the terms “tumor” or “tumor tissue” refer to an abnormal mass of tissue that results from excessive cell division, in certain cases tissue comprising cells which express, over-express, or abnormally express a hyperproliferative cell protein. A tumor or tumor tissue comprises “tumor cells” which are neoplastic cells with abnormal growth properties and no useful bodily function. Tumors, tumor tissue and tumor cells may be benign or malignant.
As used herein, the term “malignancy” refers to a non-benign tumor or a cancer. As used herein, the term “cancer” connotes a type of hyperproliferative disease which includes a malignancy characterized by deregulated or uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers are noted below and include: squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer. The term “cancer” includes primary malignant cells or tumors (e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original malignancy or tumor) and secondary malignant cells or tumors (e.g., those arising from metastasis, the migration of malignant cells or tumor cells to secondary sites that are different from the site of the original tumor). Cancers conducive to treatment methods of the present invention involves cells which express, over-express, or abnormally express a target cell antigen.
The method of the present invention may be used to treat premalignant conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those disorders described above. Such uses are indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976). Such conditions in which cells begin to express, over-express, or abnormally express a target cell antigen, are particularly treatable by the methods of the present invention.
Additional pre-neoplastic disorders which can be treated by the method of the invention include, but are not limited to, benign dysproliferative disorders (e.g., benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps, colon polyps, and esophageal dysplasia), leukoplakia, keratoses, Bowen's disease, Farmer's Skin, solar cheilitis, and solar keratosis.
In preferred embodiments, the method of the invention is used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed herein.
Additional hyperproliferative diseases, disorders, and/or conditions include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, emangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
Several assay systems, cell lines and animal models are known in the art for evaluating the effects of the LIGHT targeting agents and anti-HER2 antibody molecules described herein. For example, cell lines expressing different levels of one or more of HER2, LTbR and HVEM can be used. Exemplary human cell lines expressing high levels of HER2 include, but are not limited to, BT474, SKBR3, N87 and SKOV3. Exemplary cell lines expressing moderate levels of HER2 include rat Tubo and human HT29. Exemplary cell lines expressing low or undetectable levels of HER2 include human MCF7, MDA-MB-231, MDA-MB-468, as well as mouse TSA and 4T1. Examples of cell lines that express LIGHT receptors include HT29, N87 and WiDr (which express high levels of LTβR); BT474, SKBR3, MCF7, MDA-MB-231, MDA-MB-468, SKOV3 and Tubo (all of which express moderate levels of LTβR); and mouse TSA and 4T1 (which express low levels of LTβR). Cell lines expressing moderate levels of HVEM include MCF7, MDA-MB-231 and HT29. Xenograft animal models for testing the molecules of the invention are described in the Examples below.
Pharmaceutical Compositions, Dosages, Modes of Administration
The molecules (also referred to herein as “active compounds”) of the invention can be incorporated into pharmaceutical compositions. Such compositions typically include the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.
A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
When a therapeutically effective amount of a molecule of the invention is administered by intravenous, cutaneous or subcutaneous injection, binding agent will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to binding agent an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additive known to those of skill in the art.
For administration by inhalation, the molecules of the invention are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays, including, for example, radiographic tumor imaging. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.
The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.
The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions comprising antibodies or a cocktail thereof are administered to a patient not already in the disease state or in a pre-disease state to enhance the patient's resistance. Such an amount is defined to be a “prophylactic effective dose.” In this use, the precise amounts again depend upon the patient's state of health and general immunity, but generally range from 0.1 to 25 mg per dose, especially 0.5 to 2.5 mg per dose. A relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives.
In therapeutic applications, a relatively high dosage (e.g., from about 1 to 400 mg/kg of binding molecule, e.g., antibody per dose, with dosages of from 5 to 25 mg being more commonly used for radioimmunoconjugates and higher doses for cytotoxin-drug conjugated molecules) at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patent can be administered a prophylactic regime.
In one embodiment, a subject can be treated with a nucleic acid molecule encoding a LIGHT targeting molecule or an antibody molecule (collectively referred to herein as “binding molecules” or “molecules”) (e.g., in a vector). Doses for nucleic acids encoding polypeptides range from about 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per patient. Doses for infectious viral vectors vary from 10-100, or more, virions per dose.
Therapeutic agents can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or intramuscular means for prophylactic and/or therapeutic treatment. In some methods, agents are injected directly into a particular tissue where cancer-expressing cells have accumulated, for example intracranial injection. Intramuscular injection or intravenous infusions are preferred for administration of antibody. In some methods, particular therapeutic antibodies are injected directly into the cranium. In some methods, antibodies are administered as a sustained release composition or device, such as a Medipad® device.
Molecules of the invention can optionally be administered in combination with other agents that are effective in treating the disorder or condition in need of treatment (e.g., prophylactic or therapeutic). The LIGHT-targeting molecule, or anti-HER2 antibody molecule, alone or in combination with another agent (e.g., a chemotherapeutic agent as described herein), can be administered to a subject, e.g., a mammal, suffering from a hyperproliferative condition and/or disorder, in an amount sufficient to elicit at least one LIGHT-associated biological activity, in the subject.
While a great deal of clinical experience has been gained with 131I and 90Y, other radiolabels are known in the art and have been used for similar purposes. Still other radioisotopes are used for imaging. For example, additional radioisotopes which are compatible with the scope of the instant invention include, but are not limited to, 123I, 125I, 32P, 57Co, 64Cu, 67Cu, 77Br, 81Rb, 81Kr, 87Sr, 113In, 127Cs, 129Cs, 132I, 197Hg, 203Pb, 206Bi, 177Lu, 186Re, 212Pb, 212Bi, 47Sc, 105Rh, 109Pd, 153Sm, 188Re, 199Au, 225Ac, 211At, and 213Bi. In this respect alpha, gamma and beta emitters are all compatible with in the instant invention. Further, in view of the instant disclosure it is submitted that one skilled in the art could readily determine which radionuclides are compatible with a selected course of treatment without undue experimentation. To this end, additional radionuclides which have already been used in clinical diagnosis include 125I, 123I, 99Tc, 43K, 52Fe, 67Ga, 68Ga, as well as 111In. Antibodies have also been labeled with a variety of radionuclides for potential use in targeted immunotherapy (Peirersz et al. Immunol. Cell Biol. 65: 111-125 (1987)). These radionuclides include 188Re and 186Re as well as 199Au and 67Cu to a lesser extent. U.S. Pat. No. 5,460,785 provides additional data regarding such radioisotopes and is incorporated herein by reference.
While molecules of the invention may be administered as described immediately above, it must be emphasized that in other embodiments conjugated and unconjugated binding molecules may be administered to otherwise healthy patients as a first line therapeutic agent.
However, selected embodiments of the invention comprise the administration of molecules of the invention to patients or in combination or conjunction with one or more adjunct therapies such as radiotherapy or chemotherapy (i.e. a combined therapeutic regimen). As used herein, the administration of binding molecules of the invention in conjunction or combination with an adjunct therapy means the sequential, simultaneous, coextensive, concurrent, concomitant or contemporaneous administration or application of the therapy and the disclosed binding molecules. Those skilled in the art will appreciate that the administration or application of the various components of the combined therapeutic regimen may be timed to enhance the overall effectiveness of the treatment. For example, chemotherapeutic agents could be administered in standard courses of treatment followed within a few weeks by radioimmunoconjugates described herein. Conversely, cytotoxin-conjugated binding molecules could be administered intravenously followed by tumor localized external beam radiation. In yet other embodiments, binding molecules may be administered concurrently with one or more selected chemotherapeutic agents in a single office visit. A skilled artisan (e.g. an experienced oncologist) would be readily be able to discern effective combined therapeutic regimens without undue experimentation based on the selected adjunct therapy and the teachings of the instant specification.
In this regard it will be appreciated that the combination of a binding molecule (with or without cytotoxin) and the chemotherapeutic agent may be administered in any order and within any time frame that provides a therapeutic benefit to the patient. That is, the chemotherapeutic agent and binding molecule may be administered in any order or concurrently. In selected embodiments, binding molecules of the present invention will be administered to patients that have previously undergone chemotherapy. In yet other embodiments, binding molecules of the present invention will be administered substantially simultaneously or concurrently with the chemotherapeutic treatment. For example, the patient may be given the binding molecule while undergoing a course of chemotherapy. In preferred embodiments the binding molecule will be administered within 1 year of any chemotherapeutic agent or treatment. In other preferred embodiments the polypeptide will be administered within 10, 8, 6, 4, or 2 months of any chemotherapeutic agent or treatment. In still other preferred embodiments the binding molecule will be administered within 4, 3, 2 or 1 week of any chemotherapeutic agent or treatment. In yet other embodiments the binding molecule will be administered within 5, 4, 3, 2 or 1 days of the selected chemotherapeutic agent or treatment. It will further be appreciated that the two agents or treatments may be administered to the patient within a matter of hours or minutes (i.e. substantially simultaneously).
With respect to these aspects of the invention, exemplary chemotherapeutic agents that are compatible with the instant invention include alkylating agents, vinca alkaloids (e.g., vincristine and vinblastine), procarbazine, methotrexate and prednisone. The four-drug combination MOPP (mechlethamine (nitrogen mustard), vincristine (Oncovin), procarbazine and prednisone) is very effective in treating various types of lymphoma and comprises a preferred embodiment of the present invention. In MOPP-resistant patients, ABVD (e.g., adriamycin, bleomycin, vinblastine and dacarbazine), Ch1VPP (chlorambucil, vinblastine, procarbazine and prednisone), CABS (lomustine, doxorubicin, bleomycin and streptozotocin), MOPP plus ABVD, MOPP plus ABV (doxorubicin, bleomycin and vinblastine) or BCVPP (carmustine, cyclophosphamide, vinblastine, procarbazine and prednisone) combinations can be used. Arnold S. Freedman and Lee M. Nadler, Malignant Lymphomas, in Harrison's Principles of Internal Medicine 1774-1788 (Kurt J. Isselbacher et al., eds., 13th ed. 1994) and V. T. DeVita et al., (1997) and the references cited therein for standard dosing and scheduling. These therapies can be used unchanged, or altered as needed for a particular patient, in combination with one or more antibodies or immunospecific fragments thereof of the present invention.
For patients with intermediate- and high-grade malignancies, who fail to achieve remission or relapse, salvage therapy is used. Salvage therapies employ drugs such as cytosine arabinoside, cisplatin, carboplatin, etoposide and ifosfamide given alone or in combination. In relapsed or aggressive forms of certain neoplastic disorders the following protocols are often used: IMVP-16 (ifosfamide, methotrexate and etoposide), MIME (methyl-gag, ifosfamide, methotrexate and etoposide), DHAP (dexamethasone, high dose cytarabine and cisplatin), ESHAP (etoposide, methylpredisolone, HD cytarabine, cisplatin), CEPP(B) (cyclophosphamide, etoposide, procarbazine, prednisone and bleomycin) and CAMP (lomustine, mitoxantrone, cytarabine and prednisone) each with well known dosing rates and schedules.
The amount of chemotherapeutic agent to be used in combination with the binding molecules of the present invention may vary by subject or may be administered according to what is known in the art. See for example, Bruce A Chabner et al., Antineoplastic Agents, in Goodman & Gilman's The Pharmacological Basis of Therapeutics 1233-1287 (Joel G. Hardman et al., eds., 9th ed. (1996)).
In another embodiment, a binding molecule of the present invention is administered in conjunction with a biologic. Biologics useful in the treatment of cancers are known in the art and a binding molecule of the invention may be administered, for example, in conjunction with such known biologics. For example, the FDA has approved the following biologics for the treatment of breast cancer: Herceptin® (trastuzumab, Genentech Inc., South San Francisco, Calif.; a humanized monoclonal antibody that has anti-tumor activity in HER2-positive breast cancer); Faslodex® (fulvestrant, AstraZeneca Pharmaceuticals, LP, Wilmington, Del.; an estrogen-receptor antagonist used to treat breast cancer); Arimidex® (anastrozole, AstraZeneca Pharmaceuticals, LP; a nonsteroidal aromatase inhibitor which blocks aromatase, an enzyme needed to make estrogen); Aromasin® (exemestane, Pfizer Inc., New York, N.Y.; an irreversible, steroidal aromatase inactivator used in the treatment of breast cancer); Femara® (letrozole, Novartis Pharmaceuticals, East Hanover, N.J.; a nonsteroidal aromatase inhibitor approved by the FDA to treat breast cancer); and Nolvadex® (tamoxifen, AstraZeneca Pharmaceuticals, LP; a nonsteroidal antiestrogen approved by the FDA to treat breast cancer). Other biologics with which the binding molecules of the invention may be combined include: Avastin® (bevacizumab, Genentech Inc.; the first FDA-approved therapy designed to inhibit angiogenesis); and Zevalin® (ibritumomab tiuxetan, Biogen Idec, Cambridge, Mass.; a radiolabeled monoclonal antibody currently approved for the treatment of B-cell lymphomas).
In addition, the FDA has approved the following biologics for the treatment of colorectal cancer: Avastin®; Erbitux® (cetuximab, ImClone Systems Inc., New York, N.Y., and Bristol-Myers Squibb, New York, N.Y.; is a monoclonal antibody directed against the epidermal growth factor receptor (EGFR)); Gleevec® (imatinib mesylate; a protein kinase inhibitor); and Ergamisol® (levamisole hydrochloride, Janssen Pharmaceutica Products, LP, Titusville, N.J.; an immunomodulator approved by the FDA in 1990 as an adjuvant treatment in combination with 5-fluorouracil after surgical resection in patients with Dukes' Stage C colon cancer).
For use in treatment of Non-Hodgkin's Lymphomas currently approved therapies include: Bexxar® (tositumomab and iodine I-131 tositumomab, GlaxoSmithKline, Research Triangle Park, N.C.; a multi-step treatment involving a mouse monoclonal antibody (tositumomab) linked to a radioactive molecule (iodine I-131)); Intron® A (interferon alfa-2b, Schering Corporation, Kenilworth, N.J.; a type of interferon approved for the treatment of follicular non-Hodgkin's lymphoma in conjunction with anthracycline-containing combination chemotherapy (e.g., cyclophosphamide, doxorubicin, vincristine, and prednisone [CHOP])); Rituxan® (rituximab, Genentech Inc., South San Francisco, Calif., and Biogen Idec, Cambridge, Mass.; a monoclonal antibody approved for the treatment of non-Hodgkin's lymphoma; Ontak® (denileukin diftitox, Ligand Pharmaceuticals Inc., San Diego, Calif.; a fusion protein consisting of a fragment of diphtheria toxin genetically fused to interleukin-2); and Zevalin® (ibritumomab tiuxetan, Biogen Idec; a radiolabeled monoclonal antibody approved by the FDA for the treatment of B-cell non-Hodgkin's lymphomas).
For treatment of Leukemia, exemplary biologics which may be used in combination with the binding molecules of the invention include Gleevec®; Campath®-1H (alemtuzumab, Berlex Laboratories, Richmond, Calif.; a type of monoclonal antibody used in the treatment of chronic Lymphocytic leukemia). In addition, Genasense (oblimersen, Genta Corporation, Berkley Heights, N.J.; a BCL-2 antisense therapy under development to treat leukemia may be used (e.g., alone or in combination with one or more chemotherapy drugs, such as fludarabine and cyclophosphamide) may be administered with the claimed binding molecules.
For the treatment of lung cancer, exemplary biologics include Tarceva® (erlotinib HCL, OSI Pharmaceuticals Inc., Melville, N.Y.; a small molecule designed to target the human epidermal growth factor receptor 1 (HER1) pathway).
For the treatment of multiple myeloma, exemplary biologics include Velcade® Velcade (bortezomib, Millennium Pharmaceuticals, Cambridge Mass.; a proteasome inhibitor). Additional biologics include Thalidomid® (thalidomide, Clegene Corporation, Warren, N.J.; an immunomodulatory agent and appears to have multiple actions, including the ability to inhibit the growth and survival of myeloma cells and anti-angiogenesis).
Other exemplary biologics include the MOAB IMC-C225, developed by ImClone Systems, Inc., New York, N.Y.
Diagnostic or Prognostic Methods Using Binding Molecules and Nucleic Acid Amplification Assays
Binding molecules can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and/or conditions associated with the aberrant expression and/or activity of a target cell antigen, e.g., HER2 or CD23. Expression of these targets may be in tumor tissue and other neoplastic conditions. Binding molecules are useful for diagnosis, treatment, prevention and/or prognosis of hyperproliferative disorders in mammals, preferably humans. Exemplary disorders are disclosed herein. Thus, the invention provides a diagnostic method useful during diagnosis of a cancers and other hyperproliferative disorders, which involves measuring the expression level of target protein or transcript in tissue or other cells or body fluid from an individual and comparing the measured expression level with a standard target expression levels in normal tissue or body fluid, whereby an increase in the expression level compared to the standard is indicative of a disorder.
One embodiment provides a method of detecting the presence of abnormal hyperproliferative cells, e.g., precancerous or cancerous cells, in a fluid or tissue sample, comprising assaying for the expression of the target in tissue or body fluid samples of an individual and comparing the presence or level of target expression in the sample with the presence or level of target expression in a panel of standard tissue or body fluid samples, where detection of target expression or an increase in target expression over the standards is indicative of aberrant hyperproliferative cell growth.
More specifically, the present invention provides a method of detecting the presence of abnormal hyperproliferative cells in a body fluid or tissue sample, comprising (a) assaying for the expression of target in tissue or body fluid samples of an individual using target-specific antibody molecules of the present invention, and (b) comparing the presence or level of target expression in the sample with a the presence or level of target expression in a panel of standard tissue or body fluid samples, whereby detection of target expression or an increase in target expression over the standards is indicative of aberrant hyperproliferative cell growth.
With respect to cancer, the presence of a relatively high amount of target protein in biopsied tissue from an individual may indicate the presence of a tumor or other malignant growth, may indicate a predisposition for the development of such malignancies or tumors, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
Target-specific antibody molecules of the present invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In) and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. Suitable assays are described in more detail elsewhere herein.
One aspect of the invention is a method for the in vivo detection or diagnosis of a hyperproliferative disease or disorder associated with aberrant expression of the target in a subject, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled antibody or fragment thereof of the present invention, which specifically binds to target; b) waiting for a time interval following the administering for permitting the labeled binding molecule to preferentially concentrate at sites in the subject where target is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of target. Background level can be determined by various methods including comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.
It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of, e.g., 99Tc. The labeled binding molecule, e.g., antibody or antibody fragment, will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).
Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 7 to 10 days.
Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography. Antibody labels or markers for in vivo imaging of target expression include those detectable by X-radiography, nuclear magnetic resonance imaging (NMR), MRI, CAT-scans or electron spin resonance imaging (ESR). In a related embodiment to those described above, monitoring of an already diagnosed disease or disorder is carried out by repeating any one of the methods for diagnosing the disease or disorder, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
Where a diagnosis of a disorder, including diagnosis of a tumor, has already been made according to conventional methods, detection methods as disclosed herein are useful as a prognostic indicator, whereby patients continuing to exhibiting enhanced target expression will experience a worse clinical outcome relative to patients whose expression level decreases nearer the standard level.
By “assaying the expression level of the tumor associated target polypeptide” is intended qualitatively or quantitatively measuring or estimating the level of target polypeptide in a first biological sample either directly (e.g., by determining or estimating absolute protein level) or relatively (e.g., by comparing to the cancer associated polypeptide level in a second biological sample). Preferably, target polypeptide expression level in the first biological sample is measured or estimated and compared to a standard target polypeptide level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having the disorder. As will be appreciated in the art, once the “standard” target polypeptide level is known, it can be used repeatedly as a standard for comparison.
By “biological sample” is intended any biological sample obtained from an individual, cell line, tissue culture, or other source of cells potentially expressing target. As indicated, biological samples include body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid), and other tissue sources which contain cells potentially expressing target. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.
In an additional embodiment, antibodies, or immunospecific fragments of antibodies directed to a conformational epitope of target may be used to quantitatively or qualitatively detect the presence of target gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluoresence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
Cancers that may be diagnosed, and/or prognosed using the methods described above include but are not limited to, stomach cancer, renal cancer, brain cancer, bladder cancer, colon cancer, lung cancer, breast cancer, pancreatic cancer, ovarian cancer, and prostate cancer.
Immunoassays
Target-specific antibodies or immunospecific fragments thereof disclosed herein may be assayed for immunospecific binding by any method known in the art. The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, Vol. 1 (1994), which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).
The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by Scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest is conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second antibody.
Target-specific antibodies may, additionally, be employed histologically, as in immunofluorescence, immunoelectron microscopy or non-immunological assays, for in situ detection of cancer antigen gene products or conserved variants or peptide fragments thereof. In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled target-specific antibody or fragment thereof, preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.
Surface plasmon resonance (SPR) as performed on BIAcore offers a number of advantages over conventional methods of measuring the affinity of antibody-antigen interactions: (i) no requirement to label either antibody or antigen; (ii) antibodies do not need to be purified in advance, cell culture supernatant can be used directly; (iii) real-time measurements, allowing rapid semi-quantitative comparison of different monoclonal antibody interactions, are enabled and are sufficient for many evaluation purposes; (iv) biospecific surface can be regenerated so that a series of different monoclonal antibodies can easily be compared under identical conditions; (v) analytical procedures are fully automated, and extensive series of measurements can be performed without user intervention. BIAapplications Handbook, version AB (reprinted 1998), BIACORE code No. BR-1001-86; BIAtechnology Handbook, version AB (reprinted 1998), BIACORE code No. BR-1001-84.
Epitope specificity is an important characteristic of a monoclonal antibody. Epitope mapping with BIAcore, in contrast to conventional techniques using radioimmunoassay, ELISA or other surface adsorption methods, does not require labeling or purified antibodies, and allows multi-site specificity tests using a sequence of several monoclonal antibodies. Additionally, large numbers of analyses can be processed automatically.
Peptide inhibition is another technique used for epitope mapping. This method can complement pair-wise antibody binding studies, and can relate functional epitopes to structural features when the primary sequence of the antigen is known. Peptides or antigen fragments are tested for inhibition of binding of different MAbs to immobilized antigen. Peptides which interfere with binding of a given MAb are assumed to be structurally related to the epitope defined by that MAb.
The Examples that follow are set forth to aid in the understanding of the inventions but are not intended to, and should not be construed to limit its scope in any way.
EXAMPLES
Example 1
Selection of hHER2/ErbB2 Specific Fabs from Phage Libraries
Recombinant human HER2 ectodomain was used to screen a human naïve phagemid Fab library containing 3.5×1010 unique clones (Nat Biotechnol. 2005 March; 23(3):344-8.) Biotinylated hHer2-Fc protein was captured on steptavidin-coated magnetic beads prior to incubation with the phage library. Selections were performed as described previously, with depletion on hEGFR-Fc to eliminate Fc specific binders as well as EGFR cross-reactive binders (Nat Biotechnol. 2005 March; 23(3):344-8). After 3 rounds of panning, the 479 by gene III stump was removed by M/u/digestion, and the vector was religated for soluble Fab expression in TG1 cells. ELISA analysis of 920 clones yielded 224 positive clones, containing 79 unique sequences. Unique clones were purified and binding was reconfirmed at a single concentration to recombinant human hHER2 ectodomain by ELISA as well as by FACS on CHO cells stably transfected with full-length human HER2 (see below for cell line construction). Based on binding data, 24 unique clones (65A03, 65B03, 65C10, 65H09, 66A12, 66C01, 67A02, 67C12, 67F10, 67F11, 68B11, 68D03, 69A09, 69E02, 69F02, 70C01, 70C08, 70D11, 71A03, 71A06, 71F08, 71F10, 71H02 and 72H10) were selected for further analysis.
Example 2
Binding Activity of Fabs to hHER2/ErbB2 Measured by Flow Cytometry
The ability of Fabs to bind to the wild type hHER2/ErbB2 was determined by flow cytometry using CHO cell line stably transfected with hHER2/ErbB2.
CHO cells (Chinese Hamster Ovary cells) stably transfected with hHER2 expression vector and selected in G418 containing medium. G418 resistant clones were pooled and split 24 hours prior to the setup of the assay to obtain 70% confluent monolayer. Routinely, CHO cell line was maintained within 20 passages. Cells were lifted with cell dissociation buffer (Gibco catalog #13151-014), counted, washed and adjusted to 1×106 cells/ml and one ml of cells were then added to each tube (12×75 mm tube Falcon catalog #352054). Cells were pelleted and supernatant removed by centrifugation at 1200 rpm for 5 min and 100 μl of diluted antibodies were then added to the cell pellet. Purified Fabs were tested at a starting concentration of either 210 or 60 μg/ml with 1:3 dilutions in FACS buffer, down to 0.001 μg/ml. FACS buffer used throughout the assay was PBS (without Ca++/Mg++) containing 1% BSA (Sigma catalog #A-7906) and 0.1% Sodium Azide (Sigma catalog #S2002). Samples were allowed to incubate on ice for 1 hour and 15 minutes then were washed with 2 ml FACS buffer and centrifuged at 1200 rpm for 5 minutes at 4° C. The supernatant was aspirated and 100 μl of the secondary detection antibody was added to each corresponding tube in FACS buffer. Samples were then incubated for 30 minutes on ice, in the dark. Cells were washed as described above, then, re-suspended in 250 μl FACS buffer per tube/sample.
Cell bound Fabs were detected using FITC-conjugated affinity-purified F(ab′)2 Fragment specific goat anti-human-IgG (Jackson ImmunoResearch Lab catalog #109-096-006; used at 5 μg/ml), while positive murine control antibody was detected using the F(ab′)2 FITC conjugated goat anti-mouse IgG (H+L) (Jackson ImmunoResearch, catalog#115-096-062; used at 5 μg/ml). Cells were stained for live cell determination with Propidium Iodide staining solution (PI for dead cell exclusion; BD Pharmingen catalog #51-66211E or 556463; use at 1:500 final in FACS buffer). Samples were run on the FACSCalibur instrument (Becton Dickinson) with 10,000 live events collected per sample. Data analysis was done using GraphPad Prism version 4.0 software (www.graphpad.com) (GraphPad Software, Inc., 11452 El Camino Real, #215, San Diego, Calif. 92130 USA).
Once samples have been run and geometric means determined, antibody concentration (X axis) vs. geometric mean (Y axis) was graphed to the log10, using Graphpad Prism (Prism Graph) graphing program. Data sets were then transformed (X value data set=antibody concentration) to X=Log(X) and graphed using a nonlinear regression curve fit, Sigmoidal dose-response. EC50 values and R2 values were generated using the Prism Graph software.
24 Fabs identified from the screening in Example 1 were tested at multiple concentrations by FACS on human HER2-CHO cells and untransfected CHO cells to confirm specificity (data not shown and Table 1)
12 Fabs (65B03, 65C10, 65H09, 66A12, 66C01, 67A02, 67C12, 67F10, 67F11, 69A09, 71A06 and 71F10) showed good binding activity to wild type HER2/ErbB expressed on CHO cells. The Fabs were tested on the MDA-MB-468 tumor cell line (EGFR+, HER2−); no binding was detected, demonstrating that these Fabs are specific to HER2. A full titration was performed on MCF7 and SKBR3 tumor cell lines, and human Her2-CHO and murine Her2-CHO cell lines in order to calculate an approximate EC50 (data not shown, see summary in Table 1). The 12 Fabs were also tested on CHO cells stably transfected with full-length murine HER2 and cyno HER2; One Fab (71F10) was found to bind murine HER2 and nine were found to bind to cyno HER2 (Table 1).
Example 3
Binding Activity of Fabs to Human and Mouse HER2/ErbB2 Measured by ELISA
The ability of Fabs to bind to the wild type hHER2/ErbB2 and mHER2/ErbB2 was determined by Enzyme-Linked ImmunoSorbent Assay (ELISA).
96 well microtiter Immulon II plates (Fisher, Cat# 14245-61) were coated with 100 ul/well of 2 ug/ml unlabeled Gt-anti-hu IgG or unlabeled Gt-anti-hu Kappa (SouthernBiotech, Cat# 2040-01; 2060-01) in Na2CO3/NaHCO3 buffer, pH 9.5 overnight at 4° C. and dumped coating buffer out of plate. In the next step, 100 ul dilution buffer (0.5% nonfat dry milk in PBS plus 0.01% thimerosal) and 100 ul individual supernatant or purified protein in dilution buffer were added to duplicate wells and incubated for 1 h at 37° C. After washing with tap water, 100 ul of a 1/10,000 dilution in dilution buffer of Gt-anti-hu Kappa-HRP (SouthernBiotech, Cat# 2060-05) was added to the wells and incubated for 1 h at 37° C. After washing, 100 ul/well of a HRPO Substrate combined TMB Peroxidase Substrate/Peroxidase Solution B (Kirdgaard and Perry Labs, Cat. 50-76-00) was added. The reaction was stopped with 100 ul of 2M H2SO4 after 5 to 10 min. The OD was measured at 450 nm and 540 nm using a Molecular Devices plate reader and binding curves were generated.
Twenty two of the twenty four Fabs showed moderate/strong binding activity to wild type hHER2/ErbB2 (data not shown and Table 1). Only Fab 71F10 showed moderate/strong binding activity to wild type mHER2/ErbB2 (data not shown and Table 1).
Example 4
Antibody Cross-reactivity
The Cynomolgous Monkey HER2 cDNA was amplified by RT-PCR from kidney mRNA of Cynomologous monkey kidney (BioChain Institute, Inc, Hayward, Calif.). PCR primers are: for cyno HER2 extracellular domain forward primer, GAGCCATGGGGCCGGAGCCGCAGTGAGCACCATG (SEQ ID NO:159); reverse primer,
TCGGGGCTTCTGCGGACTTGGCCTTCTGGTTCAC (SEQ ID NO:160); for the intracelluar domain: forward primer: GCCCAACCAGGCGCAGATGCGGATCCTGAAAGAG (SEQ ID NO:161); reverse primer: CCAGATCCAAGCACCTTCACCTTCCTCAGCTCCG (SEQ ID NO:162). The amplified PCR products were cloned into a TA cloning vector, pCR2.1 (Invitrogen) and sequenced. The full length cyno HER2 cDNA was assembled at the BsmB I site from both extracellular and intracellular domain sequences. The plamid is termed pCR2.1cynoHER2.
To express cyno HER2 in cell lines, the HER2 cDNA was digested from pCR2.1cynoHER2 using NotI and HindIII and cloned into a lentiviral vector plasmid under the control of the hCMV promoter. Lentiviral vector was produced and the culture supernatant was used to transduce HEK 293 cells.
To establish a CHO cell line that expresses mouse HER2, a mouse HER2 coding sequence was PCR amplified from a plasmid containing mouse HER2 cDNA (MGC:62447 IMAGE:570). PCR primers: forward primer: CATGGCGGCCGCCCGGAGCCGCAGTGATCATC (SEQ ID NO:163); and reverse primer: CGATGCGGCCGCGGATGTCTGCACATGTGACC (SEQ ID NO:164). The PCR product was inserted into a mammalian expression vector pV90. The resultant plasmid was termed pKJS462.21. pKJS462.21 DNA was transfected into DG44-1 CHO cells and selected for stable integration by culturing in alpha minus MEM with 10% dialyzed FBS (Hyclone#SH30079.03). The bulk stable population was subcloned and a single clone expressing high levels of HER2, termed, 3G11B, was selected for Fab cross reactivity analysis for mHer2 using FACS. This cell line was generally referred as CHO/mHer2.
Cross reactivity to rat Her2 was analyzed by FACS analysis using Tubo cancer cells (derived from rat HER2 transgenic mouse).
Lentiviral Vector Plasmid Construction
pLenti6/TR from Invitrogen kit, V480-20, was modified by replacing the Tet Repressor gene with a multiple cloning site to bridge the BamH1 to EcoR1 sites. Sense oligo GATCCCCGGGTACCGGTCGGCGCGCCTCGAGATATCTTAATTAAG (SEQ ID NO:115) was annealed to antisense AATTCTTAATTAAGATATCTCGAGGCGCGCCGACCGGTACCCGGG (SEQ ID NO:116) and ligated into the BamH1/EcoR1 digested backbone. This plasmid (CK072) contains a multiple cloning site downstream of the CMV promoter with SV40 promoter driving a blasticidin resistance marker. The cynomolgus monkey HER2 gene was PCRed in two pieces for sequencing using oligos GAGCCATGGGGCCGGAGCCGCAGTGAGCACCATG (SEQ ID NO:117) and antisense CCAGATCCAAGC-ACCTTCACCTTCCTCAGCTCCG (SEQ ID NO:118) for the extracellular portion and GCCCAACCAGGCGCAGATGCGGATCCTGAAAGAG (SEQ ID NO:119) and antisense TCGGGGCTTCTGCGGACTTGGCCTTCTGGTTCAC (SEQ ID NO:120) for the intracellular portion. The PCR products were TA-TOPO cloned into pCR2.1 (Invitrogen K4500-01) and sequenced. Finally, the consensus cyno Her2 sequences were assembled from extracellular and intracellular plasmids (joined at the BsmB1 site), excised with SpeI/XbaI digestion, and ligated into the downstream XbaI site of CK072 described above. The resulting lentiviral vector plasmid (CK090.18) was used to generate vector for overexpressing cyno HER2 in target cell lines.
Vector Production and Transduction
293FT cells (Invitrogen #R700-07) were co-transfection with the Invitrogen Virapower packaging mix (K4975-00) and lentiviral vector plasmid CK90.18 described above, following the manufacturer's instructions for Lipofectamine 2000 (Invitrogen #11668-019). Culture supernatant was harvested 48 hours later, cellular debris pelleted at 1250 g for ten minutes, and the clarified supernatant was 0.45u filtered. Supernatants were applied to HEK293 cells in order to varify the ability of the vector to deliver the cyno Her2 transgene. Stably transduced HEK 293 cells showed high level expression of the trangene in a portion of a mixed population of positive and negative cells.
Cross Reactivity Test
A pool population of transduced and untransduced HEK293 cells were treated to the various Fabs and stained with FITC conjugated secondary antibodies for viewing as follows. Briefly, HEK 293 cells expressing cynoHer2 were plated in CC2-coated 8-well chamber slides (Nunc #154941) and allowed to attach overnight in the incubator (5% CO2, 37° C.). The growth medium was replaced with 50 to 75 ul of the Dyax Fabs (10 mg/ml) and these were allowed to bind for 90 minutes at 4oC. Cells were rinsed with dilution buffer (PBS with 10% FBS) and then fixed for 20 minutes at room temperature with 4% formaldehyde in PBS. The fixed cells were rinsed and incubated with secondary conjugated antibodies for 45 minutes at 4° C. The secondary antibody solution was a 50:50 mix of FITC conjugated goat anti human kappa chain and goat anti human IgG F(ab′)2 specfic fragment (Sigma #F3761 and Jackson Immun. #109-096-097 respectively). The wells were rinsed with dilution buffer to get rid of unbound antibodies before viewing in the Zeiss Axiovert fluorescent microscope.
Results: Nine out of twelve Fabs selected for testing showed cross-reactivity with cynoHER2 (Table 1). One Fab, 71-F10, showed moderate binding cross-reactivity to mHER2/ErbB2 (Table 1) was confirmed in this assay. 71F10 also showed cross-reactivity with rat HER2 (data not shown).
Example 5
HER2/ErbB2 Fab Epitope Mapping
Amino acid sequences of human ErbB2 ECD (SEQ ID NO:105) and Rhesus ErbB2 ECD (SEQ ID NO:106) are shown in FIG. 4 with cysteine pairing. Residues different between human and rhesus are highlighted. Potential N-glycosylation sites are underlined.
Human ErbB2-encoding DNA fragments were obtained by PCR from pLXSN-HER2. ErbB2-Fc vectors for expression in CHO cells were generated by cloning each ErbB2 DNA fragment (residues Thr1-Thr196 (Domain 1), Thr1-Arg318 (Domains 1-2), Thr1-Asn508 (Domains 1-3) or Thr1-Asn630 (Domains 1-4) of human HER2/ErbB2 ECD) in frame with a huIgG1-Fc into the PV90 vector. The protein constructs are denoted MR066 (Domain 1), MR067 (Domain 1-2), MR068 (Domain 1-3) and MR069 (full length ECD).
Proteins were transiently expressed in 293E mammalian cells. After 4 days of culture at 37° C., the cell supernatants were harvested, filtered and purified on Protein A Sepharose. Protein was eluted from column with 25 mM H3PO4/NaOH, 0.1 M NaCl, pH 2.8 and neutralized immediately with 1/20th volume 0.5 M sodium phosphate, pH 8.6. Protein was evaluated for purity and degree of aggregation by SDS-PAGE and analytical SEC. Protein concentration was determined by UV absorbance using the sequence-predicted extinction coefficient at 280 nm for each protein.
The ability of 65B03, 66C01, 67F10, 67F11, 71F10, 69A09, 66A12, 65C10, 65H09 and 67A02 Fabs and control anti-HER2 antibody to bind MR066, MR067, MR068 and MR069, was determined by ELISA. Nunc Maxisorp ELISA plates were coated with 5 ug/ml rabbit pAbs anti-human Fc gamma in PBS overnight at 4 degrees. Plates were washed one time with PBST (PBS, 0.05% tween 20) and blocked with 1% BSA in PBS for one hour at room temperature. Plates were washed one time with PBST and incubated with 1 ug/ml HER2-Fc fusion protein for one hour. Plates were washed three times with PBST and dilutions of Fab (four 5-fold serial dilutions, starting at 1 ug/ml) in block were added and incubated for ninety minutes. Plates were washed three times with PBST and incubated with 1:2,000 HRP-labelled goat anti-human kappa (Southern Biotech cat#2060-05) and HRP-labelled goat anti-human lambda (Southern Biotech cat#2070-05) in block for one hour at room temperature. Plates were washed three times with PBST, developed with TMB solution for 5 minutes, and stopped with equal volume 1 NH2SO4. Absorbance values were read at 450 nm. The results are shown in FIG. 5.
Example 6
Construction of 71F10 Fab-hLIGHT Fusions with Delta4; G4Sdelta4 (SEQ ID NO: 147) and (G4S)4 (SEQ ID NO: 134) Linkers
The protein sequences of the 71F10 mature heavy chain variable domain (VH) and Light chain variable domain (VL) are shown in SEQ ID NO:11 and 13, respectively. The CDRs, (complementarity determining regions, based upon the Kabat designations) of 71F10 heavy chain and light chain are shown in SEQ ID NOs:47-49 and 74-76, respectively. Alignment of partial amino acid sequences between 71F10 VH (residues 1-97 of SEQ ID NO:11) and HV3-23 VH (residues 1-97 of SEQ ID NO:107) shows 93% (91/97) identities and 95% (93/97) positives (parameters: Length=98, Score=186 bits (471), and Expect=3e-050). HV3-23 refers to the human germline HV3-23 sequence with Genbank accession number M99660. Nucleotide sequences of 71F10 VH and VL are shown in SEQ ID NOs:156 and 14, respectively. The nucleotide sequence of 71F10 VH (SEQ ID NO:156) is codon-optimized for mammalian cell expression. The amino acid and nucleotide sequences of HV3-23 VH are shown in SEQ ID NOs:107 and 108, respectively.
The scheme of 71F10 Fab-hLIGHT construction is described as follows.
71F10 VL region was synthesized by PCR amplification using the oligonucleotide primers described in 110-F1 (SEQ ID NO:121), C06-5′ (SEQ ID NO:122) and 039-VLR (SEQ ID NO:123). The 5′ VL PCR primers consisted of a forward primer 110-F1 (SEQ ID NO:121) including a Not I restriction endonuclease site (GCGGCCGC (SEQ ID NO:180)) followed by sequences encoding a partial Light chain signal peptide and an internal forward primer C06-5′ (SEQ ID NO:122) encoding a partial Light chain signal peptide followed by sequences complementary to the amino terminus of 71F10 VL. The 3′ VL PCR primer 039-VLR (SEQ ID NO:123) included a BsiW I restriction endonuclease site (CGTACG (SEQ ID NO:181)) and sequences encoding the carboxyl terminus of 71F10 VL. The 71F10 VL region was amplified in two sequential PCR reactions through the common overlapping sequences encoding the Light chain signal peptide using these three PCR primers from plasmid DNA CPG169 containing the 71F10VL. The 71F10 VL gene fragment was cloned into the Not I/Bsiw I digested the modified pV100 vector which contained a BsiW I site at the amino terminus of the human kappa domain. Correct sequences were confirmed by DNA sequence analysis.
71F10 VH region was recoded by replacing with the consensus codons from human immunoglobulin VH database without change in polypeptide sequence and eliminating the putative cryptic splice donor and acceptor sites by synonymous codon exchange. The recoded 71F10 VH region was synthesized by assembly PCR amplification using the oligonucleotide primers described in VH-FR1-F (SEQ ID NO:124), VH-FR2-R (SEQ ID NO:125), VH-FR3-F (SEQ ID NO:126), 71F10VH-CDR1-F (SEQ ID NO:127), 71F10VH-CDR2-F (SEQ ID NO:128), 71F10VH-CDR3+FR4-R (SEQ ID NO:129), VH-pV90-F (SEQ ID NO:130), and VH-pV90-R (SEQ ID NO:131). In the first PCR step, two sets of three oligodeoxynucleotides (oligos) were annealed and elongated to produce a half-length product. The 5′ half of the recoded 71F10 VH was generated by PCR using the forward 5′ primer VH-FR1-F (SEQ ID NO:124) which consists of nucleotide sequences encoding the 71F10 VH framwork1 region and the forward 5′ primer 71F10VH-CDR1-F (SEQ ID NO:125) which consists of nucleotide sequences encoding the 71F10 VH CDR1 region and the reverse 3′ primer VH-FR2-R (SEQ ID NO:126) which consists of nucleotide sequences encoding the 71F10 VH framework 2 region. The 3′ half of the recoded 71F10 VH was generated by PCR using the forward 5′ primer 71F10VH-CDR2-F (SEQ ID NO:127) which consists of nucleotide sequences encoding the 71F10 VH CDR2 region and the forward 5′ primer DyaxVH-FR3-F (SEQ ID NO:128) which consists of nucleotide sequences encoding the 71F10 VH framework 3 region and the reverse 3′ primer 71F10VH-CDR3+FR4-R (SEQ ID NO:129) which consists of nucleotide sequences encoding the 71F10 VH CDR3 and framework 4 regions. In the second PCR step, the full-length recoded 71F10 VH gene sequence is selectively amplified from two mixtures of first PCR step using primers specific for the desired full-length product. The 5′ forward primer, VH-pV90-F (SEQ ID NO:130) which contained a unique Mlu I restriction endonuclease site (ACGCGT (SEQ ID NO:160)) followed by sequences encoding the last three amino acids of the heavy chain signal peptide followed by sequences complementary to the amino terminus of the recoded 71F10 VH and the 3′ reverse primer, VH-pV90-R (SEQ ID NO:131) which contained a Nhe I site (GCTAGC (SEQ ID NO:182)) and sequence encoding complementary to the carboxyl terminus of the recoded 71F10 VH. The recoded 71F10 VH gene fragment was cloned into the Mlu I/NheI digested the modified pV90 vector which contained a synthetic heavy-chain leader sequence and a Mlu I site followed by a BamH I site at the amino terminus of the IgG1 CH1 domain. Correct sequences were confirmed by DNA sequence analysis.
Three different linkers (SEQ ID NOs:132-134) were used to connect the 71F10 Fab heavy chain to the amino terminus of the huLIGHT and the amino acid sequences of these linkers are shown in Delta 4 (SEQ ID NO:132), G4Sdelta4 (SEQ ID NO:133), and (G4S)4 (SEQ ID NO:134). The optimal linker length was determined as follow:
1. The initial 3D structure of Fab was built through homology modeling using Modeller 9 based on crystal structure of human IgG(pdb: 1 HZH). Five pairs of Cysteines were constrained to form disulfide bond. The 3D LIGHT/LTbR structure was constructed based on crystal structure of TNFbeta/TNF-R p55(pdb: 1TNR).
2. Trimeric Fab structure was constructed with C3 symmetry using in-house program.
3. Energetic analysis was carried out by changing distance between trimeric Fab and LIGHT/LTbR with C3 axis aligned. d is the distance between the one point (intersection point of Fab axis and C3 axis) and closed point (intersection of surface and C3 axis) of LIGHT/LTbR. The vdW interaction energy was calculated with each structure. 25A is the minimum distance; otherwise there will be significant repulsion between trimeric Fab and LIGHT; The interaction energy is zero beyond 40A. In our model for linker optimization, 40A was chosen as the starting geometry.
4. Four different linkers were constructed and evaluated using Modeller: (G4S)2 (SEQ ID NO: 151), (G4S)4 (SEQ ID NO: 134), delta4 (natural LIGHT linker), G4S+delta (SEQ ID NO: 147). The results indicate that (G4S)2 (SEQ ID NO: 151) is too short while the other three are linkers of appropriate length.
The 71F10 Fab-hLIGHT fusion heavy chains were constructed in the PCR reactions using the 5′ forward+3′ reverse PCR primers and internal overlapping PCR primer sets encoding three different linkers from plasmid DNAs N5KG1 containing the huIgG1 and pABF046 containing the huLIGHT. The oligonucleotide primers were described as MB-04F (SEQ ID NO:135), 130-R1 (SEQ ID NO:136), 130-F2 (SEQ ID NO:137), 130-R2 (SEQ ID NO:138), 131-R2 (SEQ ID NO:139), 132-F2 (SEQ ID NO:140), and 132-R2 (SEQ ID NO:141). Briefly, the 5′ forward primer, MB-04F (SEQ ID NO:135) which included an Age I site (ACCGGT (SEQ ID NO:161)) followed by sequences complementary to the IgG1 CH1 region. The 3′ reverse primer, 130-R1 (SEQ ID NO:136) which contained a BamH I site (GGATCC (SEQ ID NO:177)) and sequence encoding complementary to the carboxyl terminus of huLIGHT. The 5′ forward primers, 130-F2 (SEQ ID NO:137) and 132-F2 (SEQ ID NO:138) of the internal overlapping PCR primer sets included the three partial linkers followed by sequences complementary to the amino terminus huLIGHT. The 3′ reverse primers, 130-R2 (SEQ ID NO:139), 131-R2 (SEQ ID NO:140), and 132-R2 (SEQ ID NO:141) of the internal overlapping PCR primer sets contained sequences encoding a partial IgG1 hinge followed by the three partial linkers. The PCR fragments were then finally assembled in a second PCR reaction using sequences encoding the overlapping three linkers and digested with the Age I and BamH I restriction endonucleases and ligated into the Age I/BamH I digested 71F10 IgG1 heavy chain vector. This resulted in fusion products of the 71F10 Fab heavy chain to the amino terminus of the huLIGHT. Correct sequences were confirmed by DNA sequence analysis.
The 71F10 light chain vector (pBIIB71F10-129) was commonly used in the 71F10Fab-hLIGHT fusions and the DNA (SEQ ID NO:110) and amino acid (SEQ ID NO:109) are disclosed herein. Heavy chain DNA and amino acid sequences for the 71F10Fab-hLIGHT fusions (pBIIB71F10-130, pBIIB71F10-131, and pBIIB71F10-132) and their physical and chemical parameters are described herein as SEQ ID NOs:2-4 and 6-8, respectively.
The 71F10 agly IgG heavy-chain (pBIIB71F10-137) was constructed using QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene Cat# 200513) using the 71F10 IgG heavy-chain (pBIIB71F10-134) as a template. A threonine amino acid was changed to an alanine amino acid at position 299 (kabat number) in CH2 domain by primer 137-F which contains a desired mutation. DNA and amino acid sequences for the 71F10 IgG heavy-chain (pBIIB71F10-134) are shown in SEQ ID NO:157 and 158, respectively. DNA and amino acid sequences for the 71F10 agly IgG heavy-chain (pBIIB71F10-137) are shown in SEQ ID NOs:153 and 154, respectively. The 71F10 light chain vector (pBIIB71F10-129) was common used in the 71F10 IgG and 71F10 agly IgG antibodies.
The oligonucleotide (forward 5′ PCR primer 137-F) used for Site-Directed Mutagenesis of the 71F10 agly IgG heavy-chain is 5′-GAGGAGCAGTACAACAGCGCCTACCGTGTGGTCAGCGTC-3′ (SEQ ID NO:155) which includes a desired point mutation codon (underlined).
Example 7
Construction of Anti-CD23 Fab-hLIGHT Fusion, BIIB CD23-204
Anti-CD23 VH region was synthesized by PCR amplification using the oligonucleotide primers described in 204-F (SEQ ID NO:142) and DyaxVH-pV90-R (SEQ ID NO:143). The 5′ forward primer, 204-F (SEQ ID NO:142) which contains a unique Mlu I restriction endonuclease site (ACGCGT (SEQ ID NO:160) followed by sequences encoding the last three amino acids of the heavy chain signal peptide followed by sequences complementary to the amino terminus of the anti-CD23 VH. The 3′ reverse primer, DyaxVH-pV90-R (SEQ ID NO:143) which contained a Nhe I site (GCTAGC (SEQ ID NO:182) and sequence encoding complementary to the carboxyl terminus of the anti-CD23 VH. The anti-CD23 VH region was amplified in a PCR reaction from plasmid DNA pBIIB CD23-121 containing the anti-CD23 VH gene. The anti-CD23 VH gene fragment was cloned into the Mlu I/Nhe I digested the pBIIB71F10-132. Correct sequences were confirmed by DNA sequence analysis. The resultant vector is termed pBIIBCD23-204. The anti-CD23 light chain vector (pBIIBCD23-178) was used in the anti-CD23Fab-hLIGHT fusion. DNA and amino acid sequences for the light chain of the anti-CD23 Fab-hLIGHT fusion are shown in SEQ ID NOs:104 and 103, respectively. DNA and amino acid sequences for the heavy chain of the anti-CD23 Fab-hLIGHT fusion (pBIIBCD23-204) are shown in SEQ ID NOs:173 and 174, respectively. The amino acid and nucleotide sequences corresponding to heavy chain of 71F10 Fab are shown starting from the N-terminus; followed by amino acids corresponding to the linking group (amino acids 224 to 243); followed by the amino acids corresponding to human LIGHT extracellular domain (amino acids 244 to 391).
A second anti-CD23Fab-hLIGHT fusion was also made with a (G4S)3 (SEQ ID NO: 148) linker for evaluation (data not shown). DNA and amino acid sequences for the light chain of the anti-CD23 Fab-hLIGHT fusion with a (G4S)3 linker are shown in SEQ ID NOs:104 and 103, respectively. DNA and amino acid sequences for the heavy chain of the anti-CD23 FAb-hLIGHT fusion with a (G4S)3 linker are shown as SEQ ID NOs:102 and 101, respectively. The amino acid and nucleotide sequences corresponding to heavy chain of 71F10 Fab are shown starting from the N-terminus; followed by amino acids corresponding to the linking group (amino acids 222 to 236); followed by the amino acids corresponding to human LIGHT extracellular domain (amino acids 237 to 384).
Example 8
Expression of 71F10 Fab-hLIGHT Fusions in CHO Cells
A summary of 71F10 constructs used for expression is shown in FIG. 6.
Transient Expression of 71F10 Fab-hLIGHT in CHO Cells
The host DG44 suspension cells were maintained in CD-CHO (25%) and DMEM/F12 (75%), and two identical transfections were carried out. For each transfection, the cells were seeded at 7.5×105 cells/well in a six-well plates and cultured for 24 hours; then the cells were transfected with 1 μg of pBIIB71F10-129 (Light chain in pV100) and 1 μg of pBIIB71F10-130, pBIIB71F10-131, pBIIB71F10-132, pBIIB71F10-134 or pBIIB71F10-137 (heavy chain in pV90) using FuGENE transfection reagent (Roche) according to the manufacturing protocol. The RR399 vector which contains the 71F10 IgG1 agly heavy chain with original VH sequence was used as a control in CHO cell expression. 48 hour later, the supernatant was harvested and the titer was determined by Octet (ForteBio) with a surrogate standard according to quantitation method from manufacturing protocol. The titers of 71F10Fab/Hlight fusions from transcient transfection are 0.06 ug/ml (pBIIB71F10-137), 1.6 ug/ml (pBIIB71F10-134), 0.9 ug/ml (pBIIB71F10-130), 0.2 ug/ml (pBIIB71F10-131), and 1.0 ug/ml (pBIIB71F10-132).
Stable Expression of 71F10 Fab-hLIGHT in CHO Cells
The host DG44 suspension cells were maintained in CHO-SSFMII (Invitrogen), and two identical transfections were carried out. For each transfection, the cells were seeded at 7.5×105 cells/well in a six-well plates and cultured for 24 hours; then the cells were transfected with 1 μg of pBIIB71F10-129 (Light chain in pV100) and 1 μg of pBIIB71F10-130, pBIIB71F10-131, pBIIB71F10-132, pBIIB71F10-134 or pBIIB71F10-137 (heavy chain in pV90) using FuGENE transfection reagent (Roche) according to the manufacturing protocol. 48 hour later, cells from two transfections were combined and splited into three T-75 flasks that each contained 15 ml CHO-SSFMII supplemented with 400 μg/ml of genticin (Invitrogen). After two or three week selection, the cells either sorted or as an unsorted pool were scaled up in CHO-SSFMII with 400 μg/ml of genticin and later in BCM16 with 400 μg/ml of genticin for production run. In the end of the run, the supernatant was harvested and the titer was determined by Octet (ForteBio) with a surrogate standard.
Octet Assay
Analysis of Fab-LIGHT fusions by biolayer interferometry (Octet QK System, ForteBio, Inc. Menlo Park, Calif.). Anti-human Fc-specific biosensors (Fortebio SKU 18-0001) were hydrated in OB (1×PBS supplemented with 1 mg/ml BSA, 0.05% NaN3, and 0.02% TWEEN 20) for at least five minutes prior to assays. For each assay, the respective receptor Fc fusion was diluted to 10 μg/ml in OB and allowed to bind to the biosensors for five minutes. The sensors were then incubated in the indicated culture supernatant for an additional five minutes, and subsequently transferred to fresh wells containing. As a control, the behavior of the 71F10 FAb (10 μg/ml in OB) was also examined in this assay. Dissociation constants (kd) were calculated using software provided by the manufacturer and off-rates compared to the kd of the 71F10 Fab.
Example 9
Purification of 71F10 Fab-hLIGHT Fusion Proteins
The fusion protein produced in CHO cells were purified and characterized by methods described below.
Protein A Capture: Pre-equilibrate the Protein A column with 1×PBS (equilibration buffer) at 100-150 cm/hr with 3 column volumes. Load the supernatant at 150 cm/hr with a maximum of 10 mg of 71F10 Fab-hLIGHT per milliliter of resin. After loading, wash the column with 5 column volumes of equilibration buffer. Then, step elute in an upflow direction with 100 mM Glycine, pH 3.0. Collect desired fractions and titrate to neutral pH with 2M Tris base. Dialyze collected fractions against 1×PBS and concentrate material to prepare for the size exclusion step.
SUPERDEX(r) 200 (Size Exclusion) aggregate removal step involved equilibration of SUPERDEX(r) 200 with 1×PBS with 1.5 column volumes at a flow rate of 36 cm/hr followed by loading of protein and collecting desired fractions.
Identity testing performed as follows
1). Intact mass analysis by mass spectrometry where molecular mass measurements were performed on an electrospray mass spectrometer (ESI-MSD). Prior to analysis, the sample was reduced to remove disulfide bonds. The deconvoluted mass spectrum represents the masses of the heavy and Light chains.
2). N-terminal sequence analysis was performed by Edman degradation using an ABI protein sequencer equipped with an on-line PTH analyzer. The sequences for the initial amino acids of the Light chain and heavy chain were identified.
3). Peptide mapping with mass spectrometric analysis: tryptic or/and EndoLysC peptide maps were performed to obtain complete sequence coverage by analysis of the LC/MS data generated from each peptide. In addition, determination of sites and amounts of oxidation and deamidation were detected.
Purity testing was performed by; 1) SDS-Page or CE-SDS: Reduced and non-reduced samples, this technique is used to measure antibody fragmentation, aggregation and impurities, 2) SEC-HPLC with LS and R1 technique was used to measure aggregation and fragmentation and LIGHT scattering determines the molar mass of sample components. 3) SDS gel or capillary IEF method was used to determine the isoelectric focusing pattern and pI distribution of charge isoforms that can result from C- and N-terminal heterogeneity and/or deamidation.
Finally, endotoxin concentrations were measured by the Limulus amoebocyte lysate (LAL) kinetic turbidometric method.
Example 10
Binding Activity of 71F10 Fab-hLIGHT Fusion Proteins
The concentration of 71F10 Fab-Light fusion proteins and the 71F10 mAb expressed transiently by 293E cells was quantified by ELISA, using purified 71F10 Fab as a standard. Wells of 96-well plates were coated by incubation overnight at 4-8° C. with goat antibodies against human kappa chain (80 ul/well of a 5 ug/ml solution in PBS). Wells were emptied, filled completely with a solution of PBS with 1% BSA (blocking buffer) and incubated at room temperature for 1 hour. Wells were emptied, washed twice PBS containing 0.05% Tween 20 (PBST) and filled with the samples (80 ul/well of 3× dilution series of the standard and supernatants in blocking buffer) prepared in another 96-well plate. After a 2-hour incubation at room temperature, the wells were emptied, washed three times with PBST and filled with a solution of HRP-conjugated goat pAbs anti-human kappa chain (80 ul/well of a 2000-fold dilution in blocking buffer). After 1-hour incubation, the wells were emptied and filled with a solution of HRP substrate prepared freshly. The color was left to develop for 3 minutes and was stopped by addition of an equal volume of 1N H2SO4. Absorbance was measured at 450 nm using a Plate Reader (SpectraMax, Molecular Devices). The data was processed and graphs were created using SoftMax Pro software.
A similar ELISA format was used to compare the binding of control anti-HER2 antibody, control anti-HER2 antibody Fab, 71F10 Fab, 71F10 mAb and 71F10 Fab-Light fusion proteins to human (FIG. 7) or murine (FIG. 8) HER2-Fc. control anti-HER2 antibody was obtained from Pharmaceuticals Buyers International, Inc. The Fab of control anti-HER2 antibody was prepared by limited papain digestion and purified using standard technology. Human and murine HER2-Fc (homodimeric fusion proteins consisting of the extracellular domain of HER2 and the Fc fragment of human IgG1) were obtained from R&D Systems. The ELISA was done using the same protocol as described before with the exception that the wells were coated with murine or human HER2-Fc instead of goat anti-human kappa antibodies.
Example 11
Characterization of 71F10 Fab-hLIGHT Trimer
The Fab, the mAb and Fab-Light fusion proteins were purified by chromatography on protein A Sepharose and Size Exclusion to isolate material substantially devoid of aggregates. The purified proteins were quantified by UV absorbance at 280 nm using the sequence-predicted extinction coefficient. Purity was evaluated by SDS-PAGE and size-exclusion HPLC. The molecular mass was measured by SEC/static light scattering analysis using a BioSeptember 3000 column (Phenomenex) in PBS with a Waters Alliance HPLC instrument coupled to a refractive index detector (Waters) and light scattering detector (PD2000, Precision Detectors). The average molecular masses were determined using the Precision Detector software (FIG. 9).
Example 12
SEC/LS Analysis of 71F10 Fab-hLIGHT/shuLTBR Complex
Soluble human LTBR (shuLTBR) was prepared by limited proteolytic digestion of human LTBR-Ig and purified by chromatography on protein A Sepharose and Fractogel TMAE. 71F10 Fab-(G4S)4-LIGHT (SEQ ID NO: 134) was incubated overnight at 4-8° C. with a 5-fold molar excess of shuLTBR. Controls with each protein were also included. The samples were analyzed by SEC using a BioSeptember 3000 column (Phenomenex) in PBS with a Waters Alliance HPLC instrument. Results show the formation of a higher molecular weight complex, demonstrating that Fab-Light is capable of binding the receptor (data not shown). The precision of the light scattering analysis data did not allow calculation of the exact stoechiometry which was 2 or 3 shuLTBR for 1 Fab-Light trimer.
Example 13
Binding of Purified LIGHT Fusion Protein, BIIB71F10-132, to Receptors Measured by ELISA
The binding of 71F10 Fab or BIIB71F10-132 LIGHT fusion protein (dilution series) to human or murine HER2-Fc was tested by ELISA. Human and murine ErbB2-Fc were obtained from R&D Systems. Wells of 96-well plates were coated with 2 ug/ml or 0.2 ug/ml of human or murine HER2-Fc by incubation overnight at 4-8° C. with either of these six proteins (80 ul/well of a 0.2 or 2 ug/ml solution in PBS). Wells were emptied, filled completely with a solution of PBS with 1% BSA (blocking buffer) and incubated at room temperature for 1 hour. Wells were emptied, washed twice PBS containing 0.05% Tween 20 (PBST) and filled with the samples (80 ul/well of 3× dilution series of the standard and supernatants in blocking buffer) prepared in another 96-well plate. After a 2-hour incubation at room temperature, the wells were emptied, washed three times with PBST and filled with a solution of HRP-conjugated goat pAbs anti-human kappa chain (80 ul/well of a 2000-fold dilution in blocking buffer). After 1-hour incubation, the wells were emptied and filled with a solution of HRP substrate prepared freshly. The color was left to develop for 3 minutes and was stopped by addition of an equal volume of 1N H2SO4. Absorbance was measured at 450 nm using a Plate Reader (SpectraMax, Molecular Devices). The data was processed and graphs were created using SoftMax Pro software. Results demonstrated that the HER2 binding activity of the LIGHT fusion is HER2 receptor density dependent, i.e. higher affinity when HER2 antigen is abundantly presented on the coated plate (2 ug/ml). Results are shown in FIGS. 7 and 8.
The binding of Flag-huLIGHT (control) or BIIB71F10-132 LIGHT fusion (dilution series) to human or murine LTBR-Ig was tested by ELISA (FIGS. 10A and 10B and Table 2) using the protocol described above except that plates were coated with 2 ug/ml or 0.2 ug/ml of human or murine LTBR-Ig; and HRP-conjugated anti-Flag mAb or goat anti-human kappa pAb was used. Human and murine LTBR-Ig were expressed and purified in house. At high receptor density, 71F10 Fab-hLIGHT showed high affinity to both hLTbR and mLTbR. At low receptor density, 71F10 Fab-hLIGHT showed high affinity to hLTbR and moderate affinity to mLTbR.
The binding of Flag-huLIGHT (control) or BIIB71F10-132 (dilution series) to human or murine HVEM-Ig (Herpes virus entry mediator-Ig) was tested by ELISA (FIGS. 11A and 11B and Table 2) using the protocol described above except that plates were coated with 2 ug/ml or 0.2 ug/ml of human or murine LTBR-Ig; and HRP-conjugated anti-Flag mAb or goat anti-human kappa pAb was used. Human and murine HVEM-Ig were expressed and purified in house.
Binding activity of 71F10-hLIGHT fusion proteins is summarized in Table 2.
Example 14
LIGHT Fusion Proteins Bind to High Her2 Expressing SKBR3 Cells
Methods: SK-BR-3 cell line was obtained from the American Type Culture Collection (Rockville, Md.). Cells were cultured in McCoy's 5a medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1.5 mM L-glutamine. PE-conjugated goat anti-mouse F(ab′)2 specific antibody was purchased from Jackson ImmunoResearch (West Grove, Pa.). SK-BR-3 cells were exposed to various concentrations of 71F10-LIGHT fusion proteins (BIIB71F10-130, BIIB71F10-131 and BIIB71F10-132). Control anti-HER2 Fab, 71F10 Fab and 71F10 IgG (BIIB71F10-134) were used as controls. Samples were washed in FACS buffer (PBS containing 5% FBS and 0.02% sodium azide), and counterstained with PE-conjugated goat anti-mouse F(ab′)2 specific antibody. Cells were finally washed and resuspended in FACS buffer with 1% paraformaldehyde. Fixed samples were subjected to analysis on a FACScan flow microfluorometer (Becton Dickinson, Sunnyvale, Calif.).
Results: Trimeric 71F10 Fab-hLIGHT showed significantly higher binding activity to SKBR3 cells than that of 71F10 Fab, presumably due to increased avidity (FIG. 12).
Example 15
Binding of hLIGHT Fusion Proteins to CHO/hHER2 Cells can be Blocked by Either LTbR-Ig or HER2-Fc
Methods: Original CHO cell line was obtained from the American Type Culture Collection and modified to express hHER2 as described in Example 4. Clone KS19 that expresses high levels of HER2 was chosen for the experiments. KS19 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS in 6 well culture plates. KS19 cells were exposed to BIIB71F10-130, BIIB71F10-131, BIIB71F10-132 and BIIB71F10-MAB at a concentration of 0.2 nM for 1 h. In separate groups, fusion proteins and BIIB71F10-MAB were pre-incubated with hLTbR-Ig (20 mg/ml) or HER2-Fc (20 mg/ml). Mouse anti-LTbR mAb (AC H16), hLIGHT, and 71F10 Fab were used as controls (data not shown). Samples were washed in FACS buffer, and counterstained with PE-conjugated goat anti-mouse F(ab′)2 specific antibody. Cells were finally washed and subjected to analysis on a FACScan flow microfluorometer.
Results: 71F10-hLIGHT fusion proteins binding to CHO/hHER2 cells can be blocked by either LTbR-Ig or HER2-Fc (FIG. 13). These results suggested that both LIGHT and HER2 specific Fab are capable of binding to their receptors on the CHO cell surface. The binding is specific and can be competed by its receptor fusion proteins, i.e. LTbR-Ig or HER2-Fc proteins. The level of blocking effect depends on the receptor numbers on the cell surface. It appears that hHER2 receptor is much more aboudant than LTbR on KS10 cells.
Example 16
LIGHT Fusion Proteins can Simultaneously Engage Both HER2 and hLIGHT Receptors, LTbR and HVEM, on Cell Surface
Methods: KS19 cells were exposed to BIIB71F10-130, BIIB71F10-131, BIIB71F10-132 and BIIB71F10-134, IgG at a concentration of 0.2 nM for 1 h at 4° C. After washed with FACS buffer, groups of cell were incubated with FACS buffer alone, hLTbR-Ig (20 ug/ml) or HVEM-Fc (20 ug/ml) for 30 min and then washed again. For group incubated with FACS buffer, cells were counterstained with PE-conjugated goat anti-mouse F(ab′)2 specific antibody; for groups contacted with hLTbR-Ig or HER2-Fc, cells were staining with PE-conjugated goat anti-human Fc specific antibody (Jackson ImmunoResearch) for 30 min. Cells were finally washed and fixed in FACS buffer with 1% paraformaldehyde. Samples were subjected to analysis on a FACScan flow microfluorometer (FIG. 14).
Results: Both ends (71F10 Fab and LIGHT) of three fusion proteins are functional simultaneously, with 71F10 Fab site recognizing HER2 and LIGHT end binding to LTβR-Ig or HVEM-Ig.
Example 17
In Vitro Enhancement of T Cell Proliferation by 71F10 Fab-hLIGHT Fusion Proteins
Methods: Flat-bottom 96-well plates were pre-coated with sub-optimal concentration of anti-CD3 mAb (0.25 mg/well) at 4° C. overnight. Nylon wool column-purified T cells isolated from naïve Balb/c mice were subsequently cultured in the presented of various concentrations of hLIGHT, BIIB71F10-134 IgG and 71F10-LIGHT fusion proteins (BIIB71F10-130, BIIB71F10-131 and BIIB71F10-132) at 37° C. for 72 h. Anti-CD28 mAb was used as a control. [3H]TdR (1 uCi/well) was added for the last 18 h. Plates were harvested using a Tomtec Harvester Mach III M cell harvester (Hamden, Conn.), and the radioactivity was measured using a MicroBeta liquid scintillation and luminescence counter (Wallac, Turku, Finland). Augmentation of T cell proliferation was determined by plotting the percentage of [3H]TdR incorporation compared with cells treated with anti-CD3 mAb alone that were taken as 100%.
Results: hLIGHT fusion proteins enhanced primary mouse T cell proliferation in the presence of sub-optimal anti-CD3 antibody (FIG. 15). These results suggest that the fusion proteins remain as active co-stimulatory molecule for T cell proliferation. In the absence of anti-CD3, LIGHT fusion proteins showed no activity on T cell proliferation.
Example 18
Inhibition of Tumor Cell Growth by 71F10 Fab-hLIGHT Fusion Proteins
SKBR3 Tumor Cell Growth Inhibition by Recombinant Hlight Proteins
Methods: Human breast cancer cell line, SKBR-3, cells were seeded in flat-bottom 96-well plates at 1.0×104 cells/well in McCoy's 5a and allowed to adhere overnight. The cells were then treated with various concentrations of hLIGHT. As control groups, hLIGHT were pre-incubated with hLTβR-Ig (20 ug/ml) to block LIGHT binding activity to LTbR on tumor cell surface. An agonistic anti-LTβR mAb (CBE11) and control anti-HER2 antibody were used as controls. The plates were incubated for 72 h and then pulsed for 6 h with [3H]TdR (1 μCi/well). Plates were harvested and [3H]TdR incorporation was then measured.
Results: Treatment with recombinant hLIGHT proteins lead to growth inhibition of SKBR-3 cells. The inhibitory effects of hLIGHT can be specifically blocked by the addition of LTbR-Fc—suggesting that this inhibitory activity depends on hLIGHT/LTbR interaction. In contrast, the agonistic antibody CBE11 did not show any inhibition any the tested concentrations and control anti-HER2 antibody demonstrated growth inhibition at higher antibody concentrations (>2 nM) (FIG. 16).
SKBR-3 Cell Growth Inhibition by hLIGHT Fusion Proteins
The growth inhibitory activity of three LIGHT fusion proteins were tested on SKBR-3 cells following the procedure described in above section. In brief, SK-BR-3 cells were seeded in flat-bottom 96-well plates (1.0×104/well) overnight. The cells were then treated with increasing concentration of BIIB71F10-130, -131, -132 and -134 (BIIB71F10 mAb control). Again control anti-HER2 antibody was used. The plates were incubated for 72 h at 37° C. in a 5% CO2 incubator and then pulsed with [3H]TdR for 6 h. Plates were harvested and measured using a MicroBeta liquid scintillation and luminescence counter.
Results indicated that all three hLIGHT fusion proteins showed potent inhibitory activity of SKBR-3 cell growth. However, unlike seen in hLIGHT where inhibition correlated with hLIGHT concentration (above), all three LIGHT fusion proteins only inhibited cell growth at lower concentrations and lost their inhibitory activity at higher concentrations. This pattern of growth inhibition was described as “U” shaped cell growth inhibition (FIG. 17). It was hypothesized that this is the balancing result of LIGHT induced inhibition and trimeric Fab triggered resistance or growth promoton of these cells. It was also noted that the control antibody or BIIB71F10-134 mAb (IgG) showed neither growth promoting nor inhibitory effect.
Mechanism of Action Study of LIGHT Fusion Proteins in SKBR-3 Cells
SKBR-3 cell growth inhibition experiment was setup following procedure described above. The treatment employed only one fusion protein, BIIB71F10-132. In addition to BIIB71F10-132 LIGHT fusion protein treatment, BIIB71F10-132 was also pre-incubated with LTbR-Ig (20 ug/ml) or Her2-Fc (20 ug/ml) respectively at RT for 30 min, then were used for treatment. Again, control anti-HER2 antibody and recombinant hLIGHT was used as additional control. Results were shown in FIG. 18. Again BIIB71F10-132 treatment alone showed “U” shaped inhibition curve (FIG. 17). In contrast, blockade of Her2 binding activity of the LIGHT fusion protein by preincubation with Her2-Fc abrogated the tail of “U” shape curve, i.e. suppressed the growth promoting signal from trimeric Fab and showed similar growth inhibition as seen with hLIGHT. Whereas blockade of LIGHT function by LTβR-Fc completely blocked the inhibitory activity of the LIGHT fusion protein—suggesting the inhibitory effect was LIGHT/LTβR interaction dependent.
hLIGHT Fusion Proteins Growth Inhibition of BT474 Cells
BT474 cells, at a concentration of 0.1×106 cells/ml in RPMI1640 with 10% FBS, were plated (100 μl/well) in flat-bottom 96-well plates and allowed to adhere overnight. The cells were then treated with 200 μl of different concentrations of BIIB71F10-132 alone, 71F10 mAb alone, hLIGHT alone, various concentrations of 71F10 mAb mixed with hLIGHT (200 nM) or 71F10 mAb (2.5 nM) in combination with different concentration of hLIGHT (200, 20, 2, 0.2 and 0.02 nM). Control anti-HER2 antibody was used. The plates were incubated for 72 h and then pulsed for 6 h with [3H]TdR. The radioactivity was measured using a MicroBeta liquid scintillation and luminescence counter.
Results: 71F10 Fab-hLIGHT fusion proteins, hLIGHT as well as BIIB71F10-134 (71F10 mAb) all showed potent growth inhibition in breast cancer line—BT474 cells (FIG. 19). When the targeting mAb, 71F10 mab was added together with hLIGHT, no significant synergy was observed in these cells. In comparison with the SKBR-3 cells results, no “U” shaped growth inhibition curve was observed. These results suggest that different cell line respond very differently to LIGHT fusion protein treatment.
Example 19
Internalization of 71F10-hLIGHT Fusion Proteins
Internalization Assay (Fabs)
SKBR3 cells were plated in CC2-coated 8-well chamber slides (Nunc #154941) and allowed to attach over night in the incubator (5% CO2, 37° C.). The growth medium was replaced with 50 ul to 75 ul of the Dyax Fabs (10 ug/ml) and they were allowed to bind for 15 minutes at 4oC. Santa Cruz anti human Neu monoclonal antibody (9G6), Sc-08 was used as a positive control since it rapidly internalizes upon cross-linking with a secondary antibody. One set of slides was moved to 37° C. for one hour. The SC08-treated wells were changed to solution containing Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Invitrogen A-11029) and 37° C. incubation continued for one additional hour. Wells were washed with dilution buffer (PBS with 10% FBS). Cells were fixed for 10 minutes at room temperature with 4% formaldehyde in PBS. Slides were washed with dilution buffer. Cells were permeabilized with 0.2% Triton X in PBS at room temperature for 15 minutes. Slides were washed and then incubated for 45 minutes at 4oC with a 50:50 mix of FITC conjugated goat anti human kappa chain and goat anti human IgG F(ab′)2 specific fragment (Sigma #F3761 and Jackson Immun. #109-096-097 respectively). Slides were washed with dilution buffer, the chambers removed, and Vectashield with DAPI (Vector Laboratories #H-1200) was added before coverslipping. Stacks of images were captured using the Leica Confocal microscope and ones that focus on a central plain were selected for the figures.
Binding Assay
SKBR3 cells were plated in CC2-coated 8-well chamber slides (Nunc #154941) and allowed to attach over night in the incubator (5% CO2, 37° C.). One well was treated with Adenoviral vector delivering the murine LIGHT gene for a positive control. The next day, the growth medium was replaced with 100 ml of test antibodies (10 mg/ml) and they were allowed to bind for one hour at 4oC. Cells were washed with dilution buffer (PBS with 10% FBS) and 100 ml LTβR-human Fc fusion was added and let bind for one hour at 4° C. Cells were washed and then the slides were incubated with FITC-conjugated goat anti human Fc (gamma) fragment specific antibody (Jackson Immun. #109-096-098) for one hour at 4° C. Slides were washed with dilution buffer, the chambers removed, and Vectashield with DAPI (Vector Laboratories #H-1200) was added before coverslipping. Stacks of images were captured using the Leica Confocal microscope and ones that focus on a central plain were selected for the figures.
Results: 71F10-hLIGHT fusion proteins display functional hLIGHT on tumor cell surface (data not shown).
Internalization Assay (Fab-LIGHT Fusion Proteins)
Methods: Similar to above procedure of binding assay, SKBR3 and BT474 cells were plated in CC2-coated 8-well chamber slides (Nunc #154941) and allowed to attach over night in the incubator (5% CO2, 37° C.). The growth medium was replaced with 0.1 ml of the treatment antibodies (1 ug/ml SC08 and 100 nM fusions and control anti-HER2 antibody) and let bind for 15 minutes at 4° C. Santa Cruz anti human Neu monoclonal antibody (9G6), Sc-08 was used as a positive control since it rapidly internalizes upon cross-linking with a secondary antibody. One set of slides was moved to 37° C. for 90 minutes. The SC08-treated wells were changed to solution containing Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Invitrogen A-11029) and 37° C. incubation continued for 30 minutes more. Wells were washed with dilution buffer (PBS with 10% FBS). Cells were fixed for 10 minutes at room temperature with 4% formaldehyde in PBS. Slides were washed with dilution buffer. Cells were permeabilized with 0.2% Triton X in PBS at room temperature for 15 minutes. Slides were washed and then incubated for 45 minutes at 4° C. with a 50:50 mix of FITC conjugated goat anti human kappa chain and goat anti human IgG F(ab′)2 specific fragment (Sigma #F3761 and Jackson Immun. #109-096-097 respectively). Slides were washed with dilution buffer, the chambers removed, and Vectashield with DAPI (Vector Laboratories #H-1200) was added before coverslipping. Stacks of images were captured using the Leica Confocal microscope and ones that focus on a central plain were selected.
Results: None of the 71F10 Fab-hLIGHT fusion proteins (BIIB71F10-130, BIIB71F10-131, BIIB71F10-132) showed internalization (Confocal Microscopy on SKBR3) under experimental conditions.
Example 20
Induction of Proinflammatory Genes by hLIGHT Fusion Proteins
Quantitative PCR Analysis of Proinflammatory Genes Induced by LIGHT Fusion Proteins
Methods: HT29 cells were seeded in a 6-well plate to be approximately 80% confluent at time of treatment. 24 hours later, they were refed with fresh media or fresh media with 4 nM BIIB71F10-130 and allowed to incubate for 24 hours more. At the time of harvest, the cells were estimated at 1E6/well. The RNA was isolated using Qiagen RNeasy mini extraction kit (#74104) according to the manufacturer's instructions including a Dnase treatment to remove any genomic contamination. First strand cDNA synthesis was performed using a kit from SuperArray Bioscience Corporation (#C-03). QPCR was done using SuperArray “Human Inflammatory Cytokines and Receptors” 96-well array plates (#PAHS-011), the RT Sybr Green PCR Master Mix (SuperArray #PA-012) and cycled according to manufacturer's recommendations on an ABI 7300 real time PCR cycler. Data from the thermocycler were entered into the “RT2 PCR Array Data Analysis Software” that SuperArray supplies with the arrays. Briefly, after the data are normalized for controls of the various steps of sample preparation (reverse transcription and PCR) as well as a panel of five housekeeping genes, they are presented as treated divided by untreated data that manifests as a “fold change” in gene expression.
Results: Treatment of HT29 cells with LIGHT fusion #130 led to a significant change in the expression of pro-inflammatory chemokines and cytokines. Most of the 84 genes assayed were upregulated and a few were down-regulated when we compare the treated to the untreated transcripts. This indicates that when the LIGHT end of the fusion molecule engages the LTbR, it initiates an alteration of gene expression to a pro-inflammatory state. A sampling of genes that show a high fold change is presented in Table 3.
Stimulatory Effect of LIGHT Fusion Proteins on IP-10 and IL-8 Secretion
Methods: HT29 cells were plated at 3×104 per well in a 96-well plate and allowed to attach overnight. The cells were refed with dilutions of reagents in media containing 100 u/ml IFNg (total volume of 100 ul/well). 24 hours later the supernatants were collected and spun to pellet cellular debris. The clarified supernatants were stored overnight on ice. The R+D Systems Quantikine ELISA kits for Human CXCL8/IL-8 (#D8000C) and Human IP-10 (#DIP100) were used according to the manufacturer's instructions. Samples were diluted a total of 1:90 for the IL-8 ELISA and 1:30 for the IP-10 ELISA. Data above are plotted in ng/ml of IP-10 or IL-8 over the molar treatment concentrations.
Results: 71F10 Fab-hLIGHT fusion proteins stimulate IP-10 and IL-8 secretion in HT29 cells (FIG. 20).
Example 21
HER2 Signaling Analysis of Anti-Her2 Fabs, mAbs and LIGHT Fusion Proteins
Effect of on Phosphorylation of HER2, Akt and MAPK
Methods: 5×105 MCF7, SKBR3, BT474 or N87 cells/well were plated in 6-well plates in 1 mL volume. After 24 hrs 1 mL 200 nM solution of indicated reagent (Fab, mAb, control anti-HER2 antibody or LIGHT fusion proteins) was added to wells to give 2 mL of 100 nM solution. After desirable incubation time (1, 6, 24 or 72 hours), media were removed and cells were washed 1× with PBS. Cells lysed with 500 uL RIPA buffer (20 mM MOPS pH 7.0, 150 mM Sodium Chloride, 1% NP40, 1% Deoxycholate, 0.1% SDS) containing protease and phosphatase inhibitors (Mini-complete and PhosStop, respectively, Roche Applied Bioscience cat#11836153001 and 04906837001, respectively). Lysates collected in microcentrifuge tubes and stored at 4° C. Total protein concentrations determined using Micro BCA Protein Assay kit (Pierce cat#23235). 10 ug of total protein loaded per well on NuPAGE® Novex 4-12% Bis-Tris Gel (Invitrogen cat#. Gels run with NuPAGE® MOPS SDS Running Buffer (Invitrogen cat#NP0001) for 1 hr at 200V. Gels blotted onto PVDF membranes (Invitrogen cat#LC2002) using transfer buffer (12.5 mM Tris, 96 mM Glycine, 20% Methanol) run at 30V for 1 hr. Membranes rinse in TBS-T (1× Tris-buffered with 0.1% Tween20) and block in StartingBlock T20 (TBS) Blocking Buffer (Pierece cat#37543) for 1 hr at R/T with gentle shaking. Primary antibodies (Her2 (#2242), phosphoHer2 (#2249), Akt (#4691), phosphoAkt (#4060), MAPK (#4695), phosphoMAPK (#4370) all from Cell Signaling Technology, Beverly, Mass.) added to blocking buffer. Blots incubated with antibodies overnight at 4° C. with gentle shaking. Blot was washed thoroughly in TBS-T. HRP-conjugated donkey anti-rabbit IgG (H+L) (JacksonImmuno cat#711-035-152) secondary antibody was added in blocking buffer at a concentration of 1:10000. Blots incubated at R/T for one hour with gentle shaking. After incubation blots were thoroughly washed with TBS-T and ECL Plus reagent (GE Healthcare Life Science cat#RPN2132) was added as per instructions. Blots were exposed to X-ray film (Kodak BioMax XAR cat#165-1454) and developed. Alternatively, blots were incubated with detecting antibodies overnight at 4° C. with gentle shaking. Blots were washed thoroughly in TBS-T. Goat anti-mouse IgG (H+L), DyLight 680 conjugated (Pierce cat#35518) and goat anti-rabbit IgG (H+L), DyLight 800 conjugated (Pierce cat#35571) secondary antibodies were added to blocking buffer at a concentration of 1:10000 in one container and incubated for one hour at R/T with gentle shaking. Blots were then washed thoroughly with TBS-T. Blots visualized on Licor Odyssey Infrared Scanner.
Results:
None of the anti-HER2 Fabs appears to induce HER2, MAP kinase and AKT phosphorylation as observed at 6 or 72 hours compared to control anti-HER2 antibody (data not shown).
After converting to full antibody, BIIB71F10-134, showed weak agonistic activity in activating MAP kinase in the absence of Heregulin in MCF7 cells (mAb concentration 1-100 nM). No significant in total or phosphorylated Her2 and Akt was detected after BIIB71F10-134 treatment. LIGHT fusion protein, BIIB71F10-132, treatment also induced MAP kinase phosphorylation in the absence of Heregulin in MCF7 cells. Induction of MAP kinase phosphrylation can be detected from 2.5 pM to 200 nM of BIIB71F10-132 fusion protein treatment (data not shown). Similar MAP kinase activation was also detected in SKBR3 cells, but at much lower levels (data not shown).
Furthermore, MAP kinase activation by BIIB71F10-132 fusion protein in MCF7 is presumably through the trimerization of HER2 upon LIGHT fusion treatment. This MAP kinase activation is not a result of LIGHT pathway activation because blockage of LIGHT function by LTbR-Ig of the fusion protein produced identical results as observed in LIGHT fusion treatment alone. These results suggest that trimeric 71F10 appears to be agonistic and is able to activate HER2 signal transduction under current experimental conditions.
Effect on HER2 Heterodimerization with HER3
Methods: 5×105 MCF7, SKBR3 or N87 cells were plated in 6-well plates. After 24 hrs testing reagents (Fab, full antibodies, or LIGHT fusion proteins) were added at 50 nM and cells were incubated with them for one or 72 hrs. After antibody incubation heregulin beta (100 ng/mL) was added to media and cells were incubated for an additional 30 min. Cells were washed 1× with PBS and lysed with 250 uL of RIPA buffer (20 mM MOPS pH 7.0, 150 mM Sodium Chloride, 1% NP40, 1% Deoxycholate, 0.1% SDS) containing protease and phosphatase inhibitors (Mini-complete and PhosStop, respectively, Roche Applied Bioscience cat#11836153001 and 04906837001, respectively). Lysates were collected in microcentrifuge tubes and stored at 4° C. 100 uL of lysate was added to 900 uL of RIPA buffer not containing protease and phosphotase inhibitors. 1 ug of rabbit anti-HER3 (Santa Cruz Biotechnology cat#sc-285) added to each sample, along with 25 uL of Protein A sepharose (GE Healthcare cat#17-5280-01). Mixtures were rocked overnight at 4° C. After overnight incubation sepharose beads were pelleted by centrifugation at 3000 rpm for one minute. The supernatant was removed by aspiration and the pellet washed with 500 uL PBS. This procedure was repeated two more times. After the final supernatant was removed 30 uL of 5×SDS sample reducing buffer (11.5% SDS, 50% Glycerol, 0.3M Tris, 0.025% Phenol Red, 25% beta-mercaptoethanol) was added to pellet, pellet disrupted and heated to 95° C. for five minutes. The sample was then centrifuged at 14000 rpm for 3 minutes and the supernatant loaded on a NuPAGE® Novex 4-12% Bis-Tris Gel (Invitrogen). Gels were run with NuPAGE® MOPS SDS Running Buffer (Invitrogen cat#NP0001) for 1 hr at 200V. Gels were blotted onto PVDF membranes (Invitrogen cat#LC2002) using transfer buffer (12.5 mM Tris, 96 mM Glycine, 20% Methanol) run at 30V for 1 hr. Membranes were rinsed in TBS-T (1× Tris-buffered with 0.1% Tween20) and blocked in StartingBlock T20 (TBS) Blocking Buffer (Pierce cat#37543) for 1 hr at R/T with gentle shaking. A rabbit-anti Human HER2 antibody (Cell Signal Technologies cat#2242) was added to the blocking buffer. Blots incubated with antibody overnight at 4° C. with gentle shaking. Blots were washed thoroughly in TBS-T. HRP-conjugated donkey anti-rabbit IgG (H+L) (JacksonImmuno cat#711-035-152) secondary antibody was added in blocking buffer at a concentration of 1:20000. Blots were incubated at R/T for one hour with gentle shaking. After incubation blots were thoroughly washed with TBS-T and ECL Plus reagent (GE Healthcare Life Science cat#RPN2132) was added as per instructions. Blots were exposed to X-ray film (Kodak BioMax XAR cat#165-1454) and developed.
Results:
Fab 65H09 appears to increase heterodimerization with HER3 in the absence of Heregulin, but that effect is abrogated by Heregulin treatment. This can be explained by that 65H09 binding site at Her2 overlaps with the interaction site of Heregulin to HER2. Since Heregulin has higher affinity than Fab 65H09, the facilitation effect of 65H09 to HER2/HER3 heterodimerization was eliminated. All other Fabs show no effect on HER2/HER3 heterodimerization (data not shown).
It appears that Fab 71F10 decreased HER2/HER3 heterodimerization at 1 hour in the presence of HER3 ligand Heregulin in N87 cells. But the effect is not replicated in SKBR3 cells (data not shown). The effect of LIGHT fusion proteins on Her2/Her3 heterodimerization remain to be tested using the same procedure described above.
Effect of Fabs on HER2 Heterodimerization with EGFR
Methods: 5×105 MCF7, SKBR3 or B87 cells were plated in 6-well plates. After 24 hrs antibodies were added at 50 nM and cells were incubated with them for one or 72 hrs. After antibody incubation human epidermal growth factor (100 ng/mL) was added and cells were incubated for an additional 30 min. Cells were washed 1× with PBS and lysed with 250 uL of RIPA buffer (20 mM MOPS pH 7.0, 150 mM Sodium Chloride, 1% NP40, 1% Deoxycholate, 0.1% SDS) containing protease and phosphatase inhibitors (Mini-complete and PhosStop, respectively, Roche Applied Bioscience cat#11836153001 and 04906837001, respectively). Lysates were collected in microcentrifuge tubes and stored at 4° C. 100 uL of lysate was added to 900 uL of RIPA buffer not containing protease and phosphotase inhibitors. 1 ug of mouse anti human EGFR (BD Bioscinces Pharmingen cat#610016) added to each sample, along with 25 uL of Protein A sepharose (GE Healthcare cat#17-5280-01). Mixtures were rocked overnight at 4° C. After overnight incubation sepharose beads were pelleted by centrifugation at 3000 rpm for one minute. The supernatant was removed by aspiration and the pellet washed with 500 uL PBS. This procedure was repeated two more times. 30 uL of 5×SDS sample reducing buffer (11.5% SDS, 50% Glycerol, 0.3M Tris, 0.025% Phenol Red, 25% beta-mercaptoethanol) was added to pellet, pellet disrupted and heated to 95° C. for five minutes. The sample was then centrifuged at 14000 rpm for 3 minutes and the supernatant loaded on a NuPAGE® Novex 4-12% Bis-Tris Gel (Invitrogen). Gels were run with NuPAGE® MOPS SDS Running Buffer (Invitrogen cat#NP0001) for 1 hr at 200V. Gels were blotted onto PVDF membranes (Invitrogen cat#LC2002) using transfer buffer (12.5 mM Tris, 96 mM Glycine, 20% Methanol) run at 30V for 1 hr. Membranes were rinsed in TBS-T (1× Tris-buffered with 0.1% Tween20) and blocked in StartingBlock T20 (TBS) Blocking Buffer (Pierce cat#37543) for 1 hr at R/T with gentle shaking. A rabbit anti Human HER2 antibody (Cell Signal Technologies cat#2242) was added to the blocking buffer. Blots incubated with antibody overnight at 4° C. with gentle shaking. Blots were washed thoroughly in TBS-T. HRP-conjugated donkey anti-rabbit IgG (H+L) (JacksonImmuno cat#711-035-152) secondary antibody was added in blocking buffer at a concentration of 1:20000. Blots were incubated at R/T for one hour with gentle shaking. After incubation blots were thoroughly washed with TBS-T and ECL Plus reagent (GE Healthcare Life Science cat#RPN2132) was added as per instructions. Blots were exposed to X-ray film (Kodak BioMax XAR cat#165-1454) and developed.
Results:
Results indicated that Fab 66A12 facilitates EGFR/HER2 heterodimerization in SKBR3 cells, whereas all other Fabs show no activity. Similarly control anti-HER2 antibody Fab was also able to facilitate EGFR/HER2 heterodimerization as observed in 66A12 (data not shown). The effect of LIGHT fusion proteins on EGFR/HER2 heterodimerization remain to be evaluated using the same procedure.
Example 22
Tumor Models for Evaluation of LIGHT Fusion Activity Against Primary Tumor and Metastasis
LIGHT fusion protein potential display its anti-tumor activity through the combination of HER2 pathway blockade and activation of LTβR pathway. Therefore LTβR and HER2 double positive tumor models are desirable for testing LIGHT fusion proteins. These tumor models include human tumor cell lines such as BT474, SKBR-3, MCF7, MDA-MB-231, MDA-MB-468, N87, SKOV3, HT29, WiDr; and mouse breast tumor cell lines Tubo, TSA and 4T1 cells.
BT474, MCF7, MDA-MB-231, MDA-MB-468 human breast tumor cell lines, N87 human gastric cancer cell line, and SKOV3 human ovarian cancer cell line were cultured in RPMI 1640 with 10% fetal bovine serum in a humidified atmosphere of 5% CO2 at 37° C. SKBR3 human breast tumor cell line, HT29, Widr human colon adenocarcinoma were cultured in McCoy's 5a medium containing 1.5 mM L-glutamine, and 10% fetal bovine serum. Mouse mammary gland tumor cell lines Tubo, TSA and 4T1 were cultured in DMEM supplied with 10% fetal bovine serum. Medium was changed every 3 days. Cells were removed from culture flasks for passage by washing once with PBS, followed by a 5-min incubation with 0.5 mM EDTA and 0.05% trypsin at PH 7.4. Their viability was determined by microscopic examination of cells stained by 0.1% trypan blue. Viable cells were inoculated into mice in a volume of 0.1 ml of PBS.
To establish xenograft models of LTβR and HER2 double positive tumor, female athymic nude (nu/nu) BALB/c mice, 6-8 weeks of age, were inoculated subcutaneously into the right flank skin with 2×106 viable cultured human tumor cells in 0.1 ml of PBS. Mice with established s.c. tumors were evaluated by perpendicular bidimensional tumor measurements twice weekly with a Vernier caliper. Mice were euthanized and scored as dead from lethal tumor progression when the s.c. tumor diameter exceeded 14 mm. Tumor volume was calculated using the formula, V=xy2/2. where x and y are the two perpendicular diameters (length and width). Because MCF7 tumors do not grow without estradiol, all mice inoculated with MCF7 tumors received s.c. 17β-estradiol pellet (Innovative Research of America, Sarasota, Fla.) implants 7 days prior to tumor inoculation.
To establish syngeneic models, female Balb/c (H-2Kd) mice, were inoculated subcutaneously into the right flank skin with 0.5×106 viable cultured mouse tumor cells (Tubo, TSA or 4T1) in 0.1 ml of PBS. Tumor volume was calculated as described above.
LIGHT fusion protein can also be tested in engineered tumor cell lines to over express human or mouse Her2 such as MCF7/hHer2, 4T1/mHer2, TSA/mHer2. The overexpression of Her2 in these cells attempts to mimic the Her2 overexpression in human breast cancer and made it more feasible to test anti-HER2 LIGHT fusion proteins tumor targeting capability. These modified cell lines will be grown in the same culture medium of their parental cell lines but including selection drugs such as G418 and Blasticydin.
Example 23
In Vivo Inhibition of Tumor Growth Using Combination Therapy
Method: The efficacy of LIGHT fusion protein in inhibiting tumor growth in combination with chemotherapeutic agents (e.g., Docetaxel, Paclitaxel, Doxcirubicin, Cyclophosphamide, Fluorouracil (5FU), Gemcitabine and Vinorelbine) can be tested in a xenograft model (e.g., BT474 or MCF model) or a model of primary tumor segments. The efficacy of LIGHT-Fab fusion protein administered intraperitoneally (i.p.) two times per week at 30 mg/kg for 7 weeks or one time per week at 60 mg/kg for 5 weeks can be evaluated in combination with gemcitabine administered according to the current standard of care (i.e., 80 mg/kg every 3 days for 4 weeks). Gemcitabine alone, LIGHT-Fab fusion protein alone, and sham injections of the delivery vehicle alone can be administered as negative controls. Tumor volume at the start of the therapy was approximately 200 mm3. Primary tumor regression, hLIGHT fusion accumulation in tumors, infiltrating lymphocytes, CD4, CD8, Tregs and tumor re-challenge can be evaluated.
Example 24
Antitumor Activity of Fab-LIGHT Fusion Proteins in Xenograft Tumor Models
LIGHT fusion protein is believed to display its antitumor activity at least in part through the combination of tumor cell target inhibition (e.g. HER2) and activation of LIGHT receptors (namely LTβR and HVEM) on tumor cell surface. Therefore both HER2 and LIGHT receptor(s) double positive tumor models can be used for testing LIGHT fusion proteins. As examples, HT29 and N87 xenograft tumor models were selected for testing LIGHT fusion protein antitumor activity in vivo.
HT29, a tumor cell line derived from human colorectal adenocarcinoma, was purchased from ATCC (cat# HTB-38) and passaged afterwards. These cells express HER2 at medium levels (IHC score 2+) as determined by Western blot and immunohistochemical staining (data not shown). HT29 cells also express both LTβR and HVEM receptors for LIGHT. The growth of HT29 cells is not HER2 dependent although these cells do express HER2. For example, these cells do not exhibit growth arreast after anti-HER2 antibody treatment (data not shown). Therefore the antitumor activity of 71-F10-hLIGHT fusion proteins is mainly dependent on the LIGHT-directed killing activity to the tumor cells as shown by in vitro proliferation assays (data not shown). In contrast, N87 is a tumor cell line derived from human gastric carcinoma (ATCC, #CRL5822) and forms subcutaneous tumors in SCID mice. N87 cells over-express HER2 to high levels (IHC score 3+) and respond to anti-HER2 antibody treatment, which lead to growth arrest and cell death (data not shown). Therefore, blocking of HER2 and cell growth arrest/cell death triggered by LIGHT can contribute to 71F10-hLIGHT antitumor activity.
Both nu/nu athymic female mice and SCID female mice were obtained from Charles River Laboratories (Wilmington, Mass.) at 6-8 weeks of age. 2×106 (HT-29) and 5×106 (N87) tumor cells were inoculated subcutaneously in the right flank of nu/nu and SCID mice, respectively. Mice with established tumors (50-200 mm3) were selected for study (N=7 to 10 per treatment group). Tumor dimensions were measured using calipers and tumor volumes were calculated using the equation for an ellipsoid sphere (L×W2)/2=mm3, where L and W refer to the larger and smaller dimensions collected at each measurement. The test fusion proteins or antibodies were formulated and administered intravenously (IV) or via the intraperitoneal cavity (IP) at a dose volume of 6 mL/kg. The vehicle alone was administered to control groups. Animals were dosed three days per week (TIW—Monday, Wednesday, Friday) for six to eight consecutive weeks. For monoclonal antibodies, the treatment was twice a week (BIW—Monday and Thursday). Animals were weighed and the tumors were measured twice per week. Mice were followed until tumor volumes in the control group reached approximately 1000 mm3 and were sacrificed by CO2 euthanasia. The mean tumor volumes of each group were calculated. The change in mean treated tumor volume was divided by the change in mean control tumor volume, multiplied by 100 and subtracted from 100% to give the tumor growth inhibition for each group. Statistical analysis was performed using the standard T-test and using GraphPad Prism© Software.
Example 25
The Anti-HER2-LIGHT Fusion Protein Demonstrated Potent Anti-Tumor Activity in HT29 Xenograft Model
Results of 71F10-hLIGHT activity in HT29 tumor model is summarized as FIG. 21. Significant antitumor activity of 71F10-hLIGHT was shown in 10 mg/kg 71F10-hLIGHT fusion protein treatment group (p<0.05). The T/C (treated group vs. control group) value is less than 42% (data not shown). Although not reaching statistical significance, the 1 mg/kg and 5 mg/kg groups also showed clear anti-tumor activity in a dose dependent manner compared to the control groups. No difference was observed between IP vs IV delivery of 71F10-hLIGHT proteins for anti-tumor efficacy (data not shown). In contrast, the anti-HER2 monoclonal antibody BIIB71-F10 treatment alone did not show any antitumor effect. As discussed in Example 24, the growth of HT29 cells is not HER2-dependent although these cells do express HER2, which might contribute the lack of efficacy in the BIIB-71F10 Mab treatment. Furthermore, these results showed that the antitumor efficacy of HT29 tumors is from the LIGHT side of the 71F10-LIGHT fusion molecule since no activity was observed from 71F10 Mab treatment. The direct killing activity of LIGHT was further demonstrated in the control fusion molecule, anti-CD23-hLIGHT treatment groups. CD23 is not expressed on HT29 cells and therefore no “targeting effect” should be expected from its treatment. Only mild anti-tumor activity was detected in 5 and 10 mg/kg anti-CD23-hLIGHT groups, but there was no dose dependency (i.e. 5 and 10 mg/kg data overlapped) for its efficacy. These results suggested that free LIGHT fusion molecule in circulation is probably sufficient to induce tumor growth arrest.
HER2-targeted 71F10-hLIGHT showed much more potent anti-tumor activity than that of the non-targeted anti-CD23-hLIGHT control molecule. The weaker anti-tumor activity of anti-CD23-hLIGHT is not due to reagent difference in quality or pharmacokinetic difference since both 71F10-hLIGHT and anti-CD23-hLIGHT have equal potency as shown by in vitro killing assays using HT29 cells and identical PK values (data not shown). Therefore, the more potent activity of 71F10-hLIGHT can result from the necessity of targeting LIGHT to tumor cells for its maximal anti-tumor activity. These results provided additional evidence to support the role of LIGHT/LTβR/HVEM receptor oligomerization on cell surface in obtaining maximal signaling strength of TNF family members.
Example 26
The Anti-HER2-LIGHT Fusion Protein Demonstrated Potent Anti-tumor Activity in HER2-dependent N87 Xenograft Tumor Model
To test the synergy between anti-HER2 therapy and LIGHT-directed killing activity, N87 tumor model was employed in this experiment. As mentioned above, N87 cells over-express HER2 to high levels (IHC score 3+) and respond to anti-HER2 antibody such as control anti-HER2 antibody treatment that results in cell growth rest and cell death (data not shown). N87 tumors were grown on the flank of SCID mice and treated when tumor size reached 50-200 mm3. The treatments were similar to those used in Example 25, but included Trastuzumab or control anti-HER2 antibody (Roch/Genentech). The results were summarized in FIG. 22. 71F10-hLIGHT demonstrated significant (p<0.001) anti-tumor activity and suppressed tumor growth (T/C<40%) in a dose dependent manner. Similar to the observations from Example 25, anti-CD23-hLIGHT was less efficacious than 71F10-hLIGHT at the same dose (5 mg/kg) and showed significant efficacy in N87 models (p<0.02). The targeting antibody alone, BIIB71-F10 (without the LIGHT moiety) did not reach statistical significance in reducing tumor growth (note BIIB71-F10 antibody does not cross block with control anti-HER2 antibody). Control anti-HER2 antibody treatment showed significant growth retardation throughout the course of treatments (p<0.02). However, N87 tumors treated with control anti-HER2 antibody relapsed and resumed growth at Day 50. In contrast, tumors treated with 71F10-hLIGHT showed continued growth inhibition and even trend of tumor regression. These observations underscore some of the molecular and cellular differences between anti-HER2 antibody and anti-HER2-LIGHT fusion molecules in their respective anti-tumor activity. For example, aside from the mechanisms involving the blocking of HER2 receptor and cell growth arrest and death triggered by LIGHT, LIGHT may have stimulated the innate cells such as NK cells and macrophage to assist tumor cell killing. This mechanism is consistent with the known biological function of LIGHT and the observation that there was a slight delay in tumor response to 71F10-hLIGHT fusion protein treatment. The involvement of innate immune cells in anti-tumor activity can be further investigated by immunohistochemical staining of tumor sections and cell depletion experiments. Regardless the mechanism of action, 71F10-hLIGHT fusion protein displayed differences in treating HER2-dependent tumors compared to anti-HER2 antibodies, and offers an alternative therapy to overcome resistance to anti-HER2 antibody therapies.
Example 27
LIGHT Fusion Treatment Stimulates Host Anti-tumor Immune Responses
LIGHT is a potent co-stimulatory molecule for T cell activity, therefore targeting LIGHT to tumor tissue via Fab-LIGHT evokes host anti-tumor responses and reduces or eradicates tumor metastasis. The immune responses can be tested in immune competent animals. Syngeneic mouse tumor models, such as mouse breast tumor cells TSA, 4T1 and Tubo (Her2+) cells can be employed. Briefly, tumor cells can be prepared as single cell suspension. 1-5×105 tumor cells can be inoculated at the right flank of Balb/c mice. When the s.c. tumor area is established and reaches 50-100 mm3, animals can be treated with 71F10-hLIGHT fusion protein and controls at various doses and schedules for two weeks. At the end of the treatment and two weeks post treatment, tumor tissues and draining lymph nodes can be harvested to analyze immune cells infiltration, i.e. tumor residing CD4, CD8, NK cells and macrophages. For histology analyses lymphoid-like structure in tumor tissues can be examined and intratumoral levels of SLC (CCL21) can be determined for lymphocytes trafficking. The draining lymph nodes and lung tissues can be collected at one month after treatment for metastasis analyses. The fusion protein treatment group can have increased number of tumor infiltrating lymphacytes, upregulation of cytokines/adhesion molecules and result in reduced metastatic tumors.
The effect of LIGHT fusion protein induced-immunity in protecting animals from tumor rechallenge can also be determined. Both primary growth of re-challenged tumor and metastasis can be monitored.
Example 28
Construction of Anti-IGFR Fab-LIGHT Fusion Protein, C06-hLIGHT
Monoclonal antibody C06 VH region was synthesized by PCR amplification using the oligonucleotide primers described as DyaxVH-pV90-F (SEQ ID NOs:157), 256-R (SEQ ID NO:158), and 165-R2 (SEQ ID NO:159). The 5′ forward primer, DyaxVH-pV90-F which contains a unique Mlu I restriction endonuclease site (ACGCGT (SEQ ID NO:160) followed by sequences encoding the last three amino acids of the heavy chain signal peptide followed by sequences complementary to the amino terminus of the recoded C06 VH. The 3′ reverse primers consisted of an internal reverse primer, 256-R encoding the carboxyl terminus of C06 VH and a second reverse primer 165-R2 encoding a partial human IgG1 CH1 domain and an Age I site (ACCGGT (SEQ ID NO:161)). The recoded C06 VH region was amplified in two sequential PCR reactions through the common overlapping sequences encoding the human IgG1 CH1 domain using these three PCR primers from plasmid DNA pBIIBC06-030 containing the recoded Dyax anti-IGF1R C06 VH gene. The C06 VH gene fragment was cloned into the Mlu I/Age I digested the pBIIB71F10-132. Correct sequences were confirmed by DNA sequencing. Heavy chain DNA and amino acid sequences for the C06Fab-hLIGHT fusion (pBIIBC06-256) are shown as SEQ ID NOs:162 and 163, respectively. The amino acid and nucleotide sequences corresponding to heavy chain of C06Fab are shown starting from the N-terminus; followed by amino acids corresponding to the linking group (amino acids 226 to 245); followed by the amino acids corresponding to human LIGHT extracellular domain (amino acids 246 to 393). The amino acid sequences for C06 VH CDRs 1-3 are shown in SEQ ID NOs:164-166, respectively.
The C06 light chain vector (pBIIBC06-117) was used in the C06Fab-hLIGHT fusion and DNA and amino acid sequences are shown as SEQ ID NOs:167 and 168, respectively. The amino acid sequences for C06 VL CDRs 1-3 are shown in SEQ ID NOs:169-171, respectively.
C06-hLIGHT fusion protein was produced by transient transfection of CHO cells as described in Example 8. The protein titer in the medium was determined by Octet (ForteBio) and used for ELISA assay to assess binding activities to IGFR and LIGHT receptor LTβR. Anti-HER271F10-hLIGHT was used as a control. Results were summarized in FIGS. 23A and 23B. C06-hLIGHT showed specific binding to both IGFR (FIG. 23A) and LTβR (FIG. 23B) and demonstrated its bi-functional activity.
Example 29
Construction of Dimeric Form of Fab-LIGHT Fusion Protein
Dimeric form of Fab-LIGHT fusion protein was generated by including the full CH1 hinge region of IgG1. Dimer is stabilized by the formation of double disulfide bonds. The human IgG1 hinge sequence which contains two cysteine residues for interchain disulfide bonds formation was inserted in front of the (G4S)4 linker region of in 71F10-hLIGHT fusion heavy chain (FIG. 24). The 71F10 Fab-hLIGHT dimeric fusion heavy chain was constructed in the PCR reaction using the 5′ forward plus 3′ reverse PCR primers and an internal overlapping PCR primer set encoding the human IgG1 hinge sequence from plasmid DNAs containing the human IgG1 and hLIGHT. The primer sequences were shown as MB-04F (SEQ ID NO:135), 130-R1 (SEQ ID NO:136), 255-mF (SEQ ID NO:175) and 255-mR (SEQ ID NO:176). Briefly, the 5′ forward primer, MB-04F that has an AgeI site (ACCGGT (SEQ ID NO161)) followed by sequences complementary to the IgG1 CH1 region. The 3′ reverse primer, 130-R1 which contained a BamH I site (GGATCC (SEQ ID NO:177)) and sequence encoding complementary to the carboxyl terminus of hLIGHT. The 5′ forward primer, 255-mF and the 3′ reverse primer 255-mR of the internal overlapping PCR primer set included the sequences encoding the human IgG1 hinge region followed by the partial (G4S)4 linker. The PCR fragments were assembled together in a second PCR reaction using sequences encoding the overlapping the huIgG1 hinge and (G4S)4 linker sequence. The final PCR products were digested with the Age I and BamH I and ligated into the Age I/BamH I digested pBIIB71F10-134 vector which contains the BIIB71-F10 IgG1 heavy chain sequence. DNA sequences of the construct were confirmed by DNA sequencing. Heavy chain DNA and amino acid sequences for the 71F10 Fab-hLIGHT dimeric fusion (pBIIB71F10-255) are shown as SEQ ID NOs:178 and 179, respectively. The amino acid and nucleotide sequences corresponding to heavy chain of 71F10 Fab are shown starting from the N-terminus; followed by amino acids corresponding to the CH1 hinge region of IgG1 (amino acids 225 to 232 of SEQ ID NO:172); followed by amino acids corresponding to the linking group (amino acids 233 to 252 of SEQ ID NO:172); followed by the amino acids corresponding to human LIGHT extracellular domain (amino acids 253 to 400 of SEQ ID NO:172).
The 71F10 light chain vector (pBIIB71F10-129) was used in the 71F10 Fab-hLIGHT dimeric fusion and DNA and amino acid sequences are shown as SEQ ID NOs:110 and 109, respectively.
Dimeric 71F10 Fab-hLIGHT fusion protein was produced by transient transfection of CHO cells with pBIIB71F10-255 vector as described in Example 8. The protein titer in the medium was determined by Octet (ForteBio) and used for ELISA assay to assess binding activities to HER2 target and LIGHT receptor LTβR. AntiHer2 71F10-hLIGHT was used as a control. Results were summarized in FIGS. 25A and 25B. In FIG. 25A, 2-fold serial dilution of medium was added to the plates coated with 2 ug/ml rhErbB2-Fc, and binding was detected by goat-anti-human kappa-HRP (1:10000). In FIG. 25B, 2-fold serial dilution of medium was added to the plates coated with 2 ug/ml hHER2-Fc, and binding was detected by streptavidin-HRP (1:4000) after incubation with 1 ug/ml of Biotin-hLTβR-Fc. C06-hLIGHT showed specific binding to both IGFR (FIG. 25A) and LTβR (FIG. 25B) and demonstrated its bi-functional activity.
Deposits
CHO cells expressing the following Fab-LIGHT fusions were deposited on behalf of Biogen IDEC Inc., 14 Cambridge Center, Cambridge, Mass. 02142, with the Amercian Type Tissue Culture Collection (ATCC®) on Sep. 24, 2009 pursuant to the requirements of the Budapest Treaty:
ATCC ® Patent Deposit Designation
CHO cells: 71F10 fablight #1
PTA-10355
CHO cells: 65H09 fablight 5F6
PTA-10356
CHO cells: 67F11 fablight #4
PTA-10357
CHO cells: 65C10 fablight sorted pool
PTA-10358
Equivalents
All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
TABLE 1
human Her2
murine Her2
cyno Her2
CHO FACS
CHO FACS
CHO FACS
Antibody
EC50 (nM)
Binding
Binding
BIIB65-B03
15
no
yes
BIIB65-C10
6
no
yes
BIIB65-H09
20
no
yes
BIIB66-A12
60
no
yes
BIIB66-C01
12
no
yes
BIIB67-A02
100
no
no
BIIB67-C12
n.d.
no
no
BIIB67-F10
2
no
yes
BIIB67-F11
7
no
yes
BIIB69-A09
2
no
yes
BIIB71-A06
6
no
no
BIIB71-F10
60
yes
yes
TABLE 2
Binding activity summary of LIGHT fusion proteins
Octet Kinetic
ELISA
FACS
off-rates (kd),
EC50 (nM)
EC50 (nM)
x ~1e−4 sec−1
Construct Name
Her2
LTbR
HVEM
Her2
LTbR
HVEM
Her2
LTbR
HVEM
BIIB71F10 Mab
0.07
ND
ND
2
ND
ND
ND
BIIB71F10-130
0.05
0.01
0.01
0.5
0.2
0.5
1.12
1.68
1.22
BIIB71F10-131
0.05
0.01
0.01
0.5
0.2
0.5
3.07
1.83
ND
BIIB71F10-132
0.05
0.01
0.01
0.5
0.2
0.5
1.17
1.97
1.60
BIIBCD23-121
ND
ND
ND
ND
ND
ND
0.83
2.82
ND—not determined
TABLE 3
Examples of pro-inflammatory genes in HT29 cells that are
effected by treatment with BIIB71F10-130 as measured by
quantitative reverse transcriptase PCR
Fold Difference
Test Sample/
Gene
Control Sample
CCL2 (MCP-1)
1.05E+03
CCL21
5.46E+02
CCL5 (RANTES)
1.22E+03
CCL7 (MCP-3)
−5.27E+02
CCR4
1.65E+03
CXCL10 (IP-10)
5.27E+03
CXCL11 (I-TAC/IP9)
1.73E+03
CXCL2
1.13E+03
CXCL3
1.09E+03
CXCL5 (ENA-78/LIX)
1.81E+03
CXCL9
6.75E+02
IFNA2
1.93E+02
IL10RA
3.44E+03
IL5
9.40E+02
IL8
3.73E+02
LTB
2.91E+03
TNF
7.37E+03
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12610204
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biogen idec ma inc.
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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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424/134.1
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Apr 1st, 2022 05:10PM
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Apr 1st, 2022 05:10PM
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Biogen
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:biib
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Biogen
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Nov 25th, 2014 12:00AM
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Jul 5th, 2013 12:00AM
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https://www.uspto.gov?id=US08894999-20141125
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Use of DR6 and p75 antagonists to promote survival of cells of the nervous system
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The present invention relates to Death Receptor-6 (DR6) proteins which are members of the tumor necrosis factor (TNF) receptor family, and have now been shown to be important for regulating apoptosis in cells of the nervous system. In addition, it has been discovered that p75 is a ligand for DR6. As a result, this invention relates to methods for inhibiting the interaction of DR6 and p75 using DR6 and/or p75 antagonists. In addition, the methods described herein include methods of promoting survival of cells of the nervous system using DR6 antagonists, optionally in combination with p75 antagonists, and methods of treating neurodegenerative conditions by the administration of a DR6 antagonists, optionally in combination with a p75 antagonist.
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8894999
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1. A method of inhibiting the binding of DR6 to p75 comprising contacting a DR6 polypeptide and/or p75 polypeptide with a DR6 antagonist under conditions wherein binding of DR6 to p75 is inhibited.
2. The method of claim 1, wherein said contacting occurs in vitro.
3. The method of claim 1, wherein said contacting occurs in vivo.
4. A method of treating a condition associated with oligodendrocyte death or lack of differentiation comprising administering a therapeutically effective amount of a DR6 antagonist, wherein said DR6 antagonist is an antibody or antigen-binding fragment thereof that can specifically bind to DR6 and inhibit the binding of DR6 to p75.
5. The method of claim 4, wherein said DR6 antagonist is used in combination with a p75 antagonist.
6. The method of claim 4, wherein said DR6 antibody or antigen-binding fragment thereof does not prevent binding of DR6 to APP.
7. The method of claim 4, wherein the antibody or antigen-binding fragment binds to an epitope in amino acids 133-189 of SEQ ID NO:2.
8. The method of claim 4, wherein said DR6 antagonist is administered by a route selected from the group consisting of topical administration, intraocular administration, intravitreal administration, parenteral administration, intrathecal administration, subdural administration, subcutaneous administration or via a capsule implant.
9. The method of claim 8, wherein said route is parenteral administration.
10. A method of treating a condition associated with oligodendrocyte death or lack of differentiation comprising administering a therapeutically effective amount of a DR6 antagonist, wherein said DR6 antagonist comprises a DR6 antibody or antigen-binding fragment thereof, and wherein said DR6 antibody, or antigen-binding fragment thereof can specifically bind to the same DR6 epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M50-H01, M51-H09, M53-E04, M53-F04, M62-B02, M63-E10, M66-B03, M67-G02, M72-F03, and M73-C04 or a reference monoclonal antibody selected from the group consisting of 1P1D6.3, 1P2F2.1, and 1P5D10.2.
11. The method of claim 10, wherein the antibody or antigen-binding fragment thereof comprises
a heavy chain variable region (VH) comprising VH-CDR1, VH-CDR2, and VH-CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 128, 129, and 130 respectively and
a light chain variable region (VL) comprising VL-CDR1, VL-CDR2, and VL-CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 133, 134, and 135 respectively.
12. The method of claim 10, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising the amino acids of SEQ ID NO: 132.
13. The method of claim 10, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acids of SEQ ID NO: 127.
14. The method of claim 10, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising the amino acids of SEQ ID NO: 132 and a heavy chain variable region (VH) comprising the amino acids of SEQ ID NO: 127.
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14
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REFERENCE TO RELATED APPLICATIONS
Related applications U.S. Ser. No. 13/131,231, §371(c) Date Feb. 2, 2012, PCT/US2009/065755, filed Nov. 24, 2009, and U.S. 61/117,917, filed Nov. 25, 2008 are herein incorporated by reference in their entireties.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB
The content of the electronically submitted Sequence Listing (Name: sequencelisting.ascii.txt; Size: 115,276; and Date of Creation: Jan. 4, 2010) is herein incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to neurobiology, neurology and pharmacology. More particularly, it relates to methods for promoting survival of cells of the nervous system using Death Receptor-6 (DR6) antagonists, optionally in combination with p75 antagonists. The invention also relates to methods of treating neurodegenerative conditions by the administration of a DR6 antagonist, optionally in combination with a p75 antagonist. The invention also relates to methods of preventing the interaction of DR6 and p75 using DR6 and/or p75 antagonists.
2. Background
Apoptosis (i.e. programmed cell death) has been shown to play an important role in numerous diseases of the nervous system including both acute and chronic injuries. For example, the role of apoptosis has been demonstrated in Alzheimer's disease, Parkinson's disease, Huntington's disease, motor neuron disease (e.g. amyotrophic lateral sclerosis, which is also called ALS or Lou Gehrig's disease), multiple sclerosis, neuronal trauma and cerebral ischemia (e.g. stroke).
Many studies have been directed to understanding the molecular mechanisms of apoptosis, and these studies have led to the discovery of a family of receptors called the death receptors. Eight death receptors, which are characterized by a cytoplasmic death domain, have been identified thus far. The death receptors have been grouped into two different families. Members of the first family recruit a death inducing signaling complex (DISC), which promotes apoptotic signaling. Members of the second family recruit a different set of molecules to transduce apoptotic signals. Interestingly, members of the second family also transduce cell survival signals.
Death receptor 6 (DR6) is a member of the second family of death receptors. DR6 is widely expressed, but appears to function differently in different cell types. DR6 mRNA has been observed in heart, brain, placental, pancreas, lymph node, thymus and prostate tissues. Lower levels have been observed in other cell types including skeletal muscle, kidney and testes, but little or no expression has previously been observed in adult liver or any lines of hematopoeitic origin. Interestingly, it has been observed that DR6 is capable of inducing apoptosis in only a subset of cells tested. For example, overexpression of DR6 in HeLa S3 cervical carcinoma cells resulted in apoptosis in a death-domain-dependent manner (Pan et al. FEBS 431:351-356 (1998)). In addition, Nikoleav et al. (Nature 457:981-990 (2009)) have shown that beta-amyloid precursor protein (APP) is a DR6 ligand and suggested that the binding of an APP fragment to DR6 triggers degeneration of neuronal cell bodies and axons. In contrast, DR6 did not induce cell death in MCF7 (a human breast adenocarcinoma line) cells (Pan et al. FEBS 431:351-356 (1998)). The characteristics that differentiate a cell's response to DR6 expression and signaling have not yet been identified.
Drugs that can specifically modulate apoptosis may be useful for treating diseases involving neuronal cell death, for example, because neurons may have less capacity to regenerate than other cell types. However, currently available anti-apoptotic drugs have low specificity and selectivity and as a result, produce undesirable side effects. Such side effects might be reduced or avoided by for example, targeting anti-apoptotic drugs specifically to the desired site of action. Alternatively, characterization of death receptors such as DR6 that specifically act in a particular subset of cell types has the potential to provide a more specific therapeutic effect.
BRIEF SUMMARY OF THE INVENTION
The present invention is based on the discovery that DR6 is specifically able to induce apoptosis in cells of the nervous system and that p75 is a ligand for DR6. It has also been discovered that antagonists of DR6 and p75, including anti-DR6 antibodies, are able to inhibit the interaction of DR6 and p75 and to inhibit death of cells of the nervous system. Accordingly, antagonists of DR6 and/or p75 can be useful for therapy in which modulation of DR6 expression or activity is advantageous.
Based on the discoveries described herein, an isolated antibody or antigen-binding fragment thereof that can specifically bind to a DR6 polypeptide, such that the antibody promotes survival of cells of the nervous system is described. Also described is an isolated antibody or antigen-binding fragment thereof that specifically binds to DR6, wherein the antibody promotes proliferation, differentiation or survival of oligodendrocytes. DR6 antibodies also include an isolated antibody or antigen-binding fragment thereof which specifically binds to DR6, wherein the antibody promotes myelination. In some embodiments, the DR6 antibody inhibits binding of DR6 to p75. In some embodiments, the DR6 antibody inhibits binding of DR6 to p75 but does not inhibit binding of DR6 to beta-amyloid precursor protein (APP).
In some embodiments, the DR6 antibody is an isolated antibody or antigen-binding fragment thereof that specifically binds to the same DR6 epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M50-H01, M51-H09, M53-E04, M53-F04, M62-B02, M63-E10, M66-B03, M67-G02, M72-F03, and M73-C04 or a reference monoclonal antibody selected from the group consisting of 1P1D6.3, 1P2F2.1, and 1P5D10.2.
In some embodiments, the DR6 antibody an isolated antibody or antigen-binding fragment thereof which specifically binds to DR6, wherein said antibody or fragment thereof competitively inhibits a reference monoclonal Fab antibody fragment selected from the group consisting of M50-H01, M51-H09, M53-E04, M53-F04, M62-B02, M63-E10, M66-B03, M67-G02, M72-F03, and M73-C04 or a reference monoclonal antibody selected from the group consisting of 1P1D6.3, 1P2F2.1, and 1P5D10.2 from binding to DR6.
In some embodiments, the DR6 antibody is an isolated antibody or antigen-binding fragment thereof that specifically binds to DR6, wherein said antibody or fragment thereof is comprises an antigen binding domain identical to that of a monoclonal Fab antibody fragment selected from the group consisting of M50-H01, M51-H09, M53-E04, M53-F04, M62-B02, M63-E10, M66-B03, M67-G02, M72-F03, and M73-C04 or a reference monoclonal antibody selected from the group consisting of 1P1D6.3, 1P2F2.1, and 1P5D10.2.
In some embodiments, the DR6 antibody is an isolated antibody or fragment thereof that specifically binds to DR6, wherein the heavy chain variable region (VH) of said antibody or fragment thereof comprises an amino acid sequence at least 90% identical to a reference amino acid sequence selected from the group consisting of: SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:47, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:77, SEQ ID NO:87, SEQ ID NO: 97, SEQ ID NO:107, SEQ ID NO:117, and SEQ ID NO:127.
In some embodiments, the DR6 antibody is an isolated antibody or fragment thereof that specifically binds to DR6, wherein the light chain variable region (VL) of said antibody or fragment thereof comprises an amino acid sequence at least 90% identical to a reference amino acid sequence selected from the group consisting of: SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:32, SEQ ID NO:42, SEQ ID NO:52, SEQ ID NO:62, SEQ ID NO:72, SEQ ID NO:82, SEQ ID NO:92, SEQ ID NO:102 SEQ ID NO:112, SEQ ID NO:122, and SEQ ID NO:132.
In some embodiments, the DR6 antibody is an isolated antibody or fragment thereof that specifically binds to DR6, wherein the VH of said antibody or fragment thereof comprises an amino acid sequence identical, except for 20 or fewer conservative amino acid substitutions, to a reference amino acid sequence selected from the group consisting of: SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:47, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:77, SEQ ID NO:87, SEQ ID NO: 97, SEQ ID NO:107, SEQ ID NO:117, and SEQ ID NO:127.
In some embodiments, the DR6 antibody is an isolated antibody or fragment thereof that specifically binds to DR6, wherein the VL of said antibody or fragment thereof comprises an amino acid sequence identical, except for 20 or fewer conservative amino acid substitutions, to a reference amino acid sequence selected from the group consisting of: SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:32, SEQ ID NO:42, SEQ ID NO:52, SEQ ID NO:62, SEQ ID NO:72, SEQ ID NO:82, SEQ ID NO:92, SEQ ID NO:102 SEQ ID NO:112, SEQ ID NO:122, and SEQ ID NO:132.
In some embodiments, the DR6 antibody is an isolated antibody or fragment thereof that specifically binds to DR6, wherein the VH of said antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:47, SEQ ID NO:57, SEQ ID NO:67, SEQ ID NO:77, SEQ ID NO:87, SEQ ID NO: 97, SEQ ID NO:107, SEQ ID NO:117, and SEQ ID NO:127.
In some embodiments, the DR6 antibody is an isolated antibody or fragment thereof that specifically binds to DR6, wherein the VL of said antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:32, SEQ ID NO:42, SEQ ID NO:52, SEQ ID NO:62, SEQ ID NO:72, SEQ ID NO:82, SEQ ID NO:92, SEQ ID NO:102 SEQ ID NO:112, SEQ ID NO:122, and SEQ ID NO:132.
In some embodiments, the DR6 antibody is an isolated antibody or fragment thereof that specifically binds to DR6, wherein the VH and VL of said antibody or fragment thereof comprise, respectively, amino acid sequences at least 90% identical to reference amino acid sequences selected from the group consisting of: SEQ ID NO:7 and SEQ ID NO:12; SEQ ID NO:17 and SEQ ID NO:22; SEQ ID NO:27 and SEQ ID NO:32; SEQ ID NO:37 and SEQ ID NO:42; SEQ ID NO:47 and SEQ ID NO:52; SEQ ID NO:57 and SEQ ID NO:62; SEQ ID NO:67 and SEQ ID NO:72; SEQ ID NO:77 and SEQ ID NO:82; SEQ ID NO:87 and SEQ ID NO:92; SEQ ID NO:97 and SEQ ID NO:102; SEQ ID NO:107 and SEQ ID NO:112; SEQ ID N0117 and SEQ ID NO:122; and SEQ ID NO:127 and SEQ ID NO:132.
In some embodiments, the DR6 antibody is an isolated antibody or fragment thereof that specifically binds to DR6, wherein the VH and VL of said antibody or fragment thereof comprise, respectively, amino acid sequences identical, except for 20 or fewer conservative amino acid substitutions each, to reference amino acid sequences selected from the group consisting of: SEQ ID NO:7 and SEQ ID NO:12; SEQ ID NO:17 and SEQ ID NO:22; SEQ ID NO:27 and SEQ ID NO:32; SEQ ID NO:37 and SEQ ID NO:42; SEQ ID NO:47 and SEQ ID NO:52; SEQ ID NO:57 and SEQ ID NO:62; SEQ ID NO:67 and SEQ ID NO:72; SEQ ID NO:77 and SEQ ID NO:82; SEQ ID NO:87 and SEQ ID NO:92; SEQ ID NO:97 and SEQ ID NO:102; SEQ ID NO:107 and SEQ ID NO:112; SEQ ID N0117 and SEQ ID NO:122; and SEQ ID NO:127 and SEQ ID NO:132.
In some embodiments, the DR6 antibody is an isolated antibody or fragment thereof that specifically binds to DR6, wherein the VH and VL of said antibody or fragment thereof comprise, respectively, amino acid sequences selected from the group consisting of: SEQ ID NO:7 and SEQ ID NO:12; SEQ ID NO:17 and SEQ ID NO:22; SEQ ID NO:27 and SEQ ID NO:32; SEQ ID NO:37 and SEQ ID NO:42; SEQ ID NO:47 and SEQ ID NO:52; SEQ ID NO:57 and SEQ ID NO:62; SEQ ID NO:67 and SEQ ID NO:72; SEQ ID NO:77 and SEQ ID NO:82; SEQ ID NO:87 and SEQ ID NO:92; SEQ ID NO:97 and SEQ ID NO:102; SEQ ID NO:107 and SEQ ID NO:112; SEQ ID N0117 and SEQ ID NO:122; and SEQ ID NO:127 and SEQ ID NO:132.
In some embodiments, the DR6 antibody is an isolated antibody or fragment thereof that specifically binds to DR6, wherein the VH of said antibody or fragment thereof comprises a Kabat heavy chain complementarity determining region-1 (VH-CDR1) amino acid sequence identical, except for two or fewer amino acid substitutions, to a reference VH-CDR1 amino acid sequence selected from the group consisting of: SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ID NO: 48, SEQ ID NO: 58, SEQ ID NO: 68, SEQ ID NO: 78, SEQ ID NO: 88, SEQ ID NO: 98, SEQ ID NO: 108 SEQ ID NO: 118, and SEQ ID NO: 128. In one embodiment, the VH-CDR1 amino acid sequence is selected from the group consisting of: SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ID NO: 48, SEQ ID NO: 58, SEQ ID NO: 68, SEQ ID NO: 78, SEQ ID NO: 88, SEQ ID NO: 98, SEQ ID NO: 108 SEQ ID NO: 118, and SEQ ID NO: 128.
In some embodiments, the DR6 antibody is an isolated antibody or fragment thereof that specifically binds to DR6, wherein the VH of said antibody or fragment thereof comprises a Kabat heavy chain complementarity determining region-2 (VH-CDR2) amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VH-CDR2 amino acid sequence selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 69, SEQ ID NO: 79, SEQ ID NO: 89, SEQ ID NO: 99, SEQ ID NO: 109, SEQ ID NO: 119 and SEQ ID NO: 129. In one embodiment, the VH-CDR2 amino acid sequence is selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 69, SEQ ID NO: 79, SEQ ID NO: 89, SEQ ID NO: 99, SEQ ID NO: 109, SEQ ID NO: 119 and SEQ ID NO: 129.
In some embodiments, the DR6 antibody is an isolated antibody or fragment thereof that specifically binds to DR6, wherein the VH of said antibody or fragment thereof comprises a Kabat heavy chain complementarity determining region-3 (VH-CDR3) amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VH-CDR3 amino acid sequence selected from the group consisting of: SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 30, SEQ ID NO: 40, SEQ ID NO: 40, SEQ ID NO: 60, SEQ ID NO: 70, SEQ ID NO: 80, SEQ ID NO: 90, SEQ ID NO: 100, SEQ ID NO: 110, SEQ ID NO: 120 and SEQ ID NO:130. In one embodiment, the VH-CDR3 amino acid sequence is selected from the group consisting of: SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 30, SEQ ID NO: 40, SEQ ID NO: 40, SEQ ID NO: 60, SEQ ID NO: 70, SEQ ID NO: 80, SEQ ID NO: 90, SEQ ID NO: 100, SEQ ID NO: 110, SEQ ID NO: 120 and SEQ ID NO:130.
In some embodiments, the DR6 antibody is an isolated antibody or fragment thereof that specifically binds to DR6, wherein the VL of said antibody or fragment thereof comprises a Kabat light chain complementarity determining region-1 (VL-CDR1) amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VL-CDR1 amino acid sequence selected from the group consisting of: SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 53, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 83, SEQ ID NO: 93, SEQ ID NO: 103, SEQ ID NO: 113, SEQ ID NO: 123 and SEQ ID NO: 133. In one embodiment, the VL-CDR1 amino acid sequence is selected from the group consisting of: SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 53, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 83, SEQ ID NO: 93, SEQ ID NO: 103, SEQ ID NO: 113, SEQ ID NO: 123 and SEQ ID NO: 133.
In some embodiments, the DR6 antibody is an isolated antibody or fragment thereof that specifically binds to DR6, wherein the VL of said antibody or fragment thereof comprises a Kabat light chain complementarity determining region-2 (VL-CDR2) amino acid sequence identical, except for two or fewer amino acid substitutions, to a reference VL-CDR2 amino acid sequence selected from the group consisting of: SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 34, SEQ ID NO: 44, SEQ ID NO: 54, SEQ ID NO: 64, SEQ ID NO: 74, SEQ ID NO: 84, SEQ ID NO: 94, SEQ ID NO: 104, SEQ ID NO: 114, SEQ ID NO: 124, and SEQ ID NO: 134. In one embodiment, the VL-CDR2 amino acid sequence is selected from the group consisting of: SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 34, SEQ ID NO: 44, SEQ ID NO: 54, SEQ ID NO: 64, SEQ ID NO: 74, SEQ ID NO: 84, SEQ ID NO: 94, SEQ ID NO: 104, SEQ ID NO: 114, SEQ ID NO: 124, and SEQ ID NO: 134.
In some embodiments, the DR6 antibody is an isolated antibody or fragment thereof that specifically binds to DR6, wherein the VL of said antibody or fragment thereof comprises a Kabat light chain complementarity determining region-3 (VL-CDR3) amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VL-CDR3 amino acid sequence selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 25, SEQ ID NO: 35, SEQ ID NO: 45, SEQ ID NO: 55, SEQ ID NO: 65, SEQ ID NO: 75, SEQ ID NO: 85, SEQ ID NO: 95, SEQ ID NO: 105, SEQ ID NO: 115, SEQ ID NO: 125, and SEQ ID NO: 135. In one embodiment, the VL-CDR3 amino acid sequence is selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 25, SEQ ID NO: 35, SEQ ID NO: 45, SEQ ID NO: 55, SEQ ID NO: 65, SEQ ID NO: 75, SEQ ID NO: 85, SEQ ID NO: 95, SEQ ID NO: 105, SEQ ID NO: 115, SEQ ID NO: 125, and SEQ ID NO: 135.
In some embodiments, the DR6 antibody is an isolated antibody or fragment thereof that specifically binds to DR6, wherein the VH of said antibody or fragment thereof comprises VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 8, 9, and 10; SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70; SEQ ID NOs: 78, 79, and 80; SEQ ID NOs: 88, 89, and 90; SEQ ID NOs: 98, 99, and 100; SEQ ID NOs: 108, 109, and 110; SEQ ID NOs: 118, 119, and 120; and SEQ ID NOs: 128, 129, and 130, except for one, two, three, or four amino acid substitutions in at least one of said VH-CDRs.
In some embodiments, the DR6 antibody is an isolated antibody or fragment thereof that specifically binds to DR6, wherein the VH of said antibody or fragment thereof comprises VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 8, 9, and 10; SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70; SEQ ID NOs: 78, 79, and 80; SEQ ID NOs: 88, 89, and 90; SEQ ID NOs: 98, 99, and 100; SEQ ID NOs: 108, 109, and 110; SEQ ID NOs: 118, 119, and 120; and SEQ ID NOs: 128, 129, and 130.
In some embodiments, the DR6 antibody is an isolated antibody or fragment thereof that specifically binds to DR6, wherein the VL of said antibody or fragment thereof comprises VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 13, 14, and 15; SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 53, 54, and 55; SEQ ID NOs: 63, 64, and 65; SEQ ID NOs: 73, 74, and 75; SEQ ID NOs: 83, 84, and 85; SEQ ID NOs: 93, 94, and 95; SEQ ID NOs: 103, 104, and 105; SEQ ID NOs: 113, 114, and 115; SEQ ID NOs: 123, 124, and 125; and SEQ ID NOs: 133, 134, and 135, except for one, two, three, or four amino acid substitutions in at least one of said VL-CDRs.
In some embodiments, the DR6 antibody is an isolated antibody or fragment thereof that specifically binds to DR6, wherein the VL of said antibody or fragment thereof comprises VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 13, 14, and 15; SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 53, 54, and 55; SEQ ID NOs: 63, 64, and 65; SEQ ID NOs: 73, 74, and 75; SEQ ID NOs: 83, 84, and 85; SEQ ID NOs: 93, 94, and 95; SEQ ID NOs: 103, 104, and 105; SEQ ID NOs: 113, 114, and 115; SEQ ID NOs: 123, 124, and 125; and SEQ ID NOs: 133, 134, and 135.
In various embodiments of the above-described antibodies or fragments thereof, the VH framework regions and/or VL framework regions are human, except for five or fewer amino acid substitutions.
In some embodiments, the above-described antibodies or fragments thereof bind to a linear epitope or a non-linear conformation epitope.
In some embodiments, the above-described antibodies or fragments thereof are multivalent, and comprise at least two heavy chains and at least two light chains.
In some embodiments, the above-described antibodies or fragments thereof are multispecific. In further embodiments, the above-described antibodies or fragments thereof are bispecific.
In various embodiments of the above-described antibodies or fragments thereof, the heavy and light chain variable domains are murine. In further embodiments, the heavy and light chain variable domains are from a monoclonal antibody selected from the group consisting of 1P1D6.3, 1P2F2.1, and 1P5D10.2.
In various embodiments of the above-described antibodies or fragments thereof, the heavy and light chain variable domains are fully human. In further embodiments, the heavy and light chain variable domains are from a monoclonal Fab antibody fragment selected from the group consisting of M50-H01, M51-H09, M53-E04, M53-F04, M62-B02, M63-E10, M66-B03, M67-G02, M72-F03, and M73-C04.
In various embodiments, the above-described antibodies or fragments thereof are humanized.
In various embodiments, the above-described antibodies or fragments thereof are chimeric.
In various embodiments, the above-described antibodies or fragments thereof are primatized.
In various embodiments, the above-described antibodies or fragments thereof are fully human.
In certain embodiments, the above-described antibodies or fragments thereof are Fab fragments, Fab′ fragments, F(ab)2 fragments, or Fv fragments. In certain embodiments, the above-described antibodies are single chain antibodies. In certain embodiments, the antibodies or fragments thereof are conjugated to a polymer. In certain embodiments, the polymer is a polyalkylene glycol. In further embodiments, the polyalkylene glycol is polyethylene glycol (PEG).
In certain embodiments, the above-described antibodies or fragments thereof comprise light chain constant regions selected from the group consisting of a human kappa constant region and a human lambda constant region.
In certain embodiments, the above-described antibodies or fragments thereof comprise a heavy chain constant region or fragment thereof. In further embodiments, the heavy chain constant region or fragment thereof is selected from the group consisting of human IgG4, IgG4 agly, IgG1, and IgG1agly.
In some embodiments, the above-described antibodies or fragments thereof specifically bind to a DR6 polypeptide or fragment thereof, or a DR6 variant polypeptide, with an affinity characterized by a dissociation constant (KD) which is less than the KD for said reference monoclonal antibody. In further embodiments, the dissociation constant (KD) is no greater than 5×10−2 M, 10−2 M, 5×10−3 M, 10−3 M, 5×10−4 M, 10−4 M, 5×10−5 M, 10−5 M, 5×10−6 M, 10−6 M, 5×10−7 M, 10−7 M, 5×10−8 M, 10−8 M, 5×10−9 M, 10−9 M, 5×10−10 M, 10−10 M, 5×10−11 M, 10−11 M, 5×10−12 M, 10−12 M, 5×10−13 M, 10−13 M, 5×10−14 M, 10−14 M, 5×10−15 M, or 10−15 M.
In some embodiments, the above-described antibodies or fragments thereof preferentially bind to a human DR6 polypeptide or fragment thereof, relative to a murine DR6 polypeptide or fragment thereof.
In addition, the methods described herein relate generally to methods of promoting survival and preventing apoptosis of cells of the nervous system. In certain embodiments, the methods include a method of promoting survival of cells of the nervous system comprising contacting said cells with a DR6 antagonist. In some particular embodiments, the cells of the nervous system cells are cells of the central nervous system, such as brain cells, spinal cord cells, or cell lines derived from such cells. In some embodiments, the cells of the central nervous system are cortical neurons, oligodendrocytes, microglia or astrocytes, or cell lines derived from such cells. In some embodiments the cells of the nervous system are neurons, for example cortical neurons, motor neurons and dorsal root ganglion (DRG) neurons or cell lines derived from such cells. In some embodiments the cells are glial cells including microglia and macroglia or cell lines derived from such cells. Examples of macroglial cells include astrocytes, oligodendrocytes, ependymocytes and radial glial cells. In some embodiments, the cells are precursors of these cells such as oligodendrocyte precursor cells or cell lines derived from such cells. In some embodiments, the cells of the nervous system are peripheral nervous system cells. In some embodiments, the peripheral nervous system cells are dorsal root ganglion neurons, schwann cells, or cell lines derived from such cells.
The methods described herein also provide a method of promoting oligodendrocyte proliferation, differentiation or survival comprising contacting oligodendrocyte cells or oligodendrocyte precursor cells with a DR6 antagonist. In some embodiments, the method is a method of treating a condition associated with oligodendrocyte death or lack of differentiation, comprising administering a therapeutically effective amount of a DR6 antagonist.
The methods described herein also provide a method of promoting myelination comprising contacting a mixture of neuronal cells and oligodendrocyte cells or oligodendrocyte precursor cells with a DR6 antagonist. In some embodiments, the method is a method of treating a condition associated with dysmyelination or demyelination comprising administering a therapeutically effective amount of a DR6 antagonist.
The method also relates generally to methods of treating conditions associated with death of cells of the nervous system. In another embodiment, the method is a method of treating a condition associated with death of cells of the nervous system comprising administering an effective amount of a DR6 antagonist to a mammal in need thereof. In some particular embodiments the condition associated with death of cells of the nervous system can be Alzheimer's disease, Parkinson's disease, Huntington's disease, motor neuron disease (e.g. amyotrophic lateral sclerosis, which is also called ALS or Lou Gehrig's disease), multiple sclerosis, neuronal trauma or cerebral ischemia (e.g. stroke). In some particular embodiments, the condition is neuropathic pain.
The methods described herein also include methods of inhibiting the binding of DR6 to p75 comprising contacting a DR6 polypeptide and/or p75 polypeptide with a DR6 antagonist under conditions wherein binding of DR6 to p75 is inhibited.
In various embodiments of the above methods, the DR6 antagonist can be any molecule which interferes with the ability of DR6 to negatively regulate survival of cells of the nervous system. In certain embodiments, the DR6 antagonist is selected from the group consisting of a soluble DR6 polypeptide, a DR6 antagonist compound, a DR6 antagonist antibody or fragment thereof, a DR6 antagonist polynucleotide (e.g. RNA interference), a DR6 aptamer, or a combination of two or more DR6 antagonists.
In certain embodiments, the DR6 antagonist polypeptide is a soluble DR6 polypeptide. Certain soluble DR6 polypeptides as described herein include, but are not limited to soluble DR6 polypeptides which comprise the DR6 extracellular domain or one or more of the DR6 TNFR-like cysteine-rich motifs. In some embodiments, the soluble DR6 polypeptide lacks one or more of a DR6 TNFR-like cysteine-rich motif, a transmembrane domain, a death domain or a cytoplasmic domain. In some embodiments, the DR6 antagonist polypeptide comprises amino acids 1 to 349 of SEQ ID NO:2 (DR6); 40 to 349 of SEQ ID NO:2; or 41 to 349 of SEQ ID NO:2.
In some embodiments, the soluble DR6 antagonist is a fusion polypeptide comprising a non-DR6-heterologous polypeptide. In some embodiments, the non-DR6 heterologous polypeptide is selected from the group consisting of an immunoglobulin polypeptide or fragment thereof, a serum albumin polypeptide, a targeting polypeptide, a reporter polypeptide, and a purification-facilitating polypeptide. In some embodiments, the antibody Ig polypeptide is a hinge and an Fc polypeptide.
In alternative embodiments the DR6 antagonist is an antibody or fragment thereof as described above. In other embodiments, the DR6 antagonist is an an antibody or fragment thereof which binds to a DR6 polypeptide comprising one or more of the following domains (i) a DR6 extracellular domain, and (ii) a DR6 TNFR-like cysteine-rich motif. Additionally, the DR6 antagonist antibody or fragment thereof can specifically bind to an epitope within a polypeptide comprising a DR6 polypeptide as described herein. The DR6 antagonist can also be an antigen-binding fragment of such antibodies or a combination of two or more antibodies or fragments thereof.
In other embodiments, the DR6 antagonist is a DR6 antagonist polynucleotide such as an antisense polynucleotide, an aptamer, a ribozyme, a small interfering RNA (siRNA), or a small-hairpin RNA (shRNA).
In additional embodiments, the DR6 antagonist is a DR6 aptamer. A DR6 aptamer is a small polypeptide or a polynucleotide which binds DR6 and promotes nervous system cell survival or prevents cell apoptosis.
In some embodiments of the above methods, the DR6 antagonist is administered to a subject by a method comprising (a) introducing into a nervous system cell a polynucleotide that encodes the DR6 antagonist through operable association with an expression control sequence; and (b) allowing expression of said DR6 antagonist. In some embodiments the nervous system cells are in a mammal and said introducing comprises (a) administering to said mammal a polynucleotide which encodes a DR6 antagonist through operable association with an expression control sequence. In some embodiments, the cultured host cell is derived from the mammal to be treated. In certain embodiments, the polynucleotide is introduced into the host cell or nervous system cell via transfection, electroporation, viral transduction or direct microinjection.
In certain embodiments the DR6 antagonist is a polynucleotide that can be administered to a mammal, at or near the site of the disease, disorder or injury. In some embodiments, the polynucleotide is administered as an expression vector. In certain embodiments, the vector is a viral vector which is selected from the group consisting of an adenoviral vector, an alphavirus vector, an enterovirus vector, a pestivirus vector, a lentiviral vector, a baculoviral vector, a herpesvirus vector (e.g. an Epstein Barr viral vector, or a herpes simplex viral vector) a papovaviral vector, a poxvirus vector (e.g. a vaccinia viral vector) and a parvovirus. In some embodiments, the vector is administered by a route selected from the group consisting of topical administration, intraocular administration, and parenteral administration (e.g. intravenous, intraarterial, intramuscular, intracardiac, subcutaneous, intradermal, intrathecal, intraperitoneal).
According to the methods described herein, the DR6 antagonist can be used in combination with a p75 antagonist. The p75 antagonist can be used simultaneously or sequentially.
The methods described herein also include methods of inhibiting the binding of DR6 to p75 comprising contacting a p75 polypeptide and/or DR6 polypeptide with a p75 antagonist under conditions wherein binding of DR6 to p75 is inhibited. The p75 antagonist can be (i) a p75 antagonist compound; (ii) p75 antagonist polypeptide; (iii) a p75 antagonist antibody or fragment thereof; (iv) a p75 antagonist polynucleotide; (v) a p75 aptamer; or (vi) a combination of two or more of said p75 antagonists.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
FIGS. 1A-D—DR6 is regulated during development and expressed in the CNS. Graphs displaying results of DR6 quantitative PCR. DR6 mRNA levels from E18, P1, P7, P14, P21 and adult rat brain (FIG. 1A) and spinal cord (FIG. 1B) are expressed as a ratio of the DR6 mRNA level/DR6 mRNA level at E18. Protein expression in the cortex (CX), hippocampus (HP), striatum (ST), mid brain (MB), cerebellum (CB) and spinal cord (SC) based on Western blot using anti-DR6 antibody (FIG. 1C). RT-PCR of DR6 and GAPDH from lysates of oligodendrocyte precursor cells (OPCs), cortical neurons, microglias and astrocytes (FIG. 1D).
FIGS. 2A-B—DR6 is expressed in oligodendrocytes. RT-PCR of DR6 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from lysates of A2B5, O4 and MBP positive oligodendrocytes (FIG. 2A). The DR6 RT-PCR was performed without RT enzyme as a negative control (−RT DR6). Western blot showing expression of DR6 in A2B5, O4, and MBP-positive oligodendrocytes (FIG. 2B).
FIG. 3—DR6 is regulated in Alzheimer's disease. Graph displaying quantitative PCR of DR6 mRNA levels in Alzheimer's disease brains and control brains. Levels are expressed as a ratio of Alzheimer's Disease brain mRNA level/average control brain mRNA level.
FIG. 4—DR6 is regulated in response to neuronal injury. Graph displaying quantitative PCR of DR6 mRNA levels in control neurons and neurons subjected to axotomy. DR6 mRNA levels are expressed as a ratio of the mRNA level in axotomy sample/mRNA level in control sample.
FIGS. 5A-D—Overexpression of DR6 induces neuronal death. Images of cortical neurons infected with lentivirus expressing FL-DR6 and control cortical neurons (FIG. 5A). Graph displaying absorbance measured in an XTT assay for cell viability performed on untreated cells, cells infected with full-length DR6 (DR6-FL) lentivirus, dominant negative DR6 (DR6-DN) lentivirus and GFP lentivirus (FIG. 5B). Graph displaying free rhodamine measured in an assay for caspase-3 activity in untreated cells, DR6-FL, DR6-DN and GFP lentivirus infected cells (FIG. 5C). Western blots of DR6-FL, DR6-DN and GFP lentivirus infected cells probed with anti-activated caspase-3, anti-βIII-tubulin and anti-GFP antibodies (FIG. 5D).
FIGS. 6A-B—Overexpression of DR6 induces death of OPCs. Graph depicting percent cytotoxicity on cells infected with DR6-FL, DR6-DN or GFP lentivirus (FIG. 6A). Graph depicting absorbance as measured in an XTT assay on cells treated with rotenone and cells infected with DR6-FL, DR6-DN or GFP lentivirus (FIG. 6B). Graph depicting absorbance measured in an LDH assay for cell viability in cells infected with DR6-FL, DR6-DN or GFP lentivirus (FIG. 6C).
FIGS. 7A-B—Blocking DR6 signaling pathway promotes oligodendrocyte survival and differentiation. Western blots of cell lysates from DR6 FL and DR6 DN infected oligodendrocytes probed with anti-MBP, anti-MOG, anti-GFP and anti-β-actin antibodies (FIG. 7A). An ELISA measuring the level of CNPase in DR6 FL and DR6 DN infected oligodendrocytes (FIG. 7B).
FIGS. 8A-B—Soluble DR6 blocks full-length DR6 from inducing neuronal cell death. Time lapse images of cultured E18 cerebral cortical neurons that were treated with control human Fc (top panels), infected with FL-DR6 and treated with control human-Fc (middle panels) and infected with FL-DR6 and treated with DR6-Fc (bottom panels) (FIG. 8A). Graph depicting number of surviving cells after treatment with DR6-Fc or control Fc (FIG. 8B).
FIGS. 9A-B—Soluble DR6 promotes aged DRG neuron survival and neurite outgrowth in neurons expressing DR6-FL. Graph depicting neurons bearing processes after treatment with DR6-Fc, control Fc, or no treatment (FIG. 9A). Graph depicting neurons bearing large and complex processes after treatment with DR6-Fc, control Fc, or no treatment (FIG. 9B).
FIGS. 10A-B—DR6-induced neuronal death is inhibited by DR6-Fc. Western blots of untreated and DR6-FL lentivirus infected cells incubated in media containing of 0, 1, 3, or 30 μg/ml of recombinant soluble DR6 (DR6 Fc) probed with anti-activated caspase-3, anti-GFP and anti-β-actin antibodies (FIG. 10A). Graph depicting level of activated caspase-3 in cultures treated with DR6-Fc (FIG. 10B).
FIGS. 11A-D—DR6 RNAi promotes neuron survival. Graphs depicting percent cytotoxicity as measured in an LDH assay on cells treated with DR6 or control siRNAs (FIG. 11A). Graphs depicting percent cytotoxicity as measured in an XTT assay on cells exposed to DR6 or control siRNAs and treated with increasing concentrations of Abeta42 (FIG. 11B), glutamate (FIG. 11C) or TNF alpha (FIG. 11D).
FIGS. 12A-B—DR6 siRNA promotes oligodendrocyte differentiation. RT-PCR of DR6 and GAPDH from lysates of oligodendrocytes treated with control siRNA or DR6 siRNA (FIG. 12A). The DR6 RT-PCR reaction was also performed without the RT enzyme as a negative control (DR6-RT). Western blots of DR6 siRNA or control siRNA treated cells probed with anti-MBP, anti-MOG and anti-β-actin antibodies (FIG. 12B).
FIGS. 13A-C—Anti-DR6 antibodies bind to rat, mouse and human DR6. Graphs depict FACS analysis performed to assess the ability of anti-DR6 antibodies (1D10, 2F2 and 5D10) to bind to DR6 produced from 293T cells transfected with rat (FIG. 13A), human (FIG. 13B) or mouse DR6 (FIG. 13C).
FIGS. 14A-C—Blocking DR6 by anti-DR6 antibodies promotes oligodendrocyte differentiation and inhibits apoptosis. Graph depicting percent caspase 3 activity in oligodendrocyte cultures treated with anti-DR6 antibodies. (FIG. 14A). Graph depicting percent MBP+ cells in oligodendrocyte cultures treated with anti-DR6 antibodies. (FIG. 14B). Western blot of cell cultures treated with anti-DR6 antibody and probed with rabbit anti-cleaved caspase-3 (1:1000, Cell Signaling), mouse anti-MBP antibody (SMI 94 and SMI 99, 1:4000, Convance) and rabbit anti-β-actin antibodies (1:2000, Sigma) (FIG. 14C).
FIG. 15—Blocking DR6 by anti-DR6 antibodies promotes oligodendrocyte/DRG myelination in co-culture. Western blot of co-cultures of oligodendrocytes and DRG neurons treated with anti-DR6 antibody and probed with mouse anti-MBP antibody (SMI 94 and SMI 99, 1:4000, Convance), mouse anti-MOG antibody (1:500) and rabbit anti-β-actin antibodies (1:2000, Sigma).
FIG. 16A-B—Blocking DR6 by anti-DR6 antibodies promote remyelination in rat brain slice culture. Images of p17 brain slices no treatment and after treatment with bioactive lipid lysophosphatidylcholine (LPC) and anti-DR6 antibody (FIG. 16A). Graph depicting black gold stainging intensity after no treatment and treatment with bioactive lipid lysophosphatidylcholine (LPC) and anti-DR6 antibody (FIG. 16B).
FIGS. 17A-B—Anti-DR6 antibodies promote functional recovery in rat EAE model. Graph depicting EAE scores (measured as a functional recovery of the EAE disease) in MOG induced EAE rats treated with anti-DR6 antibody beginning at day 14 of MOG injection and occurring twice a week for 2 weeks. EAE score was measured as functional recovery of the EAE disease. Graphs depicting nerve conduction velocities in rat EAE model treated with control or anti-DR6 antibody.
FIGS. 18A-B—Lymphocyte and whole blood cell numbers are not affected by anti-DR6 antibody treatment in EAE rats. Graphs depicting total lymphocyte number (FIG. 18A) and whole blood cell number (FIG. 18B) determined at the end of EAE study (day 32).
FIG. 19—DR6 antibodies inhibit T-cell infiltration into spinal cord in EAE rats. Graph depicting results of IHC staining of spinal cord tissue using an anti-CD4 antibody to visualize T cell infiltration in the EAE mice. Stainings were performed at the end of the EAE study.
FIG. 20A-C—TNFα promotes neuron death and upregulates DR6. Graph depicting percent apoptotic cells after treatment with TNFα (FIG. 20A). Graph depicting % apoptotic cells versus total number of cells (DAPI+) after treatment with TNFα (FIG. 20B). Graph depicting % DR6+ cells versus total number of cells (DAPI+) after treatment with TNFα (FIG. 20C).
FIGS. 21A-D—TNFα upregulates DR6 and induces neuron death through NFkB signaling. Western blot of cortical neurons treated with TNFα for 18 and 24 hours and probed with anti-DR6, anti-NFκB, anti-IκBα, and anti-β-actin antibodies (FIG. 21A). Graph depicting quantitative amount of DR6 after treatment with TNFα (FIG. 21B). Graph depicting quantitative amount of NFκB after treatment with TNFα (FIG. 21C). Graph depicting quantitative amount of IκBα after treatment with TNFα (FIG. 21D).
FIG. 22A-D—DR6 RNAi decreases NFκB expression in neurons. Graph depicting DR6 mRNA level after no treatment and treatment with DR6 RNAi and control RNAi (FIG. 22A). Western blot of cortical neurons treated with TNFα, and further treated with DR6 RNAi and probed with anti-NFκB, anti-IκBα, and anti-β-actin antibodies (FIG. 22B). Graph depicting quantitative amount of NFκB after no treatment and treatment with DR6 RNAi (FIG. 22C). Graph depicting quantitative amount of IκBα after no treatment and treatment with DR6 RNAi (FIG. 22D).
FIG. 23A-B—Anti-DR6 antibodies promote schwann cell myelination. Western blot showing MBP and beta-actin levels in schwann cell and DRG neuron co-cultures after treatment with anti-DR6 or control antibodies (FIG. 23A). Bar graph depicting quantitation of MBP levels as compared to beta-actin levels in the co-cultures (FIG. 23B).
FIG. 24—Levels of DR6 and phosphorylated-AKT are inversely related. Western blot showing DR6, p-AKT, AKT and β-actin levels in cultured rat cortical neurons.
FIGS. 25A-B—DR6 and p75 interact. Western blots showing DR6 and p75 protein levels that result from immunopreciptation of DR6 from cells recombinantly expressing combinations of DR6, p75, and TrkA (negative control) proteins (FIG. 25A). Bar graph depicting quantification of binding of p75 to the surface of cells recombinantly expressing DR6 or pV90 (negative control) (FIG. 25C). Western blots showing DR6 and p75 protein levels the result from immunopreciptation of DR6 from human fetal spinal cord isolates (FIG. 25B).
FIG. 26—DR6 and p75 are co-expressed in mouse brain. Bar graph showing quantification of DR6 and p75 mRNA levels in various regions of the mouse brain.
FIGS. 27A-B—DR6 antibody 5D10 blocks the interaction of DR6 and p75. Western blot showing p75 and DR6 proteins that immunoprecipitate with anti-DR6 antibodies 2A9 and 5D10 (FIG. 27A). Image showing CHO cells transfected with a control vector or a vector encoding p75 and exposed to either alkaline phosphatase-DR6 alone, or alkaline phosphatase-DR6 in combination with an anti-DR6 antibody (FIG. 27B).
FIGS. 28A-B—Antibodies that block the DR6-p75 interaction bind to the Cys3/Cys4 domain of DR6. Bar graph showing level of expression of recombinant DR6 deletion mutants (FIG. 28A). Western blot showing amounts of myc-DR6 fusion proteins that immunoprecipitate using an anti-myc antibody (positive control), anti-DR6 antibody 5D10, and anti-DR6 antibody 2A9 (FIG. 28B). Bar graph showing quantitation of interaction of DR6 deletion proteins with anti-DR6 antibodies 5D10, 2A9, 1D6, 2F2, and A4A (FIG. 28C). MOPC-21 indicates a mouse monoclonal control antibody.
FIG. 29—The Cys3/Cys4 region of DR6 is important for binding to p75. Western blot showing the amounts of DR6 deletion protein and p75 protein that immunprepitate with DR6.
FIG. 30—Anti-DR6 antibodies 5D10 and M53E04 bind to human DR6. Graphs depict FACS analysis performed to assess the ability of anti-DR6 antibodies to bind to human and rat DR6. Anti-myc antibody 9E10 staining results show human and rat DR6 protein expression.
FIG. 31—Blocking DR6 by anti-DR6 antibodies promotes oligodendrocyte/DRG myelination in co-culture. Western blot of co-cultures of oligodendrocytes and DRG neurons treated with anti-DR6 antibodies (M53E04 and 5D10), an anti-LINGO-1 antibody (Li81), and a control antibody and probed with anti-MBP antibody, anti-MOG antibody, and anti-β-actin antibody.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application including the definitions will control. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents and other references mentioned herein are incorporated by reference in their entireties for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
Although methods and materials similar or equivalent to those described herein can be used in practice or testing of the methods described herein, suitable methods and materials are described below. The materials, methods and examples are illustrative only and are not intended to be limiting. Other features and advantages of the antibodies and methods described herein will be apparent from the detailed description and from the claims.
In order to further define this invention, the following terms and definitions are provided.
It is to be noted that the term “a” or “an” entity, refers to one or more of that entity; for example, “an immunoglobulin molecule,” is understood to represent one or more immunoglobulin molecules. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
Throughout this specification and claims, the word “comprise,” or variations such as “comprises” or “comprising,” indicate the inclusion of any recited integer or group of integers but not the exclusion of any other integer or group of integers.
As used herein, the term “consists of,” or variations such as “consist of” or “consisting of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, but that no additional integer or group of integers can be added to the specified method, structure or composition.
As used herein, the term “consists essentially of,” or variations such as “consist essentially of” or “consisting essentially of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not materially change the basic or novel properties of the specified method, structure or composition.
As used herein, a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutic result can be, e.g., lessening of symptoms, prolonged survival, improved mobility, or the like. A “therapeutically effective amount” can achieve any one of the desired therapeutic results or any combination of multiple desired therapeutic results A therapeutic result need not be a “cure”.
As used herein, a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
As used herein, a “polynucleotide” can contain the nucleotide sequence of the full length cDNA sequence, including the untranslated 5′ and 3′ sequences, the coding sequences, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence. The polynucleotide can be composed of any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotides can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. Polynucleotides can also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
A polypeptide can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and can contain amino acids other than the 20 gene-encoded amino acids (e.g. non-naturally occurring amino acids). The polypeptides described herein can be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification can be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide can contain many types of modifications. Polypeptides can be branched, for example, as a result of ubiquitination, and they can be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides can result from posttranslation natural processes or can be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, Proteins—Structure And Molecular Properties, 2nd Ed., T. E. Creighton, W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)
The terms “fragment,” “variant,” “derivative” and “analog” when referring to a Death Receptor-6 (DR6) antagonist include any antagonist molecules which promote nervous system cell survival. Soluble DR6 polypeptides can include DR6 proteolytic fragments, deletion fragments and in particular, fragments which more easily reach the site of action when delivered to an animal. Polypeptide fragments further include any portion of the polypeptide which comprises an antigenic or immunogenic epitope of the native polypeptide, including linear as well as three-dimensional epitopes. Soluble DR6 polypeptides can comprise variant DR6 regions, including fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants can occur naturally, such as an allelic variant. By an “allelic variant” is intended alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants can be produced using art-known mutagenesis techniques. Soluble DR6 polypeptides can comprise conservative or non-conservative amino acid substitutions, deletions or additions. DR6 antagonists can also include derivative molecules. For example, soluble DR6 polypeptides can include DR6 regions which have been altered so as to exhibit additional features not found on the native polypeptide. Examples include fusion proteins and protein conjugates.
A “polypeptide fragment” refers to a short amino acid sequence of a DR6 polypeptide. Protein fragments can be “free-standing,” or comprised within a larger polypeptide of which the fragment forms a part of region. Representative examples of polypeptide fragments, include, for example, fragments comprising about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 60 amino acids, about 70 amino acids, about 80 amino acids, about 90 amino acids, and about 100 amino acids in length.
As used herein, the term “antigen binding molecule” (“ABM”) refers in its broadest sense to a molecule that specifically binds an antigenic determinant. It is understood by those of skill in the art that fragments of mature antibodies can bind specifically to an antigen. Accordingly, an antigen binding molecule, as the term is used herein, includes, but is not limited to, fragments of mature antibodies that bind specifically to a target antigen. An ABM need not contain a constant region. If one or more constant region(s) is present, in particular embodiments, the constant region is substantially identical to human immunoglobulin constant regions, e.g., at least about 85-90%, or about 95% or more identical. The ABMs can be glycoengineered to enhance antibody dependent cellular cytotoxicity.
Antibody or Immunoglobulin.
In one embodiment, the DR6 antagonists for use in the treatment methods disclosed herein are “antibody” or “immunoglobulin” molecules, or immunospecific fragments thereof, e.g., naturally occurring antibody or immunoglobulin molecules or engineered antibody molecules or fragments that bind antigen in a manner similar to antibody molecules. The terms “antibody” and “immunoglobulin” are used interchangeably herein. As used herein, the term antibody or immunoglobulin is intended to include whole antibody molecules, including monoclonal, polyclonal and multispecific (e.g., bispecific) antibodies as well as antibody fragments having the Fc region and retaining binding specificity, and fusion proteins that include a region equivalent to the Fc region of an immunoglobulin and that retain binding specificity. Also encompassed are antibody fragments that retain binding specificity including, but not limited to, VH fragments, VL fragments, Fab fragments, F(ab′)2 fragments, scFv fragments, Fv fragments, minibodies, diabodies, triabodies, and tetrabodies (see, e.g., Hudson and Souriau, Nature Med. 9: 129-134 (2003)). Also encompassed are humanized, primatized and chimeric antibodies.
As will be discussed in more detail below, the term “immunoglobulin” comprises five broad classes of polypeptides that can be distinguished biochemically. Although all five classes are clearly useful in the methods described, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000. The four chains are typically joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region.
Both the light and heavy chains are divided into regions of structural and functional homology. The terms “constant” and “variable” are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino-terminus of the antibody. The N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.
Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (γ, μ, α, δ, ε)) with some subclasses among them (e.g., γ1-γ4). It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant invention.
As indicated above, the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH domain of an antibody combine to form the variable region that defines a three dimensional antigen binding site. This quaternary antibody structure forms the antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three complementary determining regions (CDRs) on each of the VH and VL chains. In some instances, e.g., certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, a complete immunoglobulin molecule can consist of heavy chains only, with no light chains. See, e.g., Hamers Casterman et al., Nature 363:446 448 (1993).
In naturally occurring antibodies, the six “complementarity determining regions” or “CDRs” present in each antigen binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding domain as the antibody assumes its three dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen binding domains, referred to as “framework” regions, show less inter-molecular variability. The framework regions largely adopt a β-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the β-sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined (see, “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which are incorporated herein by reference in their entireties).
In the case where there are two or more definitions of a term which is used and/or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term “complementarity determining region” (“CDR”) to describe the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. This particular region has been described by Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference, where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.
TABLE 1
CDR DEFINITIONS1
Kabat
Chothia
VH CDR1
31-35
26-32
VH CDR2
50-65
52-58
VH CDR3
95-102
95-102
VL CDR1
24-34
26-32
VL CDR2
50-56
50-52
VL CDR3
89-97
91-96
1Numbering of all CDR definitions in Table 1 is according to the numbering conventions set forth by Kabat et al. (see below).
Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of “Kabat numbering” to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in a C35 antibody or antigen-binding fragment, variant, or derivative thereof of are according to the Kabat numbering system.
In camelid species, however, the heavy chain variable region, referred to as VHH, forms the entire CDR. The main differences between camelid VHH variable regions and those derived from conventional antibodies (VH) include (a) more hydrophobic amino acids in the light chain contact surface of VH as compared to the corresponding region in VHH, (b) a longer CDR3 in VHH, and (c) the frequent occurrence of a disulfide bond between CDR1 and CDR3 in VHH.
In one embodiment, an antigen binding molecule comprises at least one heavy or light chain CDR of an antibody molecule. In another embodiment, an antigen binding molecule comprises at least two CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule comprises at least three CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule comprises at least four CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule comprises at least five CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule comprises at least six CDRs from one or more antibody molecules. Exemplary antibody molecules comprising at least one CDR that can be included in the subject antigen binding molecules are known in the art and exemplary molecules are described herein.
Antibodies or immunospecific fragments thereof for use in the methods described herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab′ and F(ab′)2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to binding molecules disclosed herein). ScFv molecules are known in the art and are described, e.g., in U.S. Pat. No. 5,892,019. Immunoglobulin or antibody molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
Antibody fragments, including single-chain antibodies, can comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Antigen-binding fragments can also comprise any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. Antibodies or immunospecific fragments thereof for use in the diagnostic and therapeutic methods disclosed herein can be from any animal origin including birds and mammals. In certain embodiments, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region can be condricthoid in origin (e.g., from sharks). As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.
As used herein, the term “heavy chain portion” includes amino acid sequences derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain portion comprises at least one of: a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, a binding polypeptide can comprise a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide comprises a polypeptide chain comprising a CH3 domain. Further, a binding polypeptide can lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As set forth above, it will be understood by one of ordinary skill in the art that these domains (e.g., the heavy chain portions) can be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule.
In certain embodiments, DR6 antagonist antibodies or immunospecific fragments thereof for use in the treatment methods disclosed herein, the heavy chain portions of one polypeptide chain of a multimer are identical to those on a second polypeptide chain of the multimer. Alternatively, heavy chain portion-containing monomers for use in the methods described herein are not identical. For example, each monomer can comprise a different target binding site, forming, for example, a bispecific antibody.
The heavy chain portions of a binding polypeptide for use in the diagnostic and treatment methods disclosed herein can be derived from different immunoglobulin molecules. For example, a heavy chain portion of a polypeptide can comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, a heavy chain portion can comprise a hinge region derived, in part, from an IgG1 molecule and, in part, from an IgG3 molecule. In another example, a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgG1 molecule and, in part, from an IgG4 molecule.
As used herein, the term “light chain portion” includes amino acid sequences derived from an immunoglobulin light chain. Typically, the light chain portion comprises at least one of a VL or CL domain.
An isolated nucleic acid molecule encoding a non-natural variant of a polypeptide derived from an immunoglobulin (e.g., an immunoglobulin heavy chain portion or light chain portion) can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of the immunoglobulin such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. For example, conservative amino acid substitutions are made at one or more non-essential amino acid residues.
Antibodies or immunospecific fragments thereof for use in the treatment methods disclosed herein can also be described or specified in terms of their binding affinity to a polypeptide. For example, binding affinities include those with a dissociation constant or Kd less than 5×10−2 M, 10−2 M, 5×10−3 M, 10−3 M, 5×10−4 M, 10−4 M, 5×10−5 M, 10−5 M, 5×10−6 M, 10−6 M, 5×10−7 M, 10−7 M, 5×10−8 M, 10−8 M, 5×10−9 M, 10−9 M, 5×10−10 M, 10−10 M, 5×10−11 M, 10−11 M, 5×10−12 M, 10−12 M, 5×10−13 M, 10−13 M, 5×10−14 M, 10−14 M, 5×10−15 M, or 10−15 M.
Antibodies or immunospecific fragments thereof for use in the treatment methods disclosed herein act as antagonists of DR6 as described herein. For example, an antibody for use in the methods described herein can function as an antagonist, blocking or inhibiting the suppressive activity of the DR6 polypeptide.
As used herein, the term “chimeric antibody” will be held to mean any antibody wherein the immunoreactive region or site is obtained or derived from a first species and the constant region (which can be intact, partial or modified) is obtained from a second species. In certain embodiments the target binding region or site will be from a non-human source (e.g. mouse or primate) and the constant region is human.
As used herein, the term “engineered antibody” refers to an antibody in which the variable domain in either the heavy and light chain or both is altered by at least partial replacement of one or more CDRs from an antibody of known specificity and, if necessary, by partial framework region replacement and sequence changing. Although the CDRs can be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs will be derived from an antibody of different class and/or an antibody from a different species. An engineered antibody in which one or more “donor” CDRs from a non-human antibody of known specificity is grafted into a human heavy or light chain framework region is referred to herein as a “humanized antibody.” In some cases it is not necessary to replace all of the CDRs with the complete CDRs from the donor variable region to transfer the antigen binding capacity of one variable domain to another. Rather, in some cases, it is only be necessary to transfer those residues that are necessary to maintain the activity of the target binding site. Given the explanations set forth in, e.g., U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370, it will be well within the competence of those skilled in the art, either by carrying out routine experimentation or by trial and error testing to obtain a functional engineered or humanized antibody.
As used herein, the term humanized is used to refer to an antigen-binding molecule derived from a non-human antigen-binding molecule, for example, a murine antibody, that retains or substantially retains the antigen-binding properties of the parent molecule but which is less immunogenic in humans. This can be achieved by various methods including (a) grafting the entire non-human variable domains onto human constant regions to generate chimeric antibodies, (b) grafting only the non-human CDRs onto human framework and constant regions with or without retention of critical framework residues (e.g., those that are important for retaining good antigen binding affinity or antibody functions), or (c) transplanting the entire non-human variable domains, but “cloaking” them with a human-like section by replacement of surface residues. Such methods are disclosed in Jones et al., Morrison et al., Proc. Natl. Acad. Sci., 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988); Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun., 31(3):169-217 (1994), all of which are incorporated by reference in their entirety herein. There are generally 3 complementarity determining regions, or CDRs, (CDR1, CDR2 and CDR3) in each of the heavy and light chain variable domains of an antibody, which are flanked by four framework subregions (i.e., FR1, FR2, FR3, and FR4) in each of the heavy and light chain variable domains of an antibody: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A discussion of humanized antibodies can be found, inter alia, in U.S. Pat. No. 6,632,927, and in published U.S. Application No. 2003/0175269, both of which are incorporated herein by reference in their entirety.
As used herein, the terms “linked,” “fused” or “fusion” are used interchangeably. These terms refer to the joining together of two more elements or components, by whatever means including chemical conjugation or recombinant means. An “in-frame fusion” refers to the joining of two or more open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct reading frame of the original ORFs. Thus, the resulting recombinant fusion protein is a single protein containing two ore more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature.) Although the reading frame is thus made continuous throughout the fused segments, the segments can be physically or spatially separated by, for example, in-frame linker sequence.
In the context of polypeptides, a “linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminal direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide.
The term “expression” as used herein refers to a process by which a gene produces a biochemical, for example, an RNA or polypeptide. The process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product and the translation of such mRNA into polypeptide(s). If the final desired product is biochemical, expression includes the creation of that biochemical and any precursors.
By “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; bears; and so on. In certain embodiments, the mammal is a human subject.
The term “RNA interference” or “RNAi” refers to the silencing or decreasing of gene expression by siRNAs. It is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by siRNA that is homologous in its duplex region to the sequence of the silenced gene. The gene can be endogenous or exogenous to the organism, present integrated into a chromosome or present in a transfection vector that is not integrated into the genome. The expression of the gene is either completely or partially inhibited. RNAi can also be considered to inhibit the function of a target RNA; the function of the target RNA can be complete or partial.
Death Receptor-6 (DR6/TNFRSF21)
It has been discovered that DR6 is expressed in cells of the nervous system including neurons and oligodendrocyte precursor cells and that DR6 can induce cell death in these cells.
DR6 is a polypeptide consisting of 655 amino acids. In certain embodiments, the human polypeptide is encoded by an mRNA comprising the nucleotides of SEQ ID NO:1 (Accession Number: NM—014452). In certain embodiments, the human DR6 polypeptide sequence comprises the amino acids of SEQ ID NO:2 (Accession Number: O75509). Mouse DR6 is also a 655 amino acid polypeptide. In certain embodiments, mouse DR6 is encoded by an mRNA comprising the nucleotides of SEQ ID NO:3 (Accession Number: NM—178589). In certain embodiments, the mouse DR6 polypeptide sequence comprises the amino acid sequence of SEQ ID NO:4 (Accession Number: NP—848704).
Table 2 lists DR6 domains and other regions according to the amino acid residue number based on the sequence of SEQ ID NO:2. As one of skill in the art will appreciate, the beginning and ending residues of the domains listed below can vary depending upon the computer modeling program used, the method used for determining the domain, minor sequence variations etc.
TABLE 2
Domain or Region
Beginning Residue
Ending Residue
Signal Sequence
1
40 or 41
Extracellular Domain
41 or 42
349 or 350
TNFR-like Cysteine-Rich Motif-1
50
88
TNFR-like Cysteine-Rich Motif-2
90
131
TNFR-like Cysteine-Rich Motif-3
133
167
TNFR-like Cysteine-Rich Motif-4
170
211
Transmembrane
350 or 351
367-370
Cytoplasmic
368-371
655
Death Domain
415
498
Leucine Zipper Motif
497
526
P75/TNR16
It has also been discovered that p75 neurotrophin receptor is a ligand for DR6. P75, also known as tumor necrosis factor receptor superfamily member 16 (TNR16 or TNFRSF16) or nerve growth factor receptor (NGFR), is a polypeptide consisting of 427 amino acids. The human polypeptide sequence is Accession Number NP—002498 (SEQ ID NO: 165) and the nucleic acid sequence is Accession Number NM—002507 (SEQ ID NO: 166). The p75 protein, like the DR6 protein, includes an extracellular region containing four TNFR Cysteine-Rich motifs, a transmembrane region, and an intracellular region containing a death domain. It has previously been shown that p75 is a low affinity receptor which can bind to NGF, BDNF, NT-3, and NT-4. Mi et al. Nat. Neuroscience 7:221-228 (2004). In addition, p75 is a component of the LINGO-1/Nogo-66 receptor signaling pathway and can mediate survival and death of neuronal cells. Id.
Methods of Using Antagonists of DR6 and p75
In one embodiment, the method is a method for promoting survival of cells of the nervous system comprising contacting said cells with a DR6 antagonist. Another embodiment provides methods for promoting oligodendrocyte proliferation, differentiation or survival comprising contacting oligodendrocyte cells or oligodendrocyte precursor cells with a DR6 antagonist. Another embodiment provides methods for promoting myelination comprising contacting a mixture of neuronal cells and oligodendrocytes or oligodendrocyte precursor cells with a DR6 antagonist. Yet another embodiment provides methods of inhibiting the binding of DR6 and p75 comprising contacting a DR6 polypeptide and/or a p75 polypeptide with a DR6 antagonist under conditions wherein binding of DR6 to p75 is inhibited. Similarly, the methods described herein also include methods of inhibiting the binding of DR6 to p75 comprising contacting a DR6 polypeptide and/or a p75 polypeptide with a p75 antagonist.
A DR6 antagonist can be a DR6 antagonist polypeptide, a DR6 antagonist compound, a DR6 antibody, a DR6 antagonist polynucleotide, a DR6 aptamer or a combination of two or more DR6 antagonists. Additional embodiments include methods for treating a condition associated with death of cells of the nervous system comprising administering a therapeutically effective amount of a DR6 antagonist.
A p75 antagonist can be a p75 antagonist polypeptide, a p75 antagonist compound, a p75 antibody, a p75 antagonist polynucleotide, a p75 aptamer, or a combination of two or more p75 antagonists. Additional embodiments include methods for treating a condition associated with death of cells of nervous system comprising administering a therapeutically effective amount of a DR6 antagonist in combination with a p75 antagonist.
In some particular embodiments the condition associated with death of nervous system cells can be Alzheimer's disease, Parkinson's disease, Huntington's disease, motor neuron disease (e.g. amyotrophic lateral sclerosis, which is also called ALS or Lou Gehrig's disease), multiple sclerosis, neuronal trauma or cerebral ischemia (e.g. stroke). Another embodiment provides methods for treating a disease of neuronal degeneration comprising administering a therapeutically effective amount of a DR6 antagonist.
Cells of the nervous system include both cells of the central nervous system (or “CNS cells” and cells of the peripheral nervous system (or “PNS cells”). CNS cells include cells in the brain and cells in the spinal cord, as well as cell lines derived from such cells. PNS cells include, for example, dorsal root ganglion neurons, schwann cells, motor neurons and cell lines derived from such cells. Cells of the nervous system include neurons such as cortical neurons, motor neurons, dorsal root ganglion (DRG) neurons and cell lines derived from such cells. Cells of the nervous also include neuronal support cells or glial cells including microglia and macroglia as well as cell lines derived from such cells. Examples of macroglial cells include astrocytes, oligodendrocytes, ependymocytes and radial glial cells. Cells of the nervous system also include precursors of these cells such as oligodendrocyte precursor cells and cell lines derived from such cells.
DR6 Antagonist Polypeptides
DR6 antagonists to be used herein include those polypeptides which block, inhibit or interfere with the biological function of naturally occurring DR6. Specifically, soluble DR6 polypeptides include fragments, variants, or derivative thereof of a soluble DR6 polypeptide. Table 1 above describes the various domains of a human DR6 polypeptide. Similar domain structures can be deduced for DR6 polypeptides of other species, e.g., mouse or rat DR6. Soluble DR6 polypeptides typically lack the transmembrane domain of the DR6 polypeptide, and optionally lack the cytoplasmic domain of the DR6 polypeptide. For example, certain soluble human DR6 polypeptides lack amino acids 351-367 of SEQ ID NO:2, which comprises the transmembrane domain of human DR6. Another soluble human DR6 polypeptide lacks both the transmembrane domain and the intracellular domain (amino acids 350-655 of SEQ ID NO:2). Additionally, certain soluble DR6 polypeptides comprise one or more of the TNFR-like cysteine rich motifs and/or the entire extracellular domain (corresponding to amino acids 40 to 349 of SEQ ID NO:2, 40 to 350 of SEQ ID NO:2, 41 to 349 of SEQ ID NO:2 or 41 to 350 of SEQ ID NO:2) of the DR6 polypeptide. As one of skill in the art would appreciate, the entire extracellular domain of DR6 can comprise additional or fewer amino acids on either the C-terminal or N-terminal end of the extracellular domain polypeptide. The soluble antagonist DR6 polypeptide can or can not include the signal sequence.
Additional soluble DR6 polypeptides for use in the methods described herein include, but are not limited to, a soluble DR6 polypeptide comprising, consisting essentially of, or consisting of amino acids 1 to 40 of SEQ ID NO:2; 1 to 41 of SEQ ID NO:2; 65 to 105 of SEQ ID NO:2; 106 to 145 of SEQ ID NO:2; 146 to 185 of SEQ ID NO:2; and 186 to 212 of SEQ ID NO:2; or fragments, variants, or derivatives of such polypeptides.
Further soluble DR6 polypeptides for use in the methods described herein include, but are not limited to, a soluble DR6 polypeptide comprising, consisting essentially of, or consisting of amino acids 1 to 40 of SEQ ID NO:2; 1 to 41 of SEQ ID NO:2; 1 to 64 of SEQ ID NO:2; 1 to 105 of SEQ ID NO:2; 1 to 145 of SEQ ID NO:2; 1 to 185 of SEQ ID NO:2; 1 to 212 of SEQ ID NO:2; 1 to 349 of SEQ ID NO:2; or fragments, variants, or derivatives of such polypeptides.
Still further soluble DR6 polypeptides for use in the methods described herein include, but are not limited to, a DR6 polypeptide comprising, consisting essentially of, or consisting of amino acids 41 to 64 of SEQ ID NO:2; 41 to 105 of SEQ ID NO:2; 41 to 145 of SEQ ID NO:2; 41 to 185 of SEQ ID NO:2; 41 to 212 of SEQ ID NO:2; 41 to 349 of SEQ ID NO:2; 41 to 350 of SEQ ID NO:2; 42 to 64 of SEQ ID NO:2; 42 to 105 of SEQ ID NO:2; 42 to 145 of SEQ ID NO:2; 42 to 185 of SEQ ID NO:2; 42 to 212 of SEQ ID NO:2; 42 to 349 of SEQ ID NO:2; and 42 to 350 of SEQ ID NO:2; or fragments, variants, or derivatives of such polypeptides.
Additional soluble DR6 polypeptide for us in the methods described herein include, but are not limited to, a soluble DR6 polypeptide comprising, consisting essentially of, or consisting of amino acids 65 to 105 of SEQ ID NO:2; 65 to 212 of SEQ ID NO:2; 65 to 349 of SEQ ID NO:2; 106 to 145 of SEQ ID NO:2; 106 to 212 of SEQ ID NO:2; 106 to 349 of SEQ ID NO:2; 146 to 185 of SEQ ID NO:2; 146 to 212 of SEQ ID NO:2; 146 to 349 of SEQ ID NO:2; 186 to 212 of SEQ ID NO:2; 186 to 349 of SEQ ID NO:2; and 213 to 349 of SEQ ID NO:2; or fragments, variants, or derivatives of such polypeptides.
A variant DR6 polypeptide can also vary in sequence from the corresponding wild-type polypeptide. In particular, certain amino acid substitutions can be introduced into the DR6 sequence without appreciable loss of a DR6 biological activity. In exemplary embodiments, a variant DR6 polypeptide contains one or more amino acid substitutions, and/or comprises an amino acid sequence which is at least 70%, 80%, 85%, 90%, 95%, 98% or 99% identical to a reference amino acid sequence selected from the group consisting of: amino acids 41 to 349 of SEQ ID NO:2 or equivalent fragments of SEQ ID NO:4. A variant DR6 polypeptide differing in sequence from any given fragment of SEQ ID NO:2 or SEQ ID NO:4 can include one or more amino acid substitutions (conservative or non-conservative), one or more deletions, and/or one or more insertions. In certain embodiments, the soluble DR6 polypeptide promotes survival of cells of the neuronal system such as neurons and OPCs, e.g., in a mammal.
A soluble DR6 polypeptide can comprise a fragment of at least six, e.g., ten, fifteen, twenty, twenty-five, thirty, forty, fifty, sixty, seventy, one hundred, or more amino acids of SEQ ID NO:2 or SEQ ID NO:4. In addition, a soluble or dominant negative DR6 polypeptide can comprise at least one, e.g., five, ten, fifteen or twenty conservative amino acid substitutions. Corresponding fragments of soluble DR6 polypeptides at least 70%, 75%, 80%, 85%, 90%, or 95% identical to a reference DR6 polypeptide of SEQ ID NO:2 or SEQ ID NO:4 are also contemplated.
By “a DR6 reference amino acid sequence,” or “reference amino acid sequence” is meant the specified sequence without the introduction of any amino acid substitutions. As one of ordinary skill in the art would understand, if there are no substitutions, the “isolated polypeptide” comprises an amino acid sequence which is identical to the reference amino acid sequence.
Conservative substitutions include substitutions within the following groups: valine, alanine and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. The non-polar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above-mentioned polar, basic or acidic groups by another member of the same group can be deemed a conservative substitution.
Non-conservative substitutions include those in which (i) a residue having an electropositive side chain (e.g., Arg, H is or Lys) is substituted for, or by, an electDR6egative residue (e.g., Glu or Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g., Ala, Leu, Ile, Phe or Val), (iii) a cysteine or proline is substituted for, or by, any other residue, or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, Ile, Phe or Trp) is substituted for, or by, one having a smaller side chain (e.g., Ala, Ser) or no side chain (e.g., Gly).
As known in the art, “sequence identity” between two polypeptides is determined by comparing the amino acid sequence of one polypeptide to the sequence of a second polypeptide. When discussed herein, whether any particular polypeptide is at least about 70%, 75%, 80%, 85%, 90% or 95% identical to another polypeptide can be determined using methods and computer programs/software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence described herein, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.
As would be well understood by a person of ordinary skill in the art, the DR6 fragments such as those listed above can vary in length, for example, by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at either end (either longer or shorter) based, for example, on alternate predictions of the DR6 domain regions. In addition, any of the fragments listed above can further include a secretory signal peptide at the N-terminus, e.g., amino acids 1 to 40 of SEQ ID NO:2 or amino acids 1 to 41 of SEQ ID NO:2. Other secretory signal peptides, such as those described elsewhere herein, can also be used. Corresponding fragments of soluble DR6 polypeptides at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:2, SEQ ID NO:4, or fragments thereof described herein are also contemplated.
Soluble DR6 polypeptides for use in the methods described herein can include any combination of two or more soluble DR6 polypeptides. Accordingly, soluble DR6 polypeptide dimers, either homodimers or heterodimers, are contemplated. Two or more soluble DR6 polypeptides as described herein can be directly connected, or can be connected via a suitable peptide linker. Such peptide linkers are described elsewhere herein.
Soluble DR6 polypeptides for use in the methods described herein can be cyclic. Cyclization of the soluble DR6 polypeptides reduces the conformational freedom of linear peptides and results in a more structurally constrained molecule. Many methods of peptide cyclization are known in the art. For example, “backbone to backbone” cyclization by the formation of an amide bond between the N-terminal and the C-terminal amino acid residues of the peptide. The “backbone to backbone” cyclization method includes the formation of disulfide bridges between two ω-thio amino acid residues (e.g. cysteine, homocysteine). Certain soluble DR6 peptides described herein include modifications on the N- and C-terminus of the peptide to form a cyclic DR6 polypeptide. Such modifications include, but are not limited, to cysteine residues, acetylated cysteine residues, cysteine residues with a NH2 moiety and biotin. Other methods of peptide cyclization are described in Li & Roller. Curr. Top. Med. Chem. 3:325-341 (2002), which is incorporated by reference herein in its entirety.
Cyclic DR6 polypeptides for use in the methods described herein include, but are not limited to, C1LSPX9X10X11C2 (SEQ ID NO:5) where X1 is lysine, arginine, histidine, glutamine, or asparagine, X2 is lysine, arginine, histidine, glutamine, or asparagine, X3 is lysine, arginine, histidine, glutamine, or asparagine, C1 optionally has a moiety to promote cyclization (e.g. an acetyl group or biotin) attached and C2 optionally has a moiety to promote cyclization (e.g. an NH2 moiety) attached.
In some embodiments, the DR6 antagonist polypeptide inhibits binding of DR6 to p75. In some embodiments, the DR6 antagonist polypeptide inhibits binding of DR6 to p75, but does not prevent DR6 binding to APP.
DR6 Antagonist Compounds
DR6 antagonists in the methods described herein include any chemical or synthetic compound which inhibits or decreases the activity of DR6 compared to the activity of DR6 in the absence of the antagonist compound. The DR6 antagonist compound can be one that inhibits binding of DR6 to p75. The DR6 antagonist compound can also be one that inhibits binding of DR6 to p75 but does not prevent binding of DR6 to APP.
One of ordinary skill in the art would know how to screen and test for DR6 antagonist compounds which would be useful in the methods described herein, for example by screening for compounds that modify nervous system cell survival using assays described elsewhere herein.
DR6 Antibodies or Immunospecific Fragments Thereof
DR6 antagonists for use in the methods described herein also include DR6-antigen binding molecules, DR-specific antibodies or antigen-binding fragments, variants, or derivatives which are antagonists of DR6 activity. For example, binding of certain DR6 antigen binding molecules or DR6 antibodies to DR6, as expressed in neurons inhibit apoptosis or promote cell survival.
In certain embodiments, the antibody is an antibody or antigen-binding fragment, variant or derivative of that specifically binds to DR6, wherein the antibody promotes survival of cells of the nervous system. In certain embodiments, the antibody is an antibody or antigen-binding fragment, variant or derivative of that specifically binds to DR6, wherein the antibody promotes proliferation, differentiation or survival of oligodendrocytes. In certain embodiments, the DR6 antibody is an antibody or antigen-binding fragment, variant or derivative thereof that specifically binds to DR6, wherein the antibody promotes myelination. In other embodiments, the DR6 antibody is an antibody or antigen-binding fragment, variant or derivative thereof that inhibits binding of DR6 to p75. In other embodiments, the DR6 antibody is an antibody or antigen-binding fragment, variant or derivative thereof that inhibits binding of DR6 to p75 but does not prevent binding of DR6 to APP.
DR6 antibodies include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, scFvs, diabodies, triabodies, tetrabodies, minibodies, domain-deleted antibodies, Fab fragments, F(ab′)2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies described herein) and epitope-binding fragments of any of the above. The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
Certain DR6 antagonist antibodies for use in the methods described herein specifically or preferentially binds to a particular DR6 polypeptide fragment or domain, for example, a DR6 polypeptide, fragment, variant, or derivative as described herein. Such DR6 polypeptide fragments include, but are not limited to, a DR6 polypeptide comprising, consisting essentially of, or consisting of one or more TNFR-like cysteine-rich motifs of DR6. Such fragments include for example, fragments comprising, consisting essentially of or consisting of amino acids 65 to 105 of SEQ ID NO:2; 106 to 145 of SEQ ID NO:2; 146 to 185 of SEQ ID NO:2; 186 to 212 of SEQ ID NO:2; 65 to 145 of SEQ ID NO:2; 65 to 185 of SEQ ID NO:2; 65 to 212 of SEQ ID NO:2; 106 to 185 of SEQ ID NO:2; 106 to 212 of SEQ ID NO:2; and 146 to 212 of SEQ ID NO:2. Such fragments also include amino acids 134-189 of SEQ ID NO:2; 168-189 of SEQ ID NO:2; and 134-168 of SEQ ID NO:2. Corresponding fragments of a variant DR6 polypeptide at least 70%, 75%, 80%, 85%, 90% or 95% identical to amino acids 65 to 105 of SEQ ID NO:2; 106 to 145 of SEQ ID NO:2; 146 to 185 of SEQ ID NO:2; 186 to 212 of SEQ ID NO:2; 65 to 145 of SEQ ID NO:2; 65 to 185 of SEQ ID NO:2; 65 to 212 of SEQ ID NO:2; 106 to 185 of SEQ ID NO:2; 106 to 212 of SEQ ID NO:2; 146 to 212 of SEQ ID NO:2; 134-189 of SEQ ID NO:2; 168-189 of SEQ ID NO:2; and 134-168 of SEQ ID NO:2 are also contemplated. In some embodiments, the DR6 antibody, antigen-binding fragment, variant, or derivative thereof requires both the Cys3 and Cys4 regions of DR6 to interact with DR6.
In other embodiments, the antibody is an antibody, or antigen-binding fragment, variant, or derivative thereof which specifically or preferentially binds to at least one epitope of DR6, where the epitope comprises, consists essentially of, or consists of at least about four to five amino acids of SEQ ID NO:2 or SEQ ID NO:4, at least seven, at least nine, or between at least about 15 to about 30 amino acids of SEQ ID NO:2 or SEQ ID NO:4. The amino acids of a given epitope of SEQ ID NO:2 or SEQ ID NO:4 as described can be, but need not be contiguous or linear. In certain embodiments, the at least one epitope of DR6 comprises, consists essentially of, or consists of a non-linear epitope formed by the extracellular domain of DR6 as expressed on the surface of a cell or as a soluble fragment, e.g., fused to an IgG Fc region. Thus, in certain embodiments the at least one epitope of DR6 comprises, consists essentially of, or consists of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, between about 15 to about 30, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous or non-contiguous amino acids of SEQ ID NO:2, or SEQ ID NO:4, where non-contiguous amino acids form an epitope through protein folding.
In other embodiments, the antibody is an antibody, or antigen-binding fragment, variant, or derivative thereof which specifically or preferentially binds to at least one epitope of DR6, where the epitope comprises, consists essentially of, or consists of, in addition to one, two, three, four, five, six or more contiguous or non-contiguous amino acids of SEQ ID NO:2 or SEQ ID NO:4 as described above, and an additional moiety which modifies the protein, e.g., a carbohydrate moiety can be included such that the DR6 antibody binds with higher affinity to modified target protein than it does to an unmodified version of the protein. Alternatively, the DR6 antibody does not bind the unmodified version of the target protein at all.
In certain aspects, the antibody is an antibody, or antigen-binding fragment, variant, or derivative thereof which specifically binds to a DR6 polypeptide or fragment thereof, or a DR6 variant polypeptide, with an affinity characterized by a dissociation constant (KD) which is less than the KD for a given reference monoclonal antibody.
In certain embodiments, an antibody, or antigen-binding fragment, variant, or derivative thereof binds specifically to at least one epitope of DR6 or fragment or variant described above, i.e., binds to such an epitope more readily than it would bind to an unrelated, or random epitope; binds preferentially to at least one epitope of DR6 or fragment or variant described above, i.e., binds to such an epitope more readily than it would bind to a related, similar, homologous, or analogous epitope; competitively inhibits binding of a reference antibody which itself binds specifically or preferentially to a certain epitope of DR6 or fragment or variant described above; or binds to at least one epitope of DR6 or fragment or variant described above with an affinity characterized by a dissociation constant KD of less than about 5×10−2 M, about 10−2 M, about 5×10−3 M, about 10−3 M, about 5×10−4 M, about 10−4 M, about 5×10−5 M, about 10−5 M, about 5×10−6 M, about 10−6 M, about 5×10−7 M, about 10−7 M, about 5×10−8 M, about 10−8 M, about 5×10−9 M, about 10−9 M, about 5×10−10 M, about 10−10 M, about 5×10−11 M, about 10−11 M, about 5×10−12 M, about 10−12 M, about 5×10−13 M, about 10−13 M, about 5×10−14 M, about 10−14 M, about 5×10−15 M, or about 10−15 M. In a particular aspect, the antibody or fragment thereof preferentially binds to a human DR6 polypeptide or fragment thereof, relative to a murine DR6 polypeptide or fragment thereof. In another particular aspect, the antibody or fragment thereof preferentially binds to one or more DR6 polypeptides or fragments thereof, e.g., one or more mammalian DR6 polypeptides.
As used in the context of antibody binding dissociation constants, the term “about” allows for the degree of variation inherent in the methods utilized for measuring antibody affinity. For example, depending on the level of precision of the instrumentation used, standard error based on the number of samples measured, and rounding error, the term “about 10−2 M” might include, for example, from 0.05 M to 0.005 M.
In specific embodiments, an antibody, or antigen-binding fragment, variant, or derivative thereof binds DR6 polypeptides or fragments or variants thereof with an off rate (k(off)) of less than or equal to 5×10−2 sec−1, 10−2 sec−1, 5×10−3 sec−1 or 10−3 sec−1. Alternatively, an antibody, or antigen-binding fragment, variant, or derivative thereof binds DR6 polypeptides or fragments or variants thereof with an off rate (k(off)) of less than or equal to 5×10−4 sec−1, 10−4 sec−1, 5×10−5 sec−1, or 10−5 sec−1, 5×10−6 sec−1, 10−6 sec−1, 5×10−7 sec−1 or 10−7 sec−1.
In other embodiments, an antibody, or antigen-binding fragment, variant, or derivative thereof binds DR6 polypeptides or fragments or variants thereof with an on rate (k(on)) of greater than or equal to 103 M−1 sec−1, 5×103 M−1 sec−1, 104 M−1 sec−1 or 5×104 M−1 sec−1. Alternatively, an antibody, or antigen-binding fragment, variant, or derivative thereof binds DR6 polypeptides or fragments or variants thereof with an on rate (k(on)) greater than or equal to 105 M−1 sec−1, 5×105 M−1 sec−1, 106 M−1 sec−1, or 5×106 M−1 sec−1 or 107 M−1 sec−1.
In one embodiment, the DR6 antibody includes DR6 antibodies, or antigen-binding fragments, variants, or derivatives thereof which at least the antigen-binding domains of certain monoclonal antibodies, and fragments, variants, and derivatives thereof shown in Tables 3 and 4. Table 3 lists human anti-DR6 Fab regions identified from a phage display library. Table 4 lists mouse anti-DR6 antibodies derived from hybridomas.
TABLE 3
DR6-specific human Fabs.
Fab
1.
M50-H01
2.
M51-H09
3.
M53-E04
4.
M53-F04
5.
M62-B02
6.
M63-E10
7.
M66-B03
8.
M67-G02
9.
M72-F03
10.
M73-C04
TABLE 4
DR6-specific Murine Monoclonal Antibodies.
Murine Antibody
1
1P1D6.3
2
1P2F2.1
3
1P5D10.2
As used herein, the term “antigen binding domain” includes a site that specifically binds an epitope on an antigen (e.g., an epitope of DR6). The antigen binding domain of an antibody typically includes at least a portion of an immunoglobulin heavy chain variable region and at least a portion of an immunoglobulin light chain variable region. The binding site formed by these variable regions determines the specificity of the antibody.
In some embodiments, the DR6 antibody is a DR6 antibody, or antigen-binding fragment, variant or derivatives thereof, where the DR6 antibody specifically binds to the same DR6 epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M50-H01, M51-H09, M53-E04, M53-F04, M62-B02, M63-E10, M66-B03, M67-G02, M72-F03, and M73-C04 or a reference monoclonal antibody selected from the group consisting of 1P1D6.3, 1P2F2.1, and 1P5D10.2.
In some embodiments, the DR6 antibody is a DR6 antibody, or antigen-binding fragment, variant or derivatives thereof, where the DR6 antibody competitively inhibits a reference monoclonal Fab antibody fragment selected from the group consisting of M50-H01, M51-H09, M53-E04, M53-F04, M62-B02, M63-E10, M66-B03, M67-G02, M72-F03, and M73-C04 or a reference monoclonal antibody selected from the group consisting of 1P1D6.3, 1P2F2.1, and 1P5D10.2 from binding to DR6.
In some embodiments, the DR6 antibody is a DR6 antibody, or antigen-binding fragment, variant or derivatives thereof, where the DR6 antibody comprises an antigen binding domain identical to that of a monoclonal Fab antibody fragment selected from the group consisting of M50-H01, M51-H09, M53-E04, M53-F04, M62-B02, M63-E10, M66-B03, M67-G02, M72-F03, and M73-C04 or a reference monoclonal antibody selected from the group consisting of 1P1D6.3, 1P2F2.1, and 1P5D10.2.
In some embodiments, the DR6 antibody is not an antibody selected from the group consisting of 3F4.48, 4B6.9.7 or 1E5.57 as described in International Publication No. WO2008/080045, filed Dec. 21, 2007. In some embodiments, the DR6 antibody is not antibody selected from the group consisting of antibodies that competitively inhibit binding of 3F4.48, 4B6.9.7 or 1E5.57 to DR6.
In some embodiments, the DR6 antibody is an antagonist antibody.
Methods of making antibodies are well known in the art and described herein. Once antibodies to various fragments of, or to the full-length DR6 without the signal sequence, have been produced, determining which amino acids, or epitope, of DR6 to which the antibody or antigen binding fragment binds can be determined by epitope mapping protocols as described herein as well as methods known in the art (e.g. double antibody-sandwich ELISA as described in “Chapter 11—Immunology,” Current Protocols in Molecular Biology, Ed. Ausubel et al., v.2, John Wiley & Sons, Inc. (1996)). Additional epitope mapping protocols can be found in Morris, G. Epitope Mapping Protocols, New Jersey: Humana Press (1996), which are both incorporated herein by reference in their entireties. Epitope mapping can also be performed by commercially available means (i.e. ProtoPROBE, Inc. (Milwaukee, Wis.)).
Additionally, antibodies produced which bind to any portion of DR6 can then be screened for their ability to act as an antagonist of DR6 for example, promoting survival of cells of the nervous system, treating a condition associated with death of cells of the nervous and preventing apoptosis of cells of the nervous system Antibodies can be screened for these and other properties according to methods described in detail in the Examples. Other functions of antibodies described herein can be tested using other assays as described in the Examples herein.
In one embodiment, a DR6 antagonist for use in the methods described herein is an antibody molecule, or immunospecific fragment thereof. Unless it is specifically noted, as used herein a “fragment thereof” in reference to an antibody refers to an immunospecific fragment, i.e., an antigen-specific fragment.
In one embodiment, a binding molecule or antigen binding molecule for use in the methods described herein comprises a synthetic constant region wherein one or more domains are partially or entirely deleted (“domain-deleted antibodies”). Certain methods described herein comprise administration of a DR6 antagonist antibody, or immunospecific fragment thereof, in which at least a fraction of one or more of the constant region domains has been deleted or otherwise altered so as to provide desired biochemical characteristics such as reduced effector functions, the ability to non-covalently dimerize, increased ability to localize at the site of action, reduced serum half-life, or increased serum half-life when compared with a whole, unaltered antibody of approximately the same immunogenicity. For example, certain antibodies for use in the treatment methods described herein are domain deleted antibodies which comprise a polypeptide chain similar to an immunoglobulin heavy chain, but which lack at least a portion of one or more heavy chain domains.
In certain embodiments compatible modified antibodies will comprise domain deleted constructs or variants wherein the entire CH2 domain has been removed (ΔCH2 constructs). For other embodiments a short connecting peptide can be substituted for the deleted domain to provide flexibility and freedom of movement for the variable region. Those skilled in the art will appreciate that such constructs can be desirable under certain circumstances due to the regulatory properties of the CH2 domain on the catabolic rate of the antibody. Domain deleted constructs can be derived using a vector (e.g., from Biogen IDEC Incorporated) encoding an IgG1 human constant domain (see, e.g., WO 02/060955A2 and WO02/096948A2). This exemplary vector was engineered to delete the CH2 domain and provide a synthetic vector expressing a domain deleted IgG1 constant region.
In certain embodiments, modified antibodies for use in the methods disclosed herein are minibodies. Minibodies can be made using methods described in the art (see, e.g., see e.g., U.S. Pat. No. 5,837,821 or WO 94/09817A1).
In one embodiment, a DR6 antagonist antibody or fragment thereof for use in the treatment methods disclosed herein comprises an immunoglobulin heavy chain having deletion or substitution of a few or even a single amino acid as long as it permits association between the monomeric subunits. For example, the mutation of a single amino acid in selected areas of the CH2 domain can be enough to substantially reduce Fc binding and thereby increase localization to the intended site of action. Similarly, it can be desirable to simply delete that part of one or more constant region domains that control the effector function (e.g. complement binding) to be modulated. Such partial deletions of the constant regions can improve selected characteristics of the antibody (serum half-life) while leaving other desirable functions associated with the subject constant region domain intact. Moreover, as alluded to above, the constant regions of the disclosed antibodies can be synthetic through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct. In this respect it can be possible to disrupt the activity provided by a conserved binding site (e.g. Fc binding) while substantially maintaining the configuration and immunogenic profile of the modified antibody. Yet other embodiments comprise the addition of one or more amino acids to the constant region to enhance desirable characteristics such as effector function or provide for more cytotoxin or carbohydrate attachment. In such embodiments it can be desirable to insert or replicate specific sequences derived from selected constant region domains.
In certain DR6 antagonist antibodies or immunospecific fragments thereof for use in the therapeutic methods described herein, the Fc portion can be mutated to decrease effector function using techniques known in the art. For example, modifications of the constant region can be used to modify disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility. The resulting physiological profile, bioavailability and other biochemical effects of the modifications can easily be measured and quantified using well know immunological techniques without undue experimentation.
The methods described herein also provide the use of antibodies that comprise, consist essentially of, or consist of, variants (including derivatives) of antibody molecules (e.g., the VH regions and/or VL regions) described herein, which antibodies or fragments thereof immunospecifically bind to a DR6 polypeptide. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a binding molecule, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions. In various embodiments, the variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the reference VH region, VHCDR1, VHCDR2, VHCDR3, VL region, VLCDR1, VLCDR2, or VLCDR3. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity.
For example, it is possible to introduce mutations only in framework regions or only in CDR regions of an antibody molecule. Introduced mutations can be silent or neutral missense mutations, i.e., have no, or little, effect on an antibody's ability to bind antigen. These types of mutations can be useful to optimize codon usage, or improve a hybridoma's antibody production. Alternatively, non-neutral missense mutations can alter an antibody's ability to bind antigen. The location of most silent and neutral missense mutations is likely to be in the framework regions, while the location of most non-neutral missense mutations is likely to be in CDR, though this is not an absolute requirement. One of skill in the art would be able to design and test mutant molecules with desired properties such as no alteration in antigen binding activity or alteration in binding activity (e.g., improvements in antigen binding activity or change in antibody specificity). Following mutagenesis, the encoded protein can routinely be expressed and the functional and/or biological activity of the encoded protein can be determined using techniques described herein or by routinely modifying techniques known in the art.
In one embodiment, an antibody is a bispecific binding molecule, binding polypeptide, or antibody, e.g., a bispecific antibody, minibody, domain deleted antibody, or fusion protein having binding specificity for more than one epitope, e.g., more than one antigen or more than one epitope on the same antigen. In one embodiment, a bispecific antibody has at least one binding domain specific for at least one epitope on DR6. A bispecific antibody can be a tetravalent antibody that has two target binding domains specific for an epitope of DR6 and two target binding domains specific for a second target. Thus, a tetravalent bispecific antibody can be bivalent for each specificity.
Modified forms of antibodies or immunospecific fragments thereof for use in the diagnostic and therapeutic methods disclosed herein can be made from whole precursor or parent antibodies using techniques known in the art. Exemplary techniques are discussed in more detail herein.
DR6 antagonist antibodies or immunospecific fragments thereof for use in the diagnostic and treatment methods disclosed herein can be made or manufactured using techniques that are known in the art. In certain embodiments, antibody molecules or fragments thereof are “recombinantly produced,” i.e., are produced using recombinant DNA technology. Exemplary techniques for making antibody molecules or fragments thereof are discussed in more detail elsewhere herein.
DR6 antagonist antibodies or fragments thereof for use in the methods described herein can be generated by any suitable method known in the art.
Polyclonal antibodies can be produced by various procedures well known in the art. For example, a DR6 immunospecific fragment can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. Various adjuvants can be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.
Monoclonal antibodies can 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 can 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 Elsevier, N.Y., 563-681 (1981) (said references incorporated by reference in their entireties). The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. 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. Thus, the term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma and recombinant and phage display technology.
Using art recognized protocols, in one example, antibodies are raised in mammals by multiple subcutaneous or intraperitoneal injections of the relevant antigen (e.g., purified DR6 antigens or cells or cellular extracts comprising such antigens) and an adjuvant. This immunization typically elicits an immune response that comprises production of antigen-reactive antibodies from activated splenocytes or lymphocytes. While the resulting antibodies can be harvested from the serum of the animal to provide polyclonal preparations, it is often desirable to isolate individual lymphocytes from the spleen, lymph nodes or peripheral blood to provide homogenous preparations of monoclonal antibodies (MAbs). In certain specific embodiments, the lymphocytes are obtained from the spleen.
In this well known process (Kohler et al., Nature 256:495 (1975)) the relatively short-lived, or mortal, lymphocytes from a mammal which has been injected with antigen are fused with an immortal tumor cell line (e.g. a myeloma cell line), thus, producing hybrid cells or “hybridomas” which are both immortal and capable of producing the genetically coded antibody of the B cell. The resulting hybrids are segregated into single genetic strains by selection, dilution, and regrowth with each individual strain comprising specific genes for the formation of a single antibody. They produce antibodies which are homogeneous against a desired antigen and, in reference to their pure genetic parentage, are termed “monoclonal.”
Typically, hybridoma cells thus prepared are seeded and grown in a suitable culture medium that contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. Those skilled in the art will appreciate that reagents, cell lines and media for the formation, selection and growth of hybridomas are commercially available from a number of sources and standardized protocols are well established. Generally, culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies against the desired antigen. In certain embodiments, the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by in vitro assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). After hybridoma cells are identified that produce antibodies of the desired specificity, affinity and/or activity, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp. 59-103 (1986)). It will further be appreciated that the monoclonal antibodies secreted by the subclones can be separated from culture medium, ascites fluid or serum by conventional purification procedures such as, for example, protein-A, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.
Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, Fab and F(ab′)2 fragments can 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.
Those skilled in the art will also appreciate that DNA encoding antibodies or antibody fragments (e.g., antigen binding sites) can also be derived from antibody phage libraries. In a particular, such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can 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. Exemplary methods are set forth, for example, in EP 368 684 B1; U.S. Pat. No. 5,969,108, Hoogenboom, H. R. and Chames, Immunol. Today 21:371 (2000); Nagy et al. Nat. Med. 8:801 (2002); Huie et al., Proc. Natl. Acad. Sci. USA 98:2682 (2001); Lui et al., J. Mol. Biol. 315:1063 (2002), each of which is incorporated herein by reference. Several publications (e.g., Marks et al., Bio/Technology 10:779-783 (1992)) have described the production of high affinity human antibodies by chain shuffling, as well as combinatorial infection and in vivo recombination as a strategy for constructing large phage libraries. In another embodiment, Ribosomal display can be used to replace bacteriophage as the display platform (see, e.g., Hanes et al., Nat. Biotechnol. 18:1287 (2000); Wilson et al., Proc. Natl. Acad. Sci. USA 98:3750 (2001); or Irving et al., J. Immunol. Methods 248:31 (2001)). In yet another embodiment, cell surface libraries can be screened for antibodies (Boder et al., Proc. Natl. Acad. Sci. USA 97:10701 (2000); Daugherty et al., J. Immunol. Methods 243:211 (2000)). Such procedures provide alternatives to traditional hybridoma techniques for the isolation and subsequent cloning of monoclonal antibodies.
In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL regions are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of lymphoid tissues) or synthetic cDNA libraries. In certain embodiments, the DNA encoding the VH and VL regions are joined together by an scFv linker by PCR and cloned into a phagemid vector (e.g., p CANTAB 6 or pComb 3 HSS). The vector is electroporated in E. coli and the E. coli is infected with helper phage. Phage used in these methods are typically filamentous phage including fd and M13 and the VH or VL regions are usually recombinantly fused to either the phage gene III or gene VIII. Phage expressing an antigen binding domain that binds to an antigen of interest (i.e., a DR6 polypeptide or a fragment thereof) can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
Additional examples of phage display methods that can be used to make the antibodies 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; each of which is incorporated herein by reference in its entirety.
As described in the above references, after phage selection, the antibody coding regions from the phage can 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. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can 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).
In another embodiment, DNA encoding desired monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). In certain embodiments, isolated and subcloned hybridoma cells serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells or myeloma cells that do not otherwise produce immunoglobulins. More particularly, the isolated DNA (which can be synthetic as described herein) can be used to clone constant and variable region sequences for the manufacture antibodies as described in Newman et al., U.S. Pat. No. 5,658,570, filed Jan. 25, 1995, which is incorporated by reference herein. Essentially, this entails extraction of RNA from the selected cells, conversion to cDNA, and amplification by PCR using Ig specific primers. Suitable primers for this purpose are also described in U.S. Pat. No. 5,658,570. As will be discussed in more detail below, transformed cells expressing the desired antibody can be grown up in relatively large quantities to provide clinical and commercial supplies of the immunoglobulin.
In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains can be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs can be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody. The framework regions can be naturally occurring or consensus framework regions, e.g., human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278:457-479 (1998) for a listing of human framework regions). In certain embodiments, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds to at least one epitope of a desired polypeptide, e.g., DR6. In further embodiments, one or more amino acid substitutions can be made within the framework regions, for example, to improve binding of the antibody to its antigen. Additionally, such methods can be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are contemplated and within the skill of the art.
In certain embodiments, a DR6 antagonist antibody or immunospecific fragment thereof for use in the treatment methods disclosed herein will not elicit a deleterious immune response in the animal to be treated, e.g., in a human. In one embodiment, DR6 antagonist antibodies or immunospecific fragments thereof for use in the treatment methods disclosed herein be modified to reduce their immunogenicity using art-recognized techniques. For example, antibodies can be humanized, primatized, deimmunized, or chimeric antibodies can be made. These types of antibodies are derived from a non-human antibody, typically a murine or primate antibody, that retains or substantially retains the antigen-binding properties of the parent antibody, but which is less immunogenic in humans. This can be achieved by various methods, including (a) grafting the entire non-human variable domains onto human constant regions to generate chimeric antibodies; (b) grafting at least a part of one or more of the non-human complementarity determining regions (CDRs) into a human framework and constant regions with or without retention of critical framework residues; or (c) transplanting the entire non-human variable domains, but “cloaking” them with a human-like section by replacement of surface residues. Such methods are disclosed in Morrison et al., Proc. Natl. Acad. Sci. 81:6851-6855 (1984); Morrison et al., Adv. Immunol. 44:65-92 (1988); Verhoeyen et al., Science 239:1534-1536 (1988); Padlan, Molec. Immun. 28:489-498 (1991); Padlan, Molec. Immun. 31:169-217 (1994), and U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,190,370, all of which are hereby incorporated by reference in their entirety.
De-immunization can also be used to decrease the immunogenicity of an antibody. As used herein, the term “de-immunization” includes alteration of an antibody to modify T cell epitopes (see, e.g., WO9852976A1, WO0034317A2). For example, VH and VL sequences from the starting antibody are analyzed and a human T cell epitope “map” from each V region showing the location of epitopes in relation to complementarity-determining regions (CDRs) and other key residues within the sequence. Individual T cell epitopes from the T cell epitope map are analyzed in order to identify alternative amino acid substitutions with a low risk of altering activity of the final antibody. A range of alternative VH and VL sequences are designed comprising combinations of amino acid substitutions and these sequences are subsequently incorporated into a range of binding polypeptides, e.g., DR6 antagonist antibodies or immunospecific fragments thereof for use in the diagnostic and treatment methods disclosed herein, which are then tested for function. Typically, between 12 and 24 variant antibodies are generated and tested. Complete heavy and light chain genes comprising modified V and human C regions are then cloned into expression vectors and the subsequent plasmids introduced into cell lines for the production of whole antibody. The antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identified.
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 human 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); Takeda et al., Nature 314:452-454 (1985), Neuberger et al., Nature 312:604-608 (1984); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entireties. 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. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, e.g., 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.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), 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. 5,565,332).
Yet another highly efficient means for generating recombinant antibodies is disclosed by Newman, Biotechnology 10: 1455-1460 (1992). Specifically, this technique results in the generation of primatized antibodies that contain monkey variable domains and human constant sequences. This reference is incorporated by reference in its entirety herein. Moreover, this technique is also described in commonly assigned U.S. Pat. Nos. 5,658,570, 5,693,780 and 5,756,096 each of which is incorporated herein by reference.
Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.
Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes, that are incapable of endogenous immunoglobulin production (see e.g., U.S. Pat. Nos. 6,075,181, 5,939,598, 5,591,669 and 5,589,369 each of which is incorporated herein by reference). For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. The human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a desired target polypeptide. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B-cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.) and GenPharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
Another means of generating human antibodies using SCID mice is disclosed in U.S. Pat. No. 5,811,524 which is incorporated herein by reference. It will be appreciated that the genetic material associated with these human antibodies can also be isolated and manipulated as described herein.
Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/Technology 12:899-903 (1988)). See also, U.S. Pat. No. 5,565,332, which discloses, for example, selection from random libraries where only VHCDR3 is unvaried.
Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,694,778; Bird, Science 242:423-442 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-554 (1989)) can be used. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain antibody. Techniques for the assembly of functional Fv fragments in E. coli can also be used (Skerra et al., Science 242:1038-1041 (1988)). Examples of techniques which can 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); and Shu et al., PNAS 90:7995-7999 (1993).
In another embodiment, lymphocytes can be selected by micromanipulation and the variable genes isolated. For example, peripheral blood mononuclear cells can be isolated from an immunized mammal and cultured for about 7 days in vitro. The cultures can be screened for specific IgGs that meet the screening criteria. Cells from positive wells can be isolated. Individual Ig-producing B cells can be isolated by FACS or by identifying them in a complement-mediated hemolytic plaque assay. Ig-producing B cells can be micromanipulated into a tube and the VH and VL genes can be amplified using, e.g., RT-PCR. The VH and VL genes can be cloned into an antibody expression vector and transfected into cells (e.g., eukaryotic or prokaryotic cells) for expression.
Alternatively, antibody-producing cell lines can be selected and cultured using techniques well known to the skilled artisan. Such techniques are described in a variety of laboratory manuals and primary publications. In this respect, techniques suitable for use in the methods as described below are described in Current Protocols in Immunology, Coligan et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991) which is herein incorporated by reference in its entirety, including supplements.
Antibodies for use in the therapeutic methods disclosed herein can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression techniques as described herein.
It will further be appreciated that the alleles, variants and mutations of antigen binding DNA sequences can be used in the methods described herein.
In one embodiment, cDNAs that encode the light and the heavy chains of the antibody can be made, either simultaneously or separately, using reverse transcriptase and DNA polymerase in accordance with well known methods. PCR can be initiated by consensus constant region primers or by more specific primers based on the published heavy and light chain DNA and amino acid sequences. As discussed above, PCR also can be used to isolate DNA clones encoding the antibody light and heavy chains. In this case the libraries can be screened by consensus primers or larger homologous probes, such as mouse constant region probes.
DNA, typically plasmid DNA, can be isolated from the cells using techniques known in the art, restriction mapped and sequenced in accordance with standard, well known techniques set forth in detail, e.g., in the foregoing references relating to recombinant DNA techniques. Of course, the DNA can be synthetic at any point during the isolation process or subsequent analysis.
Recombinant expression of an antibody, or fragment, derivative or analog thereof, e.g., a heavy or light chain of an antibody which is a DR6 antagonist, requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (e.g., containing the heavy or light chain variable domain), has been obtained, the vector for the production of the antibody molecule can be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also considered herein are replicable vectors comprising a nucleotide sequence encoding an antibody molecule, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors can include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody can be cloned into such a vector for expression of the entire heavy or light chain.
The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody for use in the methods described herein. Thus, host cells containing a polynucleotide encoding an antibody, or a heavy or light chain thereof, operably linked to a heterologous promoter are also described herein. In certain embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains can be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
A variety of host-expression vector systems can be utilized to express antibody molecules for use in the methods described elsewhere herein.
The host cell can be co-transfected with two expression vectors, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors can contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector can be used which encodes both heavy and light chain polypeptides. In such situations, the light chain is advantageously placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy and light chains can comprise cDNA or genomic DNA.
Once an antibody molecule has been recombinantly expressed, it can be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Alternatively, a method for increasing the affinity of antibodies is disclosed in US 2002 0123057 A1.
Furthermore, as described in more detail below, any of the DR6 antibodies or antibody fragments as described herein can be conjugated (covalently linked) to one or more polymers. In one particular embodiment, an antibody fragment that recognizes a specific epitope, for example, a Fab, F(ab)2, Fv fragment or single chain antibody can be conjugated to a polymer. Examples of polymers suitable for such conjugation include polypeptides, sugar polymers and polyalkylene glycol chains (as described in more detail below). The class of polymer generally used is a polyalkylene glycol. Polyethylene glycol (PEG) is most frequently used. PEG moieties, e.g., 1, 2, 3, 4 or 5 PEG polymers, can be conjugated to DR6 antibodies or fragments thereof to increase serum half life. PEG moieties are non-antigenic and essentially biologically inert. PEG moieties used can be branched or unbranched.
Polynucleotides Encoding DR6 Antibodies
The polynucleotides described herein include nucleic acid molecules encoding DR6 antibodies, or antigen-binding fragments, variants, or derivatives thereof.
In one embodiment, the polynucleotide an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH), where at least one of the CDRs of the heavy chain variable region or at least two of the VH-CDRs of the heavy chain variable region are at least 80%, 85%, 90% or 95% identical to reference heavy chain VH-CDR1, VH-CDR2, or VH-CDR3 amino acid sequences from monoclonal DR6 antibodies disclosed herein. Alternatively, the VH-CDR1, VH-CDR2, and VH-CDR3 regions of the VH are at least 80%, 85%, 90% or 95% identical to reference heavy chain VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences from monoclonal DR6 antibodies disclosed herein. Thus, according to this embodiment a heavy chain variable region has VH-CDR1, VH-CDR2, or VH-CDR3 polypeptide sequences related to the polypeptide sequences shown in Table 5.
In another embodiment, the polynucleotide is an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region (VL), where at least one of the VL-CDRs of the light chain variable region or at least two of the VL-CDRs of the light chain variable region are at least 80%, 85%, 90% or 95% identical to reference light chain VL-CDR1, VL-CDR2, or VL-CDR3 amino acid sequences from monoclonal DR6 antibodies disclosed herein. Alternatively, the VL-CDR1, VL-CDR2, and VL-CDR3 regions of the VL are at least 80%, 85%, 90% or 95% identical to reference light chain VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences from monoclonal DR6 antibodies disclosed herein. Thus, according to this embodiment a light chain variable region has VL-CDR1, VL-CDR2, or VL-CDR3 polypeptide sequences related to the polypeptide sequences shown in Table 5.
TABLE 5
DR6 Antibody Sequence SEQ ID NOs
VH
VH
VH
VH
VH
VL
VL
VL
VL
VL
Antibody
PN
PP
CDR1
CDR2
CDR3
PN
PP
CDR1
CDR2
CDR2
M50-H01
6
7
8
9
10
11
12
13
14
15
M51-H09
16
17
18
19
20
21
22
23
24
25
M53-E04
26
27
28
29
30
31
32
33
34
35
M53-F04
36
37
38
39
40
41
42
43
44
45
M62-B02
46
47
48
49
50
51
52
53
54
55
M63-E10
56
57
58
59
60
61
62
63
64
65
M66-B03
66
67
68
69
70
71
72
73
74
75
M67-G02
76
77
78
79
80
81
82
83
84
85
M72-F03
86
87
88
89
90
91
92
93
94
95
M73-C04
96
97
98
99
100
101
102
103
104
105
1P1D6.3
106
107
108
109
110
111
112
113
114
115
1P2F2.1
116
117
118
119
120
121
122
123
124
125
1P5D10.2
126
127
128
129
130
131
132
133
134
135
As known in the art, “sequence identity” between two polypeptides or two polynucleotides is determined by comparing the amino acid or nucleic acid sequence of one polypeptide or polynucleotide to the sequence of a second polypeptide or polynucleotide. When discussed herein, whether any particular polypeptide is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to another polypeptide can be determined using methods and computer programs/software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.
In certain embodiments, an antibody or antigen-binding fragment comprising the VH encoded by the polynucleotide specifically or preferentially binds to DR6. In certain embodiments the nucleotide sequence encoding the VH polypeptide is altered without altering the amino acid sequence encoded thereby. For instance, the sequence can be altered for improved codon usage in a given species, to remove splice sites, or the remove restriction enzyme sites. Sequence optimizations such as these are described in the examples and are well known and routinely carried out by those of ordinary skill in the art.
In another embodiment, the polynucleotide is isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH) in which the VH-CDR1, VH-CDR2, and VH-CDR3 regions have polypeptide sequences which are identical to the VH-CDR1, VH-CDR2, and VH-CDR3 groups shown in Table 5. In certain embodiments, an antibody or antigen-binding fragment comprising the VH encoded by the polynucleotide specifically or preferentially binds to DR6.
In some embodiments, the polynucleotide is an isolated polynucleotide comprising a nucleic acid which encodes an antibody VH polypeptide, where the VH polypeptide comprises VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 8, 9, and 10; SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70; SEQ ID NOs: 78, 79, and 80; SEQ ID NOs: 88, 89, and 90; SEQ ID NOs: 98, 99, and 100; SEQ ID NOs: 108, 109, and 110; SEQ ID NOs: 118, 119, and 120; and SEQ ID NOs: 128, 129, and 130; and where an antibody or antigen binding fragment thereof comprising the VH-CDR3 specifically binds to DR6.
In certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VH encoded by one or more of the polynucleotides described above specifically or preferentially binds to the same DR6 epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M50-H01, M51-H09, M53-E04, M53-F04, M62-B02, M63-E10, M66-B03, M67-G02, M72-F03, and M73-C04 or a reference monoclonal antibody selected from the group consisting of 1P1D6.3, 1P2F2.1, and 1P5D10.2, or will competitively inhibit such a monoclonal antibody or fragment from binding to DR6.
In certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VH encoded by one or more of the polynucleotides described above specifically or preferentially binds to a DR6 polypeptide or fragment thereof, or a DR6 variant polypeptide, with an affinity characterized by a dissociation constant (KD) no greater than 5×10−2 M, 10−2 M, 5×10−3 M, 10−3 M, 5×10−4 M, 10−4 M, 5×10−5 M, 10−5 M, 5×10−6 M, 10−6 M, 5×10−7 M, 10−7 M, 5×10−8 M, 10−8 M, 5×10−9 M, 10−9 M, 5×10−10 M, 10−10 M, 5×10−11 M, 10−11 M, 5×10−12 M, 10−12 M, 5×10−13 M, 10−13 M, 5×10−14 M, 10−14 M, 5×10−15 M, or 10−15 M.
In certain embodiments, an antibody or antigen-binding fragment comprising the VL encoded by the polynucleotide specifically or preferentially binds to DR6.
In another embodiment, the polynucleotide is an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region (VL) in which the VL-CDR1, VL-CDR2, and VL-CDR3 regions have polypeptide sequences which are identical to the VL-CDR1, VL-CDR2, and VL-CDR3 groups shown in Table 5. In certain embodiments, an antibody or antigen-binding fragment comprising the VL encoded by the polynucleotide specifically or preferentially binds to DR6.
In a further aspect, the polynucleotide is an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region (VL) in which the VL-CDR1, VL-CDR2, and VL-CDR3 regions are encoded by nucleotide sequences which are identical to the nucleotide sequences which encode the VL-CDR1, VL-CDR2, and VL-CDR3 groups shown in Table 5. In certain embodiments, an antibody or antigen-binding fragment comprising the VL encoded by the polynucleotide specifically or preferentially binds to DR6.
In some embodiments, the polynucleotide is an isolated polynucleotide comprising a nucleic acid which encodes an antibody VL polypeptide, wherein said VL polypeptide comprises VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 13, 14, and 15; SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 53, 54, and 55; SEQ ID NOs: 63, 64, and 65; SEQ ID NOs: 73, 74, and 75; SEQ ID NOs: 83, 84, and 85; SEQ ID NOs: 93, 94, and 95; SEQ ID NOs: 103, 104, and 105; SEQ ID NOs: 113, 114, and 115; SEQ ID NOs: 123, 124, and 125; and SEQ ID NOs: 133, 134, and 135; and wherein an antibody or antigen binding fragment thereof comprising said VL-CDR3 specifically binds to DR6.
In certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VL encoded by one or more of the polynucleotides described above specifically or preferentially binds to the same DR6 epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M50-H01, M51-H09, M53-E04, M53-F04, M62-B02, M63-E10, M66-B03, M67-G02, M72-F03, and M73-C04 or a reference monoclonal antibody selected from the group consisting of 1P1D6.3, 1P2F2.1, and 1P5D10.2 or will competitively inhibit such a monoclonal antibody or fragment from binding to DR6.
In certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VL encoded by one or more of the polynucleotides described above specifically or preferentially binds to a DR6 polypeptide or fragment thereof, or a DR6 variant polypeptide, with an affinity characterized by a dissociation constant (KD) no greater than 5×10−2 M, 10−2 M, 5×10−3 M, 10−3 M, 5×10−4 M, 10−4 M, 5×10−5 M, 10−5 M, 5×10−6 M, 10−6 M, 5×10−7 M, 10−7 M, 5×10−8 M, 10−8 M, 5×10−9 M, 10−9 M, 5×10−10 M, 10−10 M, 5×10−11 M, 10−11 M, 5×10−12 M, 10−12 M, 5×10−13 M, 10−13 M, 5×10−14 M, 10−14 M, 5×10−15 M, or 10−15 M.
In a further embodiment, the polynucleotide can be an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding a VH at least 80%, 85%, 90% 95% or 100% identical to a reference VH polypeptide sequence selected from the group consisting of SEQ ID NOs: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117 and 127. In certain embodiments, an antibody or antigen-binding fragment comprising the VH encoded by the polynucleotide specifically or preferentially binds to DR6.
In another aspect, the polynucleotide can be an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VH having a polypeptide sequence selected from the group consisting of SEQ ID NOs: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117 and 127. In certain embodiments, an antibody or antigen-binding fragment comprising the VH encoded by the polynucleotide specifically or preferentially binds to DR6.
In a further embodiment, the polynucleotide can be an isolated polynucleotide comprising, consisting essentially of, or consisting of a VH-encoding nucleic acid at least 80%, 85%, 90% 95% or 100% identical to a reference nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, and 126. In certain embodiments, an antibody or antigen-binding fragment comprising the VH encoded by such polynucleotides specifically or preferentially binds to DR6.
In another aspect, the polynucleotide can be an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VH, where the amino acid sequence of the VH is selected from the group consisting of SEQ ID NOs: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117 and 127. The polynucleotide can also be an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VH, where the sequence of the nucleic acid is selected from the group consisting of SEQ ID NOs: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, and 126. In certain embodiments, an antibody or antigen-binding fragment comprising the VH encoded by such polynucleotides specifically or preferentially binds to DR6.
In certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VH encoded by one or more of the polynucleotides described above specifically or preferentially binds to the same DR6 epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M50-H01, M51-H09, M53-E04, M53-F04, M62-B02, M63-E10, M66-B03, M67-G02, M72-F03, and M73-C04 or a reference monoclonal antibody selected from the group consisting of 1P1D6.3, 1P2F2.1, and 1P5D10.2, or will competitively inhibit such a monoclonal antibody or fragment from binding to DR6, or will competitively inhibit such a monoclonal antibody from binding to DR6.
In certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VH encoded by one or more of the polynucleotides described above specifically or preferentially binds to a DR6 polypeptide or fragment thereof, or a DR6 variant polypeptide, with an affinity characterized by a dissociation constant (KD) no greater than 5×10−2 M, 10−2 M, 5×10−3 M, 10−3 M, 5×10−4 M, 10−4 M, 5×10−5 M, 10−5 M, 5×10−6 M, 10−6 M, 5×10−7 M, 10−7 M, 5×10−8 M, 10−8 M, 5×10−9 M, 10−9 M, 5×10−10 M, 10−10 M, 5×10−11 M, 10−11 M, 5×10−12 M, 10−12 M, 5×10−13 M, 10−13 M, 5×10−14 M, 10−14 M, 5×10−15 M, or 10−15 M.
In a further embodiment, the polynucleotide can be an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding a VL at least 80%, 85%, 90% 95% or 100% identical to a reference VL polypeptide sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122 and 132. In a further embodiment, the polynucleotide can be an isolated polynucleotide comprising, consisting essentially of, or consisting of a VL-encoding nucleic acid at least 80%, 85%, 90% 95% or 100% identical to a reference nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, and 131. In certain embodiments, an antibody or antigen-binding fragment comprising the VL encoded by such polynucleotides specifically or preferentially binds to DR6.
In another aspect, the polynucleotide can be an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VL having a polypeptide sequence selected from the group consisting of SEQ ID NOs: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122 and 132. The polynucleotide can be an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VL, where the sequence of the nucleic acid is selected from the group consisting of SEQ ID NOs: 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, and 131. In certain embodiments, an antibody or antigen-binding fragment comprising the VL encoded by such polynucleotides specifically or preferentially binds to DR6.
In certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VL encoded by one or more of the polynucleotides described above specifically or preferentially binds to the same DR6 epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M50-H01, M51-H09, M53-E04, M53-F04, M62-B02, M63-E10, M66-B03, M67-G02, M72-F03, and M73-C04 or a reference monoclonal antibody selected from the group consisting of 1P1D6.3, 1P2F2.1, and 1P5D10.2, or will competitively inhibit such a monoclonal antibody or fragment from binding to DR6.
In certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VL encoded by one or more of the polynucleotides described above specifically or preferentially binds to a DR6 polypeptide or fragment thereof, or a DR6 variant polypeptide, with an affinity characterized by a dissociation constant (KD) no greater than 5×10−2 M, 10−2 M, 5×10−3 M, 10−3 M, 5×10−4 M, 10−4 M, 5×10−5 M, 10−5 M, 5×10−6 M, 10−6 M, 5×10−7 M, 10−7 M, 5×10−8 M, 10−8 M, 5×10−9 M, 10−9 M, 5×10−10 M, 10−10 M, 5×10−11 M, 10−11 M, 5×10−12 M, 10−12 M, 5×10−13 M, 10−13 M, 5×10−14 M, 10−14 M, 5×10−15 M, or 10−15 M.
Any of the polynucleotides described above can further include additional nucleic acids, encoding, e.g., a signal peptide to direct secretion of the encoded polypeptide, antibody constant regions as described herein, or other heterologous polypeptides as described herein.
Also, as described in more detail elsewhere herein, the compositions include compositions comprising the polynucleotides comprising one or more of the polynucleotides described above. In one embodiment, the compositions includes compositions comprising a first polynucleotide and second polynucleotide wherein said first polynucleotide encodes a VH polypeptide as described herein and wherein said second polynucleotide encodes a VL polypeptide as described herein. Specifically a composition which comprises, consists essentially of, or consists of a VH polynucleotide, and a VL polynucleotide, wherein the VH polynucleotide and the VL polynucleotide encode polypeptides, respectively at least 80%, 85%, 90% 95% or 100% identical to reference VH and VL polypeptide amino acid sequences selected from the group consisting of SEQ ID NOs: 7 and 12, 17 and 22, 27 and 32, 37 and 42, 47 and 52, 57 and 62, 67 and 72, 77 and 82, 87 and 92, 97 and 102, 107 and 112, 117 and 122 and 127 and 132. Or alternatively, a composition which comprises, consists essentially of, or consists of a VH polynucleotide, and a VL polynucleotide at least 80%, 85%, 90% 95% or 100% identical, respectively, to reference VL and VL nucleic acid sequences selected from the group consisting of SEQ ID NOs: 6 and 11, 16 and 21, 26 and 31, 36 and 41, 46 and 51, 56 and 61, 66 and 71, 76 and 81, 86 and 91, 96 and 101, 106 and 111, 116 and 121, and 126 and 131. In certain embodiments, an antibody or antigen-binding fragment comprising the VH and VL encoded by the polynucleotides in such compositions specifically or preferentially binds to DR6.
The polynucleotides described herein also include fragments of the polynucleotides, as described elsewhere. Additionally polynucleotides which encode fusion polynucleotides, Fab fragments, and other derivatives, as described herein, are also contemplated.
The polynucleotides can be produced or manufactured by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
Alternatively, a polynucleotide encoding a DR6 antibody, or antigen-binding fragment, variant, or derivative thereof can be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the antibody can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, such as poly A+RNA, isolated from, any tissue or cells expressing the antibody or other DR6 antibody, such as hybridoma cells selected to express an antibody) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody or other DR6 antibody. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.
Once the nucleotide sequence and corresponding amino acid sequence of the DR6 antibody, or antigen-binding fragment, variant, or derivative thereof is determined, its nucleotide sequence can be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1990) and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, NY (1998), which are both incorporated by reference herein in their entireties), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.
A polynucleotide encoding a DR6 antibody, or antigen-binding fragment, variant, or derivative thereof can be composed of any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, a polynucleotide encoding DR6 antibody, or antigen-binding fragment, variant, or derivative thereof can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, a polynucleotide encoding a DR6 antibody, or antigen-binding fragment, variant, or derivative thereof can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide encoding a DR6 antibody, or antigen-binding fragment, variant, or derivative thereof can also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
An isolated polynucleotide encoding a non-natural variant of a polypeptide derived from an immunoglobulin (e.g., an immunoglobulin heavy chain portion or light chain portion) can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of the immunoglobulin such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions can be made at one or more non-essential amino acid residues.
DR6 Antibody Polypeptides
Isolated polypeptides which make up DR6 antibodies, and polynucleotides encoding such polypeptides are also described herein. DR6 antibodies comprise polypeptides, e.g., amino acid sequences encoding DR6-specific antigen binding regions derived from immunoglobulin molecules. A polypeptide or amino acid sequence “derived from” a designated protein refers to the origin of the polypeptide having a certain amino acid sequence. In certain cases, the polypeptide or amino acid sequence which is derived from a particular starting polypeptide or amino acid sequence has an amino acid sequence that is essentially identical to that of the starting sequence, or a portion thereof, wherein the portion consists of at least 10-20 amino acids, at least 20-30 amino acids, at least 30-50 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the starting sequence.
In one embodiment, the polypeptide can be an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH), where at least one of VH-CDRs of the heavy chain variable region or at least two of the VH-CDRs of the heavy chain variable region are at least 80%, 85%, 90% or 95% identical to reference heavy chain VH-CDR1, VH-CDR2 or VH-CDR3 amino acid sequences from monoclonal DR6 antibodies disclosed herein. Alternatively, the VH-CDR1, VH-CDR2 and VH-CDR3 regions of the VH are at least 80%, 85%, 90% or 95% identical to reference heavy chain VH-CDR1, VH-CDR2 and VH-CDR3 amino acid sequences from monoclonal DR6 antibodies disclosed herein. Thus, according to this embodiment a heavy chain variable region has VH-CDR1, VH-CDR2 and VH-CDR3 polypeptide sequences related to the groups shown in Table 5, supra. While Table 5 shows VH-CDRs defined by the Kabat system, other CDR definitions, e.g., VH-CDRs defined by the Chothia system, are also described. In certain embodiments, an antibody or antigen-binding fragment comprising the VH specifically or preferentially binds to DR6.
In another embodiment, the polypeptide can be an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH) in which the VH-CDR1, VH-CDR2 and VH-CDR3 regions have polypeptide sequences which are identical to the VH-CDR1, VH-CDR2 and VH-CDR3 groups shown in Table 5. In certain embodiments, an antibody or antigen-binding fragment comprising the VH specifically or preferentially binds to DR6.
In another embodiment, the polypeptide can be an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH) in which the VH-CDR1, VH-CDR2 and VH-CDR3 regions have polypeptide sequences which are identical to the VH-CDR1, VH-CDR2 and VH-CDR3 groups shown in Table 5, except for one, two, three, four, five, or six amino acid substitutions in any one VH-CDR. In larger CDRs, e.g., VH-CDR-3, additional substitutions can be made in the CDR, as long as the a VH comprising the VH-CDR specifically or preferentially binds to DR6. In certain embodiments the amino acid substitutions are conservative. In certain embodiments, an antibody or antigen-binding fragment comprising the VH specifically or preferentially binds to DR6.
In some embodiments, the polypeptide can be an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH) in which the VH-CDR1, VH-CDR2 and VH-CDR3 regions have polypeptide sequences selected from the group consisting of: SEQ ID NOs: 8, 9, and 10; SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70; SEQ ID NOs: 78, 79, and 80; SEQ ID NOs: 88, 89, and 90; SEQ ID NOs: 98, 99, and 100; SEQ ID NOs: 108, 109, and 110; SEQ ID NOs: 118, 119, and 120; and SEQ ID NOs: 128, 129 and 130, except for one, two, three, four, five or six amino acid substitutions in at least one of said VH-CDRs.
In some embodiments, the polypeptide can be an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH) in which the VH-CDR1, VH-CDR2 and VH-CDR3 regions have polypeptide sequences selected from the group consisting of: SEQ ID NOs: 8, 9, and 10; SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70; SEQ ID NOs: 78, 79, and 80; SEQ ID NOs: 88, 89, and 90; SEQ ID NOs: 98, 99, and 100; SEQ ID NOs: 108, 109, and 110; SEQ ID NOs: 118, 119, and 120; and SEQ ID NOs: 128, 129 and 130.
In a further embodiment, the polypeptide can be an isolated polypeptide comprising, consisting essentially of, or consisting of a VH polypeptide at least 80%, 85%, 90% 95% or 100% identical to a reference VH polypeptide amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, and 127. In certain embodiments, an antibody or antigen-binding fragment comprising the VH polypeptide specifically or preferentially binds to DR6.
In another aspect, the polypeptide can be an isolated polypeptide comprising, consisting essentially of, or consisting of a VH polypeptide selected from the group consisting of SEQ ID NOs: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, and 127. In certain embodiments, an antibody or antigen-binding fragment comprising the VH polypeptide specifically or preferentially binds to DR6.
In certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a one or more of the VH polypeptides described above specifically or preferentially binds to the same DR6 epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M50-H01, M51-H09, M53-E04, M53-F04, M62-B02, M63-E10, M66-B03, M67-G02, M72-F03, and M73-C04 or a reference monoclonal antibody selected from the group consisting of 1P1D6.3, 1P2F2.1, and 1P5D10.2, or will competitively inhibit such a monoclonal antibody or fragment from binding to DR6.
In certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of one or more of the VH polypeptides described above specifically or preferentially binds to a DR6 polypeptide or fragment thereof, or a DR6 variant polypeptide, with an affinity characterized by a dissociation constant (KD) no greater than 5×10−2 M, 10−2 M, 5×10−3 M, 10−3 M, 5×10−4 M, 10−4 M, 5×10−5 M, 10−5 M, 5×10−6 M, 10−6 M, 5×10−7 M, 10−7 M, 5×10−8 M, 10−8 M, 5×10−9 M, 10−9 M, 5×10−10 M, 10−10 M, 5×10−11 M, 10−11 M, 5×10−12 M, 10−12 M, 5×10−13 M, 10−13 M, 5×10−14 M, 10−14 M, 5×10−15 M, or 10−15 M.
In another embodiment, the polypeptide can be an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain variable region (VL), where at least one of the VL-CDRs of the light chain variable region or at least two of the VL-CDRs of the light chain variable region are at least 80%, 85%, 90% or 95% identical to reference light chain VL-CDR1, VL-CDR2 or VL-CDR3 amino acid sequences from monoclonal DR6 antibodies disclosed herein. Alternatively, the VL-CDR1, VL-CDR2 and VL-CDR3 regions of the VL are at least 80%, 85%, 90% or 95% identical to reference light chain VL-CDR1, VL-CDR2 and VL-CDR3 amino acid sequences from monoclonal DR6 antibodies disclosed herein. Thus, according to this embodiment a light chain variable region has VL-CDR1, VL-CDR2 and VL-CDR3 polypeptide sequences related to the polypeptides shown in Table 5, supra. While Table 5 shows VL-CDRs defined by the Kabat system, other CDR definitions, e.g., VL-CDRs defined by the Chothia system, are also described. In certain embodiments, an antibody or antigen-binding fragment comprising the VL polypeptide specifically or preferentially binds to DR6.
In another embodiment, the polypeptide can be an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain variable region (VL) in which the VL-CDR1, VL-CDR2 and VL-CDR3 regions have polypeptide sequences which are identical to the VL-CDR1, VL-CDR2 and VL-CDR3 groups shown in Table 5. In certain embodiments, an antibody or antigen-binding fragment comprising the VL polypeptide specifically or preferentially binds to DR6.
In another embodiment, the polypeptide can be an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VL) in which the VL-CDR1, VL-CDR2 and VL-CDR3 regions have polypeptide sequences which are identical to the VL-CDR1, VL-CDR2 and VL-CDR3 groups shown in Table 5, except for one, two, three, four, five, or six amino acid substitutions in any one VL-CDR. In larger CDRs, additional substitutions can be made in the VL-CDR, as long as the a VL comprising the VL-CDR specifically or preferentially binds to DR6. In certain embodiments the amino acid substitutions are conservative. In certain embodiments, an antibody or antigen-binding fragment comprising the VL specifically or preferentially binds to DR6.
In some embodiments, the polypeptide can be an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VL) in which the VL-CDR1, VL-CDR2 and VL-CDR3 regions have polypeptide sequences selected from the group consisting of: SEQ ID NOs: 13, 14, and 15; SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 53, 54, and 55; SEQ ID NOs: 63, 64, and 65; SEQ ID NOs: 73, 74, and 75; SEQ ID NOs: 83, 84, and 85; SEQ ID NOs: 93, 94, and 95; SEQ ID NOs: 103, 104, and 105; SEQ ID NOs: 113, 114 and 115; SEQ ID NOs: 123, 124, and 125; and SEQ ID NOs: 133, 134 and 135, except for one, two, three, four, five or six amino acid substitutions in at least one of said VL-CDRs.
In some embodiments, the polypeptide can be an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VL) in which the VL-CDR1, VL-CDR2 and VL-CDR3 regions have polypeptide sequences selected from the group consisting of: SEQ ID NOs: 13, 14, and 15; SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 53, 54, and 55; SEQ ID NOs: 63, 64, and 65; SEQ ID NOs: 73, 74, and 75; SEQ ID NOs: 83, 84, and 85; SEQ ID NOs: 93, 94, and 95; SEQ ID NOs: 103, 104, and 105; SEQ ID NOs: 113, 114 and 115; SEQ ID NOs: 123, 124, and 125; and SEQ ID NOs: 133, 134 and 135.
In a further embodiment, the polypeptide can be an isolated polypeptide comprising, consisting essentially of, or consisting of a VL polypeptide at least 80%, 85%, 90% 95% or 100% identical to a reference VL polypeptide sequence selected from the group consisting of SEQ ID NOs: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, and 132. In certain embodiments, an antibody or antigen-binding fragment comprising the VL polypeptide specifically or preferentially binds to DR6.
In another aspect, the polypeptide can be an isolated polypeptide comprising, consisting essentially of, or consisting of a VL polypeptide selected from the group consisting of SEQ ID NOs: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, and 132. In certain embodiments, an antibody or antigen-binding fragment comprising the VL polypeptide specifically or preferentially binds to DR6.
In certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, one or more of the VL polypeptides described above specifically or preferentially binds to the same DR6 epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M50-H01, M51-H09, M53-E04, M53-F04, M62-B02, M63-E10, M66-B03, M67-G02, M72-F03, and M73-C04 or a reference monoclonal antibody selected from the group consisting of 1P1D6.3, 1P2F2.1, and 1P5D10.2, or will competitively inhibit such a monoclonal antibody or fragment from binding to DR6.
In certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a one or more of the VL polypeptides described above specifically or preferentially binds to a DR6 polypeptide or fragment thereof, or a DR6 variant polypeptide, with an affinity characterized by a dissociation constant (KD) no greater than 5×10−2 M, 10−2 M, 5×10−3 M, 10−3 M, 5×10−4 M, 10−4 M, 5×10−5 M, 10−5 M, 5×106 M, 10−6 M, 5×10−7 M, 10−7 M, 5×10−8 M, 10−8 M, 5×10−9 M, 10−9 M, 5×10−10 M, 10−10 M, 5×10−11 M, 10−11 M, 5×10−12 M, 10−12 M, 5×10−13 M, 10−13 M, 5×10−14 M, 10−14 M, 5×10−15 M, or 10−15 M.
In other embodiments, an antibody or antigen-binding fragment thereof comprises, consists essentially of or consists of a VH polypeptide, and a VL polypeptide, where the VH polypeptide and the VL polypeptide, respectively are at least 80%, 85%, 90% 95% or 100% identical to reference VH and VL polypeptide amino acid sequences selected from the group consisting of SEQ ID NOs: 7 and 12, 17 and 22, 27 and 32, 37 and 42, 47 and 52, 57 and 62, 67 and 72, 77 and 82, 87 and 92, 97 and 102, 107 and 112, 117 and 122 and 127 and 132. In certain embodiments, an antibody or antigen-binding fragment comprising these VH and VL polypeptides specifically or preferentially binds to DR6.
Any of the polypeptides described above can further include additional polypeptides, e.g., a signal peptide to direct secretion of the encoded polypeptide, antibody constant regions as described herein, or other heterologous polypeptides as described herein. Additionally, polypeptides include polypeptide fragments as described elsewhere. Additionally polypeptides include fusion polypeptide, Fab fragments, and other derivatives, as described herein.
Also, as described in more detail elsewhere herein, the present compositions include compositions comprising the polypeptides described above.
It will also be understood by one of ordinary skill in the art that DR6 antibody polypeptides as disclosed herein can be modified such that they vary in amino acid sequence from the naturally occurring binding polypeptide from which they were derived. For example, a polypeptide or amino acid sequence derived from a designated protein can be similar, e.g., have a certain percent identity to the starting sequence, e.g., it can be 60%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the starting sequence.
Furthermore, nucleotide or amino acid substitutions, deletions, or insertions leading to conservative substitutions or changes at “non-essential” amino acid regions can be made. For example, a polypeptide or amino acid sequence derived from a designated protein can be identical to the starting sequence except for one or more individual amino acid substitutions, insertions, or deletions, e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more individual amino acid substitutions, insertions, or deletions. A polypeptide or amino acid sequence derived from a designated protein can be identical to the starting sequence except for one or more individual amino acid substitutions, insertions, or deletions, e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more individual amino acid substitutions, insertions, or deletions. In other embodiments, a polypeptide or amino acid sequence derived from a designated protein can be identical to the starting sequence except for two or fewer, three or fewer, four or fewer, five or fewer, six or fewer, seven or fewer, eight or fewer, nine or fewer, ten or fewer, fifteen or fewer, or twenty or fewer individual amino acid substitutions, insertions, or deletions. In certain embodiments, a polypeptide or amino acid sequence derived from a designated protein has one to five, one to ten, one to fifteen, or one to twenty individual amino acid substitutions, insertions, or deletions relative to the starting sequence.
Certain DR6 antibody polypeptides comprise, consist essentially of, or consist of an amino acid sequence derived from a human amino acid sequence. However, certain DR6 antibody polypeptides comprise one or more contiguous amino acids derived from another mammalian species. For example, a DR6 antibody can include a primate heavy chain portion, hinge portion, or antigen binding region. In another example, one or more murine-derived amino acids can be present in a non-murine antibody polypeptide, e.g., in an antigen binding site of a DR6 antibody. In another example, the antigen binding site of a DR6 antibody is fully murine. In certain therapeutic applications, DR6-specific antibodies, or antigen-binding fragments, variants, or analogs thereof are designed so as to not be immunogenic in the animal to which the antibody is administered.
In certain embodiments, a DR6 antibody polypeptide comprises an amino acid sequence or one or more moieties not normally associated with an antibody. Exemplary modifications are described in more detail below. For example, a single-chain fv antibody fragment can comprise a flexible linker sequence, or can be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label).
An DR6 antibody polypeptide can comprise, consist essentially of, or consist of a fusion protein. Fusion proteins are chimeric molecules which comprise, for example, an immunoglobulin antigen-binding domain with at least one target binding site, and at least one heterologous portion, i.e., a portion with which it is not naturally linked in nature. The amino acid sequences can normally exist in separate proteins that are brought together in the fusion polypeptide or they can normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. Fusion proteins can be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.
The term “heterologous” as applied to a polynucleotide or a polypeptide, means that the polynucleotide or polypeptide is derived from a distinct entity from that of the rest of the entity to which it is being compared. For instance, as used herein, a “heterologous polypeptide” to be fused to a DR6 antibody, or an antigen-binding fragment, variant, or analog thereof is derived from a non-immunoglobulin polypeptide of the same species, or an immunoglobulin or non-immunoglobulin polypeptide of a different species.
A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in an immunoglobulin polypeptide can be replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.
Alternatively, in another embodiment, mutations can be introduced randomly along all or part of the immunoglobulin coding sequence, such as by saturation mutagenesis, and the resultant mutants can be incorporated into DR6 antibodies for use in the diagnostic and treatment methods disclosed herein and screened for their ability to bind to the desired antigen, e.g., DR6.
Fusion Polypeptides and Antibodies
DR6 polypeptides and antibodies for use in the treatment methods disclosed herein can further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus. For example, DR6 antagonist polypeptides or antibodies can be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.
DR6 antagonist polypeptides and antibodies for use in the treatment methods disclosed herein can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and can contain amino acids other than the 20 gene-encoded amino acids.
DR6 antagonists include fusion proteins comprising, consisting essentially of, or consisting of a DR6 antagonist polypeptide or antibody fusion that inhibits DR6 function. In certain embodiments, the heterologous polypeptide to which the DR6 antagonist polypeptide or antibody is fused is useful for function or is useful to target the DR6 antagonist polypeptide or antibody. In certain embodiments, a soluble DR6 antagonist polypeptide, e.g., a DR6 polypeptide comprising the extracellular domain (corresponding to amino acids 1 to 349 or 41 to 349 of SEQ ID NO: 2), or any other soluble DR6 polypeptide fragment, variant or derivative described herein, is fused to a heterologous polypeptide moiety to form a DR6 antagonist fusion polypeptide. DR6 antagonist fusion proteins and antibodies can be used to accomplish various objectives, e.g., increased serum half-life, improved bioavailability, in vivo targeting to a specific organ or tissue type, improved recombinant expression efficiency, improved host cell secretion, ease of purification, and higher avidity. Depending on the objective(s) to be achieved, the heterologous moiety can be inert or biologically active. Also, it can be chosen to be stably fused to the DR6 antagonist polypeptide or antibody or to be cleavable, in vitro or in vivo. Heterologous moieties to accomplish these other objectives are known in the art.
As an alternative to expression of a DR6 antagonist fusion polypeptide or antibody, a chosen heterologous moiety can be preformed and chemically conjugated to the DR6 antagonist polypeptide or antibody. In most cases, a chosen heterologous moiety will function similarly, whether fused or conjugated to the DR6 antagonist polypeptide or antibody. Therefore, in the following discussion of heterologous amino acid sequences, unless otherwise noted, it is to be understood that the heterologous sequence can be joined to the DR6 antagonist polypeptide or antibody in the form of a fusion protein or as a chemical conjugate.
Pharmacologically active polypeptides such as DR6 antagonist polypeptides or antibodies often exhibit rapid in vivo clearance, necessitating large doses to achieve therapeutically effective concentrations in the body. In addition, polypeptides smaller than about 60 kDa potentially undergo glomerular filtration, which sometimes leads to nephrotoxicity. Fusion or conjugation of relatively small polypeptides such as DR6 antagonist polypeptides or antibodies can be employed to reduce or avoid the risk of such nephrotoxicity. Various heterologous amino acid sequences, i.e., polypeptide moieties or “carriers,” for increasing the in vivo stability, i.e., serum half-life, of therapeutic polypeptides are known.
Due to its long half-life, wide in vivo distribution, and lack of enzymatic or immunological function, essentially full-length human serum albumin (HSA), or an HSA fragment, is commonly used as a heterologous moiety. Through application of methods and materials such as those taught in Yeh et al., Proc. Natl. Acad. Sci. USA 89:1904-08 (1992) and Syed et al., Blood 89:3243-52 (1997), HSA can be used to form a DR6 antagonist fusion polypeptide or antibody or polypeptide/antibody conjugate that displays pharmacological activity by virtue of the DR6 moiety while displaying significantly increased in vivo stability, e.g., 10-fold to 100-fold higher. The C-terminus of the HSA can be fused to the N-terminus of the DR6 polypeptide. Since HSA is a naturally secreted protein, the HSA signal sequence can be exploited to obtain secretion of a soluble DR6 fusion protein into the cell culture medium when the fusion protein is produced in a eukaryotic, e.g., mammalian, expression system.
In certain embodiments, DR6 antagonist polypeptides or antibodies for use in the methods described herein further comprise a targeting moiety. Targeting moieties include a protein or a peptide which directs localization to a certain part of the body, for example, to the brain or compartments therein. In certain embodiments, DR6 antagonist polypeptides or antibody for use in the methods described herein are attached or fused to a brain targeting moiety. The brain targeting moieties are attached covalently (e.g., direct, translational fusion, or by chemical linkage either directly or through a spacer molecule, which can be optionally cleavable) or non-covalently attached (e.g., through reversible interactions such as avidin, biotin, protein A, IgG, etc.). In other embodiments, a DR6 antagonist polypeptide or antibody for use in the methods described herein is attached to one more brain targeting moieties. In additional embodiments, the brain targeting moiety is attached to a plurality of DR6 antagonist polypeptides or antibodies for use in the methods described herein.
A brain targeting moiety associated with a DR6 antagonist polypeptide or antibody enhances brain delivery of such a DR6 antagonist polypeptide or antibody. A number of polypeptides have been described which, when fused to a protein or therapeutic agent, delivers the protein or therapeutic agent through the blood brain barrier (BBB). Non-limiting examples include the single domain antibody FC5 (Abulrob et al. (2005) J. Neurochem. 95, 1201-1214); mAB 83-14, a monoclonal antibody to the human insulin receptor (Pardridge et al. (1995) Pharmacol. Res. 12, 807-816); the B2, B6 and B8 peptides binding to the human transferrin receptor (hTfR) (Xia et al. (2000) J. Virol. 74, 11359-11366); the OX26 monoclonal antibody to the transferrin receptor (Pardridge et al. (1991) J. Pharmacol. Exp. Ther. 259, 66-70); and SEQ ID NOs: 1-18 of U.S. Pat. No. 6,306,365. The contents of the above references are incorporated herein by reference in their entirety.
Enhanced brain delivery of a DR6 antagonist composition is determined by a number of means well established in the art. For example, administering to an animal a radioactively, enzymatically or fluorescently labeled DR6 antagonist polypeptide or antibody linked to a brain targeting moiety; determining brain localization; and comparing localization with an equivalent radioactively labeled DR6 antagonist polypeptide o antibody that is not associated with a brain targeting moiety. Other means of determining enhanced targeting are described in the above references.
The signal sequence is a polynucleotide that encodes an amino acid sequence that initiates transport of a protein across the membrane of the endoplasmic reticulum. Signal sequences useful for constructing an immunofusin include antibody light chain signal sequences, e.g., antibody 14.18 (Gillies et al., J. Immunol. Meth. 125:191-202 (1989)), antibody heavy chain signal sequences, e.g., the MOPC141 antibody heavy chain signal sequence (Sakano et al., Nature 286:5774 (1980)). Alternatively, other signal sequences can be used. See, e.g., Watson, Nucl. Acids Res. 12:5145 (1984). The signal peptide is usually cleaved in the lumen of the endoplasmic reticulum by signal peptidases. This results in the secretion of an immunofusin protein containing the Fc region and the DR6 polypeptide.
In some embodiments, the DNA sequence can encode a proteolytic cleavage site between the secretion cassette and the DR6 polypeptide. Such a cleavage site can provide, e.g., for the proteolytic cleavage of the encoded fusion protein, thus separating the Fc domain from the target protein. Useful proteolytic cleavage sites include amino acid sequences recognized by proteolytic enzymes such as trypsin, plasmin, thrombin, factor Xa, or enterokinase K.
The secretion cassette can be incorporated into a replicable expression vector. Useful vectors include linear nucleic acids, plasmids, phagemids, cosmids and the like. An exemplary expression vector is pdC, in which the transcription of the immunofusin DNA is placed under the control of the enhancer and promoter of the human cytomegalovirus. See, e.g., Lo et al., Biochim. Biophys. Acta 1088:712 (1991); and Lo et al., Protein Engineering 11:495-500 (1998). An appropriate host cell can be transformed or transfected with a DNA that encodes a DR6 polypeptide and used for the expression and secretion of the DR6 polypeptide. Host cells that are typically used include immortal hybridoma cells, myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, Hela cells, and COS cells.
In one embodiment, a DR6 polypeptide is fused to a hinge and Fc region, i.e., the C-terminal portion of an Ig heavy chain constant region. Potential advantages of a DR6-Fc fusion include solubility, in vivo stability, and multivalency, e.g., dimerization. The Fc region used can be an IgA, IgD, or IgG Fc region (hinge-CH2-CH3). Alternatively, it can be an IgE or IgM Fc region (hinge-CH2-CH3-CH4). An IgG Fc region is generally used, e.g., an IgG1 Fc region or IgG4 Fc region. In one embodiment, a sequence beginning in the hinge region just upstream of the papain cleavage site which defines IgG Fc chemically (i.e. residue 216, taking the first residue of heavy chain constant region to be 114 according to the Kabat system), or analogous sites of other immunoglobulins is used in the fusion. The precise site at which the fusion is made is not critical; particular sites are well known and can be selected in order to optimize the biological activity, secretion, or binding characteristics of the molecule. Materials and methods for constructing and expressing DNA encoding Fc fusions are known in the art and can be applied to obtain DR6 fusions without undue experimentation. Some methods described herein employ a DR6 fusion protein such as those described in Capon et al., U.S. Pat. Nos. 5,428,130 and 5,565,335.
In some embodiments, fully intact, wild-type Fc regions display effector functions that can be unnecessary and undesired in an Fc fusion protein used in the methods described herein. Therefore, certain binding sites can be deleted from the Fc region during the construction of the secretion cassette. For example, since coexpression with the light chain is unnecessary, the binding site for the heavy chain binding protein, Bip (Hendershot et al., Immunol. Today 8:111-14 (1987)), is deleted from the CH2 domain of the Fc region of IgE, such that this site does not interfere with the efficient secretion of the immunofusin. Transmembrane domain sequences, such as those present in IgM, also are generally deleted.
In certain embodiments, the IgG1 Fc region is used. Alternatively, the Fc region of the other subclasses of immunoglobulin gamma (gamma-2, gamma-3 and gamma-4) can be used in the secretion cassette. The IgG1 Fc region of immunoglobulin gamma-1 includes at least part of the hinge region, the CH2 region, and the CH3 region. In some embodiments, the Fc region of immunoglobulin gamma-1 is a CH2-deleted-Fc, which includes part of the hinge region and the CH3 region, but not the CH2 region. A CH2-deleted-Fc has been described by Gillies et al., Hum. Antibod. Hybridomas 1:47 (1990). In some embodiments, the Fc region of one of IgA, IgD, IgE, or IgM, is used.
DR6-Fc fusion proteins can be constructed in several different configurations. In one configuration the C-terminus of the DR6 polypeptide is fused directly to the N-terminus of the Fc hinge moiety. In a slightly different configuration, a short polypeptide, e.g., 2-10 amino acids, is incorporated into the fusion between the N-terminus of the DR6 moiety and the C-terminus of the Fc moiety. Such a linker provides conformational flexibility, which can improve biological activity in some circumstances. If a sufficient portion of the hinge region is retained in the Fc moiety, the DR6-Fc fusion will dimerize, thus forming a divalent molecule. A homogeneous population of monomeric Fc fusions will yield monospecific, bivalent dimers. A mixture of two monomeric Fc fusions each having a different specificity will yield bispecific, bivalent dimers.
Soluble DR6 polypeptides can be fused to heterologous peptides to facilitate purification or identification of the soluble DR6 moiety. For example, a histidine tag can be fused to a soluble DR6 polypeptide to facilitate purification using commercially available chromatography media.
A “linker” sequence is a series of one or more amino acids separating two polypeptide coding regions in a fusion protein. A typical linker comprises at least 5 amino acids. Additional linkers comprise at least 10 or at least 15 amino acids. In certain embodiments, the amino acids of a peptide linker are selected so that the linker is hydrophilic. The linker (Gly-Gly-Gly-Gly-Ser)3 (G4S)3 (SEQ ID NO:136) is a useful linker that is widely applicable to many antibodies as it provides sufficient flexibility. Other linkers include (Gly-Gly-Gly-Gly-Ser)2 (G4S)2 (SEQ ID NO:137), Glu Ser Gly Arg Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser (SEQ ID NO:138), Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr (SEQ ID NO:139), Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr Gln (SEQ ID NO:140), Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Val Asp (SEQ ID NO:141), Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu Gly Lys Gly (SEQ ID NO:142), Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser Leu Asp (SEQ ID NO:143), and Glu Ser Gly Ser Val Ser Ser Glu Glu Leu Ala Phe Arg Ser Leu Asp (SEQ ID NO:144). Examples of shorter linkers include fragments of the above linkers, and examples of longer linkers include combinations of the linkers above, combinations of fragments of the linkers above, and combinations of the linkers above with fragments of the linkers above.
DR6 polypeptides can be fused to a polypeptide tag. The term “polypeptide tag,” as used herein, is intended to mean any sequence of amino acids that can be attached to, connected to, or linked to a DR6 polypeptide and that can be used to identify, purify, concentrate or isolate the DR6 polypeptide. The attachment of the polypeptide tag to the DR6 polypeptide can occur, e.g., by constructing a nucleic acid molecule that comprises: (a) a nucleic acid sequence that encodes the polypeptide tag, and (b) a nucleic acid sequence that encodes a DR6 polypeptide. Exemplary polypeptide tags include, e.g., amino acid sequences that are capable of being post-translationally modified, e.g., amino acid sequences that are biotinylated. Other exemplary polypeptide tags include, e.g., amino acid sequences that are capable of being recognized and/or bound by an antibody (or fragment thereof) or other specific binding reagent. Polypeptide tags that are capable of being recognized by an antibody (or fragment thereof) or other specific binding reagent include, e.g., those that are known in the art as “epitope tags.” An epitope tag can be a natural or an artificial epitope tag. Natural and artificial epitope tags are known in the art, including, e.g., artificial epitopes such as FLAG, Strep, or poly-histidine peptides. FLAG peptides include the sequence Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO:145) or Asp-Tyr-Lys-Asp-Glu-Asp-Asp-Lys (SEQ ID NO:146) (Einhauer, A. and Jungbauer, A., J. Biochem. Biophys. Methods 49:1-3:455-465 (2001)). The Strep epitope has the sequence Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (SEQ ID NO:147). The VSV-G epitope can also be used and has the sequence Tyr-Thr-Asp-Ile-Glu-Met-Asn-Arg-Leu-Gly-Lys (SEQ ID NO:148). Another artificial epitope is a poly-His sequence having six histidine residues (His-His-His-His-His-His) (SEQ ID NO:149). Naturally-occurring epitopes include the influenza virus hemagglutinin (HA) sequence Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala-Ile-Glu-Gly-Arg (SEQ ID NO:150) recognized by the monoclonal antibody 12CA5 (Murray et al., Anal. Biochem. 229:170-179 (1995)) and the eleven amino acid sequence from human c-myc (Myc) recognized by the monoclonal antibody 9E10 (Glu-Gln-Lys-Leu-Leu-Ser-Glu-Glu-Asp-Leu-Asn) (SEQ ID NO:151) (Manstein et al., Gene 162:129-134 (1995)). Another useful epitope is the tripeptide Glu-Glu-Phe which is recognized by the monoclonal antibody YL 1/2. (Stammers et al. FEBS Lett. 283:298-302 (1991)).
In certain embodiments, the DR6 polypeptide and the polypeptide tag can be connected via a linking amino acid sequence. As used herein, a “linking amino acid sequence” can be an amino acid sequence that is capable of being recognized and/or cleaved by one or more proteases. Amino acid sequences that can be recognized and/or cleaved by one or more proteases are known in the art. Exemplary amino acid sequences are those that are recognized by the following proteases: factor VIIa, factor IXa, factor Xa, APC, t-PA, u-PA, trypsin, chymotrypsin, enterokinase, pepsin, cathepsin B,H,L,S,D, cathepsin G, renin, angiotensin converting enzyme, matrix metalloproteases (collagenases, stromelysins, gelatinases), macrophage elastase, Cir, and Cis. The amino acid sequences that are recognized by the aforementioned proteases are known in the art. Exemplary sequences recognized by certain proteases can be found, e.g., in U.S. Pat. No. 5,811,252.
In some methods, a soluble DR6 fusion construct is used to enhance the production of a soluble DR6 moiety in bacteria. In such constructs a bacterial protein normally expressed and/or secreted at a high level is employed as the N-terminal fusion partner of a soluble DR6 polypeptide. See, e.g., Smith et al., Gene 67:31 (1988); Hopp et al., Biotechnology 6:1204 (1988); La Vallie et al., Biotechnology 11:187 (1993).
By fusing a soluble DR6 moiety at the amino and carboxy termini of a suitable fusion partner, bivalent or tetravalent forms of a soluble DR6 polypeptide can be obtained. For example, a soluble DR6 moiety can be fused to the amino and carboxy termini of an Ig moiety to produce a bivalent monomeric polypeptide containing two soluble DR6 moieties. Upon dimerization of two of these monomers, by virtue of the Ig moiety, a tetravalent form of a soluble DR6 protein is obtained. Such multivalent forms can be used to achieve increased binding affinity for the target. Multivalent forms of soluble DR6 also can be obtained by placing soluble DR6 moieties in tandem to form concatamers, which can be employed alone or fused to a fusion partner such as Ig or HSA.
DR6 Antagonist Conjugates
DR6 antagonist polypeptides and antibodies for use in the treatment methods disclosed herein include derivatives that are modified, i.e., by the covalent attachment of any type of molecule such that covalent attachment does not prevent the DR6 antagonist polypeptide or antibody from inhibiting the biological function of DR6. For example, but not by way of limitation, the DR6 antagonist polypeptides and antibodies can be modified e.g., by glycosylation, acetylation, pegylation, phosphylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative can contain one or more non-classical amino acids.
DR6 antagonist polypeptides and antibodies can be modified by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in the DR6 antagonist polypeptide or antibody, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini, or on moieties such as carbohydrates. It will be appreciated that the same type of modification can be present in the same or varying degrees at several sites in a given DR6 antagonist polypeptide or antibody. Also, a given DR6 antagonist polypeptide or antibody can contain many types of modifications. DR6 antagonist polypeptides or antibodies can be branched, for example, as a result of ubiquitination, and they can be cyclic, with or without branching. Cyclic, branched, and branched cyclic DR6 antagonist polypeptides and antibodies can result from posttranslation natural processes or can be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, Proteins—Structure And Molecular Properties, T. E. Creighton, W. H. Freeman and Company, New York 2nd Ed., (1993); Posttranslational Covalent Modification Of Proteins, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992)).
Any of a number of cross-linkers that contain a corresponding amino-reactive group and thiol-reactive group can be used to link DR6 antagonist polypeptides to a heterologous fusion partner. Examples of suitable linkers include amine reactive cross-linkers that insert a thiol-reactive maleimide, e.g., SMCC, AMAS, BMPS, MBS, EMCS, SMPB, SMPH, KMUS, and GMBS. Other suitable linkers insert a thiol-reactive haloacetate group, e.g., SBAP, SIA, SIAB. Linkers that provide a protected or non-protected thiol for reaction with sulfhydryl groups to product a reducible linkage include SPDP, SMPT, SATA, and SATP. Such reagents are commercially available (e.g., Pierce Chemicals).
Conjugation does not have to involve the N-terminus of a soluble DR6 polypeptide or the thiol moiety on serum albumin. For example, soluble DR6-albumin fusions can be obtained using genetic engineering techniques, wherein the soluble DR6 moiety is fused to the serum albumin gene at its N-terminus, C-terminus, or both.
Soluble DR6 polypeptides or DR6 antibodies can be polypeptides or antibodies wherein one or more polymers are conjugated (covalently linked) to the DR6 polypeptide or antibody. Examples of polymers suitable for such conjugation include polypeptides (discussed above), sugar polymers and polyalkylene glycol chains. Typically, but not necessarily, a polymer is conjugated to the soluble DR6 polypeptide or DR6 antibody for the purpose of improving one or more of the following: solubility, stability, or bioavailability.
The class of polymer generally used for conjugation to a DR6 antagonist polypeptide or antibody is a polyalkylene glycol. Polyethylene glycol (PEG) is most frequently used. PEG moieties, e.g., 1, 2, 3, 4 or 5 PEG polymers, can be conjugated to each DR6 antagonist polypeptide or antibody to increase serum half life, as compared to the DR6 antagonist polypeptide or antibody alone. PEG moieties are non-antigenic and essentially biologically inert. PEG moieties can be branched or unbranched.
The number of PEG moieties attached to the DR6 antagonist polypeptide or antibody and the molecular weight of the individual PEG chains can vary. In general, the higher the molecular weight of the polymer, the fewer polymer chains attached to the polypeptide. Usually, the total polymer mass attached to the DR6 antagonist polypeptide or antibody is from 20 kDa to 40 kDa. Thus, if one polymer chain is attached, the molecular weight of the chain is generally 20-40 kDa. If two chains are attached, the molecular weight of each chain is generally 10-20 kDa. If three chains are attached, the molecular weight is generally 7-14 kDa.
The polymer, e.g., PEG, can be linked to the DR6 antagonist polypeptide or antibody through any suitable, exposed reactive group on the polypeptide. The exposed reactive group(s) can be, e.g., an N-terminal amino group or the epsilon amino group of an internal lysine residue, or both. An activated polymer can react and covalently link at any free amino group on the DR6 antagonist polypeptide or antibody. Free carboxylic groups, suitably activated carbonyl groups, hydroxyl, guanidyl, imidazole, oxidized carbohydrate moieties and mercapto groups of the DR6 antagonist polypeptide or antibody (if available) also can be used as reactive groups for polymer attachment.
In a conjugation reaction, from about 1.0 to about 10 moles of activated polymer per mole of polypeptide, depending on polypeptide concentration, is typically employed. Usually, the ratio chosen represents a balance between maximizing the reaction while minimizing side reactions (often non-specific) that can impair the desired pharmacological activity of the DR6 antagonist polypeptide or antibody. In certain embodiments, at least 50% of the biological activity (as demonstrated, e.g., in any of the assays described herein or known in the art) of the DR6 antagonist polypeptide or antibody is retained. In further embodiments, nearly 100% is retained.
The polymer can be conjugated to the DR6 antagonist polypeptide or antibody using conventional chemistry. For example, a polyalkylene glycol moiety can be coupled to a lysine epsilon amino group of the DR6 antagonist polypeptide or antibody. Linkage to the lysine side chain can be performed with an N-hydroxylsuccinimide (NHS) active ester such as PEG succinimidyl succinate (SS-PEG) and succinimidyl propionate (SPA-PEG). Suitable polyalkylene glycol moieties include, e.g., carboxymethyl-NHS and norleucine-NHS, SC. These reagents are commercially available. Additional amine-reactive PEG linkers can be substituted for the succinimidyl moiety. These include, e.g., isothiocyanates, nitrophenylcarbonates (PNP), epoxides, benzotriazole carbonates, SC-PEG, tresylate, aldehyde, epoxide, carbonylimidazole and PNP carbonate. Conditions are usually optimized to maximize the selectivity and extent of reaction. Such optimization of reaction conditions is within ordinary skill in the art.
PEGylation can be carried out by any of the PEGylation reactions known in the art. See, e.g., Focus on Growth Factors 3:4-10 (1992), and European patent applications EP0154316 and EP0401384. PEGylation can be carried out using an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer).
PEGylation by acylation generally involves reacting an active ester derivative of polyethylene glycol. Any reactive PEG molecule can be employed in the PEGylation. PEG esterified to N-hydroxysuccinimide (NHS) is a frequently used activated PEG ester. As used herein, “acylation” includes without limitation the following types of linkages between the therapeutic protein and a water-soluble polymer such as PEG: amide, carbamate, urethane, and the like. See, e.g., Bioconjugate Chem. 5:133-140, 1994. Reaction parameters are generally selected to avoid temperature, solvent, and pH conditions that would damage or inactivate the soluble DR6 polypeptide.
Generally, the connecting linkage is an amide and typically at least 95% of the resulting product is mono-, di- or tri-PEGylated. However, some species with higher degrees of PEGylation can be formed in amounts depending on the specific reaction conditions used. Optionally, purified PEGylated species are separated from the mixture, particularly unreacted species, by conventional purification methods, including, e.g., dialysis, salting-out, ultrafiltration, ion-exchange chromatography, gel filtration chromatography, hydrophobic exchange chromatography, and electrophoresis.
PEGylation by alkylation generally involves reacting a terminal aldehyde derivative of PEG with DR6 antagonist polypeptide or antibody in the presence of a reducing agent. In addition, one can manipulate the reaction conditions to favor PEGylation substantially only at the N-terminal amino group of a DR6 antagonist polypeptide or antibody, i.e. a mono-PEGylated protein. In either case of mono-PEGylation or poly-PEGylation, the PEG groups are typically attached to the protein via a —CH2-NH— group. With particular reference to the —CH2- group, this type of linkage is known as an “alkyl” linkage.
Derivatization via reductive alkylation to produce an N-terminally targeted mono-PEGylated product exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization. The reaction is performed at a pH that allows one to take advantage of the pKa differences between the epsilon-amino groups of the lysine residues and that of the N-terminal amino group of the protein. By such selective derivatization, attachment of a water-soluble polymer that contains a reactive group, such as an aldehyde, to a protein is controlled: the conjugation with the polymer takes place predominantly at the N-terminus of the protein and no significant modification of other reactive groups, such as the lysine side chain amino groups, occurs.
The polymer molecules used in both the acylation and alkylation approaches are selected from among water-soluble polymers. The polymer selected is typically modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization can be controlled as provided for in the present methods. An exemplary reactive PEG aldehyde is polyethylene glycol propionaldehyde, which is water stable, or mono C1-C10 alkoxy or aryloxy derivatives thereof (see, e.g., Harris et al., U.S. Pat. No. 5,252,714). The polymer can be branched or unbranched. For the acylation reactions, the polymer(s) selected typically have a single reactive ester group. For reductive alkylation, the polymer(s) selected typically have a single reactive aldehyde group. Generally, the water-soluble polymer will not be selected from naturally occurring glycosyl residues, because these are usually made more conveniently by mammalian recombinant expression systems.
Methods for preparing a PEGylated soluble DR6 polypeptide or antibody generally includes the steps of (a) reacting a DR6 antagonist polypeptide or antibody with polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions whereby the molecule becomes attached to one or more PEG groups, and (b) obtaining the reaction product(s). In general, the optimal reaction conditions for the acylation reactions will be determined case-by-case based on known parameters and the desired result. For example, a larger the ratio of PEG to protein, generally leads to a greater the percentage of poly-PEGylated product.
Reductive alkylation to produce a substantially homogeneous population of mono-polymer/soluble DR6 polypeptide or DR6 antibody generally includes the steps of: (a) reacting a soluble DR6 protein or polypeptide with a reactive PEG molecule under reductive alkylation conditions, at a pH suitable to permit selective modification of the N-terminal amino group of the polypeptide or antibody; and (b) obtaining the reaction product(s).
For a substantially homogeneous population of mono-polymer/soluble DR6 polypeptide or DR6 antibody, the reductive alkylation reaction conditions are those that permit the selective attachment of the water-soluble polymer moiety to the N-terminus of the polypeptide or antibody. Such reaction conditions generally provide for pKa differences between the lysine side chain amino groups and the N-terminal amino group. For purposes described herein, the pH is generally in the range of 3-9, typically 3-6.
Soluble DR6 polypeptides or antibodies can include a tag, e.g., a moiety that can be subsequently released by proteolysis. Thus, the lysine moiety can be selectively modified by first reacting a His-tag modified with a low-molecular-weight linker such as Traut's reagent (Pierce) which will react with both the lysine and N-terminus, and then releasing the His tag. The polypeptide will then contain a free SH group that can be selectively modified with a PEG containing a thiol-reactive head group such as a maleimide group, a vinylsulfone group, a haloacetate group, or a free or protected SH.
Traut's reagent can be replaced with any linker that will set up a specific site for PEG attachment. For example, Traut's reagent can be replaced with SPDP, SMPT, SATA, or SATP (Pierce). Similarly one could react the protein with an amine-reactive linker that inserts a maleimide (for example SMCC, AMAS, BMPS, MBS, EMCS, SMPB, SMPH, KMUS, or GMBS), a haloacetate group (SBAP, SIA, SIAB), or a vinylsulfone group and react the resulting product with a PEG that contains a free SH.
In some embodiments, the polyalkylene glycol moiety is coupled to a cysteine group of the DR6 antagonist polypeptide or antibody. Coupling can be effected using, e.g., a maleimide group, a vinylsulfone group, a haloacetate group, or a thiol group.
Optionally, the soluble DR6 polypeptide or antibody is conjugated to the polyethylene-glycol moiety through a labile bond. The labile bond can be cleaved in, e.g., biochemical hydrolysis, proteolysis, or sulfhydryl cleavage. For example, the bond can be cleaved under in vivo (physiological) conditions.
The reactions can take place by any suitable method used for reacting biologically active materials with inert polymers, generally at about pH 5-8, e.g., pH 5, 6, 7, or 8, if the reactive groups are on the alpha amino group at the N-terminus. Generally the process involves preparing an activated polymer and thereafter reacting the protein with the activated polymer to produce the soluble protein suitable for formulation.
In some embodiments, the antibodies or polypeptides are fusion proteins comprising a DR6 antibody, or antigen-binding fragment, variant, or derivative thereof, and a heterologous polypeptide. The heterologous polypeptide to which the antibody is fused can be useful for function or is useful to target the DR6 polypeptide expressing cells. In one embodiment, a fusion protein comprises, consists essentially of, or consists of, a polypeptide having the amino acid sequence of any one or more of the VH regions of an antibody or the amino acid sequence of any one or more of the VL regions of an antibody or fragments or variants thereof, and a heterologous polypeptide sequence. In another embodiment, a fusion protein for use in the diagnostic and treatment methods disclosed herein comprises, consists essentially of, or consists of a polypeptide having the amino acid sequence of any one, two, three of the VH-CDRs of a DR6-specific antibody, or fragments, variants, or derivatives thereof, or the amino acid sequence of any one, two, three of the VL-CDRs of a DR6-specific antibody, or fragments, variants, or derivatives thereof, and a heterologous polypeptide sequence. In one embodiment, the fusion protein comprises a polypeptide having the amino acid sequence of a VH-CDR3 of a DR6-specific antibody, or fragment, derivative, or variant thereof, and a heterologous polypeptide sequence, which fusion protein specifically binds to at least one epitope of DR6. In another embodiment, a fusion protein comprises a polypeptide having the amino acid sequence of at least one VH region of a DR6-specific antibody and the amino acid sequence of at least one VL region of a DR6-specific antibody or fragments, derivatives or variants thereof, and a heterologous polypeptide sequence. In one embodiment, the VH and VL regions of the fusion protein correspond to a single source antibody (or scFv or Fab fragment) which specifically binds at least one epitope of DR6. In yet another embodiment, a fusion protein for use in the diagnostic and treatment methods disclosed herein comprises a polypeptide having the amino acid sequence of any one, two, three or more of the VH CDRs of a DR6-specific antibody and the amino acid sequence of any one, two, three or more of the VL CDRs of a DR6-specific antibody, or fragments or variants thereof, and a heterologous polypeptide sequence. In some embodiments, two, three, four, five, six, or more of the VH-CDR(s) or VL-CDR(s) correspond to single source antibody (or scFv or Fab fragment). Nucleic acid molecules encoding these fusion proteins are also encompassed.
DR6 Polynucleotide Antagonists
Specific embodiments comprise a method of promoting nervous system cell survival by contacting the cells with a DR6 polynucleotide antagonist. The polynucleotide antagonist can be any polynucleotide that encodes a DR6-antagonist polypeptide. The polynucleotide antagonist can also be a nucleic acid molecule which specifically binds to a polynucleotide which encodes DR6. The DR6 polynucleotide antagonist prevents expression of DR6 (knockdown). In certain embodiments, the DR6 polynucleotide antagonist promotes nervous system cell survival or inhibits nervous system cell apoptosis. DR6 polynucleotide antagonists include, but are not limited to antisense molecules, ribozymes, siRNA, shRNA and RNAi. Typically, such binding molecules are separately administered to the animal (see, for example, O'Connor, J. Neurochem. 56:560 (1991), but such binding molecules can also be expressed in vivo from polynucleotides taken up by a host cell and expressed in vivo. See also Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).
RNAi refers to the expression of an RNA which interferes with the expression of the targeted mRNA. Specifically, the RNAi silences a targeted gene via interacting with the specific mRNA (e.g. DR6) through a siRNA (short interfering RNA). The ds RNA complex is then targeted for degradation by the cell. Additional RNAi molecules include Short hairpin RNA (shRNA); also short interfering hairpin. The shRNA molecule contains sense and antisense sequences from a target gene connected by a loop. The shRNA is transported from the nucleus into the cytoplasm, it is degraded along with the mRNA. Pol III or U6 promoters can be used to express RNAs for RNAi.
RNAi is mediated by double stranded RNA (dsRNA) molecules that have sequence-specific homology to their “target” mRNAs (Caplen et al., Proc Natl Acad Sci USA 98:9742-9747, 2001). Biochemical studies in Drosophila cell-free lysates indicates that the mediators of RNA-dependent gene silencing are 21-25 nucleotide “small interfering” RNA duplexes (siRNAs). Accordingly, siRNA molecules are advantageously used in the methods described herein. The siRNAs are derived from the processing of dsRNA by an RNase known as DICER (Bernstein et al., Nature 409:363-366, 2001). It appears that siRNA duplex products are recruited into a multi-protein siRNA complex termed RISC (RNA Induced Silencing Complex). Without wishing to be bound by any particular theory, it is believed that a RISC is guided to a target mRNA, where the siRNA duplex interacts sequence-specifically to mediate cleavage in a catalytic fashion (Bernstein et al., Nature 409:363-366, 2001; Boutla et al., Curr Biol 11:1776-1780, 2001).
RNAi has been used to analyze gene function and to identify essential genes in mammalian cells (Elbashir et al., Methods 26:199-213, 2002; Harborth et al., J Cell Sci 114:4557-4565, 2001), including by way of non-limiting example neurons (Krichevsky et al., Proc Natl Acad Sci USA 99:11926-11929, 2002). RNAi is also being evaluated for therapeutic modalities, such as inhibiting or blocking the infection, replication and/or growth of viruses, including without limitation poliovirus (Gitlin et al., Nature 418:379-380, 2002) and HIV (Capodici et al., J Immunol 169:5196-5201, 2002), and reducing expression of oncogenes (e.g., the bcr-abl gene; Scherr et al., Blood September 26 epub ahead of print, 2002). RNAi has been used to modulate gene expression in mammalian (mouse) and amphibian (Xenopus) embryos (respectively, Calegari et al., Proc Natl Acad Sci USA 99:14236-14240, 2002; and Zhou, et al., Nucleic Acids Res 30:1664-1669, 2002), and in postnatal mice (Lewis et al., Nat Genet 32:107-108, 2002), and to reduce transgene expression in adult transgenic mice (McCaffrey et al., Nature 418:38-39, 2002). Methods have been described for determining the efficacy and specificity of siRNAs in cell culture and in vivo (see, e.g., Bertrand et al., Biochem Biophys Res Commun 296:1000-1004, 2002; Lassus et al., Sci STKE 2002(147):PL13, 2002; and Leirdal et al., Biochem Biophys Res Commun 295:744-748, 2002).
Molecules that mediate RNAi, including without limitation siRNA, can be produced in vitro by chemical synthesis (Hohjoh, FEBS Lett 521:195-199, 2002), hydrolysis of dsRNA (Yang et al., Proc Natl Acad Sci USA 99:9942-9947, 2002), by in vitro transcription with T7 RNA polymerase (Donzeet et al., Nucleic Acids Res 30:e46, 2002; Yu et al., Proc Natl Acad Sci USA 99:6047-6052, 2002), and by hydrolysis of double-stranded RNA using a nuclease such as E. coli RNase III (Yang et al., Proc Natl Acad Sci USA 99:9942-9947, 2002).
siRNA molecules can also be formed by annealing two oligonucleotides to each other, typically have the following general structure, which includes both double-stranded and single-stranded portions:
Wherein N, X and Y are nucleotides; X hydrogen bonds to Y; “:” signifies a hydrogen bond between two bases; x is a natural integer having a value between 1 and about 100; and m and n are whole integers having, independently, values between 0 and about 100. In some embodiments, N, X and Y are independently A, G, C and T or U. Non-naturally occurring bases and nucleotides can be present, particularly in the case of synthetic siRNA (i.e., the product of annealing two oligonucleotides). The double-stranded central section is called the “core” and has base pairs (bp) as units of measurement; the single-stranded portions are overhangs, having nucleotides (nt) as units of measurement. The overhangs shown are 3′ overhangs, but molecules with 5′ overhangs are also contemplated. Also contemplated are siRNA molecules with no overhangs (i.e., m=0 and n=0), and those having an overhang on one side of the core but not the other (e.g., m=0 and n≧1, or vice-versa).
Initially, RNAi technology did not appear to be readily applicable to mammalian systems. This is because, in mammals, dsRNA activates dsRNA-activated protein kinase (PKR) resulting in an apoptotic cascade and cell death (Der et al, Proc. Natl. Acad. Sci. USA 94:3279-3283, 1997). In addition, it has long been known that dsRNA activates the interfeDR6 cascade in mammalian cells, which can also lead to altered cell physiology (Colby et al, Annu. Rev. Microbiol. 25:333, 1971; Kleinschmidt et al., Annu. Rev. Biochem. 41:517, 1972; Lampson et al., Proc. Natl. Acad. Sci. USA 58L782, 1967; Lomniczi et al., J. Gen. Virol. 8:55, 1970; and Younger et al., J. Bacteriol. 92:862, 1966). However, dsRNA-mediated activation of the PKR and interfeDR6 cascades requires dsRNA longer than about 30 base pairs. In contrast, dsRNA less than 30 base pairs in length has been demonstrated to cause RNAi in mammalian cells (Caplen et al., Proc. Natl. Acad. Sci. USA 98:9742-9747, 2001). Thus, it is expected that undesirable, non-specific effects associated with longer dsRNA molecules can be avoided by preparing short RNA that is substantially free from longer dsRNAs.
References regarding siRNA: Bernstein et al., Nature 409:363-366, 2001; Boutla et al., Curr Biol 11:1776-1780, 2001; Cullen, Nat Immunol. 3:597-599, 2002; Caplen et al., Proc Natl Acad Sci USA 98:9742-9747, 2001; Hamilton et al., Science 286:950-952, 1999; Nagase et al., DNA Res. 6:63-70, 1999; Napoli et al., Plant Cell 2:279-289, 1990; Nicholson et al., Mamm. Genome 13:67-73, 2002; Parrish et al., Mol Cell 6:1077-1087, 2000; Romano et al., Mol Microbiol 6:3343-3353, 1992; Tabara et al., Cell 99:123-132, 1999; and Tuschl, Chembiochem. 2:239-245, 2001.
Paddison et al. (Genes & Dev. 16:948-958, 2002) have used small RNA molecules folded into hairpins as a means to effect RNAi. Accordingly, such short hairpin RNA (shRNA) molecules are also advantageously used in the methods described herein. The length of the stem and loop of functional shRNAs varies; stem lengths can range anywhere from about 25 to about 30 nt, and loop size can range between 4 to about 25 nt without affecting silencing activity. While not wishing to be bound by any particular theory, it is believed that these shRNAs resemble the dsRNA products of the DICER RNase and, in any event, have the same capacity for inhibiting expression of a specific gene. The shRNA can be expressed from a lentiviral vector (e.g., pLL3.7).
Antisense technology can be used to control gene expression through antisense DNA or RNA, or through triple-helix formation. Antisense techniques are discussed for example, in Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance, Lee et al., Nucleic Acids Research 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1300 (1991). The methods are based on binding of a polynucleotide to a complementary DNA or RNA.
For example, the 5′ coding portion of a polynucleotide that encodes DR6 can be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of the target protein. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the target polypeptide.
In one embodiment, antisense nucleic acids specific for the DR6 gene are produced intracellularly by transcription from an exogenous sequence. For example, a vector or a portion thereof, is transcribed, producing an antisense nucleic acid (RNA). Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells. Expression of the antisense molecule can be by any promoter known in the art to act in vertebrate, e.g., human cells, such as those described elsewhere herein.
Absolute complementarity of an antisense molecule is not required. A sequence complementary to at least a portion of an RNA encoding DR6, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; or triplex. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the larger the hybridizing nucleic acid, the more base mismatches it can contain and still form a stable duplex (or triplex as the case can be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
Oligonucleotides that are complementary to the 5′ end of a messenger RNA, e.g., the 5′ untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3′ untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., Nature 372:333-335 (1994). Thus, oligonucleotides complementary to either the 5′- or 3′-non-translated, non-coding regions could be used in an antisense approach to inhibit translation of DR6. Oligonucleotides complementary to the 5′ untranslated region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the methods described herein. Antisense nucleic acids are typically at least six nucleotides in length and include, for example, oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.
Polynucleotides for use the therapeutic methods disclosed herein can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide can include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci. 84:648-652 (1987)); PCT Publication No. WO88/09810, published Dec. 15, 1988) or the blood-brain barrier (see, e.g., PCT Publication No. WO89/10134, published Apr. 25, 1988), hybridization-triggered cleavage agents. (See, e.g., Krol et al., BioTechniques 6:958-976 (1988)) or intercalating agents. (See, e.g., Zon, Pharm. Res. 5:539-549 (1988)). To this end, the oligonucleotide can be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
An antisense oligonucleotide for use in the therapeutic methods disclosed herein can comprise at least one modified base moiety such as, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N-6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N-6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′ methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3(3-amino-3-N2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
An antisense oligonucleotide for use in the therapeutic methods disclosed herein can also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
In yet another embodiment, an antisense oligonucleotide for use in the therapeutic methods disclosed herein comprises at least one modified phosphate backbone such as, but not limited to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
In yet another embodiment, an antisense oligonucleotide for use in the therapeutic methods disclosed herein is an α-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual situation, the strands run parallel to each other (Gautier et al., Nucl. Acids Res. 15:6625-6641 (1987)). The oligonucleotide is a 2′-O-methylribonucleotide (Inoue et al., Nucl. Acids Res. 15:6131-6148 (1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett. 215:327-330 (1987)).
Polynucleotides can be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides can be synthesized by the method of Stein et al., Nucl. Acids Res. 16:3209 (1988), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451 (1988)), etc.
Polynucleotide compositions for use in the therapeutic methods disclosed herein further include catalytic RNA, or a ribozyme (See, e.g., PCT International Publication WO 90/11364, published Oct. 4, 1990; Sarver et al., Science 247:1222-1225 (1990). Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5′-UG-3′. The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, Nature 334:585-591 (1988). In certain embodiments, the ribozyme is engineered so that the cleavage recognition site is located near the 5′ end of the target mRNA; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.
As in the antisense approach, ribozymes for use in the diagnostic and therapeutic methods disclosed herein can be composed of modified oligonucleotides (e.g. for improved stability, targeting, etc.) and can be delivered to cells which express DR6 in vivo. DNA constructs encoding the ribozyme can be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA. One method of delivery involves using a DNA construct “encoding” the ribozyme under the control of a stDR6g constitutive or inducible promoter, such as, for example, pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous DR6 messages and inhibit translation. Since ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
DR6 Aptamer Antagonists
In another embodiment, the DR6 antagonist for use in the methods described herein is an aptamer. An aptamer can be a nucleotide or a polypeptide which has a unique sequence, has the property of binding specifically to a desired target (e.g., a polypeptide), and is a specific ligand of a given target. Nucleotide aptamers include double stranded DNA and single stranded RNA molecules that bind to DR6. In certain embodiments, the DR6 aptamer antagonist promotes proliferation, differentiation, or survival of oligodendrocytes; promotes, oligodendrocyte-mediated myelination of neurons, or prevents demyelination, e.g., in a mammal.
Nucleic acid aptamers are selected using methods known in the art, for example via the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process. SELEX is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules as described in e.g. U.S. Pat. Nos. 5,475,096, 5,580,737, 5,567,588, 5,707,796, 5,763,177, 6,011,577, and 6,699,843, incorporated herein by reference in their entirety. Another screening method to identify aptamers is described in U.S. Pat. No. 5,270,163 (also incorporated herein by reference). The SELEX process is based on the capacity of nucleic acids for forming a variety of two- and three-dimensional structures, as well as the chemical versatility available within the nucleotide monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric, including other nucleic acid molecules and polypeptides. Molecules of any size or composition can serve as targets.
The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve desired binding affinity and selectivity. Starting from a mixture of nucleic acids, which can comprise a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding; partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules; dissociating the nucleic acid-target complexes; amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand enriched mixture of nucleic acids. The steps of binding, partitioning, dissociating and amplifying are repeated through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
Nucleotide aptamers can be used, for example, as diagnostic tools or as specific inhibitors to dissect intracellular signaling and transport pathways (James, Curr. Opin. Pharmacol. 1:540-546 (2001)). The high affinity and specificity of nucleotide aptamers makes them good candidates for drug discovery. For example, aptamer antagonists to the toxin ricin have been isolated and have IC50 values in the nanomolar range (Hesselberth J R et al., J Biol Chem 275:4937-4942 (2000)). Nucleotide aptamers can also be used against infectious disease, malignancy and viral surface proteins to reduce cellular infectivity.
Nucleotide aptamers for use in the methods described herein can be modified (e.g., by modifying the backbone or bases or conjugated to peptides) as described herein for other polynucleotides.
Using the protein structure of DR6, screening for aptamers that act on DR6 using the SELEX process would allow for the identification of aptamers that inhibit DR6-mediated processes.
Polypeptide aptamers for use in the methods described herein are random peptides selected for their ability to bind to and thereby block the action of DR6. Polypeptide aptamers can include a short variable peptide domain attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the peptide aptamer to levels comparable to an antibody's (nanomolar range). See, e.g., Hoppe-Seyler F et al., J Mol Med 78(8):426-430 (2000). The length of the short variable peptide is typically about 10 to 20 amino acids, and the scaffold can be any protein which has good solubility and compacity properties. One non-limiting example of a scaffold protein is the bacterial protein Thioredoxin-A. See, e.g., Cohen B A et al., PNAS 95(24): 14272-14277 (1998).
Polypeptide aptamers are peptides or small polypeptides that act as dominant inhibitors of protein function. Peptide aptamers specifically bind to target proteins, blocking their functional ability (Kolonin et al. (1998) Proc. Natl. Acad. Sci. 95: 14,266-14,271). Peptide aptamers that bind with high affinity and specificity to a target protein can be isolated by a variety of techniques known in the art. Peptide aptamers can be isolated from random peptide libraries by yeast two-hybrid screens (Xu, C. W., et al. (1997) Proc. Natl. Acad. Sci. 94:12,473-12,478) or by ribosome display (Hanes et al. (1997) Proc. Natl. Acad. Sci. 94:4937-4942). They can also be isolated from phage libraries (Hoogenboom, H. R., et al. (1998) Immunotechnology 4:1-20) or chemically generated peptide libraries. Additionally, polypeptide aptamers can be selected using the selection of Ligand Regulated Peptide Aptamers (LiRPAs). See, e.g., Binkowski B F et al., (2005) Chem & Biol 12(7): 847-855, incorporated herein by reference. Although the difficult means by which peptide aptamers are synthesized makes their use more complex than polynucleotide aptamers, they have unlimited chemical diversity. Polynucleotide aptamers are limited because they utilize only the four nucleotide bases, while peptide aptamers would have a much-expanded repertoire (i.e., 20 amino acids).
Peptide aptamers for use in the methods described herein can be modified (e.g., conjugated to polymers or fused to proteins) as described for other polypeptides elsewhere herein.
P75 Antagonists
Antagonists of p75 to be used in accordance with the methods described herein include, for example, (i) p75 antagonists compounds; (ii) p75 antagonist polypeptides; (iii) p75 antagonist antibodies or fragments thereof; (iv) −75 antagonist polynucleotides; (v) p75 aptamers; and (vi) combinations of two or more of said p75 antagonists. In some embodiments, the p75 antagonist inhibits interaction of p75 with DR6.
P75 antagonists are known in the art, and one of ordinary skill in the art would know how to screen for and test p75 antagonists which would inhibit the interaction of p75 and DR6. For example, a cyclic decapeptide antagonist of p75 is described in Turner et al. J. Neuroscience Research 78: 193-199 (2004), which is herein incorporated by reference in its entirety.
Vectors and Host Cells
Host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, express a DR6 and/or p75 antagonist polypeptide or antibody in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing DR6 and/or p75 antagonist polypeptide or antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing DR6 and/or p75 antagonist polypeptide or antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing DR6 and/or p75 antagonist polypeptide or antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing DR6 and/or p75 antagonist polypeptide or antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Bacterial cells such as Escherichia coli, or eukaryotic cells, e.g., for the expression of DR6 and/or p75 antagonist polypeptide or whole recombinant antibody molecules, are used for the expression of a recombinant DR6 and/or p75 antagonist polypeptide or antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for DR6 and/or p75 antagonist polypeptide or antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).
In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the DR6 and/or p75 antagonist polypeptide or antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of the DR6 and/or p75 antagonist polypeptide or antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified can be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the DR6 and/or p75 antagonist polypeptide or antibody coding sequence can be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is typically used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The DR6 and/or p75 antagonist polypeptide or antibody coding sequence can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems can be utilized. In cases where an adenovirus is used as an expression vector, the DR6 and/or p75 antagonist polypeptide or antibody coding sequence of interest can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the DR6 and/or p75 antagonist polypeptide or antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation signals can also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).
In addition, a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
For long-term, high-yield production of recombinant proteins, stable expression is typically used. For example, cell lines which stably express the DR6 and/or p75 antagonist polypeptide or antibody molecule can be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells can be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which stably express the DR6 and/or p75 antagonist polypeptide or antibody molecule.
A number of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 1980) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); TIB TECH 11(5):155-215 (May, 1993); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their entireties.
The expression levels of a DR6 and/or p75 antagonist polypeptide or antibody can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Academic Press, New York, Vol. 3. (1987)). When a marker in the vector system expressing a DR6 and/or p75 antagonist polypeptide or antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody or other polypeptide gene, production of the antibody or other polypeptide will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).
Vectors comprising nucleic acids encoding DR6 and/or p75 antagonists, e.g., soluble polypeptides, antibodies, antagonist polynucleotides, or aptamers, can be used to produce antagonists for use in the methods described herein. The choice of vector and expression control sequences to which such nucleic acids are operably linked depends on the functional properties desired, e.g., protein expression, and the host cell to be transformed.
Expression control elements useful for regulating the expression of an operably linked coding sequence are known in the art. Examples include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, and other regulatory elements. When an inducible promoter is used, it can be controlled, e.g., by a change in nutrient status, or a change in temperature, in the host cell medium.
The vector can include a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra-chromosomally in a bacterial host cell. Such replicons are well known in the art. In addition, vectors that include a prokaryotic replicon can also include a gene whose expression confers a detectable marker such as a drug resistance. Examples of bacterial drug-resistance genes are those that confer resistance to ampicillin or tetracycline.
Vectors that include a prokaryotic replicon can also include a prokaryotic or bacteriophage promoter for directing expression of the coding gene sequences in a bacterial host cell. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment to be expressed. Examples of such plasmid vectors are pUC8, pUC9, pBR322 and pBR329 (BioRad), pPL and pKK223 (Pharmacia). Any suitable prokaryotic host can be used to express a recombinant DNA molecule encoding a protein used in the methods described herein.
For the purposes described herein, numerous expression vector systems can be employed. For example, one class of vector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. Additionally, cells which have integrated the DNA into their chromosomes can be selected by introducing one or more markers which allow selection of transfected host cells. The marker can provide for prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper. The selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation. The neomycin phosphotransferase (neo) gene is an example of a selectable marker gene (Southern et al., J. Mol. Anal. Genet. 1:327-341 (1982)). Additional elements can also be needed for optimal synthesis of mRNA. These elements can include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals.
In one embodiment, a proprietary expression vector of Biogen IDEC, Inc., referred to as NEOSPLA (U.S. Pat. No. 6,159,730) can be used. This vector contains the cytomegalovirus promoter/enhancer, the mouse beta globin major promoter, the SV40 origin of replication, the bovine growth hormone polyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2, the dihydrofolate reductase gene and leader sequence. This vector has been found to result in very high level expression upon transfection in CHO cells, followed by selection in G418 containing medium and methotrexate amplification. Of course, any expression vector which is capable of eliciting expression in eukaryotic cells can be used. Examples of suitable vectors include, but are not limited to plasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAX1, and pZeoSV2 (available from Invitrogen, San Diego, Calif.), and plasmid pCI (available from Promega, Madison, Wis.). Additional eukaryotic cell expression vectors are known in the art and are commercially available. Typically, such vectors contain convenient restriction sites for insertion of the desired DNA segment. Exemplary vectors include pSVL and pKSV-10 (Pharmacia), pBPV-1, pml2d (International Biotechnologies), pTDT1 (ATCC 31255), retroviral expression vector pMIG and pLL3.7, adenovirus shuttle vector pDC315, and AAV vectors. Other exemplary vector systems are disclosed e.g., in U.S. Pat. No. 6,413,777.
In general, screening large numbers of transformed cells for those which express suitably high levels of the antagonist is routine experimentation which can be carried out, for example, by robotic systems.
Frequently used regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and enhancers derived from retroviral LTRs, cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdmlP)), polyoma and strong mammalian promoters such as native immunoglobulin and actin promoters. For further description of viral regulatory elements, and sequences thereof, see e.g., Stinski, U.S. Pat. No. 5,168,062; Bell, U.S. Pat. No. 4,510,245; and Schaffner, U.S. Pat. No. 4,968,615.
The recombinant expression vectors can carry sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., Axel, U.S. Pat. Nos. 4,399,216; 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to a drug, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Frequently used selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr− host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
Vectors encoding DR6 and/or p75 antagonists can be used for transformation of a suitable host cell. Transformation can be by any suitable method. Methods for introduction of exogenous DNA into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules can be introduced into mammalian cells by viral vectors.
Host cells for expression of a DR6 and/or p75 antagonist for use in a method described herein can be prokaryotic or eukaryotic. Exemplary eukaryotic host cells include, but are not limited to, yeast and mammalian cells, e.g., Chinese hamster ovary (CHO) cells (ATCC Accession No. CCL61), NIH Swiss mouse embryo cells NIH-3T3 (ATCC Accession No. CRL1658), and baby hamster kidney cells (BHK). Other useful eukaryotic host cells include insect cells and plant cells. Exemplary prokaryotic host cells are E. coli and Streptomyces.
Transformation of host cells can be accomplished by conventional methods suited to the vector and host cell employed. For transformation of prokaryotic host cells, electroporation and salt treatment methods can be employed (Cohen et al., Proc. Natl. Acad. Sci. USA 69:2110-14 (1972)). For transformation of vertebrate cells, electroporation, cationic lipid or salt treatment methods can be employed. See, e.g., Graham et al., Virology 52:456-467 (1973); Wigler et al., Proc. Natl. Acad. Sci. USA 76:1373-76 (1979).
In certain embodiments, the host cell line used for protein expression is of mammalian origin; those skilled in the art are credited with ability to determine particular host cell lines which are best suited for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to NSO, SP2 cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVI with SV40 T antigen), R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/O (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAH (human lymphocyte) and 293 (human kidney). Host cell lines are typically available from commercial services, the American Tissue Culture Collection or from published literature.
Expression of polypeptides from production cell lines can be enhanced using known techniques. For example, the glutamine synthetase (GS) system is commonly used for enhancing expression under certain conditions. See, e.g., European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 and European Patent Application No. 89303964.4.
Gene Therapy
A DR6 and/or p75 antagonist can be produced in vivo in a mammal, e.g., a human patient, using a gene-therapy approach to treatment of a nervous-system disease, disorder or injury in which promoting survival, proliferation and differentiation of oligodendrocytes or promoting myelination of neurons would be therapeutically beneficial. This involves administration of a suitable DR6 and/or p75 antagonist-encoding nucleic acid operably linked to suitable expression control sequences. Generally, these sequences are incorporated into a viral vector. Suitable viral vectors for such gene therapy include an adenoviral vector, an alphavirus vector, an enterovirus vector, a pestivirus vector, a lentiviral vector, a baculoviral vector, a herpesvirus vector, an Epstein Barr viral vector, a papovaviral vector, a poxvirus vector, a vaccinia viral vector, adeno-associated viral vector and a herpes simplex viral vector. The viral vector can be a replication-defective viral vector. Adenoviral vectors that have a deletion in its E1 gene or E3 gene are typically used. When an adenoviral vector is used, the vector usually does not have a selectable marker gene.
Pharmaceutical Compositions
The DR6 and/or p75 antagonists used in the methods described herein can be formulated into pharmaceutical compositions for administration to mammals, including humans. The pharmaceutical compositions used in the methods described herein comprise pharmaceutically acceptable carriers, including, e.g., ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
The compositions used in the methods described herein can be administered by any suitable method, e.g., parenterally, intraventricularly, orally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. As described previously, DR6 and/or p75 antagonists used in the methods described herein act in the nervous system to promote survival and prevent apoptosis of nervous system cells. Accordingly, in certain methods described herein, the DR6 and/or p75 antagonists are administered in such a way that they cross the blood-brain barrier. This crossing can result from the physico-chemical properties inherent in the DR6 and/or p75 antagonist molecule itself, from other components in a pharmaceutical formulation, or from the use of a mechanical device such as a needle, cannula or surgical instruments to breach the blood-brain barrier. Where the DR6 and/or p75 antagonist is a molecule that does not inherently cross the blood-brain barrier, e.g., a fusion to a moiety that facilitates the crossing, suitable routes of administration are, e.g., intrathecal or intracranial, e.g., directly into a chronic lesion of MS. Where the DR6 and/or p75 antagonist is a molecule that inherently crosses the blood-brain barrier, the route of administration can be by one or more of the various routes described below.
Sterile injectable forms of the compositions described herein can be aqueous or oleaginous suspension. These suspensions can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile, injectable preparation can also be a sterile, injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a suspension in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purposes of formulation.
Parenteral formulations can be a single bolus dose, an infusion or a loading bolus dose followed with a maintenance dose. These compositions can be administered at specific fixed or variable intervals, e.g., once a day, or on an “as needed” basis.
Certain pharmaceutical compositions used in the methods described herein can be orally administered in an acceptable dosage form including, e.g., capsules, tablets, aqueous suspensions or solutions. Certain pharmaceutical compositions also can be administered by nasal aerosol or inhalation. Such compositions can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and/or other conventional solubilizing or dispersing agents.
The amount of a DR6 and/or p75 antagonist that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated, the type of antagonist used and the particular mode of administration. The composition can be administered as a single dose, multiple doses or over an established period of time in an infusion. Dosage regimens also can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response).
In some cases, the methods described herein use a “therapeutically effective amount” or a “prophylactically effective amount” of a DR6 and/or p75 antagonist. Such a therapeutically or prophylactically effective amount can vary according to factors such as the disease state, age, sex, and weight of the individual. A therapeutically or prophylactically effective amount is also one in which any toxic or detrimental effects are outweighed by the therapeutically beneficial effects.
A specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the particular DR6 and/or p75 antagonist used, the patient's age, body weight, general health, sex, and diet, and the time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. Judgment of such factors by medical caregivers is within the ordinary skill in the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.
In the methods described herein the DR6 and/or p75 antagonists are generally administered directly to the nervous system, intracerebroventricularly, or intrathecally, e.g. into a chronic lesion. Compositions for administration according to the methods described herein can be formulated so that a dosage of 0.001-10 mg/kg body weight per day of the DR6 and/or p75 antagonist is administered. In some embodiments, the dosage is 0.01-1.0 mg/kg body weight per day. In some embodiments, the dosage is 0.001-0.5 mg/kg body weight per day.
For treatment with a DR6 and/or p75 antagonist antibody, the dosage can range, e.g., from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), of the host body weight. For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg, for example, at least 1 mg/kg. Doses intermediate in the above ranges can also be used. Subjects can be administered such doses daily, on alternative days, weekly or according to any other schedule determined by empirical analysis. An exemplary treatment entails administration in multiple dosages over a prolonged period, for example, of at least six months. Additional exemplary treatment regimes entail administration once per every two weeks or once a month or once every 3 to 6 months. Exemplary dosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kg weekly. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated.
In certain embodiments, a subject can be treated with a nucleic acid molecule encoding a DR6 and/or p75 antagonist polynucleotide. Doses for nucleic acids range from about 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per patient. Doses for infectious viral vectors vary from 10-100, or more, virions per dose.
Supplementary active compounds also can be incorporated into the compositions used in the methods described herein. For example, a soluble polypeptide or a fusion protein can be coformulated with and/or coadministered with one or more additional therapeutic agents.
The delivery methods encompass any suitable delivery method for a DR6 and/or p75 antagonist to a selected target tissue, including bolus injection of an aqueous solution or implantation of a controlled-release system. Use of a controlled-release implant reduces the need for repeat injections.
The DR6 and/or p75 antagonists described herein can be directly infused into the brain. Various implants for direct brain infusion of compounds are known and are effective in the delivery of therapeutic compounds to human patients suffering from neurological disorders. These include chronic infusion into the brain using a pump, stereotactically implanted, temporary interstitial catheters, permanent intracranial catheter implants, and surgically implanted biodegradable implants. See, e.g., Gill et al., supra; Scharfen et al., “High Activity Iodine-125 Interstitial Implant For Gliomas,” Int. J. Radiation Oncology Biol. Phys. 24(4):583-591 (1992); Gaspar et al., “Permanent 125I Implants for Recurrent Malignant Gliomas,” Int. J. Radiation Oncology Biol. Phys. 43(5):977-982 (1999); chapter 66, pages 577-580, Bellezza et al., “Stereotactic Interstitial Brachytherapy,” in Gildenberg et al., Textbook of Stereotactic and Functional Neurosurgery, McGraw-Hill (1998); and Brem et al., “The Safety of Interstitial Chemotherapy with BCNU-Loaded Polymer Followed by Radiation Therapy in the Treatment of Newly Diagnosed Malignant Gliomas: Phase I Trial,” J. Neuro-Oncology 26:111-23 (1995).
The compositions can also comprise a DR6 and/or p75 antagonist dispersed in a biocompatible carrier material that functions as a suitable delivery or support system for the compounds. Suitable examples of sustained release carriers include semipermeable polymer matrices in the form of shaped articles such as suppositories or capsules. Implantable or microcapsular sustained release matrices include polylactides (U.S. Pat. No. 3,773,319; EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-56 (1985)); poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate (Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981); Langer, Chem. Tech. 12:98-105 (1982)) or poly-D-(−)-3hydroxybutyric acid (EP 133,988).
In some embodiments of the methods described herein, a DR6 and/or p75 antagonist is administered to a patient by direct infusion into an appropriate region of the brain. See, e.g., Gill et al., Nature Med. 9: 589-95 (2003). Alternative techniques are available and can be applied to administer a DR6 and/or p75 antagonist. For example, stereotactic placement of a catheter or implant can be accomplished using the Riechert-Mundinger unit and the ZD (Zamorano-Dujovny) multipurpose localizing unit. A contrast-enhanced computerized tomography (CT) scan, injecting 120 ml of omnipaque, 350 mg iodine/ml, with 2 mm slice thickness can allow three-dimensional multiplanar treatment planning (STP, Fischer, Freiburg, Germany). This equipment permits planning on the basis of magnetic resonance imaging studies, merging the CT and MRI target information for clear target confirmation.
The Leksell stereotactic system (Downs Surgical, Inc., Decatur, Ga.) modified for use with a GE CT scanner (General Electric Company, Milwaukee, Wis.) as well as the Brown-Roberts-Wells (BRW) stereotactic system (Radionics, Burlington, Mass.) can be used for this purpose. Thus, on the morning of the implant, the annular base ring of the BRW stereotactic frame can be attached to the patient's skull. Serial CT sections can be obtained at 3 mm intervals though the (target tissue) region with a graphite rod localizer frame clamped to the base plate. A computerized treatment planning program can be run on a VAX 11/780 computer (Digital Equipment Corporation, Maynard, Mass.) using CT coordinates of the graphite rod images to map between CT space and BRW space.
The methods of treatment of nervous system disorders associated with increased cell death as described herein are typically tested in vitro, and then in vivo in an acceptable animal model, for the desired therapeutic or prophylactic activity, prior to use in humans. Suitable animal models, including transgenic animals, are will known to those of ordinary skill in the art. For example, in vitro assays to demonstrate the survival effect of the DR6 and/or p75 antagonists are described herein. The effect of the DR6 and/or p75 antagonists on apoptosis can be tested in vitro as described in the Examples. Finally, in vivo tests can be performed by creating transgenic mice which express the DR6 and/or p75 antagonist or by administering the DR6 and/or p75 antagonist to mice or rats in models as described herein.
The practices described herein will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning: A Laboratory Manual (3-Volume Set), J. Sambrook, D. W. Russell, Cold Spring Harbor Laboratory Press (2001); Genes VIII, B. Lewin, Prentice Hall (2003); PCR Primer, C. W. Dieffenbach and G. S. Dveksler, CSHL Press (2003); DNA Cloning, D. N. Glover ed., Volumes I and II (1985); Oligonucleotide Synthesis Methods and Applications (Methods in Molecular Biology), P. Herdewijn (Ed.), Humana Press (2004); Culture of Animal Cells: A Manual of Basic Technique, 4th edition, R. I. Freshney, Wiley-Liss (2000); Oligonucleotide Synthesis, M. J. Gait (Ed.), (1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization, B. D. Hames & S. J. Higgins eds. (1984); Nucleic Acid Hybridization, M. L. M. Anderson, Springer (1999); Animal Cell Culture and Technology, 2nd edition, M. Butler, BIOS Scientific Publishers (2004); Immobilized Cells and Enzymes: A Practical Approach (Practical Approach Series), J. Woodward, Irl Pr (1992); Transcription And Translation, B. D. Hames & S. J. Higgins (Eds.) (1984); Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc., (1987); Immobilized Cells And Enzymes, IRL Press, (1986); A Practical Guide To Molecular Cloning, 3rd edition, B. Perbal, John Wiley & Sons Inc. (1988); the treatise, Methods In Enzymology, Academic Press, Inc., N.Y.; Gene Transfer Vectors For Mammalian Cells, J. H. Miller and M. P. Calos eds., Cold Spring Harbor Laboratory (1987); Methods In Enzymology, Vols. 154 and 155, Wu et al. (Eds.); Immunochemical Methods In Cell And Molecular Biology, Mayer and Walker, (Eds.), Academic Press, London (1987); Handbook Of Experimental Immunology, Volumes I-IV, D. M. Weir and C. C. Blackwell (Eds.), (1986); Immunology Methods Manual: The Comprehensive Sourcebook of Techniques (4 Volume Set), 1st edition, I. Lefkovits, Academic Press (1997); Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press (2002); and in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989).
General principles of antibody engineering are set forth in Antibody Engineering: Methods and Protocols (Methods in Molecular Biology), B. L. Lo (Ed.), Humana Press (2003); Antibody engineering, R. Kontermann and S. Dubel (Eds.), Springer Verlag (2001); Antibody Engineering, 2nd edition, C. A. K. Borrebaeck (Ed.), Oxford Univ. Press (1995). General principles of protein engineering are set forth in Protein Engineering, A Practical Approach, Rickwood, D., et al. (Eds.), IRL Press at Oxford Univ. Press, Oxford, Eng. (1995). General principles of antibodies and antibody-hapten binding are set forth in: Antibodies: A Laboratory Manual, E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press (1988); Nisonoff, A., Molecular Immunology, 2nd edition, Sinauer Associates, Sunderland, Mass. (1984); and Steward, M. W., Antibodies, Their Structure and Function, Chapman and Hall, New York, N.Y. (1984). Additionally, standard methods in immunology known in the art and not specifically described are generally followed as in Current Protocols in Immunology, John Wiley & Sons, New York; Stites et al. (Eds.), Immunochemical Protocols (Methods in Molecular Biology), 2nd edition, J. D. Pound (Ed.), Humana Press (1998), Weir's Handbook of Experimental Immunology, 5th edition, D. M. Weir (Ed.), Blackwell Publishers (1996), Methods in Cellular Immunology, 2nd edition, R. Fernandez-Botran, CRC Press (2001); Basic and Clinical Immunology, 8th edition, Appleton & Lange, Norwalk, Conn. (1994) and Mishell and Shiigi (Eds.), Selected Methods in Cellular Immunology, W.H. Freeman and Co., New York (1980).
Standard reference works setting forth general principles of immunology include Current Protocols in Immunology, John Wiley & Sons, New York; Klein, J.; Kuby Immunology, 4th edition, R. A. Goldsby, et al., H. Freeman & Co. (2000); Basic and Clinical Immunology, M. Peakman, et al., Churchill Livingstone (1997); Immunology, 6th edition, I. Roitt, et al., Mosby, London (2001); Cellular and Molecular Immunology, 5th edition; A. K. Abbas, A. H. Lichtman, Elsevier—Health Sciences Division (2005); Immunology Methods Manual: The Comprehensive Sourcebook of Techniques (4 Volume Set), 1st edition, I. Lefkovits, Academic Press (1997) Immunology, 5th edition, R. A. Goldsby, et al., W. H. Freeman (2002); Monoclonal Antibodies: Principles and Practice, 3rd Edition, J. W. Goding, Academic Press (1996); Immunology: The Science of Self-Nonself Discrimination, John Wiley & Sons, New York (1982); Kennett, R., et al. (Eds.), Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses, Plenum Press, New York (1980); Campbell, A., “Monoclonal Antibody Technology” in Burden, R., et al. (Eds.), Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Elsevere, Amsterdam (1984).
All of the references cited above, as well as all references cited herein, are incorporated herein by reference in their entireties.
EXAMPLES
Example 1
DR6 is Expressed in the Nervous System
Tissue sections from adult mouse cerebral cortex and rat spinal cord were examined for expression of DR6 protein. Tissue sections were first penetrated with PBS containing 1% Triton X-100 (Sigma) for 30 minutes followed by incubation in blocking solution (PBS containing 0.1% Triton X-100 and 10% normal goat serum (NGS)) for 1 hour at room temperature. For primary antibody labeling, sections were incubated in blocking medium containing rabbit anti-DR6 (Santa Cruz, sc-13106, 1:200) and mouse anti-neuronal class III β-tubulin (Covance, MMS-435P, 1:500) at 4° C. overnight. After three PBS rinses, sections were incubated in 5% NGS-PBS containing Alexa 594 anti-rabbit antibody (Invitrogen) (1:500) at room temperature for 1 hour. The results show that the colocalization of DR6 and neuronal class III β-tubulin, which indicates that DR6 is expressed in neurons (data not shown).
To understand the role of DR6 in the nervous system, DR6 mRNA expression levels were evaluated to determine if they were developmentally regulated across rat brain tissues using quantitative real-time polymerase chain reaction after reverse transcription (RT-PCR). mRNA was extracted from whole brain and spinal cord homogenates taken at embryonic day 18 (E18), postnatal days 1 (P1), 7 (P7), 14 (P14), and 21 (P21) and from adults. All mRNA were extracted using Absolutely RNA miniprep kit following the manufacturer's instructions (Stratagene). Purified RNA (High Capacity cDNA Archive Kit, Applied Biosystems) was then used to generate cDNA of DR6. The cDNAs served as the template for quantitative real-time PCR (Q-PCR), and TaqMan Gene Expression system (Mx3000P) was used to quantify the DR6 using Mm00446361_m1 premixed primer set with MGB probes (Applied Biosystems). As seen in FIGS. 1A-B, DR6 expression level is low at E18, peaks at postnatal day 7 or 14, then reaches lower levels in both brain and spinal cord after maturation. The developmental transcription profile agreed with protein expression profile based on Western blot using anti-DR6 antibody (FIG. 1C). Immunohistochemical staining of human and rat brain tissues section revealed that DR6 is expressed in both human and rat neurons based on colocalization with the βIII-tubulin neuron marker (data not shown).
In addition, several other cell types were examined for expression of DR6 mRNA. mRNA was extracted from purified cultures of P2 oligodendrocyte progenitor cells (OPCs), E18 cortical neurons, P2 microglias and P42 cerebral cortex astrocytes. All mRNA were extracted using Absolutely RNA miniprep kit following the manufacturer's instructions (Stratagene). Purified RNA (High Capacity cDNA Archive Kit, Applied Biosystems) was then used to generate cDNA of DR6 and of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a control. FIG. 1D shows that DR6 is expressed in all four cell types examined.
The expression of DR6 is temporally regulated in the oligodendrocyte lineage. Three different approaches were used to examine the DR6 expression in oligodendrocytes. First, semi-quantitive RT-PCR was performed to determine the mRNA level from three different stages of purified populations of oligodendrocyte (A2B5, O4 and MBP). As shown in the FIG. 2A, DR6 mRNA was detected through all stage of the oligodendrocyte lineage with equivalent mRNA levels found in A2B5+, O4+ and MBP+ oligodendrocytes. Second, Western blot was performed to determine the DR6 protein level in the three different stage of oligodendrocytes. As shown in the FIG. 2B, DR6 protein is detectable in all three stages of oligodendrocyte. Interestingly, it is 5-fold higher in pre-myelinating (O4+) oligodendrocytes stage than earlier progenitor A2B5 and 10 fold higher than mature oligodendrocyte (MBP positive) suggesting that premyelinaitng oligodendrocytes are the predominant DR6 expressing cells. Third, the presence of DR6 protein in oligodendrocytes was confirmed using immunohistochemistry to show that A2B5+, O4+ and MBP+ oligodendrocytes were labeled by an anti-DR6 antibody (data not shown). Again, O4 positive cells show much more intensive fluorescence staining than MBP positive cells suggesting more DR6 expression in the premyelinating stage (O4 positive) than the mature oligodendrocytes. Pre-adsorption of the anti-DR6 antibody by addition of competing DR6-Fc completely ablated the signal.
Example 2
DR6 is Overexpressed in Brains of Patients with Alzheimer's Disease
Levels of DR6 mRNA in the brains of Alzheimer's disease patients were also examined. Quantitative real-time PCR was performed using six snap-frozen brain tissue blocks from four different donors with Alzheimer's disease. These results were compared to those obtained using three brain tissue blocks from 2 donors without neurological disease.
As shown in FIG. 3, DR6 is expressed 1.2- to 1.8-fold higher in Alzheimer's samples (2 frontal lobes, 1 temporal lobe, 1 basal ganglion and 2 unspecified regions collected from 4 individual donors) when compared to 3 normal brain samples (FIG. 3). To determine if the up-regulated DR6 is neurons specific, immunohistochemistry staining was performed. Cells in the center of αβ amyloid plaque are DR6 positive. The cells near the plaque have significantly brighter DR6 staining, suggesting higher levels of DR6 expression. The mRNA extraction, cDNA production and Q-PCR were performed as described in Example 1.
Example 3
DR6 is Upregulated after Axotomy
The effect of axotomy on DR6 mRNA and protein levels was also examined. For these experiments, embryonic DRG neurons were prepared as previously described (Mi et al., Nat. Neurosci. 8:745-51 (2005)). Briefly, DRGs were first dissected out from 2-week-old E16 Sprague Dawley rats (Charles River) and incubated in 0.25% Trypsin/EDTA (Invitrogen) at 37° C. for 30 minutes. An equal volume of DMEM (Invitrogen) containing 20% fetal bovine serum (Invitrogen) was then added to the digestion mixture to stop the reaction. Cell pellets that were collected after spinning down at 1,000 rpm at room temperature for 5 minutes were mechanically dissociated by gently passing through a plastic pipette until no large fragments were visible. About 1×105 DRG neurons were spotted in the center of each well of 4 well chamber-slides (LabTek) that were coated with 100 μg/ml Poly-D-lysine (Sigma). Cells were allowed to attach in growth medium (Neurobasal medium containing B27 supplement (Invitrogen) and 100 ng/ml nerve growth factor (BD Biosciences) at 37° C. in humidified air with 5% CO2. The next morning, the medium was replaced with fresh growth medium and cells were treated with 20 μM of fluorodeoxyuridine for 3 days to remove proliferating glial cells. Cultures were then maintained in growth medium at 37° C. in humidified air with 5% CO2 with fresh medium change every 3-4 days.
These cultured axons were severed by blade, and then at 0, 24 and 48 hours after injury, mRNAs were extracted using the Absolutely RNA miniprep kit following manufacturer's instructions (Stratagene). Quantitation, as shown in FIG. 4, revealed that DR6 mRNA levels were high at both 24 and 48 hours after axotomy compared to control uninjured axon cultures.
The mRNA extraction, cDNA production and Q-PCR were performed as described in Example 1.
Example 4
DR6 is Upregulated in Motor Neurons at the Lesion Site after Spinal Cord Injury
To create a model of spinal cord injury, dorsal hemisection of rat spinal cords were performed as described by Ji et al. (Mol Cell Neurosci. 33: 311-20 (2006)). The spinal cord tissues were fixed and stained using an anti-DR6 antibody. A significantly higher level of DR6 positive motor neurons was detected in rats with spinal cord injuries than in uninjured rats.
Example 5
Overexpression of DR6 Induces Neuronal Death
Cell Culture
Cerebral cortical neurons grown in cell cultures were infected with lentivirus expressing DR6, and the effects on cell death were examined. Cerebral cortical neurons were prepared from E18 Sprague Dawley rats (Charles River). Briefly, cerebral cortices from E18 rat embryos were dissected out, minced and incubated in 0.25% Trypsin/EDTA (Invitrogen) at 37° C. for 10 minutes. The cells were triturated after adding 60 μg/ml DNase I (Sigma) and 10% fetal bovine serum (Invitrogen) to stop the reaction. Cell pellets collected after spinning down at 1,000 rpm at room temperature for 5 minutes were then mechanically dissociated by gently passing through a plastic pipette until no large fragments were visible. All surfaces of tissue culture plates (Costar) were coated with 100 μg/ml Poly-D-lysine (Sigma) prior to cell seeding. The plating densities for different experimental setups were as follows: 1×106/well of a 12 well plate for western blots, 1×105/well of a 24 well plate for time-lapse imaging and 2×104/well of a 96 well plate for LDH assay, homogeneous caspase assay and Q-PCR analysis. Cells were maintained in Neurobasal medium containing B27 supplement (Invitrogen) at 37° C. in humidified air with 5% CO2 with fresh medium change every 3-4 days.
Protein Expression Constructs
DNA encoding full-length DR6 (amino acids 1-655) was inserted into the Not I sites of HRST-IRESeGFP lentivirus vector. The sequence of full-length human DR6 in lentivirus was obtained and is the sequence of SEQ ID NO:154. Nucleotides 124-153 of SEQ ID NO:154 encode a Myc tag that is used to check protein expression. The nucleotide sequence encodes a full-length Myc-tagged human DR6 polypeptide of the sequence of SEQ ID NO:155. Amino acids 42-51 of SEQ ID NO:155 are the Myc tag.
DNA encoding dominant negative DR6 (amino acids 1-370) was inserted into the Not I sites of HRST-IRESeGFP lentivirus vector. The sequence of dominant negative DR6 in lentivirus is provided as SEQ ID NO: 156. Nucleotides 124-153 of SEQ ID NO:156 encode a Myc tag that is used to check protein expression. The polypeptide sequence of dominant negative human DR6 polypeptide is provided as SEQ ID NO: 157. Amino acids 42-51 of SEQ ID NO:157 are the Myc tag.
Infections
The resulting plasmids and a GFP control plasmid were transfected into 293 cells to produce lentivirus as previously described (Rubinson et al. Nat. Genet. 33:401-6 (2003)), and the cortical neurons were infected with lentivirus at a multiplicity of infection (MOI) of 1. Ectopic expression of FL-DR6 induced cortical neurons apoptosis as visualized by cell morphology and cell count (FIG. 5A) compared to control infected cells after 92 hours.
DR6 induced cell death was further verified by XTT assay in parallel cultures that monitors mitochondrial activities of all living cells. The XTT assay is a colorimetric way to determine cell number by measuring mitochondrial activity. FL-DR6 infected cortical neurons exhibited a 2 fold reduction in XTT reading reflecting a significant decrease in cell number caused by DR6 overexpression-induced neuronal death (FIG. 5B). To determine if the Death domain (DD) is required for the cell death, a dominant-negative DR6 lentivirus (DN-DR6) which does not contain the DD, was introduced into cortical neurons by infection. As shown in FIG. 5B, DN-DR6 failed to induce apoptosis in cultured neurons suggesting that DR6 death domain is essential for its neuronal death induction.
The effect of DR6 on caspase-3, a key mediator of apoptosis, was also analyzed. Cortical neurons were infected as described above, and cell lysates were collected 48 hours after infection. A fluorometric homogenous caspase assay kit (Roche, 03005372001) was used to measure caspase-3 activity. Cells were assayed 48 hours after infection. As shown in FIG. 5C, free Rhodamine levels increased 2 fold after infection with the full-length DR6 lentivirus compared to uninfected cells, cells infected with the GFP control lentivirus or cells infected with the dominant negative DR6 lentivirus. This indicates that DR6, but not dominant negative DR6, increases the activity of caspase-3.
These results were confirmed by western blot. Cell lysates were subjected to PAGE, and the separated proteins were probed with anti-DR6 antibody (Santa Cruz). As shown in FIG. 5D, active caspase-3 levels were higher in cells infected with the full-length DR6 lentivirus than with cells infected with the dominant negative DR6 lentivirus or the GFP control lentivirus. In contrast, levels of a control protein (βIII-tubulin) were similar in all three infections. Efficacy of each of the infections was comparable as demonstrated by similar levels of GFP produced by the lentivirus vector in each of the infections. (See FIG. 5D).
Each of these results suggests that DR6 is able to induce cell death in cortical neurons.
Example 6
DR6-FL Overexpression Induces Death of OPCs
The effect of DR6 on the viability of oligodendrocyte precursor cells (OPCs) was also examined. In these experiments, cultures of enriched oligodendrocytes were prepared as previously described (Mi et al., Nat. Neurosci. 8:745-51 (2005)). Briefly, forebrains from P2 Sprague Dawley rats (Charles River) were dissected out, minced and incubated in 0.01% Trypsin (Sigma) and 10 μg/ml DNase (Sigma) at 37° C. for 15 minutes. Dissociated cells were plated into 100 μg/ml poly-D-lysine T75 tissue culture flasks and were grown at 37° C. for 10 days in Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal bovine serum (Invitrogen). To get enriched oligodendrocyte progenitors, flasks were shaken at 200 rpm at 37° C. overnight, resulting in a population of 95% purity. Oligodendrocyte progenitor cells were then plated at 2×104/well of a 96 well plate and maintained in DMEM (Invitrogen) containing 10 ng/ml platelet-derived growth factor (PDGFAA) and 10 ng/ml fibroblast growth factor (FGF) at 37° C. in humidified air with 5% CO2.
OPCs were then infected with GFP, full-length DR6 and dominant negative DR6 lentivirus as described in Example 5, and 48 hours after infection, the number of dying cells was assessed. Untreated cells and cells treated with 2% Triton X-100 were also examined as negative and positive controls, respectively. First, cells were visualized using phase contrast microscopy and the total number of cells, as well as the number of dying cells, was counted. The percentage of dying cells is represented graphically in FIG. 6A. Infection with full-length DR6 lentivirus led to a significant increase in the percentage of dying cells as compared to cells infected with dominant negative DR6 lentivirus or GFP lentivirus.
The effect of DR6 on the viability of OPCs was also evaluated using XTT and LDH assays. The XTT assay was performed as described in Example 5. Cells were treated with rotenone, an NADH inhibitor, as a positive control. As shown in FIG. 6B, cells that were infected with either dominant negative DR6 lentivirus or GFP lentivirus showed similar levels of cell viability. In contrast, cells treated with rotenone or infected with full-length DR6 lentivirus showed significantly lower levels of cell viability.
Similar results were obtained using an LDH assay. LDH is an enzyme that is released upon cell lysis, so colorimetric assays for LDH activity can be used to measure cell damage. The LDH assay was performed using a cytotoxicity detection kit (LDH, Roche, 11644793001) by following the manufacture's instruction. The results are displayed graphically in FIG. 6C and demonstrate that infection of OPCs with full-length DR6 lentivirus result in significantly greater cytotoxicity as compared to infection with either dominant negative DR6 lentivirus or GFP lentivirus.
Each of these experiments suggest that full-length DR6 is able to induce cell death in OPCs, in addition to cortical neurons.
Example 7
Blocking DR6 Signaling Pathway Promotes Oligodendrocyte Survival and Differentiation
Since oligodendrocyte survival is critical for their terminal differentiation, the ability of DR6 antagonists to promote oligodendrocyte survival, differentiation and myelination was evaluated. To address this issue, DR6-DN (deletion of death domain) was used to block DR6 function in oligodendrocytes. As shown in the FIG. 7A, cells infected with DR6 DN exhibited 5-fold higher levels of MBP+ cells and higher MBP and MOG protein determined by Western blot analysis using mouse anti-MBP antibody (SMI 94 and SMI 99, 1:4000, Convance), mouse anti-MOG antibody (1:500) and rabbit anti-beta actin antibody (1:2000, Sigma) (FIG. 7A). Cell infection by lentivirus was confirmed by Western blot detection of the GFP protein co-expression marker (FIG. 7A). In addition, a CNPase assay (FIG. 7B) was performed to measure the level of CNPase, a marker for both immature and mature oligodendrocytes, in an ELISA format. As shown in FIG. 7B, the culture of DR6 FL infected oligodendrocytes expressed decreased CNPase activity compared to the control. In contrast, blocking the DR6 signaling pathways with DR6 DN increased CNPase activity (FIG. 7B). These data support the notion that endogenous DR6 negatively regulates oligodendrocyte survival and differentiation.
Example 8
DR6-Induced Neuronal Death is Reversed by DR6-Fc
Cortical neurons were infected with full-length DR6 lentivirus as described in Example 5. However, these cells were incubated in media that was supplemented with increasing amounts of recombinant soluble DR6. Recombinant soluble DR6 was produced by fusing amino acids 1-349 of DR6 to an Fc sequence. The nucleotide sequence of SEQ ID NO: 158 was used in these experiments. Nucleotides 1-1047 of SEQ ID NO: 158 encode DR6 amino acids, and nucleotides 1051-1731 of SEQ ID NO: 158 encode Fc amino acids. Nucleotides 1048-1050 of SEQ ID NO: 158 were inserted due to cloning procedures.
The sequence of the soluble DR6 polypeptide is provided as SEQ ID NO: 159. Amino acids 1-349 of SEQ ID NO: 159 are DR6 amino acids. Amino acids 351-576 of SEQ ID NO:159 are Fc amino acids, and amino acid 350 is an amino acid that was inserted due to cloning procedures.
The soluble DR6 coding sequence was inserted into a lenti viral vector and then used to produce and purify recombinant soluble DR6 from 293 cells. To directly monitor the survival effects of DR6-Fc on FL-DR6 expressing neurons, time-lapse images were obtained. In the presence of DR6-Fc, FL-DR6 failed to induce cortical neuron death (FIG. 8A-B).
The effect of soluble DR6 on DRG neurons was also examined. DRG neurons were first dissected out from adult Sprague Dawley rats (Charles River) and incubated in 0.25% Trypsin/EDTA (Invitrogen) at 37° C. for 30 minutes. An equal volume of DMEM (Invitrogen) containing 20% fetal bovine serum (Invitrogen) was then added to the digestion mixture to stop the reaction. Cell pellets were collected after spinning down at 1,000 rpm at room temperature for 5 minutes and were mechanically dissociated by gently passing through a plastic pipette until no large fragments were visible. About 1×105 DRG neurons were spotted in the center of each well of 4 well chamber-slides (LabTek) that were coated with 100 μg/ml Poly-D-lysine (Sigma). Cells were allowed to attach in growth medium (Neurobasal medium containing B27 supplement (Invitrogen) and 100 ng/ml nerve growth factor (BD Biosciences)) at 37° C. in humidified air with 5% CO2. The next morning, fresh growth medium was replaced, and cells were treated with 20 μM of fluorodeoxyuridine for 3 days to remove proliferating glial cells. Cultures were then maintained in growth medium at 37° C. in humidified air with 5% CO2 with fresh medium change every 3-4 days. After 7 days of culture, DRGs were treated with either control Fc or soluble DR6-Fc for 3 days. DRGs were fixed with 4% paraformaldehyde, and then stained with mouse anti-neuronal class III β-tubulin (Covance, MMS-435P, 1:500). Soluble DR6-Fc increased the total number of neurons bearing processes. In addition, the number of neurons with large and complex processes increased when treated with soluble DR6-Fc (FIG. 9A-B).
In another experiment, cortical neurons were incubated in media containing 0, 1, 3, 10 or 30 μg/ml soluble DR6 protein. After 48 hours, cell lysates were collected and levels of activated caspase-3 protein were measured using rabbit anti-cleaved caspase-3 antibody (91:1000; Cell Signaling). As shown in FIG. 10A, activated caspase-3 was not detectable in uninfected cells, but cells infected with full-length DR6 lentivirus showed high levels of activated caspase-3. However, levels of activated caspase-3 decreased when infected cells were incubated in media containing soluble DR6 (FIG. 10 A-B). Cell lysates were also probed with anti-GFP and anti-β-actin antibodies to control for efficacy of infection and quantity and quality of cell lysates, respectively. These data support the notion that blocking DR6 expression promotes neuronal survival and axon integrity.
Example 9
The Effect of DR6-Fc on Cell Death in Animal Model of Alzheimer's Disease
The effect of DR6-Fc in vivo can be studied using a mouse model of Alzheimer's disease, for example, APPswe/PS-1ΔE9 mice (Park et al., J. Neurosci 26:1386-1395 (2006)) from Jackson laboratories (Bar Harbor, Me.) (Stock #04462).
Mice are divided into several treatment groups. The first groups serves as a normal control. Each additional group is treated with DR6-Fc, for example, by intracranial injection or by systemic administration. The amount administered varies for each treatment group. For example, a group can receive 1, 10, 25, 50, 75, 100, 200, 300, 400 or 500 μg/kg per day. Administration can be a one time administration or can occur repeatedly for a specified period of time. Administration can occur before the onset of Alzheimer's symptoms, such that a delay or lack of development of symptoms is indicative of successful prevention and/or treatment. Alternatively, administration can begin after the onset of symptoms (i.e. at 7 months of age), such that a decrease or lack of increase in symptoms is indicative of successful treatment.
Efficacy of treatment can be evaluated symptomatically in live mice, for example by comparison of treated and untreated mice in a water mice. Efficacy can also be evaluated by molecular, biochemical and histological analysis of tissues, such as brain tissue, from sacrificed mice. For example, the number of apoptotic cells, e.g. cortical neurons, in a predetermined size and region of the brain can be compared in treated and untreated mice. The number of apoptotic cells can be determined using any known method in the art including for example, the TUNEL (TdT-mediated dUTP Nick-End Labeling) assay or anti-PARP (poly(ADP-ribose) polymerase) staining. In addition, the total number of surviving cortical neurons in a predetermined size and region of the brain can be compared in treated and untreated mice.
Example 10
The Effect of DR6-Fc on Cell Death in Animal Model of ALS
The effect of DR6-Fc in vivo can also be studied using an animal model of Amyotrophic Lateral Sclerosis (ALS), for example, mice, rats, flies, or worms expressing a mutant superoxide dismutase (SOD1).
For example, mice expressing mutant SOD1 (G37R) are treated with DR6-Fc by any mode of administration such as parenteral administration, subcutaneous administration etc. The amounts and times of administration can be varied. Administration can occur before the onset of ALS symptoms (i.e. at 7 to 9 months of age), such that a delay or lack of development of symptoms is indicative of successful prevention and/or treatment. In addition, administration can begin after the onset of symptoms, such that a decrease or lack of increase in symptoms is indicative of successful treatment.
Efficacy of treatment can be evaluated symptomatically, for example by comparison of muscle strength or longevity of treated and untreated mice. Efficacy can also be evaluated by molecular, biochemical and histological analysis of tissues, such as sections of motor neurons, from sacrificed mice. For example, the number of apoptotic cells, e.g. motor neurons, from a predetermined location, for example, along the spinal cord, can be compared in treated and untreated mice. The number of apoptotic cells can be determined using any known method in the art including for example, the TUNEL (TdT-mediated dUTP Nick-End Labeling) assay or anti-PARP (poly(ADP-ribose) polymerase) staining. In addition, the total number of surviving motor neurons in a predetermined size and region of the spinal cord can be compared in treated and untreated mice.
Example 11
DR6 RNAi Promotes Neuron Survival
Neocortical neurons were removed from embryonic 18 rats, and three million cells were transfected with either 200 nM DR6 siRNAs or scramble control siRNAs using Rat Neuron Nucleofector Kit (Amaxa Inc.). The DR6 siRNAs were a mixture of 4 siRNAs obtained from Dharmacon. The sequences of the 4 siRNAs were: AGAAACGGCUCCUUUAUUA (SEQ ID NO:160), GGAAGGACAUCUAUCAGUU (SEQ ID NO:161), GGCCGAUGAUUGAGAGAUU (SEQ ID NO:162), GCAGUUGGAAACAGACAAA (SEQ ID NO:163). The sequence of the control siRNA was: GGUGACAUGAUCGACAGCCAU (SEQ ID NO:164).
Transfected cells were plated in one 96-well plate and cultured for 6 days. At day 7, half of the culture media (100 μl) was removed and replaced with 100 μl fresh Neurobasal media containing different concentrations of glutamate, Aβ42 or TNFα. Triplicate cultures were set up for each treatment condition. Cultures were treated for 24 hours. 100 μl of supernatant was removed from each well, and LDH assays were performed using a cytotoxicity kit (LDH Cytotoxicity Detection Kit, Clontech Laboratories, Inc.) according to the manufacturer's instruction. As shown in FIG. 11, knocking down DR6 using RNAi promotes neocortical neuronal survival. Moreover, reducing DR6 expression attenuates Aβ42, glutamate, and TNFα-induced neuronal cytotoxicity. The data also suggests that blocking DR6 expression using RNAi promotes neuronal survival and prevents neuronal death.
Example 12
Blocking DR6 Signaling Pathway by siRNA Promotes Oligodendrocyte Differentiation
The effect of DR6 antagonism by DR6 RNAi on oligodendrocyte survival, differentiation and myelination was also evaluated. In these experiments, A2B5 cells were transfected with DR6 RNAi or control RNAi. The cells were harvested and half of the lysis was used for RT-PCR and analyzed by 15% agarose gel. The rest of the cell lysis was used for MBP and MOG western. As shown in FIGS. 12A-B, cells exposed to DR6 RNAi exhibited 2 fold higher levels of MBP+ cells, and higher MBP and MOG protein levels were shown by Western blot analysis (FIG. 12B). Cell infection by lentivirus was confirmed by Western blot detection of the GFP protein co-expression marker. These data further support the notion that endogenous DR6 negatively regulates oligodendrocyte survival and differentiation.
Example 13
Generation of Phage-Display-Derived Fab Antibodies
Recombinant human DR6 ectodomain was used to screen a human naïve phagemid Fab library containing 3.5×1010 unique clones (Nat Biotechnol. 2005 March; 23(3):344-8.) Biotinylated AP-DR6 protein was captured on steptavidin-coated magnetic beads prior to incubation with the phage library. Selections were performed as described previously, with depletion on a AP-p75 to eliminate AP specific binders (Nat Biotechnol. 2005 March; 23(3):344-8). After 3 rounds of panning, the 479 bp gene III stump was removed by MluI digestion, and the vector was religated for soluble Fab expression in TG1 cells. ELISA analysis of 2496 clones yielded 212 positive clones, containing 49 unique sequences. Unique clones were purified and binding was reconfirmed at a single concentration to recombinant human DR6 ectodomain by ELISA as well as by FACS on 293E cells transiently transfected with full-length human DR6. Twenty-four unique clones were selected from this analysis for further characterization. The 24 Fabs were tested at multiple concentrations by ELISA on human DR6-Fc to confirm specificity for the DR6 ectodomain versus the AP-DR6 fusion protein, as well as by FACS on full-length human DR6-293E cells, full-length rat DR6-293E cells, and untransfected 293E cells to check for species cross-reactivity. Based on specificity and cross-reactivity data, ten Fabs were selected.
Example 14
Cloning of Murine Anti-Human DR6 Monoclonal Antibody Variable Domains
Total cellular RNA from murine hybridoma cells was prepared using a Qiagen RNeasy mini kit following the manufacturer's recommended protocol. cDNAs encoding the variable regions of the heavy and light chains were cloned by RT-PCR from total cellular RNA, using random hexamers for priming of first strand cDNA. For PCR amplification of the murine immunoglobulin variable domains with intact signal sequences, a cocktail of degenerate forward primers hybridizing to multiple murine immunoglobulin gene family signal sequences and a single back primer specific for the 5′ end of the murine constant domain were used. The PCR products were gel-purified and subcloned into Invitrogen's pCR2.1TOPO vector using their TOPO cloning kit following the manufacturer's recommended protocol. Inserts from multiple independent subclones were sequenced to establish a consensus sequence. Deduced mature immunoglobulin N-termini were identical to those determined by Edman degradation of the purified immunoglobulins from the hybridomas. Assignment to specific subgroups is based upon BLAST analysis using consensus immunoglobulin variable domain sequences from the Kabat database. CDRs are designated using the Kabat definitions.
Shown below as SEQ ID NO:107 is the 1P1D6.3 mature heavy chain variable domain protein sequence, with CDRs (Kabat definitions) underlined:
1
QVQLQQSGTE LARPGASVKL SCKASGYTFT DYYLNWMKQG TGQGLEWIGE
51
IYPGGDHTYY NEKFKGKATL TADKSSNTAF MQLSSLTSED SAVYFCTRGV
101
IKWGQGTLVT VSL
This is a murine subgroup II(A) heavy chain. The DNA sequence of the 1P1D6.3 heavy chain variable domain (from pYL466) is provided as SEQ ID NO:106.
Shown below as SEQ ID NO:112 is the 1P1D6.3 mature light chain variable domain protein sequence, with CDRs underlined:
1
DILMTQSPPS MSVSLGDTVS ITCHASQGIS SNIGWLQQKP GKSFKGLIYH
51
GSTLEDGVPS RFSGSGSGAE FSLTISSLES EDFADYYCVQ YAQFPYTFGG
101
GTKLEIK
This is a murine subgroup V kappa light chain. The DNA sequence of the mature light chain variable domain (from pYL469) is provided as SEQ ID NO:111.
Shown below as SEQ ID NO:117 is the mature 1P2F2.1 heavy chain variable domain protein sequence, with CDRs underlined:
1
QVQLQQSGPE VARPGASVKL SCKASGYTFT DYYLNWVKQR TGQGLEWIGE
51
IYPGNNHTYY NEKFKGKATL TADNSSSTAY LQFSSLTSED SAVYFCTRGV
101
IKWGQGTLVT VSV
This is a murine subgroup II(A) heavy chain. Note the potential N-linked glycosylation site in CDR2 indicated as double underlined above. The DNA sequence of the 1P2F2.1 heavy chain variable domain (from pYL467) is provided as SEQ ID NO:116.
The heavy chains of 1P1D6.3 and 1P2F2.1 are related, sharing 89.4% identity at the protein level, with identical CDR1 and CDR3 sequences. IgBLAST analyses suggest that they were derived from the same recombinational event. Shown below is the alignment of the heavy chains of 1P1D6.3 (top) and 1P2F2.1 (bottom):
Shown below as SEQ ID NO:122 is the 1P2F2.1 mature light chain variable domain protein sequence, with CDRs underlined:
1
DILMTQSPSS MSVSLGDTVS ITCHASQGIR NSIGWLQQKP GKSFKGLIYH
51
ATTLEDGVPS RFTGSGSGAD FSLTISSLES EDFADYYCVQ YAQFPYTFGG
101
GTKLEIK
This is a murine subgroup V kappa light chain. The DNA sequence of the mature light chain variable domain (from pYL470) is provided as SEQ ID NO:121.
The light chains of 1P1D6.3 and 1P2F2.1 are related, sharing 92.5% identity at the protein level, with identical CDR3 sequences. IgBLAST analyses suggest that they were derived from the same recombinational event. Shown below is the alignment of the light chains of 1P1D6.3 (top) and 1P2F2.1 (bottom):
Shown below as SEQ ID NO:127 is the mature 1P5D10.2 heavy chain variable domain protein sequence, with CDRs underlined:
1
EVQLVESGGG LVKPGGSLKL SCAASGFTFS DYYMYWVRQT PEKRLEWVAT
51
ISDGGLYTYY QDSVKGRFTI SRDNAKNNLY LQMSSLKSED TAMYYCARED
101
DYDGDFYTMD YWGQGTSVTV SS
This is a murine subgroup III(D) heavy chain. The DNA sequence of the 1P5D10.2 heavy chain variable domain (from pYL468) is provided as SEQ ID NO:126.
Shown below as SEQ ID NO:132 is the 1P5D10.2 mature light chain variable domain protein sequence, with CDRs underlined:
1
QIVLTQSPAI MSASPGEKVT ITCSASSSVS YMHWFQQKPG TSPKLWIYST
51
SNLASGVPAR FSGSGSGTSY SLTISRMEAE DAATYYCQQR SSYPLTFGAG
101
TKLELK
This is a murine subgroup VI kappa light chain. The DNA sequence of the mature 1P5D10.2 light chain variable domain (from pYL471) is provided as SEQ ID NO:131.
Example 15
Anti-DR6 Antibodies Bind to Rat, Mouse and Human DR6
Six million HEK293 cells were transfected with 10 ug of plasmid DNA, which encoded full length human, rat, or mouse DR6. Three days after transfection, approximately 50,000 cells in 200 μL of PBS, 1% BSA, 0.1% NaN3 (FACS buffer) were analyzed. Cells were pelleted and resuspended in 150 μL of serial dilutions of anti-DR6 antibodies in FACS buffer. Samples were incubated for 1 hour on ice with occasional agitation and then washed three times. Bound DR6 antibody was visualized with PE-labeled goat F(ab)2 anti-human Fab (for Dyax Fabs) or anti-mouse IgG specific antibody (for monoclonal antibodies) (Jackson Labs). The results, shown in FIG. 13, demonstrate that 5D10 and 1E6 antibodies each bind to human, rat, and mouse DR6. The results of binding assays using 5D10 and M53E04 are shown in FIG. 30.
Example 16
Blocking DR6 by Anti-DR6 Antibodies Promotes Oligodendrocyte Differentiation and Inhibit Apoptosis
To further validate the role of DR6 function in oligodendrocyte survival, anti-DR6 antibodies were used to block DR6 function in the oligodendrocytes culture. As shown in FIGS. 14A-B, anti-DR6 antibody treatment reduced caspase 3+ cells about 3 fold (FIG. 14A) and increased MBP+ cells by 10 fold (FIG. 14B). These results were confirmed by the Western blot analysis (FIG. 14C) where the anti-DR6 antibody reduced the caspase 3 production about 3 fold. In contrast, a 10 fold increase of MBP protein production was seen in the cell cultures treated with the anti-DR6 antibody (FIG. 14C). The results of an oligodendrocyte-DRG co-culture assay using DR6 antibodies M53E04 and 5D10 are shown in FIG. 31 and compared to the results obtained using anti-LINGO-1 antibody Li81 as a positive control.
Example 17
Blocking DR6 by Anti-DR6 Antibodies Promotes Oligodendrocyte/DRG Myelination in Co-Culture
To test the hypothesis that blocking DR6 promotes myelination, co-cultures of rat primary oligodendrocytes and DRG neurons were used to ascertain the effects of anti-DR6 antibody on myelination. Such co-cultures normally exhibit low basal levels of myelination which was profoundly enhanced by the addition of anti-DR6 antibody. The cocultures were infected with lentivirus at a multiplicity of infection of two per cell (2 MOI). Treatment with anti-DR6 antibody for 10 days resulted in robust axonal myelination as evident by the presence of MBP+ myelinated axons, 10 fold higher than control Ig treated cells culture. Western blot analysis using mouse anti-MBP antibody (SMI 94 and SMI 99, 1:4000, Convance), mouse anti-MOG antibody (1:500), and rabbit anti-beta actin antibody (1:2000, Sigma) demonstrates that anti-DR6 antibody promotes myelination in a dosage dependent manner, the higher concentration of anti-DR6 antibody added to the co-culture, the higher level of MBP and MOG protein was produce (FIG. 15). These studies demonstrate that blocking DR6 function promotes oligodendrocyte survival, differentiation and myelination.
Example 18
Blocking DR6 by Anti-DR6 Antibodies Promote Remylination in Rat Brain Slice Culture
The brain slice culture system provides a powerful in vitro model for the analyses of the pathology of demyelination and mechanisms of remyelination. Three day treatment of P17 brain slices with the bioactive lipid, lysophosphatidylcholine (LPC), results in a rapid and near-complete demyelination as visualized by the absence of black gold staining for myelination (FIG. 16A). Exposure to anti-DR6 antibody for 4 days after LPC removal resulted in 15-fold more black gold staining, whereas the control antibody treatment had no effect (FIGS. 16A-B). We next determined whether remyelination could be achieved in vivo in the adult LPC induced demyelination model. LPC was injected into the dorsal columns of the 9 week old young adult (250 gms) rat spinal cords at day 0, followed by anti-DR6 antibody administration 3 days later. The extent of the LPC induced lesion and remyelination was next determined by black gold staining. Myelinated white matter appears dark red in the black gold stained sections and demyelinated lesions appear as pale red or white. Sections from control antibody-treated animals (n=3) showed large lesions with extensive areas of demyelination, whereas substantially smaller lesions were apparent in the anti-DR6 treated group (n=3) 7 days after LPC injection. The black gold staining pattern of anti-DR6-treated and control lesions differed. In anti-DR6 treated lesions, lace-like structures were present throughout the lesion indicative of remyelination. Both brain slice culture and in vivo lysolecithin studies demonstrated that blocking DR6 function by anti-DR6 antibody promote remyelination.
Example 19
Anti-DR6 Antibodies Promote Functional Recovery in Rat EAE Model
Adult 9-week old Brown Norway rats (150 g) were anaesthetized with isoflourine, followed by an injection at the base of the tail with 200 μl of cocktail solution containing: 100 μl of CFA (complete Freund's adjuvant from Chondrex Inc.) and 100 μl of 100 μg recombinant rat MOG corresponding to the N-terminal sequence of rat MOG (amino acids 1-125) in DPBS (MP Biomedicals, LCC). Animals developed signs of EAE 10-15 days after injections. After MOG induction, each animal was assessed by a behavioral test based on motor functions. EAE scores were used as a surrogate clinical metric for demyelination. Rats were scored for clinical signs of EAE daily. The signs were scored as follows: grade 0.5, distal paresis of the tail; grade 1, complete tail paralysis; grade 1.5, paresis of the tail and mild hind leg paresis; grade 2.0, unilateral severe hind leg paresis; grade 2.5, bilateral severe hind limb paresis; grade 3.0, complete bilateral hind limb paralysis; grade 3.5, complete bilateral hind limb paralysis and paresis of one front limb; and grade 4, complete paralysis (tetraplegia), moribund state, or death. After 15 day immunization, rats were randomly assigned to one of two groups. One group of rats (n=10) was injected with isotype control antibodies, and another group (n=10) was injected with DR6 antibodies. The rats were injected twice a week at 6 mg/kg for a total of 5 treatments. EAE scores were measured daily, and the statistical significance was assessed using an unpaired t-test (two-tailed). As shown in FIG. 17A, the EAE scores in DR6 antibody treated rats were significantly lower compared with control animals. For electrophysiological recordings, after 40 day immunization, motor potentials (MEPs) were induced by magnetic cortical stimulation (Magstim) and recorded from the gatrocnemius muscles (Cadwell). The onset of the first, usually negative, deflection was taken as the cortical MEP latency. As shown in FIG. 17B, the DR6-treated rats showed faster nerve conduction velocity.
Example 20
Lymphocyte Number is not Affected in EAE Rats Treated by Anti-DR6 Antibodies
EAE is a complex model for demyelination, as it involves both immune and neurological components. To determine whether treatment with anti-DR6 antibodies affects lymphocytes, after 40 day MOG immunization, peripheral blood was drawn from the facial vein of EAE rats treated with anti-DR6 antibody or control antibody. The total whole blood cells and subset numbers were measured using Hemavet. As shown in FIG. 18, the total lymphocyte numbers and the percentage of lymphocytes in total white blood cells did not show significant differences between the control and anti-DR6 antibody treated animals.
Example 21
Anti-DR6 Antibodies Inhibit T-Cell Infiltration into Spinal Cord in EAE Rats
To determine whether anti-DR6 antibodies affect T cell infiltration into the CNS, EAE rats were euthanized with CO2, then perfused with 0.1 M phosphate buffer after six weeks of MOG immunizations. The lumbar region of spinal cords were dissected out and fixed in 4% paraformaldehyde overnight at 4° C. followed by incubation in 25% sucrose in 0.1 M PBS. 15 μm transverse and longitude frozen sections (Leica microtome) were cut and standard fluoresce immunohistochemistry was performed using anti-CD4 antibodies (BD Pharmingen). Images were taken under Leica fluorescence microscopy and analyzed using Openlab. As shown in FIG. 19, systemic administration of anti-DR6 antibodies significantly reduced the infiltration of T cells into the spinal cord of the EAE rats, suggesting that the decreased T cell infiltration at least partially explains the decreased severity of the EAE symptoms (lower EAE scores) in anti-DR6 treated animals.
Example 22
TNFalpha Promotes Neuron Death Through NFκb
TNF has been reported to induce DR6 expression in tumor cells to activate apoptosis by activating the NF-κB, Caspase-3 pathway and down regulating Iκb protein level. In order to determine if TNFalpha induces DR6 expression in cortical neurons, neurons were exposed to 24 hour treatment with TNFalpha, and immunohistochemistry staining was performed. The results demonstrated that DR6 expression was increased significantly. TNFalpha induced neuronal death and was well correlated with DR6 positive cells (FIG. 20A-C). The study was confirmed by Western blot (FIG. 21A). Treatment with TNFalpha induced a 2 fold increase in DR6 expression after a 24 hour treatment, which also correlated with a 10 fold NF-κB increase and a 2 fold Iκb protein down regulation (FIGS. 21A-D). DR6 RNAi transfected neurons showed a 2 fold reduction of DR6 and NFκb level (FIGS. 22B and C). In contrast, DR6 RNAi transected neurons showed increased (2 fold) Iκb protein expression (FIGS. 22A-D). (Control and DR6 siRNAs used were as described in Example 11). These data suggest that DR6 up-regulation correlated with NFκb expression and inversely correlated with Iκb expression.
Example 23
DR6 Antagonists Promote Schwann Cell Myelination of DRG Axons
In order to determine if DR6 antagonists affect schwann cells, the effect of anti-DR6 antibodies on schwann cell and DR6 neuron co-cultures was examined. In these experiments, DRG neurons from E16 rats were plated (50,000/well) in 4 well slides with Neurobasal medium plus B27 and NGF. The cultures were treated by FDUR for 4-6 days to removed the dividing cells. After day 7, the DRG cells were treated with Neurobasal medium plus B27 and NGF (100 ng/ml) for an additional 7-10 days. Then, purified schwann cells (50,000/well) were added to the DRG neurons (50,000/well) in Neurobasal medium with B27 and 100 ng/ml NGF. The medium was changed weekly. The co-cultures were harvested and assayed by IHC or Western blot for MBP protein after 10 days. IHC staining and Western blots (FIG. 23) both showed increased levels of MBP protein in cultures treated with anti-DR6 antibodies compared to cultures treated with a control antibody. These data indicate that DR6 antagonists promote schwann cell myelination of neurons.
Example 24
DR6 is Upregulated in Apoptotic Cortical Neurons
In order to examine DR6 expression in neocortical neurons, neurons were first separated from E18 Sprague Dawley rats (Charles River). Briefly, cerebral cortices from E18 rat embryos were dissected out, minced, and incubated in 0.25% Trypsin/EDTA (Invitrogen) at 37° C. for 10 minutes. The cells were then triturated after adding 20 ug/ml DNase I (Sigma) and 10% fetal bovine serum (Invitrogen) to stop the reaction. Cell pellets were collected and then mechanically dissociated by gently passing through a plastic pipette until no large fragments were visible. Cells were plated in 8-well slide chambers (NUNC) that were pre-coated with 100 ug/ml Poly-D-Lysine (Sigma) at 4×104 cells per well. Cells were maintained in Neurobasal medium containing B27 supplement (Invitrogen) at 37° C. in humidified air with 5% CO2 with fresh medium changed every 3-4 days. Cells were cultured for 3 weeks and fixed with 4% paraformaldehyde in PBS for 30 minutes. After three washes with PBS, cells were penetrated with PBS containing 1% Triton X-100 (PBST, Sigma) for 30 minutes followed by incubation in blocking solution (PBS containing 0.1% Triton X-100 and 10% normal goat serum (NGS)) for 30 minutes at room temperature. For primary labeling, cells were then incubated in blocking medium containing rabbit anti-DR6 (Santa Cruz, sc-13106, 1:200) and mouse anti-neuronal class III β-tubulin (Covance, MMS-435P, 1:500) at 4° C. overnight. After three PBST rinses, cells were incubated in 5% NGS-PBS containing Alexa 594 anti-rabbit IgG (1:500) and Alexa 488 anti-mouse IgG (1:500) secondary antibodies at room temperature for 1 hour in the dark. After three PBST washes, cells were mounted with antifade with DAPI reagents (Invitrogen) and observed under fluorescence microscope. Apoptosis, shown by nuclear condensation, was visible in neocortical neurons after 3 weeks in culture. Levels of DR6 were compared in apoptotic and non-apoptotic neurons. The expression level of DR6 was up-regulated in the apoptotic neurons compared to non-apoptotic neurons. These results suggest than an increase in DR6 expression can contribute to aged neuron apoptosis.
Example 25
DR6 Antagonists Promote Axon Integrity
In order to examine the effect of DR6 antagonists on the axonal integrity of neocortical neurons, neurons were separated from E18 Sprague Dawley rats (Charles River) and cultured in neurobasal medium containing B27 supplement (Invitrogen) at 37° C. in humidified air with 5% CO2 as described in Example 24. After 7 days of culture, cells were treated with β-amyloid (aggregated Aβ-42) at a concentration of (50 μg/ml) and either 10 μg/ml soluble DR6-Fc or 10 μg/ml soluble control antibody. After 48 hours, neurons were fixed with 4% paraformaldehyde and then stained with mouse anti-neuronal class III β-tubulin (Covance, MMS-435P, 1:500) as described in Example 24. Treatment with β-amyloid induced neuronal cell death and axon degeneration as compared to untreated controls. However, soluble DR6-Fc treatment significantly attenuated β-amyloid induced axonal degeneration and neuronal cell death. Soluble DR6-Fc treatment also led to increased survival of neurons and decreased axon beading, which is typical of axon degeneration morphology. These results suggest that DR6 antagonists diminish the negative effects of β-amyloid on neocortical neurons.
Example 26
DR6 and AKT Expression are Inversely Correlated
Phosphorylated AKT (phospho-AKT) is a well-known survival signal. Therefore, the relationship of DR6 and phospho-AKT levels was examined in neocortical neurons. In these experiments, neocortical neurons were separated as described above. After 3, 7, 14, and 20 days of culture, neocortical neurons were lysed in 80 μl lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton X-100, and 10% glycerol) for 30 minutes at 4° C. After centrifugation at 14,000×g for 15 minutes, the supernatants were boiled in Laemmli sample buffer, subjected to 4-20% SDS-PAGE, and analyzed by Western blotting with rabbit anti-phospho-AKT antibody (1:500, cell signaling), rabbit anti-AKT antibody (1:1000, cell signaling), goat anti-DR6 antibody (1:1000, Santa Cruz), and rabbit anti-beta actin antibody (1:2000, Sigma). Primary antibodies were visualized using anti-rabbit IgG-HRP (1:5000) and anti-goat IgG-HRP (1:5000, Bio-Rad) accordingly. The expression levels of DR6 were high in day 7 cultures and low in day 20 cultures (FIG. 24). In contrast, the levels of phospho-AKT were low at day 7 and high at day 20 (FIG. 24). The inverse correlation of DR6 and phospho-AKT levels suggests that DR6 could induce cell death through the AKT signaling pathway.
Example 27
DR6 and p75 Form a Complex
In order to determine if p75 could interact with DR6, recombinant cell lines expressing (i) DR6, p75, and TrkA, (ii) DR6 and TrkA, (iii) p75 and TrkA, and (iv) DR6 and p75 were created. In these experiments, human TrkA was expressed with a Flag tag fused to the N-terminal of the mature TrkA protein (amino acids 34-796). Rat p75 was expressed with a His tag fused to the N-terminus of the mature p75 protein (amino acids 30-431), and human DR6 was expressed with a Myc tag fused to the N-terminus of the mature DR6 protein (amino acids 32-655). DR6 was immunoprecipitated from cell lysates using a Myc antibody, and the levels of DR6 and p75 in the samples was assessed. Western blots (FIG. 25 A) show that p75 co-immunoprecipitated with DR6 in the presence or absence of TrkA.
In addition, cells containing a vector encoding DR6 or a negative control vector (pV90) were assayed for binding to p75. Alkaline phosphatase-p75 protein was added to the cells and cell surface binding was measured. The results are shown in FIG. 25B.
Similar results were obtained using samples obtained from human fetal spinal cords obtained from BioChain®. Immunoprepitation experiments demonstrated that p75 co-immunoprecipiates with DR6 (FIG. 25 C).
These data demonstrate that DR6 and p75 form a complex both in cell culture assays and in human samples.
Example 28
The Expression Patterns of DR6 and p75 Overlap
The expression levels of DR6 and p75 mRNA were obtained from the publically available database mouse.brain-map.org, made available by the Allen Institute for Brain Science. Both DR6 and p75 were highly expressed in various regions of the brain, and expression levels were well correlated (FIG. 26). These results suggest that DR6 and p75 co-localize, and therefore can interact and function together in vivo.
Example 29
DR6 Antibodies can Block Interaction of DR6 with p75
In order to determine if DR6 antibodies block the interaction of DR6 with p75, recombinant DR6 was immunoprecipitated from cells using either the 2A9 anti-DR6 antibody or the 5D10 anti-DR6 antibody. Western analysis demonstrated that while both anti-DR6 antibodies were able to pull down DR6 protein, p75 only co-immunoprecipitated with DR6 when the 2A9 antibody was used (FIG. 27A). Thus, the 5D10 DR6 antibody disrupted interaction of DR6 with p75.
The ability of 5D10 to disrupt the interaction of DR6 and p75 was also confirmed by a functional assay. In this assay cells CHO cells containing either a control vector or a vector encoding p75 were incubated with alkaline phosphatase-DR6 and assayed for binding. High levels of cell surface binding were observed when the p75-expressing cells were incubated with DR6. However, the addition of 5D10 prevented DR6 from binding to p75-expressing cells (FIG. 27B). These results indicate that the DR6 antibody 5D10 can disrupt binding of DR6 to p75 and promote cell survival.
Example 30
The TNFR-Cys Repeats 3 and 4 of DR6 Bind to Antibodies that Disrupt the DR6-p75 Interaction
In order to identify domains of DR6 that bind to DR6 antibodies that disrupt the DR6-p75 interaction, cells expressing DR6 deletion constructs were created. The deletion constructs were tagged with Myc. The constructs tested included deletions of amino acids 168-189 of SEQ ID NO:2 (#123); amino acids 134-168 of SEQ ID NO:2 (#124); amino acids 109-131 of SEQ ID NO:2 (#134); amino acids 49-108 of SEQ ID NO:2 (#234); amino acids 133-189 of SEQ ID NO:2 (#12); amino acids 49-131 of SEQ ID NO:2 (#34); and amino acids 49-108 and 168-189 of SEQ ID NO:2 (#23).
FACS analysis was used to demonstrate that each of the deletion constructs expressed in cells (FIG. 28A). Then, samples obtained from the recombinant cells were immunoprecipitated with an anti-Myc antibody or an anti-DR6 antibody. The immunoprecipitates were assayed for p75 protein by Western, and the results showed that antibody 5D10 binds to the Cys3 and Cys4 domain of DR6 (amino acids 133-189) (FIG. 28B). Antibody 2A9 binds to the Cyst domain of DR6 (amino acids 49-131) (FIG. 28B). The binding of a panel of DR6 antibodies to DR6 deletion constructs was also assayed (FIG. 28C). The results demonstrated that 5D10 and 4A4 bind to the Cys3 and Cys4 domains of DR6, and 2A9, 1D6, and 2F2 bind to the Cys1 domain of DR6.
Example 31
The TNFR-Cys Repeats 3 and 4 of DR6 Bind to p75
In order to identify the domains of DR6 that bind to p75, cells expressing the DR6 deletion constructs described in Example 30 were assayed for binding to p75. Recombinant cells were incubated with p75, and then cell lysates were immunoprecipitated using an anti-Myc antibody. Levels of p75 were assessed by Western, and the results demonstrated that deletion of the Cys3 and Cys4 domain of DR6 (amino acids 133-189) resulted in a decreased ability of DR6 to interact with p75.
The present invention is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the invention, and any compositions or methods which are functionally equivalent are within the scope of this invention. Indeed, 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 accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections can set forth one or more but not all exemplary embodiments of the present invention as contemplated by the inventor(s), and thus, are not intended to limit the present invention and the appended claims in any way.
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13936084
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biogen idec ma inc.
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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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424/130.1
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Apr 1st, 2022 05:10PM
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Apr 1st, 2022 05:10PM
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Biogen
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:biib
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Biogen
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Apr 20th, 2010 12:00AM
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Jun 28th, 2004 12:00AM
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https://www.uspto.gov?id=US07700097-20100420
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Purification and preferential synthesis of binding molecules
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The instant invention describes methods of separating or preferentially synthesizing dimers which are linked via at least one interchain disulfide linkage from dimers which are not linked via at least one interchain disulfide linkage from a mixture comprising the two types of polypeptide dimers. These forms can be separated from each other using hydrophobic interaction chromatography. In addition, the invention pertains to connecting peptides that result in the preferential biosynthesis of dimers that are linked via at least one interchain disulfide linkage or that are not linked via at least one interchain disulfide linkage. The invention also pertains to compositions in which a majority of the dimers are linked via at least one interchain disulfide linkage or are not linked via at least one interchain disulfide linkage. The invention still further pertains to novel binding molecules, e.g., comprising connecting peptides of the invention.
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7700097
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1. A composition comprising polypeptide dimers having at least two antigen binding sites and at least two polypeptide chains, wherein said at least two polypeptide chains each comprise a complete Ig heavy chain, and a chimeric hinge,
wherein said chimeric hinge connects the CH1 and the CH2 domain of the Ig heavy chain, and
wherein greater than 50% of the polypeptide dimers comprise polypeptide chains that are linked via at least one interchain disulfide linkage, and
wherein amino acids at positions 226-242 (Kabat numbering) comprise: (i) the human IgG1 upper hinge region sequence EPKSCDKTHT (SEQ ID NO:2) or the human IgG4 upper hinge region sequence ESKYGPP (SEQ ID NO:50) at Kabat hinge positions 226-238; (ii) a cysteine residue (C) at Kabat hinge position 239; (iii) a proline residue (P) at Kabat hinge position 240; (444 iv) a proline (P) or serine (5) residue at Kabat hinge position 241; (v) the human IgG3 middle hinge sequence CPEPKSCDTPPPCPR (SEQ ID NO:37) at Kabat hinge positions 241EE-241SS; and (vi) a cysteine residue (C) at Kabat hinge position 242.
2. The composition of claim 1, wherein greater than 90% of the polypeptide dimers are linked via at least one interchain disulfide linkage.
3. The composition of claim 1, wherein the chimeric hinge further comprises a gly-ser linker.
4. The composition of claim 3, wherein the gly-ser linker consists of the amino acid sequence GGGSSGGGSG (SEQ ID NO:1).
5. The composition of claim 1, wherein the polypeptide dimers are linked via two or more interchain disulfide linkages.
6. The composition of claim 1, wherein the heavy chain is from an antibody of an isotype selected from the group consisting of: IgG1, IgG2, IgG3, and IgG4.
7. The composition of claim 1, wherein the polypeptide dimers comprise four polypeptide chains and wherein two of the polypeptide chains comprise at least one heavy chain and a chimeric hinge.
8. The composition of claim 1, wherein at least one antigen binding site binds to an antigen expressed by tumor cells.
9. The composition of claim 1 wherein the chimeric hinge is the amino acid sequence of SEQ ID NO:8.
10. The composition of claim 1, wherein the chimeric hinge is the amino acid sequence of SEQ ID NO:9.
11. The composition of claim 1, wherein the chimeric hinge is the amino acid sequence of SEQ ID NO:53.
12. A connecting peptide comprising the amino acid sequence SEQ ID NO:8.
13. A connecting peptide comprising the amino acid sequence SEQ ID NO:9.
14. A connecting peptide comprising the amino acid sequence SEQ ID NO:10.
15. A connecting peptide comprising the amino acid sequence SEQ ID NO:11.
16. A connecting peptide comprising the amino acid sequence SEQ ID NO:12.
17. A connecting peptide comprising the amino acid sequence SEQ ID NO:13.
18. A connecting peptide comprising the amino acid sequence SEQ ID NO:14.
19. A connecting peptide comprising the amino acid sequence SEQ ID NO:15.
20. A connecting peptide comprising the amino acid sequence SEQ ID NO:53.
21. A connecting peptide consisting of the amino acid sequence SEQ ID NO:7.
22. A connecting peptide consisting of amino acid sequence SEQ ID NO:8.
23. A connecting peptide consisting of amino acid sequence SEQ ID NO:9.
24. A connecting peptide consisting of amino acid sequence SEQ ID NO:10.
25. A connecting peptide consisting of amino acid sequence SEQ ID NO:11.
26. A connecting peptide consisting of amino acid sequence SEQ ID NO:12.
27. A connecting peptide consisting of amino acid sequence SEQ ID NO:13.
28. A connecting peptide consisting of amino acid sequence SEQ ID NO:14.
29. A connecting peptide consisting of amino acid sequence SEQ ID NO:15.
30. A connecting peptide consisting of amino acid sequence SEQ ID NO:53.
31. A composition comprising polypeptide dimers having at least two antigen binding sites and at least two polypeptide chains, wherein said at least two polypeptide chains each comprise (i) an Ig heavy chain lacking modified to lacking a CH2 domain, and (ii) a synthetic connecting peptide comprising a chimeric hinge,
wherein said connecting peptide is selected from the group consisting of SEQ ID NO: 7-15 and 53 and wherein said connecting peptide connects said heavy chain to at least one of said binding sites, and
wherein greater than 50% of the polypeptide dimers comprise polypeptide chains that are linked via at least one interchain disulfide linkage.
32. The composition of claim 6, wherein the heavy chain is from an IgG3 molecule.
33. The composition of claim 6, wherein the heavy chain is from an IgG4 molecule.
34. The composition of claim 31, wherein greater than 90% of the polypeptide dimers are linked via at least one interchain disulfide linkage.
35. The composition of claim 31, wherein the heavy chain is from an antibody of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.
36. The composition of claim 31, wherein the polypeptide dimers comprise four polypeptide chains and wherein two of the polypeptide chains comprise at least one heavy chain and a synthetic connecting peptide.
37. The composition of claim 31, wherein at least one antigen binding site binds to an antigen expressed by tumor cells.
38. The composition of claim 31, wherein the heavy chain is from an antibody of an IgG3 isotype.
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38
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RELATED APPLICATIONS
This application claims priority to U.S. Ser. No. 60/483,877, titled “Purification and Preferential Synthesis of Polypeptides,” filed on Jun. 27, 2003 and to U.S. Ser. No. 60/508,810, titled “Purification and Preferential Synthesis of Antigen Binding Polypeptides,” filed Oct. 3, 2003. This application also claims priority to U.S. Ser. No. 60/515,351, titled “Modified Antibody Molecules Comprising Connecting Peptides,” Oct. 28, 2003 and to U.S. Ser. No. 60/516,030, titled “Modified Antibody Molecules Comprising Connecting Peptides,” filed Oct. 30, 2003. This application is also related to U.S. Ser. No. 10/880,028, titled “Modified Binding Molecules Comprising Connecting Peptides” filed on Jun. 28, 2004. The contents of these applications are incorporated in their entirety by this reference.
BACKGROUND OF THE INVENTION
Antibodies are dimeric molecules; each monomer making up the dimer comprises one light and one heavy chain. Solutions of antibody molecules exist in two forms associated with hinge heterogeneity. Using SDS-PAGE analysis of purified Mab MAb, typically the two forms are observed as two protein bands, a major band (MW approximately 150-160 kDa) and a minor band (MW approximately 75-80 kDa). This latter form is typically observed after SDS-PAGE analysis of purified IgG4 preparations, but can be identified at much lower frequencies in all IgG isotypes, including purified, recombinant MAbs (Angal et al. 1993. Mol. Immunol. 30:105; Norderhaug et al. 1990. Eur. J. Immunol. 21:2370). The larger molecular weight isoform, referred to as Form A, contains covalent interchain disulfide bonds at positions corresponding to 239 and 242, Kabat numbering system (positions 226 and 229, EU numbering system) (Kabat, E, Wu, T T, Perry, H M, Gottesman, K S, Foeller, C: Sequences of Proteins of Immunological Interest. Bethesda, US Department of Health and Human Services, NIH, 1991). The second isoform, Form B, is thought to contain no covalent linkages between the two heavy chains and an intrachain disulfide bond between the two neighboring cysteine residues as evidenced by the 75-80 kDa seen in non-reducing SDS-PAGE electrophoresis. The two heavy chains of Form B are presumably held together by strong non-covalent (e.g., ionic) interactions associated with the CH3 domain region of the molecule. These mixtures of A and B forms are not present in solutions of MAb fragments that contain an intact hinge, but lack a CH3 domain, such as, for example, F(ab)2 fragments. Typically, genetically engineered or enzymatically digested F(ab)2 MAb preparations lack the B-form, since the molecule lacks the necessary domains for maintaining non-covalent interactions (e.g., hydrogen bonding). However, they are present in MAb preparations that do contain a CH3 domain, such as IgG4, the CH2 domain deleted MAb fragments (e.g., as described in 02/060955 A2) and minibodies (see, e.g., Hu et al. 1996. Cancer Research 56:3055) as well as in IgG4 molecule.
The application of protein engineering techniques to therapeutic antibody design has also produced a number of antibody formats that have been shown to have altered, and in some cases, improved pharmacodynamic, biodistribution, and activity profiles. Some altered antibody molecules have been made in which the number of cysteine residues in the hinge region is reduced to one to facilitate assembly of antibody molecules as it is only necessary to form a single disulfide bond. This also provides a specific target for attaching the hinge region either to another hinge region or to an effector or reporter molecule (U.S. Pat. No. 5,677,425). The number of cysteine residues in the antibody hinge has also been increased (U.S. Pat. No. 5,677,425). Other mutated antibodies have been constructed in which the IgG1 hinge region and the CH2 domain have been replaced with the human IgG3 hinge region. (WO 97/11370). These molecules contain 11 sulfhydryl groups for substitution of multiple haptens via thiol groups.
CH2 domain deleted antibodies have a molecular mass of approximately 120 kDa and have been shown to penetrate tumors significantly better than full length IgG. Minibodies, which also have deletion of the CH2 domain, have similar characteristics. These domain deleted molecules accumulate at tumor sites more efficiently than other MAb fragments, such as F(ab)′2s, but without the unfavorable pharmacodynamic profiles seen with intact IgG antibody. CH2 domain deleted antibodies consist of a VLCL light chain and a VH1 heavy chain domain and a portion of the hinge region (e.g., the upper and middle hinge) genetically fused (either directly or through a synthetic peptide spacer) to a CH3 domain. As an example, the biosynthesis of recombinant CH2 domain deleted ddCC49, a domain deleted antibody that recognizes the tumor associated TAG72 antigen expressed on a variety of human carcinomas, produces the A and B forms in approximately 50:50 distribution in cell cultures. Cells engineered to express alternative forms of CH2 domain deleted antibodies, for example, tetravalent CH2 domain deleted antibodies, minibodies, or tetravalent minibodies also express a mixture consisting of A and B forms and/or monomeric half-mer molecules.
Form A and Form B are extremely difficult to separate even after MAb purification, since they are composed of identical amino acids and, therefore, have identical molecular weight and similar physical and chemical properties. They cannot be separated by standard gel filtration, affinity chromatography, or ion exchange chromatography typically used to purify antibody molecules, including recombinant MAb proteins. Current manufacturing processes discard at least 50% of the total antibody produced, having a negative impact on overall yield. Moreover, the presence of the two isoforms increases efforts required for downstream processing. Thus, a method of separating forms A and B or of increasing biosynthesis of one or the other form of antibody would be of great benefit.
SUMMARY OF THE INVENTION
The invention is based, at least in part, on the finding that in a composition comprising a mixture of dimeric polypeptide molecules comprising different isoforms (molecules comprising two heavy chain portions in which a fraction of the molecules comprise two heavy chain portions that are linked via at least one interchain disulfide linkage (Form A) and a portion of the molecules comprise two heavy chain portions that are not linked via at least one interchain disulfide linkage (Form B)) one form or the other can be preferentially obtained, e.g., by separation using-hydrophobic interaction chromatography or by inclusion of synthetic connecting peptides which result in the preferential biosynthesis of either Form A or Form B. The connecting peptides of the invention can be included in any dimeric molecule that tends to form both Form A and Form B, e.g., antibody molecules, domain deleted antibody molecules (e.g., lacking all or part of a CH2 domain), minibodies, diabodies, fusion proteins, etc. In a preferred embodiment, the formation of Form A is enhanced. Form A polypeptide dimers show enhanced stability in vitro and enhanced biodistribution in vivo.
Accordingly, in one aspect, the invention pertains to a composition comprising polypeptide dimers having at least two binding sites and at least two polypeptide chains, wherein said at least two polypeptide chains comprise at least one heavy chain portion and a synthetic connecting peptide, wherein greater than 50% of the polypeptide dimers are linked via at least one interchain disulfide linkage.
In one embodiment, greater than 90% of the polypeptide dimers are linked via at least one interchain disulfide linkage.
In one embodiment, the polypeptide chains comprise a CH3 domain genetically fused to a VL, VH or CH1 via the connecting peptide.
In one embodiment, the polypeptide chains lack all or part of a CH2 domain.
In one embodiment, the polypeptide dimers are linked via two or more interchain disulfide linkages.
In one embodiment, the heavy chain portion is derived from an antibody of an isotype selected from the group consisting of: IgG1, IgG2, IgG3, and IgG4.
In one embodiment, the heavy chain portion comprises an amino acid sequence derived from a hinge region selected from the group consisting of: a γ1 hinge, a γ2 hinge, a γ3 hinge, and a γ4 hinge.
In one embodiment, wherein the heavy chain portion comprises a chimeric hinge.
In one embodiment, the synthetic connecting peptide comprises at least a portion of an IgG1 hinge domain, at least a portion of an IgG3 hinge domain.
In one embodiment, the binding sites are individually selected from the group consisting of: an antigen binding site, a ligand binding portion of a receptor, and a receptor binding portion of a ligand.
In one embodiment, the polypeptide dimer comprises four polypeptide chains.
In another embodiment, two of the polypeptide chains comprise at least one heavy chain portion and a synthetic connecting peptide.
In another aspect, the invention pertains to a composition comprising polypeptide dimers having at least two binding sites and at least two polypeptide chains, wherein said at least two polypeptide chains comprise at least one heavy chain portion and a synthetic connecting peptide, wherein greater than about 50% of the polypeptide dimers are linked via at least one interchain disulfide linkage and wherein the connecting peptide comprises a proline residue at position 243 of the Kabat numbering system.
In one embodiment, greater than 90% of the polypeptide dimers are linked via at least one interchain disulfide linkage.
In one embodiment, the synthetic connecting peptide further comprises a cysteine residue at position 239 or 242 of the Kabat numbering system.
In one embodiment, at least one of the polypeptide chains comprises a CH3 domain linked to a VL, VH or CH1 domain via the connecting peptide.
In one embodiment, the synthetic connecting peptide further comprises an alanine residue at position 244 and a proline residue at position 245 of the Kabat numbering system.
In another aspect, the invention pertains to a method of treating a subject that would benefit from treatment with a binding molecule comprising administering a composition of the invention to the subject such that treatment occurs.
In one embodiment, the subject is suffering from cancer.
In one embodiment, the subject is suffering from lymphoma.
In one embodiment, the subject is suffering from an autoimmune disease or disorder.
In one embodiment, the subject is suffering from an inflammatory disease or disorder.
In one embodiment, the binding sites are individually selected from the group consisting of: an antigen binding site, a ligand binding portion of a receptor, and a receptor binding portion of a ligand.
In another aspect, the invention pertains to a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain of the invention.
In another aspect, the invention pertains to a host cell comprising the nucleic acid molecule of claim 23.
In one embodiment, the polypeptide dimers comprise four polypeptide chains and wherein two of the polypeptide chains comprise at least one heavy chain portion and a synthetic connecting peptide.
In one aspect, the invention pertains to a connecting peptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs: 8-15 and 53.
In one aspect, the invention pertains to a connecting peptide consisting of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 8-15 and 53.
In one aspect, the invention pertains to a nucleic acid molecule encoding a polypeptide chain, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: SEQ ID NOs:16, 20, 21, 38, 42, 46 and 47.
In one aspect, the invention pertains to a nucleic acid molecule encoding a polypeptide chain, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: SEQ ID NOs:24 and 25.
In one aspect, the invention pertains to a domain deleted antibody molecule comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs: 18, 22, 23, 40, and 44.
In one aspect, the invention pertains to a domain deleted antibody molecule comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs: 26 and 27.
In one aspect, the invention pertains to an antibody molecule comprising the amino acid sequence shown in SEQ ID NO: 31.
In one aspect, the invention pertains to an antibody molecule comprising the amino acid sequence shown in SEQ ID NO: 35.
In one aspect, the invention pertains to a method for separating a first and a second polypeptide dimer wherein the first polypeptide dimer comprises at least two binding sites and at least two polypeptide chains, wherein said at least two polypeptide chains comprises a heavy chain portion and wherein the first polypeptide dimer is linked via at least one disulfide linkage and wherein the second polypeptide dimer comprises at least two binding sites and at least two polypeptide chains, wherein said at least two polypeptide chains comprises a heavy chain portion and wherein the second polypeptide dimer is not linked via at least one disulfide linkage, comprising the steps of:
i) contacting a mixture comprising the first and the second polypeptide dimers with a medium that separates the polypeptide dimers based on hydrophobic interaction under conditions that allow the second polypeptide dimmer to bind to the medium; and
ii) collecting the first polypeptide dimer
to thereby separate the first and the collecting step comprises contacting the medium with a solution having a conductivity of approximately 120 mS/cm at approximately neutral pH.
In one embodiment the conductivity is approximately 116 mS/cm.
In one embodiment the method further comprises contacting the medium with a solution having a conductivity of approximately 140 mS/cm and at approximately neutral pH prior to step ii) such that both the first and second polypeptide dimers bind to the medium.
In one embodiment, the polypeptide chains comprise a CH3 domain linked to a VL, VH or CH1 domain via a synthetic connecting peptide.
In one embodiment, the heavy chain portions of the first and second polypeptide dimers are derived from an antibody of an isotype selected from the group consisting of: IgG1, IgG2, IgG3, and IgG4.
In one embodiment, the heavy chain portions comprises an amino acid sequence derived from a hinge region selected from the group consisting of: a γ1 hinge, a γ2 hinge a γ3 hinge, and a γ4 hinge.
In one embodiment, the binding sites are individually selected from the group consisting of: an antigen binding site, a ligand binding portion of a receptor, and a receptor binding portion of a ligand.
In one embodiment, at least one binding site of a polypeptide dimer comprises at least one CDR from an antibody selected from the group consisting of: 2B8, Lym 1, Lym 2, LL2, Her2, B1, MB1, BH3, B4, B72.3, CC49, and 5E10.
In one embodiment, at least one binding site of a polypeptide dimer comprises the receptor binding portion of a ligand.
In one embodiment, at least one binding site of a polypeptide dimer comprises the ligand binding portion of a receptor.
In another aspect, the invention pertains to a method of treating a subject that would benefit from treatment with a binding molecule comprising administering a composition of the invention to the subject such that treatment occurs.
In one embodiment, the polypeptide dimers comprise four polypeptide chains and wherein two of the polypeptide chains comprise at least one heavy chain portion.
In another aspect, the invention pertains to a method for separating a first properly folded antibody molecule from a second improperly folded antibody molecule, wherein each of the first and second antibody molecules comprises four polypeptide chains, wherein at least two of the chains comprise at least one heavy chain portion, and at least two of the chains comprise at least one light chain portion, the method comprising the steps of:
i) contacting a mixture comprising the first and second antibody molecules with a medium that separates antibody molecules based on hydrophobic interaction;
ii) contacting the medium with a solution having a conductivity of approximately 120 mS/cm and at an approximately neutral pH, such that the first antibody molecule is not bound to the medium and the second antibody molecule is bound to the medium, thereby separating the first and second antibody molecules.
In one embodiment, the invention pertains to a method for increasing the amount of a first polypeptide dimer relative to the amount of a second polypeptide dimer produced by a cell, wherein the first and second polypeptide dimers comprise at least two binding sites and at least two polypeptide chains, said at least two polypeptide chains comprising a heavy chain portion, wherein the first dimer is linked via at least one disulfide linkage and wherein the second dimer is not linked via at least one disulfide linkage, the method comprising the step of engineering said polypeptide chain to include a synthetic connecting peptide, such that the amount of the first polypeptide dimer produced by the cell is increased relative to the amount of the second polypeptide dimer.
In one embodiment, the invention pertains to a composition comprising a first polypeptide prepared by the methods of the invention.
In one embodiment, the polypeptide dimers comprise four polypeptide chains and wherein two of the polypeptide chains comprise at least one heavy chain portion and a synthetic connecting peptide.
In one embodiment, the invention pertains to a polypeptide comprising a synthetic connecting peptide which comprises the amino acid sequence of SEQ ID NO: 37, wherein the polypeptide is not a naturally occurring IgG3 molecule.
In one embodiment, the molecule is an IgG4 molecule.
In one embodiment, the polypeptide binds to VLA-4.
In one aspect, the invention pertains to a method for increasing the amount of dimers linked via at least one disulfide linkage in a population of IgG4 molecules produced by a cell, comprising the step of causing the cell to express an IgG4 molecule comprising a synthetic connecting peptide, such that the amount dimers linked via at least one disulfide linkage in a population of IgG4 molecules is increased.
In one aspect, the invention pertains to composition comprising polypeptide dimers having at least two binding sites and at least two polypeptide chains, wherein said at least two polypeptide chains comprise at least one heavy chain portion and lacks all or part of a CH2 domain, wherein greater than 50% of the polypeptide dimers are linked via at least one interchain disulfide linkage.
In one aspect, the invention pertains to composition of claim 1 or 31.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows Form A which appears as a 120 kDa dimer and Form B which appears as a 60 kDa monomer in domain deleted antibodies.
FIGS. 2A and B show densitometer plots of non-reducing SDS-PAGE gels for ddCC49 (with a CH2 spacer) and ddCC49 (with a Gly/Ser spacer), respectively.
FIGS. 3A-D shows that the G1/G3/Pro243Ala244Pro245+[Gly/Ser] (FIG. 3D) and G1/G3/Pro243+[Gly/Ser] (FIG. 3C) hinges resulted in the production of primarily Form A CH2 domain-deleted huCC49 antibody with low or essentially no detectable Form B. In contrast, CH2 domain-deleted huCC49 Cys242Ser:Pro243 (FIG. 3A) and CH2 domain-deleted huCC49 Cys242Ser:Pro243Ala244Pro245 (FIG. 3B) resulted in a moderate to significant preference of the Form B isoform, respectively.
FIG. 4A (SEQ ID NO: 16) shows the single-stranded DNA sequence of heavy chain CH2 domain-deleted huCC49 Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide. FIG. 4B (SEQ ID NO: 17) shows the single-stranded DNA sequence of light chain CH2 domain-deleted huCC49.
FIG. 5 shows SDS-PAGE gel of huCC49 G1/G3/PAP purified using only a Protein G column, showing that the antibody eluted essentially as a single peak at ≧96% purity without further HIC purification.
FIG. 6 shows SDS-PAGE gel of huCC49 V2 G1/G3/PAP purified using only a Protein G column, showing that the antibody eluted essentially as a single peak at ≧96% purity without further HIC purification.
FIGS. 7A-C shows peptide mapping HPLC-MS of hinge-engineered HuCC49□CH2 antibodies. FIG. 7A is Endo Lys-C non-reduced. FIG. 7B is Endo Lys-C reduced. FIG. 7C is Tryptic. G1/G3: PAP fragment H146-208 shows a shift in retention time relative to PAP and HuCC49□CH2 resulting from a Leu→Met mutation due to a PCR artifact. This was corrected in all subsequent constructs.
FIG. 8A (SEQ ID NO: 18) shows the amino acid sequence of heavy chain CH2 domain-deleted huCC49 Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide. FIG. 8B (SEQ ID NO: 19) shows the amino acid sequence of light chain CH2 domain-deleted huCC49.
FIG. 9 (SEQ ID NO: 20) shows the single-stranded DNA sequence of heavy chain CH2 domain-deleted huCC49 G1/G3/Pro243+[Gly/Ser] hinge connecting peptide.
FIG. 10 (SEQ ID NO: 21) shows the single-stranded DNA sequence of heavy chain CH2 domain-deleted huCC49 containing G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
FIG. 11 (SEQ ID NO: 22) shows the amino acid sequence of heavy chain CH2 domain-deleted huCC49 G1/G3/Pro243+[Gly/Ser] hinge connecting peptide.
FIG. 12 (SEQ ID NO: 23) shows the amino acid sequence of heavy chain CH2 domain-deleted huCC49 containing G1/G3/Pro243Ala244Pro245+[GlySer] hinge connecting peptide.
FIG. 13A (SEQ ID NO: 24) shows the DNA sequence of heavy chain CH2 domain-deleted huCC49 V2 containing G1/G3/Pro243Ala244Pro245+[GlySer] hinge connecting peptide. FIG. 13B (SEQ ID NO: 25) shows the DNA sequence of light chain CH2 domain-deleted huCC49 V2.
FIG. 14A (SEQ ID NO: 26) shows the amino acid sequence of heavy chain CH2 domain-deleted huCC49 V2 containing G1/G3/Pro243Ala244Pro245+[GlySer] hinge connecting peptide. FIG. 14B (SEQ ID NO: 27) shows the amino acid sequence of light chain CH2 domain-deleted huCC49 V2.
FIG. 15 shows a chromatogram of the HIC purification of CH2 domain-deleted huCC49 A and B forms.
FIG. 16 shows purified CH2 domain-deleted huCC49 Forms A and B shown in lanes 3 and 4, respectively.
FIG. 17A-B shows the separation of the two CH2 domain-deleted huCC49 isoforms at a preparative scale. The first two peaks comprise the isocratic elution of Form A, the second peak shows the eluted Form B, while the third peak contains impurities, which are removed from the stationary phase during cleaning.
FIG. 18 shows that the CH2 domain-deleted huCC49 fractions eluted isocratically using 0.73 M Ammonium Sulfate/20 mM Sodium Phosphate, pH 4.0 (lanes 6 to 8) contain predominantly Form A (purity>90%).
FIG. 19A shows an alignment of the light chain variable regions of murine CC49 (SEQ ID NO:28), LEN (SEQ ID NO:29) humanized CC49 V1 (version 1) (SEQ ID NO:30), and humanized CC49 V2 (version 2; which comprises one amino acid substitution in the light chain as compared to humanized CC49 V1, see underlined amino acid) (SEQ ID NO:31) and FIG. 19B shows alignment of the heavy chain variable regions of murine CC49 (SEQ ID NO:32), 21/28′ CL (SEQ ID NO:33), humanized CC49 V1 (SEQ ID NO:34), and humanized CC49 V2 (which comprises two amino acid substitutions in the heavy chain as compared to humanized CC49, see underlined amino acids (SEQ ID NO:35).
FIG. 20 shows the results of a competitive binding assay to bovine submaxillary mucine, a source of the TAG-72 antigen, by time-resolved fluorometric immunoassay.
FIG. 21 shows the tumor to organ ratio comparing 90Y/CHx-DTPA conjugated domain deleted CC49 constructs in LS174T tumor xenografts.
FIG. 22 shows the biodistribution of Human CC49 V2 comprising the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide.
FIG. 23 shows the biodistribution of HuCC49 [gly/ser].
FIG. 24 shows that domain deleted CC49 G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO: 9) appears more stable toward glutathione (GSH) reduction, as is parent CC49, than domain deleted CC49 with a Gly-Ser hinge linker.
FIG. 25A (SEQ ID NO: 46) shows the DNA sequence of the heavy chain CH2 domain-deleted anti-CD20 IgG1 C2B8 containing the G1/G3/Pro243Ala244Pro245+[GlySer] hinge connecting peptide. FIG. 25B (SEQ ID NO: 47) shows the corresponding amino acid sequence of heavy chain C2B8 containing the G1/G3/Pro243Ala244Pro245+[GlySer] hinge connecting peptide.
FIG. 26A (SEQ ID NO: 48) shows the DNA sequence of the light chain CH2 domain-deleted anti-CD20 IgG1 C2B8. FIG. 26B (SEQ ID NO: 49) shows the corresponding amino acid sequence of light chain C2B8.
FIG. 27 shows that the inclusion of the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide (SEQ ID NO:9) into an antibody of different specificity (here the CH2 domain-deleted C2B8 antibody) results in the production of essentially all form A antibody with little or no detectable Form B (lane 3).
FIG. 28 A (SEQ ID NO: 38) shows the DNA sequence of heavy chain CH2 domain-deleted the anti-CD23 Ab 5E8 containing the G1/G3/Pro243Ala244Pro245+[GlySer] hinge connecting peptide. FIG. 28B (SEQ ID NO: 39) shows the DNA sequence of light chain CH2 domain-deleted the anti-CD23 5E8.
FIG. 29A (SEQ ID NO: 40) shows the amino acid sequence of heavy chain CH2 domain-deleted anti-CD23 antibody 5E8 containing G1/G3/Pro243Ala244Pro245+[GlySer] hinge connecting peptide. FIG. 29B (SEQ ID NO: 41) shows the amino acid sequence of light chain CH2 domain-deleted anti-CD23 antibody 5E8.
FIG. 30 shows that the inclusion of the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide (SEQ ID NO:9) into an antibody of different specificity (here the CH2 domain-deleted 5E8 antibody) results in the production of essentially all form A antibody with little or no detectable Form B (see lane 2).
FIG. 31 shows the results of a competitive binding assay to soluble CD23 by time-resolved fluorometric immunoassay.
FIG. 32 A (SEQ ID NO: 42) shows the DNA sequence of heavy chain CH2 domain-deleted chB3F6 containing G1/G3/Pro243Ala244Pro245+[GlySer] hinge connecting peptide. FIG. 32B (SEQ ID NO: 43) shows the DNA sequence of light chain CH2 domain-deleted chB3F6.
FIG. 33A (SEQ ID NO: 44) shows the amino acid sequence of heavy chain CH2 domain-deleted chB3F6 containing G1/G3/Pro243Ala244Pro245+[GlySer] hinge connecting peptide. FIG. 33B (SEQ ID NO: 45) shows the amino acid sequence of light chain CH2 domain-deleted chB3F6.
FIG. 34 shows that the inclusion of the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide (SEQ ID NO:9) into an antibody of different specificity (here the CH2 domain-deleted chB3F6 antibody) results in the production of essentially all form A antibody with little or no detectable Form B (see lane 4).
FIG. 35 shows that chimeric B3F6 (chB3F6) and chimeric B3F6 domain deleted antibody comprising a connecting peptide (B3F6ΔCH2 G1/G3/Pro243Ala244Pro245) compete equally for binding to GEO tumor cells.
DETAILED DESCRIPTION OF THE INVENTION
Human immunoglobulins (Igs), including monoclonal antibodies (MAbs), can exist in two forms that are associated with hinge heterogeneity. In native solutions, both of these forms are present as dimeric proteins (each monomer comprising one heavy chain and one light chain). One immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond (Form A) and one comprises a form in which the dimers are not linked via interchain disulfide bonds (Form B). Form B also forms a stable dimer under native conditions, but can be identified under denaturing, non-reducing conditions, in which the heavy chains dissociate yielding a 75-80 kDa molecule. These forms have been extremely difficult to separate, even after MAb affinity purification.
The frequency of appearance of the B form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the MAb molecule. In fact, a single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the B form (Angal et al. 1993. Molecular Immunology 30:105) to levels typically observed using a human IgG1 hinge. However, applying this same amino acid substitution to MAb fragments in which the CH3 domain was retained did not eliminate Form B from preparations. Typically, all recombinant CH2 domain deleted antibodies produced in cell cultures often result in hinge heterogeneity which is not corrected via similar molecular mutations in the hinge.
The instant invention advances the state of the art by providing methods of, e.g., separating a first dimeric polypeptide having from a second dimeric polypeptide wherein the first and second polypeptides comprise at least two polypeptide chains and at least two of the polypeptide chains comprise at least one heavy chain portion. In one embodiment, the polypeptides of the invention lack all or part of a CH2 domain. The monomers are linked via at least one interchain disulfide linkage (referred to herein as “Form A”) and the monomers of the second polypeptide are not linked via at least one interchain disulfide linkage (referred to herein as “Form B”). These forms can be separated from each other using hydrophobic interaction chromatography. In addition, the invention pertains to polypeptides that comprise connecting peptides. The inclusion of certain connecting peptides results in the preferential biosynthesis of polypeptide dimers that are linked via at least one interchain disulfide linkage or that are not linked via at least one interchain disulfide linkage.
Before further description of the invention, for convenience, certain terms are described below:
I. DEFINITIONS
The polypeptides of the invention are binding molecules, i.e., polypeptide molecules or the nucleic acid molecules that encode them, that comprise at least one binding domain which comprises a binding site that specifically binds to a target molecule (such as an antigen or binding partner). For example, in one embodiment, a binding molecule of the invention comprises an immunoglobulin antigen binding site or the portion of a receptor molecule responsible for ligand binding or the portion of a ligand molecule that is responsible for receptor binding. The binding molecules of the invention are polypeptides or the nucleic acid molecules which encode them.
In one embodiment, the binding molecules comprise at least two binding sites. In one embodiment, the binding molecules comprise two binding sites. In one embodiment, the binding molecules comprise three binding sites. In another embodiment, the binding molecules comprise four binding sites.
The polypeptides of the invention are multimers. For example, in one embodiment, the polypeptides of the invention are dimers. In one embodiment, the dimers of the invention are homodimers, comprising two identical monomeric subunits. In another embodiment, the dimers of the invention are heterodimers, comprising two non-identical monomeric subunits. The subunits of the dimer may comprise one or more polypeptide chains. For example, in one embodiment, the dimers comprise at least two polypeptide chains. In one embodiment, the dimers comprise two polypeptide chains. In another embodiment, the dimers comprise four polypeptide chains (e.g., as in the case of antibody molecules).
The polypeptides of the invention comprise at least one amino acid sequence derived from an immunoglobulin domain. A polypeptide or amino acid sequence “derived from” a designated protein refers to the origin of the polypeptide. Preferably, the polypeptide or amino acid sequence which is derived from a particular starting polypeptide or amino acid sequence has an amino acid sequence that is essentially identical to that of the starting sequence, or a portion thereof wherein the portion consists of at least 10-20 amino acids, preferably at least 20-30 amino acids, more preferably at least 30-50 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the starting sequence.
Preferred binding polypeptides comprise an amino acid sequence derived from a human amino acid sequence. However, binding polypeptides may comprise one or more amino acids from another mammalian species. For example, a primate heavy chain portion, hinge portion, or binding site may be included in the subject binding polypeptides and/or connecting polypeptides. Alternatively, one or more murine amino acids may be present in a binding polypeptide, e.g., in an antigen binding site of a binding molecule. Preferred binding molecules of the invention are not immunogenic.
It will also be understood by one of ordinary skill in the art that the binding molecules of the invention (e.g., the heavy chain or light chain portions or binding portions of the subject polypeptides) may be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule from which they were derived. For example, nucleotide or amino acid substitutions leading to conservative substitutions or changes at “non-essential” amino acid residues may be made.
An isolated nucleic acid molecule encoding a non-natural variant of a polypeptide derived from an immunoglobulin (e.g., an immunoglobulin heavy chain portion or light chain portion) can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of the immunoglobulin such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.
Alternatively, in another embodiment, mutations may be introduced randomly along all or part of the immunoglobulin coding sequence, such as by saturation mutagenesis, and the resultant mutants can be incorporated into polypeptides of the invention and screened for their ability to bind to the desired antigen.
As used herein, the term “heavy chain portion” includes amino acid sequences derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain portion comprises at least one of: a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. In one embodiment, a polypeptide of the invention comprises a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain. In another embodiment, a polypeptide of the invention comprises a polypeptide chain comprising a CH1 domain and a CH3 domain. In another embodiment, a polypeptide of the invention comprises a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain. In another embodiment, a polypeptide of the invention comprises a polypeptide chain comprising a CH3 domain. In one embodiment, a polypeptide of the invention lacks at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). In another embodiment, a polypeptide of the invention comprises a complete Ig heavy chain. As set forth above, it will be understood by one of ordinary skill in the art that these domains (e.g., the heavy chain portions) may be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule.
In one embodiment, at least two of the polypeptide chains of a binding molecule of the invention comprise at least one heavy chain portion derived from an antibody or immunoglobulin molecule. In one embodiment, at least two heavy chain portions of a polypeptide of the invention are present on different polypeptide chains and interact, e.g., via at least one disulfide linkage (Form A) or via non-covalent interactions (Form B) to form a dimeric polypeptide, each monomer of the dimer comprising at least one heavy chain portion.
In one embodiment, the heavy chain portions of one polypeptide chain of a dimer are identical to those on a second polypeptide chain of the dimer. In one embodiment, the monomers (or half-mers) of a dimer of the invention are identical to each other. In another embodiment, they are not identical. For example, each monomer may comprise a different target binding site.
In one embodiment, a dimer of the invention is held together by covalent interactions, e.g., disulfide bonds. In one embodiment, a dimer of the invention is held together by one or more disulfide bonds. In another embodiment, a dimer of the invention is held together by one or more, preferably two disulfide bonds. In another embodiment, a dimer of the invention is held together by one or more, preferably three disulfide bonds. In another embodiment, a dimer of the invention is held together by one or more, preferably four disulfide bonds. In another embodiment, a dimer of the invention is held together by one or more, preferably five disulfide bonds. In another embodiment a dimer of the invention is held together by one or more, preferably six disulfide bonds. In another embodiment, a dimer of the invention is held together by one or more, preferably seven disulfide bonds. In another embodiment, a dimer of the invention is held together by one or more, preferably eight disulfide bonds. In another embodiment, a dimer of the invention is held together by one or more, preferably nine disulfide bonds. In another embodiment, a dimer of the invention is held together by one or more, preferably ten disulfide bonds. In a further embodiment, a dimer of the invention is not held together by disulfide bonds, but is held together, e.g., by non-covalent interactions.
The heavy chain portions of a polypeptide may be derived from different immunoglobulin molecules. For example, a heavy chain portion of a polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, a heavy chain portion can comprise a hinge region derived, in part, from an IgG1 molecule and, in part, from an IgG3 molecule. In another example, a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgG1 molecule and, in part, from an IgG4 molecule. As used herein, the term “light chain portion” includes amino acid sequences derived from an immunoglobulin light chain. Preferably, the light chain portion comprises at least one of a VL or CL domain.
In one embodiment a polypeptide of the invention comprises an amino acid sequence or one or more moieties not derived from an Ig molecule. Exemplary modifications are described in more detail below. For example, in one embodiment, a polypeptide of the invention may comprise a flexible linker sequence. In another embodiment, a polypeptide may be modified to add a functional moiety (e.g., PEG, a drug, or a label).
In one embodiment, a binding polypeptide of the invention is a fusion protein. Fusion proteins are chimeric molecules which comprise a binding domain comprising at least one target binding site and at least one heavy chain portion. In one embodiment, a fusion protein further comprises a synthetic connecting peptide.
A “chimeric” protein comprises a first amino acid sequence linked to a second amino acid sequence with which it is not naturally linked in nature. The amino acid sequences may normally exist in separate proteins that are brought together in the fusion polypeptide or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. A chimeric protein may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship. Exemplary chimeric polypeptides include fusion proteins and the chimeric hinge connecting peptides of the invention.
The term “heterologous” as applied to a polynucleotide or a polypeptide, means that the polynucleotide or polypeptide is derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared. For instance, a heterologous polynucleotide or antigen may be derived from a different species origin, different cell type, or the same type of cell of distinct individuals.
The term “ligand binding domain” or “ligand binding portion” as used herein refers to any native receptor (e.g., cell surface receptor) or any region or derivative thereof retaining at least a qualitative ligand binding ability, and preferably the biological activity of a corresponding native receptor.
The term “receptor binding domain” or “receptor binding portion” as used herein refers to any native ligand or any region or derivative thereof retaining at least a qualitative receptor binding ability, and preferably the biological activity of a corresponding native ligand.
In one embodiment, a binding molecule of the invention is a fusion protein. A fusion proteins of the invention is a chimeric molecule that comprises a binding domain (which comprises at least one binding site) and a dimerization domain (which comprises at least one heavy chain portion). The heavy chain portion may be from any immunoglobulin, such as IgG1, IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD or IgM.
In another embodiment of the invention, a binding molecule is an “antibody-fusion protein chimera.” Such molecules comprise a molecule which combines at least one binding domain of an antibody with at least one fusion protein. Preferably, the interface between the two polypeptides is a CH3 domain of an immunoglobulin molecule.
In one embodiment, the binding molecules of the invention are “antibody” or “immunoglobulin” molecules, e.g., naturally occurring antibody or immunoglobulin molecules or genetically engineered antibody molecules that bind antigen in a manner similar to antibody molecules. As used herein, the term “immunoglobulin” includes a polypeptide having a combination of two heavy and two light chains whether or not it possesses any relevant specific immunoreactivity. “Antibodies” refers to such assemblies which have significant known specific immunoreactive activity to an antigen of interest (e.g. a tumor associated antigen). Antibodies and immunoglobulins comprise light and heavy chains, with or without an interchain covalent linkage between them. Basic immunoglobulin structures in vertebrate systems are relatively well understood.
As will be discussed in more detail below, the generic term “immunoglobulin” comprises five distinct classes of antibody that can be distinguished biochemically. All five classes of antibodies are clearly within the scope of the present invention, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, immunoglobulins comprise two identical light polypeptide chains of molecular weight approximately 23,000 Daltons, and two identical heavy chains of molecular weight 53,000-70,000. The four chains are joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region.
Both the light and heavy chains are divided into regions of structural and functional homology. The terms “constant” and “variable” are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino-terminus of the antibody. The N-terminus is a variable region and at the C-terminus is a constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.
Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class may be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (γ, μ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4). It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant invention.
As indicated above, the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH domain of an antibody combine to form the variable region that defines a three dimensional antigen binding site. This quaternary antibody structure forms the antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three complementary determining regions (CDRs) on each of the VH and VL chains.
As used herein, the term “binding site” or “binding domain” comprises a region of a polypeptide which is responsible for selectively binding to a target molecule of interest (e.g. an antigen, ligand, receptor, substrate or inhibitor) Exemplary binding domains include an antibody variable domain, a receptor binding domain of a ligand, a ligand binding domain of a receptor or an enzymatic domain.
In one embodiment, the binding molecules have at least one binding site specific for a molecule targeted for reduction or elimination, e.g., a cell surface antigen or a soluble antigen.
In preferred embodiments, the binding domain is an antigen binding site. An antigen binding site is formed by variable regions that vary from one polypeptide to another. The polypeptides of the invention comprise at least two antigen binding sites. As used herein, the term “antigen binding site” includes a site that specifically binds (immunoreacts with) an antigen (e.g., a cell surface or soluble antigen). The antigen binding site includes an immunoglobulin heavy chain and light chain variable region and the binding site formed by these variable regions determines the specificity of the antibody. In one embodiment, an antigen binding molecule of the invention comprises at least one heavy or light chain CDR of an antibody molecule (e.g., the sequence of which is known in the art or described herein). In another embodiment, an antigen binding molecule of the invention comprises at least two CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule of the invention comprises at least three CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule of the invention comprises at least four CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule of the invention comprises at least five CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule of the invention comprises at least six CDRs from one or more antibody molecules. Exemplary antibody molecules comprising at least one CDR that can be included in the subject antigen binding molecules are known in the art and exemplary molecules are described herein.
The polypeptides comprising two heavy chain portions disclosed herein may be linked to form two associated Ys so there will be four binding sites forming a “tetravalent” molecule (see e.g., WO02/096948A2)). In another embodiment, tetravalent minibodies or domain deleted antibodies can be made.
The term “specificity” includes the number of potential binding sites which specifically bind (e.g., immunoreact with) a given target. A polypeptide may be monospecific and contain one or more binding sites which specifically bind a target or a polypeptide may be multispecific and contain two or more binding sites which specifically bind the same or different targets.
In one embodiment, a binding molecule of the invention is a bispecific molecule (e.g., antibody, minibody, domain deleted antibody, or fusion protein having binding specificity for more than one molecule, e.g., more than one antigen or more than one epitope on the same antigen. In one embodiment, the bispecific molecules have at least one target binding site specific for a molecule targeted for reduction or elimination and a targeting molecule on a cell. In another embodiment, the bispecific molecules have at least one target binding site specific for a molecule targeted for reduction or elimination and at least one target binding site specific for a drug. In yet another embodiment, the bispecific molecules have at least one target binding site specific for a molecule targeted for reduction or elimination and at least one target binding site specific for a prodrug. In a preferred embodiment, the bispecific molecules are tetravalent antibodies that have two target binding sites specific for one target and two target binding sites specific for the second target. A tetravalent bispecific molecule may be bivalent for each specificity. Further description of bispecific molecules is provided below.
As used herein the term “valency” refers to the number of potential target binding sites in a polypeptide. Each target binding site specifically binds one target molecule or specific site on a target molecule. When a polypeptide comprises more than one target binding site, each target binding site may specifically bind the same or different molecules (e.g., may bind to different ligands or different antigens, or different epitopes on the same antigen).
In naturally occurring antibodies, the six CDRs present on each monomeric antibody are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding site as the antibody assumes its three dimensional configuration in an aqueous environment. The remainder of the heavy and light variable domains show less inter-molecular variability in amino acid sequence and are termed the framework regions. The framework regions largely adopt a β-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the β-sheet structure. Thus, these framework regions act to form a scaffold that provides for positioning the six CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen binding site formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to the immunoreactive antigen epitope. The position of CDRs can be readily identified by one of ordinary skill in the art.
As previously indicated, the subunit structures and three dimensional configuration of the constant regions of the various immunoglobulin classes are well known. As used herein, the term “VH domain” includes the amino terminal variable domain of an immunoglobulin heavy chain and the term “CH1 domain” includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.
As used herein the term “CH2 domain” includes the portion of a heavy chain molecule that extends, e.g., from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system; and Kabat E A et al. Sequences of Proteins of Immunological Interest. Bethesda, US Department of Health and Human Services, NIH. 1991). The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminal of the IgG molecule and comprises approximately 108 residues.
As used herein, the term “hinge region” includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al. J. Immunol. 1998 161:4083).
In one embodiment, a binding molecule of the invention comprises a connecting peptide. The connecting peptides of the invention are synthetic. As used herein the term “synthetic” with respect to polypeptides includes polypeptides which comprise an amino acid sequence that is not naturally occurring. For example, non-naturally occurring polypeptides which are modified forms of naturally occurring polypeptides (e.g., comprising a mutation such as an addition, substitution or deletion) or which comprise a first amino acid sequence (which may or may not be naturally occurring) that is linked in a linear sequence of amino acids to a second amino acid sequence (which may or may not be naturally occurring) to which it is not naturally linked in nature.
Connecting peptides of the invention connect two domains (e.g., a binding domain and a dimerization domain) of a binding molecule of the invention. For example, connecting peptides connect a heavy chain portion to a binding domain comprising a binding site. In one embodiment, a connecting peptide connects two heavy chain constant region domains, such as CH1 and CH2 domains; CH1 and CH3 domains; hinge and CH1 domains; hinge and CH3 domains; VH and hinge domains, or a CH3 domain and a non-immunoglobulin polypeptide) in a linear amino acid sequence of a polypeptide chain. Preferably, such connecting peptides provide flexibility to the polypeptide molecule and facilitate dimerization via disulfide bonding. In one embodiment, the connecting peptides of the invention are used to replace one or more heavy chain domains (e.g., at least a portion of a constant region domain (e.g., at least a portion of a CH2 domain) and/or at least a portion of the hinge region (e.g., at least a portion of the lower hinge region domain) in a domain deleted construct). For example, in one embodiment, a VH domain is fused to a CH3 domain via a connecting peptide (the C-terminus of the connecting peptide is attached to the N-terminus of the CH3 domain and the N-terminus of the connecting peptide is attached to the C-terminus of the VH domain). In another embodiment, a VL domain is fused to a CH3 domain via a connecting peptide (the C-terminus of the connecting peptide is attached to the N-terminus of the CH3 domain and the N-terminus of the connecting peptide is attached to the C-terminus of the VL domain. In another embodiment, a CH1 domain is fused to a CH3 domain via a connecting peptide (the C-terminus of the connecting peptide is attached to the N-terminus of the CH3 domain and the N-terminus of the connecting peptide is attached to the C-terminus of the CH1 domain).
In one embodiment, a synthetic connecting peptide comprises a portion of a constant region domain. For example, in one embodiment, a connecting peptide that replaces a CH2 domain can comprise a portion of the CH2 domain.
In one embodiment, a connecting peptide comprises or consists of a gly-ser linker. As used herein, the term “gly-ser linker” refers to a peptide that consists of glycine and serine residues An exemplary gly/ser linker comprises the amino acid sequence GGGSSGGGSG (SEQ ID NO:1). In one embodiment, a connecting peptide of the invention comprises at least a portion of an upper hinge region (e.g., derived from an IgG1, IgG3, or IgG4 molecule), at least a portion of a middle hinge region (e.g., derived from an IgG1, IgG3, or IgG4 molecule) and a series of gly/ser amino acid residues (e.g., a gly/ser linker such as GGGSSGGGSG (SEQ ID NO:1)). In one embodiment, the connecting peptide comprises a substitution of one or more amino acids as compared to naturally occurring IgG1 or IgG3 hinge regions. In another embodiment, a connecting peptide comprises an amino acid sequence such as described in WO 02/060955. Connecting peptides are described in more detail below.
As used herein the term “disulfide bond” includes the covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system).
It is known in the art that the constant region mediates several effector functions. For example, binding of the C1 component of complement to antibodies activates the complement system. Activation of complement is important in the opsonisation and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and may also be involved in autoimmune hypersensitivity. Further, antibodies bind to cells via the Fc region, with a Fc receptor site on the antibody Fc region binding to a Fc receptor (FcR) on a cell. There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors). Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production.
In one embodiment, the Fc portion may be mutated to decrease effector function using techniques known in the art. For example, the deletion or inactivation (through point mutations or other means) of a constant region domain may reduce Fc receptor binding of the circulating modified antibody thereby increasing tumor localization. In other cases it may be that constant region modifications consistent with the instant invention moderate compliment binding and thus reduce the serum half life and nonspecific association of a conjugated cytotoxin. Yet other modifications of the constant region may be used to modify disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility. More generally, those skilled in the art will realize that antibodies modified as described herein may exert a number of subtle effects that may or may not be readily appreciated. However the resulting physiological profile, bioavailability and other biochemical effects of the modifications, such as tumor localization, biodistribution and serum half-life, may easily be measured and quantified using well know immunological techniques without undue experimentation.
In one embodiment, modified forms of antibodies can be made from a whole precursor or parent antibody using techniques known in the art. Exemplary techniques are discussed in more detail below. In particularly preferred embodiments both the variable and constant regions of polypeptides of the invention are human. In one embodiment, fully human antibodies can be made using techniques that are known in the art. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to make antibodies are described in U.S. Pat. Nos. 6,150,584; 6,458,592; 6,420,140. Other techniques are known in the art.
A polypeptide comprising a heavy chain portion may or may not comprise other amino acid sequences or moieties not derived from an immunoglobulin molecule. Such modifications are described in more detail below. For example, in one embodiment, a polypeptide of the invention may comprise a flexible linker sequence. In another embodiment, a polypeptide may be modified to add a functional moiety such as PEG.
The polypeptides of the instant invention comprise at least two binding sites that provide for the association of the polypeptide with the selected target molecule.
In one embodiment, a binding molecule of the invention comprises an antibody molecule, e.g., an intact antibody molecule, or a fragment of an antibody molecule. In another embodiment, binding molecule of the invention is a modified or synthetic antibody molecule. In one embodiment, a binding molecule of the invention comprises all or a portion of (e.g., at least one antigen binding site from, at least one CDR from, or at least one heavy chain portion from) a monoclonal antibody, a humanized antibody, a chimeric antibody, or a recombinantly produced antibody.
In embodiments where the binding molecule is an antibody or modified antibody, the antigen binding site and the heavy chain portions need not be derived from the same immunoglobulin molecule. In this regard, the variable region may or be derived from any type of animal that can be induced to mount a humoral response and generate immunoglobulins against the desired antigen. As such, the variable region of the polypeptides may be, for example, of mammalian origin e.g., may be human, murine, non-human primate (such as cynomolgus monkeys, macaques, etc.), lupine, camelid (e.g., from camels, llamas and related species). In another embodiment, the variable region may be condricthoid in origin (e.g., from sharks).
Polypeptides of the invention can be made using techniques that are known in the art. In one embodiment, the polypeptides of the invention are antibody molecules that have been “recombinantly produced,” i.e., are produced using recombinant DNA technology. Exemplary techniques for making antibody molecules are discussed in more detail below.
In one embodiment, the polypeptides of the invention are modified antibodies. As used herein, the term “modified antibody” includes synthetic forms of antibodies which are altered such that they are not naturally occurring, e.g., antibodies that comprise at least two heavy chain portions but not two complete heavy chains (such as, domain deleted antibodies or minibodies); multispecific forms of antibodies (e.g., bispecific, trispecific, etc.) altered to bind to two or more different antigens or to different epitopes on a single antigen); heavy chain molecules joined to scFv molecules and the like. ScFv molecules are known in the art and are described, e.g., in U.S. Pat. No. 5,892,019. In addition, the term “modified antibody” includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies that bind to three or more copies of the same antigen). In another embodiment, a binding molecule of the invention is a fusion protein comprising at least one heavy chain portion lacking a CH2 domain and comprising a binding domain of a polypeptide comprising the binding portion of one member of a receptor ligand pair.
In one embodiment, the term, “modified antibody” according to the present invention includes immunoglobulins, antibodies, or immunoreactive fragments or recombinants thereof, in which at least a fraction of one or more of the constant region domains has been deleted or otherwise altered so as to provide desired biochemical characteristics such as the ability to non-covalently dimerize, increased ability to localize at the site of a tumor, or reduced serum half-life when compared with a whole, unaltered antibody of approximately the same immunogenicity. In a preferred embodiment, the polypeptides of the present invention are domain deleted antibodies which comprise a polypeptide chain similar to an immunoglobulin heavy chain, but which lack at least a portion of one or more heavy chain domains. More preferably, one entire domain of the constant region of the modified antibody will be deleted and even more preferably all or part of the CH2 domain will be deleted.
In preferred embodiments, a polypeptide of the invention will not elicit a deleterious immune response in a human. Modifications to the constant region compatible with the instant invention comprise additions, deletions or substitutions of one or more amino acids in one or more domains. That is, the polypeptides of the invention disclosed herein may comprise alterations or modifications to one or more of the three heavy chain constant domains (CH1, CH2 or CH3) and/or to the light chain constant region domain (CL).
For example, in one embodiment, the invention pertains to a humanized form of CC49 (huCC49 version 2 (V2)) having certain amino acid differences as compared to previously known humanized forms of CC49. FIGS. 19A and B show respective alignments of the light and heavy chain variable regions of murine CC49, the human antibodies LEN or 21/28′ CL, humanized CC49, and humanized CC49 V2 (which comprises one amino acid substitution in the light chain and two amino acid substitutions in the heavy chain as compared to humanized CC49, see underlined amino acids at position 43 of the light chain and positions 69 and 93 of the heavy chain, Kabat numbering system).
In one embodiment, the invention pertains to a humanized form of CC49 comprising a novel connecting peptide of the invention. For example, FIG. 13A (SEQ ID NO: 24) shows the DNA sequence of heavy chain CH2 domain-deleted huCC49 V2 containing G1/G3/Pro243Ala244Pro245-+[Gly/Ser] hinge connecting peptide. FIG. 13B (SEQ ID NO: 25) shows the DNA sequence of light chain CH2 domain-deleted huCC49 V2. FIG. 14A (SEQ ID NO: 26) shows the amino acid sequence of heavy chain CH2 domain-deleted huCC49 V2 containing G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide. FIG. 14B (SEQ ID NO: 27) shows the amino acid sequence of light chain CH2 domain-deleted huCC49 V2.
In one embodiment, the polypeptides of the invention may be modified to reduce their immunogenicity using art-recognized techniques. For example, antibodies or polypeptides of the invention can be humanized, deimmunized, or chimeric antibodies can be made. These types of antibodies are derived from a non-human antibody, typically a murine antibody, that retains or substantially retains the antigen-binding properties of the parent antibody, but which is less immunogenic in humans. This may be achieved by various methods, including (a) grafting the entire non-human variable domains onto human constant regions to generate chimeric antibodies; (b) grafting at least a part of one or more of the non-human complementarity determining regions (CDRs) into a human framework and constant regions with or without retention of critical framework residues; or (c) transplanting the entire non-human variable domains, but “cloaking” them with a human-like section by replacement of surface residues. Such methods are disclosed in Morrison et al., Proc. Natl. Acad. Sci. 81: 6851-5 (1984); Morrison et al., Adv. Immunol. 44: 65-92 (1988); Verhoeyen et al., Science 239: 1534-1536 (1988); Padlan, Molec. Immun. 28: 489-498 (1991); Padlan, Molec. Immun. 31: 169-217 (1994), and U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762 all of which are hereby incorporated by reference in their entirety.
De-immunization can also be used to decrease the immunogenicity of an antibody. As used herein, the term “de-immunization” includes alteration of an antibody to modify T cell epitopes (see, e.g., WO9852976A1, WO0034317A2). For example, VH and VL sequences from the starting antibody are analyzed and a human T cell epitope “map” from each V region showing the location of epitopes in relation to complementarity-determining regions (CDRs) and other key residues within the sequence Individual T cell epitopes from the T cell epitope map are analyzed in order to identify alternative amino acid substitutions with a low risk of altering activity of the final antibody. A range of alternative VH and VL sequences are designed comprising combinations of amino acid substitutions and these sequences are subsequently incorporated into a range of polypeptides of the invention that are tested for function. Typically, between 12 and 24 variant antibodies are generated and tested. Complete heavy and light chain genes comprising modified V and human C regions are then cloned into expression vectors and the subsequent plasmids introduced into cell lines for the production of whole antibody. The antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identified. In one embodiment, the binding molecule comprises a chimeric antibody. In the context of the present application the term “chimeric antibodies” will be held to mean any antibody wherein the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial or modified in accordance with the instant invention) is obtained from a second species. In preferred embodiments the target binding region or site will be from a non-human source (e.g. mouse) and the constant region is human. Preferably, the variable domains in both the heavy and light chains are altered by at least partial replacement of one or more CDRs and, if necessary, by partial framework region replacement and sequence changing. Although the CDRs may be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs will be derived from an antibody of different class and preferably from an antibody from a different species. It may not be necessary to replace all of the CDRs with the complete CDRs from the donor variable region to transfer the antigen binding capacity of one variable domain to another. Rather, it may only be necessary to transfer those residues that are necessary to maintain the activity of the target binding site. Given the explanations set forth in U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, it will be well within the competence of those skilled in the art, either by carrying out routine experimentation or by trial and error testing to obtain a functional antibody with reduced immunogenicity.
As used herein the term “properly folded polypeptide” includes polypeptides (e.g., antigen binding molecules such as antibodies) in which all of the functional domains comprising the polypeptide are distinctly active. As used herein, the term “improperly folded polypeptide” includes polypeptides in which at least one of the functional domains of the polypeptide is not active. In one embodiment, a properly folded polypeptide comprises polypeptide chains linked by at least one disulfide bond and, conversely, an improperly folded polypeptide comprises polypeptide chains not linked by at least one disulfide bond.
As used herein, the term “malignancy” refers to a non-benign tumor or a cancer. As used herein, the term “cancer” includes a malignancy characterized by deregulated or uncontrolled cell growth. Exemplary cancers include: carcinomas, sarcomas, leukemias, and lymphomas. The term “cancer” includes primary malignant tumors (e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original tumor) and secondary malignant tumors (e.g., those arising from metastasis, the migration of tumor cells to secondary sites that are different from the site of the original tumor).
In one embodiment, a binding molecule of the invention binds to a tumor cell. Exemplary antibodies which comprise antigen binding sites that bind to antigens expressed on tumor cells are known in the art and one or more CDRs from such antibodies can be included in a binding molecule of the invention. Exemplary antibodies include: 2B8, Lym 1, Lym 2, LL2, Her2, B1, MB1, BH3, B4, B72.3, 5E8, B3F6 and 5E10. In a preferred embodiment, a polypeptide of the invention is a C2B8 antibody which binds to CD20. In another preferred embodiment, a polypeptide of the invention is a CC49 antibody which recognizes TAG72.
In one embodiment, a binding molecule of the invention binds to a molecule which is useful in treating an autoimmune or inflammatory disease or disorder.
As used herein, the term “autoimmune disease or disorder” refers to disorders or conditions in a subject wherein the immune system attacks the body's own cells, causing tissue destruction. Autoimmune diseases include general autoimmune diseases, i.e., in which the autoimmune reaction takes place simultaneously in a number of tissues, or organ specific autoimmune diseases, i.e., in which the autoimmune reaction targets a single organ. Examples of autoimmune diseases that can be diagnosed, prevented or treated by the methods and compositions of the present invention include, but are not limited to, Crohn's disease; Inflammatory bowel disease (IBD); systemic lupus erythematosus; ulcerative colitis; rheumatoid arthritis; goodpasture's syndrome; Grave's disease; Hashimoto's thyroiditis; pemphigus vulgaris; myasthenia gravis; scleroderma; autoimmune hemolytic anemia; autoimmune thrombocytopenic purpura; polymyositis and dermatomyositis; pernicious anemia; Sjögren's syndrome; ankylosing spondylitis; vasculitis; type I diabetes mellitus; neurological disorders, multiple sclerosis, and secondary diseases caused as a result of autoimmune diseases.
As used herein the term “inflammatory disease or disorder” includes diseases or disorders which are caused, at least in part, or exacerbated by inflammation, e.g., increased blood flow, edema, activation of immune cells (e.g., proliferation, cytokine production, or enhanced phagocytosis). Exemplary disorders include those in which inflammation or inflammatory factors (e.g., matrix metalloproteinases (MMPs), nitric oxide (NO), TNF, interleukins, plasma proteins, cellular defense systems, cytokines, lipid metabolites, proteases, toxic radicals, mitochondria, apoptosis, adhesion molecules, etc.) are involved or are present in an area in aberrant amounts, e.g., in amounts which may be advantageous to alter, e.g., to benefit the subject. The inflammatory process is the response of living tissue to damage. The cause of inflammation may be due to physical damage, chemical substances, micro-organisms, tissue necrosis, cancer or other agents. Acute inflammation is short-lasting, lasting only a few days. If it is longer lasting however, then it may be referred to as chronic inflammation.
Inflammatory disorders include acute inflammatory disorders, chronic inflammatory disorders, and recurrent inflammatory disorders. Acute inflammatory disorders are generally of relatively short duration, and last for from about a few minutes to about one to two days, although they may last several weeks. The main characteristics of acute inflammatory disorders include increased blood flow, exudation of fluid and plasma proteins (edema) and emigration of leukocytes, such as neutrophils. Chronic inflammatory disorders, generally, are of longer duration, e.g., weeks to months to years or even longer, and are associated histologically with the presence of lymphocytes and macrophages and with proliferation of blood vessels and connective tissue. Recurrent inflammatory disorders include disorders which recur after a period of time or which have periodic episodes. Examples of recurrent inflammatory disorders include asthma and multiple sclerosis. Some disorders may fall within one or more categories.
Inflammatory disorders are generally characterized by heat, redness, swelling, pain and loss of function. Examples of causes of inflammatory disorders include, but are not limited to, microbial infections (e.g., bacterial, viral and fungal infections), physical agents (e.g., burns, radiation, and trauma), chemical agents (e.g., toxins and caustic substances), tissue necrosis and various types of immunologic reactions. Examples of inflammatory disorders include, but are not limited to, osteoarthritis, rheumatoid arthritis, acute and chronic infections (bacterial, viral and fungal); acute and chronic bronchitis, sinusitis, and other respiratory infections, including the common cold; acute and chronic gastroenteritis and colitis; acute and chronic cystitis and urethritis; acute respiratory distress syndrome; cystic fibrosis; acute and chronic dermatitis; acute and chronic conjunctivitis; acute and chronic serositis (pericarditis, peritonitis, synovitis, pleuritis and tendinitis); uremic pericarditis; acute and chronic cholecystis; acute and chronic vaginitis; acute and chronic uveitis; drug reactions; and burns (thermal, chemical, and electrical).
As used herein the term “medium that separates polypeptides based on hydrophobic interaction” includes a medium comprising hydrophobic ligands (e.g., alkyl or aryl groups) covalently attached to a matrix. Such a medium can be used to separate polypeptides based on interaction between a solvent and accessible non-polar groups on the surface of the polypeptides and the hydrophobic ligands of the medium. An exemplary medium is Phenyl 5PW-HR available from Tosoh Bioscience.
As used herein, the term “conductivity” includes electrical conductivity of a solution as measured in microSiemens/cm (formerly micromhos/cm). The greater the ion content of a solution, the greater the conductivity of the solution. Conductivity can be readily measured using techniques that are well known in the art (e.g., by measuring the current passing between two electrodes).
The separation methods of the invention can be used with solutions having a pH ranging from acid to neutral, e.g., from about pH 3.5 to approximately neutral. As used herein, the term “approximately neutral pH” includes pH values of approximately 7. For example, in one embodiment, a separation method of the invention can be performed using a solution (e.g., a buffer) having a pH of about 3, about 4, about 5, about 6, about 7, or about 8. Preferably, the pH of the solution is about 6 or about 7. In one embodiment, the pH of the solution is about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0.
As used herein the term “affinity matrix” includes a matrix, such as agarose, controlled pore glass, or poly(styrenedivinyl)benzene to which an affinity ligand is attached. The affinity ligand binds to the desired polypeptide and the contaminating polypeptides are not bound to the affinity ligand. The desired polypeptide can be eluted from the affinity matrix using known protocols.
As used herein the term “engineered” includes manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g. by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides or some combination of these techniques). Preferably, the binding molecules of the invention are engineered, e.g., to express a connecting peptide of the invention.
As used herein, the terms “linked,” “fused” or “fusion” are used interchangeably. These terms refer to the joining together of two more elements or components, by whatever means including chemical conjugation or recombinant means. An “in-frame fusion” refers to the joining of two or more open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct reading frame of the original ORFs. Thus, the resulting recombinant fusion protein is a single protein containing two ore more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature.) Although the reading frame is thus made continuous throughout the fused segments, the segments may be physically or spatially separated by, for example, in-frame linker sequence.
In the context of polypeptides, a “linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminal direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide.
As used herein, the phrase “subject that would benefit from administration of a binding molecule” includes subjects, such as mammalian subjects, that would benefit from administration of a binding molecule used, e.g., for detection of an antigen recognized by a binding molecule (e.g., for a diagnostic procedure) and/or from treatment with a binding molecule to reduce or eliminate the target recognized by the binding molecule. For example, in one embodiment, the subject may benefit from reduction or elimination of a soluble or particulate molecule from the circulation or serum (e.g., a toxin or pathogen) or from reduction or elimination of a population of cells expressing the target (e.g., tumor cells). As described in more detail herein, the binding molecule can be used in unconjugated form or can be conjugated, e.g., to a drug, prodrug, or an isotope.
II. SYNTHETIC CONNECTING PEPTIDES
At least one polypeptide chain of a dimer of the invention can comprise a synthetic connecting peptide of the invention. In one embodiment, at least two chains of a dimer of the invention comprise a connecting peptide. In a preferred embodiment, two chains of a dimer of the invention comprise a connecting peptide.
In one embodiment, connecting peptides can be used to join two heavy chain portions in frame in a single polypeptide chain. For example, in one embodiment, a connecting peptide of the invention can be used to fuse a CH3 domain (or synthetic CH3 domain) to a hinge region (or synthetic hinge region). In another embodiment, a connecting peptide of the invention can be used to fuse a CH3 domain (or synthetic CH3 domain) to a CH1 domain (or synthetic CH1 domain). In still another embodiment, a connecting peptide can act as a peptide spacer between the hinge region (or synthetic hinge region) and a CH2 domain (or a synthetic CH2 domain).
In another embodiment, a CH3 domain can be fused to an extracellular protein domain (e.g., a VL domain (or synthetic domain), a VH domain (or synthetic domain), a CH1 domain (or synthetic domain), a hinge domain (or synthetic hinge), or to the ligand binding portion of a receptor or the receptor binding portion of a ligand). For example, in one embodiment, a VH or VL domain is fused to a CH3 domain via a connecting peptide (the C-terminus of the connecting peptide is attached to the N-terminus of the CH3 domain and the N-terminus of the connecting peptide is attached to the C-terminus of the VH or VL domain). In another embodiment, a CH1 domain is fused to a CH3 domain via a connecting peptide (the C-terminus of the connecting peptide is attached to the N-terminus of the CH3 domain and the N-terminus of the connecting peptide is attached to the C-terminus of the CH1 domain). In another embodiment, a connecting peptide of the invention can be used to fuse a CH3 domain (or synthetic CH3 domain) to a hinge region (or synthetic hinge region) or portion thereof. In still another embodiment, a connecting peptide can act as a peptide spacer between the hinge region (or synthetic hinge region) and a CH2 domain (or a synthetic CH2 domain).
In one embodiment, a connecting peptide can comprise or consist of a gly/ser spacer. For example, a domain deleted CC49 construct having a short amino acid spacer GGSSGGGGSG (SEQ. ID No. 1) substituted for the CH2 domain and the lower hinge region (CC49.ΔCH2 [gly/ser]) can be used. In another embodiment, a connecting peptide comprises the amino acid sequence IGKTISKKAK (SEQ ID NO:36).
In another embodiment, connecting peptide can comprise at least a portion of an immunoglobulin hinge region. For example, chimeric hinge domains can be constructed which combine hinge elements derived from different antibody isotypes. In one embodiment, a connecting peptide comprises at least a portion of an IgG1 hinge region. In another embodiment, a connecting peptide can comprise at least a portion of an IgG3 hinge region. In another embodiment, a connecting peptide can comprise at least a portion of an IgG1 hinge region and at least a portion of an IgG3 hinge region. In one embodiment, a connecting peptide can comprise an IgG1 upper and middle hinge and a single IgG3 middle hinge repeat motif.
Because the numbering of individual amino acids in such connecting peptides comprising an amino acid sequence derived from an immunoglobulin hinge region may vary depending upon the length of the connecting peptide, the numbering of amino acid positions in these molecules is given using Kabat numbering see, e.g., Table 2). Table 1 shows naturally occurring hinge sequence for IgG1, IgG3, and IgG4 molecules. Table 2 shows Kabat numbering for portions of these hinge molecules and also shows Kabat numbering for connecting peptide amino acid residues presented in that table.
In one embodiment, a connecting peptide of the invention comprises a non-naturally occurring immunoglobulin hinge region domain, e.g., a hinge region domain that is not naturally found in the polypeptide comprising the hinge region domain and/or a hinge region domain that has been altered so that it differs in amino acid sequence from a naturally occurring immunoglobulin hinge region domain. In one embodiment, mutations can be made to hinge region domains to make a connecting peptide of the invention. In one embodiment, a connecting peptide of the invention comprises a hinge domain which does not comprise a naturally occurring number of cysteines, i.e., the connecting peptide comprises either fewer cysteines or a greater number of cysteines than a naturally occurring hinge molecule. In a preferred embodiment, incorporation of the connecting peptide into a polypeptide results in a composition in which greater than 50%, 60%, 70%, 80% or 90% of the dimeric molecules present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage.
In one embodiment of the invention, a connecting peptide comprises hinge region domain comprising a proline residue at an amino acid position corresponding to amino acid position 243 in the Kabat numbering system (position 230, EU numbering system). In one embodiment, a connecting peptide comprises an alanine residue at an amino acid position corresponding to position 244, Kabat numbering system (position 246, EU numbering system). In another embodiment, a connecting peptide of the invention comprises a proline residue at an amino acid position corresponding to position 245 (Kabat numbering system; position 247, EU numbering system)). In one embodiment, a connecting peptide comprises a cysteine residue at an amino acid position corresponding to position 239, Kabat numbering system (position 226, EU numbering system). In one embodiment, a connecting peptide comprises a serine residue at an amino acid position corresponding to position 239, Kabat numbering system (position 226, EU numbering system). In one embodiment, a connecting peptide comprises a cysteine residue at an amino acid position corresponding to position 242, Kabat numbering system (position 229, EU numbering system). In one embodiment, a connecting peptide comprises a serine residue at an amino acid position corresponding to position 242, Kabat numbering system (position 229, EU numbering system).
In one embodiment, the connecting peptide can be chosen to result in the preferential synthesis of a particular isoform of polypeptide, e.g., in which the two heavy chain portions are linked via disulfide bonds or are not linked via disulfide bonds. For example, as described in the instant examples, the G1/G3/Pro243+[gly/ser] linker (SEQ ID NO: 8), G1/G3/Pro243Ala244Pro245+[gly/ser] linker (SEQ ID NO: 9), Pro243+[gly/ser] linker (SEQ ID NO:15), and Pro243Ala244Pro245+[gly/ser] linker (SEQ ID NO: 14), connecting peptides resulted in the production of only Form A CH2 domain-deleted antibody with no detectable Form B. In contrast, CH2 domain-deleted Cys242Ser:Pro243 (SEQ ID NO: 12), and CH2 domain-deleted Cys242Ser:Pro243Ala244Pro245 (SEQ ID NO: 13), both resulted in a preference for the Form B form. These synthetic hinge region connecting peptides would thus be useful for favoring synthesis of Form A or B form. This is true for any isotype of antibody, (e.g., IgG1, IgG2, IgG3, or IgG4) based on the high degree of homology among the CH3 domains for all four human isotypes. (Including identical and conserved amino acid residues, IgG1 CH3 domain is 98.13% homologous to IgG2 CH3, 97.20% homologous to IgG3 CH3, and 96.26% homologous to IgG4 CH3). The parentheticals referring to connecting peptides and various binding molecules of the invention represent equivalent terminology unless otherwise indicated.
In one embodiment, a connecting peptide of the invention comprises a hinge region domain followed by a flexible gly/ser linker. Exemplary connecting peptides are shown in Table 2 and in SEQ ID NOs: 8-15, 37 and 53. It will be understood that variant forms of these exemplary connecting peptides can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence encoding a connecting peptide such that one or more amino acid substitutions, additions or deletions are introduced into the connecting peptide. For example, mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more non-essential amino acid residues such that the ability of the connecting peptide to preferentially enhance synthesis of Form A or Form B is not altered. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.
Connecting peptides of the invention can be of varying lengths. In one embodiment, a connecting peptide of the invention is from about 15 to about 50 amino acids in length. In another embodiment, a connecting peptide of the invention is from about 20 to about 45 amino acids in length. In another embodiment, a connecting peptide of the invention is from about 25 to about 40 amino acids in length. In another embodiment, a connecting peptide of the invention is from about 30 to about 35 amino acids in length. In another embodiment, a connecting peptide of the invention is from about 24 to about 27 amino acids in length. In another embodiment, a connecting peptide of the invention is from about 40 to about 42 amino acids in length.
Connecting peptides can be introduced into polypeptide sequences using techniques known in the art. For example, in one embodiment, the Splicing by Overlap Extension (SOE) method (Horton, R. M. 1993 Methods in Molecular Biology, Vol 15: PCR Protocols: Current Methods and applications. Ed. B. A. White) can be used. Modifications can be confirmed by DNA sequence analysis. Plasmid DNA can be used to transform host cells for stable production of the polypeptides produced.
In one embodiment, incorporation of one of the subject connecting peptides into a polypeptide yields a composition comprising polypeptide molecules having at least two binding sites and at least two polypeptide chains, wherein at least two of the polypeptide chains comprise a synthetic connecting peptide and wherein greater than 50% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage. In another embodiment, greater than 60% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage. In another embodiment, greater than 70% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage. In another embodiment, greater than 80% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage. In another embodiment, greater than 90% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage.
III. BINDING MOLECULES
A. Antibodies or Portions Thereof
In one embodiment, a binding molecule of the invention is an antibody molecule. Using art recognized protocols, for example, antibodies are preferably raised in mammals by multiple subcutaneous or intraperitoneal injections of the relevant antigen (e.g., purified tumor associated antigens or cells or cellular extracts comprising such antigens) and an adjuvant. This immunization typically elicits an immune response that comprises production of antigen-reactive antibodies from activated splenocytes or lymphocytes. While the resulting antibodies may be harvested from the serum of the animal to provide polyclonal preparations, it is often desirable to isolate individual lymphocytes from the spleen, lymph nodes or peripheral blood to provide homogenous preparations of monoclonal antibodies (MAbs). Preferably, the lymphocytes are obtained from the spleen.
In this well known process (Kohler et al., Nature, 256:495 (1975)) the relatively short-lived, or mortal, lymphocytes from a mammal which has been injected with antigen are fused with an immortal tumor cell line (e.g. a myeloma cell line), thus, producing hybrid cells or “hybridomas” which are both immortal and capable of producing the genetically coded antibody of the B cell. The resulting hybrids are segregated into single genetic strains by selection, dilution, and regrowth with each individual strain comprising specific genes for the formation of a single antibody. They produce antibodies which are homogeneous against a desired antigen and, in reference to their pure genetic parentage, are termed “monoclonal.”
Hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. Those skilled in the art will appreciate that reagents, cell lines and media for the formation, selection and growth of hybridomas are commercially available from a number of sources and standardized protocols are well established. Generally, culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies against the desired antigen. Preferably, the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro assay, such as a radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). After hybridoma cells are identified that produce antibodies of the desired specificity, affinity and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp 59-103 (Academic Press, 1986)). It will further be appreciated that the monoclonal antibodies secreted by the subclones may be separated from culture medium, ascites fluid or serum by conventional purification procedures such as, for example, protein-A, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.
In another embodiment, DNA encoding a desired monoclonal antibodies may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The isolated and subcloned hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells or myeloma cells that do not otherwise produce immunoglobulins. More particularly, the isolated DNA (which may be synthetic as described herein) may be used to clone constant and variable region sequences for the manufacture antibodies as described in Newman et al., U.S. Pat. No. 5,658,570, filed Jan. 25, 1995, which is incorporated by reference herein. Essentially, this entails extraction of RNA from the selected cells, conversion to cDNA, and amplification by PCR using Ig specific primers. Suitable primers for this purpose are also described in U.S. Pat. No. 5,658,570. As will be discussed in more detail below, transformed cells expressing the desired antibody may be grown up in relatively large quantities to provide clinical and commercial supplies of the immunoglobulin.
Those skilled in the art will also appreciate that DNA encoding antibodies or antibody fragments (e.g., antigen binding sites) may also be derived from antibody phage libraries, e.g., using pd phage or Fd phagemid technology. Exemplary methods are set forth, for example, in EP 368 684 B1; U.S. Pat. No. 5,969,108, Hoogenboom, H. R. and Chames. 2000. Immunol. Today 21:371; Nagy et al. 2002. Nat. Med. 8:801; Huie et al. 2001. Proc. Natl. Acad. Sci. USA 98:2682; Lui et al. 2002. J. Mol. Biol. 315:1063, each of which is incorporated herein by reference. Several publications (e.g., Marks et al. Bio/Technology 10:779-783 (1992)) have described the production of high affinity human antibodies by chain shuffling, as well as combinatorial infection and in vivo recombination as a strategy for constructing large phage libraries. In another embodiment, Ribosomal display can be used to replace bacteriophage as the display platform (see, e.g., Hanes et al. 2000. Nat. Biotechnol. 18:1287; Wilson et al. 2001. Proc. Natl. Acad. Sci. USA 98:3750; or Irving et al. 2001 J. Immunol. Methods 248:31. In yet another embodiment, cell surface libraries can be screened for antibodies (Boder et al. 2000. Proc. Natl. Acad. Sci. USA 97:10701; Daugherty et al. 2000 J. Immunol. Methods 243:211. Such procedures provide alternatives to traditional hybridoma techniques for the isolation and subsequent cloning of monoclonal antibodies.
Yet other embodiments of the present invention comprise the generation of human or substantially human antibodies in transgenic animals (e.g., mice) that are incapable of endogenous immunoglobulin production (see e.g., U.S. Pat. Nos. 6,075,181, 5,939,598, 5,591,669 and 5,589,369 each of which is incorporated herein by reference). For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of a human immunoglobulin gene array to such germ line mutant mice will result in the production of human antibodies upon antigen challenge. Another preferred means of generating human antibodies using SCID mice is disclosed in U.S. Pat. No. 5,811,524 which is incorporated herein by reference. It will be appreciated that the genetic material associated with these human antibodies may also be isolated and manipulated as described herein.
Yet another highly efficient means for generating recombinant antibodies is disclosed by Newman, Biotechnology, 10: 1455-1460 (1992). Specifically, this technique results in the generation of primatized antibodies that contain monkey variable domains and human constant sequences. This reference is incorporated by reference in its entirety herein. Moreover, this technique is also described in commonly assigned U.S. Pat. Nos. 5,658,570, 5,693,780 and 5,756,096 each of which is incorporated herein by reference.
In another embodiment, lymphocytes can be selected by micromanipulation and the variable genes isolated. For example, peripheral blood mononuclear cells can be isolated from an immunized mammal and cultured for about 7 days in vitro. The cultures can be screened for specific IgGs that meet the screening criteria. Cells from positive wells can be isolated. Individual Ig-producing B cells can be isolated by FACS or by identifying them in a complement-mediated hemolytic plaque assay. Ig-producing B cells can be micromanipulated into a tube and the VH and VL genes can be amplified using, e.g., RT-PCR. The VH and VL genes can be cloned into an antibody expression vector and transfected into cells (e.g., eukaryotic or prokaryotic cells) for expression.
Moreover, genetic sequences useful for producing the polypeptides of the present invention may be obtained from a number of different sources. For example, as discussed extensively above, a variety of human antibody genes are available in the form of publicly accessible deposits. Many sequences of antibodies and antibody-encoding genes have been published and suitable antibody genes can be chemically synthesized from these sequences using art recognized techniques. Oligonucleotide synthesis techniques compatible with this aspect of the invention are well known to the skilled artisan and may be carried out using any of several commercially available automated synthesizers. In addition, DNA sequences encoding several types of heavy and light chains set forth herein can be obtained through the services of commercial DNA synthesis vendors. The genetic material obtained using any of the foregoing methods may then be altered or synthetic to provide obtain polypeptides of the present invention.
Alternatively, antibody-producing cell lines may be selected and cultured using techniques well known to the skilled artisan. Such techniques are described in a variety of laboratory manuals and primary publications. In this respect, techniques suitable for use in the invention as described below are described in Current Protocols in Immunology, Coligan et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991) which is herein incorporated by reference in its entirety, including supplements.
It will further be appreciated that the scope of this invention further encompasses all alleles, variants and mutations of antigen binding DNA sequences.
As is well known, RNA may be isolated from the original hybridoma cells or from other transformed cells by standard techniques, such as guanidinium isothiocyanate extraction and precipitation followed by centrifugation or chromatography. Where desirable, mRNA may be isolated from total RNA by standard techniques such as chromatography on oligo dT cellulose. Suitable techniques are familiar in the art.
In one embodiment, cDNAs that encode the light and the heavy chains of the antibody may be made, either simultaneously or separately, using reverse transcriptase and DNA polymerase in accordance with well known methods. PCR may be initiated by consensus constant region primers or by more specific primers based on the published heavy and light chain DNA and amino acid sequences. As discussed above, PCR also may be used to isolate DNA clones encoding the antibody light and heavy chains. In this case the libraries may be screened by consensus primers or larger homologous probes, such as mouse constant region probes.
DNA, typically plasmid DNA, may be isolated from the cells using techniques known in the art, restriction mapped and sequenced in accordance with standard, well known techniques set forth in detail, e.g., in the foregoing references relating to recombinant DNA techniques. Of course, the DNA may be synthetic according to the present invention at any point during the isolation process or subsequent analysis.
B. Modified Antibodies
In one embodiment, a binding molecule or antigen binding molecule of the invention comprise synthetic constant regions wherein one or more domains are partially or entirely deleted (“domain-deleted antibodies”). In especially preferred embodiments compatible modified antibodies will comprise domain deleted constructs or variants wherein the entire CH2 domain has been removed (ΔCH2 constructs). For other preferred embodiments a short connecting peptide may be substituted for the deleted domain to provide flexibility and freedom of movement for the variable region. Those skilled in the art will appreciate that such constructs are particularly preferred due to the regulatory properties of the CH2 domain on the catabolic rate of the antibody.
In one embodiment, the modified antibodies of the invention are minibodies. Minibodies can be made using methods described in the art (see, e.g., see e.g., U.S. Pat. No. 5,837,821 or WO 94/09817A1).
In another embodiment, the modified antibodies of the invention are CH2 domain deleted antibodies which are known in the art. Domain deleted constructs can be derived using a vector (e.g., from IDEC Pharmaceuticals, San Diego) encoding an IgG1 human constant domain (see, e.g., WO 02/060955A2 and WO02/096948A2). This exemplary vector was engineered to delete the CH2 domain and provide a synthetic vector expressing a domain deleted IgG1 constant region. Genes encoding the murine variable region of the C2B8 antibody, 5E8 antibody, B3F6 antibody, or the variable region of the humanized CC49 antibody were then inserted in the synthetic vector and cloned. When expressed in transformed cells, these vectors provided C2B8.ΔCH2, 5E8.ΔCH2, B3F6.ΔCH2 or huCC49.ΔCH2 or respectively. These constructs exhibit a number of properties that make them particularly attractive candidates for monomeric subunits.
It will be noted that these exemplary constructs were engineered to fuse the CH3 domain directly to a hinge region of the respective polypeptides of the invention. In other constructs it may be desirable to provide a peptide spacer between the hinge region and the synthetic CH2 and/or CH3 domains. For example, compatible constructs could be expressed wherein the CH2 domain has been deleted and the remaining CH3 domain (synthetic or unsynthetic) is joined to the hinge region with a 5-20 amino acid spacer. Such a spacer may be added, for instance, to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible. For example, a domain deleted CC49 construct having a short amino acid spacer GGSSGGGGSG (SEQ. ID No. 1) substituted for the CH2 domain and the lower hinge region (CC49.ΔCH2 [gly/ser]) can be used. Other exemplary connecting peptides are shown in Table 2. These connecting peptides can be used with any of the polypeptides of the invention. Preferably, the connecting peptides are used with a polypeptide lacking a CH2 heavy chain domain. Preferably, any linker compatible with the instant invention will be relatively non-immunogenic and not inhibit the non-covalent association of the polypeptides of the invention.
In one embodiment, a polypeptide of the invention comprises an immunoglobulin heavy chain having deletion or substitution of a few or even a single amino acid as long as it permits the desired covalent or non-covalent association between the monomeric subunits. For example, the mutation of a single amino acid in selected areas of the CH2 domain may be enough to substantially reduce Fc binding and thereby increase tumor localization. Similarly, it may be desirable to simply delete that part of one or more constant region domains that control the effector function (e.g. complement binding) to be modulated. Such partial deletions of the constant regions may improve selected characteristics of the antibody (serum half-life) while leaving other desirable functions associated with the subject constant region domain intact. Moreover, as alluded to above, the constant regions of the disclosed antibodies may be synthetic through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct. In this respect it may be possible to disrupt the activity provided by a conserved binding site (e.g. Fc binding) while substantially maintaining the configuration and immunogenic profile of the modified antibody. Yet other preferred embodiments may comprise the addition of one or more amino acids to the constant region to enhance desirable characteristics such as effector function or provide for more cytotoxin or carbohydrate attachment. In such embodiments it may be desirable to insert or replicate specific sequences derived from selected constant region domains.
C. Fusion Proteins
The invention also pertains to binding molecules which comprise one or more immunoglobulin domains. The fusion proteins of the invention comprise a binding domain (which comprises at least one binding site) and a dimerization domain (which comprises at least one heavy chain portion). The subject fusion proteins may be bispecific (with one binding site for a first target and a second binding site for a second target) or may be multivalent (with two binding sites for the same target).
Exemplary fusion proteins reported in the literature include fusions of the T cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84:2936-2940 (1987)); CD4 (Capon et al., Nature 337:525-531 (1989); Traunecker et al., Nature 339:68-70 (1989); Zettmeissl et al., DNA Cell Biol. USA 9:347-353 (1990); and Byrn et al., Nature 344:667-670 (1990)); L-selectin (homing receptor) (Watson et al., J. Cell. Biol. 110:2221-2229 (1990); and Watson et al., Nature 349:164-167 (1991)); CD44 (Aruffo et al., Cell 61:1303-1313 (1990)); CD28 and B7 (Linsley et al., J. Exp. Med. 173:721-730 (1991)); CTLA-4 (Lisley et al., J. Exp. Med. 174:561-569 (1991)); CD22 (Stamenkovic et al., Cell 66:1133-1144 (1991)); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Lesslauer et al., Eur. J. Immunol. 27:2883-2886 (1991); and Peppel et al., J. Exp. Med. 174:1483-1489 (1991)); and IgE receptor a (Ridgway and Gorman, J. Cell. Biol. Vol. 115, Abstract No. 1448 (1991)).
In one embodiment a fusion protein combines the binding domain(s) of the ligand or receptor (e.g. the extracellular domain (ECD) of a receptor) with at least one heavy chain domain and a synthetic connecting peptide. In one embodiment, when preparing the fusion proteins of the present invention, nucleic acid encoding the binding domain of the ligand or receptor domain will be fused C-terminally to nucleic acid encoding the N-terminus of an immunoglobulin constant domain sequence. N-terminal fusions are also possible. In one embodiment, a fusion protein includes a CH2 and a CH3 domains. Fusions may also be made to the C-terminus of the Fc portion of a constant domain, or immediately N-terminal to the CH1 of the heavy chain or the corresponding region of the light chain.
In one embodiment, the sequence of the ligand or receptor binding domain is fused to the N-terminus of the Fc domain of an immunoglobulin molecule. It is also possible to fuse the entire heavy chain constant region to the ligand or receptor binding domain sequence. In one embodiment, a sequence beginning in the hinge region just upstream of the papain cleavage site which defines IgG Fc chemically (i.e. residue 216, taking the first residue of heavy chain constant region to be 114), or analogous sites of other immunoglobulins is used in the fusion. The precise site at which the fusion is made is not critical; particular sites are well known and may be selected in order to optimize the biological activity, secretion, or binding characteristics of the molecule. Methods for making fusion proteins are known in the art.
For bispecific fusion proteins, the fusion proteins are assembled as multimers, and particularly as heterodimers or heterotetramers. Generally, these assembled immunoglobulins will have known unit structures. A basic four chain structural unit is the form in which IgG, IgD, and IgE exist. A four chain unit is repeated in the higher molecular weight immunoglobulins; IgM generally exists as a pentamer of four basic units held together by disulfide bonds. IgA globulin, and occasionally IgG globulin, may also exist in multimeric form in serum. In the case of multimer, each of the four units may be the same or different.
Additional exemplary ligands and their receptors that may be included in the subject fusion proteins include the following:
Cytokines and Cytokine Receptors
Cytokines have pleiotropic effects on the proliferation, differentiation, and functional activation of lymphocytes. Various cytokines, or receptor binding portions thereof, can be utilized in the fusion proteins of the invention. Exemplary cytokines include the interleukins (e.g. IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-13, and IL-18), the colony stimulating factors (CSFs) (e.g. granulocyte CSF (G-CSF), granulocyte-macrophage CSF (GM-CSF), and monocyte macrophage CSF (M-CSF)), tumor necrosis factor (TNF) alpha and beta, and interferons such as interferon-α, β, or γ (U.S. Pat. Nos. 4,925,793 and 4,929,554).
Cytokine receptors typically consist of a ligand-specific alpha chain and a common beta chain. Exemplary cytokine receptors include those for GM-CSF, IL-3 (U.S. Pat. No. 5,639,605), IL-4 (U.S. Pat. No. 5,599,905), IL-5 (U.S. Pat. No. 5,453,491), IFNγ (EP0240975), and the TNF family of receptors (e.g., TNFα (e.g. TNFR-1 (EP 417, 563), TNFR-2 (EP 417,014) lymphotoxin beta receptor).
Adhesion Proteins
Adhesion molecules are membrane-bound proteins that allow cells to interact with one another. Various adhesion proteins, including leukocyte homing receptors and cellular adhesion molecules, of receptor binding portions thereof, can be incorporated in a fusion protein of the invention. Leucocyte homing receptors are expressed on leucocyte cell surfaces during inflammation and include the β-1 integrins (e.g. VLA-1, 2, 3, 4, 5, and 6) which mediate binding to extracellular matrix components, and the β2-integrins (e.g. LFA-1, LPAM-1, CR3, and CR4) which bind cellular adhesion molecules. (CAMs) on vascular endothelium. Exemplary CAMs include ICAM-1, ICAM-2, VCAM-1, and MAdCAM-1. Other CAMs include those of the selectin family including E-selectin, L-selectin, and P-selectin.
Chemokines
Chemokines, chemotactic proteins which stimulate the migration of leucocytes towards a site of infection, can also be incorporated into a fusion protein of the invention. Exemplary chemokines include Macrophage inflammatory proteins (MIP-1-α and MIP-1-β), neutrophil chemotactic factor, and RANTES (regulated on activation normally T-cell expressed and secreted).
Growth Factors and Growth Factor Receptors
Growth factors or their receptors (or receptor binding or ligand binding portions thereof) may be incorporated in the fusion proteins of the invention. Exemplary growth factors include Vascular Endothelial Growth Factor (VEGF) and its isoforms (U.S. Pat. No. 5,194,596); Fibroblastic Growth Factors (FGF), including aFGF and bFGF; atrial natriuretic factor (ANF); hepatic growth factors (HGFs; U.S. Pat. Nos. 5,227,158 and 6,099,841), neurotrophic factors such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-β platelet-derived growth factor (PDGF) (U.S. Pat. Nos. 4,889,919, 4,845,075, 5,910,574, and 5,877,016); transforming growth factors (TGF) such as TGF-alpha and TGF-beta (WO 90/14359), osteoinductive factors including bone morphogenetic protein (BMP); insulin-like growth factors-I and -II (IGF-I and IGF-II; U.S. Pat. Nos. 6,403,764 and 6,506,874); Erythropoietin (EPO); stem-cell factor (SCF), thrombopoietin (c-Mpl ligand), and the Wnt polypeptides (U.S. Pat. No. 6,159,462).
e) Hormones
Exemplary growth hormones for use as targeting agents in the fusion proteins of the invention include renin, human growth hormone (HGH; U.S. Pat. No. 5,834,598), N-methionyl human growth hormone; bovine growth hormone; growth hormone releasing factor; parathyroid hormone (PTH); thyroid stimulating hormone (TSH); thyroxine; proinsulin and insulin (U.S. Pat. Nos. 5,157,021 and 6,576,608); follicle stimulating hormone (FSH), calcitonin, luteinizing hormone (LH), leptin, glucagons; bombesin; somatropin; mullerian-inhibiting substance; relaxin and prorelaxin; gonadotropin-associated peptide; prolactin; placental lactogen; OB protein; or mullerian-inhibiting substance.
Clotting Factors
Exemplary blood coagulation factors for use as targeting agents in the fusion proteins of the invention include the clotting factors (e.g., factors V, VII, VIII, X, IX, XI, XII and XIII, von Willebrand factor); tissue factor (U.S. Pat. Nos. 5,346,991, 5,349,991, 5,726,147, and 6,596,845); thrombin and prothrombin; fibrin and fibrinogen; plasmin and plasminogen; plasminogen activators, such as urokinase or human urine or tissue-type plasminogen activator (t-PA).
Other exemplary fusion proteins are taught, e.g., in WO0069913A1 and WO0040615A2. Another exemplary molecule that may be included in a fusion protein of the invention is IGSF9.
Fusion proteins can be prepared using methods that are well known in the art (see for example U.S. Pat. Nos. 5,116,964 and 5,225,538). Ordinarily, the ligand or ligand binding partner is fused C-terminally to the N-terminus of the constant region of the heavy chain (or heavy chain portion) and in place of the variable region. Any transmembrane regions or lipid or phospholipids anchor recognition sequences of ligand binding receptor are preferably inactivated or deleted prior to fusion. DNA encoding the ligand or ligand binding partner is cleaved by a restriction enzyme at or proximal to the 5′ and 3′ends of the DNA encoding the desired ORF segment. The resultant DNA fragment is then readily inserted into DNA encoding a heavy chain constant region. The precise site at which the fusion is made may be selected empirically to optimize the secretion or binding characteristics of the soluble fusion protein. DNA encoding the fusion protein is then transfected into a host cell for expression.
In one embodiment, a nucleic acid molecule of the invention comprises a nucleotide sequence shown in FIG. 4A (SEQ ID NO 16). In another embodiment, a nucleic acid molecule of the invention comprises a nucleotide sequence shown in FIG. 4B (SEQ ID NO:17). In one embodiment, a polypeptide molecule of the invention comprises a polypeptide sequence encoded by a nucleic acid molecule comprising a nucleic acid sequence shown in FIG. 4A (SEQ ID NO: 16). In another embodiment, a polypeptide molecule of the invention comprises a polypeptide sequence encoded by a nucleic acid molecule having a nucleotide sequence shown in FIG. 4B (SEQ ID NO: 17).
In one embodiment, a polypeptide molecule of the invention comprises an amino acid sequence shown in FIG. 8A (SEQ ID NO: 18). In another embodiment, a polypeptide molecule of the invention comprises an amino acid sequence shown in FIG. 8B (SEQ ID NO: 19).
In another embodiment, a polypeptide of the invention comprises an amino acid sequence encoded by a nucleotide sequence shown in FIG. 9 (SEQ ID NO:20). In another embodiment, a nucleic acid molecule of the invention comprises a nucleotide sequence shown in FIG. 9 (SEQ ID NO:20).
In another embodiment, a polypeptide of the invention comprises an amino acid sequence encoded by a nucleotide sequence shown in FIG. 10 (SEQ ID NO: 21). In another embodiment, a nucleic acid molecule of the invention comprises a nucleotide sequence shown in FIG. 10 (SEQ ID NO: 21).
In another embodiment, a polypeptide of the invention comprises an amino acid sequence encoded by a nucleotide sequence shown in FIG. 28A (SEQ ID NO: 38). In another embodiment, a nucleic acid molecule of the invention comprises a nucleotide sequence shown in FIG. 28A (SEQ ID NO: 38).
In another embodiment, a polypeptide of the invention comprises an amino acid sequence encoded by a nucleotide sequence shown in FIG. 28B (SEQ ID NO: 39). In one embodiment, a nucleic acid molecule of the invention comprises a nucleotide sequence shown in FIG. 28B (SEQ ID NO: 39).
In another embodiment, a polypeptide of the invention comprises an amino acid sequence shown in FIG. 11 (SEQ ID NO: 22).
In another embodiment, a polypeptide of the invention comprises an amino acid sequence shown in FIG. 12 (SEQ ID NO: 23).
In another embodiment, a polypeptide of the invention comprises an amino acid sequence shown in FIG. 29A (SEQ ID NO: 40). In another embodiment, a polypeptide of the invention comprises an amino acid sequence shown in FIG. 29B (SEQ ID NO: 41).
In another embodiment, a polypeptide of the invention comprises an amino acid sequence encoded by a nucleotide sequence shown in FIG. 32A (SEQ ID NO: 42). In another embodiment, a nucleic acid molecule of the invention comprises a nucleotide sequence shown in FIG. 32A (SEQ ID NO: 42). In another embodiment, a polypeptide of the invention comprises an amino acid sequence encoded by a nucleotide sequence shown in FIG. 32B (SEQ ID NO: 43). In one embodiment, a nucleic acid molecule of the invention comprises a nucleotide sequence shown in FIG. 32B (SEQ ID NO: 43).
In another embodiment, a polypeptide of the invention comprises an amino acid sequence encoded by a nucleotide sequence shown in FIG. 13A (SEQ ID NO: 24). In one embodiment, a nucleic acid molecule of the invention comprises a nucleotide sequence shown in FIG. 13A (SEQ ID NO: 24). In another embodiment, a polypeptide of the invention comprises an amino acid sequence encoded by a nucleotide sequence shown in FIG. 13B (SEQ ID NO: 25). In one embodiment, a nucleic acid molecule of the invention comprises a nucleotide sequence shown in FIG. 13B (SEQ ID NO: 25).
In another embodiment, a polypeptide of the invention comprises an amino acid sequence shown in FIG. 14A (SEQ ID NO: 26). In another embodiment, a polypeptide of the invention comprises an amino acid sequence shown in FIG. 14B (SEQ ID NO: 27).
In another embodiment, a polypeptide of the invention comprises an amino acid sequence shown in FIG. 33A (SEQ ID NO: 44). In another embodiment, a polypeptide of the invention comprises an amino acid sequence shown in FIG. 33B (SEQ ID NO: 45).
In another embodiment, a nucleic acid molecule of the invention comprises a nucleotide sequence shown in FIG. 25A (SEQ ID NO: 46). In another embodiment, a polypeptide of the invention comprises an amino acid sequence shown in FIG. 25B (SEQ ID NO: 47).
In another embodiment, a nucleic acid molecule of the invention comprises a nucleotide sequence shown in FIG. 26A (SEQ ID NO: 48). In another embodiment, a polypeptide of the invention comprises an amino acid sequence shown in FIG. 26B (SEQ ID NO: 49).
In another embodiment, a polypeptide of the invention comprises an amino acid sequence shown in FIG. 12 (SEQ ID NO: 31).
In another embodiment, a polypeptide of the invention comprises an amino acid sequence shown in FIG. 12 (SEQ ID NO: 35).
The other nucleic acid and amino acid sequences disclosed herein in the sequence listing and Figures are also embraced by the invention.
D. Expression of Polypeptides
Following manipulation of the isolated genetic material to provide polypeptides of the invention as set forth above, the genes are typically inserted in an expression vector for introduction into host cells that may be used to produce the desired quantity of polypeptide that, in turn, provides the claimed polypeptides.
The term “vector” or “expression vector” is used herein for the purposes of the specification and claims, to mean vectors used in accordance with the present invention as a vehicle for introducing into and expressing a desired gene in a cell. As known to those skilled in the art, such vectors may easily be selected from the group consisting of plasmids, phages, viruses and retroviruses. In general, vectors compatible with the instant invention will comprise a selection marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.
For the purposes of this invention, numerous expression vector systems may be employed. For example, one class of vector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. Additionally, cells which have integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow selection of transfected host cells. The marker may provide for prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper. The selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals. In particularly preferred embodiments the cloned variable region genes are inserted into an expression vector along with the heavy and light chain constant region genes (preferably human) synthetic as discussed above. Preferably, this is effected using a proprietary expression vector of IDEC, Inc., referred to as NEOSPLA (U.S. Pat. No. 6,159,730). This vector contains the cytomegalovirus promoter/enhancer, the mouse beta globin major promoter, the SV40 origin of replication, the bovine growth hormone polyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2, the dihydrofolate reductase gene and leader sequence. As seen in the examples below, this vector has been found to result in very high level expression of antibodies upon incorporation of variable and constant region genes, transfection in CHO cells, followed by selection in G418 containing medium and methotrexate amplification. Vector systems are also taught in U.S. Pat. Nos. 5,736,137 and 5,658,570, each of which is incorporated by reference in its entirety herein. This system provides for high expression levels, e.g., >30 pg/cell/day. Other exemplary vector systems are disclosed e.g., in U.S. Pat. No. 6,413,777.
In other preferred embodiments the polypeptides of the invention of the instant invention may be expressed using polycistronic constructs such as those disclosed in copending U.S. provisional application No. 60/331,481 filed Nov. 16, 2001 and incorporated herein in its entirety. In these novel expression systems, multiple gene products of interest such as heavy and light chains of antibodies may be produced from a single polycistronic construct. These systems advantageously use an internal ribosome entry site (IRES) to provide relatively high levels of polypeptides of the invention in eukaryotic host cells. Compatible IRES sequences are disclosed in U.S. Pat. No. 6,193,980 which is also incorporated herein. Those skilled in the art will appreciate that such expression systems may be used to effectively produce the full range of polypeptides disclosed in the instant application.
More generally, once the vector or DNA sequence encoding a monomeric subunit of the polypeptide (e.g. a modified antibody) has been prepared, the expression vector may be introduced into an appropriate host cell. That is, the host cells may be transformed. Introduction of the plasmid into the host cell can be accomplished by various techniques well known to those of skill in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See, Ridgway, A. A. G. “Mammalian Expression Vectors” Chapter 24.2, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). Most preferably, plasmid introduction into the host is via electroporation. The transformed cells are grown under conditions appropriate to the production of the light chains and heavy chains, and assayed for heavy and/or light chain protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or flourescence-activated cell sorter analysis (FACS), immunohistochemistry and the like.
As used herein, the term “transformation” shall be used in a broad sense to refer to any introduction of DNA into a recipient host cell that changes the genotype and consequently results in a change in the recipient cell.
Along those same lines, “host cells” refers to cells that have been transformed with vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene. In descriptions of processes for isolation of antibodies from recombinant hosts, the terms “cell” and “cell culture” are used interchangeably to denote the source of antibody unless it is clearly specified otherwise. In other words, recovery of polypeptide from the “cells” may mean either from spun down whole cells, or from the cell culture containing both the medium and the suspended cells.
The host cell line used for protein expression is most preferably of mammalian origin; those skilled in the art are credited with ability to preferentially determine particular host cell lines which are best suited for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVI with SV40 T antigen), R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/O (mouse myeloma), P3.times.63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocyte) and 293 (human kidney). CHO cells are particularly preferred. Host cell lines are typically available from commercial services, the American Tissue Culture Collection or from published literature.
In vitro production allows scale-up to give large amounts of the desired polypeptides. Techniques for mammalian cell cultivation under tissue culture conditions are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilized or entrapped cell culture, e.g. in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges. If necessary and/or desired, the solutions of polypeptides can be purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose or (immuno-)affinity chromatography, e.g., after preferential biosynthesis of a synthetic hinge region polypeptide or prior to or subsequent to the HIC chromatography step described herein.
Genes encoding the polypeptide of the invention can also be expressed non-mammalian cells such as bacteria or yeast or plant cells. In this regard it will be appreciated that various unicellular non-mammalian microorganisms such as bacteria can also be transformed; i.e. those capable of being grown in cultures or fermentation. Bacteria, which are susceptible to transformation, include members of the enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae. It will further be appreciated that, when expressed in bacteria, the polypeptides typically become part of inclusion bodies. The polypeptides must be isolated, purified and then assembled into functional molecules. Where tetravalent forms of antibodies are desired, the subunits will then self-assemble into tetravalent antibodies (WO02/096948A2).
In addition to prokaryates, eukaryotic microbes may also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example, (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)) is commonly used. This plasmid already contains the TRP1 gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). The presence of the trp1 lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
IV. SEPARATION OF POLYPEPTIDES COMPRISING AT LEAST ONE INTERCHAIN DISULFIDE Linkage from Those Lacking Interchain Disulfide Linkages
In one aspect, the invention pertains to separation of molecules having two heavy chain portions from a mixture, where a fraction of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage and a fraction of the molecules comprise heavy chain portions that are not linked via at least one disulfide linkage by hydrophobic interaction chromatography.
Hydrophobic interaction chromatography was first developed following the observation that proteins could be retained on affinity gels which comprised hydrocarbon spacer arms but lacked the affinity ligand. Elution from HIC supports can be effected by alterations in solvent, pH, ionic strength, or by the addition of chaotropic agents or organic modifiers, such as ethylene or propylene glycol. A description of the general principles of hydrophobic interaction chromatography can be found e.g., in U.S. Pat. No. 3,917,527 and in U.S. Pat. No. 4,000,098. HIC in the context of high performance liquid chromatography (HPLC) has been used to separate antibody fragments lacking heavy chain portions (e.g., F(ab′)2) from intact antibody molecules in a single step protocol. (Morimoto, K. et al., L Biochem. Biophvs. Meth. 24: 107 (1992)).
The separation method of the invention can be performed on an unpurified population of polypeptides (e.g., culture supernatants or preparations or preparations of polypeptides isolated from prokaryotic inclusion bodies). Alternatively, the instant separation methods can be used on polypeptide mixtures obtained after one or more initial purification steps, e.g., after a preparation comprising forms A and B has been eluted from an affinity matrix.
In one embodiment, the binding molecules subjected to HIC chromatography comprise a connecting peptide of the invention.
In a preferred embodiment, HIC can be applied to mixtures that have been partially purified by other protein purification procedures. The term “partially purified” as used herein includes a protein preparation in which the protein of interest is present in at least 5% by weight, more preferably at least 10% and most preferably at least 45%. Initial or subsequent purification steps can be used to remove, e.g., immunoglobulin aggregates, misfolded species, host cell protein, residue material from preceding chromatographic steps (such as Protein A when employed). In one embodiment, HIC can be performed on polypeptides comprising a connecting peptide of the invention. Accordingly, the application of HIC can also be appreciated in the context of an overall purification protocol. Exemplary purification steps that can be used prior to or subsequent to HIC include: affinity chromatography (for example, PROSEP-A® (BioProcessing Ltd., U.K.) which consists of Protein A covalently coupled to controlled pore glass or Protein A SEPHAROSE® Fast Flow (Pharmacia) or TOYOPEARL 650M Protein A (Toso Haas)). Protein A is preferred for human γ1, γ2, or γ4 heavy chains and protein G for mouse isotypes. Bakerbond ABX™ resin can be used if the molecule comprises a CH3 domain. In addition or alternatively, ion exchange chromatography may be employed. In this regard various anionic or cationic substituents. may be attached to matrices in order to form anionic or cationic supports for chromatography. Anionic exchange substituents include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary amine (Q) groups. Cationic exchange substituents. include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S). Cellulose ion exchange resins such as DE23, DE32, DE52, CM-23, CM-32 and CM-52 are available from Whatman Ltd. Maidstone, Kent, U.K. SEPHADEX®-based and -locross-linked ion exchangers are also known. For example, DEAE-, QAE-, CM-, and SP-SEPHADEX® and DEAE-, Q-, CM- and S-SEPHAROSE® and SEPHAROSE® Fast Flow are all available from Pharmacia AB. Further, both DEAE and CM derivitized ethylene glycol-methacrylate copolymer such as TOYOPEARL DEAE-650S or M and TOYOPEARL CM-650S or M are available from Toso Haas Co., Philadelphia, Pa. Because elution from ion exchange supports usually involves addition of salt and because HIC is enhanced under increased salt concentrations, the introduction of a HIC step following an ionic exchange chromatographic step or other salt mediated purification step is preferred. Additional purification protocols may be added including but not necessarily limited to: further ionic exchange chromatography, size exclusion chromatography, viral inactivation, concentration and freeze drying, hydroxylapatite chromatography, gel electrophoresis, dialysis, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEQHAROSE™, chromatofocusing, or ammonium sulfate precipitation.
Prior to purification using the subject methods, the composition comprising the mixture of polypeptides to be separated will preferably be placed in a buffer of acidic or approximately neutral pH. This can be done, for example, by adding concentrated buffer, resuspending the sample in the buffer, exchanging the buffer (e.g., using dialysis or ultrafiltration). Alternatively, the pH of the sample buffer can simply be adjusted to be within the desired range.
Hydrophobic interactions are strongest at high ionic strength, therefore, this form of separation is conveniently performed following salt precipitations or ion exchange procedures. Adsorption of the proteins to a HIC column is favored by high salt concentrations, but the actual concentrations can vary over a wide range depending on the nature of the protein and the particular HIC ligand chosen. Various ions can be arranged in a so-called soluphobic series depending on whether they promote hydrophobic interactions (salting-out effects) or disrupt the structure of water (chaotropic effect) and lead to the weakening of the hydrophobic interaction. Cations are ranked in terms of increasing salting out effect as Ba++<; Ca++<; Mg++<; Li+<; Cs+<; Na+<; K+<; Rb+<; NH4+, while anions may be ranked in terms of increasing chaotropic effect as PO−−−<; SO4−−<; CH3COOO−<; Cl−<; Br−<; NO3−<; ClO4−<; I−<; SCN−.
In general, Na, K or NH4 sulfates effectively promote ligand-protein interaction in HIC. Salts may be formulated that influence the strength of the interaction as given by the following relationship: (NH4)2SO4>; Na2SO4>; NaCl>; NH4Cl>; NaBr>; NaSCN. In general, salt concentrations of between about 0.75 and about 2M ammonium sulfate or between about 1 and 4M NaCl are useful.
A number of chromatographic supports may be employed in the preparation of HIC columns, the most extensively used are agarose, silica and organic polymer or co-polymer resins. The hydrophobic interaction material is generally a base matrix (e.g., a hydrophilic carbohydrate (such as cross-linked agarose) or synthetic copolymer material) to which hydrophobic ligands (e.g., alkyl or aryl groups) are coupled. The preferred HIC material comprises an agarose resin substituted with phenyl groups. Exemplary HIC material includes: phenyl SEPHAROSE™, FAST FLOW with low or high substitution (Pharmacia LKB Biotechnology, AB, Sweden); phenyl SEPHAROSE™ High Performance column; phenyl or butyl-SEPHAROSE® CL-4B, butyl-SEPHAROSE® FF, octyl-SEPHAROSE® FF and phenyl-SEPHAROSE® FF (Pharmacia LKB Biotechnology AB, Sweden); Fractogel™ EMD Propyl or FRACTOGEL™ EMC Phenyl columns (E. Merck, Germany); MACROPREP™ Methyl or MACRO-PREP™ t-Butyl Supports (Bio-Rad, California); WP HI-Propyl (C3)™ column (J.T. Baker, New Jersey). Exemplary HIC materials are also available from Tosoh Corporation, Tokyo, Japan under the product names TOYOPEARL ether 650, phenyl 650, butyl 650 (Fractogel), ether-5PW-HR, or phenyl-5PW-HR; Miles-Yeda, Rehovot, Israel under the product name alkyl-agarose, wherein the alkyl group contains from 2-10 carbon atoms, and J.T. Baker, Phillipsburg, N.J. under the product name Bakerbond WP-HI-propyl. It is also possible to prepare the desired HIC column using conventional chemistry. (Sa: for example, Er-el. Z. gl all, Biochem. Biophys. Res. Comm. 49:383 (1972) or Ulbrich, V. rd gL Coll. Czech. Chem. Commum. 9:1466 (1964)).
The choice of a particular gel can be determined by the skilled artisan. In general the strength of the interaction of the protein and the HIC ligand increases with the chain length of the alkyl ligands but ligands having from about 4 to about 8 carbon atoms are suitable for most separations. A phenyl group has about the same hydrophobicity as a pentyl group, although the selectivity can be different owing to the possibility of pi-pi orbital interaction with aromatic groups on the protein. Selectively may also be affected by the chemistry of the supporting resin.
Ligand density is an important parameter in that it influences not only the strength of the interaction but the capacity of the column as well. The ligand density of the commercially available phenyl or octyl phenyl gels is on the order of 40 pmoles/ml gel bed. Gel capacity is a function of the particular protein in question as well as pH, temperature and salt type and concentration but generally can be expected to fall in the range of 3-20 mg/ml of gel.
In general, a decrease in temperature decreases the interaction with HIC material. However, any benefit that would accrue by increasing the temperature must also be weighed against adverse effects such an increase may have on the stability of the protein.
In one embodiment, the polypeptides of the invention can be eluted isocratically. In isocratic elution, all compounds begin migration through the column at onset. However, each migrates at a different rate, resulting in faster or slower elution rate. For example, as described in the instant examples, form A can be eluted with the flow through of the column.
In another embodiment, one or more polypeptides of the invention can be bound to the column and eluted, e.g., using stepwise elution or gradient elution. Elution, whether stepwise or in the form of a gradient, can be accomplished in a variety of ways: (a) by changing the salt concentration, (b) by changing the polarity of the solvent or (c) by adding detergents. By decreasing salt concentration adsorbed proteins are eluted in order of increasing hydrophobicity. Changes in polarity may be affected by additions of solvents such as ethylene or propylene glycol or (iso)propanol, thereby decreasing the strength of the hydrophobic interactions. Detergents function as displacers of proteins and have been used primarily in connection with the purification of membrane proteins.
In performing the separation, the polypeptide mixture can be contacted with the HIC material e.g., using a batch purification technique or using a column. Prior to HIC purification it may be desirable to remove any chaotropic agents or very hydrophobic substances, e.g., by passing the mixture through a precolumn.
For example, for batch purification, HIC material is prepared in or equilibrated to the desired starting buffer. A slurry of the HIC material is obtained. The polypeptide solution is contacted with the slurry to adsorb at least one of the polypeptides to be separated to the HIC material. The solution containing the polypeptides that do not bind to the HIC material is separated from the slurry, e.g., by allowing the slurry to settle and removing the supernatant. The slurry can be subjected to one or more washing steps. If desired, the slurry can be contacted with a solution of lower conductivity to desorb polypeptides that have bound to the HIC material. In order to elute bound polypeptides, the salt concentration can be decreased.
In one embodiment, the HIC material can be packed in a column. A mixture comprising the polypeptides to be separated can be applied to the column allowing at least one of the polypeptides to be separated to adsorb to the column. The polypeptides that do not adsorb to the column pass through and can be collected. In order to elute bound polypeptides, the salt concentration can be decreased, e.g., in a step-wise fashion or using a salt gradient.
Since form B is more hydrophobic than form A, it adsorbs irreversibly to the stationary phase using approximately 0.7 M (e.g., 0.73M) Ammonium Sulfate/20 mM Sodium Phosphate, pH 4.0 to pH 8.0 as the mobile phase. Form A binds to a lesser extent to the stationary phase under these conditions and is therefore eluted isocratically, i.e. it leaves the column with the flowthrough fraction. Subsequent to the isocratic elution of form A, omitting Ammonium sulfate from the mobile phase desorbs form B.
In an exemplary purification scheme, the HIC material is equilibrated in a buffer comprising a salt concentration yielding a conductivity of from between about 160 to about 110, preferably from between about 140 to about 115, even more preferably from between about 130 or about 120 to about 117 mS/cm. For example, an exemplary starting solution comprises a salt concentration of approximately 1M to 0.7M, e.g., 1M to 0.7M ammonium sulfate. In a preferred embodiment, the solution comprising the mixture of polypeptides to be separated is also brought to the same, or approximately the same conductivity (e.g., using a concentrated stock solution of salt). Under these conditions, Form A is eluted from the column at a conductivity of about 120 mS/cm. In order to elute Form B, a stepwise or linear gradient of reducing ammonium sulfate content can be applied to the column. Form B elutes at a conductivity of approximately 115 to approximately 100 mS/cm.
In one embodiment, the subject purification method yields a composition comprising polypeptide molecules having at least two binding sites and two heavy chain portions, wherein the heavy chain portions lack CH2 domains and wherein greater than 50% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage. In another embodiment, greater than 60% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage. In another embodiment, greater than 70% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage. In another embodiment, greater than 80% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage. In another embodiment, greater than 90% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage.
In one embodiment, the subject purification method yields a composition comprising recombinant polypeptide molecules having at least two binding sites and two heavy chain portions, wherein greater than 99% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage.
In one embodiment, the subject purification method yields a composition comprising polypeptide molecules having at least two binding sites and two heavy chain portions, wherein greater than 95% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage, and wherein the heavy chain portions of the polypeptides are derived from an antibody of the IgG4 isotype.
In one embodiment, the subject purification method yields a composition comprising polypeptide molecules having two light chain portions and two heavy chain portions, wherein the heavy chain portions lack CH2 domains and wherein greater than 80% of the molecules are present in a form in which the two heavy chain portions are not linked via at least one interchain disulfide linkage.
In another aspect, the instant invention also provides methods for monitoring the results of purification and/or preferential biosynthesis comprising measuring the relative amounts of Form A and Form B in a composition. Form A and Form B can be measured, e.g., as described herein using non-reducing SDS polyacrylamide gel electrophoresis or mass spectrometry.
V. LABELING OR CONJUGATION OF POLYPEPTIDES
The polypeptide molecules of the present invention may be used in non-conjugated form or may be conjugated to at least one of a variety of molecules, e.g., to facilitate target detection or for imaging or therapy of the patient. The polypeptides of the invention can be labeled or conjugated either before or after purification, when purification is performed. In particular, the polypeptides of the present invention may be conjugated to cytotoxins (such as radioisotopes, cytotoxic drugs, or toxins) therapeutic agents, cytostatic agents, biological toxins, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, immunologically active ligands (e.g., lymphokines or other antibodies wherein the resulting molecule binds to both the neoplastic cell and an effector cell such as a T cell), or PEG. In another embodiment, a polypeptide of the invention can be conjugated to a molecule that decreases vascularization of tumors. In other embodiments, the disclosed compositions may comprise polypeptides of the invention coupled to drugs or prodrugs. Still other embodiments of the present invention comprise the use of polypeptides of the invention conjugated to specific biotoxins or their cytotoxic fragments such as ricin, gelonin, pseudomonas exotoxin or diphtheria toxin. The selection of which conjugated or unconjugated polypeptide to use will depend on the type and stage of cancer, use of adjunct treatment (e.g., chemotherapy or external radiation) and patient condition. It will be appreciated that one skilled in the art could readily make such a selection in view of the teachings herein.
It will be appreciated that, in previous studies, anti-tumor antibodies labeled with isotopes have been used successfully to destroy cells in solid tumors as well as lymphomas/leukemias in animal models, and in some cases in humans. Exemplary radioisotopes include: 90Y, 125I, 131I, 123I, 111In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re and 188Re. The radionuclides act by producing ionizing radiation which causes multiple strand breaks in nuclear DNA, leading to cell death. The isotopes used to produce therapeutic conjugates typically produce high energy α- or β-particles which have a short path length. Such radionuclides kill cells to which they are in close proximity, for example neoplastic cells to which the conjugate has attached or has entered. They have little or no effect on non-localized cells. Radionuclides are essentially non-immunogenic.
With respect to the use of radiolabeled conjugates in conjunction with the present invention, polypeptides of the invention may be directly labeled (such as through iodination) or may be labeled indirectly through the use of a chelating agent. As used herein, the phrases “indirect labeling” and “indirect labeling approach” both mean that a chelating agent is covalently attached to a binding molecule and at least one radionuclide is associated with the chelating agent. Such chelating agents are typically referred to as bifunctional chelating agents as they bind both the polypeptide and the radioisotope. Particularly preferred chelating agents comprise 1-isothiocycmatobenzyl-3-methyldiothelene triaminepentaacetic acid (“MX-DTPA”) and cyclohexyl diethylenetriamine pentaacetic acid (“CHX-DTPA”) derivatives. Other chelating agents comprise P-DOTA and EDTA derivatives. Particularly preferred radionuclides for indirect labeling include 111In and 90Y.
As used herein, the phrases “direct labeling” and “direct labeling approach” both mean that a radionuclide is covalently attached directly to a polypeptide (typically via an amino acid residue). More specifically, these linking technologies include random labeling and site-directed labeling. In the latter case, the labeling is directed at specific sites on the polypeptide, such as the N-linked sugar residues present only on the Fc portion of the conjugates. Further, various direct labeling techniques and protocols are compatible with the instant invention. For example, Technetium-99m labeled polypeptides may be prepared by ligand exchange processes, by reducing pertechnate (TcO4−) with stannous ion solution, chelating the reduced technetium onto a Sephadex column and applying the polypeptides to this column, or by batch labeling techniques, e.g. by incubating pertechnate, a reducing agent such as SnCl2, a buffer solution such as a sodium-potassium phthalate-solution, and the antibodies. In any event, preferred radionuclides for directly labeling antibodies are well known in the art and a particularly preferred radionuclide for direct labeling is 131I covalently attached via tyrosine residues. Polypeptides according to the invention may be derived, for example, with radioactive sodium or potassium iodide and a chemical oxidizing agent, such as sodium hypochlorite, chloramine T or the like, or an enzymatic oxidizing agent, such as lactoperoxidase, glucose oxidase and glucose. However, for the purposes of the present invention, the indirect labeling approach is particularly preferred.
Patents relating to chelators and chelator conjugates are known in the art. For instance, U.S. Pat. No. 4,831,175 of Gansow is directed to polysubstituted diethylenetriaminepentaacetic acid chelates and protein conjugates containing the same, and methods for their preparation. U.S. Pat. Nos. 5,099,069, 5,246,692, 5,286,850, 5,434,287 and 5,124,471 of Gansow also relate to polysubstituted DTPA chelates. These patents are incorporated herein in their entirety. Other examples of compatible metal chelators are ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DPTA), 1,4,8,11-tetraazatetradecane, 1,4,8,11-tetraazatetradecane-1,4,8,11-tetraacetic acid, 1-oxa-4,7,12,15-tetraazaheptadecane-4,7,12,15-tetraacetic acid, or the like. Cyclohexyl-DTPA or CHX-DTPA is particularly preferred and is exemplified extensively below. Still other compatible chelators, including those yet to be discovered, may easily be discerned by a skilled artisan and are clearly within the scope of the present invention.
Compatible chelators, including the specific bifunctional chelator used to facilitate chelation in co-pending application Ser. Nos. 08/475,813, 08/475,815 and 08/478,967, are preferably selected to provide high affinity for trivalent metals, exhibit increased tumor-to-non-tumor ratios and decreased bone uptake as well as greater in vivo retention of radionuclide at target sites, i.e., B-cell lymphoma tumor sites. However, other bifunctional chelators that may or may not possess all of these characteristics are known in the art and may also be beneficial in tumor therapy. It will also be appreciated that, in accordance with the teachings herein, polypeptides may be conjugated to different radiolabels for diagnostic and therapeutic purposes. To this end the aforementioned co-pending applications, herein incorporated by reference in their entirety, disclose radiolabeled therapeutic conjugates for diagnostic “imaging” of tumors before administration of therapeutic antibody. “In2B8” conjugate comprises a murine monoclonal antibody, 2B8, specific to human CD20 antigen, that is attached to 111In via a bifunctional chelator, i.e., MX-DTPA (diethylenetriaminepentaacetic acid), which comprises a 1:1 mixture of 1-isothiocyanatobenzyl-3-methyl-DTPA and 1-methyl-3-isothiocyanatobenzyl-DTPA. 111In is particularly preferred as a diagnostic radionuclide because between about 1 to about 10 mCi can be safely administered without detectable toxicity; and the imaging data is generally predictive of subsequent 90Y-labeled antibody distribution. Most imaging studies utilize 5 mCi 111In-labeled antibody, because this dose is both safe and has increased imaging efficiency compared with lower doses, with optimal imaging occurring at three to six days after antibody administration. See, for example, Murray, J. Nuc. Med. 26: 3328 (1985) and Carraguillo et al., J. Nuc. Med. 26: 67 (1985).
As indicated above, a variety of radionuclides are applicable to the present invention and those skilled in the can readily determine which radionuclide is most appropriate under various circumstances. For example, 131I is a well known radionuclide used for targeted immunotherapy. However, the clinical usefulness of 131I can be limited by several factors including: eight-day physical half-life; dehalogenation of iodinated antibody both in the blood and at tumor sites; and emission characteristics (e.g., large gamma component) which can be suboptimal for localized dose deposition in tumor. With the advent of superior chelating agents, the opportunity for attaching metal chelating groups to proteins has increased the opportunities to utilize other radionuclides such as 111In and 90Y. 90Y provides several benefits for utilization in radioimmunotherapeutic applications: the 64 hour half-life of 90Y is long enough to allow antibody accumulation by tumor and, unlike e.g., 131I, 90Y is a pure beta emitter of high energy with no accompanying gamma irradiation in its decay, with a range in tissue of 100 to 1,000 cell diameters. Furthermore, the minimal amount of penetrating radiation allows for outpatient administration of 90Y-labeled antibodies. Additionally, internalization of labeled antibody is not required for cell killing, and the local emission of ionizing radiation should be lethal for adjacent tumor cells lacking the target molecule.
Those skilled in the art will appreciate that these non-radioactive conjugates may also be assembled using a variety of techniques depending on the selected agent to be conjugated. For example, conjugates with biotin are prepared e.g. by reacting the polypeptides with an activated ester of biotin such as the biotin N-hydroxysuccinimide ester. Similarly, conjugates with a fluorescent marker may be prepared in the presence of a coupling agent, e.g. those listed above, or by reaction with an isothiocyanate, preferably fluorescein-isothiocyanate. Conjugates of the polypeptides of the invention with cytostatic/cytotoxic substances and metal chelates are prepared in an analogous manner.
Preferred agents for use in the present invention are cytotoxic drugs, particularly those which are used for cancer therapy. As used herein, “a cytotoxin or cytotoxic agent” means any agent that is detrimental to the growth and proliferation of cells and may act to reduce, inhibit or destroy a cell or malignancy. Exemplary cytotoxins include, but are not limited to, radionuclides, biotoxins, enzymatically active toxins, cytostatic or cytotoxic therapeutic agents, prodrugs, immunologically active ligands and biological response modifiers such as cytokines. Any cytotoxin that acts to retard or slow the growth of immunoreactive cells or malignant cells is within the scope of the present invention.
Exemplary cytotoxins include, in general, cytostatic agents, alkylating agents, antimetabolites, anti-proliferative agents, tubulin binding agents, hormones and hormone antagonists, and the like. Exemplary cytostatics that are compatible with the present invention include alkylating substances, such as mechlorethamine, triethylenephosphoramide, cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan or triaziquone, also nitrosourea compounds, such as carmustine, lomustine, or semustine. Other preferred classes of cytotoxic agents include, for example, the maytansinoid family of drugs. Other preferred classes of cytotoxic agents include, for example, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, diynenes, and the podophyllotoxins. Particularly useful members of those classes include, for example, adriamycin, carminomycin, daunorubicin (daunomycin), doxorubicin, aminopterin, methotrexate, methopterin, mithramycin, streptonigrin, dichloromethotrexate, mitomycin C, actinomycin-D, porfiromycin, 5-fluorouracil, floxuridine, ftorafur, 6-mercaptopurine, cytarabine, cytosine arabinoside, podophyllotoxin, or podophyllotoxin derivatives such as etoposide or etoposide phosphate, melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosine and the like. Still other cytotoxins that are compatible with the teachings herein include taxol, taxane, cytochalasin B, gramicidin D, ethidium bromide, emetine, tenoposide, colchicin, dihydroxy anthracin dione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Hormones and hormone antagonists, such as corticosteroids, e.g. prednisone, progestins, e.g. hydroxyprogesterone or medroprogesterone, estrogens, e.g. diethylstilbestrol, antiestrogens, e.g. tamoxifen, androgens, e.g. testosterone, and aromatase inhibitors, e.g. aminogluthetimide are also compatible with the teachings herein. As noted previously, one skilled in the art may make chemical modifications to the desired compound in order to make reactions of that compound more convenient for purposes of preparing conjugates of the invention.
One example of particularly preferred cytotoxins comprise members or derivatives of the enediyne family of anti-tumor antibiotics, including calicheamicin, esperamicins or dynemicins. These toxins are extremely potent and act by cleaving nuclear DNA, leading to cell death. Unlike protein toxins which can be cleaved in vivo to give many inactive but immunogenic polypeptide fragments, toxins such as calicheamicin, esperamicins and other enediynes are small molecules which are essentially non-immunogenic. These non-peptide toxins are chemically-linked to the dimers or tetramers by techniques which have been previously used to label monoclonal antibodies and other molecules. These linking technologies include site-specific linkage via the N-linked sugar residues present only on the Fc portion of the constructs. Such site-directed linking methods have the advantage of reducing the possible effects of linkage on the binding properties of the constructs.
As previously alluded to, compatible cytotoxins may comprise a prodrug. As used herein, the term “prodrug” refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. Prodrugs compatible with the invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate containing prodrugs, peptide containing prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs that can be converted to the more active cytotoxic free drug. Further examples of cytotoxic drugs that can be derivatized into a prodrug form for use in the present invention comprise those chemotherapeutic agents described above.
Among other cytotoxins, it will be appreciated that polypeptides can also be associated with a biotoxin such as ricin subunit A, abrin, diptheria toxin, botulinum, cyanginosins, saxitoxin, shigatoxin, tetanus, tetrodotoxin, trichothecene, verrucologen or a toxic enzyme. Preferably, such constructs will be made using genetic engineering techniques that allow for direct expression of the antibody-toxin construct. Other biological response modifiers that may be associated with the polypeptides of the invention of the present invention comprise cytokines such as lymphokines and interferons. In view of the instant disclosure it is submitted that one skilled in the art could readily form such constructs using conventional techniques.
Another class of compatible cytotoxins that may be used in conjunction with the disclosed polypeptides are radiosensitizing drugs that may be effectively directed to tumor or immunoreactive cells. Such drugs enhance the sensitivity to ionizing radiation, thereby increasing the efficacy of radiotherapy. An antibody conjugate internalized by the tumor cell would deliver the radiosensitizer nearer the nucleus where radiosensitization would be maximal. The unbound radiosensitizer linked polypeptides of the invention would be cleared quickly from the blood, localizing the remaining radiosensitization agent n the target tumor and providing minimal uptake in normal tissues. After rapid clearance from the blood, adjunct radiotherapy would be administered in one of three ways: 1.) external beam radiation directed specifically to the tumor, 2.) radioactivity directly implanted in the tumor or 3.) systemic radioimmunotherapy with the same targeting antibody. A potentially attractive variation of this approach would be the attachment of a therapeutic radioisotope to the radiosensitized immunoconjugate, thereby providing the convenience of administering to the patient a single drug.
In one embodiment, a moiety that enhances the stability or efficacy of the polypeptide can be conjugated. For example, in one embodiment, PEG can be conjugated to the polypeptides of the invention to increase their half-life in vivo. Leong, S. R., et al. 2001. Cytokine 16:106; 2002; Adv. in Drug Deliv. Rev. 54:531; or Weir et al. 2002. Biochem. Soc. Transactions 30:512.
VI. ADMINISTRATION OF POLYPEPTIDES
Methods of preparing and administering polypeptides of the invention to a subject are well known to or are readily determined by those skilled in the art. The route of administration of the polypeptide of the invention may be oral, parenteral, by inhalation or topical. The term parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. The intravenous, intraarterial, subcutaneous and intramuscular forms of parenteral administration are generally preferred. While all these forms of administration are clearly contemplated as being within the scope of the invention, a form for administration would be a solution for injection, in particular for intravenous or intraarterial injection or drip. Usually, a suitable pharmaceutical composition for injection may comprise a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. human albumin), etc. However, in other methods compatible with the teachings herein, the polypeptides can be delivered directly to the site of the adverse cellular population thereby increasing the exposure of the diseased tissue to the therapeutic agent.
Preparations for parenteral administration includes sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. In the subject invention, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1M and preferably 0.05M phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. More particularly, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and will preferably be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity 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. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. 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. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
In any case, sterile injectable solutions can be prepared by incorporating an active compound (e.g., a polypeptide by itself or in combination with other active agents) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yields a powder of an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparations for injections are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. Further, the preparations may be packaged and sold in the form of a kit such as those described in co-pending U.S. Ser. No. 09/259,337 and U.S. Ser. No. 09/259,338 each of which is incorporated herein by reference. Such articles of manufacture will preferably have labels or package inserts indicating that the associated compositions are useful for treating a subject suffering from, or predisposed to autoimmune or neoplastic disorders. Effective doses of the compositions of the present invention, for the treatment of the above described conditions vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human but non-human mammals including transgenic mammals can also be treated. Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy. For passive immunization with an antibody, the dosage can range, e.g., from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), of the host body weight. For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg, preferably at least 1 mg/kg. Doses intermediate in the above ranges are also intended to be within the scope of the invention. Subjects can be administered such doses daily, on alternative days, weekly or according to any other schedule determined by empirical analysis. An exemplary treatment entails administration in multiple dosages over a prolonged period, for example, of at least six months. Additional exemplary treatment regimes entail administration once per every two weeks or once a month or once every 3 to 6 months. Exemplary dosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kg weekly. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated.
Polypeptides of the invention can be administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of polypeptide or target molecule in the patient. In some methods, dosage is adjusted to achieve a plasma polypeptide concentration of 1-1000 μg/ml and in some methods 25-300 μg/ml. Alternatively, binding molecules can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, humanized antibodies show the longest half-life, followed by chimeric antibodies and nonhuman antibodies. In one embodiment, the binding molecules of the invention can be administered in unconjugated form, In another embodiment, the polypeptides of the invention can be administered multiple times in conjugated form. In still another embodiment, the binding molecules of the invention can be administered in unconjugated form, then in conjugated form, or vise versa.
The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions containing the present antibodies or a cocktail thereof are administered to a patient not already in the disease state to enhance the patient's resistance. Such an amount is defined to be a “prophylactic effective dose.” In this use, the precise amounts again depend upon the patient's state of health and general immunity, but generally range from 0.1 to 25 mg per dose, especially 0.5 to 2.5 mg per dose. A relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives.
In therapeutic applications, a relatively high dosage (e.g., from about 1 to 400 mg/kg of binding molecule, e.g., antibody per dose, with dosages of from 5 to 25 mg being more commonly used for radioimmunoconjugates and higher doses for cytotoxin-drug conjugated molecules) at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patent can be administered a prophylactic regime.
In one embodiment, a subject can be treated with a nucleic acid molecule encoding a polypeptide of the invention (e.g., in a vector). Doses for nucleic acids encoding polypeptides range from about 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per patient. Doses for infectious viral vectors vary from 10-100, or more, virions per dose.
Therapeutic agents can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or intramuscular means for prophylactic and/or therapeutic treatment. The most typical route of administration of an immunogenic agent is subcutaneous although other routes can be equally effective. The next most common route is intramuscular injection. This type of injection is most typically performed in the arm or leg muscles. In some methods, agents are injected directly into a particular tissue where deposits have accumulated, for example intracranial injection. Intramuscular injection or intravenous infusion are preferred for administration of antibody. In some methods, particular therapeutic antibodies are injected directly into the cranium. In some methods, antibodies are administered as a sustained release composition or device, such as a Medipad™ device.
Agents of the invention can optionally be administered in combination with other agents that are effective in treating the disorder or condition in need of treatment (e.g., prophylactic or therapeutic).
Effective single treatment dosages (i.e., therapeutically effective amounts) of 90Y-labeled polypeptides of the invention range from between about 5 and about 75 mCi, more preferably between about 10 and about 40 mCi. Effective single treatment non-marrow ablative dosages of 131I-labeled antibodies range from between about 5 and about 70 mCi, more preferably between about 5 and about 40 mCi. Effective single treatment ablative dosages (i.e., may require autologous bone marrow transplantation) of 131I-labeled antibodies range from between about 30 and about 600 mCi, more preferably between about 50 and less than about 500 mCi. In conjunction with a chimeric antibody, owing to the longer circulating half life vis-á-vis murine antibodies, an effective single treatment non-marrow ablative dosages of iodine-131 labeled chimeric antibodies range from between about 5 and about 40 mCi, more preferably less than about 30 mCi. Imaging criteria for, e.g., the 111In label, are typically less than about 5 mCi.
While a great deal of clinical experience has been gained with 131I and 90Y, other radiolabels are known in the art and have been used for similar purposes. Still other radioisotopes are used for imaging. For example, additional radioisotopes which are compatible with the scope of the instant invention include, but are not limited to, 123I, 125I, 32P, 57Co, 64Cu, 67Cu, 77Br, 81Rb, 81Kr, 87Sr, 113In, 127Cs, 129Cs, 132I, 197Hg, 203Pb, 206Bi, 177Lu, 186Re, 212Pb, 212Bi, 47Sc, 105Rh, 109Pd, 153Sm, 188Re, 199Au, 225Ac 211At, and 213Bi. In this respect alpha, gamma and beta emitters are all compatible with in the instant invention. Further, in view of the instant disclosure it is submitted that one skilled in the art could readily determine which radionuclides are compatible with a selected course of treatment without undue experimentation. To this end, additional radionuclides which have already been used in clinical diagnosis include 125I, 123I, 99Tc, 43K, 52Fe, 67Ga, 68Ga, as well as 111In. Antibodies have also been labeled with a variety of radionuclides for potential use in targeted immunotherapy (Peirersz et al. Immunol. Cell Biol. 65: 111-125 (1987)). These radionuclides include 188Re and 186Re as well as 199Au and 67Cu to a lesser extent. U.S. Pat. No. 5,460,785 provides additional data regarding such radioisotopes and is incorporated herein by reference.
Whether or not the polypeptides of the invention are used in a conjugated or unconjugated form, it will be appreciated that a major advantage of the present invention is the ability to use these polypeptides in myelosuppressed patients, especially those who are undergoing, or have undergone, adjunct therapies such as radiotherapy or chemotherapy. That is, the beneficial delivery profile (i.e. relatively short serum dwell time, high binding affinity and enhanced localization) of the polypeptides makes them particularly useful for treating patients that have reduced red marrow reserves and are sensitive to myelotoxicity. In this regard, the unique delivery profile of the polypeptides make them very effective for the administration of radiolabeled conjugates to myelosuppressed cancer patients. As such, the polypeptides of the invention are useful in a conjugated or unconjugated form in patients that have previously undergone adjunct therapies such as external beam radiation or chemotherapy. In other preferred embodiments, the polypeptides (again in a conjugated or unconjugated form) may be used in a combined therapeutic regimen with chemotherapeutic agents. Those skilled in the art will appreciate that such therapeutic regimens may comprise the sequential, simultaneous, concurrent or coextensive administration of the disclosed antibodies and one or more chemotherapeutic agents. Particularly preferred embodiments of this aspect of the invention will comprise the administration of a radiolabeled polypeptide.
While the polypeptides may be administered as described immediately above, it must be emphasized that in other embodiments conjugated and unconjugated polypeptides may be administered to otherwise healthy patients as a first line therapeutic agent. In such embodiments the polypeptides may be administered to patients having normal or average red marrow reserves and/or to patients that have not, and are not, undergoing adjunct therapies such as external beam radiation or chemotherapy.
However, as discussed above, selected embodiments of the invention comprise the administration of polypeptides to myelosuppressed patients or in combination or conjunction with one or more adjunct therapies such as radiotherapy or chemotherapy (i.e. a combined therapeutic regimen). As used herein, the administration of polypeptides in conjunction or combination with an adjunct therapy means the sequential, simultaneous, coextensive, concurrent, concomitant or contemporaneous administration or application of the therapy and the disclosed polypeptides. Those skilled in the art will appreciate that the administration or application of the various components of the combined therapeutic regimen may be timed to enhance the overall effectiveness of the treatment. For example, chemotherapeutic agents could be administered in standard, well known courses of treatment followed within a few weeks by radioimmunoconjugates of the present invention. Conversely, cytotoxin associated polypeptides could be administered intravenously followed by tumor localized external beam radiation. In yet other embodiments, the polypeptide may be administered concurrently with one or more selected chemotherapeutic agents in a single office visit. A skilled artisan (e.g. an experienced oncologist) would be readily be able to discern effective combined therapeutic regimens without undue experimentation based on the selected adjunct therapy and the teachings of the instant specification.
In this regard it will be appreciated that the combination of the polypeptide (with or without cytotoxin) and the chemotherapeutic agent may be administered in any order and within any time frame that provides a therapeutic benefit to the patient. That is, the chemotherapeutic agent and polypeptide may be administered in any order or concurrently. In selected embodiments the polypeptides of the present invention will be administered to patients that have previously undergone chemotherapy. In yet other embodiments, the polypeptides and the chemotherapeutic treatment will be administered substantially simultaneously or concurrently. For example, the patient may be given the binding molecule while undergoing a course of chemotherapy. In preferred embodiments the binding molecule will be administered within 1 year of any chemotherapeutic agent or treatment. In other preferred embodiments the polypeptide will be administered within 10, 8, 6, 4, or 2 months of any chemotherapeutic agent or treatment. In still other preferred embodiments the polypeptide will be administered within 4, 3, 2 or 1 week of any chemotherapeutic agent or treatment. In yet other embodiments the polypeptide will be administered within 5, 4, 3, 2 or 1 days of the selected chemotherapeutic agent or treatment. It will further be appreciated that the two agents or treatments may be administered to the 30′ patient within a matter of hours or minutes (i.e. substantially simultaneously).
Moreover, in accordance with the present invention a myelosuppressed patient shall be held to mean any patient exhibiting lowered blood counts. Those skilled in the art will appreciate that there are several blood count parameters conventionally used as clinical indicators of myelosuppresion and one can easily measure the extent to which myelosuppresion is occurring in a patient. Examples of art accepted myelosuppression measurements are the Absolute Neutrophil Count (ANC) or platelet count. Such myelosuppression or partial myeloablation may be a result of various biochemical disorders or diseases or, more likely, as the result of prior chemotherapy or radiotherapy. In this respect, those skilled in the art will appreciate that patients who have undergone traditional chemotherapy typically exhibit reduced red marrow reserves. As discussed above, such subjects often cannot be treated using optimal levels of cytotoxin (i.e. radionuclides) due to unacceptable side effects such as anemia or immunosuppression that result in increased mortality or morbidity.
More specifically conjugated or unconjugated polypeptides of the present invention may be used to effectively treat patients having ANCs lower than about 2000/mm3 or platelet counts lower than about 150,000/mm3. More preferably the polypeptides of the present invention may be used to treat patients having ANCs of less than about 1500/mm3, less than about 1000/mm3 or even more preferably less than about 500/mm3. Similarly, the polypeptides of the present invention may be used to treat patients having a platelet count of less than about 75,000/mm3, less than about 50,000/mm3 or even less than about 10,000/mm3. In a more general sense, those skilled in the art will easily be able to determine when a patient is myelosuppressed using government implemented guidelines and procedures.
As indicated above, many myelosuppressed patients have undergone courses of treatment including chemotherapy, implant radiotherapy or external beam radiotherapy. In the case of the latter, an external radiation source is for local irradiation of a malignancy. For radiotherapy implantation methods, radioactive reagents are surgically located within the malignancy, thereby selectively irradiating the site of the disease. In any event, the disclosed polypeptides may be used to treat disorders in patients exhibiting myelosuppression regardless of the cause.
In this regard it will further be appreciated that the polypeptides of the instant invention may be used in conjunction or combination with any chemotherapeutic agent or agents (e.g. to provide a combined therapeutic regimen) that eliminates, reduces, inhibits or controls the growth of neoplastic cells in vivo. As discussed, such agents often result in the reduction of red marrow reserves. This reduction may be offset, in whole or in part, by the diminished myelotoxicity of the compounds of the present invention that advantageously allow for the aggressive treatment of neoplasias in such patients. In other preferred embodiments the radiolabeled immunoconjugates disclosed herein may be effectively used with radiosensitizers that increase the susceptibility of the neoplastic cells to radionuclides. For example, radiosensitizing compounds may be administered after the radiolabeled binding molecule has been largely cleared from the bloodstream but still remains at therapeutically effective levels at the site of the tumor or tumors.
With respect to these aspects of the invention, exemplary chemotherapeutic agents that are compatible with the instant invention include alkylating agents, vinca alkaloids (e.g., vincristine and vinblastine), procarbazine, methotrexate and prednisone. The four-drug combination MOPP (mechlethamine (nitrogen mustard), vincristine (Oncovin), procarbazine and prednisone) is very effective in treating various types of lymphoma and comprises a preferred embodiment of the present invention. In MOPP-resistant patients, ABVD (e.g., adriamycin, bleomycin, vinblastine and dacarbazine), ChlVPP (chlorambucil, vinblastine, procarbazine and prednisone), CABS (lomustine, doxorubicin, bleomycin and streptozotocin), MOPP plus ABVD, MOPP plus ABV (doxorubicin, bleomycin and vinblastine) or BCVPP (carmustine, cyclophosphamide, vinblastine, procarbazine and prednisone) combinations can be used. Arnold S. Freedman and Lee M. Nadler, Malignant Lymphomas, in HARRISON'S PRINCIPLES OF INTERNAL MEDICINE 1774-1788 (Kurt J. Isselbacher et al., eds., 13th ed. 1994) and V. T. DeVita et al., (1997) and the references cited therein for standard dosing and scheduling. These therapies can be used unchanged, or altered as needed for a particular patient, in combination with one or more polypeptides of the invention as described herein.
Additional regimens that are useful in the context of the present invention include use of single alkylating agents such as cyclophosphamide or chlorambucil, or combinations such as CVP (cyclophosphamide, vincristine and prednisone), CHOP (CVP and doxorubicin), C-MOPP (cyclophosphamide, vincristine, prednisone and procarbazine), CAP-BOP (CHOP plus procarbazine and bleomycin), m-BACOD (CHOP plus methotrexate, bleomycin and leucovorin), ProMACE-MOPP (prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide and leucovonn plus standard MOPP), ProMACE-CytaBOM (prednisone, doxorubicin, cyclophosphamide, etoposide, cytarabine, bleomycin, vincristine, methotrexate and leucovorin) and MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, fixed dose prednisone, bleomycin and leucovorin). Those skilled in the art will readily be able to determine standard dosages and scheduling for each of these regimens. CHOP has also been combined with bleomycin, methotrexate, procarbazine, nitrogen mustard, cytosine arabinoside and etoposide. Other compatible chemotherapeutic agents include, but are not limited to, 2-chlorodeoxyadenosine (2-CDA), 2′-deoxycoformycin and fludarabine.
For patients with intermediate- and high-grade NHL, who fail to achieve remission or relapse, salvage therapy is used. Salvage therapies employ drugs such as cytosine arabinoside, cisplatin, etoposide and ifosfamide given alone or in combination. In relapsed or aggressive forms of certain neoplastic disorders the following protocols are often used: IMVP-16 (ifosfamide, methotrexate and etoposide), MIME (methyl-gag, ifosfamide, methotrexate and etoposide), DHAP (dexamethasone, high dose cytarabine and cisplatin), ESHAP (etoposide, methylpredisolone, HD cytarabine, cisplatin), CEPP(B) (cyclophosphamide, etoposide, procarbazine, prednisone and bleomycin) and CAMP (lomustine, mitoxantrone, cytarabine and prednisone) each with well known dosing rates and schedules.
The amount of chemotherapeutic agent to be used in combination with the polypeptides of the instant invention may vary by subject or may be administered according to what is known in the art. See for example, Bruce A Chabner et al., Antineoplastic Agents, in GOODMAN & GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS 1233-1287 ((Joel G. Hardman et al., eds., 9th ed. 1996).
As previously discussed, the polypeptides of the present invention, immunoreactive fragments or recombinants thereof may be administered in a pharmaceutically effective amount for the in vivo treatment of mammalian disorders. In this regard, it will be appreciated that the disclosed antibodies will be formulated so as to facilitate administration and promote stability of the active agent. Preferably, pharmaceutical compositions in accordance with the present invention comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like. For the purposes of the instant application, a pharmaceutically effective amount of the polypeptide, immunoreactive fragment or recombinant thereof, conjugated or unconjugated to a therapeutic agent, shall be held to mean an amount sufficient to achieve effective binding to a target and to achieve a benefit, e.g., to ameliorate symptoms of a disease or disorder or to detect a substance or a cell. In the case of tumor cells, the polypeptide will be preferably be capable of interacting with selected immunoreactive antigens on neoplastic or immunoreactive cells and provide for an increase in the death of those cells. Of course, the pharmaceutical compositions of the present invention may be administered in single or multiple doses to provide for a pharmaceutically effective amount of the polypeptide.
In keeping with the scope of the present disclosure, the polypeptides of the invention may be administered to a human or other animal in accordance with the aforementioned methods of treatment in an amount sufficient to produce a therapeutic or prophylactic effect. The polypeptides of the invention can be administered to such human or other animal in a conventional dosage form prepared by combining the antibody of the invention with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. It will be recognized by one of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. Those skilled in the art will further appreciate that a cocktail comprising one or more species of polypeptides according to the present invention may prove to be particularly effective.
VI. METHODS OF USE
The molecules of the invention can be used for diagnostic or therapeutic purposes. Preferred embodiments of the present invention provide compounds, compositions, kits and methods for the diagnosis and/or treatment of disorders, e.g., neoplastic disorders in a mammalian subject in need of such treatment. Preferably, the subject is a human.
The polypeptides of the instant invention will be useful in a number of different applications. For example, in one embodiment, the subject binding molecules should be useful for reducing or eliminating cells bearing target (e.g., an epitope) recognized by a binding molecule of the invention. In another embodiment, the subject binding molecules are effective in reducing the concentration of or eliminating soluble target molecules in the circulation.
In one embodiment, tumor size, inhibiting tumor growth and/or prolonging the survival time of tumor-bearing animals. Accordingly, this invention also relates to a method of treating tumors in a human or other animal by administering to such human or animal an effective, non-toxic amount of polypeptide. One skilled in the art would be able, by routine experimentation, to determine what an effective, non-toxic amount of polypeptide would be for the purpose of treating malignancies. For example, a therapeutically active amount of a polypeptide may vary according to factors such as the disease stage (e.g., stage I versus stage IV), age, sex, medical complications (e.g., immunosuppressed conditions or diseases) and weight of the subject, and the ability of the antibody to elicit a desired response in the subject. The dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. Generally, however, an effective dosage is expected to be in the range of about 0.05 to 100 milligrams per kilogram body weight per day and more preferably from about 0.5 to 10, milligrams per kilogram body weight per day.
For purposes of clarification “mammal” refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human. “Treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disease or disorder as well as those in which the disease or disorder is to be prevented. Hence, the mammal may have been diagnosed as having the disease or disorder or may be predisposed or susceptible to the disease.
As discussed above, the polypeptides of the present invention may bind to one or more tumor molecules or molecules associated with immune disorders. In one embodiment, for neoplastic disorders, an antigen binding site (i.e. the variable region or immunoreactive fragment or recombinant thereof) of the disclosed polypeptides binds to a selected tumor associated molecule at the site of the malignancy. Similarly, in immune (including autoimmune) disorders the disclosed polypeptides will bind to selected markers on the offending cells. Given the number of reported molecules associated with neoplasias and immune disorders, and the number of related antibodies, those skilled in the art will appreciate that the presently disclosed polypeptides may therefore be derived from any one of a number of whole antibodies. More generally, polypeptides useful in the present invention may be obtained or derived from any antibody (including those previously reported in the literature) that reacts with a target or marker associated with the selected condition. Further, the parent or precursor antibody, or fragment thereof, used to generate the disclosed polypeptides may be murine, human, chimeric, humanized, non-human primate or primatized. In other preferred embodiments the polypeptides of the present invention may comprise single chain antibody constructs (such as that disclosed in U.S. Pat. No. 5,892,019 which is incorporated herein by reference) having altered constant domains as described herein. Consequently, any of these types of antibodies synthetic in accordance with the teachings herein is compatible with the instant invention.
As used herein, “tumor associated molecules” means any antigen or target molecule which is generally associated with tumor cells, i.e., occurring at the same or to a greater extent as compared with normal cells. More generally, tumor associated molecules comprise any molecule that provides for the localization of immunoreactive antibodies at a neoplastic cell irrespective of its expression on non-malignant cells. Such molecules may be relatively tumor specific and limited in their expression to the surface of malignant cells. Alternatively, such molecules may be found on both malignant and non-malignant cells. For example, CD20 is a pan B antigen that is found on the surface of both malignant and non-malignant B cells that has proved to be an extremely effective target for immunotherapeutic antibodies for the treatment of non-Hodgkin's lymphoma. In this respect, pan T cell antigens such as CD2, CD3, CD5, CD6 and CD7 also comprise tumor associated molecules within the meaning of the present invention. Still other exemplary tumor associated molecules comprise but not limited to MAGE-1, MAGE-3, MUC-1, HPV 16, HPV E6 & E7, TAG-72, CEA, L6-Antigen, CD19, CD22, CD37, CD52, HLA-DR, EGF Receptor and HER2 Receptor. In many cases immunoreative antibodies for each of these antigens have been reported in the literature. Those skilled in the art will appreciate that each of these antibodies may serve as a precursor for polypeptides of the invention in accordance with the present invention.
The polypeptides of the present invention preferably associate with, and bind to, tumor or immune associated molecules as described above. Accordingly, as will be discussed in some detail below the polypeptides of the present invention may be derived, generated or fabricated from any one of a number of antibodies that react with tumor associated molecules. In preferred embodiments the polypeptides are synthetic or domain deleted antibodies that are derived using common genetic engineering techniques whereby at least a portion of one or more constant region domains are deleted or altered so as to provide the desired biochemical characteristics such as reduced serum half-life. More particularly, as will be exemplified below, one skilled in the art may readily isolate the genetic sequence corresponding to the variable and/or constant regions of the subject antibody and delete or alter the appropriate nucleotides to provide polypeptides of the invention for use as monomeric subunits in accordance with the instant invention. It will further be appreciated that compatible polypeptides of the invention may be expressed and produced on a clinical or commercial scale using well-established protocols.
Previously reported antibodies that react with tumor associated molecules may be altered as described herein to provide the polypeptides of the present invention. Exemplary antibodies that may be used to provide antigen binding regions for, generate or derive the disclosed polypeptides include, but are not limited to 2B8 and C2B8 (Zevalin® and Rituxan®, IDEC Pharmaceuticals Corp., San Diego), Lym 1 and Lym 2 (Techniclone), LL2 (Immunomedics Corp., New Jersey), HER2 (Herceptin®, Genentech Inc., South San Francisco), B1 (Bexxar®, Coulter Pharm., San Francisco), Campath® (Millennium Pharmaceuticals, Cambridge) MB1, BH3, B4, B72.3 (Cytogen Corp.), CC49 (National Cancer Institute) and 5E10 (University of Iowa). In preferred embodiments, the polypeptides of the present invention will bind to the same tumor associated antigens as the antibodies enumerated immediately above. In particularly preferred embodiments, the polypeptides will be derived from or bind the same antigens as 2B8, C2B8, CC49 and C5E10 and, even more preferably, will comprise domain deleted antibodies (i.e., ΔCH2 antibodies).
In a first preferred embodiment, the polypeptide will bind to the same tumor associated antigen as Rituxan®. Rituxan® (also known as, rituximab, IDEC-C2B8 and C2B8) was the first FDA-approved monoclonal antibody for treatment of human B-cell lymphoma (see U.S. Pat. Nos. 5,843,439; 5,776,456 and 5,736,137 each of which is incorporated herein by reference). Y2B8 (90Y labeled 2B8; Zevalin®; ibritumomab tiuxetan) is the murine parent of C2B8. Rituxan® is a chimeric, anti-CD20 monoclonal antibody which is growth inhibitory and reportedly sensitizes certain lymphoma cell lines for apoptosis by chemotherapeutic agents in vitro. The antibody efficiently binds human complement, has strong FcR binding, and can effectively kill human lymphocytes in vitro via both complement dependent (CDC) and antibody-dependent (ADCC) mechanisms (Reff et al., Blood 83: 435-445 (1994)). Those skilled in the art will appreciate that dimeric variants (homodimers or heterodimers) of C2B8 or 2B8, synthetic according to the instant disclosure, may be used in conjugated or unconjugated forms to effectively treat patients presenting with CD20+ malignancies. More generally, it must be reiterated that the polypeptides disclosed herein may be used in either a “naked” or unconjugated state or conjugated to a cytotoxic agent to effectively treat any one of a number of disorders.
In other preferred embodiments of the present invention, the polypeptide of the invention will be derived from, or bind to, the same tumor associated antigen as CC49. As previously alluded to, CC49 binds human tumor associated antigen TAG-72 which is associated with the surface of certain tumor cells of human origin, specifically the LS174T tumor cell line. LS174T [American Type Culture Collection (herein ATCC) No. CL 188] is a variant of the LS180 (ATCC No. CL 187) colon adenocarcinoma line.
It will further be appreciated that numerous murine monoclonal antibodies have been developed which have binding specificity for TAG-72. One of these monoclonal antibodies, designated B72.3, is a murine IgG1 produced by hybridoma B72.3 (ATCC No. HB-8108). B72.3 is a first generation monoclonal antibody developed using a human breast carcinoma extract as the immunogen (see Colcher et al., Proc. Natl. Acad. Sci. (USA), 78:3199-3203 (1981); and U.S. Pat. Nos. 4,522,918 and 4,612,282 each of which is incorporated herein by reference). Other monoclonal antibodies directed against TAG-72 are designated “CC” (for colon cancer). As described by Schlom et al. (U.S. Pat. No. 5,512,443 which is incorporated herein by reference) CC monoclonal antibodies are a family of second generation murine monoclonal antibodies that were prepared using TAG-72 purified with B72.3. Because of their relatively good binding affinities to TAG-72, the following CC antibodies have been deposited at the ATCC, with restricted access having been requested: CC49 (ATCC No. HB 9459); CC 83 (ATCC No. HB 9453); CC46 (ATCC No. HB 9458); CC92 (ATTCC No. HB 9454); CC30 (ATCC No. HB 9457); CC11 (ATCC No. 9455); and CC15 (ATCC No. HB 9460). U.S. Pat. No. 5,512,443 further teaches that the disclosed antibodies may be altered into their chimeric form by substituting, e.g., human constant regions (Fc) domains for mouse constant regions by recombinant DNA techniques known in the art. Besides disclosing murine and chimeric anti-TAG-72 antibodies, Schlom et al. have also produced variants of a humanized CC49 antibody as disclosed in PCT/US99/25552 and single chain constructs as disclosed in U.S. Pat. No. 5,892,019 each of which is also incorporated herein by reference. Those skilled in the art will appreciate that each of the foregoing antibodies, constructs or recombinants, and variations thereof, may be synthetic and used to provide polypeptides in accordance with the present invention.
In addition to the anti-TAG-72 antibodies discussed above, various groups have also reported the construction and partial characterization of domain-deleted CC49 and B72.3 antibodies (e.g., Calvo et al. Cancer Biotherapy, 8(1):95-109 (1993), Slavin-Chiorini et al. Int. J. Cancer 53:97-103 (1993) and Slavin-Chiorini et al. Cancer. Res. 55:5957-5967 (1995).
In one embodiment, a binding molecule of the invention binds to the CD23 (U.S. Pat. No. 6,011,138). In a preferred embodiment, a binding molecule of the invention binds to the same epitope as the 5E8 antibody. In another embodiment, a binding molecule of the invention comprises at least one CDR from an anti-CD23 antibody, e.g., the 5E8 antibody.
In one embodiment, a binding molecule of the invention binds to the CRIPTO-I antigen (WO02/088170A2 or WO03/083041A2). In a preferred embodiment, a binding molecule of the invention binds to the same epitope as the B3F6 antibody. In another embodiment, a binding molecule of the invention comprises at least one CDR from an anti-CRIPTO-I antibody, e.g., the B3F6 antibody.
Still other preferred embodiments of the present invention comprise modified antibodies that are derived from or bind to the same tumor associated antigen as C5E10. As set forth in co-pending application Ser. No. 09/104,717, C5E10 is an antibody that recognizes a glycoprotein determinant of approximately 115 kDa that appears to be specific to prostate tumor cell lines (e.g. DU145, PC3, or ND1). Thus, in conjunction with the present invention, polypeptides (e.g. CH2 domain-deleted antibodies) that specifically bind to the same tumor associated antigen recognized by C5E10 antibodies could be produced and used in a conjugated or unconjugated form for the treatment of neoplastic disorders. In particularly preferred embodiments, the binding molecule will be derived or comprise all or part of the antigen binding region of the C5E10 antibody as secreted from the hybridoma cell line having ATCC accession No. PTA-865. The resulting binding molecule could then be conjugated to a radionuclide as described below and administered to a patient suffering from prostate cancer in accordance with the methods herein.
In general, the disclosed invention may be used to prophylactically or therapeutically treat any neoplasm comprising a marker that allows for the targeting of the cancerous cells by the binding molecule. Exemplary cancers that may be treated include, but are not limited to, prostate, gastric carcinomas such as colon, skin, breast, ovarian, lung and pancreatic. More particularly, the antibodies of the instant invention may be used to treat Kaposi's sarcoma, CNS neoplasias (capillary hemangioblastomas, meningiomas and cerebral metastases), melanoma, gastrointestinal and renal sarcomas, rhabdomyosarcoma, glioblastoma (preferably glioblastoma multiforme), leiomyosarcoma, retinoblastoma, papillary cystadenocarcinoma of the ovary, Wilm's tumor or small cell lung carcinoma. It will be appreciated that appropriate polypeptides may be derived for tumor associated molecules related to each of the forgoing neoplasias without undue experimentation in view of the instant disclosure.
Exemplary hematologic malignancies that are amenable to treatment with the disclosed invention include Hodgkins and non-Hodgkins lymphoma as well as leukemias, including ALL-L3 (Burkitt's type leukemia), chronic lymphocytic leukemia (CLL) and monocytic cell leukemias. It will be appreciated that the compounds and methods of the present invention are particularly effective in treating a variety of B-cell lymphomas, including low grade/follicular non-Hodgkin's lymphoma (NHL), cell lymphoma (FCC), mantle cell lymphoma (MCL), diffuse large cell lymphoma (DLCL), small lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL and Waldenstrom's Macroglobulinemia. It should be clear to those of skill in the art that these lymphomas will often have different names due to changing systems of classification, and that patients having lymphomas classified under different names may also benefit from the combined therapeutic regimens of the present invention. In addition to the aforementioned neoplastic disorders, it will be appreciated that the disclosed invention may advantageously be used to treat additional malignancies bearing compatible tumor associated molecules.
Besides neoplastic disorders, the polypeptides of the instant invention are particularly effective in the treatment of autoimmune disorders or abnormal immune responses. In this regard, it will be appreciated that the polypeptide of the present invention may be used to control, suppress, modulate or eliminate unwanted immune responses to both external and autoantigens. For example, in one embodiment, the antigen is an autoantigen. In another embodiment, the antigen is an allergen. In yet other embodiments, the antigen is an alloantigen or xenoantigen. Use of the disclosed polypeptides to reduce an immune response to alloantigens and xenoantigens is of particular use in transplantation, for example to inhibit rejection by a transplant recipient of a donor graft, e.g. a tissue or organ graft or bone marrow transplant. Additionally, suppression or elimination of donor T cells within a bone marrow graft is useful for inhibiting graft versus host disease.
In yet other embodiments the polypeptides of the present invention may be used to treat immune disorders that include, but are not limited to, allergic bronchopulmonary aspergillosis; Allergic rhinitis Autoimmune hemolytic anemia; Acanthosis nigricans; Allergic contact dermatitis; Addison's disease; Atopic dermatitis; Alopecia greata; Alopecia universal is; Amyloidosis; Anaphylactoid purpura; Anaphylactoid reaction; Aplastic anemia; Angioedema, hereditary; Angioedema, idiopathic; Ankylosing spondylitis; Arteritis, cranial; Arteritis, giant cell; Arteritis, Takayasu's; Arteritis, temporal; Asthma; Ataxia-telangiectasia; Autoimmune oophoritis; Autoimmune orchitis; Autoimmune polyendocrine failure; Behcet's disease; Berger's disease; Buerger's disease; bronchitis; Bullous pemphigus; Candidiasis, chronic mucocutaneous; Caplan's syndrome; Post-myocardial infarction syndrome; Post-pericardiotomy syndrome; Carditis; Celiac sprue; Chagas's disease; Chediak-Higashi syndrome; Churg-Strauss disease; Cogan's syndrome; Cold agglutinin disease; CREST syndrome; Crohn's disease; Cryoglobulinemia; Cryptogenic fibrosing alveolitis; Dermatitis herpetifomis; Dermatomyositis; Diabetes mellitus; Diamond-Blackfan syndrome; DiGeorge syndrome; Discoid lupus erythematosus; Eosinophilic fasciitis; Episcleritis; Drythema elevatum diutinum; Erythema marginatum; Erythema multiforme; Erythema nodosum; Familial Mediterranean fever; Felty's syndrome; Fibrosis pulmonary; Glomerulonephritis, anaphylactoid; Glomerulonephritis, autoimmune; Glomerulonephritis, post-streptococcal; Glomerulonephritis, post-transplantation; Glomerulopathy, membranous; Goodpasture's syndrome; Granulocytopenia, immune-mediated; Granuloma annulare; Granulomatosis, allergic; Granulomatous myositis; Grave's disease; Hashimoto's thyroiditis; Hemolytic disease of the newborn; Hemochromatosis, idiopathic; Henoch-Schoenlein purpura; Hepatitis, chronic active and chronic progressive; Histiocytosis X; Hypereosinophilic syndrome; Idiopathic thrombocytopenic purpura; Job's syndrome; Juvenile dermatomyositis; Juvenile rheumatoid arthritis (Juvenile chronic arthritis); Kawasaki's disease; Keratitis; Keratoconjunctivitis sicca; Landry-Guillain-Barre-Strohl syndrome; Leprosy, lepromatous; Loeffler's syndrome; lupus; Lyell's syndrome; Lyme disease; Lymphomatoid granulomatosis; Mastocytosis, systemic; Mixed connective tissue disease; Mononeuritis multiplex; Muckle-Wells syndrome; Mucocutaneous lymph node syndrome; Mucocutaneous lymph node syndrome; Multicentric reticulohistiocytosis; Multiple sclerosis; Myasthenia gravis; Mycosis fungoides; Necrotizing vasculitis, systemic; Nephrotic syndrome; Overlap syndrome; Panniculitis; Paroxysmal cold hemoglobinuria; Paroxysmal nocturnal hemoglobinuria; Pemphigoid; Pemphigus; Pemphigus erythematosus; Pemphigus foliaceus; Pemphigus vulgaris; Pigeon breeder's disease; Pneumonitis, hypersensitivity; Polyarteritis nodosa; Polymyalgia rheumatic; Polymyositis; Polyneuritis, idiopathic; Portuguese familial polyneuropathies; Pre-eclampsia/eclampsia; Primary biliary cirrhosis; Progressive systemic sclerosis (Scleroderma); Psoriasis; Psoriatic arthritis; Pulmonary alveolar proteinosis; Pulmonary fibrosis, Raynaud's phenomenon/syndrome; Reidel's thyroiditis; Reiter's syndrome, Relapsing polychrondritis; Rheumatic fever; Rheumatoid arthritis; Sarcoidosis; Scleritis; Sclerosing cholangitis; Serum sickness; Sezary syndrome; Sjogren's syndrome; Stevens-Johnson syndrome; Still's disease; Subacute sclerosing panencephalitis; Sympathetic ophthalmia; Systemic lupus erythematosus; Transplant rejection; Ulcerative colitis; Undifferentiated connective tissue disease; Urticaria, chronic; Urticaria, cold; Uveitis; Vitiligo; Weber-Christian disease; Wegener's granulomatosis and Wiskott-Aldrich syndrome.
This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference.
EXAMPLES
Example 1
Identification of A and B Forms
Solutions of antibody molecules comprise two different isoforms. One form, Form A comprises heavy chain molecules that are linked via at least one disulfide linkage. The other form, Form B, comprises heavy chain molecules that are not linked via at least one disulfide linkage. Form B does not appear or appears at a very low frequency in with intact gamma 1 MAbs, such as Rituxan®. However with domain deleted (dd) constructs having a similar hinge, the frequency of Form B is much higher. These forms can be distinguished using denaturing, non-reducing SDS page. In domain deleted antibody preparations, Form A appears as a 120 kDa dimer while Form B appears as a 60 kDa monomer (FIG. 1). FIGS. 2A and 2B show densitometer plots of non-reducing SDS-PAGE gels for ddCC49 (CH2) and ddCC49 (Gly/Ser), respectively.
Example 2
Identification of Hinge Region Heterogeneity in CH2 Domain Deleted MAb Fragments
Hinge domains can be subdivided into three distinct regions: upper, middle, and lower hinge regions (Roux et al. J. Immunol. 1998 161:4083). Polypeptide sequences encompassing these regions for IgG1 and IgG3 hinges are shown in Table 1. The IgG3 hinge middle region contains, in addition to the two conserved cysteine residues, a 15 amino acid motif that repeats three times. Amino acid sequences from these regions were used to design synthetic IgG1/IgG3 connecting peptides. These consisted of IgG1 upper hinge residues corresponding to positions 226 through 238, an IgG1 middle hinge corresponding to positions 239 through 241, and a single IgG3 middle hinge repeat motif corresponding to positions 241EE through 242 combined with either an added proline at position 243 or an added proline, alanine, proline at positions 243, 244, and 245, respectively (Kabat numbering system), followed by a flexible Gly/Ser spacer (Table 2). In addition, novel connecting peptides were designed consisting of a serine amino acid residue substituted for the cysteine at positions 239 or 242 combined with either an added proline at position 243 or an added proline, alanine, proline at positions 243, 244, and 245, respectively (Kabat numbering system). Pro243Ala244Pro245 and Pro 243 connecting peptides were also made. The amino acid sequence of the parent CH2 domain deleted humanized CC49 connecting peptide beginning at the first residue of the IgG1 hinge (position 226, Kabat numbering system) to the last residue of the hinge/GlySer connecting peptide is shown in Table 2. Also shown are the various connecting peptide designs by alignment to CC49 with positions of the cysteine residues indicated in Kabat numbering system.
TABLE 1
IgG1, IgG3 and IgG4 Hinge Regions
IgG
Upper Hinge
Middle Hinge
Lower Hinge
IgG1
EPKSCDKTHT
CPPCP
APELLGGP
(SEQ ID NO: 2)
(SEQ ID NO: 3)
(SEQ ID NO: 4)
IgG3
ELKTPLGDTTHT
CPRCP
APELLGGP
(SEQ ID NO: 5)
(EPKSCDTPPPCPRCP)3
(SEQ ID NO: 4)
(SEQ ID NO: 6)
IgG4
ESKYGPP
CPSCP
APEFLGGP
(SEQ ID NO: 50)
(SEQ ID NO: 51)
(SEQ ID NO: 52)
TABLE 2
Hinge Region Connecting Peptide Sequences
Kabat hinge position:
226
227
228
229
230
232
235
236
237
238
239
240
241
IgG1 hinge sequence
E
P
K
S
C
D
K
T
H
T
C
P
P
IgG4 hinge sequence
E
S
K
Y
G
P
P
C
P
S
IgG3 middle hinge sequence
Connecting peptide:
Connecting peptide sequences
G1
E
P
K
S
C
D
K
T
H
T
C
P
P
(Seq. ID NO: 7)
G1/G3/Pro243
E
P
K
S
C
D
K
T
H
T
C
P
P
(Seq. ID NO: 8)
G1/G3/Pro243Ala244Pro245
E
P
K
S
C
D
K
T
H
T
C
P
P
(Seq. ID NO: 9)
G1/Cys239Ser:Pro243
E
P
K
S
C
D
K
T
H
T
S
P
P
(Seq. ID NO: 10)
G1/Cys239Ser:Pro243Ala244
E
P
K
S
C
D
K
T
H
T
S
P
P
Pro245
(Seq. ID NO: 11)
G1/Cys242Ser:Pro243
E
P
K
S
C
D
K
T
H
T
C
P
P
(Seq. ID NO: 12)
G1/
E
P
K
S
C
D
K
T
H
T
C
P
P
Cys242Ser:Pro243Ala244Pro245
(Seq. ID NO: 13)
G1/Pro243Ala244Pro245
E
P
K
S
C
D
K
T
H
T
C
P
P
(Seq. ID NO: 14)
G1/Pro243
E
P
K
S
C
D
K
T
H
T
C
P
P
(Seq. ID NO: 15)
G4/G3/Pro243Ala244Pro245
E
S
K
Y
G
P
P
C
P
S
(Seq. ID NO: 53)
Kabat hinge position:
241EE
241FF
241GG
241HH
241II
241JJ
241KK
241LL
241MM
241NN
IgG1 hinge sequence
IgG4 hinge sequence
IgG3 middle hinge sequence
C
P
E
P
K
S
C
D
T
P
Connecting peptide:
Connecting peptide sequences
G1
G1/G3/Pro243
C
P
E
P
K
S
C
D
T
P
G1/G3/Pro243Ala244Pro245
C
P
E
P
K
S
C
D
T
P
G1/Cys239Ser:Pro243
G1/Cys239Ser:Pro243Ala244
Pro245
G1/Cys242Ser:Pro243
G1/Cys242Ser:Pro243Ala244
Pro245
G1/Pro243Ala244Pro245
G1/Pro243
G4/G3/Pro243Ala244Pro245
C
P
E
P
K
S
C
D
T
P
Kabat hinge position:
241OO
241PP
241OO
241RR
241SS
242
243
244
245
IgG1 hinge sequence
C
P
A
P
IgG4 hinge sequence
C
P
A
P
IgG3 middle hinge sequence
P
P
C
P
R
Connecting peptide:
Connecting peptide sequences
G1
C
GGGSSGGGSG
G1/G3/Pro243
P
P
C
P
R
C
P
GGGSSGGGSG
G1/G3/Pro243Ala244Pro245
P
P
C
P
R
C
P
A
P
GGGSSGGGSG
G1/Cys239Ser:Pro243
C
P
GGGSSGGGSG
G1/Cys239Ser:Pro243Ala244
C
P
A
P
GGGSSGGGSG
Pro245
G1/Cys242Ser:Pro243
S
P
GGGSSGGGSG
G1/Cys242Ser:Pro243Ala244
S
P
A
P
GGGSSGGGSG
Pro245
G1/Pro243Ala244Pro245
C
P
A
P
GGGSSGGGSG
G1/Pro243
C
P
GGGSSGGGSG
G4/G3/Pro243Ala244Pro245
P
P
C
P
R
C
P
A
P
Example 3
Construction of Connecting Polypeptides and Preferential Synthesis of Isoforms
Nucleic acid sequences encoding the hinge region connecting peptides shown in Table 2 were introduced into CH2 domain deleted huCC49 gene sequences using the Splicing by Overlap Extension (SOE) method (Horton, R. M. 1993 Methods in Molecular Biology, Vol 15: PCR Protocols: Current Methods and applications. Ed. B. A. White). Correct modifications to the hinge region were confirmed by DNA sequence analysis. Plasmid DNA was used to transform CHO DG44 cells for stable production of antibody protein.
CH2 domain deleted huCC49 antibodies containing the eight designed synthetic connecting peptides indicated in Table 2 were constructed and antibody produced in CHO DG44 cells. Supernatants were collected from isolated cell lines and concentration of antibody in the culture supernatants determined by immunoassay. Supernatants containing antibody ranging from 0 to 30 ng of total antibody protein from each cell line was analyzed by non-reducing SDS-PAGE electrophoresis followed by Western Blot with anti-human kappa HRP conjugated antibody to detect CH2 domain deleted huCC49 Form A and Form B forms. Under these conditions, Form A migrates as a single 120 kDa homodimer and Form B as a 60 kDa doublet. Also visible are kappa chain monomer and dimers. Connecting peptides shown in SEQ ID NOs: 8, 9, 14, and 15 were all found to increase the proportion of form A produced. Exemplary results are shown in FIG. 3. These results show that both the G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO:9) (FIG. 3D) and G1/G3/Pro243+[Gly/Ser] (SEQ ID NO:8) (FIG. 3C) hinges resulted primarily if not entirely in the production of Form A CH2 domain-deleted huCC49 antibody with little or no detectable Form B. In contrast CH2 domain-deleted huCC49 Cys242Ser:Pro243 (SEQ ID NO:12) (FIG. 3A) and CH2 domain-deleted huCC49 Cys242Ser:Pro243Ala244Pro245 (SEQ ID NO:13) (FIG. 3B) resulted in a moderate to significant preference of the Form B form, respectively.
Cell lines containing the Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO:14) and G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO:9) connecting peptides introduced into the huCC49 antibody sequence were used for antibody production. The Pro243Ala244Pro245+[Gly/Ser] and G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptides were also introduced into the huCC49 V2 antibody sequence and cell lines generated (The humanized CC49 version 2 sequence is described in Example 8; see FIGS. 19A and B for alignments of huCC49 and huCC49 V2 sequences for the light and heavy chains, respectively). Antibody was produced from CHO DG44 cells and purified using methods described in Example 4 below. Yields of the Form A isoform following the Protein G and HIC steps are reported in Table 3. From these results it is clear that the modifications introduced to the hinge region in the CH2 domain deleted antibodies led to the preferential synthesis of the A isoform. Following the HIC purification technique described in Example 4, purified HuCC49 Pro230Ala23 Pro232 and HuCC49 V2 Pro230Ala231Pro232 Form A material was achieved at values greater than 98%. HuCC49 G1/G3/Pro243Ala244Pro245 and HuCC49 V2 G1/G3/Pro243Ala244Pro245 Form A materials, purified using only the Protein G column, both eluted essentially as single peaks at ≧96% purity without further HIC purification (FIG. 5 and FIG. 6). All antibodies were examined by size exclusion chromatography and were found to elute as single peaks indicating that there was no significant aggregation or decomposition of antibody product.
Peptide mapping was used to determine the integrity of disulfide bond formation in the heavy chain hinge regions of CH2 domain deleted HuCC49, HuCC49 PAP, and HuCC49 G1/G3/PAP antibodies. Samples of the CH2 domain deleted CC49 antibodies were denatured, reduced and digested with trypsin as follows: aliquots of 150 ug were diluted to 100 ml in HPLC water and denatured in 6M guanidine hydrochloride, 50 mM Tris pH 8.0. The samples were reduced by the addition of 20 mM DTT and incubated for 30 minutes at 37° C. The reduced samples were alkylated with 50 mM iodoacetic acid for 30 minutes at 37° C. The alkylation reaction was quenched by the addition of excess DTT. The reduced and alkylated samples were buffer exchanged into 25 mM TRIS, 20 mM CaCl2, pH 7.5 using PD-10 columns. Trypsin was added to each sample in a 1:15 (w/w) ratio and incubated for 4 hours at 37° C. The digestion was stopped by the addition of trifluoroacetic acid (TFA) to a final concentration of 0.1%. Trypsin digested samples (15 ug) were then analyzed according to chromatographic conditions described below.
Samples of the CH2 domain deleted CC49 antibodies were analyzed by endoproteinase Lys-C digestion. Denatured and reduced samples were prepared by adding a final concentration of 4 M guanidine HCl and 25 mM DTT to 1.5 mg/mL of sample. Non-reduced samples were prepared by adding a final concentration of 4 M guanidine HCl to 1.5 mg/mL of sample. Samples were incubated for 2 hours at 37° C. Digestion buffer (50 mM Tris, pH 7.0 and 0.062 AU/ml endoproteinase Lys-C) was then added to the samples at 1:1 (v/v) and samples were incubated for 15 hours at 37° C. At 15 hours, a second aliquot of enzyme (0.29 mAU: ug Antibody) was added and samples were incubated for an additional 6 hours at 37° C. To quench the reaction, TFA was added at 0.1% final concentration. Non-reduced and reduced endoproteinase Lys-C digested samples (12 ug) were then analyzed according to the procedure described below.
HPLC/mass spectrometry analysis. Samples were analyzed on an Agilent 1100 HPLC system connected to an Agilent MSD single quadrupole mass spectrometer. A reverse phase C18 column (Vydac catalog number 218TP52) was used with an eluant system of water/0.1% TFA (v/v) (Buffer A) and acetonitrile/0.1% TFA (v/v) (Buffer B), at a flow rate of 0.2 mL/minute. A post column “TFA fixative” solution of acetonitrile and acetic acid (1:1 v/v) at 0.1 mL/minute was added to enhance ionization. The column temperature was controlled at 45° C. and the elution profile was monitored at 215 and 280 nm. The total ion chromatogram was monitored in positive ion mode. Samples were injected onto the column and the gradient was held at 0% Buffer B for five minutes. Elution was accomplished with a linear gradient of 0 to 50% Buffer B over 125 minutes, followed by a 75% Buffer B wash over 10 minutes and a 0% Buffer B re-equilibration over 30 minutes.
In the endo Lys-C reduced analysis, fragment (L52-109) was undetected for all samples (FIG. 7B). This fragment is very hydrophobic and may have not eluted from the column matrix due to strong interactions. The corresponding tryptic fragment (L68-109) was also undetected in all samples (FIG. 7C). Since these fragments contain a large number of amino acids, the percent amino acid identity was lowered to ˜89% identity. In addition, fragment (L68-119) was undetected in the endo Lys-C analysis of G1/G3/PAP bringing the identity down to ˜79%.
The endo Lys-C non-reduced analysis provided much better results (FIG. 7A). Fragment (L52-109) was detected as a disulfide linkage with fragment (L1-24) in all samples. All other disulfide linkages were detected and the total % amino acid identity was ˜99% for all samples. The G1/G3/PAP sample showed an additional heavy chain-heavy chain disulfide linkage in fragment (H232-275), below the original (H224-227) CPPC hinge region. The theoretical and observed mass values for the engineered hinge region peptides are shown in Table 4. The HuCC49ΔCH2 hinge endo Lys-C non-reduced peptide (residues H221-257) had an observed MW of 7419.4, in good agreement with the calculated mass of 7419.4 g/mol for a linked hinge containing two interchain disulfide bridges. The HuCC49ΔCH2 PAP hinge endo Lys-C non-reduced peptide (residues H221-260) had an observed MW of 7949.7 also in good agreement with the calculated mass of 7949.8 g/mol for a linked hinge containing two interchain disulfide bridges. Two hinge non-reduced peptide fragments resulted from digestion of HuCC49ΔCH2 G1/G3/PAP by endo Lys-C due to the presence of the lysine residue at Kabat position 241II in the 15 amino acid γ3 motif. Peptide fragments THTCPPCPEPK (residues H221-231) and SCDTPPPCPRCPAPGGGSSGGGSGGQPREPQVYTLPPSRDELTK (residues H232-275) had observed MWs of 2414.3 and 8782.6 in excellent agreement with the calculated masses of 2413.0 and 8782.0 g/mol, respectively. The mass data supports the assertion that the THTCPPCPEPK peptide (residues H221-231) derived from HuCC49ΔCH2 G1/G3/PAP contains two interchain disulfide bridges. Importantly, the SCDTPPPCPRCPAPGGGSSGGGSGGQPREPQVYTLPPSRDELTK peptide (residues H232-275) contains at least one interchain disulfide bridge consistent with the notion that the chimeric G1/G3/PAP hinge is participating in the formation of more than two disulfide bridges. These analyses show that HuCC49ΔCH2 PAP hinge forms two heavy chain interchain disulfide bonds. HuCC49ΔCH2 G1/G3/PAP hinge forms at least three heavy chain interchain disulfide bonds but possibly five. It is certain that fragment HuCC49ΔCH2 G1/G3/PAP residues H232-275 contains minimally one interchain disulfide bond, however it is not possible to discriminate mass differences in a hinge region containing three interchain disulfide bonds from one containing a single interchain and two intrachain disulfide bonds.
TABLE 3
The percentage of Form A antibody after affinity
chromatography (Protein G) and after HIC purification
CH2 domain deleted
% Form A Antibody
Antibody
After Protein G
After HIC purification
HuCC49
60
98
(connecting peptide SEQ
ID NO: 7)
HuCC49 PAP
83
98
(connecting peptide SEQ
ID NO: 14)
HuCC49 V2 PAP
90
99
(connecting peptide SEQ
ID NO: 14)
HuCC49 G1/G3/PAP
98
Not done
(connecting peptide SEQ
ID NO: 9)
HuCC49 V2 G1/G3/PAP
96
Not done
(connecting peptide SEQ
ID NO: 9)
TABLE 4
Peptide mapping of engineered HuCC49ΔCH2 antibody hinge region peptides.
Theoretical
Theoretical
Ob. Mass
Ob. Mass
Sample
Fragment #
A.A #
MW
Linked MW
Reduced
Non-reduced
HuCC49ΔCH2
Endo Lys-C fragment 29
(H221-257)
3711.7
7419.4
3711.5
7419.1
THTCPPCGGGSSGGGSGGQPREPQVYTLPPSRDELTK
PAP
Endo Lys-C fragment 29
(H221-260)
3976.9
7949.8
3976.8
7949.7
THTCPPCPAPGGGSSGGGSGGQPREPQVYTLPPSRDELTK
G1/G3:PAP
Endo Lys-C fragment 29
(H221-231)
1208.5
2413.0
1208.9
2414.3
THTCPPCPEPK
Endo Lys-C fragment 30
(H232-275)
4394.0
8782.0
4394.4
8782.6
SCDTPPPCPRCPAPGGGSSGGGSGGQPREPQVYTLPPSRDELTK
HuCC49ΔCH2
Trypsin fragment 39
(H221-241)
1971.8
NA
1972.6
NA
THTCPPCGGGSSGGGSGGQPR
PAP
Trypsin fragment 39
(H221-244)
2237.9
NA
2237.8
NA
THTCPPCPAPGGGSSGGGSGGQPR
G1/G3:PAP
Trypsin fragment 39
(H221-231)
1324.5
NA
1325.1
NA
THTCPPCPEPK
Trypsin fragment 40
(H232-241)
1187.5
NA
1187.5
NA
SCDTPPPCPR
Trypsin fragment 41
(H242-259)
1542.6
NA
1542.9
NA
CPAPGGGSSGGGSGGQPR
These data show that novel, engineered synthetic hinge region connecting peptides can be used to preferentially favor the formation of the A or B form. These studies also reveal the importance of the cysteine residues at position 242 (Kabat numbering system) in synthesizing the CH2 domain-deleted antibody Form A isoform. Accordingly, in one embodiment, a connecting peptide of the invention comprises a cysteine at at least one of position 239 or 242. Substituting the cysteine at either position 239 or 242 with serine (e.g., using connecting peptides shown in SEQ ID NOs:10, 11, 12, or 13) shifts CH2 domain-deleted antibody biosynthesis to the Form B form. The use of connecting peptides which increase the proportion of Form A produced will lead to a beneficial improvement in process, yield and/or stability. These synthetic hinge region connecting peptides are useful for favoring synthesis of CH2 domain deleted antibody Form A isoform for any antibody isotype, e.g., IgG1, IgG2, IgG3, or IgG4, based on the extremely high degree of homology among the CH3 domains for all four human isotypes. Including identical and conserved amino acid residues, IgG1 CH3 domain is 98.13% homologous to IgG2 CH3, 97.20% homologous to IgG3 CH3, and 96.26% homologous to IgG4 CH3.
Example 4
Purification of Form A and Form B from a Monoclonal Antibody Mixture Containing Both Isoforms
10 mL of ddCC49 supernatant was titrated with 1M Tris pH 9.0 to a final pH of 7.5. This material was filtered through a series of Sol-Vac 0.8μ and 0.4μ membranes. A 100 mL XK50 Protein G column was pre-equilibrated with 1×PBS at a flow rate of 80 ml/min. The titrated, filtered supernatant was loaded onto the column at 80 ml/min. Bound protein was washed with the equilibrium buffer for 2 column volumes and then eluted with 100 mM Glycine at pH 3.0. The fractions containing the ddCC49 peak were collected and immediately titrated with 1 M Tris pH 9.0 to a final pH of 7.0.
A Toso Biosep Phenyl 5PW-HR column was pre-equilibrated with 20 mM Phosphate pH 7.2; 1 M Ammonium Sulfate. The Protein G eluate was titrated to 1 M Ammonium Sulfate using a 3.5 M Ammonium Sulfate pH 7.2 stock and loaded at a concentration of 2 mg/ml of gel bed. Bound protein was washed with a 20 mM Phosphate pH 4 or 7.2 Ammonium Sulfate to adjust the conductivity to 116.4 mS/cm. The material eluted from this condition has an apparent molecular weight about 120 kD (Form A) on a non-reducing SDS-PAGE. The remaining bound antibody was further eluted with a linear gradient of reducing Ammonium Sulfate content in the Phosphate buffer. The latter eluted antibody apparently lacks the disulfide linkage between the heavy chains and its molecular weight is about 60 kDa (form B). FIG. 15 shows a chromatogram of the HIC purification. Purified A and B Forms are shown in lanes 3 and 4 of FIG. 16, respectively.
Both of the above purified materials can be recaptured by bringing the ammonium sulfate concentration to 1M and reloading it onto the cleaned Phenyl 5PW-HR column. Bound protein is eluted with 20 mM Phosphate pH 7.2 and dialyzed into 1×PBS.
Example 5
Comparison of Stability of Form A and Form B
The biologic activity of Forms A and B (as measured in preliminary experiments e.g., using direct binding or competition studies) revealed that Forms A and B have similar biologic activity.
The stability of Forms A and B was also compared. Purified ddCC49 molecules were concentrated to about 5 mg/ml by Amicon concentrator fitted with YM30 membrane (Millipore). The concentrated materials were equally divided into four portions for each isoforms and each fraction was put into 10K dialysis cassette (Pierce, cat#66410) for 16 h dialysis in the following buffers: 1) 10 mM Sodium Phosphate, pH3; 2) 10 mM Sodium acetate, pH 5; 3) 10 mM Sodium Phosphate, pH 7; and 4) 10 mM Sodium Borate, pH 9. After dialysis, the protein concentration of each solution was adjusted to 3 mg/ml. In addition to the pure A and B form solution, a portion of A and B solutions from each pH were mixed to create a mixture containing 50% each isoform. Total of 12 formulations were created (four pH levels times 3 antibody solutions). The solutions were filtered and filled in 3 ml Type-1 glass serum vials (West Pharmaceuticals) with gray butyl stopper.
Three temperatures, 2-8° C., 20-25° C., and 38-42° C. were chosen to store the protein solutions for stability testing. Prior to storage, 500 μl samples were drawn from each formulation for physical and chemical analyses, these zero-time point data were referred to as control. Once in storage, samples were drawn at the following schedule, 2 weeks, 1 month, 2 months and 3 months and submitted for testing immediately.
To evaluate the physical and chemical stability of the two isoforms, the following methods were used: turbidity measured at OD320, non-reducing SDS-PAGE, and size-exclusion chromatography.
Non-reducing SDS-PAGE was performed on for samples stored at 2-8° C., 20-25° C. and 38-42° C. for various time points. Both A and B form are relatively stable at pH 5 when stored at 2-8° C. However, when formulated at pH 7 and 9, both A and B forms showed degradation as indicated by increasing in number of bands that were smaller than the original major bands (120 kDa for form A and 60 kDa for form B). It was noticed that, particularly for pH 7 and 9 samples stored at low and intermediate temperatures, the intensity and number of bands that were less than 55 kDa were higher in B-isoform than A. This indicated that under these conditions the A-isoform is more stable than B-isoform. However, this seems not to be the case for A-isoform in pH 5 and stored at 20-25° C. This sample seemed to have more fragments than B-isoform. This appears to have been an artifact due to microbial contamination (discussed in more detail below). At high storage temperature, both forms at pH 9 were significantly degraded and there was almost no difference in gel patterns among the samples. Under this condition, trace amount of smear bands showed up at top of the gel which indicated the formation of aggregates. Because aggregates could be dissolved by SDS, the aggregation was investigated using the methods described in the following sections.
Table 5A through Table 5C list the turbidity data for ddCC49 stored at three different temperatures. The turbidity measures both the soluble and non-soluble aggregates and it is based on the amount of light scattered by these particles. When present, aggregates will scatter light and result in an increase in A320. As showed in Table 5A-C, the turbidity of ddCC49 molecules stored at 2-8° C. increases as pH increased for both A and B isoforms, with the former being less turbid than the latter. This trend held true for samples stored for less than a month at higher temperatures (20-25° C. and 38-40° C.). As storage time reached 3 months, the turbidity increased significantly for samples at high pH and temperature, and the difference between A and B forms diminished. These results parallel those of SDS-PAGE and indicate that both isoforms are relatively stable (in terms of not forming aggregates) at pH 3 and 5, and that A-isoform is less susceptible to aggregation than the B form.
TABLE 5A
Turbidity measured at A320 for ddCC49
samples stored at 2-8° C.
Time
A-isoform
B-isoform
Mixture
(month)
pH = 3
5
7
9
3
5
7
9
3
5
7
9
0
0.030
0.038
0.044
0.056
0.034
0.042
0.046
0.066
0.036
0.042
0.051
0.061
½
0.029
0.029
0.046
0.045
0.030
0.038
0.048
0.058
0.034
0.033
0.043
0.055
1
0.033
0.039
0.035
0.055
0.033
0.035
0.044
0.059
0.032
0.040
0.039
0.066
2
0.042
0.022
0.042
0.044
0.039
0.037
0.055
0.067
0.042
0.024
0.040
0.058
3
0.035
0.047
0.051
0.050
0.038
0.041
0.066
0.081
0.027
0.048
0.051
0.065
TABLE 5B
Turbidity measured at A320 for ddCC49
samples stored at 20-25° C.
Time
A-isoform
B-isoform
Mixture
(month)
pH = 3
5
7
9
3
5
7
9
3
5
7
9
½
0.031
0.032
0.056
0.066
0.039
0.034
0.064
0.083
0.034
0.039
0.060
0.071
1
0.025
0.043
0.055
0.090
0.034
0.042
0.070
0.084
0.028
0.039
0.055
0.094
2
0.034
0.053
0.077
0.113
0.046
0.032
0.090
0.087
0.037
0.038
0.066
0.108
3
0.036
0.056
0.156
0.143
0.029
0.060
0.121
0.125
0.044
0.050
0.101
0.142
TABLE 5C
Turbidity measured at A320 for ddCC49
samples stored at 38-42° C.
Time
A-isoform
B-isoform
Mixture
(month)
pH = 3
5
7
9
3
5
7
9
3
5
7
9
½
0.041
0.042
0.068
0.063
0.041
0.044
0.080
0.067
0.041
0.039
0.070
0.064
1
0.041
0.043
0.071
0.065
0.036
0.040
0.079
0.069
0.032
0.048
0.078
0.070
2
0.047
0.030
0.066
0.060
0.046
0.045
0.087
0.082
0.051
0.034
0.078
0.079
3
0.058
0.051
0.098
0.105
0.046
0.057
0.101
0.157
0.056
0.057
0.101
0.126
Size exclusion chromatography (SEC) is a powerful method for revealing the percent of intact molecules and the degraded products (both fragments and soluble aggregates) and is highly reproducible. In Table 5A-C the percent of intact monomer of A-isoform, B-isoform and the mixture stored at different temperatures are listed. For samples stored at 2-8° C., it is clear that Form A has a higher percentage of monomer as compared to Form B, and the mixture of Form A and Form B was somewhere in between. At this storage temperature, both forms were relatively stable at pH 3, 5 and 7 (with pH 5 being the most stable condition) for about three months. However, at pH 9 there was a significant decrease in percentage of monomer for Form B but only a slight decrease for Form A. At elevated temperatures, all samples showed a significant decrease in percent of monomer as storage time increased; the A-isoform outperformed the B-isoform. However there was an exception, the sample of A-isoform in pH 5 stored at room temperature exhibited much more degradation than the B-isoform or the mixture under similar storage conditions. A close examination of this particular A-isoform vial, the data from SDS-PAGE, and SEC of the sample suggested that microbial contamination might have caused this unexpected result. First, both the SEC and SDS-PAGE results indicated that the degradation for this sample was primarily accounted for by a increase in fragmentation, presumably resulting from microbial digestion, otherwise some degree of increase in aggregation would have been expected. Second, the fact that the mixture sample, which contained 50% each of A and B-isoform, showed a better stability profile than B-isoform indicating that a more stable A-isoform must have contributed to the higher percent of monomer. Finally, A-isoform in pH 5 stored at 2-8° C. and 38-42° C. both showed higher percent of monomer than B-isoform under similar conditions. Therefore, intermediate storage temperature should have yielded similar results. Due to the limited amount of sample, an assay for microbial contamination could not be performed.
It was also noted that for both isoforms of IDEC-159 stored in high pH (9) and at 40° C., the percent of monomer reduced to about 30%. Under these severe conditions, the stability differences between the two isoforms disappeared. This SEC result mirrors of the results found using SDS-PAGE. Both results indicate that, although some chemical and physical characteristics differ between the two isoforms, the mechanism and by-products of degradation for both isoforms are similar, if not identical.
In summary, the SEC results indicate that both A and B-isoforms have optimal pH at about 5, and that A-isoform is more stable than B-isoform in terms of retaining higher percent of intact monomer at similar storage conditions.
TABLE 6A
Percent of monomer for ddCC49
samples stored at 2-8° C.
Time
A-isoform
B-isoform
Mixture
(month)
pH = 3
5
7
9
3
5
7
9
3
5
7
9
0
98.81
99.13
98.16
97.93
97.02
97.70
96.88
93.51
97.83
98.27
97.44
95.81
½
98.98
99.16
98.25
98.00
97.15
97.87
96.96
91.95
98.15
98.49
97.68
95.59
1
98.80
99.20
97.99
97.11
97.02
97.81
96.62
88.99
98.04
98.45
97.41
94.45
2
98.74
99.01
98.00
95.67
97.15
97.69
95.50
84.84
98.06
98.34
96.81
92.17
3
98.28
98.89
97.88
95.31
96.69
98.14
95.37
85.98
97.61
98.15
96.65
89.90
TABLE 6B
Percent of monomer for ddCC49
samples stored at 20-25° C.
Time
A-isoform
B-isoform
Mixture
(month)
pH = 3
5
7
9
3
5
7
9
3
5
7
9
½
97.83
99.04
97.12
93.65
95.84
97.62
93.71
79.61
96.75
98.30
95.37
87.67
1
96.60
96.63
95.65
88.09
94.38
97.23
90.69
72.26
95.36
97.99
93.05
80.92
2
93.62
92.79
93.17
80.06
91.71
96.96
85.51
66.53
92.78
97.51
89.33
73.91
3
92.81
89.56
x
74.31
89.30
96.04
82.57
63.25
90.46
97.02
86.80
69.36
TABLE 6C
Percent of monomer for ddCC49
samples stored at 38-42° C.
Time
A-isoform
B-isoform
Mixture
(month)
pH = 3
5
7
9
3
5
7
9
3
5
7
9
½
86.31
97.50
85.06
66.42
79.85
94.29
69.68
63.64
82.09
95.70
76.24
63.95
1
78.71
95.19
73.77
51.55
66.73
89.37
54.70
50.10
68.53
92.02
62.93
49.28
2
66.64
91.63
60.45
38.43
60.29
81.08
42.98
37.09
61.33
85.81
51.08
36.68
3
57.87
86.99
52.82
30.81
43.61
74.23
36.68
29.73
46.75
80.93
44.35
30.18
Example 7
Preparative Purification of Forms A and B
IDEC-159 (ddCC49) is a CH2 domain deleted monoclonal antibody directed against TAG-72 antigen, which is expressed on the surface of tumors. IDEC-159 contains two isoforms of the antibody, called Form A and Form B. The current cell culture process for IDEC-159 produces an approximate 50:50 ratio of Form A to Form B. The form A isoform is an antibody with a deleted CH2 region in the Fc portion of the heavy chain. In addition to having a deleted CH2 region, Form B also lacks the disulfide bond linkage across the Fc region and is only held together by hydrophobic interactions and salt bridges.
The third and final chromatography step in the IDEC-159 purification process was developed to separate the two isoforms of IDEC-159. The separation is achieved by hydrophobic interaction chromatography (HIC), using a Phenyl TSKgel 5PW-HR adsorbent. Since Form B is more hydrophobic than Form A, it adsorbs irreversibly to the stationary phase using approximately 0.73 M Ammonium Sulfate/20 mM Sodium Phosphate, pH 4.0-pH 7.0 as the mobile phase. Form A binds to a lesser extent to the stationary phase under these conditions and is therefore eluted isocratically, i.e. it leaves the column with the flowthrough fraction. Subsequent to the isocratic elution of Form A, omitting Ammonium sulfate from the mobile phase desorbs Form B. The following method was used to separate the two isoforms of IDEC-159:
The column was sanitized using ≧3 CVs of 0.5 N NaOH, at ≦150 cm/hr.
The column was equilibrated using ≧5 CVs of 0.73 M Ammonium Sulfate/20 mM Sodium Phosphate, pH 4.0, at ≦150 cm/hr.
The column was loaded with room temperature TMAE Flowthrough that has been adjusted to include 0.43 volumes of 2.5 M Ammonium Sulfate/20 mM Sodium Phosphate, pH 4.0 liquid stock solution, at 5 mg per ml of resin. The antibody was loaded onto the column at pH 4.0, at ≦100 cm/hr. Collection of the antibody started when the outlet O.D. at 280 nm reaches 10 mAU.
The column was washed using 15 CVs of 0.73 M Ammonium Sulfate/20 mM Sodium Phosphate, pH 4.0, at ≦100 cm/hr. Continue antibody collection throughout the 15 CV wash, then the outlet was diverted back to waste.
The column was stripped using ≧5 CVs of 20 mM Sodium Phosphate, pH 4.0, at ≦100 cm/hr. 6. The column was cleaned with ≧3 CVs 0.5 N NaOH, at ≦150 cm/hr.
The column was equilibrated with ≧3 CVs of 0.73 M Ammonium Sulfate/20 mM Sodium Phosphate, pH 4.0, at ≦150 cm/hr.
The column was stored in ≧3 CVs of 20% Ethanol, at ≧150 cm/hr.
The separation of the two forms at a preparative scale (5 L column volume, total IDEC-159 load approximately 20 g) is shown in FIG. 17 (Panels A and B). The first two peaks comprise the isocratic elution of Form A, the second peak shows the eluted Form B, while the third peak contains impurities, which are removed from the stationary phase during cleaning.
The capability of this method to separate Forms A and B at preparative scale was also demonstrated by SDS PAGE. As shown in FIG. 18, the fractions eluted isocratically using 0.73 M Ammonium Sulfate/20 mM Sodium Phosphate, pH 4.0 (lanes 6 to 8) contain predominantly Form A (purity >90%).
Example 8
Humanization of Monoclonal Antibody CC49
Several changes to the CC49 antibody were made to create a humanized CC49 version 2 (huCC49 V2). FIG. 19 shows an alignment of the light (FIG. 19A) and heavy chain (FIG. 19B) variable regions of murine CC49, LEN or 21/28′ CL, humanized CC49, and humanized CC49 V2 (which comprises one amino acid substitution in the light chain and two amino acid substitutions in the heavy chain as compared to humanized CC49, see underlined amino acids). To further reduce potential immunogenicity of the humanized CC49 MAb, murine residues present in the antibody were examined and considered for replacement with human framework residues derived from human acceptor sequences LEN for light chain substitutions and 21/28′ CL for heavy chain substitutions. (Singer I I et al., 1993. Optimal Humanization of 1B4, an Anti-CD18 Murine Monoclonal Antibody, is Achieved by Correct Choice of Human V-Region Framework Sequences. J. Immunol. 150:2844-2857. Padlan E A, 1991. Possible Procedure For Reducing the Immunogenicity of Antibody Variable Domains While Preserving Their Ligand-Binding Properties. Molecular Immunol. 28:489-498). Framework residues considered to be important for preserving the specificity and affinity of the combining site revealed only a few differences. In the heavy chain sequence, the predicted buried residues at positions 69 (leucine) and 93 (threonine) were both substituted with the human residues isoleucine and alanine, respectively. In the light chain sequence, one residue predicted to be mostly buried at position 43 (serine) was substituted with the human residue proline.
Domain deleted forms of the V2 CC49 antibody were made and connecting peptides were inserted into the huCC49 V2 sequence. FIG. 13A (SEQ ID NO: 28) shows the DNA sequence of heavy chain CH2 domain-deleted huCC49 V2 containing G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide. FIG. 13B (SEQ ID NO: 29) shows the DNA sequence of light chain CH2 domain-deleted huCC49 V2. FIG. 14A (SEQ ID NO: 30) shows the amino acid sequence of heavy chain CH2 domain-deleted huCC49 V2 containing G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide. FIG. 14B (SEQ ID NO: 31) shows the amino acid sequence of light chain CH2 domain-deleted huCC49 V2.
Example 9
Enhanced Biodistribution Profiles of Antibodies Comprising Novel Connecting Peptides
Various forms of domain deleted antibodies (with and without connecting peptides) were tested in a competitive binding assay for their ability to bind to bovine submaxillary mucine, a source of the TAG-72 antigen, by time-resolved flourometric immunoassay using a Wallac 1420 Multilabel Counter Victor V (PerkinElmer). Competitive binding curves are shown in FIG. 20. HuCC49 PAP (containing the connecting peptide shown in SEQ ID NO: 14), HuCC49 V2 PAP (containing the connecting peptide shown in SEQ ID NO: 14), HuCC49 G1/G3:PAP (containing the connecting peptide shown in SEQ ID NO: 9), HuCC49 V2 G1/G3/PAP (containing the connecting peptide shown in SEQ ID NO: 9), and control parent HuCC49 antibodies were evaluated. Relative binding activities for all three hinge engineered antibodies are indistinguishable or within 2-3-fold of the control parent CC49 antibody.
Biodistribution of 90Y-2-(p-isothiocyanatobenzyl) (p-SCN-Bz)-cyclohexyldiethylenetriaminepentaacetic acid ligand (CHx-DTPA) conjugated HuCC49 V2 PAP (containing the connecting peptide shown in SEQ ID NO: 14) and control parent HuCC49 antibody were evaluated and compared in athymic mice bearing LS-174T human tumor xenografts. Percentage injected dose (% ID) of 90Y radiolabelled antibody per gram of tumor or normal tissue was determined at 3 and 24 hours and is shown in Table 7.
TABLE 7
7 mice/group
Blood
Spleen
Kidney
Liver
Tumor
HuCC49
3 hrs
20.1 ± 3.5
6.1 ± 1.6
11.7 ± 1.7
10.1 ± 1.8
9.3 ± 2.0
24 hrs
0.7 ± 0.2
9.5 ± 4.0
11.0 ± 2.0
12.0 ± 1.5
12.7 ± 7.1
HuCC49 V2 PAP
3 hrs
24.6 ± 3.0
4.6 ± 2.2
10.0 ± 1.4
8.4 ± 1.0
16.1 ± 5.0
24 hrs
2.0 ± 0.6
7.7 ± 1.8
6.7 ± 0.4**
11.2 ± 2.2
21.3 ± 4.8*
Data represent mean values +/− standard deviations.
*p < 0.05 unpaired t test compared to 24 hr time point HuCC49 in tumor
**p < 0.001 unpaired t test compared to 24 hr time point HuCC49 in kidney
Surprisingly, at the 24 hour time point HuCC49 V2 PAP uptake was significantly higher in the tumor (p<0.05 unpaired t Test) and, conversely, lower in the kidney (p<0.01) than control HuCC49 antibody. When the tumor to organ ratio for these antibodies was compared, the HuCC49 V2 PAP resulted in a higher tumor to organ ratio for all organs except blood (FIG. 21).
These results suggest that these novel hinges impart structural changes to antibodies that positively effect tumor localization and decrease uptake by normal organs, such as the kidney. Thus, these novel hinges are particularly useful when incorporated into therapeutic antibodies.
Example 10
Enhanced Biodistribution Profiles of Antibodies Comprising Novel Connecting Peptides: Detailed Time Course
This example confirms and extends the results presented in Example 9. The antibodies Human CC49 V2 G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO: 9) and HuCC49 (gly/ser) were diafiltered and concentrated into low metal 5 mM sodium acetate buffer, pH 5 (LMB) using a pre-rinsed Amicon Centricon 30. Centrifugation of the centricon was performed in a fixed angle rotor at 5000×g held at a temperature of 2-8° C. Each antibody was recovered by adding 50 ill of LMB to the sample reservoir, vortexing briefly, and back-spinning for 10 minutes at 1000×g. The protein concentration was determined using UV Spec analysis at 280 nm using the extinction coefficient of 1.48. Each antibody was then adjusted down to 10.5 mg/ml using LMB.
The antibodies were adjusted to ˜pH 8.6 using 1.0 M Boric Acid (pH 8.6, Chelex treated and 0.2 μm filtered). CHx-DTPA (dissolved in 1.0 M Boric Acid) was then added at a molar ratio of 3 chelates to 1 mole of antibody. The amount of Boric acid added was one-tenth the antibody volume. This mixture was then vortexed and incubated for 16 to 18 hours at room temperature. The reaction was stopped by adding the mixture to a new, pre-rinsed Centricon 30 and diafiltered into low metal 5 mM Sodium Acetate, 150 mM Sodium Chloride, pH 5 as per the previous diafiltration. The concentration of each antibody was adjusted to 3 mg/mL.
Female nude mice were inoculated s.c. with LS174T cells suspended in HBSS (Biowhittaker, Cat#10-547F) on the inside of the right thigh. Tumor sizes were measured one day prior to study start. Tumor volumes were calculated by multiplying the length times half of the squared width [L×((W2)/2)]. The mice were grouped to give an average tumor volume of ˜200 mm3.
Forty two nude mice were injected with 111In-labeled CH2 domain deleted antibody at time zero. The study tracked the distribution of the antibody over the course of seven timepoints with each timepoint consisting of six mice. Urine was collected from each mouse by holding the mouse over tared weigh paper and squeezing the bladder. Blood was taken via “eye bleed” (approximately 200 ul per mouse). For each individual mouse, any feces excreted during the blood and urine sampling was collected. Following the blood collection, the mice were sacrificed by cervical dislocation. Once each of the six mice had been sacrificed the other samples were collected via dissection. Each sample (except for the skin) was rinsed with 3% formalin, blotted dry on paper towel and then weighed. All samples were weighed using tared weigh paper.
Following the sample collections, the samples were placed into borosilicate test tubes and counted on a gamma counter along with a decay control consisting of a 1:10 dilution of the labeled antibody. The percent radioactivity associated with each organ or tissue relative to the decay control (% injected dose/g tissue or organ) was calculated and those values are presented in the FIG. 22 (CC49 V2 G1/G3/Pro243Ala244Pro245+[Gly/Ser]) and 23 (HuCC49 [gly/ser]). The example shows that antibody molecules comprising this novel connecting peptide show decreased accumulation in the kidney, slightly increased accumulation in the blood and significantly increased accululation at tumor. This profile is consistent with these molecules having increased stability in vivo and enhanced efficacy and safety.
Example 11
Antibodies Comprising Connecting Peptides Have Decreased Sensitivity to Reducing Agents
The example demonstrates that domain deleted CC49 G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO: 9) appears more stable toward glutathione (GSH) reduction, as is parent CC49, than domain deleted CC49 with a Gly-Ser hinge linker.
Briefly, 50 ug of ddCC49 (Gly-Ser), ddCC49 G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO: 9) or parent CC49 were incubated with 0, 1, 5 or 10 mM GSH for one hour at room temperature. Reaction buffers used include 100 mM PBS, pH 7.2 or 100 mM Sodium Acetate, 100 mM NaCl, pH 4.5. GSH-treated antibodies were heated with SDS and applied to a 4-20% gradient SDS-PAGE, non-reducing gel. Applied samples were allowed to migrate through the gel at a constant 120 Volts for 90 minutes at room temperature. Proteins were Coomassie stained and gels dried. Gels are shown in FIG. 24.
As shown in FIG. 24, domain deleted CC49 G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO: 9) appears more stable toward glutathione (GSH) reduction, as is parent CC49, than domain deleted CC49 with a Gly-Ser hinge linker. In addition, 100 mM Sodium Acetate at pH 4.5 further protects domain deleted CC49 G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO: 9) from GSH reduction compared to 100 mM PBS at pH 7.2. This unexpected observation of decreased sensitivity to reducing agents suggests that the G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO: 9) hinge design enables the use of chemistries using reducing agents, such as those used to prepare drug conjugates (e.g. SPDP linkers) or techniques for attaching radioisotopes to antibodies (eg. 99MTc), while maintaining the physical integrity of the antibody. This advantage with respect to reducing agent sensitivity does not appear to alter pharmacokinetic advantages of CH2-domain deleted constructs (see mouse biodistribution data in Example 10). The decreased sensitivity to reducing agents also may be predictive of increased in vivo stability.
Example 12
Anti-CD20 Antibody Comprising a Connecting Peptide
The hinge region connecting peptide G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO: 9) was introduced into a CH2 domain-deleted C2B8 antibody as described in Example 3. C2B8 is a chimeric anti-CD20 monoclonal antibody consisting of murine heavy and light chain variable domains fused to human heavy and light chain constant domains, respectively. Correct modifications to the hinge region were confirmed by DNA sequence analysis. Plasmid DNA was used to transform CHO DG44 cells for transient production of antibody protein.
Supernatant was collected from cells producing CH2 domain-deleted C2B8 antibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide and concentration of antibody in the culture supernatants determined by immunoassay. Approximately 3 ng of total antibody protein from the transient cell culture was compared to CH2 domain-deleted huCC49 MAb by non-reducing SDS-PAGE electrophoresis followed by Western Blot with anti-human IgG HRP conjugated antibody to detect CH2 domain deleted huCC49 Form A and Form B forms. Under these conditions, Form A migrates as a single 120 kDa homodimer and Form B as a 60 kDa doublet. Incorporation of the connecting peptide shown in SEQ ID NO: 9 was found to substantially increase the proportion of Form A produced. Exemplary results are shown in FIG. 27. This result shows that the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge (SEQ ID NO:9) resulted in the production of essentially all Form A CH2 domain-deleted C2B8 antibody with little or no detectable Form B, demonstrating that the utility of this hinge for producing the Form A isoform is generally applicable to antibodies of varying specificities.
Example 13
Anti-CD23 Antibody Comprising a Connecting Peptide
The hinge region connecting-peptide G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO: 9) was used to construct a CH2 domain-deleted 5E8 (5E8ΔCH2) antibody essentially as described in Example 3. 5E8 is a chimeric anti-CD23 monoclonal antibody consisting of primate heavy and light chain variable domains fused to human heavy and light chain constant domains, respectively. Correct modifications to the hinge region were confirmed by DNA sequence analysis. Nucleic acid and amino acid sequences of 5E8 light chain and heavy chain are shown in FIGS. 28 and 29, respectively. Plasmid DNA was used to transform CHO DG44 cells for production of antibody protein.
A cell line (1A7) containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO:9) connecting peptide introduced into the 5E8ΔCH2 antibody sequence was used for antibody production. Antibody was produced and purified using methods described in Example 4 above. 5E8ΔCH2 G1/G3/Pro243Ala244Pro245 antibody, purified using only the Protein G column, eluted essentially as a single peak at ≧97% purity without further HIC purification. Reduced and non-reduced purified protein samples were analyzed by SDS-PAGE electrophoresis. Under non-reducing conditions, Form A is expected to migrate as a single 120 kDa homodimer and Form B as a 60 kDa doublet. The connecting peptide shown in SEQ ID NO: 9 was found to substantially increase the proportion of Form A produced. Exemplary results are shown in FIG. 30. This result shows that the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge (SEQ ID NO:9) resulted in the production of essentially all Form A 5E8ΔCH2 antibody with little or no detectable Form B (see lane 2), demonstrating that the utility of this hinge for producing the Form A isoform is generally applicable to antibodies of varying specificities. 5E8ΔCH2 G1/G3/Pro243Ala244Pro245 antibody was examined by size exclusion chromatography and found to elute as a single peak indicating that there was no significant aggregation or decomposition of antibody product. 5E8ΔCH2 G1/G3/Pro243Ala244Pro245 antibody was further tested in a FRET (fluorescence resonance energy transfer) competitive binding assay for Cy5-labeled soluble. CD23 binding to Eu-labeled 5E8 IgG using a Delphia fluorimeter (Wallac 1420 Multilabel Counter Victor V, PerkinElmer). Competitive binding curves are shown in FIG. 31. 5E8ΔCH2 G1/G3/Pro243Ala244Pro245 (containing the connecting peptide shown in SEQ ID NO: 9), and control parent 5E8 IgG antibodies were evaluated. Relative binding activity of the hinge engineered antibody was indistinguishable from control parent 5E8 IgG antibody. From these results it is clear that introduction of the hinge region (containing the connecting peptide shown in SEQ ID NO: 9) in the 5E8ΔCH2 antibody led to the preferential synthesis of the A isoform while retaining full binding activity and supports the general utility of the engineered hinges.
Example 14
chB3F6 Antibody Comprising a Connecting Peptide
The hinge region connecting peptide G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO: 9) was used to construct a CH2 domain-deleted chimeric B3F6 (chB3F6ΔCH2) antibody essentially as described in Example 3. chB3F6 is a chimeric anti-CRIPTO monoclonal antibody consisting of murine heavy and light chain variable domains fused to human heavy and light chain constant domains, respectively. Correct modifications to the hinge region were confirmed by DNA sequence analysis. Nucleic acid and amino acid sequences of chB3F6 light chain and heavy chain are shown in are shown in FIGS. 32 and 33, respectively. Plasmid DNA was used to transform CHO DG44 cells for production of antibody protein.
A cell line (3C7) containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO:9) connecting peptide introduced into the chB3F6ΔCH2 antibody sequence was used for antibody production. Antibody was produced and purified using methods described in Example 4 above. ChB3F6ΔCH2 G1/G3/Pro243Ala244Pro245 antibody, purified using only the Protein G column, eluted essentially as a single peak at 97% purity without further HIC purification. Reduced and non-reduced purified protein samples were analyzed by SDS-PAGE electrophoresis. Under these conditions, Form A is expected to migrate as a single 120 kDa homodimer and Form B as a 60 kDa doublet. The connecting peptide shown in SEQ ID NO: 9 was found to substantially increase the proportion of Form A produced. Exemplary results are shown in FIG. 34. This result shows that the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge (SEQ ID NO:9) resulted in the production of essentially all Form A chB3F6ΔCH2 antibody with little or no detectable Form B, demonstrating that the utility of this hinge for producing the Form A isoform is generally applicable to antibodies of varying specificities. ChB3F6ΔCH2 G1/G3/Pro243Ala244Pro245 antibody was examined by size exclusion chromatography and found to elute essentially as a single peak ranging from 93-98% monomer indicative of little or no significant aggregation or decomposition of antibody product. ChB3F6ΔCH2 G1/G3/Pro243Ala244Pro245 antibody was further tested in a flow cytometry competitive binding assay with FITC-labeled B3F6 IgG binding to GEO tumor cells, a source of the CRIPTO antigen. Competitive binding curves are shown in FIG. 35. ChB3F6ΔCH2 G1/G3/Pro243Ala244Pro245 (containing the connecting peptide shown in SEQ ID NO: 9), and two control samples of chB3F6 IgG antibodies were evaluated. Relative binding activity of the hinge engineered antibody was indistinguishable from the control parent chB3F6 IgG antibodies. From these results it is clear that introduction of the hinge region (containing the connecting peptide shown in SEQ ID NO: 9) in chB3F6ΔCH2 antibody led to the preferential synthesis of the A isoform while retaining full binding activity and further supports the general utility of the engineered hinges.
Equivalents
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
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10880320
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biogen idec ma inc.
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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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424/133.1
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Apr 1st, 2022 05:10PM
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Apr 1st, 2022 05:10PM
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Biogen
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:biib
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Biogen
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Nov 1st, 2011 12:00AM
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Dec 10th, 2007 12:00AM
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https://www.uspto.gov?id=US08048635-20111101
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Measurement of soluble Tweak levels for evaluation of lupus patients
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Methods of treating patients and evaluating patients for disease stage and/or severity are disclosed.
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8048635
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1. A method for evaluating a test subject for status, stage, progression or severity of lupus nephritis, the method comprising:
obtaining a biological fluid sample from the test subject; and
determining the level of soluble Tweak (TNF-like weak inducer of apoptosis) in the sample;
wherein the sample is a urine sample, a serum sample, or a plasma sample;
and wherein an increase or decrease in the level of soluble Tweak as compared to a healthy subject or an earlier sample from the test subject correlates to the status, stage, progression or severity of lupus nephritis in the test subject.
2. The method of claim 1, wherein the level of soluble Tweak is determined in a urine sample from the subject.
3. The method of claim 1 or 2, wherein an increase in soluble Tweak in the sample relative to the earlier sample indicates increased activity or severity of the lupus nephritis and a decrease in soluble Tweak in the sample relative to the earlier sample indicates decreased activity or severity of the lupus nephritis.
4. The method of claim 3, wherein obtaining the sample and the earlier sample are separated by at least one week.
5. The method of claim 1 or 2, wherein the subject has or is suspected of having lupus.
6. The method of claim 1, wherein the level of soluble Tweak is determined by immunoassay.
7. The method of claim 3, wherein the determining the level of soluble Tweak in the sample comprises recording, in a print or computer readable record, the presence or level of the soluble Tweak as a diagnostic, staging or prognostic factor for the lupus nephritis.
8. The method of claim 3, wherein an increase in soluble Tweak levels is indicative of a renal flare or risk of a renal flare in the subject.
9. The method of claim 3, wherein an increase in soluble Tweak levels is indicative of increased severity or activity of the lupus nephritis.
10. The method of claim 3, wherein the evaluating steps are performed with a dipstick immunoassay.
11. A method for evaluating a test subject for status, stage, progression or severity of systemic lupus erythematosis (SLE), the method comprising:
obtaining a biological fluid sample from the subject; and
determining the level of soluble Tweak in the sample;
wherein an increase or decrease in the level of soluble Tweak as compared to a healthy subject or an earlier sample from the test subject correlates to the status, stage, progression or severity of SLE in the test subject.
12. The method of claim 11, wherein the sample is selected from the group consisting of a urine sample, a serum sample, a plasma sample, a synovial fluid sample, and a cerebrospinal fluid sample.
13. The method of claim 11, wherein Tweak in the sample is evaluated in a urine sample from the subject.
14. The method of claim 11, wherein soluble Tweak in the sample is measured by immunoassay.
15. The method of claim 11, wherein the soluble Tweak in the sample is measured with a dipstick immunoassay.
16. The method of claim 11, wherein the soluble Tweak in the sample is measured with an ELISA.
17. The method of claim 11, wherein the amount of soluble Tweak in the sample is measured in a sandwich assay.
18. The method of any one of claims 1, 2, 6, or 11, wherein determining the level of soluble Tweak in the sample comprises recording, in a print or computer readable record, the presence or level of the soluble Tweak as a diagnostic, staging or prognostic factor.
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18
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application, filed under 35 U.S.C. §111, is a continuation claiming priority under 35 U.S.C. §120 of International Application No. PCT/US2006/022830, filed on Jun. 12, 2006, which claims priority to U.S. Application Ser. No. 60/689,905, filed on Jun. 13, 2005. The contents of all the foregoing applications are hereby incorporated by reference.
BACKGROUND
The identification of genes and proteins linked with the severity and/or progression of disease and the development of diagnostic methods to identify and/or monitor disease progression are of considerable importance.
SUMMARY OF THE INVENTION
The invention is based, at least in part, on the discovery that Tweak and/or Tweak receptor, e.g., Fn14 (and certain proteins that are modulated, e.g., induced, by Tweak) can be used as biomarkers of disease activity in a biological fluid of a human subject. Accordingly, methods and compositions are provided for assessing, staging, and/or monitoring disease activity in a subject, e.g., assessing, staging, and/or monitoring inflammatory disease activity (e.g., lupus, fibrosis, rheumatoid arthritis, multiple sclerosis, and nephritis activity, e.g., lupus nephritis); and neurodegenerative disease activity.
Accordingly, in one aspect, the invention features a method of evaluating a subject. The method includes evaluating Tweak or Fn14 (and/or certain proteins that are increased by Tweak), in a biological fluid of a subject (such as a human), and correlating the result of the evaluation with the subject's risk, stage or status of disease activity, e.g., inflammatory disease activity. The subject can have, or be at risk for, e.g., an inflammatory condition such as lupus, rheumatoid arthritis (RA), psoriatic arthritis (PsA), multiple sclerosis, nephritis (e.g., interstitial nephritis, lupus nephritis, glomerulonephritis (GN), mesangial GN, membraneous GN, diffuse proliferative GN and/or membranoproliferative GN), stroke or a neurodegenerative disease (e.g., ALS, Parkinson's Disease, Huntington's Disease, Alzheimer's Disease), fibrosis, or cancer (e.g., solid cancers and/or hematological cancers). The term “correlating” means describing the relationship between the presence or level of the protein or nucleic acid, and the stage, status, extent, severity, or level of risk for disease. Such correlation may be displayed in a record, e.g., a print or computer readable material, e.g., an informational, diagnostic, or instructional material. The record may identify the presence or level of a Tweak or Tweak-R protein or nucleic acid as a diagnostic, staging or prognostic factor for the disease. The record may include a parameter (qualitative or quantitative) representing expression or activity of Tweak and/or TweakR, as evaluated by the method.
The evaluation is performed on a biological fluid from the subject, e.g., serum, urine, plasma, cerebrospinal fluid (CSF), or synovial fluid. Increased Tweak or TweakR protein levels in the fluid correlate (e.g., directly) with increased severity, stage and/or activity of disease. The ability to perform such an evaluation on a readily obtainable biological fluid from a subject provides a simple, quick, relatively non-invasive method for evaluating, staging, and/or diagnosing a subject, e.g., before, during and/or after a treatment is begun.
In one embodiment, increased Tweak or TweakR urinary, serum or plasma levels correlate with increased severity or activity of renal disease (e.g., more advanced stage or increased severity of nephritis, e.g., lupus nephritis) compared to a reference value. In another embodiment, increased Tweak or TweakR serum, plasma, urine or synovial fluid levels correlate with increased severity or activity of RA compared to a reference value. In another embodiment, increased Tweak or TweakR serum, plasma, or urine fluid levels correlate with increased severity or activity of lupus compared to a reference value. In another embodiment, increased Tweak or TweakR serum, plasma, urine or CSF levels correlate with increased severity or activity of MS or a neurodegenerative disease compared to a reference value. In another embodiment, increased Tweak or TweakR serum, plasma, or urine levels correlate with increased severity or activity of fibrosis compared to a reference value. A reference value can be a control value, e.g., a value for a normal subject (e.g., a subject not suspected of, or at risk for, the disease being evaluated), a value determined for a cohort of subjects, or a baseline (e.g., prior) value from the subject being evaluated.
The method can be used to stage and/or diagnose a disorder, e.g., to diagnose the stage or severity of the disorder; to evaluate the subject's response to treatment, e.g., to monitor progression or improvement in a parameter of the disorder in a subject being treated for the disorder; to evaluate the course of the disorder, e.g., to assess the risk of, or to predict a flare-up of, the disorder. In one embodiment, the evaluation is performed more than once, e.g., at periodic intervals over a period of time, e.g., to monitor progression of the disease or to monitor response to a treatment. For example, the evaluation may be performed daily, every 2 or 3 days, every week, every 2 weeks, monthly, every 6 weeks, every 2 months, every 3 months or as appropriate, over a period of time to encompass at least 2, 3, 5, 10 evaluations or more.
In some embodiments, the step of evaluating includes detecting expression or activity of a Tweak or Tweak-R protein or a nucleic acid encoding Tweak or Tweak-R (e.g., by qualitative or quantitative analysis of mRNA, cDNA, or protein), or evaluating one or more nucleotides in a nucleic acid (genomic, mRNA, or cDNA) encoding Tweak or Tweak-R. In one embodiment, the method includes using an immunoassay to detect Tweak protein, e.g., in a biological fluid, such as a urine sample, of the subject. In other embodiments, the method can include administering a labeled Tweak or Tweak-R binding agent (e.g., an antibody) to a subject, and evaluating localization of the labeled binding agent in the subject, e.g., by imaging the subject (e.g., imaging at least a portion of the kidney of the subject).
In one embodiment, the subject has nephritis, or is suspected of having nephritis. The method can be used to evaluate a treatment for renal disease, e.g., nephritis, e.g., lupus nephritis. For example, the subject is receiving a treatment for renal disease and the subject is evaluated before, during, or after receiving the treatment, e.g., multiple times during the course of treatment. The subject may have normal kidney function as defined and detected by a clinical measure, e.g., urine protein level, blood creatinine level, urine creatinine level, creatinine clearance, and/or blood urea nitrogen. In other cases, the subject has an abnormal, e.g., deficient, kidney function, e.g., as defined and detected by a clinical measure.
In one embodiment, the subject has arthritis, e.g., rheumatoid arthritis (RA) or psoriatic arthritis (PsA), or is suspected of having arthritis. The method can be used to evaluate a treatment for arthritis. For example, the subject is receiving a treatment for arthritis (e.g., an anti-TNF therapy, methotrexate or steroids) and the subject is evaluated before, during, or after receiving the treatment, e.g., multiple times during the course of treatment.
In one embodiment, the subject has lupus. The subject may have, e.g., lupus nephritis, and/or neuropsychiatric manifestations (CNS lupus) or other manifestations of lupus, e.g., low blood count, serositis or hematological manifestations. The method can be used to evaluate a treatment for lupus. For example, the subject is receiving a treatment for lupus (e.g., NSAID, corticosteroids, or a DMARD) and the subject is evaluated before, during, or after receiving the treatment, e.g., multiple times during the course of treatment.
In one embodiment, the subject has a neurodegenerative disease, e.g., MS, Parkinson's Disease, Huntington's Disease, Alzheimer's Disease or ALS, or is suspected of having a neurodegenerative disease. The method can be used to evaluate a treatment for a neurodegenerative disease. For example, the subject is receiving a treatment for a neurodegenerative disease (e.g., beta-interferon, riluzole, a cholinesterase inhibitor, copaxone, an NMDA receptor antagonist) and the subject is evaluated before, during, or after receiving the treatment, e.g., multiple times during the course of treatment.
The method can be used to identify a subject for treatment, e.g., for treatment for nephritis, lupus, fibrosis, rheumatoid arthritis, psoriatic arthritis, stroke, cancer or a neurodegenerative disease. The subject can be identified as a subject suited for treatment as a function of results of the detection, e.g., the results show similarity to, e.g., statistically significant similarity to, a reference value indicative of a subject being at a particular stage of the particular disease. For example, elevated Tweak or Tweak-R expression in the urine, serum, plasma, or CSF can be indicative of a subject who can be treated with a Tweak or Tweak-R blocking agent or other treatment for the disease.
An increase identified by the evaluation step, e.g., a statistically significant increase, or at least 20%, 25%, 30, 50, 70, 80, 100, or 110% increase, in a subject, over a reference value, e.g., a base value or control, can indicate increased activity or severity of the disease, e.g., lupus nephritis (e.g., membranous, focal proliferative, or diffuse proliferative lupus nephritis). For example, a 10%, 20%, 25% or greater increase in urine Tweak levels (e.g., relative to creatinine) compared to a negative control, baseline value, or previous evaluation in a subject indicates increased nephritis severity or activity. In one embodiment, Tweak levels greater than 0.5, 1, 2, 3, 4, or 5 pg/mg Cr can indicate that the subject has nephritis, e.g., lupus nephritis. In one embodiment, Tweak levels in the range of 2-8 pg/mg Cr (e.g., about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, or 7.5 pg/mg Cr) can indicate that the subject has stable disease; Tweak levels in the range of 8-10 pg/mg Cr can indicate that the subject is at risk for a relapse; Tweak levels greater than 10 pg/mg Cr (e.g., between 10-15 pg/mg Cr) can indicate that the subject is in active relapse.
In one embodiment, the method includes evaluating the subject (e.g., evaluating urine samples from the subject) a plurality of instances over time, e.g., over the course of a treatment, e.g., at least one day, five days, a week, four or six weeks, and so forth. The method can include determining a rate of change for the evaluated parameter, e.g., to determine disease progression or therapeutic efficacy. In one embodiment, the subject is also being treated, e.g., with a Tweak blocking agent or other treatment for the disease.
In one embodiment, the evaluation includes contacting a biological sample of the subject, preferably a urine, serum, plasma, CSF or synovial fluid sample, with an agent that detects Tweak, TweakR or a biomarker whose expression is modulated (e.g., increased) by Tweak (e.g., in mesangial cells). The agent, e.g., an antibody or nucleic acid probe, can be immobilized on a solid phase, e.g., on a microtiter well, tube, dipstick or other test device. In a preferred embodiment, the expression, presence, level, or activity is detected using a dipstick or other test device format assay.
The evaluation can include a protein-based (e.g., an immunoassay) or nucleic acid-based assay, e.g., a hybridization-based assay. In one embodiment, the evaluating step comprises performing one or more of: enzyme-linked immunoassay, radioimmunoassay, immunoblot assay (including Western blot analysis and sandwich assay), in situ hybridization, Northern blot analysis, and nucleic acid amplification, including PCR (e.g. quantitative RT-PCR). Many evaluation methods can include one or more features of the foregoing. Exemplary immunoassays can include contacting the sample with an antibody that binds to Tweak or can be adapted to use other agents that bind to Tweak, e.g., a soluble Tweak receptor. Nucleic acid-based assays can include hybridization with a nucleic acid from a Tweak-encoding sequence, e.g., from a human Tweak-encoding genomic sequence or cDNA, e.g., the coding or non-coding strand, or a primer or other oligonucleotide complementary to a region of a Tweak-encoding sequence. It is also possible to evaluate a biomarker that is modulated (e.g., increased) by Tweak.
In some embodiments, evaluation can be facilitated by a dipstick or other test device-based kit, e.g., suitable for testing by non-trained individuals, e.g., suitable for home testing. Such a screening test would provide convenience, privacy and eliminate the necessity and cost of visiting a physician for a screening test, although the dipstick or other test device kit could also be used in a clinical setting. The dipstick or other test device kit could be similar to a home pregnancy kit, known to those of skill in the art, and could provide a color indication for an increased risk, stage or severity for an inflammatory condition, e.g., nephritis, based upon the levels of a protein described herein, e.g., Tweak, TweakR or a biomarker whose expression modulated (e.g., increased) by Tweak, in the sample. Such a dipstick or other test device-based kit could be provided with a small plastic cup for collecting and retaining the sample and for conducting the test. In one scenario, the dipstick or other test device can react to produce one color if a reference level of a first protein, e.g., Tweak, is exceeded, a different color if a reference level of a second protein, e.g., a biomarker whose expression modulated (e.g., increased) by Tweak, is exceeded, and when both levels are exceeded, the two colors will combine to yield a third color that is easily distinguishable from the others. For example, a dipstick or other test device that turns yellow when a reference level of Tweak is exceeded, and turns blue when a reference level of the biomarker is exceeded will turn green when both levels are exceeded. Because a dipstick or other test device-based assay kit would be relatively resistant to temperature and humidity variations, it could easily be transported, stored and used virtually anywhere. In one embodiment, the kit includes at least 1, e.g., at least 2, 5, 10, 20, 30, or 50, test devices, e.g., dipsticks, e.g., membranes, e.g., membrane strips described herein. In one embodiment, the kit contains a container suitable for collecting a urine sample. The kit can also contain a device to obtain a tissue sample, such as a cotton swab or wooden swab.
In another embodiment, the method of the present invention may be utilized in combination with a densitometer in a device for use in a setting such as a doctor's office, a clinic or a hospital. The densitometer can provide rapid measurement of the optical density of dipstick or other test device strips that have been contacted with a bodily fluid or tissue.
In one embodiment, the method additionally includes treating the subject for the condition being evaluated, e.g., nephritis. For example, the method includes: identifying a subject at risk for or having lupus nephritis. The method can further include providing the subject a treatment for lupus, e.g., a Tweak blocking agent or other treatment suitable for treating lupus nephritis, e.g., corticosteroids or other immunosuppressive medications.
In a preferred embodiment, the subject is further evaluated for one or more of the following parameters: albumin levels; glucose levels; urine creatine; urine protein, and so forth.
In a preferred embodiment, the evaluation is used to choose a course of treatment. For example, if the subject is determined to be at risk for loss of renal function or renal disease, the treatment can include dietary restrictions.
In one embodiment, the evaluation is performed by the subject. In another embodiment, the evaluation is performed by a health care provider. In yet another embodiment, the evaluation is performed by a third party.
The method can also be used to select a patient population for treatment. A set of one or more subjects indicated for renal inflammation, e.g., relative to a reference are selected. The subjects of the set are administered a Tweak blocking agent or other treatment for renal inflammation, e.g., for renal nephritis.
Subjects can also be evaluated in response to other indications, e.g., signs of early disease, e.g., loss of renal function, when a renal disorder, e.g., early loss of renal function, is diagnosed; before, during or after a treatment for an a renal disorder, is begun or begins to exert its effects; or generally, as is needed to maintain health, e.g., kidney health, e.g., throughout the natural aging process. The period over which the agent is administered (or the period over which clinically effective levels are maintained in the subject) can be long term, e.g., for six months or more or a year or more, or short term, e.g., for less than a year, six months, one month, two weeks or less.
The method can also include obtaining a profile, e.g., the profile including parameters (qualitative or quantitative) representing expression or activity of a plurality of biomarkers, e.g., one or more of (preferably at least two of): Tweak and a biomarker whose expression modulated (e.g., increased) by Tweak (e.g., RANTES, KC, and/or IP-10). The profile can be compared to a reference profile, e.g., using multi-dimensional analysis, e.g., distance functions. A computer medium can be used that has executable code for effecting one or more the following steps: receive a subject expression profile; access a database of reference expression profiles; and either i) select a matching reference profile most similar to the subject expression profile or ii) determine at least one comparison score for the similarity of the subject expression profile to at least one reference profile. The subject expression profile, and the reference expression profiles each include a value representing the level of expression of Tweak RANTES, KC, and/or IP-10. The record can further include a subject identifier, e.g., a patient identifier, and optionally other clinical information, e.g., information that assesses a inflammatory response or autoimmune response.
Targeting of the Tweak pathway (e.g., with a Tweak pathway inhibitor such as an agent that blocks a Tweak-TweakR interaction, e.g., an anti-Tweak or anti-Fn14 blocking antibody) can be used in the treatment of nephritis, e.g., lupus nephritis.
In another aspect, the disclosure features a method of treating nephritis. The method includes: administering, to a subject (e.g., a human subject) who has or is at risk for nephritis, a Tweak blocking agent, e.g., in an amount and for a time to provide a therapeutic effect. In one embodiment, the agent is an antibody, e.g., a Tweak or Fn14 antibody, or a soluble form of Tweak receptor, e.g., a soluble Fn14.
In one embodiment, the subject has lupus. In such embodiments, the Tweak blocking agent is effective to treat the renal manifestations of the lupus, e.g., to treat lupus nephritis.
In one embodiment, the disorder is acute nephritis, chronic nephritis, glomerulonephritis (GN), primary glomerulonephritis, autoimmune nephritis, pyelonephritis, mesangial GN, membraneous GN, diffuse proliferative GN membranoproliferative GN and/or interstitial nephritis. In one embodiment the nephritis is not rapidly progressive crescentic glomerulonephritis.
In one embodiment, the agent is an antibody that is a full length IgG. In other embodiments, the agent is an antigen-binding fragment of a full length IgG, e.g., the agent is a single chain antibody, Fab fragment, F(ab′)2 fragment, Fd fragment, Fv fragment, or dAb fragment. In preferred embodiments, the antibody is a human, humanized or humaneered antibody or antigen-binding fragment thereof.
In one embodiment, the agent is a soluble form of the Tweak receptor, e.g., a polypeptide at least 95% identical to amino acids 28-X1 of SEQ ID NO:2, where amino acid X1 is selected from the group of residues 68 to 80 of SEQ ID NO:2. In some cases, the soluble form of the receptor is fused with a heterologous polypeptide, e.g., an antibody Fc region.
In one embodiment, the agent is administered in an amount sufficient to reduce urinary protein levels, delay or prevent additional kidney function deterioration, and/or improve kidney function.
In one embodiment, the agent is administered at a dose between 0.1-100 mg/kg, between 0.1-10 mg/kg, between 1 mg/kg-100 mg/kg, between 0.5-20 mg/kg, or 1-10 mg/kg. In the most typical embodiment, the dose is administered more than once, e.g., at periodic intervals over a period of time (a course of treatment). For example, the dose may be administered every 2 months, every 6 weeks, monthly, biweekly, weekly, or daily, as appropriate, over a period of time to encompass at least 2 doses, 3 doses, 5 doses, 10 doses, or more.
In one embodiment, the agent is administered in combination with another therapy for lupus or nephritis, e.g., corticosteroids or NSAIDs.
In some cases, the subject can be identified by performing a diagnostic assay described herein, e.g., by evaluating a urine sample from the subject, e.g., by evaluating Tweak levels or expression of a biomarker whose expression is increased by Tweak in mesangial cells. Exemplary biomarkers include RANTES, KC, and/or IP-10. Tweak or the biomarker can be detected by a variety of methods, including an immunoassay.
The term “treating” refers to administering a therapy in an amount, manner, and/or mode effective to improve or prevent a condition, symptom, or parameter associated with a disorder or to prevent onset, progression, or exacerbation of the disorder (including secondary damage caused by the disorder), to either a statistically significant degree or to a degree detectable to one skilled in the art. Accordingly, treating can achieve therapeutic and/or prophylactic benefits. An effective amount, manner, or mode can vary depending on the subject and may be tailored to the subject.
In another aspect, the invention features a method of evaluating a compound, e.g., screening for a compound, that modulates renal function. The method includes contacting a test compound to a Tweak or TweakR protein, a cell, a tissue or a test subject, e.g., a non-human mammal, and evaluating the protein, cell, tissue or test subject for Tweak or TweakR expression or function. A test compound that decreases or inhibits Tweak or TweakR expression or function is identified as a compound that modulates renal function, e.g., a compound useful to treat nephritis. In one embodiment, the test compound interacts with Tweak or TweakR, directly or indirectly. In a preferred embodiment, the test compound is a small molecule; a protein or peptide; an antibody; and/or a nucleotide sequence. For example, the agent can be an agent identified through a library screen.
In some embodiments, the method may include a two-step assay, e.g., a first step of contacting and evaluating a test compound against a Tweak or TweakR protein or a Tweak expressing cell, and a second step of contacting and evaluating the test compound against a non-human animal.
The method can also include evaluating expression or activity (in the cell, tissue or non-human animal) of one or more biomarkers whose expression modulated (e.g., increased) by Tweak (e.g., RANTES, KC, and/or IP-10).
In another aspect, the disclosure features a method of making a diagnostic device. The method includes supplying a substrate, e.g., a dipstick or other test device, well, tube, or strip; and adhering an reagent (e.g., an antibody) that detects one or more of Tweak, TweakR or a biomarker increased by Tweak, or providing such reagent as a solution or other formulation available for use in the test device, e.g., in a sandwich assay. In one embodiment, the reagent is applied by spraying, deposition of a liquid, or printing. The device can be supplied with instruction for its use in the evaluation of kidney disease, e.g., nephritis.
In another aspect, the invention features a computer readable record encoded with (a) a subject identifier, e.g., a patient identifier, (b) one or more results from an evaluation of the subject, e.g., a diagnostic evaluation described herein, e.g., the level of expression, level or activity of Tweak or Tweak-R, in the subject, and optionally (c) a value for or related to renal disease (such as nephritis), e.g., a value correlated with disease status or risk with regard to renal disease. In one embodiment, the invention features a computer medium having a plurality of digitally encoded data records. Each data record includes a value representing the expression, level or activity of Tweak or Tweak-R levels or activity, in a sample, and a descriptor of the sample. The descriptor of the sample can be an identifier of the sample, a subject from which the sample was derived (e.g., a patient), a diagnosis, or a treatment (e.g., a preferred treatment). In a preferred embodiment, the data record further includes values representing the level of expression, level or activity of genes other than Tweak or Tweak-R (e.g., other genes associated with renal disease, or other genes on an array). The data record can be structured as a table, e.g., a table that is part of a database such as a relational database (e.g., a SQL database of the Oracle or Sybase database environments). The invention also includes a method of communicating information about a subject, e.g., by transmitting information, e.g., transmitting a computer readable record described herein, e.g., over a computer network.
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 DRAWINGS
FIG. 1 shows that uTWEAK levels correlate with lupus nephritis (LN) activity. A) comparison of uTWEAK levels between 43 patients with active renal disease (defined as patients with rSLEDAI score of ≧4), to 35 patients with non-active renal disease (including patients with no previous renal involvement and those with previous kidney involvement, but in which the renal disease is inactive, all with rSLEDAI score of 0). B) comparison of rSLEDAI scores of 78 SLE patients with their levels of uTWEAK yields a positive (ρ=0.405), significant (P<0.001) correlation.
FIG. 2 shows that uTWEAK levels are higher during active disease, particularly renal flares. A) In a comparison between patients undergoing a flare and those with stable disease among all SLE patients (n=49), the flaring patients had significantly higher uTWEAK levels. Bars indicate median values. B) In patients with renal disease, both active and inactive (n=35), there was also a trend toward higher uTWEAK levels in flaring patients as opposed to chronic stable patients. Bars indicate median values.
FIG. 3 shows that uTWEAK levels are significantly higher in renal than in non-renal flares. A comparison among flaring SLE patients (n=31), undergoing a renal flare or a non-renal one: patients undergoing a renal flare had significantly higher uTWEAK levels than the patients undergoing a non-renal flare. Bars indicate median values.
FIG. 4 shows that uTWEAK fluctuations reflect changes in renal disease activity. A) In 4 of 6 patients, uTWEAK levels increased while the patient is undergoing a renal flare. Each line represents one patient's change in uTWEAK levels between the 2 timepoints in which the samples were obtained. B) Three uTWEAK measurements in one patient over the course of a year show fluctuations in uTWEAK levels that follow the course of the patient's renal disease activity, as defined by the rSLEDAI and the presence of a disease flare. C) uTWEAK levels over 6 months match the patient's disease activity (as determined by predefined criteria), despite a stable rSLEDAI score. For B-C: The solid line shows uTWEAK levels in pg/mg Cr, and the grey bars depict rSLEDAI scores.
FIG. 5 shows that uTWEAK levels correlate with other biomarkers. A) uTWEAK levels significantly correlated with uMCP-1 levels, ρ=0.501, P<0.001, n=51. uTWEAK levels in 80 SLE patients showed negative correlation with: B) standardized serum C3 levels, ρ=−0.262, P=0.019, and: C) Standardized serum C4 levels, ρ=−0.269, P=0.016. D) uTWEAK levels correlated with standardized anti-dsDNA levels, ρ=0.459, P=0.008, n=32.
DETAILED DESCRIPTION
It has been found that increased Tweak or TweakR urinary, serum, plasma or CSF levels correlate with increased severity, stage and/or activity of certain disorders. The methods described herein provide, inter alia, simple, quick, relatively non-invasive techniques for evaluating, staging, and/or diagnosing a subject for a disease, e.g., an inflammatory condition, e.g., before, during and/or after a treatment is begun. The condition can be, e.g., nephritis (e.g., lupus nephritis), RA, PsA, lupus, fibrosis, cancer, or a neurodegenerative disease (e.g., a neurodegenerative disease described herein).
Detection and Diagnosis of Nephritis
Although nephritis is discussed herein below as an exemplary disorder relating to the methods described herein, it is understood that the etiology and clinical characteristics of other conditions described herein are known to the skilled practitioner.
Nephritis is an inflammation of the kidneys. The two most common causes of nephritis are infection and auto-immune processes. Nephritis can be a symptom of underlying conditions such as systemic lupus erythematosus (SLE), diabetes, renal tuberculosis, or yellow fever. Lupus nephritis is an inflammation of the kidney caused by SLE. At least three potentially overlapping, immuno-pathogenic mechanisms are supported by experimental data. First, circulating immune complexes consisting chiefly of DNA and anti-DNA are deposited in the kidney. Resulting complement activation and chemotaxis of neutrophils leads to a local inflammatory process. Second, in situ formation of antigen and antibody complexes may similarly lead to complement activation and leukocyte mediated injury. Third, antibodies against specific cellular targets may produce renal injury. For example, antibodies, such as anti-ribosomal P, may bind to cytoplasmic antigens that have been translocated to the cell membrane with subsequent penetration and disruption of cellular function.
Glomerulonephritis is the most common type of nephritis and can include nephritic syndrome, nephrotic syndrome, and/or asymptomatic proteinuria and hematuria syndrome, all of which may lead to end stage renal disease (ESRD) and kidney failure. Lupus nephritis can involve various internal structures of the kidney and can include interstitial nephritis, glomerulonephritis (GN), mesangial GN, membranous GN, diffuse proliferative GN and/or membranoproliferative GN.
Nephritis can be detected or diagnosed by a variety of techniques, including urinalysis, e.g., detection of protein, casts, and/or red blood cells present in the urine; and/or BUN and/or creatinine tests to assess kidney function. Indications of lupus nephritis can also include high anti-DNA levels, high anti-dsDNA levels, low complement levels, high anti-nuclear antibody (ANA) panel titers and/or a positive lupus erythematosus test. Kidney X-rays or other imaging techniques and/or a kidney biopsy may also be performed. Any of these tests may be used in addition to the methods described herein.
Lupus nephritis (LN) remains a major cause of morbidity and mortality in SLE patients. Although the definition of renal involvement varies, overt renal disease is found in at least one-third to one-half of SLE patients, with reports of 5-year renal survival with treatment ranging from 46-95%. Early diagnosis and prompt treatment, however, may significantly improve long-term prognosis.
Lupus nephritis has been traditionally divided into six classes, defined by the World Health Organization (WHO) in 1982 (Churg and Sonbin. Classification and Atlas of Glomerular Disease. Tokyo: Igaku-Shoin; 1982) which take into account the renal histopathological changes together with activity of the nephritis. The prognosis and treatment of LN is heavily dependent upon this disease classification. Currently, the most accurate and reliable method to diagnose and prognosticate LN, both in terms of the activity and the chronicity of the renal processes, is by performing a biopsy. However, kidney biopsy, being an invasive procedure, can be accompanied by significant morbidity, and therefore is not usually performed serially. Furthermore, with an essentially “blind” needle biopsy there can be a question of how representative the limited number of glomeruli usually obtained are of the status of the kidney.
Evaluating a Subject for Tweak or Fn14
Techniques for evaluating a subject for Tweak or Fn14 (or other biomarker described herein) in a biological sample of the subject are known in the art. Such techniques can include detecting the presence, levels, expression or activity of a Tweak or Tweak-R protein, e.g., by qualitative or quantitative analysis of mRNA, cDNA, or protein, or by evaluating one or more nucleotides in a nucleic acid (genomic, mRNA, or cDNA) encoding Tweak or Tweak-R. Such techniques include methods for protein detection (e.g., Western blot or ELISA), and hybridization-based methods for nucleic acid detection (e.g., PCR or Northern blot). For example, an immunoassay can be used to detect Tweak protein, e.g., in a urine sample of the subject. In other embodiments, the method can include administering a labeled Tweak or Tweak-R binding agent (e.g., an antibody) to a subject, and evaluating localization of the labeled binding agent in the subject, e.g., by imaging the subject (e.g., imaging at least a portion of the kidney of the subject).
Kits: A kit can be used for assaying the Tweak pathway for risk, presence, stage or severity of a condition described herein, e.g., nephritis and/or lupus nephritis. The kit includes one or more reagents (e.g., an anti-Tweak antibody) capable of detecting one or more of: Tweak, TweakR or a biomarker described herein, in a biological sample of a subject, e.g., a human; and instructions for using the reagent to evaluate risk, predisposition, or prognosis for renal inflammation in a subject. Such a kit can include instructions to use (e.g., to contact the agent) with a sample from a subject (preferably a human) having lupus or other inflammatory disorder, or risk therefor. In a preferred embodiment, the instructions comprise instructions for use by or for a subject who has normal kidney function as defined by a clinical measure. The instructions can include directions to contact the agent with a urine sample of the subject.
The reagent can be attached to a solid substrate, e.g., a microtiter well, a tube, a sheet (e.g., a nitrocellulose sheet), a dipstick or other test device. Preferably, the kit includes a dipstick or other test device. In a preferred embodiment, the reagent is an antibody or other binding protein. The antibody can be attached to a detectable label, e.g., an enzyme, a calorimetric reagent, a fluorescent substance, or a radioactive isotope. The kit can include a positive and/or a negative control (e.g., a sample that includes an appropriate concentration of Tweak or the biomarker), e.g., with a stabilizer and/or preservative. The kit can also include a densitometer, or electrochemical strip.
Information about evaluating a subject can be obtained in a method that includes: supplying a test substrate (e.g., a tube, a strip, a dipstick, other test device, or a well) to which is attached an agent capable of detecting one or more of: Tweak or a biomarker described herein; and supplying instructions to contact the test substrate with a subject's urine. The method optionally includes supplying instructions for reading, evaluating or interpreting the contacted substrate, e.g., to evaluate risk for, predisposition, or presence of renal inflammation. The method can be performed by health care provider or a third person, or by the subject.
Other possible approaches include the use of electrochemical sensor strips, such as those used for home glucose testing, onto which a sample is placed, and which strips include reagents for initiating a reaction when wetted by the sample. The sensor strip is inserted into a meter that measures, e.g., diffusion-limited current of a reaction species indicative of the analyte of interest, e.g., Tweak and a biomarker whose expression modulated (e.g., increased) by Tweak (e.g., RANTES, KC, and/or IP-10). The meter then yields a display indicative of the concentration of analyte in the sample.
Arrays: Arrays are also particularly useful molecular tools for characterizing a sample, e.g., a sample from a subject. For example, an array having capture probes for multiple genes, including probes for Tweak and/or other biomarkers, or for multiple proteins, can be used in a method described herein. Altered expression of Tweak nucleic acids and/or protein can be used to evaluate a sample, e.g., a sample from a subject, e.g., to evaluate a disorder described herein.
Arrays can have many addresses, e.g., locatable sites, on a substrate. The featured arrays can be configured in a variety of formats, non-limiting examples of which are described below. The substrate can be opaque, translucent, or transparent. The addresses can be distributed, on the substrate in one dimension, e.g., a linear array; in two dimensions, e.g., a planar array; or in three dimensions, e.g., a three dimensional array. The solid substrate may be of any convenient shape or form, e.g., square, rectangular, ovoid, or circular.
Arrays can be fabricated by a variety of methods, e.g., photolithographic methods (see, e.g., U.S. Pat. Nos. 5,143,854; 5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow methods as described in U.S. Pat. No. 5,384,261), pin based methods (e.g., as described in U.S. Pat. No. 5,288,514), and bead based techniques (e.g., as described in PCT US/93/04145).
The capture probe can be a single-stranded nucleic acid, a double-stranded nucleic acid (e.g., which is denatured prior to or during hybridization), or a nucleic acid having a single-stranded region and a double-stranded region. Preferably, the capture probe is single-stranded. The capture probe can be selected by a variety of criteria, and preferably is designed by a computer program with optimization parameters. The capture probe can be selected to hybridize to a sequence rich (e.g., non-homopolymeric) region of the gene. The Tm of the capture probe can be optimized by prudent selection of the complementarity region and length. Ideally, the Tm of all capture probes on the array is similar, e.g., within 20, 10, 5, 3, or 2° C. of one another.
The isolated nucleic acid is preferably mRNA that can be isolated by routine methods, e.g., including DNase treatment to remove genomic DNA and hybridization to an oligo-dT coupled solid substrate (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y). The substrate is washed, and the mRNA is eluted.
The isolated mRNA can be reversed transcribed and optionally amplified, e.g., by rtPCR, e.g., as described in (U.S. Pat. No. 4,683,202). The nucleic acid can be an amplification product, e.g., from PCR (U.S. Pat. Nos. 4,683,196 and 4,683,202); rolling circle amplification (“RCA,” U.S. Pat. No. 5,714,320), isothermal RNA amplification or NASBA (U.S. Pat. Nos. 5,130,238; 5,409,818; and 5,554,517), and strand displacement amplification (U.S. Pat. No. 5,455,166). The nucleic acid can be labeled during amplification, e.g., by the incorporation of a labeled nucleotide. Examples of preferred labels include fluorescent labels, e.g., red-fluorescent dye Cy5 (Amersham) or green-fluorescent dye Cy3 (Amersham), and chemiluminescent labels, e.g., as described in U.S. Pat. No. 4,277,437. Alternatively, the nucleic acid can be labeled with biotin, and detected after hybridization with labeled streptavidin, e.g., streptavidin-phycoerythrin (Molecular Probes).
The labeled nucleic acid can be contacted to the array. In addition, a control nucleic acid or a reference nucleic acid can be contacted to the same array. The control nucleic acid or reference nucleic acid can be labeled with a label other than the sample nucleic acid, e.g., one with a different emission maximum. Labeled nucleic acids can be contacted to an array under hybridization conditions. The array can be washed, and then imaged to detect fluorescence at each address of the array.
The expression level of a Tweak or other biomarker can be determined using an antibody specific for the polypeptide (e.g., using a western blot or an ELISA assay). Moreover, the expression levels of multiple proteins, including Tweak and the exemplary biomarkers provided herein, can be rapidly determined in parallel using a polypeptide array having antibody capture probes for each of the polypeptides. Antibodies specific for a polypeptide can be generated by a method described herein (see “Antibody Generation”). The expression level of a TWEAK and the exemplary biomarkers provided herein can be measured in a subject (e.g., in vivo imaging) or in a biological sample from a subject (e.g., blood, serum, plasma, or synovial fluid).
A low-density (96 well format) protein array has been developed in which proteins are spotted onto a nitrocellulose membrane (Ge (2000) Nucleic Acids Res. 28, e3, I-VII). A high-density protein array (100,000 samples within 222×222 mm) used for antibody screening was formed by spotting proteins onto polyvinylidene difluoride (PVDF) (Lueking et al. (1999) Anal. Biochem. 270:103-111). See also, e.g., Mendoza et al. (1999). Biotechniques 27:778-788; MacBeath and Schreiber (2000) Science 289:1760-1763; and De Wildt et al. (2000). Nature Biotech. 18:989-994. These art-known methods and other can be used to generate an array of antibodies for detecting the abundance of polypeptides in a sample. The sample can be labeled, e.g., biotinylated, for subsequent detection with streptavidin coupled to a fluorescent label. The array can then be scanned to measure binding at each address.
The nucleic acid and polypeptide arrays of the invention can be used in wide variety of applications. For example, the arrays can be used to analyze a patient sample. The sample is compared to data obtained previously, e.g., known clinical specimens or other patient samples. Further, the arrays can be used to characterize a cell culture sample, e.g., to determine a cellular state after varying a parameter, e.g., exposing the cell culture to an antigen, a transgene, or a test compound.
The expression data can be stored in a database, e.g., a relational database such as a SQL database (e.g., Oracle or Sybase database environments). The database can have multiple tables. For example, raw expression data can be stored in one table, wherein each column corresponds to a gene being assayed, e.g., an address or an array, and each row corresponds to a sample. A separate table can store identifiers and sample information, e.g., the batch number of the array used, date, and other quality control information.
Expression profiles obtained from gene expression analysis on an array can be used to compare samples and/or cells in a variety of states as described in Golub et al. ((1999) Science 286:531). In one embodiment, expression (e.g., mRNA expression or protein expression) information for a gene encoding TWEAK and/or a gene encoding a exemplary biomarker provided herein are evaluated, e.g., by comparison a reference value, e.g., a reference value. Reference values can be obtained from a control, a reference subject. Reference values can also be obtained from statistical analysis, e.g., to provide a reference value for a cohort of subject, e.g., age and gender matched subject, e.g., normal subjects or subject who have rheumatoid arthritis or other disorder described herein. Statistical similarity to a particular reference (e.g., to a reference for a risk-associated cohort) or a normal cohort can be used to provide an assessment (e.g., an indication of risk of inflammatory disorder) to a subject, e.g., a subject who has not been diagnosed with a disorder described herein.
Subjects suitable for treatment can also be evaluated for expression and/or activity of TWEAK and/or other biomarker. Subjects can be identified as suitable for treatment (e.g., with a TWEAK blocking agent), if the expression and/or activity for TWEAK and/or the other biomarker is elevated relative to a reference, e.g., reference value, e.g., a reference value associated with normal.
Subjects who are being administered an agent described herein or other treatment can be evaluated as described for expression and/or activity of TWEAK and/or other biomarkers described herein. The subject can be evaluated at multiple times. e.g., at multiple times during a course of therapy, e.g., during a therapeutic regimen. Treatment of the subject can be modified depending on how the subject is responding to the therapy. For example, a reduction in TWEAK expression or activity or a reduction in the expression or activity of genes stimulated by TWEAK can be indicative of responsiveness.
Particular effects mediated by an agent may show a difference (e.g., relative to an untreated subject, control subject, or other reference) that is statistically significant (e.g., P value <0.05 or 0.02). Statistical significance can be determined by any art known method. Exemplary statistical tests include: the Students T-test, Mann Whitney U non-parametric test, and Wilcoxon non-parametric statistical test. Some statistically significant relationships have a P value of less than 0.05 or 0.02.
Any combination of the above methods can also be used. The above methods can be used to evaluate any genetic locus, e.g., in a method for analyzing genetic information from particular groups of individuals or in a method for analyzing a polymorphism associated with a disorder described herein, e.g., rheumatoid arthritis, e.g., in a gene encoding TWEAK or another biomarker described herein.
Tweak-Tweak Receptor Blocking Agents
Tweak pathway inhibitors to treat nephritis include Tweak/Tweak-R blocking agents. The agent may be any type of compound (e.g., small organic or inorganic molecule, nucleic acid, protein, or peptide mimetic) that can be administered to a subject.
In one embodiment, the blocking agent is a biologic, e.g., a protein having a molecular weight of between 5-300 kDa. For example, a Tweak/Tweak-R blocking agent may inhibit binding of Tweak to a Tweak receptor. A typical Tweak/Tweak-R blocking agent can bind to Tweak or a Tweak receptor, e.g., Fn14. A Tweak/Tweak-R blocking agent that binds to Tweak may block the binding site on Tweak or a Tweak receptor, alter the conformation of Tweak or a Tweak receptor, or otherwise decrease the affinity of Tweak for a Tweak receptor or prevent the interaction between Tweak and a Tweak receptor. A Tweak/Tweak-R blocking agent (e.g., an antibody) may bind to Tweak or to a Tweak receptor with a Kd of less than 10-6, 10-7, 10-8, 10-9, or 10-10 M. In one embodiment, the blocking agent binds to Tweak with an affinity at least 5, 10, 20, 50, 100, 200, 500, or 1000 better than its affinity for TNF or another TNF superfamily member (other than Tweak). In one embodiment, the blocking agent binds to the Tweak receptor with an affinity at least 5, 10, 20, 50, 100, 200, 500, or 1000-fold better than its affinity for the TNF receptor or a receptor for another TNF superfamily member. A preferred Tweak/Tweak-R blocking agent specifically binds Tweak or Tweak-R, such as a Tweak or Tweak-R specific antibody, e.g., a monoclonal antibody.
The sequence of human Tweak (SEQ ID NO:1) is shown below.
1 MAARRSQRRR GRRGEPGTAL LVPLALGLGL ALACLGLLLA
VVSLGSRASL SAQEPAQEEL
61 VAEEDQDPSE LNPQTEESQD PAPFLNRLVR PRRSAPKGRK
TRARRAIAAH YEVHPRPGQD
121 GAQAGVDGTV SGWEEARINS SSPLRYNRQI GEFIVTRAGL
YYLYCQVHFD EGKAVYLKLD
181 LLVDGVLALR CLEEFSATAA SSLGPQLRLC QVSGLLALRP
GSSLRIRTLP WAHLKAAPFL
241 TYFGLFQVH
Also included are proteins that include an amino acid sequence at least 90, 92, 95, 97, 98, 99% identical and completely identical to the mature processed region of SEQ ID NO:1 (e.g., an amino acid sequence at least 90, 92, 95, 97, 98, 99% identical or completely identical to amino acids X1-249 of SEQ ID NO:1, where amino acid X1 is selected from the group of residues 75-115 of SEQ ID NO:1, e.g., X1 is residue Arg 93 of SEQ ID NO:1) and proteins encoded by a nucleic acid that hybridizes under high stringency conditions to the DNA encoding SEQ ID NO:1. Preferably, a Tweak protein, in its processed mature form, is capable of providing at least one Tweak activity, e.g., ability to activate Fn14 and/or cell death in cortical neurons.
The sequence of human Tweak-R (SEQ ID NO:2) is shown below.
1 MARGSLRRLL RLLVLGLWLA LLRSVAGEQA PGTAPCSRGS
SWSADLDKCM DCASCRARPH
61 SDFCLGCAAA PPAPFRLLWP ILGGALSLTF VLGLLSGFLV
WRRCRRREKF TTPIEETGGE
121 GCPAVALIQ
Soluble proteins that include an amino acid sequence at least 90, 92, 95, 97, 98, 99% identical to the extracellular domain of Fn14 (and Tweak-binding fragments thereof) and proteins encoded by a nucleic acid that hybridizes under high stringency conditions to a human Fn14 protein are useful in the methods described herein. Preferably, a Fn14 protein useful in the methods described herein is a soluble Fn14 (lacking a transmembrane domain) that includes a region that binds to a Tweak protein, e.g., an amino acid sequence at least 90, 92, 95, 97, 98, or 99% identical, or completely identical, to amino acids 28-X1 of SEQ ID NO:2, where amino acid X1 is selected from the group of residues 68 to 80 of SEQ ID NO:2.
Calculations of “homology” or “sequence identity” between two sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences.
As used herein, the term “hybridizes under high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and nonaqueous methods are described in that reference and either can be used. High stringency hybridization conditions include hybridization in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C., or substantially similar conditions.
Exemplary Tweak/Tweak-R blocking agents include antibodies that bind to Tweak or Tweak-R and soluble forms of the Tweak-R that compete with cell surface Tweak-R for binding to Tweak. An example of a soluble form of the Tweak-R is an Fc fusion protein that includes at least a portion of the extracellular domain of Tweak-R (e.g., a soluble Tweak-binding fragment of Tweak-R), referred to as Tweak-R-Fc. Other soluble forms of Tweak-R, e.g., forms that do not include an Fc domain, can also be used. Antibody blocking agents are further discussed below. Other types of blocking agents, e.g., small molecules, nucleic acid or nucleic acid-based aptamers, and peptides, can be isolated by screening, e.g., as described in Jhaveri et al. Nat. Biotechnol. 18:1293 and U.S. Pat. No. 5,223,409. Exemplary assays for determining if an agent binds to Tweak or Tweak-R and for determining if an agent modulates a Tweak/Tweak-R interaction are described, e.g., in US 2004-0033225.
An exemplary soluble form of the Tweak-R protein includes a region of the Tweak-R protein that binds to Tweak, e.g., about amino acids 32-75, 31-75, 31-78, or 28-79 of SEQ ID NO:2. This region can be physically associated, e.g., fused to another amino acid sequence, e.g., an Fc domain, at its N- or C-terminus. The region from Tweak-R can be spaced by a linker from the heterologous amino acid sequence. U.S. Pat. No. 6,824,773 describes an exemplary Tweak-R fusion protein.
Antibodies
Exemplary Tweak/Tweak-R blocking agents include antibodies that bind to Tweak and/or Tweak-R. In on embodiment, the antibody inhibits the interaction between Tweak and a Tweak receptor, e.g., by physically blocking the interaction, decreasing the affinity of Tweak and/or Tweak-R for its counterpart, disrupting or destabilizing Tweak complexes, sequestering Tweak or a Tweak-R, or targeting Tweak or Tweak-R for degradation. In one embodiment, the antibody can bind to Tweak or Tweak-R at one or more amino acid residues that participate in the Tweak/Tweak-R binding interface. Such amino acid residues can be identified, e.g., by alanine scanning. In another embodiment, the antibody can bind to residues that do not participate in the Tweak/Tweak-R binding. For example, the antibody can alter a conformation of Tweak or Tweak-R and thereby reduce binding affinity, or the antibody may sterically hinder Tweak/Tweak-R binding. In one embodiment, the antibody can prevent activation of a Tweak/Tweak-R mediated event or activity (e.g., NF-kappaB activation).
As used herein, the term “antibody” refers to a protein that includes at least one immunoglobulin variable region, e.g., an amino acid sequence that provides an immunoglobulin variable domain or an immunoglobulin variable domain sequence. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term “antibody” encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab fragments, F(ab′)2 fragments, Fd fragments, Fv fragments, and dAb fragments) as well as complete antibodies, e.g., intact and/or full length immunoglobulins of types IgA, IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgE, IgD, IgM (as well as subtypes thereof). The light chains of the immunoglobulin may be of types kappa or lambda. In one embodiment, the antibody is glycosylated. An antibody can be functional for antibody-dependent cytotoxicity and/or complement-mediated cytotoxicity, or may be non-functional for one or both of these activities.
The VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (FR). The extent of the FR's and CDR's has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). Kabat definitions are used herein. Each VH and VL is typically composed of three CDR's and four FR's, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
An “immunoglobulin domain” refers to a domain from the variable or constant domain of immunoglobulin molecules. Immunoglobulin domains typically contain two beta-sheets formed of about seven beta-strands, and a conserved disulphide bond (see, e.g., A. F. Williams and A. N. Barclay (1988) Ann. Rev Immunol. 6:381-405). An “immunoglobulin variable domain sequence” refers to an amino acid sequence that can form a structure sufficient to position CDR sequences in a conformation suitable for antigen binding. For example, the sequence may include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may omit one, two or more N- or C-terminal amino acids, internal amino acids, may include one or more insertions or additional terminal amino acids, or may include other alterations. In one embodiment, a polypeptide that includes an immunoglobulin variable domain sequence can associate with another immunoglobulin variable domain sequence to form a target binding structure (or “antigen binding site”), e.g., a structure that interacts with Tweak or a Tweak receptor.
The VH or VL chain of the antibody can further include all or part of a heavy or light chain constant region, to thereby form a heavy or light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains. The heavy and light immunoglobulin chains can be connected by disulfide bonds. The heavy chain constant region typically includes three constant domains, CH1, CH2, and CH3. The light chain constant region typically includes a CL domain. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
One or more regions of an antibody can be human, effectively human, or humanized. For example, one or more of the variable regions can be human or effectively human. For example, one or more of the CDRs, e.g., HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3, can be human. Each of the light chain CDRs can be human. HC CDR3 can be human. One or more of the framework regions can be human, e.g., FR1, FR2, FR3, and FR4 of the HC or LC. In one embodiment, all the framework regions are human, e.g., derived from a human somatic cell, e.g., a hematopoietic cell that produces immunoglobulins or a non-hematopoietic cell. In one embodiment, the human sequences are germline sequences, e.g., encoded by a germline nucleic acid. One or more of the constant regions can be human, effectively human, or humanized. In another embodiment, at least 70, 75, 80, 85, 90, 92, 95, or 98% of the framework regions (e.g., FR1, FR2, and FR3, collectively, or FR1, FR2, FR3, and FR4, collectively) or the entire antibody can be human, effectively human, or humanized. For example, FR1, FR2, and FR3 collectively can be at least 70, 75, 80, 85, 90, 92, 95, 98, or 99% identical, or completely identical, to a human sequence encoded by a human germline segment.
An “effectively human” immunoglobulin variable region is an immunoglobulin variable region that includes a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human. An “effectively human” antibody is an antibody that includes a sufficient number of human amino acid positions such that the antibody does not elicit an immunogenic response in a normal human.
A “humanized” immunoglobulin variable region is an immunoglobulin variable region that is modified such that the modified form elicits less of an immune response in a human than does the non-modified form, e.g., is modified to include a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human. Descriptions of “humanized” immunoglobulins include, for example, U.S. Pat. Nos. 6,407,213 and 5,693,762. In some cases, humanized immunoglobulins can include a non-human amino acid at one or more framework amino acid positions.
Antibody Generation
Antibodies that bind to Tweak or a Tweak-R can be generated by a variety of means, including immunization, e.g., using an animal, or in vitro methods such as phage display. All or part of Tweak or a Tweak receptor can be used as an immunogen or as a target for selection. For example, Tweak or a fragment thereof, Tweak-R or a fragment thereof, can be used as an immunogen. In one embodiment, the immunized animal contains immunoglobulin producing cells with natural, human, or partially human immunoglobulin loci. In one embodiment, the non-human animal includes at least a part of a human immunoglobulin gene. For example, it is possible to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci. Using the hybridoma technology, antigen-specific monoclonal antibodies derived from the genes with the desired specificity may be produced and selected. See, e.g., XENOMOUSE™, Green et al. (1994) Nat. Gen. 7:13-21; US 2003-0070185; U.S. Pat. No. 5,789,650; and WO 96/34096.
Non-human antibodies to Tweak or a Tweak receptor can also be produced, e.g., in a rodent. The non-human antibody can be humanized, e.g., as described in EP 239 400; U.S. Pat. Nos. 6,602,503; 5,693,761; and 6,407,213, deimmunized, or otherwise modified to make it effectively human.
EP 239 400 (Winter et al.) describes altering antibodies by substitution (within a given variable region) of their complementarity determining regions (CDRs) for one species with those from another. Typically, CDRs of a non-human (e.g., murine) antibody are substituted into the corresponding regions in a human antibody by using recombinant nucleic acid technology to produce sequences encoding the desired substituted antibody. Human constant region gene segments of the desired isotype (usually gamma I for CH and kappa for CL) can be added and the humanized heavy and light chain genes can be co-expressed in mammalian cells to produce soluble humanized antibody. Other methods for humanizing antibodies can also be used. For example, other methods can account for the three dimensional structure of the antibody, framework positions that are in three dimensional proximity to binding determinants, and immunogenic peptide sequences. See, e.g., WO 90/07861; U.S. Pat. Nos. 5,693,762; 5,693,761; 5,585,089; and 5,530,101; Tempest et al. (1991) Biotechnology 9:266-271 and U.S. Pat. No. 6,407,213. Still another method is termed “humaneering” and is described, for example, in US 2005-008625.
Fully human monoclonal antibodies that bind to Tweak or a Tweak receptor can be produced, e.g., using in vitro-primed human splenocytes, as described by Boerner et al. (1991) J. Immunol. 147:86-95. They may be prepared by repertoire cloning as described by Persson et al. (1991) Proc. Nat. Acad. Sci. USA 88:2432-2436 or by Huang and Stollar (1991) J. Immunol. Methods 141:227-236; also U.S. Pat. No. 5,798,230. Large nonimmunized human phage display libraries may also be used to isolate high affinity antibodies that can be developed as human therapeutics using standard phage technology (see, e.g., Hoogenboom et al. (1998) Immunotechnology 4:1-20; Hoogenboom et al. (2000) Immunol Today 2:371-8; and US 2003-0232333).
Antibody and Protein Production
Antibodies and other proteins described herein can be produced in prokaryotic and eukaryotic cells. In one embodiment, the antibodies (e.g., scFv's) are expressed in a yeast cell such as Pichia (see, e.g., Powers et al. (2001) J. Immunol. Methods 251:123-35), Hanseula, or Saccharomyces.
Antibodies, particularly full length antibodies, e.g., IgGs, can be produced in mammalian cells. Exemplary mammalian host cells for recombinant expression include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621), lymphocytic cell lines, e.g., NS0 myeloma cells and SP2 cells, COS cells, K562, and a cell from a transgenic animal, e.g., a transgenic mammal. For example, the cell is a mammary epithelial cell.
In addition to the nucleic acid sequence encoding the immunoglobulin domain, the recombinant expression vectors may carry additional nucleic acid sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216; 4,634,665; and 5,179,017). Exemplary selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
In an exemplary system for recombinant expression of an antibody (e.g., a full length antibody or an antigen-binding portion thereof), 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 enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLP promoter regulatory element or an SV40 enhancer/AdMLP promoter regulatory element) 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, to transfect the host cells, to select for transformants, to culture the host cells, and to recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G.
Antibodies (and Fc fusions) may also include modifications, e.g., modifications that alter Fc function, e.g., to decrease or remove interaction with an Fc receptor or with Clq, or both. For example, the human IgG1 constant region can be mutated at one or more residues, e.g., one or more of residues 234 and 237, e.g., according to the numbering in U.S. Pat. No. 5,648,260. Other exemplary modifications include those described in U.S. Pat. No. 5,648,260.
For some proteins that include an Fc domain, the antibody/protein production system may be designed to synthesize antibodies or other proteins in which the Fc region is glycosylated. For example, the Fc domain of IgG molecules is glycosylated at asparagine 297 in the CH2 domain. The Fc domain can also include other eukaryotic post-translational modifications. In other cases, the protein is produced in a form that is not glycosylated.
Antibodies and other proteins can also be produced by a transgenic animal. For example, U.S. Pat. No. 5,849,992 describes a method for expressing an antibody in the mammary gland of a transgenic mammal. A transgene is constructed that includes a milk-specific promoter and nucleic acid sequences encoding the antibody of interest, e.g., an antibody described herein, and a signal sequence for secretion. The milk produced by females of such transgenic mammals includes, secreted-therein, the protein of interest, e.g., an antibody or Fc fusion protein. The protein can be purified from the milk, or for some applications, used directly.
Methods described in the context of antibodies can be adapted to other proteins, e.g., Fc fusions and soluble receptor fragments.
Nucleic Acid Antagonists
In certain implementations, nucleic acid antagonists are used to decrease expression of an endogenous gene encoding Tweak or a Tweak-R, e.g., Fn14. In one embodiment, the nucleic acid antagonist is an siRNA that targets mRNA encoding Tweak or a Tweak-R. Other types of antagonistic nucleic acids can also be used, e.g., an siRNA, a ribozyme, a triple-helix former, an aptamer, or an antisense nucleic acid.
siRNAs are small double stranded RNAs (dsRNAs) that optionally include overhangs. For example, the duplex region of an siRNA is about 18 to 25 nucleotides in length, e.g., about 19, 20, 21, 22, 23, or 24 nucleotides in length. Typically, the siRNA sequences are exactly complementary to the target mRNA. dsRNAs and siRNAs in particular can be used to silence gene expression in mammalian cells (e.g., human cells). See, e.g., Clemens et al. (2000) Proc. Natl. Acad. Sci. USA 97:6499-6503; Billy et al. (2001) Proc. Natl. Sci. USA 98:14428-14433; Elbashir et al. (2001) Nature. 411:494-8; Yang et al. (2002) Proc. Natl. Acad. Sci. USA 99:9942-9947, U.S. 20030166282, 20030143204, 20040038278, and 20030224432.
Anti-sense agents can include, for example, from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 nucleotides), e.g., about 8 to about 50 nucleobases, or about 12 to about 30 nucleobases. Anti-sense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression. Anti-sense compounds can include a stretch of at least eight consecutive nucleobases that are complementary to a sequence in the target gene. An oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted.
Hybridization of antisense oligonucleotides with mRNA (e.g., an mRNA encoding Tweak or Tweak-R) can interfere with one or more of the normal functions of mRNA. The functions of mRNA to be interfered with include all key functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in by the RNA. Binding of specific protein(s) to the RNA may also be interfered with by antisense oligonucleotide hybridization to the RNA.
Exemplary antisense compounds include DNA or RNA sequences that specifically hybridize to the target nucleic acid, e.g., the mRNA encoding Tweak or Tweak-R. The complementary region can extend for between about 8 to about 80 nucleobases. The compounds can include one or more modified nucleobases. Modified nucleobases may include, e.g., 5-substituted pyrimidines such as 5-iodouracil, 5-iodocytosine, and C5-propynyl pyrimidines such as C5-propynylcytosine and C5-propynyluracil. Other suitable modified nucleobases include N4-(C1-C12) alkylaminocytosines and N4,N4-(C1-C12) dialkylaminocytosines. Modified nucleobases may also include 7-substituted-8-aza-7-deazapurines and 7-substituted-7-deazapurines such as, for example, 7-iodo-7-deazapurines, 7-cyano-7-deazapurines, 7-aminocarbonyl-7-deazapurines. Examples of these include 6-amino-7-iodo-7-deazapurines, 6-amino-7-cyano-7-deazapurines, 6-amino-7-aminocarbonyl-7-deazapurines, 2-amino-6-hydroxy-7-iodo-7-deazapurines, 2-amino-6-hydroxy-7-cyano-7-deazapurines, and 2-amino-6-hydroxy-7-aminocarbonyl-7-deazapurines. Furthermore, N6-(C1-C12) alkylaminopurines and N6,N6-(C1-C12) dialkylaminopurines, including N6-methylaminoadenine and N6,N6-dimethylaminoadenine, are also suitable modified nucleobases. Similarly, other 6-substituted purines including, for example, 6-thioguanine may constitute appropriate modified nucleobases. Other suitable nucleobases include 2-thiouracil, 8-bromoadenine, 8-bromoguanine, 2-fluoroadenine, and 2-fluoroguanine. Derivatives of any of the aforementioned modified nucleobases are also appropriate. Substituents of any of the preceding compounds may include C1-C30 alkyl, C2-C30 alkenyl, C2-C30 alkynyl, aryl, aralkyl, heteroaryl, halo, amino, amido, nitro, thio, sulfonyl, carboxyl, alkoxy, alkylcarbonyl, alkoxycarbonyl, and the like.
Descriptions of other types of nucleic acid agents are also available. See, e.g., U.S. Pat. Nos. 4,987,071; 5,116,742; and 5,093,246; Woolf et al. (1992) Proc Natl Acad Sci USA; Antisense RNA and DNA, D. A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988); 89:7305-9; Haselhoff and Gerlach (1988) Nature 334:585-59; Helene, C. (1991) Anticancer Drug Des. 6:569-84; Helene (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14:807-15.
Artificial Transcription Factors
Artificial transcription factors can also be used to regulate expression of Tweak and/or Tweak-R. The artificial transcription factor can be designed or selected from a library, e.g., for ability to bind to a sequence in an endogenous gene encoding Tweak or Tweak-R, e.g., in a regulatory region, e.g., the promoter. For example, the artificial transcription factor can be prepared by selection in vitro (e.g., using phage display, U.S. Pat. No. 6,534,261) or in vivo, or by design based on a recognition code (see, e.g., WO 00/42219 and U.S. Pat. No. 6,511,808). See, e.g., Rebar et al. (1996) Methods Enzymol 267:129; Greisman and Pabo (1997) Science 275:657; Isalan et al. (2001) Nat. Biotechnol 19:656; and Wu et al. (1995) Proc. Natl. Acad. Sci. USA 92:344 for, among other things, methods for creating libraries of varied zinc finger domains.
Optionally, an artificial transcription factor can be fused to a transcriptional regulatory domain, e.g., an activation domain to activate transcription or a repression domain to repress transcription. In particular, repression domains can be used to decrease expression of endogenous genes encoding Tweak or Tweak-R. The artificial transcription factor can itself be encoded by a heterologous nucleic acid that is delivered to a cell or the protein itself can be delivered to a cell (see, e.g., U.S. Pat. No. 6,534,261). The heterologous nucleic acid that includes a sequence encoding the artificial transcription factor can be operably linked to an inducible promoter, e.g., to enable fine control of the level of the artificial transcription factor in the cell.
Pharmaceutical Compositions
A Tweak/Tweak-R blocking agent (e.g., an antibody or soluble Tweak-R protein, e.g., Tweak-R-Fc) can be formulated as a pharmaceutical composition, e.g., for administration to a subject to treat the nephritis. Typically, a pharmaceutical composition includes a pharmaceutically acceptable carrier. As used herein, “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. The composition can include a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt (see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19).
The Tweak/Tweak-R blocking agent can be formulated according to standard methods. Pharmaceutical formulation is a well-established art, and is further described, e.g., in Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins (2000) (ISBN: 0683306472); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed., Lippincott Williams & Wilkins Publishers (1999) (ISBN: 0683305727); and Kibbe (ed.), Handbook of Pharmaceutical Excipients American Pharmaceutical Association, 3rd ed. (2000) (ISBN: 091733096X).
In one embodiment, the Tweak/Tweak-R blocking agent (e.g., an antibody or Tweak-R-Fc) can be formulated with excipient materials, such as sodium chloride, sodium dibasic phosphate heptahydrate, sodium monobasic phosphate, and a stabilizer. It can be provided, for example, in a buffered solution at a suitable concentration and can be stored at 2-8° C.
The pharmaceutical compositions 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 preferred form can depend on the intended mode of administration and therapeutic application. Typically compositions for the agents described herein are in the form of injectable or infusible solutions.
Such compositions can be administered by a parenteral mode (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). The phrases “parenteral administration” and “administered parenterally” as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration. Sterile injectable solutions can be prepared by incorporating an agent described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating an agent described herein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of an agent described herein 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, for example, monostearate salts and gelatin.
In certain embodiments, the Tweak/Tweak-R blocking agent may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, 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 patented or generally known. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
A Tweak/Tweak-R blocking agent (e.g., an antibody or soluble Tweak-R protein) can be modified, e.g., with a moiety that improves its stabilization and/or retention in circulation, e.g., in blood, serum, or other tissues, e.g., by at least 1.5, 2, 5, 10, or 50 fold. The modified blocking agent can be evaluated to assess whether it can reach sites of damage after a stroke (e.g., by using a labeled form of the blocking agent).
For example, the Tweak/Tweak-R blocking agent (e.g., an antibody or soluble Tweak-R protein) can be associated with a polymer, e.g., a substantially non-antigenic polymer, such as a polyalkylene oxide or a polyethylene oxide. Suitable polymers will vary substantially by weight. Polymers having molecular number average weights ranging from about 200 to about 35,000 Daltons (or about 1,000 to about 15,000, and 2,000 to about 12,500) can be used.
For example, a Tweak or a TweakR binding antibody can be conjugated to a water-soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g. polyvinylalcohol or polyvinylpyrrolidone. A non-limiting list of such polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained. Additional useful polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates; carbomers; and branched or unbranched polysaccharides.
When the Tweak/Tweak-R blocking agent (e.g., an antibody or soluble Tweak-R protein) is used in combination with a second agent, the two agents can be formulated separately or together. For example, the respective pharmaceutical compositions can be mixed, e.g., just prior to administration, and administered together or can be administered separately, e.g., at the same or different times.
Administration
The Tweak/Tweak-R blocking agent (e.g., an antibody or soluble Tweak-R protein) can be administered to a subject, e.g., a human subject, by a variety of methods. For many applications, the route of administration is one of: intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneally (IP), or intramuscular injection. In some cases, administration may be directly into the CNS, e.g., intrathecal or intracerebroventricular (ICV). The blocking agent can be administered as a fixed dose, or in a mg/kg dose.
The dose can also be chosen to reduce or avoid production of antibodies against the Tweak/Tweak-R blocking agent.
The route and/or mode of administration of the blocking agent can also be tailored for the individual case, e.g., by monitoring the subject, e.g., using assessment criteria discussed herein.
Dosage regimens are adjusted to provide the desired response, e.g., a therapeutic response. For example, doses in the range of 0.1-100 mg/kg, 1 mg/kg-100 mg/kg, 0.5-20 mg/kg, 0.1-10 mg/kg or 1-10 mg/kg can be administered. A particular dose may be administered more than once, e.g., at periodic intervals over a period of time (a course of treatment). For example, the dose may be administered every 2 months, every 6 weeks, monthly, biweekly, weekly, or daily, as appropriate, over a period of time to encompass at least 2 doses, 3 doses, 5 doses, 10 doses, or more.
Dosage unit form or “fixed dose” as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and optionally in association with the other agent.
Single or multiple dosages may be given. Alternatively, or in addition, the blocking agent may be administered via continuous infusion. The treatment can continue for days, weeks, months or even years.
A pharmaceutical composition may include a “therapeutically effective amount” of an agent described herein. Such effective amounts can be determined based on the effect of the administered agent, or the combinatorial effect of agents if more than one agent is used. A therapeutically effective amount of an agent may also vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition is outweighed by the therapeutically beneficial effects.
Devices and Kits
Pharmaceutical compositions that include the Tweak/Tweak-R blocking agent (e.g., an antibody or soluble Tweak-R) can be administered with a medical device. The device can designed with features such as portability, room temperature storage, and ease of use so that it can be used in emergency situations, e.g., by an untrained subject or by emergency personnel in the field, removed to medical facilities and other medical equipment. The device can include, e.g., one or more housings for storing pharmaceutical preparations that include Tweak/Tweak-R blocking agent, and can be configured to deliver one or more unit doses of the blocking agent.
For example, the pharmaceutical composition can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. Many other devices, implants, delivery systems, and modules are also known.
A Tweak/Tweak-R blocking agent (e.g., an antibody or soluble Tweak-R protein) can be provided in a kit. In one embodiment, the kit includes (a) a container that contains a composition that includes a Tweak or a Tweak receptor blocking agent, and optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the agents for therapeutic benefit. In an embodiment, the kit includes also includes a second agent for treating stroke. For example, the kit includes a first container that contains a composition that includes the Tweak/Tweak-R blocking agent, and a second container that includes the second agent.
The informational material of the kits is not limited in its form. In one embodiment, the informational material can include information about production of the compound, molecular weight of the compound, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material relates to methods of administering the Tweak/Tweak-R blocking agent (e.g., an antibody or soluble Tweak-R protein), e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein), to treat a subject who has had a stroke or who is at risk for stroke. The information can be provided in a variety of formats, include printed text, computer readable material, video recording, or audio recording, or a information that provides a link or address to substantive material.
In addition to the blocking agent, the composition in the kit can include other ingredients, such as a solvent or buffer, a stabilizer, or a preservative. The blocking agent can be provided in any form, e.g., liquid, dried or lyophilized form, preferably substantially pure and/or sterile. When the agents are provided in a liquid solution, the liquid solution preferably is an aqueous solution. When the agents are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer, can optionally be provided in the kit.
The kit can include one or more containers for the composition or compositions containing the agents. In some embodiments, the kit contains separate containers, dividers or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of the agents. The containers can include a combination unit dosage, e.g., a unit that includes both the Tweak or a Tweak receptor blocking agent and the second agent, e.g., in a desired ratio. For example, the kit includes a plurality of syringes, ampules, foil packets, blister packs, or medical devices, e.g., each containing a single combination unit dose. The containers of the kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight.
The kit optionally includes a device suitable for administration of the composition, e.g., a syringe or other suitable delivery device. The device can be provided pre-loaded with one or both of the agents or can be empty, but suitable for loading.
Combination Therapies
The methods and compositions described herein can be used in combination with other therapies for inflammatory diseases, such as corticosteroids, NSAIDs, and dialysis.
All references, including patent documents, disclosed herein are incorporated by reference in their entirety.
EXAMPLES
Example 1
Tweak is a Biomarker for Lupus Nephritis
Methods
Patients: Patients for this study were recruited from 2 lupus centers:
1) Ohio State University College of Medicine, Columbus, Ohio, which runs the Ohio SLE Study (OSS). The OSS is a prospective longitudinal investigation of risk factors for SLE flare in recurrently active patients with renal or non-renal SLE, including patients of Caucasian (70%), African American (29%) and Asian (1%) descent; and
2) the Albert Einstein College of Medicine (AECOM) Lupus Cohort, Bronx, N.Y., which includes the Jacobi and Montefiore Medical Centers lupus clinics. Altogether, lupus clinics at Jacobi and Montefiore Medical Centers regularly follow around 350 lupus patients, of Caucasian (10%), African American (45%) and Hispanic (45%) descent. The studies at both centers have been approved by their respective IRBs.
Both lupus cohorts (OSS and AECOM) follow similar procedures. Firstly, all patients enrolled fulfill at least 4 of the 1982 revised American College of Rheumatology criteria for the diagnosis of SLE. The patients are seen regularly about once every 1-2 months, and at each visit they are clinically evaluated and receive care from a physician. Routine laboratory evaluation is usually performed at each visit, including CBC, serum chemistry, ESR, CRP, serum C3, C4, anti-nuclear antibodies (ANA) and anti-dsDNA titers, urinalysis, spot urine protein/creatinine ratio and/or a 24 hour urine collection. However, due to different clinical needs, a number of the patients included in the cohorts underwent only partial laboratory evaluation which did not include all of the above tests. All available laboratory values were used in the analysis.
At the time of the visit, each patient provides a freshly voided urine specimen. The fresh AECOM urine samples were frozen in small aliquots without further manipulation at −80° C. for later analysis, while the samples at OSS were first centrifuged to remove sediment before being frozen at −80° C. This difference in sample handling did not materially affect the results since results were generally consistent among the 2 centers.
Classification of SLE nephritis activity status. Kidney disease activity was assessed by the renal SLEDAI (rSLEDAI) score that consists of the 4 kidney-related items of the SLE Disease Activity Index (hematuria, pyuria, proteinuria and urinary casts) (Bombardier et al., The development and validation of the SLE Disease Activity Index (SLEDAI). Arthritis Rheum 1992 vol: 35:630-40). The presence of each one of the 4 parameters gives a score of 4 points; thus, the rSLEDAI score can range from 0 (non-active renal disease) to a maximal score of 16. The patients enrolled in the OSS were additionally classified as to the presence of active or chronic stable disease, and the severity of renal and non-renal flares, based on criteria described in detail by Rovin et al (Rovin et al., J Am Soc Nephrol 2005; 16:467-73). In brief, the patients were first divided into a renal disease group (based on a kidney biopsy that demonstrated immune complex-mediated glomerulonephritis, as well as evidence of major renal manifestations past or present attributable to SLE, such as 24 hr urine protein/creatinine ratio >1 and/or elevated serum creatinine of over 1.1 mg/dl in women and 1.3 mg/dl in men) and a non-renal disease group (normal serum creatinine, urine sediment with <5 red blood cells per high-power field and no casts). The patients were further divided within the groups as to whether they were undergoing a flare, and as to the nature of that flare (renal or non-renal). Renal flares were classified as mild, moderate or severe, based on laboratory tests such as abnormal urine sediment, an elevation of serum creatinine, or a worsening in proteinuria. Non-renal flares were recognized when the patient developed one or more symptoms or signs of non-renal SLE that required the managing physician to increase therapy. These non-renal flares were also sorted into mild, moderate or severe based on the severity and life-threatening potential of the disease manifestations present.
Urinary TWEAK measurement. Urinary TWEAK (uTWEAK) levels were determined by ELISA, as follows: microtiter plates were coated with the BEB3 murine monoclonal anti-TWEAK antibody at 2 μg/ml in bicarbonate buffer overnight. The plates were then blocked by 3% BSA/PBS for 6 hours, washed, and the urine samples diluted 1:3 in 3% BSA/PBS were added. In addition, serial dilutions of recombinant soluble human TWEAK were added to the plate to construct a standard curve. The plate was then incubated overnight at 4° C., washed, and a solution of pre-mixed biotinylated murine anti-TWEAK antibody P5G9 (Campbell et al., J Immunol 2006; 176:1889-98) and avidin-horseradish peroxidase (HRP) (0.5 μg/ml and 1:250 final, respectively) added for one hour at room temperature. The plate was washed, followed by the addition of a developer solution, and the optical density read after 10-20 minutes. uTWEAK assays were performed blindly, without knowledge of the patient's disease status or activity.
Urinary MCP-1 (uMCP-1) and urokinase plasminogen activator receptor (UPAR) measurement. The levels of MCP-1 and uPAR were measured by commercial ELISA kits, according to manufacturer's directions (BioSource International, Camarillo, Calif. and R&D Systems, Minneapolis, Minn., respectively)
Cytokine levels were normalized to urine creatinine concentrations measured in the same spot urine. uTWEAK and uMCP-1 are expressed as pg/mg creatinine (pg/mg Cr), while UPAR levels are expressed as ng/mg Cr.
Standardization. To compare the C3, C4 and anti-dsDNA laboratory measurements obtained at different centers and measured in different laboratories, values were standardized by dividing the value received from the laboratory for each patient by the mid normal range at that same laboratory. For example, if the range for serum C4 in a particular laboratory was 16 to 54 and the measured value was 10, the standardized C4 was calculated as 10 divided by (16+54)/2=10/35=0.29.
Statistical Analysis. Data is shown as mean±SEM. Testing among two groups was performed by the Mann-Whitney U test. Correlations were determined by the Spearman rank correlation coefficient (ρ). P<0.05 was considered significant. The statistics program used was GraphPad Prism version 4.03 (GraphPad Software, San Diego, Calif.).
Results
The AECOM and OSS cohorts are similar in most aspects, including the age, gender, prevalence of active renal disease, and complement levels. However, significant differences between the 2 cohorts are found in the serum creatinine and proteinuria measurements. This difference may be attributed to several possible factors. While the patients in the OSS cohort were recruited from a renal clinic, patients in the AECOM cohort were recruited from lupus clinics, Furthermore, as described above, one of the criteria for inclusion in the OSS cohort was a kidney biopsy. Therefore, chronic renal changes, such as elevated serum creatinine and/or proteinuria, may have been more frequent in the OSS than in the AECOM cohort. Nevertheless, as the mean rSLEDAI score in patients with active LN is not significantly different between the 2 groups, it appears that the OSS and AECOM cohorts are relatively comparable in terms of acute inflammatory renal changes.
To determine whether TWEAK correlates with LN activity, we compared uTWEAK levels of patients with active LN (rSLEDAI score ≧4) with those of patients who never had kidney involvement, or those with previous kidney involvement whose disease was quiescent, i.e. non-active renal disease (n=78). As shown in FIG. 1A, patients with active renal disease (n=43) have higher levels of uTWEAK than lupus patients with non-active or never renal disease (n=35) (21.57±4.6 versus 10.03±1.4 pg/mg Cr, P=0.001). Moreover, uTWEAK correlated significantly with rSLEDAI scores (ρ=0.405, P<0.001; FIG. 1B). uTWEAK levels displayed significant correlation with the total SLEDAI score, performed only in the AECOM cohort (n=30, ρ=0.421, P=0.022); however, this correlation was no longer significant when only non-renal components of the index were correlated with uTWEAK. When uTWEAK levels in SLE patients with chronic, stable disease (n=20) were compared with those undergoing a flare (n=31), patients with active disease had significantly higher uTWEAK levels than those with stable SLE (13.61±1.5 and 9.22±1.7 pg/mg Cr, respectively, P=0.037; FIG. 2A). Furthermore, in the more limited subgroup of lupus patients with previous renal involvement (n=35), a trend toward higher uTWEAK levels was found in patients undergoing a flare (n=23) as opposed to those with chronic stable renal disease (n=12) (14.54±1.6 and 9.39±2.0 pg/mg Cr, respectively, P=0.058; FIG. 2B). Additionally, when only patients undergoing a flare were considered separately (n=31), there were significantly higher uTWEAK levels in those patients undergoing a renal flare (n=23) as opposed to the patients undergoing a non-renal flare (n=8) (14.55±1.6 pg/mg Cr and 8.34±3.0 pg/mg Cr, respectively, P=0.03; FIG. 3). There were no significant differences between uTWEAK levels of patients with varying flare severities (mild-moderate versus severe), or with different WHO classes of LN.
To begin to determine the utility of uTWEAK to help clinically monitor renal disease activity over time, we analyzed uTWEAK levels in the 6 patients that had 2 urine samples available, between which they transformed from a chronic stable disease state to a flare. As shown in FIG. 4A, in 4 out of the 6 patients there was a marked elevation in uTWEAK levels as the flare was developing. Among these 6 patients, 3 had uTWEAK measurements at 3 different time points. One patient had a paradoxical decrease in uTWEAK levels while undergoing a flare (dotted line in FIG. 4A). The two remaining patients are depicted in FIG. 4B-C, which demonstrate how uTWEAK levels fluctuated in parallel with renal activity (as measured by rSLEDAI) and the OSS predefined criteria for flare status. Furthermore, as shown in FIG. 4C, uTWEAK levels in this patient anticipated renal disease activity even better than the rSLEDAI score. While the patient's renal disease was clinically worsening and uTWEAK levels concomitantly increased, the rSLEDAI score remained unchanged.
As urinary MCP-1 (uMCP-1) has already been recognized as a biomarker for LN and TWEAK stimulates the production of MCP-1 by mesangial cells and podocytes, we investigated the correlation between uMCP-1 and uTWEAK, and indeed found a strong correlation (ρ=0.501, P<0.001; FIG. 5A). In addition, as shown in FIG. 5B-D, uTWEAK correlated (albeit moderately) with other common serologic indicators of SLE activity such as anti-dsDNA antibodies (ρ=0.459, P=0.008), and complement components C3 and C4 (ρ=−0.262 and −0.269, respectively, P<0.02). Moreover, uTWEAK correlated with systemic inflammatory activity, as measured by the erythrocyte sedimentation rate (ρ=0.373, P=0.013) (data not shown).
We found that uTWEAK did not correlate with the degree of proteinuria (P=0.562). One likely explanation for this observation is that the source of uTWEAK is the kidneys reflecting local inflammatory activity, rather than resulting from damage to the glomerular filtration barrier and non-specific protein loss into the urine. Additional reinforcement for the above hypothesis comes from the fact that uTWEAK does not correlate with the levels of TWEAK in the serum (data not shown), indicating that uTWEAK is not simply a reflection of serum TWEAK concentrations. In addition, while urinary levels of the protein UPAR did not correlate with proteinuria (ρ=0.112, P=0.570; n=28), in contrast to uTWEAK, urinary UPAR levels did not correlate with renal disease activity (ρ=−0.024, P=0.891; n=36). This finding supports the conclusion that the correlation of uTWEAK with disease activity is specific, as not all urinary proteins correlate with renal disease activity or damage. Finally, uTWEAK negatively correlated with plasma BUN (ρ=−0.372, P=0.036), while a similar trend was noted with plasma creatinine (ρ=−0.206, P=0.066).
Example 2
Blocking Tweak Improves Glomerulonephritis
The role of TWEAK/Fn14 interactions in the pathogenesis of lupus nephritis (LN) in SLE is demonstrated.
Methods: We analyzed the effect of Fn14 deficiency on progression and severity of LN in the cGVH model of SLE. We chose this murine SLE model, as the Fn14 knockout (KO) was already bred into the C57Bl/6 (B6) background, which is susceptible to disease induction, In this model, a single injection of 108 MHC II incompatible splenocytes to unirradiated mice induces autoantibodies and renal disease characteristic of lupus within 2-4 weeks. In addition, we analyzed the effect of anti-TWEAK antibodies (Ab) on Fn14 wild type (WT) mice with cGVH induced lupus.
Results: We used B6.CH2bm12/Kheg (bm12) mice, a B6 derived mouse strain with only a three amino acid change in the I-Ab chain that is sufficient to induce strong alloreactivity between B6 and bm12. We compared B6 Fn14 WT and KO mice that were each injected with bm12 donor splenocytes. Control groups not expected to develop disease included B6 WT and Fn14 KO mice injected with B6 splenocytes. We found that titers of IgG and IgM anti-dsDNA, histone, and chromatin Ab were no different between B6 Fn14 WT and KO mice injected with alloreactive splenocytes. However, kidney disease, as assessed by proteinuria, was significantly less severe in Fn14 KO mice at 6, 8, and 10 weeks. Furthermore, kidney staining for MCP-1 and RANTES was significantly decreased in Fn14 KO as compared to Fn14 WT mice with induced lupus. Finally, we found that B6 Fn14 WT mice with induced lupus treated with the P5G9 anti-TWEAK mAb (200 mg×2/week I.P.) had significantly less proteinuria than mice treated with P1.17 (isotype matched control mAb) or PBS.
CONCLUSION
Inhibition of TWEAK signaling, by genetically deleting the Fn14 receptor or by anti-TWEAK mAb treatment, significantly improved glomerulonephritis in the cGVH model of lupus. Systemic autoantibody levels were not significantly altered.
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11953360
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biogen idec ma inc.
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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/ 7.1
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Apr 1st, 2022 05:10PM
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Apr 1st, 2022 05:10PM
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Biogen
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:biib
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Biogen
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May 20th, 2014 12:00AM
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Sep 27th, 2011 12:00AM
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https://www.uspto.gov?id=US08728475-20140520
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Methods for treating inflammatory bowel disease
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Methods of treating inflammatory disorders, such as rheumatoid arthritis, by modulating TWEAK and TNF-α are disclosed, as are other methods.
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8728475
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1. A method for treating inflammatory bowel disease comprising: administering to a human subject who has inflammatory bowel disease, a TWEAK (TNF-like weak inducer of apoptosis) blocking agent selected from the group consisting of a monoclonal, chimeric, human, or humanized antibody that binds to TWEAK, and a soluble form of a TWEAK receptor, in combination with a TNF-α blocking agent selected from the group consisting of a monoclonal, chimeric, human, or humanized antibody that binds to TNF-α, and a soluble form of a TNF-α receptor, in amounts and for a time to provide a therapeutic effect.
2. The method of claim 1, wherein the inflammatory bowel disease is ulcerative colitis.
3. The method of claim 1, wherein the inflammatory bowel disease is Crohn's disease.
4. The method of claim 1, wherein the TWEAK blocking agent and the TNF-α blocking agent are administered in an amount effective to inhibit TWEAK and TNF-α pathways in cells that generate inflammatory signals.
5. The method of claim 1, wherein the TWEAK blocking agent and the TNF-α blocking agent are administered in an amount effective to reduce transcription of genes induced by TWEAK and TNF-α in cells that generate inflammatory signals.
6. The method of claim 5, wherein the genes induced by TWEAK and TNF-α are synergistically activated by TWEAK and TNF-α.
7. The method of claim 1, wherein the TWEAK blocking agent is administered at a dosage that is equal to or less than an amount required for efficacy with the TWEAK blocking agent monotherapy.
8. The method of claim 7, wherein the TWEAK blocking agent is administered at a dosage that is at least 10% less than an amount required for efficacy with the TWEAK blocking agent monotherapy.
9. The method of claim 1, wherein the TNF-α blocking agent is administered at a dosage that is equal to or less than an amount required for efficacy with the TNF-α blocking agent monotherapy.
10. The method of claim 9, wherein the TNF-α blocking agent is administered at a dosage that is at least 10% less than an amount required for efficacy with TNF-α blocking agent monotherapy.
11. The method of claim 1, wherein the TWEAK blocking agent reduces the ability of TWEAK to bind to a TWEAK receptor.
12. The method of claim 1, wherein the TWEAK blocking agent is a monoclonal, chimeric, human, or humanized antibody that binds to TWEAK.
13. The method of claim 12, wherein the antibody that binds to TWEAK comprises a heavy chain variable domain comprising:
a) a complementarity determining region (CDR) 1 set forth as the amino acid sequence of amino acids 26 to 35 of SEQ ID NO:3 (GFTFSRYAMS);
b) a CDR 2 set forth as the amino acid sequence of amino acids 50 to 66 of SEQ ID NO:3 (EISSGGSYPYYPDTVTG); and
c) a CDR 3 set forth as the amino acid sequence of amino acids 99 to 114 of SEQ ID NO:3 (VLYYDYDGDRIEVMDY);
and a light chain variable domain comprising:
a) a CDR 1 set forth as the amino acid sequence of amino acids 24 to 39 of SEQ ID NO:4 (RSSQSLVSSKGNTYLH);
b) a CDR 2 set forth as the amino acid sequence of amino acids 55 to 61 of SEQ ID NO:4 (KVSNRFS); and
c) a CDR 3 set forth as the amino acid sequence of amino acids 94 to 102 of SEQ ID NO:4 (SQSTHFPRT).
14. The method of claim 13, wherein the antibody comprises a first amino acid sequence set forth in SEQ ID NO:5 and a second amino acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:7.
15. The method of claim 1, wherein the TWEAK blocking agent is a soluble form of a TWEAK receptor.
16. The method of claim 15, wherein the soluble form of the TWEAK receptor comprises an antibody Fc region.
17. The method of claim 1, wherein the TNF-α blocking agent reduces the ability of TNF-α to bind to a TNF-α receptor.
18. The method of claim 1, wherein the TNF-α blocking agent is a monoclonal, chimeric, human, or humanized antibody that binds to TNF-α.
19. The method of claim 18, wherein the TNF-α blocking agent is infliximab or adalimumab.
20. The method of claim 1, wherein the TNF-α blocking agent is a soluble form of a TNF-α receptor.
21. The method of claim 1, wherein the TNF-α blocking agent is etanercept.
22. The method of claim 1, wherein the TWEAK blocking agent and the TNF-α blocking agent are administered to the subject at the same time.
23. The method of claim 22, wherein the TWEAK blocking agent and the TNF-α blocking agent are administered as a co-formulation.
24. The method of claim 1, wherein the TWEAK blocking agent and the TNF-α blocking agent are administered sequentially.
25. The method of claim 24, wherein the TWEAK blocking agent is administered to a patient who has already received the TNF-α blocking agent, wherein the TNF-α blocking agent is present at a therapeutic level in the subject.
26. The method of claim 24, wherein the TNF-α blocking agent is administered to a patient who has already received the TWEAK blocking agent, wherein the TWEAK blocking agent is present at a therapeutic level in the subject.
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of application Ser. No. 11/937,687, filed Nov. 9, 2007, abandoned, which is a continuation application claiming priority under 35 U.S.C. §120 of International Application No. PCT/US2006/018077, filed on May 10, 2006, and claims the benefit of U.S. Provisional Application No. 60/679,518, filed on May 10, 2005, all of which are incorporated herein by reference in their entirety.
BACKGROUND
The tumor-necrosis factor (TNF)-related cytokines are a superfamily of proteins that have an array of functions, including ones implicated in immune regulation and apoptosis regulation. Examples of TNF superfamily members include TNF-α and TWEAK (TNF-like weak inducer of apoptosis).
SUMMARY
As further described below, the TWEAK and TNF-α pathways work independently to mediate aspects of inflammation. Blocking both of the molecular signalling pathways modulated by TWEAK and TNF-α can be used to treat a variety of inflammatory disorders. Examples of such treatments are described below.
In one aspect, the disclosure features a method of treating a subject for an inflammatory disorder. In a preferred embodiment, the inflammatory disorder is an arthritic disorder, e.g., rheumatoid arthritis, psoriatic arthritis, or Sjögren's Syndrome. The method includes: administering, to a subject, e.g., a human subject, who has or is at risk for the disorder, e.g., rheumatoid arthritis, a TWEAK blocking agent in combination with a TNF-α blocking agent. The TWEAK blocking agent and the TNF-α blocking agent can be administered in amounts and for a time to provide a therapeutic effect, e.g., an overall therapeutic effect. The effect can be additive or, in some cases, synergistic. For example, the effect of both blocking agents may be a greater total effect than the sum of the individual effects, e.g., in a particular subject.
A variety of TWEAK blocking agents can be administered to a subject to block a interaction or activity of TWEAK or a TWEAK-R. A “TWEAK blocking agent” refers to an agent (e.g., any compound, e.g., an antibody or a soluble form of the TWEAK receptor) that at least partially inhibits an interaction or activity of a TWEAK or TWEAK-R. For example, the agent at least partially inhibits an activity, e.g., binding of TWEAK to a TWEAK-R, or the agent at least partially inhibits a nucleic acid encoding TWEAK or TWEAK-R, e.g., to reduce TWEAK or TWEAK-R protein expression.
In one embodiment, the agent reduces the ability of TWEAK to bind to a TWEAK receptor, e.g., Fn14. The agent can be a blocking antibody that binds to TWEAK or to Fn14. The antibody can be a full length IgG. In one embodiment, the antibody is human, humanized, or effectively human. In one embodiment, the TWEAK blocking antibody competes with AB.D3 (an antibody that has ATCC Accession No. HB-12622) for binding with TWEAK, is a humanized antibody AB.D3, comprises at least two, three, four, five, or six CDRs of AB.D3 (or CDRs that are at least overall 85, 90, 92, 95, 97% identical to such CDRs), and/or comprises antibody AB.D3 variable domains (or one or more variable domains that are at least overall 85, 90, 92, 95, 97% identical to such variable domains).
In one embodiment, the agent is a soluble form of a TWEAK receptor, e.g., a human TWEAK receptor such as Fn14. The soluble form of the TWEAK receptor can be fused to an antibody Fc region (e.g., a human Fc region). For example, the soluble form of the TWEAK receptor includes a sequence at least 95% identical to amino acids 28-X1 of SEQ ID NO:2, where amino acid X1 is selected from the group of residues 68 to 80 of SEQ ID NO:2.
A variety of TNF-α blocking agents can be administered to a subject to block an interaction or activity of TNF-α or a TNF-α receptor, e.g., TNFR-I, or TNFR-II. A “TNF-α blocking agent” refers to an agent (e.g., any compound, e.g., an antibody or a soluble form of a TNF-α receptor) that at least partially inhibits an interaction or activity of TNF-α or a TNF-α receptor. For example, the agent at least partially inhibits an activity, e.g., binding of TNF-α to a TNF-α receptor, or the agent at least partially inhibits a nucleic acid encoding TNF-α or a TNF-α receptor, e.g., to reduce TNF-α or TNF-α receptor protein expression.
In one embodiment, the TNF-α blocking agent reduces the ability of TNF-α to bind to a TNF-α, receptor. For example, the TNF-α blocking agent includes an antibody that binds to TNF-α, TNFR-I, or TNFR-II. Exemplary antibodies include infliximab or adalimumab. The TNF-α blocking agent can include a soluble form of a TNF-α receptor and optionally a Fc domain. For example, the TNF-α blocking agent is etanercept.
As used herein, “administered in combination” means that two or more agents (e.g., the TWEAK blocking agent and the TNF-α blocking agent) are administered to a subject at the same time or within an interval, such that there is overlap of an effect of each agent on the patient. Preferably the administrations of the first and second agent are spaced sufficiently close together such that a combinatorial effect is achieved. The interval can be an interval of hours, days or weeks. Generally, the agents are concurrently bioavailable, e.g., detectable, in the subject. In a preferred embodiment, at least one administration of one of the agents, e.g., the first agent (e.g., TNF-α blocking agent), is made while the other agent, e.g., the TWEAK blocking agent, is still present at a therapeutic level in the subject.
In one embodiment, the TWEAK blocking agent is administered between an earlier and a later administration of the TNF-α blocking agent. In other embodiments, the TNF-α blocking agent is administered between an earlier and a later administration of the TWEAK blocking agent. In a preferred embodiment, at least one administration of one of the agents, e.g., the TNF-α blocking agent, is made within 1, 7, 14, 30, or 60 days of the other agent, e.g., the TWEAK blocking agent.
In one embodiment, prior to administering the TWEAK blocking agent and TNF-α blocking agent, the subject was receiving either the TWEAK blocking agent or TNF-α blocking agent, but not the other. The subject may have had a response that did not meet a predetermined threshold, e.g., a stabilization or reduction in a total Sharp score or a Sharp erosion score. In another embodiment, the subject can be one who has not been previously administered the TNF-α blocking agent nor the TWEAK blocking agent for at least 3 months (e.g., at least 6 months, 9 months, or a year prior) prior to being administered the first and second agent in combination.
In one implementation, the TWEAK blocking agent and TNF-α blocking agent are provided as a co-formulation, and the co-formulation is administered to the subject. It is further possible, e.g., at least 24 hours before or after administering the co-formulation, to administer one of the agents separately from the other. In another implementation, the agents are provided as separate formulations, and the step of administering includes sequentially administering the agents. The sequential administrations can be provided on the same day (e.g., within one hour of one another or at least 3, 6, or 12 hours apart) or on different days.
Generally, the TWEAK blocking agent and TNF-α blocking agent are each administered as a plurality of doses separated in time, e.g., according to a regimen. The regimen for one or both may have a regular periodicity. The regimen for the TNF-α blocking agent can have a different periodicity from the regimen for the TWEAK blocking agent, e.g., one can be administered more frequently than the other. The agents can be administered by any appropriate method, e.g., subcutaneously, intramuscularly, or intravenously. The subject can be administered doses of the TNF-α blocking agent and doses of the TWEAK blocking agent for greater than 14 weeks, greater than six or nine months, greater than 1, 1.5, or 2 years.
In some embodiments, each of the agents is administered at about the same dose as the dose used for monotherapy. In other embodiments, the TNF-α blocking agent is administered at a dosage that is equal to or less than an amount required for efficacy if administered alone (e.g., at least 10, 20, 30, or 40% less). Likewise, the TWEAK blocking agent can be administered at a dosage that is equal to or less than an amount required for efficacy if administered alone (e.g., at least 10, 20, 30, or 40% less). For example, in some embodiments in which the subject has previously received the TNF-α blocking agent, the subject is administered a reduced dose of the TNF-α blocking agent after receiving the TWEAK blocking agent (relative to the dose of the TNF-α blocking agent received before receiving the TWEAK blocking agent for the first time). The same or a different TNF-α blocking agent can be used in the combination as was used in the previous monotherapy.
A subject can be evaluated after receiving the first and second agent, e.g., for indicia of responsiveness. A skilled artisan can use various clinical or other indicia of effectiveness of treatment. The subject can be monitored at various times during a regimen.
In one embodiment, the TWEAK blocking agent and the TNF-α blocking agent are administered in amounts effective to inhibit the collective effects of TWEAK and TNF-α pathways in cells that generate inflammatory signals, e.g., synoviocytes, chondrocytes, osteoclasts, osteoblasts, dermal fibroblasts, monocytes, macrophages, or endothelial cells. The agents can be administered in amounts effective to reduce transcription of a set of genes induced by TWEAK and TNF-α in such cells, e.g., to reduce transcription of genes synergistically activated by TWEAK and TNF-α, e.g., one or more genes list in Table 1 in synoviocytes, chondrocytes, osteoclasts, or osteoblasts.
In some embodiments, the TWEAK blocking agent is administered in an amount that is at least 20, 30, 50, 60, or 70% less than standard dosages for TWEAK blocking agent monotherapy (or a TWEAK blocking agent therapy in the absence of TNF-α, blocking agent) for treating an adult subject for rheumatoid arthritis. For example, the TWEAK blocking agent is administered in an amount less than that required to be effective as a monotherapy.
In some embodiments, the TNF-α blocking agent is administered in an amount that is at least 20, 30, 50, 60, or 70% less than standard dosages for a TNF-α blocking agent monotherapy (or a TNF-α, blocking agent therapy in the absence of a TWEAK blocking agent) for treating an adult subject for rheumatoid arthritis. For example, the TNF-α blocking agent is administered in an amount less than that required to be effective as a monotherapy. In other embodiments, the TNF-α blocking agent and the TWEAK blocking agent are administered in the same dose as that used in monotherapy.
For example, the subject is not receiving methotrexate. In one embodiment, the subject is not receiving any other disease modifying anti-rheumatic drug (DMARD), i.e., other than the TWEAK blocking agent and the TNF-α blocking agent.
The amounts can be sufficient to result in a statistically significant reduction in joint damage as measured by the Sharp erosion score. For example, the subject can be monitored at one or more instances for a parameter indicative of the disorder.
The method can include evaluating (e.g., monitoring one or times, e.g., periodically) the subject, e.g., for symptoms of the disorder or indicia that grade disorder severity. For example, in the case of rheumatoid arthritis, it is possible to use the total Sharp score (TSS), Sharp erosion score, HAQ disability index, or radiological method.
In another aspect, the disclosure features a method that includes: administering, to a subject (e.g., a human subject) who has or is at risk for rheumatoid arthritis, a TWEAK blocking agent in combination with another DMARD (e.g., a biologic DMARD), in amounts and for a time to provide an overall therapeutic effect. Some examples of DMARDs for treating rheumatoid arthritis are described herein.
In another aspect, the invention features a method of reducing joint inflammation in a subject in need thereof. The method includes administering to a subject who suffers from joint inflammation a TNF-α blocking agent in combination with a TWEAK blocking agent, e.g., as described herein. In some cases, the subject has an arthritic disorder, e.g., rheumatoid arthritis.
Also featured is a pharmaceutical composition that includes: a TWEAK blocking agent; and a DMARD, e.g., a TNF-α blocking agent or other DMARD.
Kits can also be provided that include a TWEAK blocking agent and a DMARD (e.g., a TNF-α blocking agent or other DMARD). The agents can be provided as separate pharmaceutical compositions or a single pharmaceutical composition. The kit can further include instructions for administration to treat rheumatoid arthritis, a device for administering the agents, and/or reagents for evaluating a parameter, e.g., a clinical parameter associated with the disorder.
In another aspect, the disclosure features a method that includes: identifying a subject who has inflammation mediated by TWEAK and TNF-α, and/or increased TWEAK expression or activity, and/or increased expression or activity of a biomarker whose expression is modulated (e.g., increased) by TWEAK (see, e.g., Table 2); and administering to the subject a therapy. For example, therapy can include administering: (i) a TWEAK blocking agent; (ii) a TNF-α blocking agent; or (iii) a combination of (i) and (ii). A “TWEAK/TNF-α synergistically activated cellular program” is a cellular state characterized by properties that result from stimulation by particular doses of both TWEAK and TNF-α, but which are not attained to a comparable degree by stimulation with that dose of TWEAK in the absence of that dose of TNF-α nor by stimulation by that dose TNF-α in the absence of that dose of TWEAK. The subject can be identified by evaluating expression of one or more genes in cells that generate inflammatory signals, e.g., synoviocytes, chondrocytes, osteoclasts, osteoblasts, or dermal fibroblasts, or associated tissue, obtained from the subject. The one or more genes from Table 1 can be evaluated. The subject can also be evaluated for one or more symptoms of rheumatoid arthritis.
In another aspect, the disclosure features a method that includes: administering, to a human subject who has or is at risk for rheumatoid arthritis, and who is being or has been withdrawn from a DMARD (other than a TWEAK blocking agent), a TWEAK blocking agent, e.g., in an amount and for a time effective to provide an overall therapeutic effect. The method can be used to treat a subject has not previously received a TWEAK blocking agent or who has not recently received a TWEAK blocking agent, e.g., within the last month, six months, or year.
In one embodiment, the DMARD that is being or has been withdrawn is a TNF-α blocking agent. The subject may have an inadequate response to the TNF-α blocking agent. As used herein, an “inadequate response” refers to a response that, as assessed by a patient or a skilled clinician, exhibits insufficient efficacy or intolerable or unacceptable toxicity. Insufficient efficacy can be defined by failure to meet a predetermined level of response to treatment. For example, the TNF-α blocking agent may cause toxicity, induce an immune-compromised state, or lacks efficacy, thereby prompting its withdrawal. For example, the subject is refractory to therapy with the TNF-α blocking agent. The subject may have, e.g., tuberculosis, an opportunistic infection, glomerulonephritis, a demyelinating syndrome, a lupus-like reaction, or a pathogenic bacterial infection. In some cases, an inadequate response is indicated by an adverse event detected during treatment with the TNF-α blocking agent.
The TNF-α blocking agent may have been administered within the previous year, three months, month, two weeks, or week. In some cases, the subject may still be administered the TNF-α blocking agent, but its dosage may be reduced or may be a final dosage, e.g., a dosage provided prior to complete termination. In other cases, administration of the TNF-α blocking agent is ceased such that, upon administration of one or more doses of the TWEAK blocking agent, the subject is no longer receiving the TNF-α blocking agent.
In other embodiments, the DMARD that is being or has been withdrawn is methotrexate, parenteral gold, sulphasalazine, or hydroxychloroquinone. For example, the DMARD is other than a TNF-α blocking agent. The DMARD can be withdrawn due to toxicity, immune suppression or lack of efficacy. For example, an adverse event may be detected during treatment with the DMARD.
In another aspect, the disclosure features a method that includes: detecting an adverse event in a human subject who has rheumatoid arthritis, and is being treated with a DMARD other than a TWEAK blocking agent; and administering, to the subject, a TWEAK blocking agent in an amount and for a time effective to provide an overall therapeutic effect.
In one embodiment, the subject is being treated with a TNF-α blocking agent. The method can further include withdrawing the TNF-α blocking agent. The adverse event can include a lupus-like reaction, a bacterial or opportunistic infection, or tuberculosis.
In one aspect, the disclosure features a method of treating a subject for an inflammatory disorder, particularly one that a TNF-α blocking agent does not exacerbate. The inflammatory disorder can be rheumatoid arthritis, or a disorder other than rheumatoid arthritis. For example, the disorder can be psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease (including ulcerative colitis and Crohn's disease), psoriasis, or inflammatory myositis. Still other examples of inflammatory disorders include Langerhans-cell histiocytosis, adult respiratory distress syndrome/bronchiolitis obliterans, Wegener's granulomatosis, vasculitis, cachexia, stomatitis, idiopathic pulmonary fibrosis, dermatomyositis or polymyositis, non-infectious scleritis, chronic sarcoidosis with pulmonary involvement, myelodysplastic syndromes/refractory anemia with excess blasts, ulcerative colitis, moderate to severe chronic obstructive pulmonary disease, and giant cell arteritis. The method includes administering, to a human subject who has or is at risk for an inflammatory disorder, a TWEAK blocking agent in an amount and for a time to provide an overall therapeutic effect. The method can include administering the TWEAK blocking agent in combination with a TNF-α blocking agent, in amounts and for a time to provide an overall therapeutic effect, or administering the TWEAK blocking agent without providing (e.g., withholding) the TNF-α blocking agent. In one embodiment, the subject is less than 17 years of age, and the disorder is juvenile rheumatoid arthritis or pediatric psoriasis. The method can include other features described herein.
In another aspect, the disclosure features a method of evaluating a test compound, e.g., for ability to modulate a TWEAK and/or TNF-α response in vitro or in vivo. A TWEAK response includes modulation of TWEAK itself or modulation of a TWEAK receptor. The method includes contacting the test compound to a cell, tissue, or organism, in the presence of TWEAK and/or TNF-α, e.g., exogenous TWEAK and/or TNF-α. The method further includes evaluating whether the test compound modulates ability of the cell, tissue, or organism to respond to TWEAK and/or TNF-α, e.g., to reduce TWEAK/TNF-α mediated cellular programs. The method can include evaluating expression or activity of one or more genes in Table 1 or Table 2. The method can further include evaluating ability of the test compound to modulate a disorder, e.g., using an animal model of a human disorder described herein.
In another aspect, the disclosure features a method of evaluating a subject, e.g., a human subject. The subject can be evaluated in advance of providing one or more agents described herein, while receiving one or more such agents, or after receiving one or more such agents. The method includes evaluating cells (e.g., in a sample obtained from the subject), tissue or other material from the subject to determine if expression (including protein and mRNA expression) of one or more genes in Table 2 are altered relative to a reference value. The reference value can be a value associated with a reference value for a normal subject, a control subject, or a value determined, e.g., for a cohort of subjects. The reference value can be a reference value for the subject him or herself, e.g., at another instance, e.g., before receiving one or more agents, and so forth. The information from the evaluating can be stored on a computer-readable medium or another medium, and/or communicated, e.g., using a computer network. The method can be used to determine if the patient is or is predicted to be TWEAK responsive. For example, a patient that has an elevated level of expression of one or more genes in Table 2 can be indicated to be TWEAK responsive. The method can include providing an indication that the subject is TWEAK responsive, and optionally instructions to administer a TWEAK blocking agent. The method can further include administering the TWEAK blocking agent.
In another aspect, the disclosure features a medicament comprising a TWEAK blocking agent and a TNF-α blocking agent, e.g., for use in therapy.
In another aspect, the disclosure features use of a TWEAK blocking agent and a TNF-α blocking agent for the preparation of a medicament, e.g., for the treatment of an inflammatory disorder described herein, e.g., joint inflammation or an arthritic disorder, e.g., rheumatoid arthritis.
In another aspect, the disclosure features use of a TWEAK blocking agent for the preparation of a medicament, e.g., for the treatment of an inflammatory disorder described herein, e.g., joint inflammation or an arthritic disorder, e.g., rheumatoid arthritis in subjects who are unresponsive to therapy with another DMARD.
All patents, patent applications, and references cited herein are hereby incorporated by reference in their entireties. In the case of conflict, the present application controls.
The term “synergy” refers to a result from at least two events that is greater than the sum of the result of each event individually. ANOVAs can be used to determine a synergy factor in the following equation:
R=A+B+ε(A*B)
Exemplary values for the synergy factor ε can be greater than zero or a predetermined value, e.g., 1, 2, or more.
The term “treating” refers to administering a therapy in an amount, manner, and/or mode effective to improve or prevent a condition, symptom, or parameter associated with a disorder or to prevent onset, progression, or exacerbation of the disorder, to either a statistically significant degree or to a degree detectable to one skilled in the art. Accordingly, treating can achieve therapeutic and/or prophylactic benefits. An effective amount, manner, or mode can vary depending on the subject and may be tailored to the subject.
Reference to inhibition includes at least partial inhibition as well as other degrees of inhibition, e.g., substantial or complete.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
DESCRIPTION OF DRAWINGS
FIG. 1 is a line graph showing average arthritis index scores in a mCIA model of arthritis in mice that were treated with a combination of TWEAK and TNF-α blocking agents, a TWEAK blocking agent alone, a TNF-α blocking agent alone, a PBS control, or isotype-matched controls.
FIG. 2 is a plot showing average metatarsal height values in a mCIA model of arthritis in mice that were treated with a combination of TWEAK and TNF-α blocking agents, a TWEAK blocking agent alone, a TNF-α blocking agent alone, a PBS control, or isotype-matched controls.
FIG. 3 is a line graph showing percent body weight change in a mCIA model of arthritis in mice that were treated with a combination of TWEAK and TNF-α blocking agents, a TWEAK blocking agent alone, a TNF-α blocking agent alone, a PBS control, or isotype-matched controls.
FIG. 4 depicts two line plots showing average arthritis index values in CIA models of arthritis in animals treated with anti-TWEAK blocking antibodies (anti-TWEAK mAbs) or controls. The left panel shows results obtained using a mouse CIA model; the right panel shows results obtained using a rat CIA model.
FIG. 5 depicts two line plots showing average arthritis index values in CIA models of arthritis in animals treated with anti-TWEAK blocking antibodies (anti-TWEAK mAbs) or controls. The figures show the results of two dosing regimens: in the first, the antibody is administered at the time of arthritis induction, in the second, the antibody is administered after arthritis induction. The left panel shows results obtained using a mouse CIA model; the right panel shows results obtained using a rat CIA model.
FIG. 6 includes five bar graphs showing levels of inflammation and cartilage and bone loss in a rat CIA model of arthritis in rats treated with anti-TWEAK blocking antibodies (ABG.11) or controls. Similarly findings can be observed in a mouse model.
FIG. 7 is a plot showing serum TWEAK levels at various time points after induction of arthritis in a mCIA model and in a control mouse (DBA/1).
FIG. 8 includes four bar graphs showing the levels of MMP9, lymphotactin, IP-10, and IL-6 at various time points after induction of arthritis in the mCIA model in mice treated with anti-TWEAK blocking antibodies (P5G9 and P5G9 (Full)) or controls.
DETAILED DESCRIPTION
We have discovered that the TWEAK and TNF-α pathways independently contribute to inflammatory responses, e.g., in synovial cells present in joints, and that both TWEAK and TNF-α can independently activate similar sets of genes indicating redundancy between the two pathways. Accordingly, reducing the activity of both pathways provides an advantageous therapeutic route to ameliorating inflammation, e.g., in joints, e.g., in arthritic conditions. Concurrent blocking of both TWEAK and TNF-α pathways proved beneficial in a mouse model of rheumatoid arthritis (mCIA) and achieved results greater than blocking one of these pathways.
In addition to rheumatoid arthritis, reducing activity of both pathways may be used to treat in other disorders, e.g., other inflammatory disorders such as psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease, psoriasis, inflammatory myositis, and other disorders disclosed herein. A variety of methods can be used to reduce activity of the TWEAK and TNF-α pathways. For example, it is possible to administer a TWEAK blocking agent in combination with a TNF-α blocking agent. Examples of these and other agents are further described below.
In some implementations, therapeutic benefit can be achieved by reducing one of the two pathways. For example, a TWEAK blocking agent can be administered to a subject who has an inadequate response to a therapeutic that modulates just one of the pathways, e.g., an inadequate response to a TNF-α blocking agent or an inadequate response to a TWEAK blocking agent. A TWEAK blocking agent can also be administered to a subject who is or who is planning to withdraw from a DMARD treatment with another agent, e.g., an agent other than a TNF-α blocking agent.
TWEAK Blocking Agents
A variety of agents can be used as a TWEAK blocking agent. The agent may be any type of compound (e.g., small organic or inorganic molecule, nucleic acid, protein, or peptide mimetic) that can be administered to a subject. In one embodiment, the blocking agent is a biologic, e.g., a protein having a molecular weight of between 5-300 kDa. For example, a TWEAK blocking agent may inhibit binding of TWEAK to a TWEAK receptor or may prevent TWEAK-mediated NF-KB activation. A typical TWEAK blocking agent can bind to TWEAK or a TWEAK receptor, e.g., Fn14. A TWEAK blocking agent that binds to TWEAK or a TWEAK receptor may alter the conformation of TWEAK or a TWEAK receptor, block the binding site on TWEAK or a TWEAK receptor, or otherwise decrease the affinity of TWEAK for a TWEAK receptor or prevent the interaction between TWEAK and a TWEAK receptor.
A TWEAK blocking agent (e.g., an antibody) may bind to TWEAK or to a TWEAK receptor with a Kd of less than 10−6, 10−7, 10−8, 10−9, or 10−10 M. In one embodiment, the blocking agent binds to TWEAK with an affinity at least 5, 10, 20, 50, 100, 200, 500, or 1000-fold better than its affinity for TNF-α or another TNF superfamily member (other than TWEAK). In one embodiment, the blocking agent binds to the TWEAK receptor with an affinity at least 5, 10, 20, 50, 100, 200, 500, or 1000-fold better than its affinity for the TNF receptor or a receptor for another TNF superfamily member. A preferred TWEAK blocking agent specifically binds TWEAK or TWEAK-R.
Exemplary TWEAK protein molecules include human TWEAK (e.g., AAC51923, shown as SEQ ID NO:1)), mouse TWEAK (e.g., NP—035744.1), rat TWEAK (e.g., XP—340827.1), and Pan troglodytes TWEAK (e.g., XP—511964.1). Also included are proteins that include an amino acid sequence at least 90, 92, 95, 97, 98, 99% identical or completely identical to the mature processed region of the aforementioned TWEAK proteins (e.g., an amino acid sequence at least 90, 92, 95, 97, 98, 99% identical or completely identical to amino acids X1-249 of SEQ ID NO:1, where amino acid X1 is selected from the group of residues 75-115 of SEQ ID NO:1, e.g., X1 is residue Arg 93 of SEQ ID NO:1) and proteins encoded by a nucleic acid that hybridizes under high stringency conditions to a human, mouse, rat, or Pan troglodytes gene encoding a naturally occurring TWEAK protein. Preferably, a TWEAK protein, in its processed mature form, is capable of providing at least one TWEAK activity, e.g., ability to activate Fn14.
Exemplary Fn14 protein molecules include human Fn14 (e.g., NP—057723.1, shown as SEQ ID NO:2), mouse Fn14 (e.g., NP—038777.1), and rat Fn14 (e.g., NP 851600.1) as well as soluble proteins that include an amino acid sequence at least 90, 92, 95, 97, 98, 99% identical or completely identical to the extracellular domain of Fn14 (and TWEAK-binding fragments thereof) and proteins encoded by a nucleic acid that hybridizes under high stringency conditions to a human, mouse, rat, or Pan troglodytes gene encoding a naturally occurring Fn14 protein. Preferably, a Fn14 protein useful in the methods described herein is a soluble Fn14 (lacking a transmembrane domain) that includes a region that binds to a TWEAK protein, e.g., an amino acid sequence at least 90, 92, 95, 97, 98, or 99% identical, or completely identical, to amino acids 28-X1 of SEQ ID NO:2, where amino acid X1 is selected from the group of residues 68 to 80 of SEQ ID NO:2.
Calculations of “homology” or “sequence identity” between two sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences. Alignments of related proteins described herein are instructive for identifying amino acid positions that tolerate modification, e.g., insertion, deletion, and substitution, e.g., conservative or non-conservative substitution.
As used herein, the term “hybridizes under high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and nonaqueous methods are described in that reference and either can be used. High stringency hybridization conditions include hybridization in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C., or substantially similar conditions.
Exemplary TWEAK blocking agents include antibodies that bind to TWEAK or TWEAK-R and soluble forms of the TWEAK-R that compete with cell surface TWEAK-R for binding to TWEAK. An example of a soluble form of the TWEAK-R is an Fc fusion protein that includes at least a portion of the extracellular domain of TWEAK-R (e.g., a soluble TWEAK-binding fragment of TWEAK-R), referred to as TWEAK-R-Fc. Other soluble forms of TWEAK-R, e.g., forms that do not include an Fc domain, can also be used. Antibody blocking agents are further discussed below.
Other types of blocking agents, e.g., small molecules, nucleic acid or nucleic acid-based aptamers, and peptides, can be isolated by screening, e.g., as described in Jhaveri et al. Nat. Biotechnol. 18:1293 and U.S. Pat. No. 5,223,409. Exemplary assays for determining if an agent binds to TWEAK or TWEAK-R and for determining if an agent modulates a TWEAK/TWEAK-R interaction are described, e.g., in U.S. 2004-0033225.
An exemplary soluble form of the TWEAK-R protein includes a region of the TWEAK-R protein that binds to TWEAK, e.g., about amino acids 32-75, 31-75, 31-78, or 28-79 of SEQ ID NO:2. This region can be physically associated, e.g., fused to another amino acid sequence, e.g., an Fc domain, at its N- or C-terminus. The region from TWEAK-R can be spaced by a linker from the heterologous amino acid sequence. U.S. Pat. No. 6,824,773 describes an exemplary TWEAK-R fusion protein.
TNF-α Blocking Agents
A variety of agents can be used as a TNF-α blocking agent. The agent may be any type of compound (e.g., small organic or inorganic molecule, nucleic acid, protein, or peptide mimetic) that can be administered to a subject. In one embodiment, the blocking agent is a biologic, e.g., a protein having a molecular weight of between 5-300 kDa. For example, a TNF-α blocking agent may inhibit binding of TNF-α to a TNF-α receptor or otherwise prevent TNF-α receptor downstream signalling. A typical TNF-α blocking agent can bind to TNF-α or a TNF-α receptor, e.g., TNFR-I or TNFR-II. A TNF-α blocking agent that binds to TNF-α or a TNF-α receptor may alter the conformation of TNF-α or a TNF-α receptor, block the binding site on TNF-α or a TNF-α receptor, or otherwise decrease the affinity of TNF-α for a TNF-α receptor or prevent the interaction between TNF-α and a TNF-α receptor.
A TNF-α blocking agent (e.g., an antibody) may bind to TNF-α or to a TNF-α receptor with a Kd of less than 10−6, 10−7, 10−8, 10−9, or 10−10 M. In one embodiment, the blocking agent binds to TNF-α with an affinity at least 5, 10, 20, 50, 100, 200, 500, or 1000-fold better than its affinity for TWEAK or another TNF superfamily member (other than TNF-α). A preferred TNF-α blocking agent specifically binds TNF-α or a TNF-α-R, such as a TNF-α or TNF-α-R specific antibody.
Exemplary TNF-α blocking agents include antibodies that bind to TNF-α or TNF-α-R and soluble forms of the TNF-α-R that compete with cell surface TNF-α-R for binding to TNF-α. An example of a soluble form of the TNF-α-R is an Fc fusion protein that includes at least a portion of the extracellular domain of TNF-α-R (e.g., a soluble TNF-α-binding fragment of TNF-α-R), referred to as TNF-α-R-Fc. Other soluble forms of TNF-α-R, e.g., forms that do not include an Fc domain, can also be used. Antibody blocking agents are further discussed below.
An exemplary soluble form of a TNF-α receptor protein is ENBREL®. See e.g., Arthritis & Rheumatism (1994) Vol. 37, 5295; J. Invest. Med. (1996) Vol. 44, 235A. U.S. Pat. No. 6,572,852 describes additional examples. The recommended dose of ENBREL® for adult patients with rheumatoid arthritis, psoriatic arthritis, or ankylosing spondylitis is 50 mg per week given as one subcutaneous (SC) injection using a 50 mg/mL single-use prefilled syringe. In addition to ENBREL®, other similar and/or corresponding regions of TNF-α receptors can be physically associated, e.g., fused to another amino acid sequence, e.g., an Fc domain, at its N- or C-terminus.
Other well characterized examples of TNF-α blocking agents include: infliximab (REMICADE®), a chimeric antibody that binds to tumor necrosis factor-alpha (TNF-α) and adalimumab (HUMIRA®), a human antibody that binds to TNF-α. For example, the recommended dose of REMICADE® is 3 mg/kg given as an intravenous infusion followed with additional similar doses at 2 and 6 weeks after the first infusion then every 8 weeks thereafter.
Additional examples of TNF-α blocking agents include chimeric, humanized, human or in vitro generated antibodies (or antigen-binding fragments thereof) to TNF (e.g., human TNF-α), such as D2E7, (human TNF-α antibody, U.S. Pat. No. 6,258,562; BASF), CDP-571/CDP-870/BAY-10-3356 (humanized anti-TNF-α antibody; Celltech/F′ harmacia), cA2 (chimeric anti-TNFα antibody; REMICADE™, Centocor, also mentioned above); anti-TNF antibody fragments (e.g., CPD870); soluble fragments of the TNF receptors, e.g., p55 or p75 human TNF receptors or derivatives thereof, e.g., 75 kd TNFR-IgG (75 kD TNF receptor-IgG fusion protein, ENBREL™), p55 kd TNFR-IgG (55 kD TNF receptor-IgG fusion protein (LENERCEPT™)); enzyme antagonists, e.g., TNFα converting enzyme (TACE) inhibitors (e.g., an alpha-sulfonyl hydroxamic acid derivative, PCT Application WO 01/55112, and N-hydroxyformamide TACE inhibitor GW 3333, -005, or -022); and TNF-bp/s-TNFR (soluble TNF binding protein; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S284; Amer. J. Physiol.—Heart and Circulatory Physiology (1995) Vol. 268, pp. 37-42).
Antibodies
Exemplary TWEAK blocking agents include antibodies that bind to TWEAK and/or TWEAK-R. In one embodiment, the antibody inhibits the interaction between TWEAK and a TWEAK receptor, e.g., by physically blocking the interaction, decreasing the affinity of TWEAK and/or TWEAK-R for its counterpart, disrupting or destabilizing TWEAK complexes, sequestering TWEAK or a TWEAK-R, or targeting TWEAK or TWEAK-R for degradation. In one embodiment, the antibody can bind to TWEAK or TWEAK-R at one or more amino acid residues that participate in the binding interface between TWEAK and its receptor. Such amino acid residues can be identified, e.g., by alanine scanning. In another embodiment, the antibody can bind to residues that do not participate in the binding interface. For example, the antibody can alter a conformation of TWEAK or TWEAK-R and thereby reduce binding affinity, or the antibody may sterically hinder binding. In one embodiment, the antibody can prevent activation of a TWEAK-R mediated event or activity (e.g., NF-κB activation).
Similarly, exemplary TNF-α blocking agents include antibodies that bind to TNF-α and/or a TNF-α receptor, e.g., TNFR-I or TNFR-II. In one embodiment, the antibody inhibits the interaction between TNF-α and a TNF-α receptor, e.g., by physically blocking the interaction, decreasing the affinity of TNF-α and/or TNF-α-R for its counterpart, disrupting or destabilizing TNF-α complexes, sequestering TNF-α or a TNF-α receptor, or targeting TNF-α or TNF-α receptor for degradation. In one embodiment, the antibody can bind to TNF-α or TNF-α receptor at one or more amino acid residues that participate in the TNF-α/TNF-α receptor binding interface. Such amino acid residues can be identified, e.g., by alanine scanning. In another embodiment, the antibody can bind to residues that do not participate in the TNF-α/TNF-α receptor binding. For example, the antibody can alter a conformation of TNF-α or TNF-α receptor and thereby reduce binding affinity, or the antibody may sterically hinder TNF-α/TNF-α receptor binding.
As used herein, the term “antibody” refers to a protein that includes at least one immunoglobulin variable region, e.g., an amino acid sequence that provides an immunoglobulin variable domain or an immunoglobulin variable domain sequence. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term “antibody” encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab fragments, F(ab′)2 fragments, Fd fragments, Fv fragments, and dAb fragments) as well as complete antibodies, e.g., intact and/or full length immunoglobulins of types IgA, IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgE, IgD, IgM (as well as subtypes thereof). The light chains of the immunoglobulin may be of types kappa or lambda. In one embodiment, the antibody is glycosylated. An antibody can be functional for antibody-dependent cytotoxicity and/or complement-mediated cytotoxicity, or may be non-functional for one or both of these activities.
The VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (FR). The extent of the FR's and CDR's has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). Kabat definitions are used herein. Each VH and VL is typically composed of three CDR's and four FR's, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
An “immunoglobulin domain” refers to a domain from the variable or constant domain of immunoglobulin molecules. Immunoglobulin domains typically contain two β-sheets formed of about seven β-strands, and a conserved disulphide bond (see, e.g., A. F. Williams and A. N. Barclay (1988) Ann. Rev Immunol. 6:381-405). An “immunoglobulin variable domain sequence” refers to an amino acid sequence that can form a structure sufficient to position CDR sequences in a conformation suitable for antigen binding. For example, the sequence may include all or part of the amino acid sequence of a naturally-occurring variable domain. For example, the sequence may omit one, two, or more N- or C-terminal amino acids, internal amino acids, may include one or more insertions or additional terminal amino acids, or may include other alterations. In one embodiment, a polypeptide that includes an immunoglobulin variable domain sequence can associate with another immunoglobulin variable domain sequence to form a target binding structure (or “antigen binding site”), e.g., a structure that interacts with a target protein, e.g., TWEAK, a TWEAK receptor, TNF-α, TNFR-I, or TNFR-II.
The VH or VL chain of the antibody can further include all or part of a heavy or light chain constant region, to thereby form a heavy or light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains. The heavy and light immunoglobulin chains can be connected by disulfide bonds. The heavy chain constant region typically includes three constant domains, CH1, CH2, and CH3. The light chain constant region typically includes a CL domain. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
One or more regions of an antibody can be human, effectively human, or humanized. For example, one or more of the variable regions can be human or effectively human. For example, one or more of the CDRs, e.g., HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3, can be human. Each of the light chain CDRs can be human. HC CDR3 can be human. One or more of the framework regions can be human, e.g., FR1, FR2, FR3, and FR4 of the HC or LC. In one embodiment, all the framework regions are human, e.g., derived from a human somatic cell, e.g., a hematopoietic cell that produces immunoglobulins or a non-hematopoietic cell. In one embodiment, the human sequences are germline sequences, e.g., encoded by a germline nucleic acid. One or more of the constant regions can be human, effectively human, or humanized. In another embodiment, at least 70, 75, 80, 85, 90, 92, 95, or 98% of the framework regions (e.g., FR1, FR2, and FR3, collectively, or FR1, FR2, FR3, and FR4, collectively) or the entire antibody can be human, effectively human, or humanized. For example, FR1, FR2, and FR3 collectively can be at least 70, 75, 80, 85, 90, 92, 95, 98, or 99% identical, or completely identical, to a human sequence encoded by a human germline segment.
An “effectively human” immunoglobulin variable region is an immunoglobulin variable region that includes a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human. An “effectively human” antibody is an antibody that includes a sufficient number of human amino acid positions such that the antibody does not elicit an immunogenic response in a normal human.
A “humanized” immunoglobulin variable region is an immunoglobulin variable region that is modified such that the modified form elicits less of an immune response in a human than does the non-modified form, e.g., is modified to include a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human. Descriptions of “humanized” immunoglobulins include, for example, U.S. Pat. Nos. 6,407,213 and 5,693,762. In some cases, humanized immunoglobulins can include a non-human amino acid at one or more framework amino acid positions.
Antibody Generation
Antibodies that bind to a target protein (e.g., TWEAK, TWEAK-R, TNF-α, TNFR-I or TNFR-II) can be generated by a variety of means, including immunization, e.g., using an animal, or in vitro methods such as phage display. All or part of the target protein can be used as an immunogen or as a target for selection. In one embodiment, the immunized animal contains immunoglobulin producing cells with natural, human, or partially human immunoglobulin loci. In one embodiment, the non-human animal includes at least a part of a human immunoglobulin gene. For example, it is possible to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci. Using the hybridoma technology, antigen-specific monoclonal antibodies derived from the genes with the desired specificity may be produced and selected. See, e.g., XENOMOUSE™, Green et al. (1994) Nat. Gen. 7:13-21; U.S. 2003-0070185; U.S. Pat. No. 5,789,650; and PCT Application WO 96/34096.
Non-human antibodies to the target proteins can also be produced, e.g., in a rodent. The non-human antibody can be humanized, e.g., as described in EP 239 400; U.S. Pat. Nos. 6,602,503; 5,693,761; and 6,407,213, deimmunized, or otherwise modified to make it effectively human.
EP 239 400 (Winter et al.) describes altering antibodies by substitution (within a given variable region) of their complementarity determining regions (CDRs) for one species with those from another. Typically, CDRs of a non-human (e.g., murine) antibody are substituted into the corresponding regions in a human antibody by using recombinant nucleic acid technology to produce sequences encoding the desired substituted antibody. Human constant region gene segments of the desired isotype (usually gamma I for CH and kappa for CL) can be added and the humanized heavy and light chain genes can be co-expressed in mammalian cells to produce soluble humanized antibody.
Other methods for humanizing antibodies can also be used. For example, other methods can account for the three dimensional structure of the antibody, framework positions that are in three dimensional proximity to binding determinants, and immunogenic peptide sequences. See, e.g., PCT Application WO 90/07861; U.S. Pat. Nos. 5,693,762; 5,693,761; 5,585,089; and 5,530,101; Tempest et al. (1991) Biotechnology 9:266-271 and U.S. Pat. No. 6,407,213. Still another method is termed “humaneering” and is described, for example, in U.S. 2005-008625.
Fully human monoclonal antibodies that bind to target proteins can be produced, e.g., using in vitro-primed human splenocytes, as described by Boerner et al. (1991) J. Immunol. 147:86-95. They may be prepared by repertoire cloning as described by Persson et al. (1991) Proc. Nat. Acad. Sci. USA 88:2432-2436 or by Huang and Stollar (1991) J. Immunol. Methods 141:227-236; also U.S. Pat. No. 5,798,230. Large non-immunized human phage display libraries may also be used to isolate high affinity antibodies that can be developed as human therapeutics using standard phage technology (see, e.g., Hoogenboom et al. (1998) Immunotechnology 4:1-20; Hoogenboom et al. (2000) Immunol Today 2:371-378; and U.S. 2003-0232333).
Antibody and Protein Production
Antibodies and other proteins described herein can be produced in prokaryotic and eukaryotic cells. In one embodiment, the antibodies (e.g., scFv's) are expressed in a yeast cell such as Pichia (see, e.g., Powers et al. (2001) J. Immunol. Methods 251:123-35), Hanseula, or Saccharomyces.
Antibodies, particularly full length antibodies, e.g., IgG's, can be produced in mammalian cells. Exemplary mammalian host cells for recombinant expression include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621), lymphocytic cell lines, e.g., NS0 myeloma cells and SP2 cells, COS cells, K562, and a cell from a transgenic animal, e.g., a transgenic mammal. For example, the cell is a mammary epithelial cell.
In addition to the nucleic acid sequence encoding the immunoglobulin domain, the recombinant expression vectors may carry additional nucleic acid sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216; 4,634,665; and 5,179,017). Exemplary selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr− host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
In an exemplary system for recombinant expression of an antibody (e.g., a full length antibody or an antigen-binding portion thereof), 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 enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLP promoter regulatory element or an SV40 enhancer/AdMLP promoter regulatory element) 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, to transfect the host cells, to select for transformants, to culture the host cells, and to recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G.
Antibodies (and Fc fusions) may also include modifications, e.g., modifications that alter Fc function, e.g., to decrease or remove interaction with an Fc receptor or with C1q, or both. For example, the human IgG1 constant region can be mutated at one or more residues, e.g., one or more of residues 234 and 237, e.g., according to the numbering in U.S. Pat. No. 5,648,260. Other exemplary modifications include those described in U.S. Pat. No. 5,648,260.
For some proteins that include an Fc domain, the antibody/protein production system may be designed to synthesize antibodies or other proteins in which the Fc region is glycosylated. For example, the Fc domain of IgG molecules is glycosylated at asparagine 297 in the CH2 domain. The Fc domain can also include other eukaryotic post-translational modifications. In other cases, the protein is produced in a form that is not glycosylated.
Antibodies and other proteins can also be produced by a transgenic animal. For example, U.S. Pat. No. 5,849,992 describes a method for expressing an antibody in the mammary gland of a transgenic mammal. A transgene is constructed that includes a milk-specific promoter and nucleic acid sequences encoding the antibody of interest, e.g., an antibody described herein, and a signal sequence for secretion. The milk produced by females of such transgenic mammals includes, secreted-therein, the protein of interest, e.g., an antibody or Fc fusion protein. The protein can be purified from the milk, or for some applications, used directly.
Methods described in the context of antibodies can be adapted to other proteins, e.g., Fc fusions and soluble receptor fragments.
Nucleic Acid Blocking Agents
In certain implementations, nucleic acid blocking agents are used to decrease expression of a target protein such as TWEAK, a TWEAK-R (e.g., Fn14), TNF-α, TNFR-I or TNFR-II. These agents can be used in place of or in addition to proteinaceous TWEAK blocking agents and TNF-α blocking agents. In one embodiment, the nucleic acid antagonist is an siRNA that is directed against the mRNA produced from an endogenous gene that encodes the target protein. For example, the siRNA includes a region complementary to the mRNA. Other types of antagonistic nucleic acids can also be used, e.g., a dsRNA, a ribozyme, a triple-helix former, or an antisense nucleic acid.
siRNAs are small double stranded RNAs (dsRNAs) that optionally include overhangs. For example, the duplex region of an siRNA is about 18 to 25 nucleotides in length, e.g., about 19, 20, 21, 22, 23, or 24 nucleotides in length. Typically, the siRNA sequences are exactly complementary to the target mRNA. dsRNAs and siRNAs in particular can be used to silence gene expression in mammalian cells (e.g., human cells). See, e.g., Clemens et al. (2000) Proc. Natl. Acad. Sci. USA 97:6499-6503; Billy et al. (2001) Proc. Natl. Sci. USA 98:14428-14433; Elbashir et al. (2001) Nature. 411:494-8; Yang et al. (2002) Proc. Natl. Acad. Sci. USA 99:9942-9947, U.S. Pub. Apps. 2003-0166282; 2003-0143204; 2004-0038278; and 2003-0224432.
Anti-sense agents can include, for example, from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 nucleotides), e.g., about 8 to about 50 nucleobases, or about 12 to about 30 nucleobases. Anti-sense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression. Anti-sense compounds can include a stretch of at least eight consecutive nucleobases that are complementary to a sequence in the target gene. An oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted.
Hybridization of antisense oligonucleotides with mRNA (e.g., an mRNA encoding a target protein) can interfere with one or more of the normal functions of mRNA. The functions of mRNA to be interfered with include all key functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in by the RNA. Binding of specific protein(s) to the RNA may also be interfered with by antisense oligonucleotide hybridization to the RNA.
Exemplary antisense compounds include DNA or RNA sequences that specifically hybridize to the target nucleic acid, e.g., the mRNA encoding a target protein. The complementary region can extend for between about 8 to about 80 nucleobases. The compounds can include one or more modified nucleobases. Modified nucleobases may include, e.g., 5-substituted pyrimidines such as 5-iodouracil, 5-iodocytosine, and C5-propynyl pyrimidines such as C5-propynylcytosine and C5-propynyluracil, to mention but a few. Descriptions of a variety of nucleic acid agents are available. See, e.g., U.S. Pat. Nos. 4,987,071; 5,116,742; and 5,093,246; Woolf et al. (1992) Proc Natl Acad Sci USA; Antisense RNA and DNA, D. A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988); 89:7305-9; Haselhoff and Gerlach (1988) Nature 334:585-59; Helene, C. (1991) Anticancer Drug Des. 6:569-84; Helene (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14:807-15.
The nucleic acids described herein, e.g., an anti-sense nucleic acid described herein, can be incorporated into a gene construct to be used as a part of a gene therapy protocol to deliver nucleic acids that can be used to express and produce agents, e.g., anti-sense nucleic acids within cells. Expression constructs of such components may be administered in any biologically effective carrier, e.g. any formulation or composition capable of effectively delivering the component gene to cells in vivo. Approaches include insertion of the subject gene in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, lentivirus, and herpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids. Viral vectors transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized (e.g. antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO4 precipitation carried out in vivo.
A preferred approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing nucleic acid, e.g. a cDNA. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid.
Retrovirus vectors and adeno-associated virus vectors can be used as a recombinant gene delivery system for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in to vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are known to those skilled in the art. Examples of suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include *Crip, *Cre, *2 and *Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S. Pat. Nos. 4,868,116 and 4,980,286; PCT Applications WO 89/07136; WO 89/02468; WO 89/05345; and WO 92/07573).
Another viral gene delivery system utilizes adenovirus-derived vectors. See, for example, Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are known to those skilled in the art.
Yet another viral vector system useful for delivery of the subject gene is the adeno-associated virus (AAV). See, for example, Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al. (1989)J. Virol. 62:1963-1973).
Artificial Transcription Factors
Artificial transcription factors can also be used to regulate expression of a target protein, e.g., TWEAK, a TWEAK-R (e.g., Fn14), TNF-α, TNFR-I or TNFR-II. The artificial transcription factor can be designed or selected from a library, e.g., for ability to bind to a sequence in an endogenous gene encoding target protein, e.g., in a regulatory region, e.g., the promoter. For example, the artificial transcription factor can be prepared by selection in vitro (e.g., using phage display, U.S. Pat. No. 6,534,261) or in vivo, or by design based on a recognition code (see, e.g., PCT Application WO 00/42219 and U.S. Pat. No. 6,511,808). See, e.g., Rebar et al. (1996) Methods Enzymol 267:129; Greisman and Pabo (1997) Science 275:657; Isalan et al. (2001) Nat. Biotechnol 19:656; and Wu et al. (1995) Proc. Natl. Acad. Sci. USA 92:344 for, among other things, methods for creating libraries of varied zinc finger domains.
Optionally, an artificial transcription factor can be fused to a transcriptional regulatory domain, e.g., an activation domain to activate transcription or a repression domain to repress transcription. In particular, repression domains can be used to decrease expression of endogenous genes encoding TWEAK or TWEAK-R. The artificial transcription factor can itself be encoded by a heterologous nucleic acid that is delivered to a cell or the protein itself can be delivered to a cell (see, e.g., U.S. Pat. No. 6,534,261). The heterologous nucleic acid that includes a sequence encoding the artificial transcription factor can be operably linked to an inducible promoter, e.g., to enable fine control of the level of the artificial transcription factor in the cell.
Rheumatoid Arthritis (RA)
Rheumatoid arthritis (“RA”) is a chronic inflammatory disease that causes pain, swelling, stiffness, and loss of function, primarily in joints. RA frequently begins in the synovium, the membrane that surrounds a joint creating a protective sac. In many individuals suffering from RA, leukocytes infiltrate from the circulation into the synovium causing continuous abnormal inflammation (e.g., synovitis). Consequently, the synovium becomes inflamed, causing warmth, redness, swelling, and pain. The collagen in the cartilage is gradually destroyed, narrowing the joint space and eventually damaging bone. The inflammation causes erosive bone damage in the affected area. During this process, the cells of the synovium grow and divide abnormally, making the normally thin synovium thick and resulting in a joint that is swollen and puffy to the touch.
As RA progresses, abnormal synovial cells can invade and destroy the cartilage and bone within the joint. The surrounding muscles, ligaments, and tendons that support and stabilize the joint can become weak and unable to work normally. RA also may cause more generalized bone loss that may lead to osteoporosis, making bones fragile and more prone to fracture. All of these effects cause the pain, impairment and deformities associated with RA. Regions that can be effected include the wrists, knuckles, knees and the ball of the foot. Often, many joints may be involved, and even the spine can be affected. In about 25% of people with RA, inflammation of small blood vessels can cause rheumatoid nodules, or lumps, under the skin. These are bumps under the skin that often form close to the joints. As the disease progresses, fluid may also accumulate, particularly in the ankles. Many patients with RA also develop anemia, or a decrease in the normal number of red blood cells.
RA encompasses a number of disease subtypes, such as Felty's syndrome, seronegative RA, “classical” RA, progressive and/or relapsing RA, and RA with vasculitis. Some experts classify the disease into type 1 or type 2. Type 1, the less common form, lasts a few months at most and leaves no permanent disability. Type 2 is chronic and lasts for years, sometimes for life. RA can also manifest as subcutaneous rheumatoid nodules, visceral nodules, vasculitis causing leg ulcers or mononeuritis multiplex, pleural or pericardial effusions, lymphadenopathy, Felty's syndrome, Sjogren's syndrome, and episcleritis. These disease subtypes and also subjects showing one or more of the above symptoms can be treated using the methods described herein.
RA occurs across all races and ethnic groups. At least one genetic predisposition has been identified and, in white populations, localized to a pentapeptide in the HLA-DR 1 locus of class II histocompatibility genes.
RA can be assessed by a variety of clinical measures. Some exemplary indicia include the total Sharp score (TSS), Sharp erosion score, and the HAQ disability index. The methods herein can be used to achieve an improvement for at least one of these indicia.
Non-Responders to TNF-α Blocking agents
In one aspect, subjects who have rheumatoid arthritis, or who are at risk for RA, or who have or at risk for another disorder described herein, can be evaluated for a parameter predictive of their ability to respond to a particular agent (e.g., a biologic DMARD), e.g., their ability to respond to a TNF-α blocking agent such as etanercept, infliximab, or adalimumab. For example, the parameter can be the presence or absence of a nucleotide in a gene encoding TNF-α. Subjects who are indicated to be less or non-responsive to a particular agent can be administered an alternative agent. For example, subjects who are indicated as non-responsive to etanercept can be administered a TWEAK blocking agent.
Rheumatoid arthritis patients with the T allele of TNFA-857C/T SNP may respond better to etanercept therapy than homozygotes for the C allele. Kang et al. Rheumatology 2005 April; 44(4):547-52. Accordingly, RA patients that are homozygous for the C allele can be treated with a TWEAK blocking agent, and etanercept or other TNF-α blocking agent can be withheld, or dosages can be reduced, e.g., relative to a standard dose.
Non-Responders to RA Therapies
A variety of treatments for RA, in addition to TNF-α blocking agents, are available. Many of these are therapeutics classified as disease modifying anti-rheumatic drugs (DMARDs). Traditional DMARDS include PLAQUENIL® (hydroxychloroquine), AZULFIDINE® (sulfasalazine) or RHEUMATREX® (methotrexate). For rheumatoid arthritis, it has been observed that the withdrawal rate from DMARD treatment in rheumatoid arthritis increases with the length of time the patient has been receiving the drug and that a number of these withdrawals relate to loss of efficacy (see, e.g., Annals of the Rheumatic Diseases (2003) 62:95-96). Accordingly, a TWEAK blocking agent can also be administered to a subject who has an inadequate response to a DMARD treatment, e.g., an inadequate response to treatment with one of the following agents:
a. Nonsteroidal anti-inflammatory drugs including salicylates, such as aspirin.
b. Gold compounds. In some patients, gold may produce clinical remission and decrease the formation of new bony erosions. Parenteral preparations include gold sodium thiomalate or gold thioglucose. Gold should be discontinued when signs of toxicity appear. Minor toxic manifestations (e.g., mild pruritus, minor rash) may be eliminated by temporarily withholding gold therapy, then resuming it cautiously about 2 weeks after symptoms have subsided. However, if toxic symptoms progress, gold should be withheld. A TWEAK blocking agent can be administered when gold is being discontinued or when a gold chelating drug (such as dimercaprol) is being administered to counteract gold toxicity.
c. Hydroxychloroquine can also control symptoms of mild or moderately active RA. Toxic effects usually are mild and include dermatitis, myopathy, and generally reversible corneal opacity. However, irreversible retinal degeneration has been reported. Hydroxychloroquine can be withdrawn and replaced, e.g., with a TWEAK blocking agent, e.g., upon detection of one or more of these side effects.
d. Oral penicillamine may have a benefit similar to gold. Side effects requiring discontinuation are more common than with gold and include marrow suppression, proteinuria, nephrosis, other serious toxic effects (e.g., myasthenia gravis, pemphigus, Goodpasture's syndrome, polymyositis, a lupus-like syndrome), rash, and a foul taste. Oral penicillamine can be withdrawn and replaced, e.g., with a TWEAK blocking agent, e.g., upon detection of one or more of these side effects.
e. Steroids are highly effective short-term anti-inflammatory drugs. However, their clinical benefit for RA often diminishes with time. Steroids do not predictably prevent the progression of joint destruction. Furthermore, severe rebound often follows the withdrawal of corticosteroids in active disease. Accordingly, a TWEAK blocking agent can be administered, prior to withdrawal, during withdrawal, or subsequent to complete withdrawal. Other side effect which can trigger withdrawal and use of a TWEAK blocking agent include peptic ulcer, hypertension, untreated infections, diabetes mellitus, and glaucoma.
f. Immunosuppressive drugs can be used in management of severe, active RA. However, major side effects can occur, including liver disease, pneumonitis, bone marrow suppression, and, after long-term use of azathioprine, malignancy. Withdrawal from immunosuppressive drugs can include administering a TWEAK blocking agent, e.g., upon detection of a side effect.
Alternatively, a TWEAK blocking agent can be administered to a subject who is receiving another treatment for RA, e.g., one of the above treatments. The combination of the treatment and the TWEAK blocking agent can be used to achieve additional therapeutic benefit and, optionally, to reduce the dosage of the other treatment. As result, side effects and other issues can be mitigated.
The methods described herein, e.g., a TWEAK blocking agent monotherapy or a combination therapy (such as with TWEAK and TNF-α blocking agents), can be used to treat a subject who has one or more severe complications of RA. Such complications include joint destruction, gastrointestinal bleeding, heart failure, pericarditis, pleuritis, lung disease, anemia, low or high platelets, eye disease, cervical (neck) spine instability, neuropathy, and vasculitis.
Other Disorders
The methods described herein can also be used to treat other inflammatory, immune, or autoimmune disorders in patients, for example disorders that are not exacerbated by administration of a TNF-α blocking agent. Examples of disorders that can be treated include psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease (including ulcerative colitis and Crohn's disease), psoriasis, or inflammatory myositis. Still other examples of inflammatory disorders include Langerhans-cell histiocytosis, adult respiratory distress syndrome/bronchiolitis obliterans, Wegener's granulomatosis, vasculitis, cachexia, stomatitis, idiopathic pulmonary fibrosis, dermatomyositis or polymyositis, non-infectious scleritis, chronic sarcoidosis with pulmonary involvement, myelodysplastic syndromes/refractory anemia with excess blasts, ulcerative colitis, moderate to severe chronic obstructive pulmonary disease, and giant cell arteritis.
A subject who is at risk for, diagnosed with, or who has one of these disorders can be administered a TWEAK blocking agent in an amount and for a time to provide an overall therapeutic effect. The TWEAK blocking agent can be administered in combination with a TNF-α blocking agent or without providing (e.g., withholding) the TNF-α blocking agent. In the case of a combination therapy, the amounts and times of administration can be those that provide, e.g., an enhanced or synergistic therapeutic effect. Further, the administration of the TWEAK blocking agent (with or without the TNF-α blocking agent) can be used as a primary, e.g., first line treatment, or as a secondary treatment, e.g., for subjects who have an inadequate response to a previously administered therapy (i.e., a therapy other than one with a TWEAK block agent).
Pharmaceutical Compositions
A TWEAK blocking agent (e.g., an antibody or soluble TWEAK-R protein, e.g., TWEAK-R-Fc) can be formulated as a pharmaceutical composition, e.g., for administration to a subject to treat a disorder described herein, e.g., an inflammatory disorder such as rheumatoid arthritis or other disorder described herein. A TNF-α blocking agent can be similarly formulated, either in the same composition or as a separate composition.
Typically, a pharmaceutical composition includes a pharmaceutically acceptable carrier. As used herein, “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. The composition can include a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt (see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19).
Pharmaceutical formulation is a well-established art, and is further described, e.g., in Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins (2000) (ISBN: 0683306472); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed., Lippincott Williams & Wilkins Publishers (1999) (ISBN: 0683305727); and Kibbe (ed.), Handbook of Pharmaceutical Excipients American Pharmaceutical Association, 3rd ed. (2000) (ISBN: 091733096X).
In one embodiment, the TWEAK blocking agent (e.g., an antibody or TWEAK-R-Fc) and/or the TNF-α blocking agent is formulated with excipient materials, such as sodium chloride, sodium dibasic phosphate heptahydrate, sodium monobasic phosphate, and a stabilizer. It can be provided, for example, in a buffered solution at a suitable concentration and can be stored at 2-8° C.
The pharmaceutical compositions 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 preferred form can depend on the intended mode of administration and therapeutic application. Typically compositions for the agents described herein are in the form of injectable or infusible solutions.
Such compositions can be administered by a parenteral mode (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). The phrases “parenteral administration” and “administered parenterally” as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and infrasternal injection and infusion.
The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration. Sterile injectable solutions can be prepared by incorporating an agent described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating an agent described herein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yield a powder of an agent described herein 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, for example, monostearate salts and gelatin.
In certain embodiments, the TWEAK blocking agent and/or the TNF-α blocking agent may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, 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 patented or generally known. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
A TWEAK blocking agent (e.g., an antibody or soluble TWEAK-R protein) can be modified, e.g., with a moiety that improves its stabilization and/or retention in circulation, e.g., in blood, serum, or other tissues, e.g., by at least 1.5, 2, 5, 10, or 50 fold. The modified blocking agent can be evaluated to assess whether it can reach sites of inflammation, e.g., joints.
For example, the TWEAK blocking agent (e.g., an antibody or soluble TWEAK-R protein) can be associated with (e.g., conjugated to) a polymer, e.g., a substantially non-antigenic polymer, such as a polyalkylene oxide or a polyethylene oxide. Suitable polymers will vary substantially by weight. Polymers having molecular number average weights ranging from about 200 to about 35,000 Daltons (or about 1,000 to about 15,000, and 2,000 to about 12,500) can be used.
For example, a TWEAK blocking agent or TNF-α blocking agent can be conjugated to a water soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g., polyvinylalcohol or polyvinylpyrrolidone. A non-limiting list of such polymers includes polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained. Additional useful polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene; polymethacrylates; carbomers; and branched or unbranched polysaccharides.
When the TWEAK blocking agent (e.g., an antibody or soluble TWEAK-R protein) is used in combination with a second agent (e.g., a TNF-α blocking agent or other agent described herein), the two agents can be formulated separately or together. For example, the respective pharmaceutical compositions can be mixed, e.g., just prior to administration, and administered together or can be administered separately, e.g., at the same or different times.
Other therapeutic agents described herein can also be provided as pharmaceutical composition, e.g., by standard methods or method described herein.
Administration
The TWEAK blocking agent (e.g., an antibody or soluble TWEAK-R protein) and a TNF-α blocking agent can be administered to a subject, e.g., a human subject, by a variety of methods. For many applications, the route of administration is one of: intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneally (IP), or intramuscular injection. It is also possible to use intra-articular delivery. In some cases, administration may be directly to a site of inflammation, e.g., a joint or other inflamed site. The blocking agent can be administered as a fixed dose, or in a mg/kg dose.
The dose can also be chosen to reduce or avoid production of antibodies against the TWEAK blocking agent.
The route and/or mode of administration of the blocking agent can also be tailored for the individual case, e.g., by monitoring the subject, e.g., using tomographic imaging, neurological exam, and standard parameters associated with the particular disorder, e.g., criteria for assessing rheumatoid arthritis.
Dosage regimens are adjusted to provide the desired response, e.g., a therapeutic response or a combinatorial therapeutic effect. Generally, any combination of doses (either separate or co-formulated) of the TWEAK blocking agent (e.g., an antibody) (and optionally a second agent, e.g., a TNF-α blocking agent) can be used in order to provide a subject with the agent in bioavailable quantities. For example, doses in the range of 0.1-100 mg/kg, 0.5-100 mg/kg, 1 mg/kg-100 mg/kg, 0.5-20 mg/kg, or 1-10 mg/kg can be administered. Other doses can also be used.
Dosage unit form or “fixed dose” as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and optionally in association with the other agent. Single or multiple dosages may be given. Alternatively, or in addition, the blocking agent may be administered via continuous infusion.
The TWEAK blocking agent can be administered, e.g., once or twice daily, or about one to four times per week, or preferably weekly, biweekly, or monthly, e.g., for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, formulation, route of delivery, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a compound can include a single treatment or, preferably, can include a series of treatments. Animal models can also be used to determine a useful dose, e.g., an initial dose or a regimen.
If a subject is at risk for developing an inflammatory disorder or other disorder described herein, the blocking agent can be administered before the full onset of the disorder, e.g., as a preventative measure. The duration of such preventative treatment can be a single dosage of the blocking agent or the treatment may continue (e.g., multiple dosages). For example, a subject at risk for the disorder or who has a predisposition for the disorder may be treated with the blocking agent for days, weeks, months, or even years so as to prevent the disorder from occurring or fulminating.
A pharmaceutical composition may include a “therapeutically effective amount” of an agent described herein. Such effective amounts can be determined based on the effect of the administered agent, or the combinatorial effect of agents if more than one agent is used. A therapeutically effective amount of an agent may also vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual, e.g., amelioration of at least one disorder parameter or amelioration of at least one symptom of the disorder. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.
Devices and Kits for Therapy
Pharmaceutical compositions that include the TWEAK blocking agent (e.g., an antibody or soluble TWEAK-R) can be administered with a medical device. The device can designed with features such as portability, room temperature storage, and ease of use so that it can be used in emergency situations, e.g., by an untrained subject or by emergency personnel in the field, removed to medical facilities and other medical equipment. The device can include, e.g., one or more housings for storing pharmaceutical preparations that include TWEAK blocking agent, and can be configured to deliver one or more unit doses of the blocking agent. The device can be further configured to administer a second agent, e.g., a TNF-α blocking agent, either as a single pharmaceutical composition that also includes the TWEAK blocking agent or as two separate pharmaceutical compositions.
For example, the pharmaceutical composition can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering agents through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. Many other devices, implants, delivery systems, and modules are also known.
A TWEAK blocking agent (e.g., an antibody or soluble TWEAK-R protein) can be provided in a kit. In one embodiment, the kit includes (a) a container that contains a composition that includes a TWEAK blocking agent, and optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the agents for therapeutic benefit.
In an embodiment, the kit also includes a second agent for treating an inflammatory disorder, e.g., a TNF-α blocking agent. For example, the kit includes a first container that contains a composition that includes the TWEAK blocking agent, and a second container that includes the second agent.
The informational material of the kits is not limited in its form. In one embodiment, the informational material can include information about production of the compound, molecular weight of the compound, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material relates to methods of administering the TWEAK blocking agent (e.g., an antibody or soluble TWEAK-R protein) and/or TNF-α blocking agent, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein), to treat a subject who has had or who is at risk for an inflammatory disorder, or other disorder described herein. The information can be provided in a variety of formats, include printed text, computer readable material, video recording, or audio recording, or information that provides a link or address to substantive material.
In addition to the blocking agent, the composition in the kit can include other ingredients, such as a solvent or buffer, a stabilizer, or a preservative. The blocking agent can be provided in any form, e.g., liquid, dried or lyophilized form, preferably substantially pure and/or sterile. When the agents are provided in a liquid solution, the liquid solution preferably is an aqueous solution. When the agents are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer, can optionally be provided in the kit.
The kit can include one or more containers for the composition or compositions containing the agents. In some embodiments, the kit contains separate containers, dividers or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of the agents. The containers can include a combination unit dosage, e.g., a unit that includes both the TWEAK blocking agent and the second agent, e.g., in a desired ratio. For example, the kit includes a plurality of syringes, ampules, foil packets, blister packs, or medical devices, e.g., each containing a single combination unit dose. The containers of the kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight.
The kit optionally includes a device suitable for administration of the composition, e.g., a syringe or other suitable delivery device. The device can be provided pre-loaded with one or both of the agents or can be empty, but suitable for loading.
Nucleic Acid and Protein Analysis
Numerous methods for detecting TWEAK or TWEAK-R protein and nucleic acid as well as proteins and nucleic acids for other biomarkers described herein (including those listed in Table 1) are available to the skilled artisan, including antibody-based methods for protein detection (e.g., Western blot or ELISA), and hybridization-based methods for nucleic acid detection (e.g., PCR or Northern blot).
Arrays are particularly useful molecular tools for characterizing a sample, e.g., a sample from a subject. For example, an array having capture probes for multiple genes, including probes for TWEAK and/or other biomarkers, or for multiple proteins, can be used in a method described herein. Altered expression of TWEAK (or other biomarker provided herein) nucleic acids and/or protein can be used to evaluate a sample, e.g., a sample from a subject, e.g., to evaluate a disorder described herein.
Arrays can have many addresses, e.g., locatable sites, on a substrate. The featured arrays can be configured in a variety of formats, non-limiting examples of which are described below. The substrate can be opaque, translucent, or transparent. The addresses can be distributed, on the substrate in one dimension, e.g., a linear array; in two dimensions, e.g., a planar array; or in three dimensions, e.g., a three dimensional array. The solid substrate may be of any convenient shape or form, e.g., square, rectangular, ovoid, or circular.
Arrays can be fabricated by a variety of methods, e.g., photolithographic methods (see, e.g., U.S. Pat. Nos. 5,143,854; 5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow methods as described in U.S. Pat. No. 5,384,261), pin based methods (e.g., as described in U.S. Pat. No. 5,288,514), and bead based techniques (e.g., as described in PCT US/93/04145).
The capture probe can be a single-stranded nucleic acid, a double-stranded nucleic acid (e.g., which is denatured prior to or during hybridization), or a nucleic acid having a single-stranded region and a double-stranded region. Preferably, the capture probe is single-stranded. The capture probe can be selected by a variety of criteria, and preferably is designed by a computer program with optimization parameters. The capture probe can be selected to hybridize to a sequence rich (e.g., non-homopolymeric) region of the gene. The Tm of the capture probe can be optimized by prudent selection of the complementarity region and length. Ideally, the Tm of all capture probes on the array is similar, e.g., within 20, 10, 5, 3, or 2° C. of one another.
The isolated nucleic acid is preferably mRNA that can be isolated by routine methods, e.g., including DNase treatment to remove genomic DNA and hybridization to an oligo-dT coupled solid substrate (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y). The substrate is washed, and the mRNA is eluted.
The isolated mRNA can be reversed transcribed and optionally amplified, e.g., by rtPCR, e.g., as described in (U.S. Pat. No. 4,683,202). The nucleic acid can be an amplification product, e.g., from PCR (U.S. Pat. Nos. 4,683,196 and 4,683,202); rolling circle amplification (“RCA,” U.S. Pat. No. 5,714,320), isothermal RNA amplification or NASBA (U.S. Pat. Nos. 5,130,238; 5,409,818; and 5,554,517), and strand displacement amplification (U.S. Pat. No. 5,455,166). The nucleic acid can be labeled during amplification, e.g., by the incorporation of a labeled nucleotide. Examples of preferred labels include fluorescent labels, e.g., red-fluorescent dye Cy5 (Amersham) or green-fluorescent dye Cy3 (Amersham), and chemiluminescent labels, e.g., as described in U.S. Pat. No. 4,277,437. Alternatively, the nucleic acid can be labeled with biotin, and detected after hybridization with labeled streptavidin, e.g., streptavidin-phycoerythrin (Molecular Probes).
The labeled nucleic acid can be contacted to the array. In addition, a control nucleic acid or a reference nucleic acid can be contacted to the same array. The control nucleic acid or reference nucleic acid can be labeled with a label other than the sample nucleic acid, e.g., one with a different emission maximum. Labeled nucleic acids can be contacted to an array under hybridization conditions. The array can be washed, and then imaged to detect fluorescence at each address of the array.
The expression level of a TWEAK or other biomarker can be determined using an antibody specific for the polypeptide (e.g., using a western blot or an ELISA assay). Moreover, the expression levels of multiple proteins, including TWEAK and the exemplary biomarkers provided herein, can be rapidly determined in parallel using a polypeptide array having antibody capture probes for each of the polypeptides. Antibodies specific for a polypeptide can be generated by a method described herein (see “Antibody Generation”). The expression level of a TWEAK and the exemplary biomarkers provided herein can be measured in a subject (e.g., in vivo imaging) or in a biological sample from a subject (e.g., blood, serum, plasma, or synovial fluid).
A low-density (96 well format) protein array has been developed in which proteins are spotted onto a nitrocellulose membrane (Ge (2000) Nucleic Acids Res. 28, e3, I-VII). A high-density protein array (100,000 samples within 222×222 mm) used for antibody screening was formed by spotting proteins onto polyvinylidene difluoride (PVDF) (Lueking et al. (1999) Anal. Biochem. 270:103-111). See also, e.g., Mendoza et al. (1999). Biotechniques 27:778-788; MacBeath and Schreiber (2000) Science 289:1760-1763; and De Wildt et al. (2000) Nature Biotech. 18:989-994. These art-known methods and other can be used to generate an array of antibodies for detecting the abundance of polypeptides in a sample. The sample can be labeled, e.g., biotinylated, for subsequent detection with streptavidin coupled to a fluorescent label. The array can then be scanned to measure binding at each address.
The nucleic acid and polypeptide arrays of the invention can be used in a wide variety of applications. For example, the arrays can be used to analyze a patient sample. The sample is compared to data obtained previously, e.g., known clinical specimens or other patient samples. Further, the arrays can be used to characterize a cell culture sample, e.g., to determine a cellular state after varying a parameter, e.g., exposing the cell culture to an antigen, a transgene, or a test compound.
The expression data can be stored in a database, e.g., a relational database such as a SQL database (e.g., Oracle or Sybase database environments). The database can have multiple tables. For example, raw expression data can be stored in one table, wherein each column corresponds to a gene being assayed, e.g., an address or an array, and each row corresponds to a sample. A separate table can store identifiers and sample information, e.g., the batch number of the array used, date, and other quality control information.
Expression profiles obtained from gene expression analysis on an array can be used to compare samples and/or cells in a variety of states as described in Golub et al. ((1999) Science 286:531). In one embodiment, expression (e.g., mRNA expression or protein expression) information for a gene encoding TWEAK and/or a gene encoding a exemplary biomarker provided herein are evaluated, e.g., by comparison a reference value, e.g., a reference value. Reference values can be obtained from a control, e.g., a reference subject. Reference values can also be obtained from statistical analysis, e.g., to provide a reference value for a cohort of subject, e.g., age and gender matched subject, e.g., normal subjects or subject who have rheumatoid arthritis or other disorder described herein. Statistical similarity to a particular reference (e.g., to a reference for a risk-associated cohort) or a normal cohort can be used to provide an assessment (e.g., an indication of risk of inflammatory disorder) to a subject, e.g., a subject who has not been diagnosed with a disorder described herein.
Subjects suitable for treatment can also be evaluated for expression and/or activity of TWEAK and/or other biomarker. Subjects can be identified as suitable for treatment (e.g., with a TWEAK blocking agent), if the expression and/or activity for TWEAK and/or the other biomarker is elevated relative to a reference, e.g., reference value, e.g., a reference value associated with normal.
Subjects who are being administered an agent described herein or other treatment can be evaluated as described for expression and/or activity of TWEAK and/or other biomarkers described herein. The subject can be evaluated at multiple times. e.g., at multiple times during a course of therapy, e.g., during a therapeutic regimen. Treatment of the subject can be modified depending on how the subject is responding to the therapy. For example, a reduction in TWEAK expression or activity or a reduction in the expression or activity of genes stimulated by TWEAK can be indicative of responsiveness.
Particular effects mediated by an agent may show a difference (e.g., relative to an untreated subject, control subject, or other reference) that is statistically significant (e.g., P value<0.05 or 0.02). Statistical significance can be determined by any art known method. Exemplary statistical tests include: the Students T-test, Mann Whitney U non-parametric test, and Wilcoxon non-parametric statistical test. Some statistically significant relationships have a P value of less than 0.05 or 0.02.
Methods of Evaluating Genetic Material
There are numerous methods for evaluating genetic material to provide genetic information. These methods can be used to evaluate a genetic locus that includes a gene encoding TWEAK or a gene encoding a biomarker described herein. The methods can be used to evaluate one or more nucleotides, e.g., a coding or non-coding region of the gene, e.g., in a regulatory region (e.g., a promoter, a region encoding an untranslated region or intron, and so forth).
Nucleic acid samples can analyzed using biophysical techniques (e.g., hybridization, electrophoresis, and so forth), sequencing, enzyme-based techniques, and combinations-thereof. For example, hybridization of sample nucleic acids to nucleic acid microarrays can be used to evaluate sequences in an mRNA population and to evaluate genetic polymorphisms. Other hybridization based techniques include sequence specific primer binding (e.g., PCR or LCR); Southern analysis of DNA, e.g., genomic DNA; Northern analysis of RNA, e.g., mRNA; fluorescent probe based techniques (see, e.g., Beaudet et al. (2001) Genome Res. 11(4):600-8); and allele specific amplification. Enzymatic techniques include restriction enzyme digestion; sequencing; and single base extension (SBE). These and other techniques are well known to those skilled in the art.
Electrophoretic techniques include capillary electrophoresis and Single-Strand Conformation Polymorphism (SSCP) detection (see, e.g., Myers et al. (1985) Nature 313:495-8 and Ganguly (2002) Hum Mutat. 19(4):334-42). Other biophysical methods include denaturing high pressure liquid chromatography (DHPLC).
In one embodiment, allele specific amplification technology that depends on selective PCR amplification may be used to obtain genetic information. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucl. Acids Res. 17:2437-2448) or at the extreme 3′ end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11:238). In addition, it is possible to introduce a restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). In another embodiment, amplification can be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3′ end of the 5′ sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
Enzymatic methods for detecting sequences include amplification based-methods such as the polymerase chain reaction (PCR; Saiki, et al. (1985) Science 230:1350-1354) and ligase chain reaction (LCR; Wu. et al. (1989) Genomics 4:560-569; Barringer et al. (1990), Gene 1989:117-122; F. Barany (1991) Proc. Natl. Acad. Sci. USA 1988:189-193); transcription-based methods utilize RNA synthesis by RNA polymerases to amplify nucleic acid (U.S. Pat. Nos. 6,066,457; 6,132,997; and 5,716,785; Sarkar et al., (1989) Science 244:331-34; Stofler et al., (1988) Science 239:491); NASBA (U.S. Pat. Nos. 5,130,238; 5,409,818; and 5,554,517); rolling circle amplification (RCA; U.S. Pat. Nos. 5,854,033 and 6,143,495) and strand displacement amplification (SDA; U.S. Pat. Nos. 5,455,166 and 5,624,825). Amplification methods can be used in combination with other techniques.
Other enzymatic techniques include sequencing using polymerases, e.g., DNA polymerases and variations thereof such as single base extension technology. See, e.g., U.S. Pat. Nos. 6,294,336; 6,013,431; and 5,952,174.
Fluorescence based detection can also be used to detect nucleic acid polymorphisms. For example, different terminator ddNTPs can be labeled with different fluorescent dyes. A primer can be annealed near or immediately adjacent to a polymorphism, and the nucleotide at the polymorphic site can be detected by the type (e.g., “color”) of the fluorescent dye that is incorporated.
Hybridization to microarrays can also be used to detect polymorphisms, including SNPs. For example, a set of different oligonucleotides, with the polymorphic nucleotide at varying positions with the oligonucleotides can be positioned on a nucleic acid array. The extent of hybridization as a function of position and hybridization to oligonucleotides specific for the other allele can be used to determine whether a particular polymorphism is present. See, e.g., U.S. Pat. No. 6,066,454.
In one implementation, hybridization probes can include one or more additional mismatches to destabilize duplex formation and sensitize the assay. The mismatch may be directly adjacent to the query position, or within 10, 7, 5, 4, 3, or 2 nucleotides of the query position. Hybridization probes can also be selected to have a particular Tm, e.g., between 45-60° C., 55-65° C., or 60-75° C. In a multiplex assay, Tm's can be selected to be within 5, 3, or 2° C. of each other.
It is also possible to directly sequence the nucleic acid for a particular genetic locus, e.g., by amplification and sequencing, or amplification, cloning and sequence. High throughput automated (e.g., capillary or microchip based) sequencing apparati can be used. In still other embodiments, the sequence of a protein of interest is analyzed to infer its genetic sequence. Methods of analyzing a protein sequence include protein sequencing, mass spectroscopy, sequence/epitope specific immunoglobulins, and protease digestion.
Any combination of the above methods can also be used. The above methods can be used to evaluate any genetic locus, e.g., in a method for analyzing genetic information from particular groups of individuals or in a method for analyzing a polymorphism associated with a disorder described herein, e.g., rheumatoid arthritis, e.g., in a gene encoding TWEAK or another biomarker described herein.
EXAMPLES
Example 1
Exemplary Sequences
An exemplary sequence of a human TWEAK protein is as follows
(SEQ ID NO: 1)
MAARRSQRRR GRRGEPGTAL LVPLALGLGL ALACLGLLLA
VVSLGSRASL SAQEPAQEEL VAEEDQDPSE LNPQTEESQD
PAPFLNRLVR PRRSAPKGRK TRARRAIAAH YEVHPRPGQD
GAQAGVDGTV SGWEEARINS SSPLRYNRQI GEFIVTRAGL
YYLYCQVHFD EGKAVYLKLD LLVDGVLALR CLEEFSATAA
SSLGPQLRLC QVSGLLALRP GSSLRIRTLP WAHLKAAPFL
TYFGLFQVH
An exemplary sequence of a human Fn14 protein is as follows:
(SEQ ID NO: 2)
MARGSLRRLL RLLVLGLWLA LLRSVAGEQA PGTAPCSRGS
SWSADLDKCM DCASCRARPH SDFCLGCAAA PPAPFRLLWP
ILGGALSLTF VLGLLSGFLV WRRCRRREKF TTPIEETGGE
GCPAVALIQ
Example 2
Genes that are Synergistically Activated by TWEAK and TNF-α
Microarrays were analyzed to identify genes whose expression in human synoviocytes was induced by TWEAK and TNF-α. The following are examples of genes that are synergistically activated by TWEAK and TNF-α.
TABLE 1
Genes Synergistically Activated by TWEAK and TNF-α
AffyID
annotation
208229_at
—
216064_s_at
—
220396_at
—
222332_at
—
207999_s_at
adenosine deaminase, RNA-specific, B1 (RED1
homolog rat)
202109_at
ADP-ribosylation factor interacting protein 2
(arfaptin 2)
201444_s_at
ATPase, H+ transporting, lysosomal accessory
protein 2
210538_s_at
baculoviral IAP repeat-containing 3
221534_at
basophilic leukemia expressed protein BLES03
203773_x_at
biliverdin reductase A
205733_at
Bloom syndrome
211314_at
calcium channel, voltage-dependent, alpha 1G
subunit
217118_s_at
chromosome 22 open reading frame 9
216607_s_at
cytochrome P450, family 51, subfamily A,
polypeptide 1
213279_at
dehydrogenase/reductase (SDR family) member 1
209703_x_at
DKFZP586A0522 protein
210839_s_at
ectonucleotide pyrophosphatase/phosphodiesterase
2 (autotaxin)
210002_at
GATA binding protein 6
212241_at
glutamate receptor, ionotropic, N-methyl D-aspartate-
like 1A
208055_s_at
hect domain and RLD 4
204512_at
human immunodeficiency virus type I enhancer
binding protein 1
216510_x_at
immunoglobulin heavy constant gamma 1
(G1m marker)
201548_s_at
Jumonji, AT rich interactive domain 1B (RBP2-like)
220972_s_at
keratin associated protein 9-9
212805_at
KIAA0367
212546_s_at
KIAA0826
215680_at
KIAA1654 protein
218906_x_at
likely ortholog of kinesin light chain 2
210104_at
mediator of RNA polymerase II transcription, subunit
6 homolog (yeast)
214397_at
methyl-CpG binding domain protein 2
212713_at
microfibrillar-associated protein 4
203901_at
mitogen-activated protein kinase kinase kinase 7
interacting protein 1
213040_s_at
neuronal pentraxin receptor
202783_at
nicotinamide nucleotide transhydrogenase
211691_x_at
Ornithine decarboxylase antizyme 4 mRNA,
complete cds
205991_s_at
paired related homeobox 1
204715_at
pannexin 1
214735_at
phosphoinositide-binding protein PIP3-E
203709_at
phosphorylase kinase, gamma 2 (testis)
207709_at
protein kinase, AMP-activated, alpha 2 catalytic
subunit
213136_at
protein tyrosine phosphatase, non-receptor type 2
213524_s_at
putative lymphocyte G0/G1 switch gene
202388_at
regulator of G-protein signalling 2, 24 kDa
218441_s_at
RNA polymerase II associated protein 1
212140_at
SCC-112 protein
201471_s_at
sequestosome 1
212609_s_at
serologically defined colon cancer antigen 8
212393_at
SET binding factor 1
214931_s_at
SFRS protein kinase 2
M97935_MB_at
signal transducer and activator of transcription
1, 91 kDa
204804_at
Sjogren syndrome antigen A1 (52 kDa, ribo-
nucleoprotein autoantigen SS-A/Ro)
214925_s_at
spectrin, alpha, non-erythrocytic 1 (alpha-fodrin)
221268_s_at
sphingosine-1-phosphate phosphatase 1
212154_at
syndecan 2 (heparan sulfate proteoglycan 1, cell
surface-associated, fibroglycan)
212800_at
syntaxin 6
201449_at
TIA1 cytotoxic granule-associated RNA binding
protein
216241_s_at
transcription elongation factor A (SII), 1
201399_s_at
translocation associated membrane protein 1
210372_s_at
tumor protein D52-like 1
206959_s_at
UPF3 regulator of nonsense transcripts homolog A
(yeast)
219393_s_at
v-akt murine thymoma viral oncogene homolog 3
(protein kinase B, gamma)
205205_at
v-rel reticuloendotheliosis viral oncogene homolog B,
nuclear factor of kappa light polypeptide gene
enhancer in B-cells 3 (avian)
Probe Set Id
Gene Title
1405_i_at
Chemokine (C-C motif) ligand 5 (CCL5)
204490_s_at
CD44 antigen (homing function and Indian blood
group system) (CD44)
204655_at
RANTES (SCYA5)
205619_s_ay
mesenchyme homeo box 1 (MEOX1)
platelet-derived growth factor receptor-
like . . . (NM_006207)
fibroblast growth factor receptor 4
fibroblast growth factor 22
chemokine (C-C motif) ligand 18
Still other genes are activated by both (i) TWEAK in the absence of TNF-α and (ii) TNF-α in the absence of TWEAK.
Example 3
Effect of Combination of Blocking TWEAK and TNF in mCIA as Measured by an Average Arthritis Index
The mouse collagen-induced arthritis (mCIA) model is a commonly-used model (see e.g., Stuart et al., J. Clin. Invest. 69:673-683 (1982)) of rheumatoid arthritis. A mCIA model was used to study the effects of a combination anti-TWEAK and anti-TNF-α treatment on arthritis development. Arthritis was induced in mice via collagen immunization (CH/CFA: collagen II and complete Freud's adjuvant). Anti-TWEAK monoclonal antibody (mu anti-TWEAK mAb+hu IgG1); soluble TNF-α receptor (TNFr-hu Fc+mu IgG2a); a combination of anti-TWEAK monoclonal antibody and soluble TNF-α receptor (mu anti-TWEAK mAb+TNFr-hu Fc); or PBS or isotype-matched negative controls were administered on days 20, 23, 27, 30, and 34 after collagen immunization. Each treatment group contained ten mice. Each antibody was administered at a dose of 10 mg/kg. Arthritis was assessed using an average arthritis index (see e.g., Li et al., Arthritis Res. Ther. 6:R273-R281 (2004)). Four paws were measured per mouse using the scoring system: 0=normal paw; 1=swelling of individual digits; 2=moderate swelling and redness of ankle or wrist joints; 3=swelling and redness of at least two joints; and 4=swelling of the whole paw. The sum of the four paw scores (y axis) were plotted against the days after collagen immunization (x axis).
FIG. 1 shows the results of this study. The mice treated with the combination of anti-TWEAK and anti-TNF-α agents had a lower average arthritis index score than mice treated with either blocking agent alone or the controls. Also, as indicated on the right side of the graph under “% incidence,” the mice treated with the combination had a lower overall incidence of arthritis (60%) than mice treated with either agent alone (67% for anti-TNF-α treatment; 80% for anti-TWEAK mAb treatment) or with the controls (80% for PBS treatment; 90% for isotype matched antibody treatment).
Example 4
Effect of Combination of Blocking TWEAK and TNF in mCIA as Measured by Average Metatarsal Height
The mCIA model was used to study the effects of a combination anti-TWEAK and anti-TNF-α therapy on arthritis development as measured by average metatarsal height/paw thickness (see e.g., Campo et al., Arthritis Res. Ther. 5:R122-R131 (2003)).
Mice were treated with anti-TWEAK monoclonal antibody (mu anti-TWEAK mAb+hu IgG1); soluble TNF-α receptor (TNFRp55:hu Fc+mu IgG2a); a combination of anti-TWEAK monoclonal antibody and soluble TNF-α receptor (mu anti-TWEAK mAb+TNFRp55:hu Fc); or PBS or isotype-matched negative controls on days 20, 23, 27, 30, and 34 after collagen immunization. Each treatment group contained ten mice. Each antibody was administered at a dose of 10 mg/kg. Metatarsal height was measured using calipers 38 days after collagen immunization. The average metatarsal height (y axis) for each mouse per treatment (x axis) was plotted.
FIG. 2 shows the results of this study. Mice treated with the combination of anti-TWEAK and anti-TNF-α agents had statistically-significant lower average metatarsal height values than mice treated with either blocking agent alone or the controls (*p<0.05 for the average value per treatment when compared to the controls).
Example 5
Effect of Combination of Blocking TWEAK and TNF in mCIA as Measured by Body Weight Change
The mCIA model was used to study the effects of a combination anti-TWEAK and anti-TNF-α therapy on arthritis development as measured by percent body weight change (Campo et al., Arthritis Res. Ther. 5:R122-R131 (2003)). Mice were treated with anti-TWEAK monoclonal antibody (mu anti-TWEAK mAb+hu IgG1 (an isotype matched control for TNFRp55:hu Fc)); soluble TNF-α receptor (TNFRp55:hu Fc+mu IgG2a); a combination of anti-TWEAK monoclonal antibody and soluble TNF-α receptor (mu anti-TWEAK mAb+TNFRp55:hu Fc); or PBS or isotype-matched negative controls on days 20, 23, 27, 30, and 34 after collagen immunization. Each treatment group contained ten mice. Each antibody was administered at a dose of 10 mg/kg. Mice were weighed at various time points after collagen immunization and the percent change in body weight were calculated per treatment. The percent body weight change for each treatment (y axis) was plotted against the days after arthritis induction by collagen immunization (x axis).
FIG. 3 shows the results of this study. Mice treated with the combination of anti-TWEAK and anti-TNF-α agents had a statistically-significant smaller percent change in body weight than mice treated with either blocking agent alone or the controls (*p<0.01 for the value per treatment when compared to the controls or to the TNFRp55:hu Fc+mu IgG2a treated mice).
Example 6
TWEAK Induced Genes
We applied TWEAK doses (5 ng/ml and 50 ng/ml) to cells at both 6 and 24 hour time points, and observed that some genes are modulated by TWEAK only. These genes were not affected by application of TNF-α, even at a concentration of 5 ng/ml. Examples of such genes are:
TABLE 2
1.
NIK/mitogen-activated protein kinase kinase kinase 14(MAP3K14)
2.
Homo sapiens cDNA FLJ11796 fis, clone HEMBA1006158, highly
similar to Homo sapiens transcription factor forkhead-like 7 (FKHL7)
gene
3.
similar to glucosamine-6-sulfatases Homo sapiens serum
glucocorticoid regulated kinase (SGK), mRNA
4.
Homo sapiens REV3 (yeast homolog)-like, catalytic subunit of
DNA polymerase zeta (REV3L), mRNA.
5.
ADAM 10/a disintegrin and metalloproteinase domain 10
6.
nuclear factor (erythroid-derived 2)-like 1
7.
Homo sapiens SerArg-related nuclear matrix protein
(plenty of prolines 101-like) (SRM160), mRNA.
8.
Homo sapiens doublecortin and CaM kinase-like 1 (DCAMKL1),
mRNA.
9.
Homo sapiens Cdc42 effector protein 4; binder of Rho GTPases 4
(CEP4), mRNA.
10.
Homo sapiens mRNA; cDNA DKFZp762L106
(from clone DKFZp762L106); partial cds.
In addition, in normal human synoviocytes, CBR3 and IL8 are induced by TWEAK treatment (5 ng/ml) alone.
Example 7
Experiments with TWEAK
FIG. 4 shows that treatment with TWEAK-blocking monoclonal antibodies can lessen the development of arthritis in both mouse and rat CIA models of arthritis. The left panel shows that treatment with an anti-TWEAK antibody (murine anti-TWEAK mAb) results in a lower value in the average arthritis index, as compared to treatment with a control antibody (mIgG2a), in a mouse CIA model in which arthritis was induced with CII/CFA. The right panel shows that treatment with an anti-TWEAK antibody (anti-TWEAK mAb) results in a lower value in the average arthritis index, as compared to treatment with a control antibody (Ha 4/8) or PBS in a rat CIA model, in which arthritis was induced with collagen II and incomplete Freud's adjuvant (CII/IFA).
FIG. 5 shows that TWEAK-blocking monoclonal antibodies can be administered at the same time as (Dosing scheme 1) or after (Dosing scheme 2) the induction of arthritis by collagen immunization and still have the effect of lessening the development of arthritis in both mouse and rat CIA models of arthritis. The left panel shows that an anti-TWEAK antibody can be administered prior to or after the induction of arthritis to effect a lower value in the average arthritis index, as compared to administration of a control antibody (mIgG2a), in a mouse CIA model. The right panel shows that an anti-TWEAK antibody can be administered prior to or after the induction of arthritis to effect a lower value in the average arthritis index, as compared to administration of a control antibody (Ha 4/8) or PBS, in a rat CIA model.
FIG. 6 shows that anti-TWEAK monoclonal antibody (ABG. 11) treatment decreases inflammation in the rat CIA model, as measured by a clinical paw score and an overall inflammation score; the treatment also decreases cartilage and bone loss, as measured by the parameters of bone absorption, decrease in toluidine blue uptake, and loss of articular cartilage. Similar results were seen in the mouse CIA model.
FIG. 7 shows serum TWEAK levels in the mouse CIA model at various time points (day (D) 23, 28, 30, and 38) after induction of arthritis. TWEAK levels were elevated as compared to the levels in control mice (DBA/1).
FIG. 8 shows the levels of MMP9, lymphotactin, IP-10, and IL-6 at various time points (day 23, 30, and 40) after induction of arthritis in the mouse CIA model. Treatment with anti-TWEAK monoclonal antibody (P5G9 and P5G9 (Full, also termed dosing scheme 1)) prevented as great an increase in the levels of these proteins, as compared to the levels in mice treated with a control (mIgG2a) or in mice not immunized to develop arthritis (normal DBA). Similar results were seen in the rat CIA model.
Experiments were performed to demonstrate that inhibition of TWEAK with anti-TWEAK antibodies does not affect the adaptive immune response. After collagen immunization, mice that had been treated with anti-TWEAK monoclonal antibodies were able to mount collagen-specific B cell and T cell responses to a similar extent as mice that had been treated with a control, isotype-matched antibody (mIgG2a; data not shown).
Experiments were performed to measure the levels of Fn14 (TWEAK receptor) on primary human cell types found in a joint: fibroblast-like synoviocytes, articular chondrocytes, and osteoblasts. Fluorescence-activated cell sorting experiments using anti-Fn14 antibody (ITEM-4) or a control antibody (anti-mFc) demonstrated that Fn14 levels were elevated above background in all three cells types, with levels being higher in the synoviocytes and osteoblasts than in the chondrocytes.
Experiments were performed to demonstrate that TWEAK and TNF-α can each stimulate matrix metalloprotease production by chondrocytes. MMP-1, MMP-2, MMP-3, and MMP-9 levels all increased after treatment with TWEAK (100 ng/ml) or TNF-α (50 ng/ml).
Experiments were performed to demonstrate the agonistic, synergistic effects of TWEAK and TNF-α. Human fibroblast-like synoviocytes were treated with varying concentrations of TWEAK alone, TNF-α alone, or a combination of TWEAK and TNF-α, and the level of RANTES production with each treatment was measured by ELISA. Both TWEAK and TNF-α induced RANTES production. However, when TWEAK and TNF-α were administered in combination, a synergistic level of RANTES production resulted. Thus, TWEAK and TNF-α can synergize to induce expression of particular inflammatory genes.
Example 8
Genes Induced by TWEAK and TNF-α Combination Treatment in Normal Synoviocytes
Synoviocytes from a healthy donor were cultured in vitro and treated with 5 ng/ml TWEAK and 0.5 ng/ml TNF-α. Table 3 lists genes whose expression was affected by the treatment with TWEAK and TNF-α to a statistically significant degree. The genes are grouped by their gene ontology category.
TABLE 3
Go Ontology
Protein
P Value
Positive regulation
CASP1, CFLAR, LGLAS9, Myd88,
5.81e−018
of IκB
SECTM1, TNFSF10, TRIM38
Inflammatory
CCL3, CCL4, CCL7, CCL8,
9.94e−007
Response
CXCL9, ILRN, Myd88, TLR3
Chemotaxis
CCL3, CCL4, CCL7, CCL8,
0.0003
CXCL9, ERG1, SOCS1
Interferon Response
IFI44, WARS, IRF2
0.001
The changes were identified as statistically significant Go categories based on hypergeometric mean.
Example 9
Genes Induced by TWEAK and TNF-α Combination Treatment in RA Synoviocytes
Synoviocytes from a rheumatoid arthritis patient donor were cultured in vitro and treated with 5 ng/ml TWEAK and 0.5 ng/ml TNF-α. Table 4 lists genes whose expression was affected by the treatment with TWEAK and TNF-α to a statistically significant degree. The genes are grouped by their gene ontology category.
TABLE 4
Go Ontology
Protein
pValue
Inflammatory
CXCL10, CXCL3, PTGS2, APOL3
4.26e−005
Response
Response to Stress
CXCL10, CXCL3, PTGS2, APOL3,
5.56e−006
MDA5, MX1, PTGES, Rig-1
Response to biotic
CXCL10, CXCL3, PTGS2, APOL3,
3.69e−009
stimuli
MDA5, MX1, PTGES, Rig-1, GBp1
The changes were identified as statistically significant Go categories based on hypergeometric mean.
Example 10
P2D10 is an exemplary murine anti-TWEAK antibody. The sequence of the murine P2D10 heavy chain variable domain (SEQ ID NO:3), with CDRs underlined is:
1
EVQLVESGGG LVRPGGSLKL FCAASGFTFS RYAMSWVRQS
PEKRLEWVAE
51
ISSGGSYPYY PDTVTGRFTI SRDNAKNTLY LEMSSLKSED
TAMYYCARVL
101
YYDYDGDRIE VMDYWGQGTA VIVSS
This is a murine subgroup 3D heavy chain variable domain.
The sequence of the murine P2D10 light chain variable domain (SEQ ID NO:4), with CDRs underlined is:
1
DVVMTQSPLS LSVSLGDQAS ISCRSSQSLV SSKGNTYLHW
YLQKPGQSPK
51
FLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVAAEDLGV
YFCSQSTHFP
101
RTFGGGTTLE IK
This is a murine subgroup 2 kappa light chain.
This is an exemplary amino acid sequence of the mature huP2D10 H1 IgG1 heavy chain (SEQ ID NO:5):
1
EVQLVESGGG LVQPGGSLRL SCAASGFTFS RYAMSWVRQA
PGKGLEWVAE
51
ISSGGSYPYY PDTVTGRFTI SRDNAKNSLY LQMNSLRAED
TAVYYCARVL
101
YYDYDGDRIE VMDYWGQGTL VTVSSASTKG PSVFPLAPSS
KSTSGGTAAL
151
GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL
SSVVTVPSSS
201
LGTQTYICNV NHKPSNTKVD KKVEPKSCDK THTCPPCPAP
ELLGGPSVFL
251
FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV
EVHNAKTKPR
301
EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI
EKTISKAKGQ
351
PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE
SNGQPENNYK
401
TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL
HNHYTQKSLS
451
LSPG
This is an exemplary amino acid sequence of the mature huP2D10 L1 light chain (SEQ ID NO:6):
1
DVVMTQSPLS LPVTPGEPAS ISCRSSQSLV SSKGNTYLHW
YLQKPGQSPQ
51
FLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV
YFCSQSTHFP
101
RTFGGGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL
LNNFYPREAK
151
VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD
YEKHKVYACE
201
VTHQGLSSPV TKSFNRGEC
This is an exemplary amino acid sequence of the mature huP2D10 L2 light chain (SEQ ID NO:7):
1
DVVMTQSPLS LPVTPGEPAS ISCRSSQSLV SSKGNTYLHW
YLQKPGQSPQ
51
LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV
YYCSQSTHFP
101
RTFGGGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL
LNNFYPREAK
151
VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD
YEKHKVYACE
201
VTHQGLSSPV TKSFNRGEC
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
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13246197
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biogen idec ma inc.
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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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424/145.1
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Apr 1st, 2022 05:10PM
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Apr 1st, 2022 05:10PM
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Biogen
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:biib
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Biogen
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Mar 27th, 2012 12:00AM
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Oct 13th, 2009 12:00AM
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https://www.uspto.gov?id=US08142779-20120327
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Treatment of follicular lymphomas using inhibitors of the LT pathway
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Therapeutic uses of inhibitors of the lymphotoxin pathway to treat tumors, specifically to treat follicular lymphomas.
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8142779
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1. A method for arresting or reducing the advancement, severity or effects of a follicular lymphoma comprising the administration to a subject having a follicular lymphoma of an effective amount of an anti-LT-β antibody which inhibits the interaction between LT-β and its receptor.
2. The method of claim 1, wherein the subject is a mammal.
3. The method of claim 2, wherein the subject is a human.
4. The method according to claim 1, wherein the antibody is a monoclonal antibody directed against LT-β.
5. The method of claim 4, wherein the monoclonal antibody is humanized or chimeric.
6. The method of claim 1 comprising the administration to said subject of at least one chemotherapeutic agent.
7. The method of claim 1 further comprising the administration to said subject of an inhibitor of another TNF pathway.
8. The method of claim 7 comprising the administration of a composition which inhibits the CD40/CD40 ligand pathway.
9. The method of claim 8 comprising the administration of an anti-CD40 ligand antibody.
10. The method of claim 1 further comprising the administration to said subject of radiation treatments.
11. The method of claim 1 further comprising the administration to said subject of a bone marrow transplant.
12. The method of claim 1, wherein the antibody is administered as a single dose.
13. The method of claim 1, wherein the antibody is administered daily.
14. The method of claim 1, wherein the antibody is administered every 2 to 6 days.
15. The method of claim 1, wherein the antibody is administered weekly, biweekly or monthly.
16. The method of claim 6, wherein the antibody which inhibits the interaction between LT-β and its receptor is administered prior to the administration of the chemotherapeutic agent.
17. The method of claim 12, wherein the antibody which inhibits the interaction between LT-β and its receptor is administered simultaneously with the chemotherapeutic agent.
18. The method of claim 6, wherein the antibody which inhibits the interaction between LT-β and its receptor is administered subsequent to the administration of the chemotherapeutic agent.
19. The method of claim 1, wherein the antibody which inhibits the interaction between LT-β and its receptor further comprises a pharmaceutically acceptable carrier.
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19
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RELATED APPLICATIONS
This is a continuation of U.S. patent application Ser. No. 11/411,049, filed on Apr. 25, 2006, which is a divisional of U.S. patent application Ser. No. 09/626,219, filed on Jul. 26, 2000 as a continuation-in-part of PCT/US99/01928, which was filed on Jan. 29, 1999 and claims priority to U.S. Patent Application No. 60/073,112, filed Jan. 30, 1998 and U.S. Patent Application No. 60/073,410, filed Feb. 2, 1998. The entire disclosure of each of the aforesaid patent applications are incorporated herein by reference.
GOVERNMENT FUNDING
This invention was made with government support under National Institutes of Health Grant # AG 04980. The United States government has certain rights in the invention.
FIELD OF THE INVENTION
The present invention relates to compositions, and therapeutic uses of inhibitors of the Lymphotoxin pathway to treat tumors, specifically to treat lymphomas derived from germinal centers (follicular lymphomas).
BACKGROUND OF THE INVENTION
The tumor-necrosis factor (TNF)-related cytokines are mediators of host defense and immune regulation. Members of this family exist in membrane-anchored forms, acting locally through cell-to-cell contact, or as secreted proteins capable of diffusing to more distant targets. A parallel family of receptors signals the presence of these molecules leading to the initiation of cell death or cellular proliferation and differentiation in the target tissue. Presently, the TNF family of ligands and receptors has at least 11 recognized receptor-ligand pairs, including: TNF:TNF-R; LT-a:TNF-R; LT-α/β:LT-β-R; FasL:Fas; CD40L:CD40; CD30L:CD30; CD27L:CD27; OX40L:OX40 and 4-1BBL:4-1BB.
TNF family members can best be described as master switches in the immune system controlling both cell survival and differentiation. Only TNF and LT-a are currently recognized as secreted cytokines contrasting with the other predominantly membrane-anchored members of the TNF family. While a membrane form of TNF has been well characterized and is likely to have unique biological roles, secreted TNF functions as a general alarm signaling to cells more distant from the site of the triggering event. Thus TNF secretion can amplify an event leading to the well-described changes in the vasculature lining and the inflammatory state of cells. In contrast, the membrane bound members of the family send signals though the TNF type receptors only to cells in direct contact. For example T cells provide CD40 mediated “help” only to those B cells brought into direct contact via cognate TCR interactions. Similar cell-cell contact limitations on the ability to induce cell death apply to the well-studied Fas system.
Most membrane-associated LT-α/β complexes (“surface LT”) have a LT-α1/β2 stoichiometry. (Browning et al., Cell, 72, pp. 847-56 (1993); Browning et al., J. Immunol., 154, pp. 33-46 (1995)). Surface LT ligands do not bind TNF-R with high affinity and do not activate TNF-R signaling. The LT-β receptor (LT-β-R), does however bind these surface lymphotoxin complexes with high affinity (Crowe et al., Science, 264, pp. 707-10 (1994)).
LTβ-R signaling, like TNF-R signaling, has anti-proliferative effects and can be cytotoxic to tumor cells. In applicants' co-pending U.S. application Ser. No. 08/378,968, compositions and methods for selectively stimulating LT-β-R using LT-β-R activating agents are disclosed. LT-β-R activating agents are useful for inhibiting tumor cell growth without co-activating TNF-R-induced proinflammatory or immunoregulatory pathways.
Recent gene targeting studies suggest a role for LT-α/β in the development of secondary lymphoid organs. (Banks et al., J. Immunol., 155, pp. 1685-1693 (1995); De Togni et al., Science, 264, pp. 703-706 (1994)). Indeed, LT-α-deficient mice lack lymph nodes (LN) and Peyer's patches (PP). Moreover, their spleens have disrupted architecture and the expression of functional markers on cells of the splenic marginal zone is altered. (Banks et al., 1995; De Togni et al., Science, 264, pp. 703-706 (1994), Matsumoto at al., Science, 271, pp. 1289-1291 (1996)). None of these characteristics have been described for either of the TNF receptor knock out mice. (Erickson et al., Nature, 372, pp. 560-563 (1994); Pfeffer et al., Cell, 73, pp. 457-467 (1993); Rothe et al., Nature, 364, pp. 798-802 (1993). Applicants have recently defined a role for membrane LT-α/β complexes in secondary lymphoid organ development by showing that the progeny of mice which had been injected during gestation with a soluble form of mouse LT-β-R fused to the human IgG1 Fc portion (LT-β-R-Ig) lacked most lymph nodes and showed disrupted splenic architecture. (Rennert et al, 1996, “Surface Lymphotoxin alpha/beta complex is required for the development of peripheral lymphoid organs.” J. Exp Med, 184: 1999-2006). In another study, mice transgenic for a similar LT-β-R-Ig construct which starts to be expressed three days after birth, were shown to have LN. However, their splenic architecture was disrupted and several markers of splenic marginal zone cells were not expressed (Ettinger et al., “Disrupted splenic architecture, but normal lymph node development in mice expressing a soluble LTβ-R/IgG1 fusion protein”, Proc. Natl. Acad. Sci. U.S.A. 93: 13102-7). Together these data indicate there is a temporal requirement for membrane LT functions to mediate effects on the development of secondary lymphoid organs, but not for effects on splenic architecture.
The TNF system may also function in development of the spleen. Splenic marginal zone cells of TNF-deficient mice do not express macrophage markers or MAdCAM-1 (Alexopoulou et al., 60th Int. TNF Congress, Eur. Cytokine Network, pp. 228 (1996); Pasparakis et al., 60th Int. TNF Congress, Eur. Cytokine Network, pp. 239 (1996)). TNF-R55-deficient mice also lack MAdCAM-1 (but not MOMA-1) staining in the splenic marginal zone. (Neumann et al., J. Exp. Med., 184, pp. 259-264 (1996), Matsumoto et al., Science, 271, pp. 1289-1291 (1996)). The expression of these markers as seen in the spleen of TNF-R75-deficient mice appears normal. (Matsumoto et al., Science, 271, pp. 1289-1291 (1996)).
Lymphoid-like tissues do not only arise as a part of developmental processes but also appear under some pathological circumstances such as chronic inflammation, a process recently termed neolymphoorganogenesis. (Picker and Butcher, Annu. Rev. Immunol., 10, pp. 561-591 (1992), Kratz, et al., J. Exp. Med., 183, pp. 1461-1471 (1996)). TNF family members apparently influence such processes. Mice transgenic for the LT-α gene driven by the rat insulin promoter (RIP-LT) developed LT-induced chronic inflammatory lesions with characteristics of organized lymphoid tissues. (Kratz, et al., J. Exp. Med., 1183, pp. 1461-1471 (1996); Picarella et al., Proc. Natl. Acad. Sci., 89, pp, 10036-10040 (1992)).
The evaluation of LT function during a T cell-dependent immune response, using LT-α-deficient mice, showed the necessity of LT for GC formation, possibly for maintaining an organized follicular dendritic cell (FDCs) structure, and for humoral responses. (Banks et al., J. Immunol., 155, pp. 1685-1693 (1995); Matsumoto et al., Science, 271, pp. 1289-1291 (1996); Matsumoto et al., Nature, 382, pp. 462-466 (1996)). TNF-R55-deficient mice also lack FDCs, fail to develop GC and fail to develop an optimal antibody response to sheep red blood cells (SRBC). This suggests that TNF-R55 might be triggered by soluble LT or TNF signals for most of these responses (Le Hir et al., J. Exp. Med., 183, pp. 2367-2372 (1996), Alexopoulou et al., 60th Int. TNF Congress, Eur. Cytokine Network, pp. 228 (1996); Pasparakis et al., 60th Int. TNF Congress, Eur. Cytokine Network, pp. 239 (1996)).
The LT-β-receptor, a member of the TNF family of receptors, specifically binds to surface LT ligands. LT-β-R binds LT heteromeric complexes (predominantly LT-α1/β2 and LT-α2/β1) but does not bind TNF or LT-α (Crowe et al., Science, 264, pp. 707-10 (1994)). LT-β-R mRNAs are found in the human spleen, thymus and in general organs with immune system involvement. Although studies on LT-β-R expression are in their early stages, LT-β-R expression patterns appear to be similar to those reported for TNF-R55 except that LT-β-R is lacking on peripheral blood T and B cells and T and B cell lines.
Cell surface lymphotoxin (LT) complexes have been characterized in CD4+ T cell hybridoma cells (II-23.D7) which express high levels of LT. (Browning et al., J. Immunol., 147, pp. 1230-37 (1991); Androlewicz et al., J. Biol. Chem., 267, pp. 2542-47 (1992), both of which are herein incorporated by reference). The expression and biological roles of LTβ-R, LT subunits and surface LT complexes have been reviewed by C. F. Ware et al. “The ligands and receptors of the lymphotoxin system”, in Pathways for Cytolysis, Current Topics Microbiol. Immunol., Springer-Verlag, pp. 175-218 (1995) specifically incorporated by reference herein.
LT-α expression is induced and LT-α secreted primarily by activated T and B lymphocytes and natural killer (NK) cells. Among the T helper cells, LT-α appears to be produced by Th1 but not Th2 cells. LT-α has also been detected in melanocytes. Microglia and T cells in lesions of multiple sclerosis patients can also be stained with anti-LT-α antisera (Selmaj et al., J. Clin. Invest., 87, pp. 949-954 (1991)).
Lymphotoxin β (also called p33) is expressed on the surface of human and mouse T lymphocytes, T cell lines, B cell lines and lymphokine-activated killer (LAK) cells. LTβ is the subject of applicants' co-pending international applications PCT/US91/04588, published Jan. 9, 1992 as WO 92/00329; and PCT/US93/11669, published Jun. 23, 1994 as WO 94/13808, which are herein incorporated by reference.
Surface LT complexes are primarily expressed by activated T (helper, Th1, and killer cells) and B lymphocytes and natural killer (NK) cells as defined by FACS analysis or immunohistology using anti-LT antibodies or soluble LT-β-R-Ig fusion proteins. In applicants copending U.S. application Ser. No. 08/505,606, filed Jul. 21, 1995, compositions and methods for using soluble LT-β receptors and anti-LT-β receptor and ligand specific antibodies as therapeutics for the treatment of immunological diseases mediated by Th1 cells are disclosed. Surface LT has also been described on human cytotoxic T lymphocyte (CTL) clones, activated peripheral mononuclear lymphocytes (PML), IL-2-activated peripheral blood lymphocytes (LAK cells), pokeweed mitogen-activated or anti-CD40-activated peripheral B lymphocytes (PBL) and various lymphoid tumors of T and B cell lineage. Engagement of alloantigen-bearing target cells specifically induces surface LT expression by CD8+ and CD4+ CTL clones.
Applicants have described herein several immunological functions for surface LT, and show the effects of LT-α/β binding reagents on the generation and character of immunoglobulin responses, maintenance of the cellular organization of secondary lymphoid tissues including effects on the differentiation state of follicular dendritic cells and germinal center formation, and addressin expression levels which influence cell trafficking. Thus applicants define therapeutic applications for surface LT-α/β and LT-α receptor binding agents.
Studies have shown that B cells are activated in the lymph nodes (LN) and spleen following encounters with various antigens. In a specialized structure called a germinal center which forms in the B cell rich regions of LN and spleen, the B cells mature and memory B cells form (Tsiagbe, et al. Crit. Rev. Immunol. 16, 381-421 (1996)). B cells are capable of undergoing transformation into tumors at most points during their development (Freedman, et al, Cancer Medicine 3rd Ed., pp. 2028-2068 (1994)). Transformation of B cells leads to lymphomas and those derived from B cells in germinal centers are often called follicular lymphomas. The exact delineation of the various subsets of lymphomas is still in transition as more surface markers are found permitting a more precise designation of the cell of origin. Follicular lymphomas can be divided into a number of subgroups based on the stage or type of B cell that is proliferating and the prognosis varies depending on the cell type. Conventional chemotherapy regimes are capable of affecting a cure in many of the patients with low-grade type cells. Nonetheless a portion of these patients are resistant to chemotherapy and have a poor prognosis.
Therefore, despite the progress in treating tumors, there remains a need for a treatment for those tumors especially for those follicular lymphomas typically resistant to chemotherapy, as well as for treatment regimes with fewer side effects than existing therapies.
SUMMARY OF THE INVENTION
The present invention provides methods and compositions for the treatment of tumors such as follicular lymphomas which overcome certain problems existing with present therapies, and offers an alternative therapy for those with tumors resistant to traditional chemotherapy.
In certain embodiments, the claimed invention relates to methods of treating a subject having a follicular lymphoma comprising administering to the subject an effective amount of a composition which blocks the interaction of the LT-α/β heteromer with its receptor. Preferred compositions in various embodiments include, but are not limited to, soluble lymphotoxin-β receptors, antibodies directed against the LT-β receptor, and antibodies directed against surface LT ligand. More preferred are soluble lymphotoxin-β receptors having a ligand binding domain that can selectively bind to a surface LT ligand, such as, for example, a soluble LT-β-R form fused to a human immunoglobulin Fc domain. Additionally, preferred compositions include monoclonal antibodies which are directed against the LT-β receptor, including antibodies which are humanized, chimeric or otherwise altered.
In other embodiments of the invention, the claims encompass methods of treatment of subjects having follicular tumors wherein the blocking agents are administered until regression or arrest of tumor growth is noted. In certain embodiments, the LT pathway blocking agents are administered in combination with other agents known to be useful in treating tumors, such as, for example chemotherapy regimens. Additionally, the methods of the invention may in certain embodiments further comprise treating the subject with radiation or bone marrow transplantation.
In yet other embodiments, the claimed methods comprise the administration of LT-β-R blocking agents in conjunction with blocking agents of pathways of other members of the TNF family. For example, TNF blocking agents may be administered in conjunction with, simultaneously or concommitantly, a blocking agent of the claimed invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides methods and compositions for the treatment of tumors, such as follicular tumors, specifically follicular lymphomas.
The terms “immunoglobulin response” or “humoral response” as used herein refer to the immunological response of an animal to a foreign antigen whereby the animal produces antibodies to the foreign antigen. The Th2 class of T helper cells are important to the efficient production of high affinity antibodies.
The term “germinal center” as used herein refers to a secondary B cell follicle Which forms after antigen immunization. The appearance of this histologic site correlates with optimal memory generation, isotype switching, somatic hypermutation and thus the affinity maturation of an antibody response.
The terms “marginal zone” or “marginal-zone type area” refer to histologically described compartments of the secondary lymphoid tissues comprised primarily of marginal zone macrophages (MZM), metallophilic macrophages (MM), marginal zone B cells and reticular cells, and also T cells and dendritic cells. The arterial blood stream opens into the marginal sinuses thus giving antigens direct access to these cells and promoting cellular reactions to antigens at this site.
The term “T helper (Th) cells” as used herein, refers to a functional subclass of T cells which help to generate cytotoxic T cells and which cooperate with B cells to stimulate antibody production. Helper T cells recognize antigen in association with class II MHC molecules and provide contact dependent and contact independent (cytokine) signals to effector cells.
The term “Fc domain” of an antibody refers to a part of the molecule comprising the hinge, CH2 and CH3 domains, but lacking the antigen binding sites. The term is also meant to include the equivalent regions of an IgM or other antibody isotype.
The term “anti-LT-β receptor antibody” refers to any antibody that specifically binds to at least one epitope of the LT-β receptor.
The term “anti-LT antibody” refers to any antibody that specifically binds to at least one epitope of LT-α, LT-β or a LT-α/β complex.
The term “LT-β-R signaling” refers to molecular reactions associated with the LT-β-R pathway and subsequent molecular reactions which result therefrom.
The term “LT-β-R blocking agent” refers to an agent that can diminish ligand binding to LT-β-R, cell surface L-β-R clustering or LT-β-R signaling, or that can influence how the LT-β-R signal is interpreted within the cell.
An LT-β-R blocking agent that acts at the step of ligand-receptor binding can inhibit LT ligand binding to the LT-β-R by at least 20%. Examples of LT-β-R blocking agents include soluble LT-β-R-Fc molecules, and anti-LT-α, anti-LT-β, anti-LT-α/β and anti-LT-β-R Abs. Preferably, the anti bodies do not cross-react with the secreted form of LT-α.
The term “LT-β-R biological activity” refers to: 1) the ability of the LT-β-R molecule or derivative to compete for soluble or surface LT ligand binding with soluble or surface LT-β-R molecules; or 2) native LT activity such as the ability to stimulate an immune regulatory response or cytotoxic activity.
The term “LT ligand” refers to an LT-α/β heteromeric complex or derivative thereof that can specifically bind to the LTβ receptor.
The term “LT-β-R ligand binding domain” refers to the portion or portions of the LT-R that are involved in specific recognition of and interaction with a LT ligand.
The terms “surface LT” and “surface LT complex” refer to a complex comprising LT-α and membrane-bound LT-β subunits—including mutant, altered and chimeric forms of one or more of the subunits—which is displayed on the cell surface, “Surface LT ligand” refers to a surface LT complex or derivative thereof that can specifically bind to the LT-β receptor.
The term “subject” refers to an animal, or to one or more cells derived from an animal. Preferably, the animal is a mammal. Cells may be in any form, including but not limited to cells retained in tissue, cell clusters, immortalized, transfected or transformed cells, and cells derived from an animal that has been physically or phenotypically altered.
As discussed above, transformation of B cells leads to lymphomas, and the transformation of B cells derived from germinal centers, the specialized structures found in the B cell rich regions of lymph nodes and the spleen, are referred to as follicular lymphomas. The germinal center B cell requires a specific environment to mature and proliferate and follicular dendritic cells provide both antigen and mostly likely specific signals for the germinal center B cells that trigger maturation, survival and proliferation. Studies in SJL mice which spontaneously form reticular cell sarcomas (RCS, an early designation of these types of tumors) have led to transplantable as well as in vitro cell lines of RCS (CRCS) that serve as a model for the interactions between the host and the tumor (Lasky, et al., J. Immunol., 140, pp. 679-687, (1988) and Lasky, et al, Eur. J. Immunol., 19, pp. 365-371, (1989). These RCS arise frequently in the LN of SJL mice and are heterogeneous containing a variety of hematopoietic cells. Considerable evidence indicates that these lymphomas are germinal center derived and require various signals or factors provided by the host to survive and proliferate (Ponzio, et al., Intern. Rev, Immunol, 1, pp. 365-371, (1986) and Tsiagbe et al, J. Immunol., 150, pp. 5519-5528, (1993)). The ability to manipulate these survival signals provides a means of controlling the growth of these tumors
One cell type called the follicular dendritic cell (FDC) is believed to be of paramount importance in the formation and function of the germinal center. Many different factors have been implicated in the survival and maintenance of germinal center B cells. Notably, members of the TNF family of cytokines are surface signaling ligands are involved both from the B cell side, e.g. CD40, and on the FDC side, e.g. TNF and lymphotoxin (LT) receptors. Mice deficient for either the LT or TNF axis have defects in the FDC and hence lack germinal centers (Matsumoto et al., J. Exp. Med., 186, pp. 1997-2004, (1997)). The TNF axis is believed to be critical for the development of FDC although downstream roles probably exist. The LT axis appears to be more critical for the maintenance of FDC in a functional state. The LT system involves the signaling from various ligand positive lymphocytes to receptor positive cells that are most likely of a non-bone marrow derivation, i.e. possibly FDC derived, to maintain the FDC in their fully functional mature state. Applicants have found that blocking this pathway, with, for example, either antibodies to the LT ligands or a soluble receptor-immunoglobulin fusion protein, leads to a loss of mature FDC (Mackay and Browning, 1998 Nature, V. 395, pp 26-27, “Turning Off Follicular Dendritic Cells”). Furthermore, LT pathway inhibition leads to the loss of germinal center formation and some disorganization of the spleen (Mackay, et al., Eur. J, Immunol., 27, pp, 2033-2042, (1997)).
Applicants for the first time describe herein that LT pathway inhibitors can disrupt the interactions between a follicular B cell lymphoma and its environment, i.e. the FDC, and lead to slowed or arrested growth of tumors. Hence, such inhibitors are useful in the management of intractable lymphomas or as a primary therapy, or in addition to conventional chemotherapy regimes. Specifically, although it has been suggested in the art that activation of the LT pathway could be implicated in tumor therapies, applicants have surprisingly discovered that the transient blocking of the LT pathway can lead to slowing or arrest of the growth of tumors, including for example, follicular lymphomas.
In its broader embodiments the present invention encompasses methods of treating subjects having tumors or lymphomas, specifically, follicular lymphomas, by administering an effective amount of a composition that inhibits the LT pathway. Specific inhibiting compositions may comprise soluble LT-β receptor, fusion proteins comprising LT-β-R, antibodies to LT-β-R, and antibodies to LT ligand. Such inhibiting compositions preferably include a pharmaceutically acceptable carrier. The subject in preferred applications is a mammal, most preferably, humans.
The methods of the invention comprise administering the compositions of the invention to the subject until some tumor regression, or arrest, is noted. The time of treatment may vary widely, and treatment may continue over the course of several weeks, to several months, or in some cases even longer. One skilled in the art is capable of determining when tumor regression or arrest has occurred, and any of the known methods can be used. The use of FACS markers to subdivide B lymphomas has improved considerably, and it may be expected that lymphomas of certain subtypes will prove to be most tractable to this type of therapy.
It is also likely that other immune system regulatory molecules such as other TNT family members may be involved in maintaining the immune organ architecture and therefore contribute to providing a favorable environment for lymphoma proliferation.
Therefore, combined inhibition of the LT and other pathways may be an effective treatment for certain subjects. For example, one may use inhibitors of the LT pathway in combination with, for example, blockers of the CD40/CD40 ligand pathway. Any composition which blocks the desired pathway can be used, such as, for example, antibodies, soluble ligands or receptors. It may be preferable to administer antibodies against CD40 Ligand in combination with inhibitors of the LT pathway. When administering more than one blacker of a TNF member pathway, the compositions may be administered substantially simultaneously, or alternatively, one blocker may be administered sequential to the other. One skilled in the art can easily determine the most effective treatment for a particular subject based upon the particular tumor being treated, and the condition of the subject.
Conventional chemotherapeutic protocols may be used to eliminate remaining tumor burden subsequent to treatment with the compositions of the invention, or in some cases, may be used simultaneously with, or prior to the compositions of the invention, LT pathway inhibitors may be used to arrest lymphoma growth prior to embarking on a conventional chemotherapy regime. It is likely that loss of growth/survival promoting signals may render a lymphoma more susceptible to chemotherapeutic agents, and therefore, it is preferred to administer the LT pathway inhibitor prior to administration of traditional chemotherapeutic agents.
Example 1
Treatment of the SJL RCS Tumors with LT Pathway Inhibitor Reduces Total LN/Tumor Size
SJL mice were treated either 3 days prior to the tumor transplantation (D-3), at the time of transplantation (D0), or 3 days post transplantation (D3) with 0.3-0.4 mg of mouse LTBR-hIgG1 fusion protein via the intraperitoneal route. Tumor transplantation was performed essentially as described (Katz, et al., Cellular Immunol., 65, pp. 84-92, (1981)). 5×106 T cell depleted RCS cells were injected iv and allowed to seed the organs and grow. After 5-7 days, the mesenteric, brachial and axillary LN were dissected and their weight as a percentage of total body weight was calculated. Table I shows that LN size was reduced in all experiments. Spleen size was also reduced in 1 out of 3 experiments, but the reduction in spleen weight was not impressive. The reductions in LN weight ranged from about 50% with a single treatment to 80-90% with multiple dosing.
TABLE I
Inhibitory Effect of LTBR-Ig on RCS Growth in Normal SJL Mice.
Mice Injected with
(Day: Dose mg)
LN Wta (n)
p
Spleen Wt.a (n)
p
Experiment 1
huIgG control (Killed D5)
2.70 +/− 0.37 (7)
3.18 +/− 0.37 (7)
mLTβR-Igc (D0, +3: 0.4, 0.3)
1.42 +/− 0.15 (4)
<0001
3.29 +/− 0.18 (4)
NSb
Experiment 2
huIgG control (killed D7)
3.35 +/− 0.21 (3)
4.04 +/− 0.34 (3)
mLTβR-Ig (D0, +3; 0.3, 0.2)
2.37 +/− 0.24 (4)
0.0024
3.04 +/− 0.19 (4)
0.004
↓
Experiment 3
huIgG control (killed D6)
2.34 +/− 0.11 (5)
2.93 +/− 0.57 (5)
mLTβR-Ig (D-3: 0.4)
1.10 +/− 0.38 (3)
0.0024
2.27 +/− 1.39 (3)
NS
mLTβR-Ig (D-3, 0: 0.4, 0.3)
0.78 +/− 0.02 (3)
<0.0001
3.95 +/− 0.53 (3)
0.046
↑
mLTβR-Ig (D0: 0.3)
0.92 +/− 0.10 (3)
<0.0001
3.25 +/− 0.15 (3)
NS
aOrgan weight as percent of total body weight. Untreated LN weight is typically 0.5% of total body weight.
bNS = not significant.
cmLTBR-Ig is a fusion protein between mLTBR extracellular domain and the CH2 and CH3 region of hIgG1.
In other embodiments, it may be desirable to administer the inhibitors of the LT pathway simultaneously with, or prior or subsequent to, administration of radiation therapy. It will be apparent to those skilled in the art what the preferred therapy is based upon individual variables such as the patient's condition and the tumor being treated.
Inhibitory anti-LTβ-R Abs and other LT-β-R blocking agents can be identified using methods previously described in the art. (co-pending U.S. application Ser. No. 08/378,968).
The LTβ-R blocking agents in one embodiment of this invention comprise soluble LT-β receptor molecules. The sequence of the extracellular portion of the human LTβ-R, is known to encode the ligand binding domain (see FIG. 1). Using that sequence information in FIG. 1 and recombinant DNA techniques well known in the art, functional fragments encoding the LTβ-R ligand binding domain can be cloned into a vector and expressed in an appropriate host to produce a soluble LTβ-R molecule.
A soluble LT-β receptor comprising amino acid sequences selected from those shown in FIG. 1 may be attached to one or more heterologous protein domains (“fusion domain”) to increase the in vivo stability of the receptor fusion protein, or to modulate its biological activity or localization.
Preferably, stable plasma proteins—which typically have a half-life greater than 20 hours in the circulation—are used to construct the receptor fusion proteins. Such plasma proteins include but are not limited to: immunoglobulins, serum albumin, lipoproteins, apolipoproteins and transferrin. Sequences that can target the soluble LTβ-R molecule to a particular cell or tissue type may also be attached to the LTβ-R ligand binding domain to create a specifically-localized soluble LTβ-R fusion protein.
All or a functional portion of the LTβ-R extracellular region (FIG. 1) comprising the LTβ-R ligand binding domain may be fused to an immunoglobulin constant region like the Fc domain of a human IgG1 heavy chain (Browning et al., J. Immunol., 154, pp. 33-46 (1995)). Soluble receptor-IgG fusion proteins are preferable, and are common immunological reagents, and methods for their construction are known in the art (see e.g., U.S. Pat. No. 5,225,538 incorporated herein by reference).
A functional LTβ-R ligand binding domain may be fused to an immunoglobulin (Ig) Fc domain derived from an immunoglobulin class or subclass other than IgG1. The Fc domains of antibodies belonging to different Ig classes or subclasses can activate diverse secondary effector functions. Activation occurs when the Fc domain is bound by a cognate Fc receptor. Secondary effector functions include the ability to activate the complement system, to cross the placenta, and to bind various microbial proteins.
If it would be advantageous to harm or kill the LT ligand-bearing target cell, one could select an especially active Fc domain (IgG1) to make the LTβ-R-Fc fusion protein. Alternatively, if it would be desirable to target the LTβ-R-Fc fusion to a cell without triggering the complement system, an inactive IgG4 Fc domain could be selected.
Mutations in Fc domains that reduce or eliminate binding to Fc receptors and complement activation have been described (S. Morrison, Annu. Rev. Immunol., 10, pp. 239-65 (1992)). These or other mutations can be used, alone or in combination, to optimize the activity of the Fc domain used to construct the LTβ-R-Fc fusion protein.
Different amino acid residues forming the junction point of the receptor-Ig fusion protein may alter the structure, stability and ultimate biological activity of the soluble LT-β receptor fusion protein. One or more amino acids may be added to the C-terminus of the selected LTβ-R fragment to modify the junction point with the selected fusion domain.
The N-terminus of the LTβ-R fusion protein may also be varied by changing the position at which the selected LTβ-R DNA fragment is cleaved at its 5′ end for insertion into the recombinant expression vector. The stability and activity of each LTβ-R fusion protein may be tested and optimized using routine experimentation and the assays for selecting LTβ-R blocking agents described herein.
Using the LTβ-R ligand binding domain sequences within the extracellular domain shown in FIG. 1, amino acid sequence variants may also be constructed to modify the affinity of the soluble LT-β receptor or fusion protein for LT ligand. The soluble LTβ-R molecules of this invention can compete for surface LT ligand binding with endogenous cell surface LT-β receptors. It is envisioned that any soluble molecule comprising a LTβ-R ligand binding domain that can compete with cell surface LT-β receptors for LT ligand binding is a LT-β-R blocking agent that falls within the scope of the present invention.
In another embodiment of this invention, antibodies directed against the human LT-β receptor (anti-LT-β-R Abs) function as LTβ-R blocking agents. The anti-LTβ-R Abs of this invention can be polyclonal or monoclonal (mAbs) and can be modified to optimize their ability to block LTβ-R signaling, their in vivo bioavailability, stability, or other desired traits.
Polyclonal antibody sera directed against the human LT-β receptor are prepared using conventional techniques by injecting animals such as goats, rabbits, rats, hamsters or mice subcutaneously with a human LT-β receptor-Fc fusion protein (Example 1) in complete Freund's adjuvant, followed by booster intraperitoneal or subcutaneous injection in incomplete Freund's. Polyclonal antisera containing the desired antibodies directed against the LT-β receptor are screened by conventional immunological procedures.
Mouse monoclonal antibodies (mAbs) directed against a human LT-β receptor-Fc fusion protein are prepared as described in Example 5. A hybridoma cell line (BD.A8.AB9) which produces the mouse anti-human LT-β-R mAb BDA8 was deposited on Jan. 12, 1995 with the American Type Culture Collection (ATCC) (Rockville, Md.) according to the provisions of the Budapest Treaty, and was assigned the ATCC accession number HB11798. All restrictions on the availability to the public of the above ATCC deposits will be irrevocably removed upon the granting of a patent on this application.
Various forms of anti-LTβ-R antibodies can also be made using standard recombinant DNA techniques (Winter and Milstein, Nature, 349, pp. 293-99 (1991)). For example, “chimeric” antibodies can be constructed in which the antigen binding domain from an animal antibody is linked to a human constant domain (e.g. Cabilly et al., U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. U.S.A., 81, pp. 6851-55 (1984)). Chimeric antibodies reduce the observed immunogenic responses elicited by animal antibodies when used in human clinical treatments.
In addition, recombinant “humanized antibodies” which recognize the LT-β-R can be synthesized. Humanized antibodies are chimeras comprising mostly human IgG sequences into which the regions responsible for specific antigen-binding have been inserted (e.g. WO 94/04679). Animals are immunized with the desired antigen, the corresponding antibodies are isolated, and the portion of the variable region sequences responsible for specific antigen binding are removed. The animal-derived antigen binding regions are then cloned into the appropriate position of human antibody genes in which the antigen binding regions have been deleted. Humanized antibodies minimize the use of heterologous (inter-species) sequences in human antibodies, and are less likely to elicit immune responses in the treated subject.
Construction of different classes of recombinant anti-LT-β-R antibodies can also be accomplished by making chimeric or humanized antibodies comprising the anti-LT-β-R variable domains and human constant domains (CH1, CH2, CH3) isolated from different classes of immunoglobulins. For example, anti-LT-β-R IgM antibodies with increased antigen binding site valencies can be recombinantly produced by cloning the antigen binding site into vectors carrying the human T chain constant regions (Arulanandam et al., J. Exp. Med., 177, pp. 1439-50 (1993); Lane et al., Eur. J. Immunol., 22, pp. 2573-78 (1993); Traunecker et al., Nature, 339, pp. 68-70 (1989)).
In addition, standard recombinant DNA techniques can be used to alter the binding affinities of recombinant antibodies with their antigens by altering amino acid residues in the vicinity of the antigen binding sites. The antigen binding affinity of a humanized antibody can be increased by mutagenesis based on molecular modeling (Queen et al., Proc. Natl. Acad. Sci. U.S.A., 86, pp. 10029-33 (1989); WO 94/04679).
It may be desirable to increase or to decrease the affinity of anti-Lβ-R Abs for the LTβ-R depending on the targeted tissue type or the particular treatment schedule envisioned. For example, it may be advantageous to treat a patient with constant levels of anti-LTβ-R Abs with reduced ability to signal through the LT-β pathway for semi-prophylactic treatments. Likewise, inhibitory anti-LTβ-R Abs with increased affinity for the LTβ-R may be advantageous for short-term treatments.
Anti-Lt-β-R Antibodies as Lt-β-R Blocking Agents
Anti-LT-β-R antibodies that act as LTβ-R blocking agents may be selected by testing their ability to inhibit LTβ-R-induced cytotoxicity in tumor cells. By testing other antibodies directed against the human LTβ receptor, it is expected that additional anti-LT-β-R antibodies that function as LTβ-R blocking agents in humans can be identified using routine experimentation and the assays described herein.
Another preferred embodiment of this invention involves compositions and methods which comprise antibodies directed against LT ligand that function as LT-β-R blocking agents. As described above for the anti-LTβ-R Abs, anti-LT ligand antibodies that function as LTβ-R blocking agents can be polyclonal or monoclonal, and can be modified according to routine procedures to modulate their antigen binding properties and their immunogenicity.
The anti-LT antibodies of this invention can be raised against either one of the two LT subunits individually, including soluble, mutant, altered and chimeric forms of the LT subunit. If LT subunits are used as the antigen, preferably they are LT-β subunits. If LT-αsubunits are used, it is preferred that the resulting anti-LT-α antibodies bind to surface LT ligand and do not cross-react with secreted LT-α or modulate TNF-R activity.
Alternatively, antibodies directed against a homomeric (LT-β) or a heteromeric (LT-α/β) complex comprising one or more LT subunits can be raised and screened for activity as LT-β-R blocking agents. Preferably, LT-α1/β2 complexes are used as the antigen. As discussed above, it is preferred that the resulting anti-LT-α1/β2 antibodies bind to surface LT ligand without binding to secreted LT-α and without affecting TNF-R activity.
The production of polyclonal anti-human LT-α antibodies is described in applicants' co-pending application (WO 94/13808). Monoclonal anti-LT-α and anti-LT-β antibodies have also been described (Browning et al., J. Immunol., 154, pp. 33-46 (1995)).
Compounds
Therapeutic compounds useful for the methods of the invention include any compound that blocks the interaction of LT-β with LT-β-receptor and therefore inhibits the LT pathway. Anti-LT compounds specifically contemplated include polyclonal antibodies and monoclonal antibodies (mAbs), as well as antibody derivatives such as chimeric molecules, humanized molecules, molecules with reduced effector functions, bispecific molecules, and conjugates of antibodies).
The invention also includes anti-LT-β and anti-LT-β receptor molecules of other types, such as complete Fab fragments, F(ab′)2 compounds, VH regions, FV regions, single chain antibodies (see, e.g., WO 96/23071), polypeptides, fusion constructs of polypeptides, fusions of LT-β receptor, and small molecule compounds such as small semi-peptidic compounds or non-peptide compounds, all capable of blocking the LT pathway.
Various forms of antibodies may also be produced using standard recombinant DNA techniques (Winter and Milstein, Nature 349: 293-99, 1991). For example, “chimeric” antibodies may be constructed, in which the antigen binding domain from an animal antibody is linked to a human constant domain (an antibody derived initially from a nonhuman mammal in which recombinant DNA technology has been used to replace all or part of the hinge and constant regions of the heavy chain and/or the constant region of the light chain, with corresponding regions from a human immunoglobin light chain or heavy chain) (see, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. 81: 6851-55, 1984). Chimeric antibodies reduce the immunogenic responses elicited by animal antibodies when used in human clinical treatments.
In addition, recombinant “humanized” antibodies may be synthesized. Humanized antibodies are antibodies initially derived from a nonhuman mammal in which recombinant DNA technology has been used to substitute some or all of the amino acids not required for antigen binding with amino acids from corresponding regions of a human immunoglobin light or heavy chain (chimeras comprising mostly human IgG sequences into which the regions responsible for specific antigen-binding have been inserted)(see, e.g., PCT patent application WO 94/04679). Animals are immunized with the desired antigen, the corresponding antibodies are isolated and the portion of the variable region sequences responsible for specific antigen binding are removed. The animal-derived antigen binding regions are then cloned into the appropriate position of the human antibody genes in which the antigen binding regions have been deleted. Humanized antibodies minimize the use of heterologous (inter-species) sequences in human antibodies and are less likely to elicit immune responses in the treated subject.
Also useful in the methods and compositions of this invention are primate or primatized antibodies.
Antibody fragments and univalent antibodies may also be used in the methods and compositions of this invention. Univalent antibodies comprise a heavy chain/light chain dimer bound to the Fc (or stem) region of a second heavy chain. Fab regions refers to those portions of the chains which are roughly equivalent, or analogous, to the sequences which comprise the Y branch portions of the heavy chain and to the light chain in its entirety, and which collectively (in aggregates) have been shown to exhibit antibody activity. A Fab protein includes aggregates of one heavy and one light chain (commonly known as Fab), as well as tetramers which correspond to the two branch segments of the antibody Y, (commonly known as F(ab)2), whether any of the above are covalently or non-covalently aggregated, so long as the aggregation is capable of selectively reacting with a particular antigen or antigen family.
In addition, standard recombinant DNA techniques can be used to alter the binding affinities of recombinant antibodies with their antigens by altering amino acid residues in the vicinity of the antigen binding sites.
Subjects
The subjects for whom the methods of the invention are intended have follicular lymphomas.
Routes of Administration
The compounds of the invention may be administered in any manner which is medically acceptable. This may include injections, by parenteral routes such as intravenous, intravascular, intraarterial, subcutaneous, intramuscular, intratumor, intraperitoneal, intraventricular, intraepidural, or others as well as oral, nasal, ophthalmic, rectal, or topical. Sustained release administration is also specifically included in the invention, by such means as depot injections. Some forms of LT blocking compounds may be suitable for oral administration, and could be formulated as suspensions or pills.
Dosages and Frequency of Treatment
The amount of and frequency of dosing for any particular compound to be administered to a patient for a given immune complex disease is a judgment made by the patient's physician, based on a number of factors. The general dosage is established by preclinical and clinical trials, which involve extensive experiments to determine the beneficial and deleterious effects on the patient of different dosages of the compound. Even after such recommendations are made, the physician will often vary these dosages for different patients based on a variety of considerations, such as a patient's age, medical status, weight, sex, and concurrent treatment with other pharmaceuticals. Determining the optimal dosage for each LT blocking compound used to treat follicular lymphoma is a routine matter for those of skill in the pharmaceutical and medical arts.
Generally, the frequency of dosing would be determined by the attending physician, and might be either as a single dose, or repeated daily, at intervals of 2-6 days, weekly, biweekly, or monthly.
Combination therapies according to this invention for treatment of follicular lymphomas together with other agents targeted at such lymphomas, including, for example, radiation, chemotherapy, or other therapies known to those skilled in the art.
An LT blocking compound of the invention is administered to a patient in a pharmaceutically acceptable composition, which may include a pharmaceutically-acceptable carrier. Such a carrier is relatively non-toxic and innocuous to a patient at concentrations consistent with effective activity of the blocking compound or other active ingredients, so that any side effects ascribable to the carrier do not vitiate the beneficial effects of the active ingredients of the composition. The composition may include other compatible substances; compatible, as used herein, means that the components of the pharmaceutical composition are capable of being commingled with the LT blocking compound, and with each other, in a manner such that there is no interaction which would substantially reduce the therapeutic efficacy of the pharmaceutical. Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets, pills or lozenges, each containing a predetermined amount of the potentiating compound as a powder or granules; as liposomes; or as a suspension in an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, an emulsion or a draught.
The compositions of the invention may be provided in containers suitable for maintaining sterility, protecting the activity of the active ingredients during proper distribution and storage, and providing convenient and effective accessibility of the composition for administration to a patient. For an injectable formulation of a LT blocking compound, the composition might be supplied in a stoppered vial suitable for withdrawal of the contents using a needle and syringe. The vial would be intended for either single use or multiple uses. The composition might also be supplied as a prefilled syringe. In some instances, the contents would be supplied in liquid formulation, while in others they would be supplied in a dry or lyophilized state, which would require reconstitution with a standard or a supplied diluent to a liquid state. Where the compound is supplied as a liquid for intravenous administration, it might be provided in a sterile bag or container suitable for connection to an intravenous administration line or catheter. In instances where the blocking compound is orally administered in tablet or pill form, the compound might be supplied in a bottle with a removable cover. The containers may be labeled with information such as the type of compound, the name of the manufacturer or distributor, the indication, the suggested dosage, instructions for proper storage, or instructions for administration.
REFERENCES
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12578016
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biogen idec ma inc.
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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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424/130.1
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Apr 1st, 2022 05:10PM
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Apr 1st, 2022 05:10PM
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Biogen
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Health Care
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Pharmaceuticals & Biotechnology
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