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nasdaq:amgn
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Amgen
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May 30th, 2006 12:00AM
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May 24th, 2002 12:00AM
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https://www.uspto.gov?id=US07052916-20060530
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Polypeptide analyses using stable isotope labeling
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Methods for incorporating a stable isotope into a protein or peptide fragment are disclosed. The methods include providing isolated isotope-labeled protein or peptide fragments and analyzing the isolated labeld fragments. In another aspect of the invention, methods for quantitatively comparing peptides in two protein or peptide fragment mixtures are provided. In these methods, protein levels are measured using stable-isotope coded protein or peptide fragments.
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7052916
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1. A method for incorporating a stable isotope into a protein or peptide fragment, comprising:
(a) reacting a cysteinyl residue in a protein or peptide fragment with an affinity reagent to provide an affinity-labeled protein or peptide fragment, wherein the affinity reagent is coupled to the cysteinyl residue through a cleavable linkage;
(b) isolating the affinity-labeled protein or peptide fragment on a solid phase;
(c) releasing the protein or peptide fragment from the solid phase by cleaving the cleavable linkage; and
(d) reacting the released protein or peptide fragment with an agent comprising one or more stable isotopes to provide a stable isotopically-labeled protein or peptide fragment.
2. The method of claim 1, wherein the cleavable linkage is a disulfide linkage.
3. The method of claim 1, wherein the affinity reagent is a biotinylation reagent.
4. The method of claim 1, wherein the affinity reagent is N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide.
5. The method of claim 1, wherein the agent is N-ethyl iodoacetamide.
6. The method of claim 1, wherein the agent having one or more stable isotopes is N-ethyl-d5 iodoacetamide.
7. A method for incorporating a stable isotope into a protein or peptide fragment, comprising:
(a) reacting a cysteinyl residue in a protein or peptide fragment with an affinity reagent to provide an affinity-labeled protein or peptide fragment, wherein the affinity reagent is coupled to the cysteinyl residue through a cleavable linkage;
(b) isolating the affinity-labeled protein or peptide fragment on a solid phase;
(c) regenerating the protein or peptide fragment by release from the solid phase by cleaving the cleavable linkage; and
(d) reacting the cysteinyl residue of the regenerated protein or peptide fragment with an agent comprising one or more stable isotopes to provide a stable isotopically-labeled protein or peptide fragment.
8. The method of claim 7, wherein the cleavable linkage is a disulfide linkage.
9. The method of claim 7, wherein the affinity reagent is a biotinylation reagent.
10. The method of claim 7, wherein the affinity reagent is N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide.
11. The method of claim 7, wherein the agent is N-ethyl iodoacetamide.
12. The method of claim 7, wherein the agent having one or more stable isotopes is N-ethyl-d5 iodoacetamide.
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BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is relates to proteomics and more specifically involves analyzing polypeptides and peptides using stable isotope labeling techniques coupled with mass spectrometry.
2. Description of Related Art
Proteomics usually involves separating individual proteins using two dimensional gel electrophoresis (2D-PAGE) and comparing stain density. Proteomic analyses using 2D-PAGE can be automated, but only at significant expense requiring automated gel staining and destaining devices, imaging equipment, imaging software, spot cutting robotics, automated in-gel digestion, robotic MALDI plate spotting, and mass spectrometry. Even expensive high throughput 2D-PAGE systems are known to have difficulties with higher molecular weight proteins, membrane proteins, and highly acidic or basic proteins. Despite the high resolution separations of proteins provided by 2D-PAGE, the method still suffers from a limited dynamic range and low abundance proteins are very difficult to detect in the presence of high abundance proteins. Nevertheless, 2D-PAGE has been the state of the art for making quantitative proteomic measurements.
Reversible biotinylation of cysteinyl peptides has been utilized in a method for the rapid identification of components in a protein mixture (Spahr et al., 2000). In a representative method, a protein mixture is digested and the resulting peptide fragment's cysteine residues biotinylated with a cleavable biotinylation reagent (i.e., N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide, commonly known to as “biotin-HPDP”). The biotinylated peptides are then isolated using avidin affinity chromatography and then eluted from the avidin by treatment with dithiothreitol (DTT), which cleaves the link between the biotin and peptide fragment releasing the peptide fragment. The released peptide fragment has free sulfhydryl groups that are alkylated by treatment with iodoacetamide. The alkylated peptide fragments are then analyzed by LC/MS/MS to provide proteomic information. The method described above simplifies complex peptide mixtures for proteomic analysis.
Another analytical method involves labeling proteolytic peptides with different stable isotopes depending on the protein source (e.g., control cells versus stimulated cells). Identical peptides labeled with different isotopes have nearly equivalent chemical properties, so pairs of peptides differing only in the label will elute approximately at the same time and exhibit identical ionization efficiency. The first example of this method was the use of whole cell 15N labeling to compare wild type and mutant cell lines. This approach is limited to studies of cultured cells, and the isotope coding involves the incorporation of varying numbers of nitrogen atoms in each peptide, hence varying mass differences from peptide to peptide.
Another approach involves N-terminally labeling proteolytic peptides with isotope-coded nicotinic acid derivatives. This method has a side benefit of directing fragmentation in MS/MS. More recently, whole cell labeling with 13C lysine has been shown to be a simple way to introduce a constant mass shift in tryptic peptides.
In addition to the isotope labeling methods noted above, complex protein mixtures have also been quantitatively analyzed using isotope-coded affinity tags and mass spectrometry (Aebersold et al., 1999). The analysis is based on labeling a protein's cysteine residues with an isotope-coded affinity tag (ICAT) and subsequent analysis of the tagged protein, or fragment thereof, by mass spectrometry. The ICAT reagents employ cysteine-specific chemical reactivity, an isotope coded linker, and a biotin affinity tag, and introduce a constant mass difference for each cysteine present in the peptide. The ICAT reagent includes a reactive functional group having specificity toward sulfhydryl groups, a biotin affinity tag, and an isotope labeled linker covalently linking the sulfhydryl reactive group with the biotin tag. An advantage of this method is that complex tryptic peptide mixtures can be simplified by the selective isolation of peptides containing cysteine, which is one of the least common amino acids, thus approaching the ideal of obtaining a single peptide per protein.
In a representative method, the cysteinyl residues in a reduced protein sample representing one cell state are derivatized with one isotopic form (e.g., light form, no isotope label) of the ICAT and the equivalent groups in a second cell state are derivatized with another isotopic form (i.e., heavy form, isotope labeled). The two samples are then combined, enzymatically cleaved to produce peptide fragments, and the biotin tagged fragments isolated by avidin affinity chromatography. The isolated fragments are then released and analyzed by microLC-MS/MS. The quantity and sequence identity of the proteins from which the fragments are derived are determined by automated multistage mass spectrometry. Despite the utility of the ICAT method described above, the method requires the use of the relatively sophisticated and expensive ICAT reagent. Furthermore, the mass spectra of tagged protein fragments is obscured by high intensity ions related to the reagent.
Despite the advances in protein analysis, there exists a need for rapid, reliable, and efficient methods for analyzing complex protein mixtures. The present invention seeks to fulfill this need and provides further related advantages.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides methods for incorporating a stable isotope into a protein or peptide fragment and analyzing the isotope labeled protein or peptide fragment. More particularly, such a method involves causing a protein or peptide fragment to react with an agent that includes one or more stable isotopes to provide an isotope-labeled protein or peptide fragment. In one embodiment, the protein or peptide fragment is an isolated protein or peptide fragment that contains a reactive group capable of reacting with the agent that includes one or more stable isotopes. Such reactive groups can include a cysteinyl residue sulfhydryl group, thiol groups that are introduced into the protein or peptide fragment, or other reactive groups. Thus, the isotope-labeled protein or peptide fragment may be, but is not limited to, a cysteine containing protein or fragment or a tryptophan-containing protein or peptide fragment that is labeled through the protein's or peptide fragment's thiol-modified tryptophan residue.
In one embodiment the protein or peptide fragment for reaction with the isotope-labeled agent is obtained by reacting a cysteinyl residue in a protein or peptide fragment with an affinity reagent, isolating the affinity-labeled protein or peptide fragment on a solid phase, and then releasing the isolated protein or peptide fragment from the solid phase. In one embodiment, the released protein or peptide fragment is the same as the protein or peptide fragment reacted with the affinity reagent.
In another aspect of the invention, methods for measuring protein levels in two protein or peptide fragment mixtures are provided.
In one embodiment of the method, the cysteinyl residues of proteins in first and second protein mixtures are reacted with an affinity reagent to provide first and second affinity-labeled protein mixtures. The first and second affinity-labeled proteins are isolated on a solid phase to provide first and second isolated affinity-labeled proteins. The first and second isolated proteins are released from the solid phase to provide first and second released proteins. The first and second released proteins are then reacted with an isotope-coded agent to provide first and second isotope-coded proteins. The first released proteins are reacted with a first agent that includes one or more stable isotopes. The second released proteins are reacted with a second agent that includes no stable isotopes or fewer stable isotopes than the first agent. The first and second isotope-coded proteins are then analyzed. In one embodiment, the first and second isotope-coded proteins are combined and then analyzed by mass spectrometry. The mass spectral analysis of the first and second isotope-coded proteins provide quantitative information relating to the difference in the amounts (e.g., expression levels) of the proteins in the first and second protein mixtures.
In another embodiment of the method, the cysteinyl residues of proteins in first and second protein mixtures are reacted with an affinity reagent to provide first and second affinity-labeled protein mixtures. The first and second affinity-labeled protein mixtures are then digested to provide peptide fragments, those containing cysteinyl residues being affinity labeled. The first and second affinity-labeled peptide fragments are isolated on a solid phase to provide first and second isolated affinity-labeled peptide fragments. The first and second isolated peptide fragments are released from the solid phase to provide first and second released peptide fragments. The first and second released peptide fragments are then reacted with an isotope-coded agent to provide first and second isotope-coded peptide fragments. The first released peptide fragments are reacted with a first agent that includes one or more stable isotopes. The second released peptide fragments are reacted with a second agent that includes no stable isotopes or fewer stable isotopes than the first agent. The first and second isotope-coded peptide fragments are then analyzed. In one embodiment, the first and second isotope-coded peptide fragments are combined and then analyzed by mass spectrometry. The mass spectral analysis of the first and second isotope-coded peptide fragments provide quantitative information relating to the difference in the amounts (e.g., expression levels) of the proteins in the first and second protein mixtures.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:
FIG. 1 is a flow diagram illustrating a representative method of the invention. Sample proteins are reduced and biotinylated with a reagent that has a reducible linker. Next the samples are digested with a protease such as trypsin. The biotinylated peptides are isolated using avidin beads, and eluted with a reducing reagent. The thiols on the cysteines are then alkylated with either a light reagent (N-ethyl iodoacetamide) or a heavy reagent (N-d5-ethyl iodoacetamide), and then the two samples are mixed prior to any further HPLC fractionation or mass spectrometry.
FIG. 2 is a graph plotting the ratio (D0/D5) versus intensity (counts) illustrating increasing error with decreasing ion intensity. Bovine serum albumin was labeled 1:1 with N-ethyl and N-d5-ethyl iodoacetamide, digested with trypsin, and analyzed four times by LC/MS. The ratio of N-ethyl versus N-d5-ethyl iodoacetamide labeling (D0)/D5) for various ions is plotted against ion intensity.
FIG. 3 compares mass chromatograms of m/z 655.4, which corresponds to the quadruply-charged ion, (M+H4)+4, of the peptide VIHDHFGIVEGLMTTVHAITATQK from G3P_RABIT, derived from a representative method of the invention (top chromatogram, labeled ICRAP) and a representative isotope coded affinity tag method (bottom chromatogram, labeled ICAT). Three other non-cysteine containing peptides were found in the ICAT preparation that produced a sufficiently intense ion to trigger MS/MS data acquisition—NVLQPSSVDSQTAMVLVNAIVFK and ILELPFASGTMSMLVLLPDEVSGLEQLESIINFEK from OVAL_CHICK, and VAGTWYSLAMAASDISLLDAQSAPLR from LACB_BOVIN. These peptides were not found in the preparation derived from the method of the invention.
FIG. 4 compares mass chromatograms of heavy and light isotope labeled peptide, LAACFLDSMATLGLAAYGYGIR, from phosphorylase. The heavy isotope labeled peptide always elutes before the light isotope labeled peptide. However, for labeling with an isotope coded affinity tag method (labeled ICAT), where the heavy isotope labeling incorporates eight deuterium atoms, the difference in elution times is greater than for a representative method of the invention (labeled ICRAP) where the heavy isotope label has five deuterium atoms.
FIG. 5 compares the mass spectrum of peptide ions derived from an isotope coded affinity tag method (top panel, labeled ICAT) with the mass spectrum of peptide ions derived from the method of the invention (bottom panel, labeled ICRAP) for ICGGWQMEEADDWLR. The ICAT method promotes the formation of higher charge states compared to labeling in accordance with the present invention.
FIG. 6 compares the MS/MS spectrum of m/z 753.7, the triply charged ion for ICGGWQMEEADDWLR obtained from an isotope coded affinity tag method (top panel, labeled ICAT, labeled with the D8 ICAT reagent) with the MS/MS spectrum of the doubly charged ion at m/z 947.4 from the same peptide obtained from a representative method of the invention (bottom panel, labeled ICRAP, labeled with a DO reagent). The most abundant ion in the top panel is a fragment derived from the ICAT adduct. All sequence-specific fragment ions have been labeled as b- or y-type ions (**). Note that the spectrum of the doubly charged peptide obtained by the method of the invention exhibits a complete series of y-type ions, whereas the spectrum of the triply charged peptide obtained by the ICAT method is incomplete. This is partly due to the selection of a triply charged ion as the precursor for the ICAT spectrum.
DETAILED DESCRIPTION OF THE INVENTION
In one aspect, the present invention provides a method for incorporating a stable isotope into a protein or peptide fragment. In the method, a protein or peptide fragment is reacted with an agent that includes one or more stable isotopes to provide an isotope-labeled protein or peptide fragment.
In the method, a stable isotope is incorporated into the protein or peptide fragment through the reaction of an amino acid residue of the protein or peptide fragment with an agent that includes one or more stable isotopes. Suitable amino acid residues include any residue capable of coupling with the isotope-labeled agent and include cysteine, lysine, hydroxylysine, serine, threonine, hydroxyproline, asparagine, methionine, arginine, histidine, tryptophan, phenylalanine, tyrosine, aspartic acid, and glutamic acid. Suitable agents include those capable of coupling with an amino acid residue such as, for example, alkylating agents and acylating agents. In one embodiment, the isotope-labeled protein or peptide fragment is a cysteine-containing protein or peptide fragment that is labeled through the protein's or peptide fragment's cysteinyl residue sulfhydryl group. The peptide fragment can be obtained from proteolytic digestion of a protein.
In one embodiment of the method, the protein or peptide fragment for reaction with the isotope-labeled agent is obtained by reacting a cysteinyl residue in a protein or peptide fragment with an affinity reagent; isolating the affinity-labeled protein or peptide fragment on a solid phase; and releasing the isolated protein or peptide fragment from the solid phase. The cysteinyl residue for reaction with the agent can be made available by reducing a protein or peptide fragment.
As used herein, the term “affinity reagent” refers to a reagent that introduces one member of a specific binding pair into the protein or peptide fragment such that the resulting protein or peptide fragment, referred herein to as an “affinity-labeled” protein or peptide fragment, can be captured and isolated by a solid phase bearing the other member of the specific binding pair. Suitable specific binding pairs are known and include, for example, sugar ligand and lectin, hapten/antigenic determinant ligand and antibody, Fc ligand and protein A, nucleic acid and complementary nucleic acid oligomer, polymer, or analog, among others. In one embodiment, the specific binding pair is a biotin/avidin system. In such a system, the affinity reagent is a biotinylation reagent, the affinity-labeled protein or peptide fragment is a biotinylated protein or peptide fragment, and the solid phase is an avidin solid phase.
A biotinylation reagent useful in the method of the invention includes a biotin moiety covalently attached to a protein reactive moiety such that the protein reactive moiety when coupled to protein provides a cleavable linkage intermediate the biotin moiety and protein reactive moiety. Cleavage of the linkage facilitates the release of the protein or peptide fragment from the solid phase. In one embodiment, the cleavable linkage is a disulfide linkage.
The reagent's biotin moiety can be any one of a variety of biotin derivatives and analogs that are effective in avidin binding. Suitable biotin moieties include those moieties that enable the biotinylated peptide fragment to be isolated by avidin and related avidin proteins. Representative biotin moieties include biotin derivatives such as iminobiotin, biocytin, and caproylamidobiotin, and biotin analogs such as desthiobiotin and biotin sulfone. In one embodiment, the biotin moiety is biotin.
The reagent's protein reactive moiety is a functional group that is reactive with a protein residue's functional group. The reagent's protein reactive moiety can be reactive toward a variety of functional groups including, for example, a sulfhydryl group (cysteine), an amino group (lysine), a hydroxy group (serine), and a carboxy group (glutamic acid). In one embodiment, the reactive moiety is reactive toward a sulfhydryl group. Suitable protein reactive functional groups include acylating groups (e.g., carboxylic acids and their reactive derivatives) and alkylating groups (e.g., (α-halo carboxylic acids and their derivatives), among others In one embodiment, the protein reactive moiety is a pyridyldithiol moiety.
In one embodiment, the biotinylation reagent is N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide, Biotin-HPDP, commercially available from Pierce Chemical Co., Rockville, Ill.
As noted above, in one embodiment of the method, the affinity-labeled protein or peptide fragment is isolated on a solid phase. Suitable solid phases include any solid phase capable of capturing the affinity-labeled protein or peptide fragment. The solid phase bears the complement member of the binding pair. For biotinylated proteins and peptide fragments, the solid phase is an avidin solid phase. As used herein, the term “avidin” refers to any biotin-binding protein other than an immunoglobulin that binds biotin including both natural proteins and recombinant and genetically engineered proteins. The term includes the two common biotin-binding proteins known as “egg white or avian avidin” and “streptavidin.” Egg white or avian avidin, commonly referred to simply as avidin, is a protein that is a constituent of egg white and forms a noncovalent complex with biotin. Streptavidin is a protein isolated from the actinobacterium Streptomyces avidinii and also forms a noncovalent complex with biotin. Other bacterial sources of biotin binding proteins are also known. Both egg white avidin and streptavidin are tetrameric proteins in which the biotin binding sites are arranged in pairs on opposite faces of the avidin molecule. The term also refers to avidin derivatives including succinyl avidin, ferritin avidin, enzyme avidin and crosslinked avidin.
In one embodiment of the method, the protein or peptide fragment for reaction with the isotope-labeled agent is ultimately obtained by releasing the isolated protein or peptide fragment from the solid phase. In one embodiment the protein or peptide fragment is released from the solid phase by cleaving the cleavable linkage, for example, a disulfide linkage, incorporated into the protein or peptide fragment on reaction with the affinity reagent. Release from the solid phase provides a protein or peptide fragment having a sulfhydryl group that is reactive toward the isotope-labeled agent. In one embodiment, the released protein or peptide fragment is the same as the protein or peptide fragment reacted with the affinity reagent.
The stable isotope is incorporated into the released protein or peptide fragment by reaction with an isotope-labeled agent. The released protein or peptide fragment can be alkylated with an alkylating agent that includes one or more stable isotopes. In one embodiment the isotope-labeled agent is N-ethyl-d5-iodoacetamide. N-Ethyl-d5-iodoacetamide can be prepared as described in Example 1. In another embodiment the isotope-labeled agent is acrylamide-d3 (CD2═CD—C(═O)—NH2).
Generally, suitable isotope-labeled agents include any agent capable of forming a covalent bond to sulfur and bearing one or more stable isotopes. Because the proteins and peptide fragments are ultimately analyzed by mass spectrometry, preferred isotope-labeled agents include two or more stable isotopes. In one embodiment the agent includes at least two stable isotopes. In another embodiment the agent includes at least three stable isotopes. In a further embodiment the agent includes at least four stable isotopes. In another embodiment the agent includes at least five stable isotopes.
Stable isotopes useful in the method include carbon (i.e., 13C) and hydrogen (i.e., 2H, deuterium) stable isotopes.
In another aspect of the invention, methods for incorporating a stable isotope into a protein or peptide fragment and methods for measuring protein levels in two or more protein or peptide fragment mixtures are provided. The method for measuring protein levels in two or more protein mixtures includes incorporating a stable isotope into the protein or peptide fragment.
In one method, the cysteinyl residues of proteins in first and second protein mixtures are reacted with an affinity reagent to provide first and second affinity-labeled protein mixtures. Suitable affinity reagents include those described above. The cysteinyl residues can be made available by first treating the protein mixtures with a reducing agent. The first and second affinity-labeled proteins are isolated on a solid phase to provide first and second isolated affinity-labeled proteins. The first and second isolated proteins are released from the solid phase to provide first and second released proteins. Release from the solid phase can be through cleavage of a cleavable linkage incorporated into the protein or peptide fragment on reaction with the affinity reagent. In one embodiment, the released proteins include sulfhydryl groups. The first and second released proteins are then reacted with an isotope-labeled agent (e.g., a sulfhydryl alkylating agent) to provide first and second isotope-coded proteins. Suitable isotope-labeled agents include those described above. The first released proteins are reacted with a first agent that includes one or more stable isotopes (e.g., N-ethyl-d5 iodoacetamide). The second released proteins are reacted with a second agent that includes no stable isotopes or fewer stable isotopes than the first agent (e.g., N-ethyl iodoacetamide). The first and second isotope-coded proteins can then analyzed. In one embodiment, the first and second isotope-coded proteins are combined prior to analysis. The isotope-coded proteins can be analyzed by mass spectrometry. The mass spectral analysis of the first and second isotope-coded proteins provide quantitative information relating to the difference in the amounts (e.g., expression levels) of the proteins in the first and second protein mixtures.
In another method, isotope-coded peptide fragments are prepared and then analyzed. In the method, the cysteinyl residues of proteins in first and second protein mixtures are reacted with an affinity reagent to provide first and second affinity-labeled protein mixtures. Suitable affinity reagents include those described above. The cysteinyl residues can be made available by first treating the protein mixtures with a reducing agent. The first and second affinity-labeled protein mixtures are then digested to provide first and second peptide fragment mixtures, the fragments including first and second affinity-labeled peptide fragments. The first and second affinity-labeled peptide fragments are isolated on a solid phase to provide first and second isolated affinity-labeled peptide fragments. The first and second isolated proteins are released from the solid phase to provide first and second released peptide fragments. Release from the solid phase can be through cleavage of a cleavable linkage incorporated into the peptide fragment on reaction with the affinity reagent. In one embodiment, the released peptide fragments include sulfhydryl groups. The first and second released peptide fragments are then reacted with an isotope-labeled agent (e.g., a sulfhydryl alkylating agent) to provide first and second isotope-coded peptide fragments. Suitable isotope-labeled agents include those described above. The first released peptide fragments are reacted with a first agent that includes one or more stable isotopes (e.g., N-ethyl-d5 iodoacetamide). The second released peptide fragments are reacted with a second agent that includes no stable isotopes or fewer stable isotopes than the first agent (e.g., N-ethyl iodoacetamide). The first and second isotope-coded peptide fragments can then analyzed. In one embodiment, the first and second isotope-coded peptide fragments are combined prior to analysis to provide an isotope-coded peptide fragment mixture. The peptide fragment mixture can be analyzed by mass spectrometry. The mass spectral analysis of the first and second isotope-coded peptide fragments provide quantitative information relating to the difference in the amounts (e.g., expression levels) of the proteins in the first and second protein mixtures.
It will be-appreciated that the method of the invention includes measuring protein levels in two or more protein mixtures. The number of protein mixtures that can be analyzed by the method will depend on the complexity of the protein mixtures and the nature of the isotope-labeled agents. The greater the number of protein mixtures, the greater the number of distinct isotope-labeled agent required for the analysis. Each protein mixture merely requires a distinct isotope-labeled agent. Accordingly, the method of the invention is not limited to comparing protein levels in two protein mixtures, but is applicable to determining the protein levels in a plurality of protein mixtures.
In another embodiment of the method, the protein or peptide fragment for reaction with the isotope-labeled agent is obtained from a thiol-containing residue in a protein or peptide fragment. The thiol-containing residue can be formed by a variety of methods. For example, the thiol-containing residue can be obtained by isolating a tryptophan-containing protein or peptide fragment on a solid phase that provides a thiol-modified tryptophan residue on the release of the protein or peptide fragment from the solid phase. A suitable solid phase is a chlorodithiol (i.e., —S—S—Cl) solid phase that is reactive toward tryptophan residues. A suitable chlorodithiol solid phase is commercially available under the designation Pi3 Tryptophan Reagent from The Nest Group, Inc., Southborough, Mass. Release of the isolated tryptophan-containing protein or peptide fragment from the solid phase provides the thiol-modified tryptophan protein or peptide fragment, which can be labeled with the isotope-labeled agent to provide an isotope coded-protein or peptide fragment.
In another embodiment, the thiol-containing residue is a gamma-S labeled residue incorporated into a protein or peptide fragment through phosphorylation with ATP-gamma-S. The gamma-S modified protein or peptide fragment includes an available thiol group that can be reacted with the isotope-labeled agent to provide an isotope coded-protein or peptide fragment.
Thus, in another aspects, the invention provides a method for incorporating a stable isotope into a tryptophan-containing protein or peptide fragment, and a method for incorporating a stable isotope into a protein or peptide fragment that has been phosphorylated to provide an available thiol group. The isotope-coded tryptophan-containing protein or peptide fragment can be used to measure the amount of tryptophan-containing proteins or peptide fragments in mixtures as described above. Similarly, the isotope-coded phosphorylated protein or peptide fragment having an available thiol group can be used to measure the amount of phosphorylation in protein or peptide fragment mixtures as described above.
A representative method of the invention is schematically illustrated in FIG. 1. Referring to FIG. 1, in the representative method, sample proteins (e.g., isolated proteins from control cells and isolated proteins from treated cells) are reduced to provide available sulfhydryl groups which are then biotinylated with a reagent that having a reducible linker. Next the samples are digested with a protease such as trypsin. The biotinylated peptides are then isolated using avidin beads, and eluted from the avidin with a reducing reagent. The thiols on the cysteines are then alkylated with either a light reagent (e.g., N-ethyl iodoacetamide) or a heavy reagent (e.g., N-d5-ethyl iodoacetamide), and then the two samples are mixed prior to any further HPLC fractionation or mass spectrometry.
The method includes isotopic coded labeling of peptide fragments originating from proteins in each mixture and mass spectral analysis of the isotope-labeled peptide fragments to obtain quantitative information relating to the level of expression of the proteins in each mixture.
For illustration purposes, a representative embodiment of a method of the invention using a biotinylation reagent as an affinity reagent follows.
In the method, the cysteinyl residues of proteins in first and second protein mixtures are reacted with a biotinylation reagent to provide first and second biotinylated protein mixtures. The biotinylation reagent includes a biotin moiety attached to a protein reactive moiety through a cleavage disulfide linkage. Prior to biotinylation, the proteins in the mixtures can be treated with a reducing agent to make available sulfhydryl groups.
After biotinylation, the first and second protein mixtures are digested to provide first and second peptide fragments. The biotinylated fragments include peptide fragments containing cysteinyl residues that are biotinylated.
The first and second biotinylated peptide fragments are isolated by avidin affinity chromatography to provide first and second isolated biotinylated peptide fragments. Through their biotinylation, the peptide fragments can be isolated from other peptide fragments that do not include biotin, thereby selectively reducing the number of peptide fragments for analysis.
After capture on a solid phase, the first and second isolated peptide fragments are treated with a reducing agent to cleave the disulfide linkage covalently linking the peptide fragment to the captured biotin moiety resulting in the release of the isolated peptide fragments from avidin. Suitable reducing agents include disulfide reducing agents such as dithiothreitol (DTT).
The use of a biotinylation reagent that provides a disulfide linkage between the biotin moiety and the peptide fragment results in a highly selective and gentle elution of the isolated peptide fragment from the avidin solid phase. The advantage of this is that the non-specifically bound peptides will not be released, and will remain bound to the avidin beads, thereby resulting in a cleaner preparation of cysteine-containing peptides. Furthermore, reductive cleavage of the disulfide linkage provides the released peptide fragment with a sulfhydryl group.
To distinguish between the peptide fragments originating from the first and second protein mixtures, the first and second released peptide fragments are reacted with an isotope-coded alkylating agent to provide first and second isotope-coded peptide fragments. The first released peptide fragments are alkylated with a first alkylating agent that includes one or more stable isotopes, and the second released peptide fragments are alkylated with a second alkylating agent that includes no stable isotopes or fewer stable isotopes than the first alkylating agent.
In one embodiment the first alkylating agent is N-ethyl-d5 iodoacetamide, and the second alkylating agent is N-ethyl iodoacetamide. N-ethyl-d5-iodoacetamide can be prepared as described in Example 1. Briefly, reaction of ethyl amine with 1,1′-diiodoacetic anhydride provides ethyl iodoacetamide. Similar reaction with d5-ethylamine provides N-ethyl-d5 iodoacetamide. By selection of the amine and stable isotope labeled amine, a variety of suitable isotope-coded alkylating agents can be prepared.
In another embodiment, the first alkylating agent is acrylamide-d3 (CD2═CD—C(═O)—NH2), and the second alkylating agent is acrylamide. Both of these alkylating agent are commercially available. For larger peptides, the three mass unit difference obtained by alkylating with acrylamide/d3-acrylamide is less desirable as the interpretation of mass spectra obtained for these fragments can be complicated by the fragments' 13C isotopic clusters.
Generally, suitable isotope coded alkylating agents include any alkylating agent capable of forming a covalent bond to sulfur and bearing one or more stable isotopes. Because the peptide fragments are ultimately analyzed by mass spectrometry, preferred first and second isotope coded alkylating agents have molecular weights that differ by at least two, more preferably at least three, and even more preferably at least five mass units.
Stable isotopes include carbon (i.e., 13C) and hydrogen (i.e., 2H, deuterium) stable isotopes.
In the representative method, the first and second isotope-coded peptide fragments are then combined and further analyzed by mass spectrometry. The mass spectral analysis of the combined first and second isotope coded peptide fragments provide information relating to the difference in the amounts (e.g., expression levels) of the proteins in the first and second protein mixtures.
To test the yields of the method of the invention, two equal aliquots of bovine serum albumin (BSA) were prepared. For one of the aliquots, the protein was reduced, labeled with HPDP-biotin, biotinylated peptides were isolated on monoavidin beads, eluted with DTT, and alkylated with N-ethyl iodoacetamide (the light reagent). The other aliquot of BSA was reduced and alkylated with N-ethyl-d5-iodacetamide without going through the biotinylation and monoavidin procedures. The two samples were mixed and analyzed by MALDI-TOF mass spectrometry, where it was found that the biotinylation and avidin purification steps had an overall yield of approximately 75%. The yield of the alkylation step was estimated to be greater than 95% by first alkylating a sample of BSA with the light reagent, followed by a thiol quench and the addition of an excess of the heavy reagent. No alkylation by the heavy reagent was observed. Thus, the overall yield of this procedure is conservatively estimated to be in excess of 70%.
A representative method of the invention providing relative quantitative protein measurements is described in Example 2. A representative method for providing relative quantitative protein measurements by an isotope coded affinity tag (ICAT) method is described in Example 3. The ability of the two methods to provide relative quantitative measurements was tested using two mixtures of protein standards in which proteins in the two mixtures differed in their amounts. The results are summarized below in Table 1. In Table 1, the ratio of heavy/light label for the method of the invention is provided under the heading referred to as ICRAP (for “isotope coded reducible affinity proteomics”) and the ratio for the isotope coded affinity tag method is provided under the heading referred to as ICAT (for “isotope coded affinity tag”).
TABLE 1
Comparison of Quantitative Results of Protein Standard Mix.
Protein
Peptide
Expected
CAT
ICRAP
LCA_BOVIN
ALCSEK
1.0
.23
—
LDQWLCEK
1.0
.87
0.88
FLDDDLTDDIMCVK
.87
0.87
DDQNPHSSNICNISCDK
.0
OVAL_CHICK
YPILPEYLQCVK
0.5
.65
0.62
GSIGAASMEFCFDVFK
.88
0.47
VHHNANENIFYCPIAIMSALAMVYLGAK
0.41
ADHPFLFCIK
0.47
PHS2_RABIT
WLVLCNPGLAEIIAER
3.3
.27
2.65
ICGGWQMEEADDWLR
.17
2.66
LAACFLDSMATLGLAAYGYGIR
.46
2.64
TCAYTNHTVLPEALER
3.3
.09
2.73
LACB_BOVIN
WENDECAQK
0.25
0.28
LSFNPTQLEEQCHI
0.41
YLLFCMENSAEPEQSLACQCLVR
0.44
G3P_RABIT
VPTPNVSSVVDLTCR
2.0
2.8
IVSNASCTTNCLAPLAK
2.0
.84
2.03
Referring to Table 1, it was found that in some cases the observed ratios not only deviated from what was expected, but also that there was some scatter in ratios measured for peptides from the same protein. Some of the scatter could be due to the fact that ratios derived from lower intensity ions are likely to have a higher degree of error.
To establish the degree to which ion intensity affects errors in the ion intensity ratios, a mixture of BSA labeled 1:1 with N-ethyl iodoacetamide and N-ethyl-d5 iodoacetamide was digested with trypsin and varying amounts were repeatedly analyzed by LC/MS. The observed ratio of light reagent labeling to heavy turned out to be closer to 1.4 and, as expected, there was greater variation in the measured ratios as the ion intensity dropped (see FIG. 2). For the given instrument settings at the time of data acquisition, the estimation of errors for ions with counts less than 200 (95% confidence limit) were estimated to be +/−30%. For ions present at counts between 200 to 1500, 1500 to 3000, and 3000 to 5000, the estimated errors dropped off with increasing intensity: +/−20%, 10%, and 5%, respectively. Estimation of the anticipated errors in the measurement of intensity ratios is of critical importance when trying to determine if minor changes in protein abundance are real. Given that the relationship between ratio error and ion intensity (see FIG. 2) depends on the instrument, as well as the instrument settings, a determination of this relationship would have to be established from time to time. Other causes for the variations in labeling of peptides derived from the same protein could be due to variability in the alkylation efficiencies, perhaps as a result of steric hindrance.
In one embodiment, the method of the invention includes the use of a biotinylation reagent having a cleavable linkage. One of the advantages of using a cleavable biotinylation linker is that rather than eluting biotinylated peptides with acidic pH, which is just as likely to release non-specifically bound peptides, a gentler elution can be performed using reducing agents such as DTT. The significance of this is that non-cysteine containing peptides that bind monoavidin will appear as a single mass spectrometric peak without the presence of a heavy or light ICAT-labeled partner. The presence of such ions could easily be confused as being an ICAT-labeled peptide that is completely up or down regulated. The distinction can only be made by tandem mass spectrometric sequencing. In the data analysis of the ICAT experiments of the protein standard mixtures, a database search of the MS/MS spectra revealed the presence of four peptides that were identified but did not contain cysteine, and therefore could not be ICAT labeled (e.g., FIG. 3). None of these peptides were found in the data set obtained by the method of the invention. By reducing the level of non-specifically bound peptides, in data dependent LC/MS/MS mode the mass spectrometer will spend less time sequencing unlabeled uninteresting peptides.
Another distinction between the method of the invention and the isotope coded affinity tag (ICAT) method was the observation that peptides labeled with the heavy and light versions had greater separations for ICAT labeled peptides. The heavy ICAT reagent contains eight deuterium atoms, whereas the heavy reagent in the method of the invention has five deuterium atoms. A difference of 5 mass units between the heavy and light labeled peptides obtained by the method of the invention is sufficient in most cases to allow a clear distinction between 13C isotope clusters, yet will not result in as much of a chromatographic separation as observed in ICAT labeled peptides (see FIG. 4). The significance of this is that one has to be a bit more careful when trying to quantitate if the two isotopically labeled forms of the same peptide elute differently. Although labeled peptides obtained by the method of the invention tend to co-elute to a greater extent than ICAT labeled peptides, in practice, for both of these methods it is necessary to perform both LC/MS and LC/MS/MS experiments. The former is for obtaining good quantitative measurements, and the latter is for peptide identification.
Another concern with the ICAT reagent is that it adds approximately 450 mass units for each cysteine present in any given peptide, which can result in a substantial increase in mass for peptides with more than one cysteine. Furthermore, the addition of the ICAT reagent tends to increase the charge state for many peptides (see FIG. 5). The MS/MS spectra of doubly charged precursor ions generally provide a fragmentation pattern that gives more complete coverage of the sequence, as compared to that produced by triply-charged precursors (see FIG. 6). In comparison, labeled peptides obtained from the method of the invention add only 90 mass units per cysteine, and there is no increase in peptide charge states (see FIG. 5), which will usually result in a more favorable fragmentation pattern (see FIG. 6). Also, ICAT labeling introduces a number of functional groups that can be sites of labile fragmentation, which results in MS/MS spectra having ICAT-specific fragment ions, in addition to the sequence-specific peptide fragment ions. ICAT fragments and losses of ICAT from precursors and sequence-specific fragment ions can be observed. The ions at m/z 284 (from the light ICAT reagent) and m/z 288 are often the most prominent ions in ICAT-labeled peptide MS/MS spectra (see FIG. 6). Because there is only a limited amount of precursor ion current available for producing fragment ions, shunting a significant portion of this current into the production of uninformative fragment ions can limit the sensitivity. In contrast, labeling obtained by the method of the invention is not significantly different from the standard iodoacetamide alkylation of cysteine, which is a modification that is relatively stable upon collisional activation.
One difference between the method of the invention and the ICAT method is that in the method of the invention the isotopically labeled peptides are not mixed together until late in the procedure. This adds to the amount of work required in that two samples are separately digested with trypsin, biotinylated, isolated on avidin, released and labeled prior to mixing. In contrast, the ICAT procedure introduces the isotopic labeling early in the experiment, and then the combined samples are worked up as a single mixture. It might be argued that working up two separate samples and mixing late in the procedure is more likely to introduce artifacts, where proteins might appear to be up or down regulated, but, in fact, additional protein losses were suffered for one of the two samples. In general, this will not be a problem if two conditions can be met: (1) differential losses suffered in the separate work ups are non-specific; and (2) most proteins in the mixture are not up or down regulated. Condition number one seems reasonable; for example, it is unlikely that slightly different avidin yields or protein precipitation recoveries will be selective for specific proteins. If the second condition is met, then one could establish a ratio that represents no change in relative protein amounts. For example, if one of the samples suffered more overall protein losses in one of the precipitation steps compared to the second samples, then instead of observing a ratio of 1:1 for a protein that did not change its relative amounts, one might observe a ratio of 1:1.2. Basically, the data analysis would be to plot out a histogram of the different ratios, assume that the most common ratio represents no change in relative amounts, and then establish confidence intervals beyond which are cases where the proteins did change. In short, separate work ups for two samples is not all that likely to result in artifacts, although it is a bit more work. One additional disadvantage should be mentioned, which is that the HPDP-biotin reagent has only limited solubility. The maximum concentration of this reagent is 2 mM if a 4 mM stock solution in DMSO is diluted 1:1 with the sample. In order to obtain a sufficient molar excess of the reagent over cysteine thiols, either one may be limited in the total amount of protein that can be analyzed, or the volume of the reaction needs to be increased.
The present invention provides an advantageous method for making relative quantitative measurements of proteins in mixtures. In one embodiment of the method, cysteine-containing peptides are isolated via avidin beads using biotinylation reagent that includes a spacer arm that can be cleaved using reducing agents. The biotinylation reagent can be a commercially available reagent. The stable isotope labeling is accomplished by alkylating the released cysteine thiols using reagents (e.g., N-ethyl iodoacetamide and N-d5-ethyl iodoacetamide) that can be synthesized in a single step using inexpensive reagents. The five mass unit difference between the two alkylating agents is sufficient to avoid contributions of ion intensity from the naturally occurring 13C isotope peaks, yet minimizes the differences in reversed phase HPLC retention time for identical peptides labeled with the two reagents. The advantage of using a biotinylation reagent with a cleavable linker is that it reduces the possibility of observing unlabeled peptides that may non-specifically bind to the avidin beads. In the method, peptides are gently eluted from avidin using reducing agents, which has the affect of minimizing non-specifically bound peptides (fewer non-specifically bound peptides, including those lacking cysteine, are observed). In addition, the labeling does not greatly increase the peptide mass and charge in electrospray ionization, which has beneficial effects on fragmentation characteristics upon low energy collision induced dissociation.
In comparison to the isotope coded affinity tag (ICAT) method, the change in peptide mass introduced by the method of the invention is minimal, which has a favorable impact on the fragmentation of the peptide in MS/MS. There is no increase in the number of charges on the peptide ions when labeled in accordance with the present method, whereas introduction of an ICAT label often increases the intensity of higher charge state ions. Whereas doubly charged precursor ions tend to provide fragmentations with more complete sequence coverage, ICAT labeled precursor ions typically have three or more charges. Furthermore, the most abundant ions in MS/MS spectra of ICAT labeled peptides tend to be ICAT related fragment ions, which are absent from peptides labeled using the method of the invention.
The following examples are provided for the purpose of illustrating, not limiting, the invention.
EXAMPLES
Example 1
Synthesis of Representative Isotope Coded Alkylating Reagents: N-(Ethyl-d5) Iodoacetamide and N-Ethyl Iodoacetamide
N-(Ethyl-d5) Iodoacetamide. Ethyl-d5-amine hydrochloride (0.85 g, 9.81 mmol) was suspended in a solution of iodoacetic anhydride (3.47 g, 9.81 mmol) in dichloromethane (40 mL) and cooled to 0° C. A solution of 3.6 mL, 20.6 mmol) of N,N-diisopropylethylamine in dichloromethane (10 mL) was added dropwise over 20 minutes. The reaction was stirred at 0° C. for 30 minutes then allowed to stir to room temperature for 4 hours. The solvent was evaporated to give a yellow syrup. The syrup was dissolved in ethyl acetate (100 mL) and washed with 1N hydrochloric acid (2×50 mL), then saturated sodium bicarbonate solution (2×50 mL), then saturated sodium chloride solution (50 mL). After drying over anhydrous magnesium sulfate, the organic solution was filtered and evaporated to give a light yellow solid. Purification by flash chromatography on silica gel (50 g) using 1:1 ethyl acetate/hexane for the elution gave the desired product as a white solid (446 mg, 21%). 1H NMR: δ (chloroform-d), 6.04 (1H, br s), 3.68 (2H, s). 13C NMR: δ (chloroform-d), 166.6, 34.6, 13.3, -0.3. MS: 219 (M+H)+.
N-Ethyl Iodoacetamide. N-ethyl iodoacetamide was prepared from ethylamine hydrochloride in a manner analogous to that described above. 1H NMR: δ (chloroform-d), 6.37 (1H, br s), 3.67 (2H, s), 3.28 (2H, m), 1.13 (3H, t). 13C NMR: δ (chloroform-d), 166.9, 35.3, 14.3, -0.3. MS: 214 (M+H)+.
Example 2
Reversible Biotinylation of a Representative Protein Mixture
Preparation of a representative protein mixture. Protein standards were purchased from Sigma Chemical Co. (St. Louis, Mo.). Two protein mixtures were made containing hen ovalbumin (430 and 215 ug/ml), bovine beta-lactoglobulin (80 and 20 ug/ml), rabbit glyceraldehyde-3-phosphate dehydrogenase (49 and 98 ug/ml), rabbit phosphorylase b (97 and 323 ug/ml), and bovine alpha-lactalbumin (120 and 120 ug/ml).
Protein Reduction. Pelleted protein sample (25–200 ug) is solubilized in 100 ul of 8 M urea (stored over AG 501-X8 mixed bed resin to remove cyanate), 0.1 M TRIS pH 8.2, 1 mM EDTA, and heated at 100° C. for 5 minutes. The sample is cooled to room temperature and reduced using a final concentration of 5 mM tris(2-carboxyethyl)phosphine (TCEP) (Pierce Chemical Co., Rockford, Ill.). Nitrogen is blown over the sample and incubated for 10 minutes at 37° C. The excess reducing agent is removed using a chloroform/methanol/water extraction, and the resulting protein pellet was briefly dried using a vacuum centrifuge before immediately biotinylating.
Protein Biotinylation and Digestion. The dried pellet was solubilized in 50 ul of 8 M urea, followed by the addition of 50 ul of 0.1 M TRIS pH 8.2 containing 5 mM EDTA. The sample was incubated for 90 minutes in the dark under nitrogen following the addition of 100 ul of 4 mM biotin-HPDP (Pierce Chemical Co., Rockford, Ill.) stock solution in neat DMSO. The biotin-HPDP was removed using the chloroform/methanol/water extraction, and the pellet was dried briefly using a vacuum centrifuge before being resolubilized in 5 ul of 8 M urea. Next, 45 ul of 0.1 M TRIS pH 8.2 containing 1 mM EDTA was added to give a final urea concentration of 0.8 M before the addition of trypsin at an enzyme to substrate ratio of 1:50 by weight. Digestion proceeded overnight at 37° C. After digestion, the trypsin was partially destroyed by boiling for 20 minutes, followed by the addition of the trypsin inhibitor TLCK to a final concentration of 50 ug/ml, which was then incubated at 37° C. for 30 minutes. To avoid any residual tryptic activity, the pH was dropped to 5.2 by the addition of 2 ul of 3 M sodium acetate before mixing with the avidin beads.
Avidin purification of cysteine-containing peptides. Purification of biotinylated peptides was carried out using 50–200 ul of monoavidin beads for 50–200 ug total protein sample, and was performed batchwise in 1.5 ml eppendorf tubes. The monoavidin was washed twice using 5 bed volumes of PBS to remove preservatives, and then washed three more times using 5 bed volumes of 0.15 M sodium acetate, 0.15 M NaCl pH 5.2. The monoavidin beads were gently spun, and the supernatants removed. The reaction mixture was diluted in 0.15 M sodium acetate plus 1 mM EDTA and 0.15 M NaCl pH 5.2 to match the bed volume of monoavidin, and then combined with the avidin beads, and gently shaken on vortexer for 20 minutes at room temperature. The monoavidin beads were gently centrifuged, and the unbound supernatant was removed and saved. The monoavidin beads were washed three times in the sodium acetate pH 5 buffer and then two more times in 0.1 M TRIS pH 8.2. After the final wash, the monoavidin beads were suspended in 1 bed volume of 0.1 M TRIS pH 8.2 containing 1 mM EDTA, and dithiothreitol (DTT) was added to give a final concentration of 5 mM. The beads were gently agitated and incubated at 37° C. for 30 minutes. The samples were gently spun, and the supernatants set aside. Another bed volume of 0.1 M TRIS pH 8.2 containing 5 mm DTT and 1 mM EDTA was added to the monoavidin beads, mixed gently, centrifuged, and the supernatants removed. The two supernatants were pooled and partially vacuum centrifuged to reduce the volume to approximately 10 ul.
Alkylation of the purified cysteine-containing peptides. Alkylation of the purified cysteine-containing peptides was performed by resuspending the avidin eluates in 50 ul 8M urea followed by the addition of 50 ul of 0.1M TRIS pH 8.2. To reduce the possibility of the formation of mixed disulfides, an additional 5 mM DTT was added, and the samples were incubated at 55° C. for 20 minutes under nitrogen. The samples were cooled to room temperature and alkylating agent (either N-ethyl iodoacetamide or N-d5-ethyl iodoacetamide) was added to give a final concentration of 30 mM of the alkylating reagent. Samples were incubated under nitrogen in the dark for 30 minutes. At this point, samples labeled with different isotopes can be mixed and analyzed.
Example 3
Representative Isotope Coded Affinity Tag (ICAT) Method
ICAT reagents were supplied by Applied Biosystems Inc. (Framingham, Mass.). Dried protein (50 ug, as described above) was solubilized in 100 ul 50 mM TRIS pH 8.5 plus 0.1% SDS, and boiled for five minutes. The denatured proteins were reduced by the addition of 1 ul 1 M TCEP for 10 minutes at 37° C. Reducing agent was removed by overnight acetone precipitation at −20° C. The pellet was suspended in 100 ul TRIS/SDS (see above) and treated with 100 ug of either do or d8 ICAT reagent for 90 minutes at room temperature in the dark. The reaction was quenched by the addition of 1 ul mercaptoethanol for 30 minutes at room temperature, and separate do and d8 reactions were mixed together following the quench. Excess ICAT reagent was removed by acetone precipitation and the pelleted protein was suspended in 50 mm ammonium bicarbonate, and digested with 2 ug trypsin overnight at 37° C. with constant vortexing. The sample was boiled for 10 minutes, 100 ul 2×PBS was added, and the pH was adjusted to 5 with 5 ul 3 M sodium acetate prior to loading on the monomeric avidin column.
200 ul of a 50% slurry of monomeric avidin beads were placed in a BioRad mini column, washed twice with 1 ml of 30% acetonitrile with 0.4% TFA, followed by 1.2 ml of 2×PBS pH 7.2. To block tetrameric avidin sites, the column was washed with 0.6 ml 2 mM biotin in PBS, washed with 0.6 ml 100 mM glycine pH 2.8, and washed further with 1.2 ml 2×PBS to return the column to pH 7.2. After plugging the column, the sample (300 ul total volume) was incubated with the beads for 20 minutes with occasional mixing. The column was drained, washed with 1 ml 2×PBS, then 1 ml 1×PBS, followed with a wash of 1.25 ml of 50 mm ammonium bicarbonate in 20% methanol. The retentate was eluted with 0.8 ml of 30% acetonitrile containing 0.4% TFA.
While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.
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10154872
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immunex corporation
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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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436/86
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Mar 31st, 2022 02:17PM
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Mar 31st, 2022 02:17PM
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Amgen
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:amgn
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Amgen
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Apr 27th, 2010 12:00AM
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Nov 2nd, 2006 12:00AM
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https://www.uspto.gov?id=US07704501-20100427
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Antibodies binding to human ataxin-1-like polypeptide
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This invention relates to IMX97018, a new members of the human ataxin-1-like polypeptide family, methods of making such polypeptides, and to methods of using them to diagnose and treat neurological conditions and to identify compounds that alter IMX97018 polypeptide activities.
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7704501
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1. An isolated monoclonal antibody or monoclonal antibody fragment that binds to a polypeptide consisting of the amino acid sequence selected from the group consisting of (a) the amino acid sequence selected from the group consisting of: the amino acids 542 through 579 of SEQ ID NO:2, and the amino acids 464 through 583 of SEQ ID NO:2; (b) the amino acid sequence selected from the group consisting of: the amino acids 465 through 499 of SEQ ID NO:2, and the amino acids 431 through 499 of SEQ ID NO:2; and (c) the amino acid sequence selected from the group consisting of: the amino acids 465 through 590 of SEQ ID NO:2, and the amino acids 444 through 640 of SEQ ID NO:2.
2. The antibody of claim 1, wherein the antibody is a humanized or human antibody.
3. A hybridoma which produces the antibody of claim 1.
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3
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Serial No. 10/997,561, filed Nov. 23, 2004, now U.S. Pat. No. 7,183,380, which is a divisional of U.S. application Ser. No. 10/207,706, filed Jul. 26, 2002, now U.S. Pat. No. 6,887,687, which claims the benefit under 35 U.S.C. 119(e) of U.S provisional application Serial No. 60/309,056, filed Jul. 30, 2001. The entire disclosQres of these applications are relied upon and incorporated by reference herein.
FIELD OF THE INVENTION
This invention relates to IMX97018, a new ataxin-1-like human polypeptide, and to methods of making and using IMX97018 polypeptides.
BACKGROUND OF THE INVENTION
Ataxin-1 is one of a group of polypeptides implicated in spinocerebrellar ataxia (SCA) conditions, also called autosomal dominant cerebellar ataxias (ADCAs). SCA disorders are heritable autosomal dominant neurodegenerative conditions commonly featuring progressive ataxia, which is irregularity of muscle action due to failure of muscle coordination. Examples of other symptoms typically shown by patients diagnosed with SCA are paralysis of the ocular muscles (ophthalmoplegia) and loss of articulation of speech (dysarthria), as associated with SCA2 and SCA7; degeneration and loss of types of brain cells, as in loss of cerebellar Purkinje cells in SCA1 for example; and dementia, as associated with SCA2 and SCA6.
Several of the SCA disorders are characterized by genetic anticipation, which is the tendency of certain diseases to appear at earlier onset ages and/or with increased severity in each successive generation. In many cases, genetic anticipation has been shown to have a biological basis in the expansion in length of a stretch of triplet repeats that encode a particular amino acid. SCA1 shows genetic anticipation and is associated with expansions in the size of a polyglutamine tract in ataxin-1 polypeptide encoded by repeated CAG codons (Matilla et al., 1993, Presymptomatic analysis of spinocerebellar ataxia type 1 (SCA1) via the expansion of the SCA1 CAG-repeat in a large pedigree displaying anticipation and parental male bias, Hum Molec Genet 2: 2123-2128). Genetic anticipation has also been observed in families afflicted with several other of the SCA disorders such as SCA2, SCA3, SCA5, SCA6, SCA7, SCA8, SCA10, and in addition to the ataxin-1 gene of SCA1, expansions of CAG repeats have been found in alleles of those SCA genes that have been characterized to date: the SCA2/ataxin-2, SCA3/MJD1, SCA6/CACNA1A, and SCA7/ataxin-7 genes. Therefore, all of the SCA genes that have been studied at the molecular level indicate that expansions of CAG repeats are correlated with the genetic anticipation observed in the corresponding SCA disorder. While family history evidence has also been presented for genetic anticipation in SCA4 (Flanigan et al., 1996, Am J Hum Genet 59: 392-399), the SCA4 gene has not yet been identified and characterized.
Ataxin-1, -2, and -7, ataxin-3/MJD1, and CACNA1A polypeptides are detected in the cytoplasm of many types of neural cells, with the levels of expression varying from cell type to cell type, and with overlapping but non-identical patterns of expression displayed by these different polypeptides. The formation of nuclear inclusion bodies immunoreactive for these SCA-related polypeptides is positively correlated with the length of the polyglutamine tracts in the polypeptides. Interactions of these polypeptides with several different types of binding partners have been reported, and these interactions are believed to contribute in different ways to development of the SCA disease condition. For example, ataxin-1 polypeptide has been found to associate with cerebellar leucine-rich acidic nuclear protein (LANP) in the nuclear matrix of Purkinje cells, the primary site of the pathological effects of SCA1 (Matilla et al., 1997, Nature 389: 974-978). Association with nuclear proteins is thought to alter the conformation of ataxin-3/MJD1 polypeptide, exposing the polyglutamine tract (Perez et al., 1999, Hum Mol Genet 8: 2377-2385). Certain SCA-related polypeptides have been reported to have RNA-binding activity, either as part of the SCA-related polypeptide itself, or by binding to a polypeptide with RNA-binding activity. For example, ataxin-1 polypeptide binds RNA in vitro, with the RNA-binding capability inversely proportional to the length of the polyglutamine tract (Yue et al., 2001, Hum Mol Genet 10: 25-30), and ataxin-2 interacts with ataxin-2 binding protein 1 (A2BP1), a polypeptide containing RNA-binding motifs (Shibata et al., 2000, Hum Mol Genet 9: 1303-1313). Interestingly, in cells containing ataxin-1 with an expanded glutamine tract, down-regulation of particular neuronal genes is postulated to be an early step in SCA1 pathogenesis (Lin et al., 2000, Nat Neurosci 3: 157-163). In addition, there is evidence that polyglutamine tracts tend to self-associate, sequestering polypeptides containing them in nuclear inclusions and possibly trapping other polypeptides required for cell viability, such as CREB-binding protein (CBP) (McCampbell et al., 2000, Hum Mol Genet 9: 2197-2202). One possible outcome of expression of polyglutamine-containing polypeptides in neural cells is cell death through a non-apoptotic mechanism (Evert et al., 1999, Hum Mol Genet 8: 1169-1176).
In order to develop more effective treatments for spinocerebellar conditions and diseases, such as SCA1 and SCA4, information is needed about previously unidentified or uncharacterized SCA-related polypeptides.
SUMMARY OF THE INVENTION
The present invention is based upon the discovery of a new human ataxin-1-like polypeptide, IMX97018.
The invention provides an isolated polypeptide consisting of, consisting essentially of, or more preferably, comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:2;
(b) an amino acid sequence selected from the group consisting of: amino acid 542 through amino acid 579 of SEQ ID NO:2, and amino acid 464 through amino acid 583 of SEQ ID NO:2;
(c) an amino acid sequence selected from the group consisting of: amino acids 185 through 195 of SEQ ID NO:2, amino acids 213 through 223 of SEQ ID NO:2, amino acids 190 through 218 of SEQ ID NO:2, and amino acids 185 through 223 of SEQ ID NO:2;
(d) a fragment of the amino acid sequences of any of (a)-(c) comprising at least 20 contiguous amino acids;
(e) a fragment of the amino acid sequences of any of (a)-(c) comprising at least 30 contiguous amino acids;
(f) a fragment of the amino acid sequences of any of (a)-(c) having IMX97018 polypeptide activity;
(g) a fragment of the amino acid sequences of any of (a)-(c) comprising AXH domain amino acid sequences;
(h) amino acid sequences comprising at least 20 amino acids and sharing amino acid identity with the amino acid sequences of any of (a)-(g), wherein the percent amino acid identity is selected from the group consisting of: at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 99%, and at least 99.5%;
(i) an amino acid sequence of (h), wherein a polypeptide comprising said amino acid sequence of (h) binds to an antibody that also binds to a polypeptide comprising an amino acid sequence of any of (a)-(g); and
(j) an amino acid sequence of (h) or (i) having IMX97018 polypeptide activity.
Other aspects of the invention are isolated nucleic acids encoding polypeptides of the invention, with a preferred embodiment being an isolated nucleic acid consisting of, or more preferably, comprising a nucleotide sequence selected from the group consisting of:
(a) SEQ ID NO:1; and
(b) an allelic variant of (a).
The invention also provides an isolated genomic nucleic acid corresponding to the nucleic acids of the invention.
Other aspects of the invention are isolated nucleic acids encoding polypeptides of the invention, and isolated nucleic acids, preferably having a length of at least 15 nucleotides, and preferably at least 50% of the length of SEQ ID NO:1, that hybridize under conditions of moderate stringency to the nucleic acids encoding polypeptides of the invention. In preferred embodiments of the invention, such nucleic acids encode a polypeptide having IMX97018 polypeptide activity, or comprise a nucleotide sequence that shares nucleotide sequence identity with the nucleotide sequences of the nucleic acids of the invention, wherein the percent nucleotide sequence identity is selected from the group consisting of: at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 99%, and at least 99.5%.
Further provided by the invention are expression vectors and recombinant host cells comprising at least one nucleic acid of the invention, and preferred recombinant host cells wherein said nucleic acid is integrated into the host cell genome.
Also provided is a process for producing a polypeptide encoded by the nucleic acids of the invention, comprising culturing a recombinant host cell under conditions promoting expression of said polypeptide, wherein the recombinant host cell comprises at least one nucleic acid of the invention. A preferred process provided by the invention further comprises purifying said polypeptide. In another aspect of the invention, the polypeptide produced by said process is provided.
Further aspects of the invention are isolated antibodies that bind to the polypeptides of the invention, preferably monoclonal antibodies, also preferably humanized antibodies or humanized antibodies, and preferably wherein the antibody inhibits the activity of said polypeptides.
The invention additionally provides a method of designing an inhibitor of the polypeptides of the invention, the method comprising the steps of determining the three-dimensional structure of any such polypeptide, analyzing the three-dimensional structure for the likely binding sites of substrates, synthesizing a molecule that incorporates a predicted reactive site, and determining the polypeptide-inhibiting activity of the molecule. In a further aspect of the invention, a method is provided for identifying compounds that alter IMX97018 polypeptide activity comprising
(a) mixing a test compound with a polypeptide of the invention; and
(b) determining whether the test compound alters the IMX97018 polypeptide activity of said polypeptide.
In another aspect of the invention, a method is provided identifying compounds that inhibit the binding activity of IMX97018 polypeptides comprising
(a) mixing a test compound with a polypeptide of the invention and a binding partner of said polypeptide; and
(b) determining whether the test compound inhibits the binding activity of said polypeptide.
In preferred embodiments, the binding partner is a nuclear polypeptide; more preferably, the binding partner is a leucine-rich polypeptide, and still more preferably, the binding partner is a LANP polypeptide.
Further provided by the invention is a method for decreasing SCA-promoting activity of IMX97018polyQ polypeptides, comprising providing at least one antagonist of the polypeptides of the invention; with a preferred embodiment of the method further comprising decreasing said activities in a patient by administering at least one antagonist of the polypeptides of the invention, and with a further preferred embodiment wherein the antagonist is an antisense molecule that inhibits the activity of any of said polypeptides, and with a most preferred embodiment wherein the antagonist specifically inhibits the activity of polyglutamine-containing forms of said polypeptides.
An additional aspect of the invention is a method for promoting cell death comprising providing at least one compound selected from the group consisting of an IMX97018polyQ polypeptide and agonists of said polypeptides. Further provided by the invention is a method for inhibiting cell death activity comprising providing at least one antagonist of an IMX97018polyQ polypeptide, for example wherein the cell death activity is inhibited in neuronal cells.
The invention additionally provides a method for treating a oncologic condition comprising administering at least one compound selected from the group consisting of an IMX9701 polypeptide and agonists of said polypeptide. In additional aspects of the invention, the oncologic condition is selected from the group consisting of brain tumors, glioma, glioblastoma, astrocytoma, oligodendroglioma, ependymoma, ganglioglioma, medulloblastoma, neuroectodermal tumors, and pilocytic astrocytoma.
Also provided by the invention is a method for treating a neurological condition comprising administering at least one compound selected from the group consisting of an IMX97018polyQ polypeptide and agonists of said polypeptides, wherein the conditional is characterized by excess neurological activity. In addition, the invention provides a method for treating a neurological condition comprising administering an antagonist an IMX97018polyQvpolypeptide. In further aspects of the invention, the neurological condition is selected from the group consisting of dementia, including AIDS-related dementia and Alzheimer's.
In a further aspect of the invention, a method is provided for treating a neuromuscular condition comprising administering an antagonist of an IMX97018polyQ polypeptide, for example wherein the neuromuscular condition is ataxia.
In other aspects of the invention, a method is provided for treating a neurological condition comprising administering an antagonist of the polypeptide of the invention; with a preferred embodiment wherein the neurological condition is SCA4.
A further embodiment of the invention provides a use for antagonists of the polypeptides of the invention in the preparation of a medicament for treating a neurological condition; with a preferred embodiment wherein the neurological condition is SCA4.
An additional aspect of the invention provides methods for mapping and diagnosing genetic disorders linked to human chromosome 16, wherein the disorder is preferably SCA4.
DETAILED DESCRIPTION OF THE INVENTION
Similarities of IMX97018 Structure to Ataxin-1 Polypeptides
We have identified IMX97018, a new ataxin-1-like polypeptide having structural features characteristic of mammalian ataxin-1 polypeptides; the amino acid sequence of an IMX97018 polypeptide is provided in SEQ ID NO:2, and an alignment showing the sequence similarities between IMX97018 and other ataxin-I polypeptides is presented in Table 1 in Example 1 below. Collectively, the set of polypeptides comprising IMX97018 and the ataxin-1 polypeptides presented in Table 1, along with ataxin-1 homologues from other species, are referred to as ‘ataxin-1-like’ polypeptides. The ataxin-1-like polypeptides shown in Table 1 display a high degree of similarity to each other, with the mammalian ataxin-1 polypeptides extremely similar to each other, and IMX97018 polypeptide sharing about 39% amino acid identity with human ataxin-1 and the mammalian ataxin-1 homologues.
The typical structural elements common to ataxin-1 polypeptides include an AXH domain, an ataxin-1 self-association domain, and an RNA-binding domain, and in some forms of ataxin-1 polypeptides, a polyglutamine tract resulting from expansion of CAG triplet repeats in the coding sequence (see Table 1 below). The AXH domain has been identified as a domain of 120 amino acids (SEQ ID NO:6) common to ataxin-1 polypeptides from several species, and also some HMG-box-containing polypeptides (HMG box containing protein 1 [Homo sapiens], GenBank AAB71862; HMG-box containing protein 1 [Homo sapiens], GenBank.NP—036389; and HMG-box containing protein 1 [Rattus norvegicus], GenBank NP 037353; interestingly, HMG-box-containing polypeptides have been implicated in regulation of transcription initiation). Table 1 in Example 1 shows the location of the AXH domain within ataxin-1 and IMX97018 polypeptides, from amino acid 464 through amino acid 583 of SEQ ID NO:2, with a particularly strong match between the AXH domain and the IMX97018 polypeptide from amino acid 542 through amino acid 579 of SEQ ID NO:2. A region within the ataxin-1 polypeptide sufficient for self-association in a yeast two-hybrid assay system is present at approximately amino acids 495 through 605 of human ataxin-1 (SEQ ID NO:3) (Burright et al., 1997, Hum Molec Genet 6: 513-518), which corresponds to amino acids 431 through 499 of IMX97018 polypeptide (SEQ ID NO:2). This ataxin-1 self-association region is distinct from expanded polyglutamine tracts that are also implicated in self-association of ataxin-1-like polypeptides. A region of ataxin-1 polypeptide required for RNA-binding activity extends from amino acid 541 through amino acid 767 of SEQ ID NO:4 (Yue et al., 2001, Hum Mol Genet 10: 25-30); amino acids 444 through 640 of IMX97018 polypeptide (SEQ ID NO:2) align with this portion of ataxin-1. The portion of IMX97018 polypeptide that shows the greatest degree of similarity to the ataxin-1 self-association region are approximately amino acids 465 through 499 of SEQ ID NO:2, and the portion of IMX97018 polypeptide that shows the greatest degree of similarity to the ataxin-1 RNA-binding region are approximately amino acids 465 through 590 of SEQ ID NO:2; these portions of IMX97018 polypeptide are also those that approximately correspond to the AXH domain (amino acid 464 through amino acid 583 of SEQ ID NO:2). The human ataxin-1 polypeptide amino acid sequence shown as SEQ ID NO:3 has a polyglutamine tract of 28 Gln residues with two interspersed His residues from amino acid 197 to amino acid 226 of SEQ ID NO:3. The murine and rat ataxin-1 amino acid sequences shown as SEQ ID NO:4 and SEQ ID NO:5 have only two Gln residues at the position corresponding to the polyglutamine tract in human ataxin-1. While IMX97018 does not have a polyglutamine tract, it does have two glutamine residues encoded by CAG codons in the region corresponding to the polyglutamine tract of human ataxin-1: amino acid 190 and amino acid 218 of SEQ ID NO:2. The region of SEQ ID NO:2 corresponding to the polyglutamine tract of human ataxin-1 therefore preferably includes at least one of these Gln residues, or amino acids 185 through 195 of SEQ ID NO:2, amino acids 213 through 223 of SEQ ID NO:2, amino acids 190 through 218 of SEQ ID NO:2, or amino acids 185 through 223 of SEQ ID NO:2. IMX97018 polypeptides of the invention include isolated naturally occurring polypeptides having polyglutamine tracts, and IMX97018 polypeptides produced so as to include a polyglutamine tract; such polyglutamine-containing IMX97018 polypeptides are referred to as ‘IMX97018polyQ polypeptides’ herein. Preferably, such polyglutamine tracts are greater than 30 contiguous glutamine and/or histidine residues in length; such polyglutamine tracts having more than 30 residues are referred to as expanded polyglutamine tracts. More preferably, such polyglutamine tracts are 50 or more (or 60 or more, or 70 or more, or 80 or more, or 90 or more, or 100 or more, or 150 or more, or 200 or more, or 300 or more) contiguous glutamine and/or histidine residues in length. In IMX97018 polypeptides having polyglutamine tracts, the polyglutamine sequence is preferably inserted in the polyglutamine region of the IMX97018 polypeptide, i.e. at a position between amino acids 185 through 223 of SEQ ID NO:2; and more preferably between amino acids 185 through 195, amino acids 213 through 223, or amino acids 190 through 218 of SEQ ID NO:2; and most preferably at the glutamine residue at amino acid 190 or at amino acid 218 of SEQ ID NO:2.
Therefore, IMX97018 polypeptide has an overall structure consistent with other ataxin-1-like polypeptides. The skilled artisan will recognize that the boundaries of the regions of IMX97018 polypeptides described above are approximate and that the precise boundaries of such domains, as for example the boundaries of the region corresponding to the human ataxin-1 polyglutamine tract, can also differ from member to member within ataxin-1-like polypeptide family.
Biological Activities and Functions of IMX97018 Polypeptides
Typical biological activities or functions associated with ataxin-1 and ataxin-1-like polypeptides include RNA-binding activity and self-association activity. For ataxin-1 and ataxin-1-like polypeptides comprising expanded polyglutamine tracts, activities associated with such polypeptides include promoting the formation of nuclear inclusions; binding to nuclear polypeptides, for example to leucine-rich polypeptides such as LANP; down-regulating certain genes expressed in neural tissue; and promoting cell death, preferably through a non-apoptotic mechanism. The RNA-binding and self-association activities of ataxin-1 are associated with a portion of the C-terminal portion of the polypeptide which includes the AXH domain. Thus, for uses requiring RNA-binding activity, preferred IMX97018 polypeptides include those having the RNA-binding region, that is, amino acids 465 through 590 of SEQ ID NO:2 and more preferably amino acids 465 through 583 of SEQ ID NO:2. For uses requiring self-association activity, preferred IMX97018 polypeptides include those having the self-association region, that is, amino acids 465 through 499 of SEQ ID NO:2. Preferred IMX97018 polypeptides further include oligomers or fusion polypeptides comprising at least one AXH domain portion of one or more IMX97018 polypeptides, and fragments of any of these polypeptides that have RNA-binding activity or self-association activity. The RNA-binding activity of IMX97018 polypeptides can be determined, for example, in an assay that measures the amount of radiolabeled IMX97018 polypeptide that binds to agarose beads coated with a ribohomopolymer such as poly(rG). IMX97018 polypeptides (those without expanded polyglutamine tracts) having RNA-binding activity preferably have at least 25% (more preferably, at least 50%, and most preferably, at least 75%) of the RNA-binding activity of ataxin-1 having a 30-glutamine tract as measured in Yue et al., 2001, Hum Mol Genet 10: 25-30 (see FIG. 4). Ataxin-1-like polypeptides with expanded polyglutamine tracts (i.e. having more than 30 contiguous glutamine and/or histidine residues) are expected to have less RNA-binding activity in such assays than ataxin-1 having a 30-glutamine tract. The self-association activity of IMX97018 polypeptides can be determined, for example, in a yeast two-hybrid assay in which both the DNA-binding fusion protein and the activation-domain fusion protein contain IMX97018 polypeptides comprising the self-association region. IMX97018 polypeptides having self-association activity preferably have at least 25% (more preferably, at least 50%, and most preferably, at least 75%) of the self-association activity of ataxin-1 having a 30-glutamine tract as measured in a quantitative assay, such as the ONPG assay of Burright et al., 1997, Hum Molec Genet 6: 513-518 (see Table 2 on p. 514 of Burright et al.).
IMX97018 polypeptides, such as those having expanded polyglutamine tracts, which have nuclear inclusion formation activity promote the formation of IMX97018-immunoreactive nuclear aggregates. Additional activities of IMX97018 polypeptides, such as those having expanded polyglutamine tracts, include binding to leucine-rich nuclear polypeptides; down-regulation of certain genes expressed in neural tissue; and promotion of cell death. These activities are associated with the region of IMX97018 polypeptides corresponding to the polyglutamine tract of human ataxin-1 polypeptide. Thus, for uses requiring nuclear inclusion formation activity, leucine-rich nuclear polypeptide binding activity, neural gene down-regulation activity, or promotion of cell death activity, preferred IMX97018 polypeptides include those having the polyglutamine region and exhibiting one or more of the above biological activities; further preferred IMX97018 polypeptides include those having the an expanded polyglutamine tract and exhibiting one or more of the above biological activities. Preferred IMX97018 polypeptides further include oligomers or fusion polypeptides comprising at least one polyglutamine region of one or more IMX97018 polypeptides, and fragments of any of these polypeptides that have one or more of the above polyglutamine-associated activities. The nuclear inclusion formation activity and the cell death promotion activity of IMX97018 polypeptides can be determined, for example, through immunohistochemical and microscopic imaging techniques, such as confocal microscopy as used by Skinner et al. (1998, Nature 389: 971-974), or electron microscopy as used by Evert et al. (1999, Hum Mol Genet 8: 1169-1176). IMX97018 polypeptides having nuclear inclusion formation activity preferably promote nuclear inclusion formation in at least 10% (more preferably, at least 25%, and most preferably, at least 50%) of the percentage of cells having nuclear inclusions, when such cells express human ataxin-1 with a polyglutamine tract of 82 residues, when measured as in Skinner et al., 1998, Nature 389: 971-974. IMX97018 polypeptides having cell death promotion activity preferably promote cell death in at least 10% (more preferably, at least 25%, and most preferably, at least 50%) of the percentage of necrotic cells, when such cells express human ataxin-1 with a polyglutamine tract of 70 residues, when measured as in Evert et al., 1999, Hum Mol Genet 8: 1169-1176. The neural gene down-regulation activity of IMX97018 polypeptides can be determined, for example, through assays such as Northern blots of RNA from neural cells expressing IMX97018 polypeptides, or quantitative PCR from such cells, where the probe(s) or primers are specific for particular genes expressed in neural cells. IMX97018 polypeptides having neural gene down-regulation activity preferably have at least 10% (more preferably, at least 25%, and most preferably, at least 50%) of the neural gene down-regulation activity of human ataxin-1 with a polyglutamine tract of 82 residues, as measured by quantification of Northern blot band intensity.
The term “IMX97018 polypeptide activity,” as used herein, includes any one or more of the following: RNA-binding activity, self-association activity, nuclear inclusion formation promoting activity, leucine-rich nuclear protein binding activity, neural gene down-regulation activity, and cell death promoting activity, as well as the ex vivo and in vivo activities of IMX97018 family polypeptides. The degree to which individual members of the IMX97018 polypeptide family and fragments and other derivatives of these polypeptides exhibit these activities can be determined by standard assay methods, particularly assays such as those referred to above. Exemplary assays are disclosed herein; those of skill in the art will appreciate that other, similar types of assays can be used to measure IMX97018 family biological activities.
An aspect of the biological activity of IMX97018 polypeptides is the ability of members of this polypeptide family to bind particular binding partners such as nuclear polypeptides, particularly leucine-rich nuclear polypeptides, and most particularly LANP polypeptides, with this binding activity associated with the region of IMX97018 polypeptide corresponding to the polyglutamine tract of ataxin-1, and in another embodiment, with the polyglutamine tract in IMX97018 polypeptides having such a polyglutamine tract. The term “binding partner,” as used herein, includes ligands, receptors, substrates, antibodies, other IMX97018 polypeptides, the same IMX97018 polypeptide (in the case of homotypic interactions), and any other molecule that interacts with an IMX97018 polypeptide through contact or proximity between particular portions of the binding partner and the IMX97018 polypeptide. Because the polyglutamine region or tract of IMX97018 polypeptides binds to leucine-rich polypeptides, the polyglutamine region or tract when expressed as a separate fragment from the rest of an IMX97018 polypeptide, or as a soluble polypeptide, fused for example to an immunoglobulin Fc domain, is expected to disrupt the binding of IMX97018 polypeptides to their binding partners. By binding to one or more binding partners, the separate polyglutamine region or tract polypeptide likely prevents binding by native IMX97018 polypeptide(s), and so acts in a dominant negative fashion to inhibit the biological activities mediated via binding of IMX97018 polypeptides to binding partners such as leucine-rich nuclear polypeptides. Particularly suitable assays to detect or measure the binding between IMX97018 polypeptides and their binding partners are yeast two-hybrid assays and in vitro binding assays.
IMX97018 polypeptides are involved in neurological diseases or conditions, that share as a common feature disruption of neural cell morphology and neural cell death in their etiology. More specifically, the following neurological conditions are those that are known or are likely to involve the biological activities of IMX97018 polypeptides: spinocerebellar ataxia; particularly spinocerebellar ataxia type 4 (SCA4). Blocking or inhibiting the interactions between members of the IMX97018 polypeptide family and their substrates, ligands, receptors, binding partners, and or other interacting polypeptides is an aspect of the invention and provides methods for treating or ameliorating these diseases and conditions through the use of inhibitors of IMX97018 polypeptide activity. Examples of such inhibitors or antagonists are described in more detail below. For certain conditions involving too little IMX97018 polypeptide activity, methods of treating or ameliorating these conditions comprise increasing the amount or activity of IMX97018 polypeptides by providing isolated IMX97018 polypeptides or active fragments or fusion polypeptides thereof, or by providing compounds (agonists) that activate endogenous or exogenous IMX97018 polypeptides.
Additional uses for IMX97018 polypeptides include diagnostic reagents for diseases linked to human chromosome 16 and/or neurological disorders such as ataxic conditions, and research reagents for investigation of polyglutamine-containing polypeptides.
IMX97018 Polypeptides
An IMX97018 polypeptide is a polypeptide that shares a sufficient degree of amino acid identity or similarity to the IMX97018 polypeptide of SEQ ID NO:2 to (A) be identified by those of skill in the art as a polypeptide likely to share particular structural domains and/or (B) have biological activities in common with the UMX97018 polypeptide of SEQ ID NO:2 and/or (C) bind to antibodies that also specifically bind to other IMX97018 polypeptides. IMX97018 polypeptides can be isolated from naturally occurring sources, or have the same structure as naturally occurring IMX97018 polypeptides, or can be produced to have structures that differ from naturally occurring IMX97018 polypeptides. Polypeptides derived from any IMX97018 polypeptide by any type of alteration (for example, but not limited to, insertions, deletions, or substitutions of amino acids; changes in the state of glycosylation of the polypeptide; refolding or isomerization to change its three-dimensional structure or self-association state; and changes to its association with other polypeptides or molecules) are also IMX97018 polypeptides. Therefore, the polypeptides provided by the invention include polypeptides characterized by amino acid sequences similar to those of the IMX97018 polypeptides described herein, but into which modifications are naturally provided or deliberately engineered. A polypeptide that shares biological activities in common with IMX97018 polypeptides is a polypeptide having IMX97018 polypeptide activity. Examples of biological activities exhibited by IMX97018 polypeptides include, without limitation, RNA-binding activity, self-association activity, nuclear inclusion formation promoting activity, leucine-rich nuclear protein binding activity, neural gene down-regulation activity, and cell death promoting activity.
The present invention provides both full-length and mature forms of IMX97018 polypeptides. Full-length polypeptides are those having the complete primary amino acid sequence of the polypeptide as initially translated. The amino acid sequences of full-length polypeptides can be obtained, for example, by translation of the complete open reading frame (“ORF”) of a cDNA molecule. Several full-length polypeptides can be encoded by a single genetic locus if multiple mRNA forms are produced from that locus by alternative splicing or by the use of multiple translation initiation sites. The “mature form” of a polypeptide refers to a polypeptide that has undergone post-translational processing steps such as cleavage of the signal sequence or proteolytic cleavage to remove a prodomain. Multiple mature forms of a particular full-length polypeptide may be produced, for example by cleavage of the signal sequence at multiple sites, or by differential regulation of proteases that cleave the polypeptide. The mature form(s) of such polypeptide can be obtained by expression, in a suitable mammalian cell or other host cell, of a nucleic acid molecule that encodes the full-length polypeptide. The sequence of the mature form of the polypeptide may also be determinable from the amino acid sequence of the full-length form, through identification of signal sequences or protease cleavage sites. The IMX97018 polypeptides of the invention also include those that result from post-transcriptional or post-translational processing events such as alternate mRNA processing which can yield a truncated but biologically active polypeptide, for example, a naturally occurring soluble form of the polypeptide. Also encompassed within the invention are variations attributable to proteolysis such as differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the polypeptide (generally from 1-5 terminal amino acids).
The invention further includes IMX97018 polypeptides with or without associated native-pattern glycosylation. Polypeptides expressed in yeast or mammalian expression systems (e.g., COS-1 or CHO cells) can be similar to or significantly different from a native polypeptide in molecular weight and glycosylation pattern, depending upon the choice of expression system. Expression of polypeptides of the invention in bacterial expression systems, such as E. coli, provides non-glycosylated molecules. Further, a given preparation can include multiple differentially glycosylated species of the polypeptide. Glycosyl groups can be removed through conventional methods, in particular those utilizing glycopeptidase. In general, glycosylated polypeptides of the invention can be incubated with a molar excess of glycopeptidase (Boehringer Mannheim).
Species homologues of IMX97018 polypeptides and of nucleic acids encoding them are also provided by the present invention. As used herein, a “species homologue” is a polypeptide or nucleic acid with a different species of origin from that of a given polypeptide or nucleic acid, but with significant sequence similarity to the given polypeptide or nucleic acid, as determined by those of skill in the art. Species homologues can be isolated and identified by making suitable probes or primers from polynucleotides encoding the amino acid sequences provided herein and screening a suitable nucleic acid source from the desired species. The invention also encompasses allelic variants of IMX97018 polypeptides and nucleic acids encoding them; that is, naturally-occurring alternative forms of such polypeptides and nucleic acids in which differences in amino acid or nucleotide sequence are attributable to genetic polymorphism (allelic variation among individuals within a population).
Fragments of the IMX97018 polypeptides of the present invention are encompassed by the present invention and can be in linear form or cydlized using known methods, for example, as described in Saragovi et al., BiotIechnology 10, 773-778 (1992) and in McDowell et at, J. Amer. Chem. Soc. 114 9245-9253 (1992). Polypeptides and polypeptide fragments of the present invention, and nucleic acids encoding them, include polypeptides and nucleic acids with amino acid or nucleotide sequence lengths that are at least 25% (more preferably at least 50%, or at least 60%, or at least 70%, and most preferably at least 80%) of the length of an 1MX97018 polypeptide and have at least 60% sequence identity (mote preferably at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, or at least 99%, and most preferably at least 99.5%) with that 1MX97018 polypeptide or encoding nucleic acid, where sequence identity is determined by comparing the amino acid sequences of the polypeptides when aligned so as to maximize overlap and identity while minimizing sequence gaps. Also included in the present invention are polypeptides and polypeptide fragments, and nucleic acids encoding tern, that contain or encode a segment preferably comprising at least 8, or at least 10, or preferably at least 15, or more preferably at least 20, or still more preferably at least 30, or most preferably at least 40 contiguous amino acids. Such polypeptides and polyp eptide fragments may also contain a segment that shares at least 70% sequence identity (more preferably at least 70%, at least 75%, at least 8013/0, at least 85%, at least 90%, at least 95%, at least 97.5%, or at least 99%, and most preferably at least 99.5%) with any such segment of any IMX97018 polypeptide, where sequence identity is determined by comparing the amino acid sequences of the polypeptides when aligned so as to maximize overlap and identity while minimizing sequence gaps. The percent identity of two amino acid or two nucleic acid sequences can be determined by visual inspection and mathematical calculation, or more preferably, the comparison is done by comparing sequence information using a computer program. An exemplary, preferred computer program is the Genetics Computer Group ((3CC; Madison, WI) Wisconsin package version 10.0 program, ‘Gap’ (Devereux C at, 1984, Nuci. Acids Res. 12: 387). The preferred detfaultparameters for the ‘GAP’ program includes: (1) The GCG implementation of a unary comparison matrix (containing a value of 1 for identifies and 0 for non-identities) for nucleotides, and the weighted amino acid comparison matrix of Gribskov and Burgess, Nuci. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, edt, Atlas of .Polypeptide Sequence and Structure, National Biomedical Research Foundation, pp. 353-35 8, 1979; or other comparable comparison matrices; (2) a penalty of 30 for each gap and an additional penalty of 1 for each symbol in each gap for amino acid sequences, or penalty of 50 for each gap and an additional penalty of 3 for each symbol in each gap for nucleotide sequences; (3) no penalty for end gaps; and (4) no maximum penalty for long gaps. Other programs used by those skilled in the art of sequence comparison can also be used, such as, for example, the BLASTN program version 2.0.9, available for use via the National Library of Medicine website or the UW-BLAST 2.0 algorithm. In addition, the BLAST algorithm uses the BLOSUM62 amino acid scoring matrix, and optional parameters that can be used are as foflows: (A) inclusion of a filter to mask segments of the query sequence that have low compositional complexity (as determined by the SEQ program of Wootton and Federhen (Computers and Chemistry. 1993); also see Wootton and Federhen, 1996, Analysis of compositionally biased regions in sequence databases, Methods Enrymot 266: 554-71) or segments consisting of short-periodicity internal repeats (as determined by the XNU program of Claverie and States (Computers and Chemistry. 1993)), and (B) a statistical significance threshold for reporting matches against database sequences, or fl-score (the expected probability of matches being found merely by chance, according to the stochastic model of Karlin and Altschul (1990); if the statistical significance ascribed to a match is greater than this E-score threshold, the match will not be reported.); preferred fl-score threshold values are 0.5, or in order of increasing preference, 0.25, 0.1, 0.05, 0.01, 0.001, 0.0001, le-5, le-lO, le-15, le -20, le-25, le.-30, le-40, le-SO, le-75, or le-100.
The present invention also provides for soluble forms of IMX97018 polypeptides comprising certain fragments or domains of these polypeptides, and particularly those comprising the AXH domain, the polyglutamine region or tract, or one or more fragments of these domains. Soluble polypeptides are polypeptides that are capable of being secreted from the cells in which they are expressed. Soluble IMX97018 polypeptides include those forms of IMX97018 polypeptide that are capable of being secreted from a cell, such as those to which a signal peptide has been fused to the N-terminal end, and preferably those that retain IMX97018 polypeptide activity. Soluble IMX97018 polypeptides further include oligomers or fusion polypeptides, and fragments of any of these polypeptides that have IMX97018 polypeptide activity. A secreted soluble polypeptide can be identified (and distinguished from its non-soluble counterparts) by separating intact cells which express the desired polypeptide from the culture medium, e.g., by centrifugation, and assaying the medium (supernatant) for the presence of the desired polypeptide. The presence of the desired polypeptide in the medium indicates that the polypeptide was secreted from the cells and thus is a soluble form of the polypeptide. The use of soluble forms of IMX97018 polypeptides is advantageous for many applications. Purification of the polypeptides from recombinant host cells is facilitated, since the soluble polypeptides are secreted from the cells. Moreover, soluble polypeptides are generally more suitable for parenteral administration and for many enzymatic procedures.
In another aspect of the invention, preferred polypeptides comprise various combinations of IMX97018 polypeptide domains, such as the AXH domain and the polyglutamine region or tract. Accordingly, polypeptides of the present invention and nucleic acids encoding them include those comprising or encoding two or more copies of a domain such as the AXH domain domain, two or more copies of a domain such as the polyglutamine region or tract, or at least one copy of each domain, and these domains can be presented in any order within such polypeptides.
Further modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques. Modifications of interest in the polypeptide sequences can include the alteration, substitution, replacement, insertion or deletion of a selected amino acid. For example, one or more of the cysteine residues can be deleted or replaced with another amino acid to alter the conformation of the molecule, an alteration which may involve preventing formation of incorrect intramolecular disulfide bridges upon folding or renaturation. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Pat. No. 4,518,584). As another example, N-glycosylation sites in the polypeptide extracellular domain can be modified to preclude glycosylation, allowing expression of a reduced carbohydrate analog in mammalian and yeast expression systems. N-glycosylation sites in eukaryotic polypeptides are characterized by an amino acid triplet Asn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr. Appropriate substitutions, additions, or deletions to the nucleotide sequence encoding these triplets will result in prevention of attachment of carbohydrate residues at the Asn side chain. Alteration of a single nucleotide, chosen so that Asn is replaced by a different amino acid, for example, is sufficient to inactivate an N-glycosylation site. Alternatively, the Ser or Thr can by replaced with another amino acid, such as Ala. Known procedures for inactivating N-glycosylation sites in polypeptides include those described in U.S. Pat. No. 5,071,972 and EP 276,846. Additional variants within the scope of the invention include polypeptides that can be modified to create derivatives thereof by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives can be prepared by linking the chemical moieties to functional groups on amino acid side chains or at the N-terminus or C-terminus of a polypeptide. Conjugates comprising diagnostic (detectable) or therapeutic agents attached thereto are contemplated herein. Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the polypeptide or a substantial equivalent thereof. One example is a variant that binds with essentially the same binding affinity as does the native form. Binding affinity can be measured by conventional procedures, e.g., as described in U.S. Pat. No. 5,512,457 and as set forth herein.
Other derivatives include covalent or aggregative conjugates of the polypeptides with other polypeptides or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions. Examples of fusion polypeptides are discussed below in connection with oligomers. Further, fusion polypeptides can comprise peptides added to facilitate purification and identification. Such peptides include, for example, poly-His or the antigenic identification peptides described in U.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988. One such peptide is the FLAG® peptide, which is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant polypeptide. A murine hybridoma designated 4E11 produces a monoclonal antibody that binds the FLAG® peptide in the presence of certain divalent metal cations, as described in U.S. Pat. No. 5,011,912. The 4E11 hybridoma cell line has been deposited with the American Type Culture Collection under accession no. HB 9259. Monoclonal antibodies that bind the FLAG® peptide are available from Eastman Kodak Co., Scientific Imaging Systems Division, New Haven, Conn.
Encompassed by the invention are oligomers or fusion polypeptides that contain an IMX97018 polypeptide, one or more fragments of IMX97018 polypeptides, or any of the derivative or variant forms of IMX97018 polypeptides as disclosed herein. In particular embodiments, the oligomers comprise soluble IMX97018 polypeptides. Oligomers can be in the form of covalently linked or non-covalently-linked multimers, including dimers, trimers, or higher oligomers. In one aspect of the invention, the oligomers maintain the binding ability of the polypeptide components and provide therefor, bivalent, trivalent, etc., binding sites. In an alternative embodiment the invention is directed to oligomers comprising multiple IMX97018 polypeptides joined via covalent or non-covalent interactions between peptide moieties fused to the polypeptides, such peptides having the property of promoting oligomerization. Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote oligomerization of the polypeptides attached thereto, as described in more detail below.
In embodiments where variants of the IMX97018 polypeptides are constructed to include a membrane-spanning domain, they will form a Type I membrane polypeptide.
Immunoglobulin-based Oligomers. The polypeptides of the invention or fragments thereof can be fused to molecules such as immunoglobulins for many purposes, including increasing the valency of polypeptide binding sites. For example, fragments of an IMX97018 polypeptide can be fused directly or through linker sequences to the Fc portion of an immunoglobulin. For a bivalent form of the polypeptide, such a fusion could be to the Fc portion of an IgG molecule. Other immunoglobulin isotypes can also be used to generate such fusions. For example, a polypeptide-IgM fusion would generate a decavalent form of the polypeptide of the invention. The term “Fc polypeptide” as used herein includes native and mutein forms of polypeptides made up of the Fc region of an antibody comprising any or all of the CH domains of the Fc region. Truncated forms of such polypeptides containing the hinge region that promotes dimerization are also included. Preferred Fc polypeptides comprise an Fc polypeptide derived from a human IgG1 antibody. As one alternative, an oligomer is prepared using polypeptides derived from immunoglobulins. Preparation of fusion polypeptides comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., by Ashkenazi et al. (PNAS USA 88:10535, 1991); Byrn et al. (Nature 344:677, 1990); and Hollenbaugh and Aruffo (“Construction of Immunoglobulin Fusion Polypeptides”, in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11, 1992). Methods for preparation and use of immunoglobulin-based oligomers are well known in the art. One embodiment of the present invention is directed to a dimer comprising two fusion polypeptides created by fusing a polypeptide of the invention to an Fc polypeptide derived from an antibody. A gene fusion encoding the polypeptide/Fc fusion polypeptide is inserted into an appropriate expression vector. Polypeptide/Fc fusion polypeptides are expressed in host cells transformed with the recombinant expression vector, and allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between the Fc moieties to yield divalent molecules. One suitable Fc polypeptide, described in PCT application WO 93/10151, is a single chain polypeptide extending from the N-terminal hinge region to the native C-terminus of the Fc region of a human IgG1 antibody. Another useful Fc polypeptide is the Fc mutein described in U.S. Pat. No. 5,457,035 and in Baum et al., (EMBO J. 13:3992-4001, 1994). The amino acid sequence of this mutein is identical to that of the native Fc sequence presented in WO 93/10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 has been changed from Gly to Ala. The mutein exhibits reduced affinity for Fc receptors. The above-described fusion polypeptides comprising Fc moieties (and oligomers formed therefrom) offer the advantage of facile purification by affinity chromatography over Polypeptide A or Polypeptide G columns. In other embodiments, the polypeptides of the invention can be substituted for the variable portion of an antibody heavy or light chain. If fusion polypeptides are made with both heavy and light chains of an antibody, it is possible to form an oligomer with as many as four IMX97018 extracellular regions.
Peptide-linker Based Oligomers. Alternatively, the oligomer is a fusion polypeptide comprising multiple IMX97018 polypeptides, with or without peptide linkers (spacer peptides). Among the suitable peptide linkers are those described in U.S. Pat. Nos. 4,751,180 and 4,935,233. A DNA sequence encoding a desired peptide linker can be inserted between, and in the same reading frame as, the DNA sequences of the invention, using any suitable conventional technique. For example, a chemically synthesized oligonucleotide encoding the linker can be ligated between the sequences. In particular embodiments, a fusion polypeptide comprises from two to four soluble IMX97018 polypeptides, separated by peptide linkers. Suitable peptide linkers, their combination with other polypeptides, and their use are well known by those skilled in the art.
Leucine-Ziplers. Another method for preparing the oligomers of the invention involves use of a leucine zipper. Leucine zipper domains are peptides that promote oligomerization of the polypeptides in which they are found. Leucine zippers were originally identified in several DNA-binding polypeptides (Landschulz et al., Science 240:1759, 1988), and have since been found in a variety of different polypeptides. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. The zipper domain (also referred to herein as an oligomerizing, or oligomer-forming, domain) comprises a repetitive heptad repeat, often with four or five leucine residues interspersed with other amino acids. Use of leucine zippers and preparation of oligomers using leucine zippers are well known in the art.
Other fragments and derivatives of the sequences of polypeptides which would be expected to retain polypeptide activity in whole or in part and may thus be useful for screening or other immunological methodologies can also be made by those skilled in the art given the disclosures herein. Such modifications are believed to be encompassed by the present invention.
Nucleic Acids Encoding IMX97018 Polypeptides
Encompassed within the invention are nucleic acids encoding IMX97018 polypeptides, such as SEQ ID NO:1, which encodes the IMX97018 polypeptide of SEQ ID NO:2. These nucleic acids can be identified in several ways, including isolation of genomic or cDNA molecules from a suitable source. Nucleotide sequences corresponding to the amino acid sequences described herein, to be used as probes or primers for the isolation of nucleic acids or as query sequences for database searches, can be obtained by “back-translation” from the amino acid sequences, or by identification of regions of amino acid identity with polypeptides for which the coding DNA sequence has been identified. The well-known polymerase chain reaction (PCR) procedure can be employed to isolate and amplify a DNA sequence encoding an IMX97018 polypeptide or a desired combination of IMX97018 polypeptide fragments. Oligonucleotides that define the desired termini of the combination of DNA fragments are employed as 5′ and 3′ primers. The oligonucleotides can additionally contain recognition sites for restriction endonucleases, to facilitate insertion of the amplified combination of DNA fragments into an expression vector. PCR techniques are described in Saiki et al., Science 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds., Academic Press, Inc., San Diego (1989), pp. 189-196; and PCR Protocols: A Guide to Methods and Applications, Innis et. al., eds., Academic Press, Inc. (1990).
Nucleic acid molecules of the invention include DNA and RNA in both single-stranded and double-stranded form, as well as the corresponding complementary sequences. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. The nucleic acid molecules of the invention include full-length genes or cDNA molecules as well as a combination of fragments thereof. The nucleic acids of the invention are preferentially derived from human sources, but the invention includes those derived from non-human species, as well.
An “isolated nucleic acid” is a nucleic acid that has been separated from adjacent genetic sequences present in the genome of the organism from which the nucleic acid was isolated, in the case of nucleic acids isolated from naturally-occurring sources. In the case of nucleic acids synthesized enzymatically from a template or chemically, such as PCR products, cDNA molecules, or oligonucleotides for example, it is understood that the nucleic acids resulting from such processes are isolated nucleic acids. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. In one preferred embodiment, the nucleic acids are substantially free from contaminating endogenous material. The nucleic acid molecule has preferably been derived from DNA or RNA isolated at least once in substantially pure form and in a quantity or concentration enabling identification, manipulation, and recovery of its component nucleotide sequences by standard biochemical methods (such as those outlined in Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)). Such sequences are preferably provided and/or constructed in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, that are typically present in eukaryotic genes. Sequences of non-translated DNA can be present 5′ or 3′ from an open reading frame, where the same do not interfere with manipulation or expression of the coding region.
“An isolated nucleic acid consisting essentially of a nucleotide sequence” means that the nucleic acid may have, in addition to said nucleotide sequence, additional material covalently linked to either or both ends of the nucleic acid molecule, said additional material preferably between 1 and 100,000 additional nucleotides covalently linked to either end, each end, or both ends of the nucleic acid molecule, and more preferably between 1 and 10,000 additional nucleotides covalently linked to either end, each end, or both ends of the nucleic acid molecule, and most preferably between 10 and 1,000 additional nucleotides covalently linked to either end, each end, or both ends of the nucleic acid molecule. An isolated nucleic acid consisting essentially of a nucleotide sequence may be an expression vector or other construct comprising said nucleotide sequence. “An isolated nucleic acid consisting essentially of a nucleotide sequence” further is meant to exclude isolated human chromosomes or isolated contigs such as GenBank accession number AC009127.
The present invention also includes nucleic acids that hybridize under moderately stringent conditions, and more preferably highly stringent conditions, to nucleic acids encoding IMX97018 polypeptides described herein. The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by Sambrook, Fritsch, and Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11; and Current Protocols in Molecular Biology, 1995, Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4), and can be readily determined by those having ordinary skill in the art based on, for example, the length and/or base composition of the DNA. One way of achieving moderately stringent conditions involves the use of a prewashing solution containing 5×SSC, 0.5% SDS, 1.0 m−1 M EDTA (pH 8.0), hybridization buffer of about 50% formamide, 6×SSC, and a hybridization temperature of about 55 degrees C. (or other similar hybridization solutions, such as one containing about 50% formamide, with a hybridization temperature of about 42 degrees C.), and washing conditions of about 60 degrees C., in 0.5×SSC, 0.1% SDS. Generally, highly stringent conditions are defined as hybridization conditions as above, but with washing at approximately 68 degrees C., 0.2×SSC, 0.1% SDS. SSPE (1×SSPE is 0.15M NaCl, 10 mM NaH.sub.2 PO.sub.4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is 0.15M NaCl and 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete. It should be understood that the wash temperature and wash salt concentration can be adjusted as necessary to achieve a desired degree of stringency by applying the basic principles that govern hybridization reactions and duplex stability, as known to those skilled in the art and described further below (see, e.g., Sambrook et al., 1989). When hybridizing a nucleic acid to a target nucleic acid of unknown sequence, the hybrid length is assumed to be that of the hybridizing nucleic acid. When nucleic acids of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the nucleic acids and identifying the region or regions of optimal sequence complementarity. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5 to 10.degrees C. less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm (degrees C.)=2(# of A+T bases)+4(# of #G+C bases). For hybrids above 18 base pairs in length, Tm (degrees C.)=81.5+16.6(log10[Na+])+0.41(% G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1×SSC=0.165M). Preferably, each such hybridizing nucleic acid has a length that is at least 15 nucleotides (or more preferably at least 18 nucleotides, or at least 20 nucleotides, or at least 25 nucleotides, or at least 30 nucleotides, or at least 40 nucleotides, or most preferably at least 50 nucleotides), or at least 25% (more preferably at least 50%, or at least 60%, or at least 70%, and most preferably at least 80%) of the length of the nucleic acid of the present invention to which it hybridizes, and has at least 60% sequence identity (more preferably at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, or at least 99%, and most preferably at least 99.5%) with the nucleic acid of the present invention to which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing nucleic acids when aligned so as to maximize overlap and identity while minimizing sequence gaps as described in more detail above.
The present invention also provides genes corresponding to the nucleic acid sequences disclosed herein. “Corresponding genes” or “corresponding genomic nucleic acids” are the regions of the genome that are transcribed to produce the mRNAs from which cDNA nucleic acid sequences are derived and can include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes can therefore include but are not limited to coding sequences, 5′ and 3′ untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements. Corresponding genomic nucleic acids can include 10000 basepairs (more preferably, 5000 basepairs, still more preferably, 2500 basepairs, and most preferably, 1000 basepairs) of genomic nucleic acid sequence upstream of the first nucleotide of the genomic sequence corresponding to the initiation codon of the IMX97018 coding sequence, and 10000 basepairs (more preferably, 5000 basepairs, still more preferably, 2500 basepairs, and most preferably, 1000 basepairs) of genomic nucleic acid sequence downstream of the last nucleotide of the genomic sequence corresponding to the termination codon of the IMX97018 coding sequence. The corresponding genes or genomic nucleic acids can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials. An “isolated gene” or “an isolated genomic nucleic acid” is a genomic nucleic acid that has been separated from the adjacent genomic sequences present in the genome of the organism from which the genomic nucleic acid was isolated.
Methods for Making and Purifying IMX97018 Polypeptides
Methods for making IMX97018 polypeptides are described below. Expression, isolation, and purification of the polypeptides and fragments of the invention can be accomplished by any suitable technique, including but not limited to the following methods. The isolated nucleic acid of the invention can be operably linked to an expression control sequence such as the pDC409 vector (Giri et al., 1990, EMBO J., 13: 2821) or the derivative pDC412 vector (Wiley et al., 1995, Immunity 3: 673). The pDC400 series vectors are useful for transient mammalian expression systems, such as CV-1 or 293 cells. Alternatively, the isolated nucleic acid of the invention can be linked to expression vectors such as pDC312, pDC316, or pDC317 vectors. The pDC300 series vectors all contain the SV40 origin of replication, the CMV promoter, the adenovirus tripartite leader, and the SV40 polyA and termination signals, and are useful for stable mammalian expression systems, such as CHO cells or their derivatives. Other expression control sequences and cloning technologies can also be used to produce the polypeptide recombinantly, such as the pMT2 or pED expression vectors (Kaufman et al., 1991, Nucleic Acids Res. 19: 4485-4490; and Pouwels et al., 1985, Cloning Vectors. A Laboratory Manual, Elsevier, New York) and the GATEWAY Vectors (lifetech.com/Content/Tech-Online/molecular_biology/manuals_pps/11797016.pdf; Life Technologies; Rockville, Md.). In the GATEWAY system the isolated nucleic acid of the invention, flanked by attB sequences, can be recombined through an integrase reaction with a GATEWAY vector such as pDONR201 containing attP sequences. This provides an entry vector for the GATEWAY system containing the isolated nucleic acid of the invention. This entry vector can be further recombined with other suitably prepared expression control sequences, such as those of the pDC400 and pDC300 series described above. Many suitable expression control sequences are known in the art. General methods of expressing recombinant polypeptides are also described in R. Kaufman, Methods in Enzymology 185, 537-566 (1990). As used herein “operably linked” means that the nucleic acid of the invention and an expression control sequence are situated within a construct, vector, or cell in such a way that the polypeptide encoded by the nucleic acid is expressed when appropriate molecules (such as polymerases) are present. As one embodiment of the invention, at least one expression control sequence is operably linked to the nucleic acid of the invention in a recombinant host cell or progeny thereof, the nucleic acid and/or expression control sequence having been introduced into the host cell by transformation or transfection, for example, or by any other suitable method. As another embodiment of the invention, at least one expression control sequence is integrated into the genome of a recombinant host cell such that it is operably linked to a nucleic acid sequence encoding a polypeptide of the invention. In a further embodiment of the invention, at least one expression control sequence is operably linked to a nucleic acid of the invention through the action of a trans-acting factor such as a transcription factor, either in vitro or in a recombinant host cell.
In addition, a sequence encoding an appropriate signal peptide (native or heterologous) can be incorporated into expression vectors. The choice of signal peptide or leader can depend on factors such as the type of host cells in which the recombinant polypeptide is to be produced. To illustrate, examples of heterologous signal peptides that are functional in mammalian host cells include the signal sequence for interleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195; the signal sequence for interleukin-2 receptor described in Cosman et al., Nature 312:768 (1984); the interleukin-4 receptor signal peptide described in EP 367,566; the type I interleukin-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; and the type II interleukin-1 receptor signal peptide described in EP 460,846. A DNA sequence for a signal peptide (secretory leader) can be fused in frame to the nucleic acid sequence of the invention so that the DNA is initially transcribed, and the mRNA translated, into a fusion polypeptide comprising the signal peptide. A signal peptide that is functional in the intended host cells is one that promotes insertion of the polypeptide into cell membranes, and most preferably, promotes extracellular secretion of the polypeptide from that host cell. The signal peptide is preferably cleaved from the polypeptide upon membrane insertion or secretion of polypeptide from the cell. The skilled artisan will also recognize that the position(s) at which the signal peptide is cleaved can differ from that predicted by computer program, and can vary according to such factors as the type of host cells employed in expressing a recombinant polypeptide. A polypeptide preparation can include a mixture of polypeptide molecules having different N-terminal amino acids, resulting from cleavage of the signal peptide at more than one site.
Established methods for introducing DNA into mammalian cells have been described (Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990, pp. 15-69). Additional protocols using commercially available reagents, such as Lipofectamine lipid reagent (Gibco/BRL) or Lipofectamine-Plus lipid reagent, can be used to transfect cells (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987). In addition, electroporation can be used to transfect mammalian cells using conventional procedures, such as those in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1-3, Cold Spring Harbor Laboratory Press, 1989). Selection of stable transformants can be performed using methods known in the art, such as, for example, resistance to cytotoxic drugs. Kaufman et al., Meth. in Enzymology 185:487-511, 1990, describes several selection schemes, such as dihydrofolate reductase (DHFR) resistance. A suitable strain for DHFR selection is CHO strain DX-B 11, which is deficient in DHFR (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980). A plasmid expressing the DHFR cDNA can be introduced into strain DX-B11, and only cells that contain the plasmid can grow in the appropriate selective media. Other examples of selectable markers that can be incorporated into an expression vector include cDNAs conferring resistance to antibiotics, such as G418 and hygromycin B. Cells harboring the vector can be selected on the basis of resistance to these compounds.
Alternatively, IMX97018 gene products can be obtained via homologous recombination, or “gene targeting,” techniques. Such techniques employ the introduction of exogenous transcription control elements (such as the CMV promoter or the like) in a particular predetermined site on the genome, to induce expression of the endogenous nucleic acid sequence of interest (see, for example, U.S. Pat. No. 5,272,071). The location of integration into a host chromosome or genome can be easily determined by one of skill in the art, given the known location and sequence of the gene. In a preferred embodiment, the present invention also contemplates the introduction of exogenous transcriptional control elements in conjunction with an amplifiable gene, to produce increased amounts of the gene product, again, without the need for isolation of the gene sequence itself from the host cell.
A number of types of cells can act as suitable host cells for expression of the polypeptide. Mammalian host cells include, for example, the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or their derivatives such as Veggie CHO and related cell lines which grow in serum-free media (Rasmussen et al., 1998, Cytotechnology 28: 31), HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) (McMahan et al., 1991, EMBO J. 10: 2821, 1991), human embryonic kidney cells such as 293, 293 EBNA or MSR 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HL-60, U937, HaK or Jurkat cells. Optionally, mammalian cell lines such as HepG2/3B, KB, NIH 3T3 or S49, for example, can be used for expression of the polypeptide when it is desirable to use the polypeptide in various signal transduction or reporter assays. Alternatively, it is possible to produce the polypeptide in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Suitable yeasts include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous polypeptides. Suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous polypeptides. If the polypeptide is made in yeast or bacteria, it may be desirable to modify the polypeptide produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional polypeptide. Such covalent attachments can be accomplished using known chemical or enzymatic methods. The polypeptide can also be produced by operably linking the isolated nucleic acid of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBac® kit), and such methods are well known in the art, as described in Summers and Smith, Tex. Agricultural Experiment Station Bulletin No. 1555 (1987), and Luckow and Summers, Bio/Technology 6:47 (1988). Cell-free translation systems could also be employed to produce polypeptides using RNAs derived from nucleic acid constructs disclosed herein. A host cell that comprises an isolated nucleic acid of the invention, preferably operably linked to at least one expression control sequence, is a “recombinant host cell”.
The polypeptide of the invention can be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant polypeptide. The resulting expressed polypeptide can then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as selective precipitation with various salts, gel filtration, and ion exchange chromatography. The purification of the polypeptide can also include an affinity column containing agents which will bind to the polypeptide; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography using an antibody that specifically binds one or more IMX97018 epitopes. Alternatively, the polypeptide of the invention can also be expressed in a form which will facilitate purification. For example, it can be expressed as a fusion polypeptide, that is, it may be fused with maltose binding polypeptide (MBP), glutathione-5-transferase (GST), thioredoxin (TRX), a polyHis peptide, and/or fragments thereof. Kits for expression and purification of such fusion polypeptides are commercially available from New England BioLabs (Beverly, Mass.), Pharmacia (Piscataway, N.J.) and InVitrogen, respectively. The polypeptide can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope. One such epitope (FLAG®) is commercially available from Kodak (New Haven, Conn.). Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the polypeptide. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant polypeptide. The polypeptide thus purified is substantially free of other mammalian polypeptides and is defined in accordance with the present invention as an “isolated polypeptide”; such isolated polypeptides of the invention include isolated antibodies that bind to IMX97018 polypeptides, fragments, variants, binding partners etc. The polypeptide of the invention can also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the polypeptide.
It is also possible to utilize an affinity column comprising a polypeptide-binding polypeptide of the invention, such as a monoclonal antibody generated against polypeptides of the invention, to affinity-purify expressed polypeptides. These polypeptides can be removed from an affinity column using conventional techniques, e.g., in a high salt elution buffer and then dialyzed into a lower salt buffer for use or by changing pH or other components depending on the affinity matrix utilized, or be competitively removed using the naturally occurring substrate of the affinity moiety, such as a polypeptide derived from the invention. In this aspect of the invention, polypeptide-binding polypeptides, such as the anti-polypeptide antibodies of the invention or other polypeptides that can interact with the polypeptide of the invention, can be bound to a solid phase support such as a column chromatography matrix or a similar substrate suitable for identifying, separating, or purifying cells that express polypeptides of the invention on their surface. Adherence of polypeptide-binding polypeptides of the invention to a solid phase contacting surface can be accomplished by any means, for example, magnetic microspheres can be coated with these polypeptide-binding polypeptides and held in the incubation vessel through a magnetic field. Suspensions of cell mixtures are contacted with the solid phase that has such polypeptide-binding polypeptides thereon. Cells having polypeptides of the invention on their surface bind to the fixed polypeptide-binding polypeptide and unbound cells then are washed away. This affinity-binding method is useful for purifying, screening, or separating such polypeptide-expressing cells from solution. Methods of releasing positively selected cells from the solid phase are known in the art and encompass, for example, the use of enzymes. Such enzymes are preferably non-toxic and non-injurious to the cells and are preferably directed to cleaving the cell-surface binding partner. Alternatively, mixtures of cells suspected of containing polypeptide-expressing cells of the invention first can be incubated with a biotinylated polypeptide-binding polypeptide of the invention. The resulting mixture then is passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin provides the binding of the polypeptide-binding cells to the beads. Use of avidin-coated beads is known in the art. See Berenson, et al. J. Cell. Biochem., 10D:239 (1986). Wash of unbound material and the release of the bound cells is performed using conventional methods.
The polypeptide can also be produced by known conventional chemical synthesis. Methods for constructing the polypeptides of the present invention by synthetic means are known to those skilled in the art. The synthetically-constructed polypeptide sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with IMX97018 polypeptides can possess biological properties in common therewith, including IMX97018 polypeptide activity. Thus, they can be employed as biologically active or immunological substitutes for natural, purified polypeptides in screening of therapeutic compounds and in immunological processes for the development of antibodies.
The desired degree of purity depends on the intended use of the polypeptide. A relatively high degree of purity is desired when the polypeptide is to be administered in vivo, for example. In such a case, the polypeptides are purified such that no polypeptide bands corresponding to other polypeptides are detectable upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be recognized by one skilled in the pertinent field that multiple bands corresponding to the polypeptide can be visualized by SDS-PAGE, due to differential glycosylation, differential post-translational processing, and the like. Most preferably, the polypeptide of the invention is purified to substantial homogeneity, as indicated by a single polypeptide band upon analysis by SDS-PAGE. The polypeptide band can be visualized by silver staining, Coomassie blue staining, or (if the polypeptide is radiolabeled) by autoradiography.
Antagonists and Agonists of IMX97018 Polypeptides
Any method which neutralizes IMX97018 polypeptides or inhibits expression of the IMX97018 genes (either transcription or translation) can be used to reduce the biological activities of IMX97018 polypeptides. In particular embodiments, antagonists inhibit the binding of at least one IMX97018 polypeptide to cells, thereby inhibiting biological activities induced by the binding of those IMX97018 polypeptides to the cells. In certain other embodiments of the invention, antagonists can be designed to reduce the level of endogenous IMX97018 gene expression, e.g., using well-known antisense or ribozyme approaches to inhibit or prevent translation of IMX97018 mRNA transcripts; triple helix approaches to inhibit transcription of IMX97018 family genes; or targeted homologous recombination to inactivate or “knock out” the IMX97018 genes or their endogenous promoters or enhancer elements. Such antisense, ribozyme, and triple helix antagonists can be designed to reduce or inhibit either unimpaired, or if appropriate, mutant IMX97018 gene activity. Techniques for the production and use of such molecules are well known to those of skill in the art.
Antisense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing polypeptide translation. Antisense approaches involve the design of oligonucleotides (either DNA or RNA) that are complementary to an IMX97018 mRNA. The antisense oligonucleotides will bind to the complementary target gene mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required. A sequence “complementary” to a portion of a nucleic acid, as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the nucleic acid, forming a stable duplex (or triplex, as appropriate). In the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA can thus be tested, or triplex formation can be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Preferred oligonucleotides are complementary to the 5′ end of the message, e.g., the 5′ untranslated sequence up to and including the AUG initiation codon. However, oligonucleotides complementary to the 5′- or 3′-non-translated, non-coding regions of the IMX97018 gene transcript, or to the coding regions, could be used in an antisense approach to inhibit translation of endogenous IMX97018 mRNA. Antisense nucleic acids should be at least six nucleotides in length, and are preferably 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. The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. Chimeric oligonucleotides, oligonucleosides, or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of nucleotides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound (see, e.g., U.S. Pat. No. 5,985,664). Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”. 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., 1989, Proc Natl Acad Sci U.S.A. 86: 6553-6556; Lemaitre et al., 1987, Proc Natl Acad Sci 84: 648-652; PCT Publication No. WO88/09810), or hybridization-triggered cleavage agents or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5: 539-549). The antisense molecules should be delivered to cells which express the IMX97018 transcript in vivo. A number of methods have been developed for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue or cell derivation site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface), can be administered systemically. However, it is often difficult to achieve intracellular concentrations of the antisense sufficient to suppress translation of endogenous mRNAs. Therefore a preferred approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter. The use of such a construct to transfect target cells in the patient will result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous IMX97018 gene transcripts and thereby prevent translation of the IMX97018 mRNA. For example, a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense 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 mammalian cells.
Ribozyme molecules designed to catalytically cleave IMX97018 mRNA transcripts can also be used to prevent translation of IMX97018 mRNA and expression of IMX97018 polypeptides. (See, e.g., PCT International Publication WO90/11364 and U.S. Pat. No. 5,824,519). The ribozymes that can be used in the present invention include hammerhead ribozymes (Haseloff and Gerlach, 1988, Nature, 334:585-591), RNA endoribonucleases (hereinafter “Cech-type ribozymes”) such as the one which occurs naturally in Tetrahymena Thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (International Patent Application No. WO 88/04300; Been and Cech, 1986, Cell, 47:207-216). As in the antisense approach, the ribozymes can be composed of modified oligonucleotides (e.g. for improved stability, targeting, etc.) and should be delivered to cells which express the IMX97018 polypeptide in vivo. A preferred method of delivery involves using a DNA construct “encoding” the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous IMX97018 messages and inhibit translation. Because ribozymes, unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
Alternatively, endogenous IMX97018 gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the target gene (i.e., the target gene promoter and/or enhancers) to form triple helical structures that prevent transcription of the target IMX97018 gene. (See generally, Helene, 1991, Anticancer Drug Des., 6(6), 569-584; Helene, et al., 1992, Ann. N.Y. Acad. Sci., 660, 27-36; and Maher, 1992, Bioassays 14(12), 807-815).
Anti-sense RNA and DNA, ribozyme, and triple helix molecules of the invention can be prepared by any method known in the art for the synthesis of DNA and RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis. Oligonucleotides 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., 1988, Nucl. Acids Res. 16:3209. Methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451). Alternatively, RNA molecules can be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
Endogenous target gene expression can also be reduced by inactivating or “knocking out” the target gene or its promoter using targeted homologous recombination (e.g., see Smithies, et al., 1985, Nature 317, 230-234; Thomas and Capecchi, 1987, Cell 51, 503-512; Thompson, et al., 1989, Cell 5, 313-321). For example, a mutant, non-functional target gene (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous target gene (either the coding regions or regulatory regions of the target gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express the target gene in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the target gene. Such approaches are particularly suited in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive target gene (e.g., see Thomas and Capecchi, 1987 and Thompson, 1989, supra), or in model organisms such as Caenorhabditis elegans where the “RNA interference” (“RNAi”) technique (Grishok, Tabara, and Mello, 2000, Genetic requirements for inheritance of RNAi in C. elegans, Science 287 (5462): 2494-2497), or the introduction of transgenes (Demburg et al., 2000, Transgene-mediated cosuppression in the C. elegans germ line, Genes Dev. 14 (13): 1578-1583) are used to inhibit the expression of specific target genes. However this approach can be adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate vectors such as viral vectors.
Organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the nucleic acid sequences disclosed herein are provided. The desired change in gene expression can be achieved through the use of antisense nucleic acids or ribozymes that bind and/or cleave the mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol. Sci. 15(7): 250-254; Lavarosky et al., 1997, Biochem. Mol. Med. 62(1): 11-22; and Hampel, 1998, Prog. Nucleic Acid Res. Mol. Biol. 58: 1-39). Transgenic animals that have multiple copies of the gene(s) corresponding to the nucleic acid sequences disclosed herein, preferably produced by transformation of cells with genetic constructs that are stably maintained within the transformed cells and their progeny, are provided. Transgenic animals that have modified genetic control regions that increase or reduce gene expression levels, or that change temporal or spatial patterns of gene expression, are also provided (see European Patent No. 0 649 464 B1). In addition, organisms are provided in which the gene(s) corresponding to the nucleic acid sequences disclosed herein have been partially or completely inactivated, through insertion of extraneous sequences into the corresponding gene(s) or through deletion of all or part of the corresponding gene(s). Partial or complete gene inactivation can be accomplished through insertion, preferably followed by imprecise excision, of transposable elements (Plasterk, 1992, Bioessays 14(9): 629-633; Zwaal et al., 1993, Proc. Natl. Acad. Sci. USA 90(16): 7431-7435; Clark et al., 1994, Proc. Natl. Acad. Sci. USA 91(2): 719-722), or through homologous recombination, preferably detected by positive/negative genetic selection strategies (Mansour et al., 1988, Nature 336: 348-352; U.S. Pat. Nos. 5,464,764; 5,487,992; 5,627,059; 5,631,153; 5,614,396; 5,616,491; and 5,679,523). These organisms with altered gene expression are preferably eukaryotes and more preferably are mammals. Such organisms are useful for the development of non-human models for the study of disorders involving the corresponding gene(s), and for the development of assay systems for the identification of molecules that interact with the polypeptide product(s) of the corresponding gene(s).
Also encompassed within the invention are IMX97018 polypeptide variants with partner binding sites that have been altered in conformation so that (1) the IMX97018 variant will still bind to its partner(s), but a specified small molecule will fit into the altered binding site and block that interaction, or (2) the IMX97018 variant will no longer bind to its partner(s) unless a specified small molecule is present (see for example Bishop et al., 2000, Nature 407: 395-401). Nucleic acids encoding such altered IMX97018 polypeptides can be introduced into organisms according to methods described herein, and can replace the endogenous nucleic acid sequences encoding the corresponding IMX97018 polypeptide. Such methods allow for the interaction of a particular IMX97018 polypeptide with its binding partners to be regulated by administration of a small molecule compound to an organism, either systemically or in a localized manner.
The IMX97018 polypeptides themselves can also be employed in inhibiting a biological activity of IMX97018 in in vitro or in vivo procedures. Encompassed within the invention are AXH domains of IMX97018 polypeptides that act as “dominant negative” inhibitors of native IMX97018 polypeptide function when expressed as fragments or as components of fusion polypeptides. For example, a purified polypeptide domain of the present invention can be used to inhibit binding of IMX97018 polypeptides to endogenous binding partners. Such use effectively would block IMX97018 polypeptide interactions and inhibit IMX97018 polypeptide activities. In still another aspect of the invention, an antisense inhibitor is used to inhibit activation of the endogenous IMX97018 polypeptide.
In an alternative aspect, the invention further encompasses the use of agonists of IMX97018 polypeptide activity to treat or ameliorate the symptoms of a disease for which increased IMX97018 polypeptide activity is beneficial. Such diseases include but are not limited to neurological disorders such as ataxias. In a preferred aspect, the invention entails administering compositions comprising an IMX97018 nucleic acid or an IMX97018 polypeptide to cells in vitro, to cells ex vivo, to cells in vivo, and/or to a multicellular organism such as a vertebrate or mammal. Preferred therapeutic forms of IMX97018 are soluble forms, as described above. In still another aspect of the invention, the compositions comprise administering an IMX97018-encoding nucleic acid for expression of an IMX97018 polypeptide in a host organism for treatment of disease. Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant (e.g., decreased) endogenous activity of an IMX97018 family polypeptide. Furthermore, the invention encompasses the administration to cells and/or organisms of compounds found to increase the endogenous activity of IMX97018 polypeptides. One example of compounds that increase IMX97018 polypeptide activity are agonistic antibodies, preferably monoclonal antibodies, that bind to IMX97018 polypeptides or binding partners, which may increase IMX97018 polypeptide activity by causing constitutive intracellular signaling (or “ligand mimicking”), or by preventing the binding of a native inhibitor of IMX97018 polypeptide activity.
Antibodies to IMX97018 Polypeptides
Antibodies that are immunoreactive with the polypeptides of the invention are provided herein. Such antibodies specifically bind to the polypeptides via the antigen-binding sites of the antibody (as opposed to non-specific binding). In the present invention, specifically binding antibodies are those that will specifically recognize and bind with IMX97018 polypeptides, homologues, and variants, but not with other molecules. In one preferred embodiment, the antibodies are specific for the polypeptides of the present invention and do not cross-react with other polypeptides. In this manner, the IMX97018 polypeptides, fragments, variants, fusion polypeptides, etc., as set forth above can be employed as “immunogens” in producing antibodies immunoreactive therewith.
More specifically, the polypeptides, fragment, variants, fusion polypeptides, etc. contain antigenic determinants or epitopes that elicit the formation of antibodies. These antigenic determinants or epitopes can be either linear or conformational (discontinuous). Linear epitopes are composed of a single section of amino acids of the polypeptide, while conformational or discontinuous epitopes are composed of amino acids sections from different regions of the polypeptide chain that are brought into close proximity upon polypeptide folding (Janeway and Travers, Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed. 1996)). Because folded polypeptides have complex surfaces, the number of epitopes available is quite numerous; however, due to the conformation of the polypeptide and steric hindrances, the number of antibodies that actually bind to the epitopes is less than the number of available epitopes (Janeway and Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nd ed. 1996)). Epitopes can be identified by any of the methods known in the art. Thus, one aspect of the present invention relates to the antigenic epitopes of the polypeptides of the invention. Such epitopes are useful for raising antibodies, in particular monoclonal antibodies, as described in more detail below. Additionally, epitopes from the polypeptides of the invention can be used as research reagents, in assays, and to purify specific binding antibodies from substances such as polyclonal sera or supernatants from cultured hybridomas. Such epitopes or variants thereof can be produced using techniques well known in the art such as solid-phase synthesis, chemical or enzymatic cleavage of a polypeptide, or using recombinant DNA technology.
As to the antibodies that can be elicited by the epitopes of the polypeptides of the invention, whether the epitopes have been isolated or remain part of the polypeptides, both polyclonal and monoclonal antibodies can be prepared by conventional techniques. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988); Kohler and Milstein, (U.S. Pat. No. 4,376,110); the human B-cell hybridoma technique (Kozbor et al., 1984, J. Immunol. 133:3001-3005; Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030); and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Hybridoma cell lines that produce monoclonal antibodies specific for the polypeptides of the invention are also contemplated herein. Such hybridomas can be produced and identified by conventional techniques. The hybridoma producing the mAb of this invention can be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production. One method for producing such a hybridoma cell line comprises immunizing an animal with a polypeptide; harvesting spleen cells from the immunized animal; fusing said spleen cells to a myeloma cell line, thereby generating hybridoma cells; and identifying a hybridoma cell line that produces a monoclonal antibody that binds the polypeptide. For the production of antibodies, various host animals can be immunized by injection with one or more of the following: an IMX97018 polypeptide, a fragment of an IMX97018 polypeptide, a functional equivalent of an IMX97018 polypeptide, or a mutant form of an IMX97018 polypeptide. Such host animals can include but are not limited to rabbits, guinea pigs, mice, and rats. Various adjuvants can be used to increase the immunologic response, depending on the host species, including but 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 hemocyanin, dinitrophenol, and potentially useful human adjutants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. The monoclonal antibodies can be recovered by conventional techniques. Such monoclonal antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
In addition, techniques developed for the production of “chimeric antibodies” (Takeda et al., 1985, Nature, 314: 452-454; Morrison et al., 1984, Proc Natl Acad Sci USA 81: 6851-6855; Boulianne et al., 1984, Nature 312: 643-646; Neuberger et al., 1985, Nature 314: 268-270) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a porcine mAb and a human immunoglobulin constant region. The monoclonal antibodies of the present invention also include humanized versions of murine monoclonal antibodies. Such humanized antibodies can be prepared by known techniques and offer the advantage of reduced immunogenicity when the antibodies are administered to humans. In one embodiment, a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody. Alternatively, a humanized antibody fragment can comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick et al. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS 14:139, Can, 1993). Useful techniques for humanizing antibodies are also discussed in U.S. Pat. No. 6,054,297. Procedures to generate antibodies transgenically can be found in GB 2,272,440, U.S. Pat. Nos. 5,569,825 and 5,545,806, and related patents. Preferably, for use in humans, the antibodies are human or humanized; techniques for creating such human or humanized antibodies are also well known and are commercially available from, for example, Medarex Inc. (Princeton, N.J.) and Abgenix Inc. (Fremont, Calif.). In another preferred embodiment, fully human antibodies for use in humans are produced by screening a library of human antibody variable domains using either phage display methods (Vaughan et al., 1998, Nat Biotechnol. 16(6): 535-539; and U.S. Pat. No. 5,969,108), ribosome display methods (Schaffitzel et al., 1999, J Immunol Methods 231(1-2): 119-135), or mRNA display methods (Wilson et al., 2001, Proc Natl Acad Sci USA 98(7): 3750-3755).
Antigen-binding antibody fragments that recognize specific epitopes can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab′)2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the (ab′)2 fragments. Alternatively, Fab expression libraries can be constructed (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-546) can also be adapted to produce single chain antibodies against IMX97018 gene products. 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 polypeptide. Such single chain antibodies can also be useful intracellularly (i.e., as ‘intrabodies), for example as described by Marasco et al. (J. Immunol. Methods 231:223-238, 1999) for genetic therapy in HIV infection. In addition, antibodies to the IMX97018 polypeptide can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” the IMX97018 polypeptide and that may bind to the IMX97018 polypeptide's binding partners using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, 1993, FASEB J 7(5):437-444; and Nissinoff, 1991, J. Immunol. 147(8):2429-2438).
Antibodies that are immunoreactive with the polypeptides of the invention include bispecific antibodies (i.e., antibodies that are immunoreactive with the polypeptides of the invention via a first antigen binding domain, and also immunoreactive with a different polypeptide via a second antigen binding domain). A variety of bispecific antibodies have been prepared, and found useful both in vitro and in vivo (see, for example, U.S. Pat. No. 5,807,706; and Cao and Suresh, 1998, Bioconjugate Chem 9: 635-644). Numerous methods of preparing bispecific antibodies are known in the art, including the use of hybrid-hybridomas such as quadromas, which are formed by fusing two differed hybridomas, and triomas, which are formed by fusing a hybridoma with a lymphocyte (Milstein and Cuello, 1983, Nature 305: 537-540; U.S. Pat. No. 4,474,893; and U.S. Pat. No. 6,106,833). U.S. Pat. No. 6,060,285 discloses a process for the production of bispecific antibodies in which at least the genes for the light chain and the variable portion of the heavy chain of an antibody having a first specificity are transfected into a hybridoma cell secreting an antibody having a second specificity. Chemical coupling of antibody fragments has also been used to prepare antigen-binding molecules having specificity for two different antigens (Brennan et al., 1985, Science 229: 81-83; Glennie et al., J Immunol., 1987, 139:2367-2375; and U.S. Pat. No. 6,010,902). Bispecific antibodies can also be produced via recombinant means, for example, by using the leucine zipper moieties from the Fos and Jun proteins (which preferentially form heterodimers) as described by Kostelny et al. (J. Immunol. 148:1547-4553; 1992). U.S. Pat. No. 5,582,996 discloses the use of complementary interactive domains (such as leucine zipper moieties or other lock and key interactive domain structures) to facilitate heterodimer formation in the production of bispecific antibodies. Tetravalent, bispecific molecules can be prepared by fusion of DNA encoding the heavy chain of an F(ab′)2 fragment of an antibody with either DNA encoding the heavy chain of a second F(ab′)2 molecule (in which the CH1 domain is replaced by a CH3 domain), or with DNA encoding a single chain FV fragment of an antibody, as described in U.S. Pat. No. 5,959,083. Expression of the resultant fusion genes in mammalian cells, together with the genes for the corresponding light chains, yields tetravalent bispecific molecules having specificity for selected antigens. Bispecific antibodies can also be produced as described in U.S. Pat. No. 5,807,706. Generally, the method involves introducing a protuberance (constructed by replacing small amino acid side chains with larger side chains) at the interface of a first polypeptide and a corresponding cavity (prepared by replacing large amino acid side chains with smaller ones) in the interface of a second polypeptide. Moreover, single-chain variable fragments (sFvs) have been prepared by covalently joining two variable domains; the resulting antibody fragments can form dimers or trimers, depending on the length of a flexible linker between the two variable domains (Kortt et al., 1997, Protein Engineering 10:423-433).
Screening procedures by which such antibodies can be identified are well known, and can involve immunoaffinity chromatography, for example. Antibodies can be screened for agonistic (i.e., ligand-mimicking) properties. Such antibodies, upon binding to cell surface IMX97018, induce biological effects (e.g., transduction of biological signals) similar to the biological effects induced when the IMX97018 binding partner binds to cell surface IMX97018. Agonistic antibodies can be used to induce IMX97018-mediated cell stimulatory pathways or intercellular communication. Bispecific antibodies can be identified by screening with two separate assays, or with an assay wherein the bispecific antibody serves as a bridge between the first antigen and the second antigen (the latter is coupled to a detectable moiety). Bispecific antibodies that bind IMX97018 polypeptides of the invention via a first antigen binding domain will be useful in diagnostic applications. Examples of polypeptides (or other antigens) that the inventive bispecific antibodies bind via a second antigen binding domain include LANP, other leucine-rich polypeptides, and polyglutamine-containing polypeptides.
Also provided herein are conjugates comprising a detectable (e.g., diagnostic) or therapeutic agent, attached to the antibody. Examples of such agents are presented above. The conjugates find use in in vitro or in vivo procedures. The antibodies of the invention can also be used in assays to detect the presence of the polypeptides or fragments of the invention, either in vitro or in vivo. The antibodies also can be employed in purifying polypeptides or fragments of the invention by immunoaffinity chromatography.
Rational Design of Compounds that Interact with IMX97018 Polypeptides
The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact, e.g., inhibitors, agonists, antagonists, etc. Any of these examples can be used to fashion drugs which are more active or stable forms of the polypeptide or which enhance or interfere with the function of a polypeptide in vivo (Hodgson J (1991) Biotechnology 9:19-21). In one approach, the three-dimensional structure of a polypeptide of interest, or of a polypeptide-inhibitor complex, is determined by x-ray crystallography, by nuclear magnetic resonance, or by computer homology modeling or, most typically, by a combination of these approaches. Both the shape and charges of the polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Less often, useful information regarding the structure of a polypeptide may be gained by modeling based on the structure of homologous polypeptides. In both cases, relevant structural information is used to design analogous IMX97018-like molecules, to identify efficient inhibitors, or to identify small molecules that bind IMX97018 polypeptides. Useful examples of rational drug design include molecules which have improved activity or stability as shown by Braxton S and Wells J A (1992 Biochemistry 31:7796-7801) or which act as inhibitors, agonists, or antagonists of native peptides as shown by Athauda S B et al (1993 J Biochem 113:742-746). The use of IMX97018 polypeptide structural information in molecular modeling software systems to assist in inhibitor design and in studying inhibitor-IMX97018 polypeptide interaction is also encompassed by the invention. A particular method of the invention comprises analyzing the three dimensional structure of IMX97018 polypeptides for likely binding sites of substrates, synthesizing a new molecule that incorporates a predictive reactive site, and assaying the new molecule as described further herein.
It is also possible to isolate a target-specific antibody, selected by functional assay, as described further herein, and then to solve its crystal structure. This approach, in principle, yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass polypeptide crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original antigen. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides would then act as the pharmacore.
Assays of IMX97018 Polypeptide Activities
The purified IMX97018 polypeptides of the invention (including polypeptides, polypeptides, fragments, variants, oligomers, and other forms) are useful in a variety of assays. For example, the IMX97018 molecules of the present invention can be used to identify binding partners of IMX97018 polypeptides, which can also be used to modulate intercellular communication, cell stimulation, or immune cell activity. Alternatively, they can be used to identify non-binding-partner molecules or substances that modulate intercellular communication, cell stimulatory pathways, or immune cell activity.
Assays to Identify Binding Partners. Polypeptides of the IMX97018 family and fragments thereof can be used to identify binding partners. For example, they can be tested for the ability to bind a candidate binding partner in any suitable assay, such as a conventional binding assay. To illustrate, the IMX97018 polypeptide can be labeled with a detectable reagent (e.g., a radionuclide, chromophore, enzyme that catalyzes a colorimetric or fluorometric reaction, and the like). The labeled polypeptide is contacted with cells expressing the candidate binding partner. The cells then are washed to remove unbound labeled polypeptide, and the presence of cell-bound label is determined by a suitable technique, chosen according to the nature of the label.
One example of a binding assay procedure is as follows. A recombinant expression vector containing the candidate binding partner cDNA is constructed; the candidate binding partner can be part of a fusion protein construct that includes a leader peptide and/or a transmembrane domain, so that the candidate binding partner when expressed is located on the exterior of the cell surface. CV1-EBNA-1 cells in 10 cm2 dishes are transfected with this recombinant expression vector. CV-1/EBNA-1 cells (ATCC CRL 10478) constitutively express EBV nuclear antigen-1 driven from the CMV Immediate-early enhancer/promoter. CV1-EBNA-1 was derived from the African Green Monkey kidney cell line CV-1 (ATCC CCL 70), as described by McMahan et al., (EMBO J. 10:2821, 1991). The transfected cells are cultured for 24 hours, and the cells in each dish then are split into a 24-well plate. After culturing an additional 48 hours, the transfected cells (about 4×104 cells/well) are washed with BM-NFDM, which is binding medium (RPMI 1640 containing 25 mg/ml bovine serum albumin, 2 mg/ml sodium azide, 20 mM Hepes pH 7.2) to which 50 mg/ml nonfat dry milk has been added. The cells then are incubated for 1 hour at 37° C. with various concentrations of, for example, a soluble polypeptide/Fc fusion polypeptide made as set forth above. Cells then are washed and incubated with a constant saturating concentration of a 125I-mouse anti-human IgG in binding medium, with gentle agitation for 1 hour at 37° C. After extensive washing, cells are released via trypsinization. The mouse anti-human IgG employed above is directed against the Fc region of human IgG and can be obtained from Jackson Immunoresearch Laboratories, Inc., West Grove, Pa. The antibody is radioiodinated using the standard chloramine-T method. The antibody will bind to the Fc portion of any polypeptide/Fc polypeptide that has bound to the cells. In all assays, non-specific binding of 125I-antibody is assayed in the absence of the Fc fusion polypeptide/Fc, as well as in the presence of the Fc fusion polypeptide and a 200-fold molar excess of unlabeled mouse anti-human IgG antibody. Cell-bound 125I-antibody is quantified on a Packard Autogamma counter. Affinity calculations (Scatchard, Ann. N Y Acad. Sci. 51:660, 1949) are generated on RS/1 (BBN Software, Boston, Mass.) run on a Microvax computer. Binding can also be detected using methods that are well suited for high-throughput screening procedures, such as scintillation proximity assays (Udenfriend et al., 1985, Proc Natl Acad Sci USA 82: 8672-8676), homogeneous time-resolved fluorescence methods (Park et al., 1999, Anal Biochem 269: 94-104), fluorescence resonance energy transfer (FRET) methods (Clegg R M, 1995, Curr Opin Biotechnol 6: 103-110), or methods that measure any changes in surface plasmon resonance when a bound polypeptide is exposed to a potential binding partner, using for example a biosensor such as that supplied by Biacore AB (Uppsala, Sweden). Compounds that can be assayed for binding to IMX97018 polypeptides include but are not limited to small organic molecules, such as those that are commercially available—often as part of large combinatorial chemistry compound ‘libraries’—from companies such as Sigma-Aldrich (St. Louis, Mo.), Arqule (Woburn, Mass.), Enzymed (Iowa City, Iowa), Maybridge Chemical Co. (Trevillett, Cornwall, UK), MDS Panlabs (Bothell, Wash.), Pharmacopeia (Princeton, N.J.), and Trega (San Diego, Calif.). Preferred small organic molecules for screening using these assays are usually less than 10K molecular weight and can possess a number of physicochemical and pharmacological properties which enhance cell penetration, resist degradation, and/or prolong their physiological half-lives (Gibbs, J., 1994, Pharmaceutical Research in Molecular Oncology, Cell 79(2): 193-198). Compounds including natural products, inorganic chemicals, and biologically active materials such as proteins and toxins can also be assayed using these methods for the ability to bind to IMX97018 polypeptides.
Yeast Two-Hybrid or “Interaction Trap” Assays. Where the IMX97018 polypeptide binds or potentially binds to another polypeptide (such as, for example, in a receptor-ligand interaction), the nucleic acid encoding the IMX97018 polypeptide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify nucleic acids encoding the other polypeptide with which binding occurs or to identify inhibitors of the binding interaction. Polypeptides involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
Competitive Binding Assays. Another type of suitable binding assay is a competitive binding assay. To illustrate, biological activity of a variant can be determined by assaying for the variant's ability to compete with the native polypeptide for binding to the candidate binding partner. Competitive binding assays can be performed by conventional methodology. Reagents that can be employed in competitive binding assays include radiolabeled IMX97018 and intact cells expressing IMX97018 (endogenous or recombinant) on the cell surface. For example, a radiolabeled soluble IMX97018 fragment can be used to compete with a soluble IMX97018 variant for binding to cell surface receptors. Instead of intact cells, one could substitute a soluble binding partner/Fc fusion polypeptide bound to a solid phase through the interaction of Polypeptide A or Polypeptide G (on the solid phase) with the Fc moiety. Chromatography columns that contain Polypeptide A and Polypeptide G include those available from Pharmacia Biotech, Inc., Piscataway, N.J.
Diagnostic and Other Uses of IMX97018 Polypeptides and Nucleic Acids
The nucleic acids encoding the IMX97018 polypeptides provided by the present invention can be used for numerous diagnostic or other useful purposes. The nucleic acids of the invention can be used to express recombinant polypeptide for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding polypeptide is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on Southern gels; as chromosome markers or tags (when labeled) to identify chromosome 16q22.3 or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential polyglutamine-related genetic disorders, such as spinocerebellar ataxias; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to “subtract-out” known sequences in the process of discovering other novel nucleic acids; for selecting and making oligomers for attachment to a “gene chip” or other support, including for examination of expression patterns; to raise anti-polypeptide antibodies using DNA immunization techniques; as an antigen to raise anti-DNA antibodies or elicit another immune response, and for gene therapy. Uses of IMX97018 polypeptides and fragmented polypeptides include, but are not limited to, the following: purifying polypeptides and measuring the activity thereof; delivery agents; therapeutic and research reagents; molecular weight and isoelectric focusing markers; controls for peptide fragmentation; identification of unknown polypeptides; and preparation of antibodies. Any or all nucleic acids suitable for these uses are capable of being developed into reagent grade or kit format for commercialization as products. Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without limitation “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987
Probes and Primers. Among the uses of the disclosed IMX97018 nucleic acids, and combinations of fragments thereof, is the use of fragments as probes or primers. Such fragments generally comprise at least about 17 contiguous nucleotides of a DNA sequence. In other embodiments, a DNA fragment comprises at least 30, or at least 60, contiguous nucleotides of a DNA sequence. The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by Sambrook et al., 1989 and are described in detail above. Using knowledge of the genetic code in combination with the amino acid sequences set forth above, sets of degenerate oligonucleotides can be prepared. Such oligonucleotides are useful as primers, e.g., in polymerase chain reactions (PCR), whereby DNA fragments are isolated and amplified. In certain embodiments, degenerate primers can be used as probes for non-human genetic libraries. Such libraries would include but are not limited to cDNA libraries, genomic libraries, and even electronic EST (express sequence tag) or DNA libraries. Homologous sequences identified by this method would then be used as probes to identify non-human IMX97018 homologues.
Chromosome Mapping. The nucleic acids encoding IMX97018 polypeptides, and the disclosed fragments and combinations of these nucleic acids, can be used by those skilled in the art using well-known techniques to identify human chromosome 16, and in particular the 16q22.3 region of that chromosome, to which these nucleic acids map. Useful techniques include, but are not limited to, using the sequence or portions, including oligonucleotides, as a probe in various well-known techniques such as radiation hybrid mapping (high resolution), in situ hybridization to chromosome spreads (moderate resolution), and Southern blot hybridization to hybrid cell lines containing individual human chromosomes (low resolution).
Diagnostics and Gene Therapy. The nucleic acids encoding IMX97018 polypeptides, and the disclosed fragments and combinations of these nucleic acids can be used by one skilled in the art as a positional marker to map other genes of unknown location using well-known techniques. The nucleic acid encoding IMX97018 polypeptide has been located to a particular position on human chromosome 16q22.3 and can be used to map genetic disorders relative to that location, or to more precisely map human genetic disorders already known to be linked to chromosome 16. For example, the following genetic disorders are located near the chromosomal position of IX97018-encoding sequences, but the genes associated with these disorders have not yet been identified: Zonular Cataract (also called Perinuclear Cataract, Lamellar Cataract, Marner Cataract); Wilms Tumor Gene 3 (WT3); Acute Myelogenous Leukemia (AMLCR2), North American Indian Childhood Cirrhosis (CIRHIA, NAIC). As discussed below, another genetic disorder that maps closely to IMX97018-encoding sequences is Spinocerebellar Ataxia 4 (SCA4). IMX97018-encoding nucleic acids can be used to analyze genetic abnormalities associated with the SCA4 disorder, for example, enabling one of skill in the art to distinguish SCA4 kindreds in which chromosomal regions comprising IMX97018-encoding sequences are rearranged or deleted. There is substantial utility in nucleic acids that can be used to confirm or to eliminate SCA4 as a genetic factor for a kindred presenting with hereditary ataxia. For example, elimination of the known forms of SCA is the first step in diagnosing a family with a hereditary ataxia (see, for example, Devos et al., 2001, Neurology 56: 234-238). Further, assuming that the SCA4 gene overlaps with or corresponds to UMX97018-encoding sequences, the IMX97018-encoding nucleic acids can be used to identify genetic alterations in the SCA4 gene at the nucleotide level. Additionally, the IMX97018-encoding nucleic acids can be used in developing treatments for any disorder mediated (directly or indirectly) by defective, or insufficient amounts of, the genes corresponding to the nucleic acids of the invention. Disclosure herein of native nucleotide sequences permits the detection of defective genes, and the replacement thereof with normal genes. Defective genes can be detected in in vitro diagnostic assays, and by comparison of a native nucleotide sequence disclosed herein with that of a gene derived from a person suspected of harboring a defect in this gene.
Methods of Screening for Binding Partners. The IMX97018 polypeptides of the invention each can be used as reagents in methods to screen for or identify binding partners. For example, the IMX97018 polypeptides can be attached to a solid support material and may bind to their binding partners in a manner similar to affinity chromatography. In particular embodiments, a polypeptide is attached to a solid support by conventional procedures. As one example, chromatography columns containing functional groups that will react with functional groups on amino acid side chains of polypeptides are available (Pharmacia Biotech, Inc., Piscataway, N.J.). In an alternative, a polypeptide/Fc polypeptide (as discussed above) is attached to protein A- or protein G-containing chromatography columns through interaction with the Fc moiety. Purified IMX97018 polypeptides are bound to a solid phase such as a column chromatography matrix or a similar suitable substrate. For example, magnetic microspheres can be coated with the polypeptides and held in an incubation vessel through a magnetic field. Suspensions of cell mixtures containing potential binding-partner-expressing cells are contacted with the solid phase having the polypeptides thereon. Cells expressing the binding partner on the cell surface bind to the fixed polypeptides, and unbound cells are washed away. Alternatively, IMX97018 polypeptides can be conjugated to a detectable moiety, then incubated with cells to be tested for binding partner expression. After incubation, unbound labeled matter is removed and the presence or absence of the detectable moiety on the cells is determined. In a further alternative, mixtures of cells suspected of expressing the binding partner are incubated with biotinylated polypeptides. Incubation periods are typically at least one hour in duration to ensure sufficient binding. The resulting mixture then is passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin provides binding of the desired cells to the beads. Procedures for using avidin-coated beads are known (see Berenson, et al. J. Cell. Biochem., 10D:239, 1986). Washing to remove unbound material, and the release of the bound cells, are performed using conventional methods. In some instances, the above methods for screening for or identifying binding partners may also be used or modified to isolate or purify such binding partner molecules or cells expressing them.
Measuring Biological Activity. Polypeptides also find use in measuring the biological activity of IMX97018-binding polypeptides in terms of their binding affinity. The polypeptides thus can be employed by those conducting “quality assurance” studies, e.g., to monitor shelf life and stability of polypeptide under different conditions. For example, the polypeptides can be employed in a binding affinity study to measure the biological activity of a binding partner polypeptide that has been stored at different temperatures, or produced in different cell types. The polypeptides also can be used to determine whether biological activity is retained after modification of a binding partner polypeptide (e.g., chemical modification, truncation, mutation, etc.). The binding affinity of the modified polypeptide is compared to that of an unmodified binding polypeptide to detect any adverse impact of the modifications on biological activity of the binding polypeptide. The biological activity of a binding polypeptide thus can be ascertained before it is used in a research study, for example.
Carriers and Delivery Agents. The polypeptides also find use as carriers for delivering agents attached thereto to cells comprising identified binding partners. In one method of the invention, the IMX97018 polypeptide is covalently linked to a protein-transduction domain (PTD) such as, but not limited to, TAT, Antp, or VP22 (Schwarze et al., 2000, Cell Biology 10: 290-295). The polypeptides thus can be used to deliver diagnostic or therapeutic agents into such cells in in vitro or in vivo procedures. Detectable (diagnostic) and therapeutic agents that can be attached to a polypeptide include, but are not limited to, toxins, other cytotoxic agents, drugs, radionuclides, chromophores, enzymes that catalyze a colorimetric or fluorometric reaction, and the like, with the particular agent being chosen according to the intended application. Among the toxins are ricin, abrin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, ribosomal inactivating polypeptides, mycotoxins such as trichothecenes, and derivatives and fragments (e.g., single chains) thereof. Radionuclides suitable for diagnostic use include, but are not limited to, 123I, 131I, 99mTc, 111In, and 76Br. Examples of radionuclides suitable for therapeutic use are 131I, 211At, 77Br, 186Re, 188Re, 212Pb, 212Bi, 109Pd, 64Cu, and 67Cu. Such agents can be attached to the polypeptide by any suitable conventional procedure. The polypeptide comprises functional groups on amino acid side chains that can be reacted with functional groups on a desired agent to form covalent bonds, for example. Alternatively, the polypeptide or agent can be derivatized to generate or attach a desired reactive functional group. The derivatization can involve attachment of one of the bifunctional coupling reagents available for attaching various molecules to polypeptides (Pierce Chemical Company, Rockford, Ill.). A number of techniques for radiolabeling polypeptides are known. Radionuclide metals can be attached to polypeptides by using a suitable bifunctional chelating agent, for example. Conjugates comprising polypeptides and a suitable diagnostic or therapeutic agent (preferably covalently linked) are thus prepared. The conjugates are administered or otherwise employed in an amount appropriate for the particular application.
Treating Diseases with IMX97018 Polypeptides and Antagonists Thereof
The IMX97018 polypeptides, fragments, variants, antagonists, agonists, antibodies, and binding partners of the invention are likely to be useful for treating medical conditions and diseases including, but not limited to, neurological and SCA conditions as described further herein. The therapeutic molecule or molecules to be used will depend on the etiology of the condition to be treated and the biological pathways involved, and variants, fragments, and binding partners of IMX97018 polypeptides may have effects similar to or different from IMX97018 polypeptides. For example, an antagonist of the SCA-promoting activity of IMX97018 polypeptides containing polyglutamine tracts (‘IMX97018polyQ polypeptides’) can be selected for treatment of conditions involving expansion of polyglutamine tracts, but a particular fragment of a given IMX97018 polypeptide itself, such as a fragment comprising the AXH domain, may also act as an effective dominant negative antagonist of that activity. Therefore, in the following paragraphs “IMX97018 polypeptides or antagonists” refers to all IMX97018 polypeptides, fragments, variants, antagonists, agonists, antibodies, and binding partners etc. of the invention, and it is understood that a specific molecule or molecules can be selected from those provided as embodiments of the invention by individuals of skill in the art, according to the biological and therapeutic considerations described herein.
Antagonists of IMX97018polyQ polypeptides and IMX97018 polypeptides and agonists are useful for treating SCA-related disorders including Spinocerebellar atrophy I (SCA1), Olivopontocerebellar atrophy I (OPCA1), Menzel type OPCA, Spinocerebellar atrophy IV (SCA4), and Spinocerebellar ataxia, autosomal dominant, with sensory axonal neuropathy; ataxia; progressive loss of muscle coordination and tone; dementia; Alzheimer's disease, disorders of vertebral disk or column development; Bell's palsy; transmissible dementia, including Creutzfeld-Jacob disease; demyelinating neuropathy; Guillain-Barre syndrome; myasthenia gravis; silent cerebral ischemia; sleep disorders, including insomnia, narcolepsy, and sleep apnea; chronic neuronal degeneration; stroke, including cerebral ischemic diseases; and neurological conditions such as those associated with the following symptoms: progressive cerebellar ataxia, cerebellar ataxia of gait and limbs, supranuclear ophthalmoplegia, pyramidal or extrapyramidal signs, mild dementia, peripheral neuropathy, macular and retinal degeneration, upper motor neuron signs, extensor plantar responses, involuntary choreiform movements, abnormal eye movements, abnormal saccade amplitude or velocity, presence of gaze-evoked nystagmus, hypermetria, lower bulbar palsies, hyperreflexia, scanning and explosive speech, incoordination, slow motor-nerve conduction, atrophy of the cerebellum, pons and olives, degeneration of lower cranial nerve nuclei, and atrophy of the dorsal columns and spinocerebellar tracts, abnormal deep tendon reflexes, reduced aspartic acid and/or markedly elevated taurine content in brain tissue, reduction in platelet glutamate dehydrogenase activity, lack of activation of GDH by ADP in either the presence or the absence of Triton, neuronal loss from the pars compacta of the substantia nigra or in the locus coeruleus, severe atrophy of the dentatorubral pathways, severe loss of Purkinje cells and degeneration of the olivocerebellar pathways, atrophy of the nucleus pontis, marked atrophy of Clarke columns and the spinocerebellar tracts, diplopia, severe spasticity or pronounced peripheral neuropathy, impaired temperature discrimination, abnormal peripheral and central motor conduction times in motor evoked potentials, pontine and cerebellar atrophy, enlargement of the fourth ventricle, gait disturbance, difficulty with fine motor tasks, dysarthria, vibratory and joint position sense loss, pinprick-sensation loss, loss of ankle-jerk reflexes, loss of knee-jerk reflexes, and complete areflexia. In addition, provided herein is the use of antagonists of IMX97018polyQ polypeptides and IMX97018 polypeptides and agonists to treat AIDS-related neurological conditions, such as AIDS dementia complex. Antagonists of IMX97018polyQ polypeptides are also useful for treating polyglutamine-related disorders including fragile X syndrome, myotonic dystrophy, Kennedy spinal and bulbar muscular atrophy, and Huntington disease.
Antagonists of IMX97018 polypeptides and IMX97018polyQ polypeptides and agonists are useful for promoting cell death, and particularly for promoting neural cell death via a non-apoptotic mechanism. Provided herein are methods for using antagonists of IMX97018 polypeptides, IMX97018polyQ polypeptides, IMX97018polyQ polypeptide agonists, compositions or combination therapies to treat various neural oncologic disorders including brain tumors, glioma, glioblastoma, astrocytoma, oligodendroglioma, ependymoma, ganglioglioma, medulloblastoma, neuroectodermal tumors, and pilocytic astrocytoma.
Administration of IMX97018 Polypeptides and Antagonists Thereof
This invention provides compounds, compositions, and methods for treating a patient, preferably a mammalian patient, and most preferably a human patient, who is suffering from a medical disorder, and in particular an IM4×97018-mediated disorder. Such IMX97018-mediated disorders include conditions caused (directly or indirectly) or exacerbated by binding between IMX97018 and a binding partner. For purposes of this disclosure, the terms “illness,” “disease,” “medical condition,” “abnormal condition” and the like are used interchangeably with the term “medical disorder.” The terms “treat”, “treating”, and “treatment” used herein includes curative, preventative (e.g., prophylactic) and palliative or ameliorative treatment. For such therapeutic uses, IMX97018 polypeptides and fragments, IMX97018 nucleic acids encoding the IMX97018 family polypeptides, and/or agonists or antagonists of the IMX97018 polypeptide such as antibodies can be administered to the patient in need through well-known means. Compositions of the present invention can contain a polypeptide in any form described herein, such as native polypeptides, variants, derivatives, oligomers, and biologically active fragments. In particular embodiments, the composition comprises a soluble polypeptide or an oligomer comprising soluble IMX97018 polypeptides.
Therapeutically Effective Amount. In practicing the method of treatment or use of the present invention, a therapeutically effective amount of a therapeutic agent of the present invention is administered to a patient having a condition to be treated, preferably to treat or ameliorate diseases associated with the activity of an IMX97018 family polypeptide. “Therapeutic agent” includes without limitation any of the EMX97018 polypeptides, fragments, and variants; nucleic acids encoding the IMX97018 family polypeptides, fragments, and variants; agonists or antagonists of the IMX97018 polypeptides such as antibodies; IMX97018 polypeptide binding partners; complexes formed from the IMX97018 family polypeptides, fragments, variants, and binding partners, etc. As used herein, the term “therapeutically effective amount” means the total amount of each therapeutic agent or other active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual therapeutic agent or active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. As used herein, the phrase “administering a therapeutically effective amount” of a therapeutic agent means that the patient is treated with said therapeutic agent in an amount and for a time sufficient to induce an improvement, and preferably a sustained improvement, in at least one indicator that reflects the severity of the disorder. An improvement is considered “sustained” if the patient exhibits the improvement on at least two occasions separated by one or more days, or more preferably, by one or more weeks. The degree of improvement is determined based on signs or symptoms, and determinations can also employ questionnaires that are administered to the patient, such as quality-of-life questionnaires. Various indicators that reflect the extent of the patient's illness can be assessed for determining whether the amount and time of the treatment is sufficient. The baseline value for the chosen indicator or indicators is established by examination of the patient prior to administration of the first dose of the therapeutic agent. Preferably, the baseline examination is done within about 60 days of administering the first dose. If the therapeutic agent is being administered to treat acute symptoms, the first dose is administered as soon as practically possible after the injury has occurred. Improvement is induced by administering therapeutic agents such as IMX97018 polypeptides or antagonists until the patient manifests an improvement over baseline for the chosen indicator or indicators. In treating chronic conditions, this degree of improvement is obtained by repeatedly administering this medicament over a period of at least a month or more, e.g., for one, two, or three months or longer, or indefinitely. A period of one to six weeks, or even a single dose, often is sufficient for treating injuries or other acute conditions. Although the extent of the patient's illness after treatment may appear improved according to one or more indicators, treatment may be continued indefinitely at the same level or at a reduced dose or frequency. Once treatment has been reduced or discontinued, it later may be resumed at the original level if symptoms should reappear.
Dosing. One skilled in the pertinent art will recognize that suitable dosages will vary, depending upon such factors as the nature and severity of the disorder to be treated, the patient's body weight, age, general condition, and prior illnesses and/or treatments, and the route of administration. Preliminary doses can be determined according to animal tests, and the scaling of dosages for human administration is performed according to art-accepted practices such as standard dosing trials. For example, the therapeutically effective dose can be estimated initially from cell culture assays. The dosage will depend on the specific activity of the compound and can be readily determined by routine experimentation. A dose can 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, while minimizing toxicities. Such information can be used to more accurately determine useful doses in humans. Ultimately, the attending physician will decide the amount of polypeptide of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of polypeptide of the present invention and observe, the patient's response. Larger doses of polypeptide of the present invention can be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 ng to about 100 mg (preferably about 0.1 ng to about 10 mg, more preferably about 0.1 microgram to about 1 mg) of polypeptide of the present invention per kg body weight. In one embodiment of the invention, IMX97018 polypeptides or antagonists are administered one time per week to treat the various medical disorders disclosed herein, in another embodiment is administered at least two times per week, and in another embodiment is administered at least three times per week. If injected, the effective amount of IMX97018 polypeptides or antagonists per adult dose ranges from 1-20 mg/m2, and preferably is about 5-12 mg/m2. Alternatively, a flat dose can be administered, whose amount may range from 5-100 mg/dose. Exemplary dose ranges for a flat dose to be administered by subcutaneous injection are 5-25 mg/dose, 25-50 mg/dose and 50-100 mg/dose. In one embodiment of the invention, the various indications described below are treated by administering a preparation acceptable for injection containing IMX97018 polypeptides or antagonists at 25 mg/dose, or alternatively, containing 50 mg per dose. The 25 mg or 50 mg dose can be administered repeatedly, particularly for chronic conditions. If a route of administration other than injection is used, the dose is appropriately adjusted in accord with standard medical practices. In many instances, an improvement in a patient's condition will be obtained by injecting a dose of about 25 mg of IMX97018 polypeptides or antagonists one to three times per week over a period of at least three weeks, or a dose of 50 mg of IMX97018 polypeptides or antagonists one or two times per week for at least three weeks, though treatment for longer periods may be necessary to induce the desired degree of improvement. For incurable chronic conditions, the regimen can be continued indefinitely, with adjustments being made to dose and frequency if such are deemed necessary by the patient's physician. The foregoing doses are examples for an adult patient who is a person who is 18 years of age or older. For pediatric patients (age 4-17), a suitable regimen involves the subcutaneous injection of 0.4 mg/kg, up to a maximum dose of 25 mg of IMX97018 polypeptides or antagonists, administered by subcutaneous injection one or more times per week. If an antibody against an IMX97018 polypeptide is used as the IMX97018 polypeptide antagonist, a preferred dose range is 0.1 to 20 mg/kg, and more preferably is 1-10 mg/kg. Another preferred dose range for an anti-IMX97018 polypeptide antibody is 0.75 to 7.5 mg/kg of body weight. Humanized antibodies are preferred, that is, antibodies in which only the antigen-binding portion of the antibody molecule is derived from a non-human source. Such antibodies can be injected or administered intravenously.
Formulations. Compositions comprising an effective amount of an IMX97018 polypeptide of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant, sources), in combination with other components such as a physiologically acceptable diluent, carrier, or excipient, are provided herein. The term “Pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). Formulations suitable for administration include aqueous and non-aqueous sterile injection solutions which can contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which can include suspending agents or thickening agents. The polypeptides can be formulated according to known methods used to prepare pharmaceutically useful compositions. They can be combined in admixture, either as the sole active material or with other known active materials suitable for a given indication, with pharmaceutically acceptable diluents (e.g., saline, Tris-HCl, acetate, and phosphate buffered solutions), preservatives (e.g., thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/or carriers. Suitable formulations for pharmaceutical compositions include those described in Remington's Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Company, Easton, Pa. In addition, such compositions can be complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, etc., or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and U.S. Pat. No. 4,737,323. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application, so that the characteristics of the carrier will depend on the selected route of administration. In one preferred embodiment of the invention, sustained-release forms of IMX97018 polypeptides are used. Sustained-release forms suitable for use in the disclosed methods include, but are not limited to, IMX97018 polypeptides that are encapsulated in a slowly-dissolving biocompatible polymer (such as the alginate microparticles described in U.S. Pat. No. 6,036,978), admixed with such a polymer (including topically applied hydrogels), and or encased in a biocompatible semi-permeable implant.
Combinations of Therapeutic Compounds. AN IMX97018 polypeptide of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other polypeptides. As a result, pharmaceutical compositions of the invention may comprise a polypeptide of the invention in such multimeric or complexed form. The pharmaceutical composition of the invention may be in the form of a complex of the polypeptide(s) of present invention along with polypeptide or peptide antigens. The invention further includes the administration of IMX97018 polypeptides or antagonists concurrently with one or more other drugs that are administered to the same patient in combination with the IMX97018 polypeptides or antagonists, each drug being administered according to a regimen suitable for that medicament. “Concurrent administration” encompasses simultaneous or sequential treatment with the components of the combination, as well as regimens in which the drugs are alternated, or wherein one component is administered long-term and the other(s) are administered intermittently. Components can be administered in the same or in separate compositions, and by the same or different routes of administration. Examples of components that can be administered concurrently with the pharmaceutical compositions of the invention are: cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-17, IL-18, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin, or inhibitors or antagonists of any of these factors. The pharmaceutical composition can further contain other agents which either enhance the activity of the polypeptide or compliment its activity or use in treatment. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with polypeptide of the invention, or to minimize side effects. Conversely, an IMX97018 polypeptide or antagonist of the present invention may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent. Additional examples of drugs to be administered concurrently include but are not limited to antivirals, antibiotics, analgesics, corticosteroids, antagonists of inflammatory cytokines, non-steroidal anti-inflammatories, pentoxifylline, thalidomide, and disease-modifying antirheumatic drugs (DMARDs) such as azathioprine, cyclophosphamide, cyclosporine, hydroxychloroquine sulfate, methotrexate, leflunomide, minocycline, penicillamine, sulfasalazine and gold compounds such as oral gold, gold sodium thiomalate, and aurothioglucose. Additionally, IMX97018 polypeptides or antagonists can be combined with a second IMX97018 polypeptide/antagonist, including an antibody against an IMX97018 polypeptide, or an IMX97018 polypeptide-derived peptide that acts as a competitive inhibitor of a native IMX97018 polypeptide.
Routes of Administration. Any efficacious route of administration can be used to therapeutically administer IMX97018 polypeptides or antagonists thereof, including those compositions comprising nucleic acids. Parenteral administration includes injection, for example, via intra-articular, intravenous, intramuscular, intralesional, intraperitoneal or subcutaneous routes by bolus injection or by continuous infusion, and also includes localized administration, e.g., at a site of disease or injury. Other suitable means of administration include sustained release from implants; aerosol inhalation and/or insufflation; eyedrops; vaginal or rectal suppositories; buccal preparations; oral preparations, including pills, syrups, lozenges, ice creams, or chewing gum; and topical preparations such as lotions, gels, sprays, ointments or other suitable techniques. Alternatively, polypeptideaceous IMX97018 polypeptides or antagonists may be administered by implanting cultured cells that express the polypeptide, for example, by implanting cells that express IMX97018 polypeptides or antagonists. Cells may also be cultured ex vivo in the presence of polypeptides of the present invention in order to modulate cell proliferation or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes. The polypeptide of the instant invention may also be administered by the method of protein transduction. In this method, the IMX97018 polypeptide is covalently linked to a protein-transduction domain (PTD) such as, but not limited to, TAT, Antp, or VP22 (Schwarze et al., 2000, Cell Biology 10: 290-295). The PTD-linked peptides can then be transduced into cells by adding the peptides to tissue-culture media containing the cells (Schwarze et al., 1999, Science 285:1569; Lindgren et al., 2000, TiPS 21: 99; Derossi et al., 1998, Cell Biology 8: 84; WO 00/34308; WO 99/29721; and WO 99/10376). In another embodiment, the patient's own cells are induced to produce IMX97018 polypeptides or antagonists by transfection in vivo or ex vivo with a DNA that encodes IMX97018 polypeptides or antagonists. This DNA can be introduced into the patient's cells, for example, by injecting naked DNA or liposome-encapsulated DNA that encodes IMX97018 polypeptides or antagonists, or by other means of transfection. Nucleic acids of the invention can also be administered to patients by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA). When IMX97018 polypeptides or antagonists are administered in combination with one or more other biologically active compounds, these can be administered by the same or by different routes, and can be administered simultaneously, separately or sequentially.
Oral Administration. When a therapeutically effective amount of polypeptide of the present invention is administered orally, polypeptide of the present invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention can additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% polypeptide of the present invention, and preferably from about 25 to 90% polypeptide of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils can be added. The liquid form of the pharmaceutical composition can further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of polypeptide of the present invention, and preferably from about 1 to 50% polypeptide of the present invention.
Intravenous Administration. When a therapeutically effective amount of polypeptide of the present invention is administered by intravenous, cutaneous or subcutaneous injection, polypeptide of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable polypeptide 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 polypeptide of the present invention, 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 can also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art. The duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the polypeptide of the present invention will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.
Tissue Administration. For compositions of the present invention which are useful for neural tissue disorders, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition can desirably be encapsulated or injected in a viscous form for delivery to the site of tissue damage. Topical administration may be suitable for wound healing and tissue repair. Therapeutically useful agents other than a polypeptide of the invention or antagonist thereof which may also optionally be included in the composition as described above, can alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention. The composition can include a matrix capable of delivering the polypeptide- or antagonist-containing composition to the site of tissue damage, providing a structure for the developing tissue and optimally capable of being resorbed into the body. Such matrices can be formed of materials presently in use for other implanted medical applications. The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions will define the appropriate formulation. Potential matrices for the compositions can be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen. Further matrices are comprised of pure polypeptides or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or other ceramics Matrices can be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. The bioceramics can be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability. Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns. In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the polypeptide compositions from disassociating from the matrix. A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethyl-cellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). The amount of sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt % based on total formulation weight, which represents the amount necessary to prevent desorption of the polypeptide or antagonist from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the polypeptide or antagonist the opportunity to assist the activity of the progenitor cells. In further compositions, polypeptides of the invention or antagonists thereof may be combined with other agents beneficial to the treatment of the wound or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-alpha and TGF-beta), and insulin-like growth factor (IGF). The dosage regimen of a polypeptide-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the polypeptides, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue, the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage can vary with the type of matrix used in the reconstitution and with inclusion of other polypeptides in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of tissue growth and/or repair, for example, X-rays, histomorphometric determinations, and tetracycline labeling.
Veterinary Uses. In addition to human patients, IMX97018 polypeptides and antagonists are useful in the treatment of disease conditions in non-human animals, such as pets (dogs, cats, birds, primates, etc.), domestic farm animals (horses cattle, sheep, pigs, birds, etc.), thoroughbred horses, or any animal that suffers from an IMX97018-mediated condition. In such instances, an appropriate dose can be determined according to the animal's body weight. For example, a dose of 0.2-1 mg/kg may be used. Alternatively, the dose is determined according to the animal's surface area, an exemplary dose ranging from 0.1-20 mg/m2, or more preferably, from 5-12 mg/m2. For small animals, such as dogs or cats, a suitable dose is 0.4 mg/kg. In a preferred embodiment, IMX97018 polypeptides or antagonists (preferably constructed from genes derived from the same species as the patient), is administered by injection or other suitable route one or more times per week until the animal's condition is improved, or it can be administered indefinitely.
Manufacture of Medicaments. The present invention also relates to the use of IMX97018 polypeptides, fragments, and variants; nucleic acids encoding the IMX97018 family polypeptides, fragments, and variants; agonists or antagonists of the IMX97018 polypeptides such as antibodies; IMX97018 polypeptide binding partners; complexes formed from the IMX97018 family polypeptides, fragments, variants, and binding partners, etc, in the manufacture of a medicament for the prevention or therapeutic treatment of each medical disorder disclosed herein.
EXAMPLES
The following examples are intended to illustrate particular embodiments and not to limit the scope of the invention.
Example 1
Identification of IMX97018, a New Human Ataxin-1-Like Polypeptide
A partial cDNA sequence was identified in a cDNA library prepared from human dendritic cells (described in U.S. Pat. No. 6,017,729, issued Jan. 25, 2000). That cDNA was used to identify a human chromosome 16 genomic contig (AC009127.8) that does include the entire IMX97018 coding sequence, shown as SEQ ID NO:1. The entire IMX97018 coding sequence was determined based on comparisons of human ataxin-1 polypeptide with all possible reading frames of AC009127, using the GCG program TFASTA. Nucleotides 1 through 2067 of SEQ ID NO:1 encode the IMX97018 polypeptide, the amino acid sequence of which is shown as SEQ ID NO:2. The approximate position of the single exon containing IMX97018 coding sequence in the AC009127.8 contig is shown in the table below, along with its location relative to SEQ ID NO:1; note that the coding sequence is in the inverse orientation with respect to the contig, and that the 5′ and 3′ untranslated regions may extend further along the contig sequence beyond those portions that correspond to SEQ ID NO:1, as indicated by the parentheses around the AC009127.8 positions in the table.
Corresponding position of IMX97018 gene exon in human contig AC009127.8 and in SEQ ID NO:1:
Position in AC009127.8
Position in SEQ ID NO: 1
Exon 1
(162604)-(160538)
1-2067
The human genomic region corresponding to the AC009127.8 contig, 16q22, also includes the genetic map location for SCA4. SCA4 was mapped by Flanigan et al. (1996, Am J Hum Genet 59: 392-399) to a 6 centiMorgan region around the D16S397 marker on 16q22.1, and bounded on the distal end by D16S512 at 16q22.3. The AC009127.8 contig maps between D16S397 and D16S512 within 16q22.3. Therefore, the ataxin-1-like IMX97018 polypeptide is considered to be a strong candidate for the product of the SCA4 gene.
Additional variations of IMX97018 polypeptides are provided as naturally occurring genomic variants of the IMX97018 sequences disclosed herein; such variations may be incorporated into an IMX97018 polypeptide or nucleic acid individually or in any combination. As one example, the GGC codon encoding the Gly residue at position 428 of SEQ ID NO:2 is present as an AGC codon encoding a Ser residue in a naturally occurring variant of SEQ ID NO: 1, likely representing an allelic variation.
The amino acid sequence of IMX97018 (SEQ ID NO:2) was compared with the amino acid sequences of ataxin-1-like polypeptides—human (‘Hs’), mouse (‘Mm’), and rat (‘Rn’) ataxin-1 (SEQ ID NO:3-SEQ ID NO:5, respectively)—using the GCG “pretty” multiple sequence alignment program, with amino acid similarity scoring matrix=blosum62, gap creation penalty=8, and gap extension penalty=1. An alignment of these sequences is shown in Table 1, and includes consensus residues which are identical among all four of the amino acid sequences in the alignment. The capitalized residues in the alignment are those which match the consensus residues. The numbering of amino acid residues in Table 1 corresponds to the position of those residues in the IMX97018 amino acid sequence (SEQ ID NO:2). The AXH ataxin-1 and HMG-box-containing conserved domain sequence (SEQ ID NO:6) has been optimally aligned with the Table 1 amino acid sequences, and residues within the AXH sequence that match the Table 1 consensus residues are capitalized.
Amino acid substitutions and other alterations (deletions, insertions, etc.) to IMX97018 amino acid sequences (e.g. SEQ ID NO:2) are predicted to be more likely to alter or disrupt IMX97018 polypeptide activities if they result in changes to the capitalized residues of the amino acid sequences as shown in Table 1, and particularly if those changes do not substitute an amino acid of similar structure (such as substitution of any one of the aliphatic residues—Ala, Gly, Leu, Ile, or Val—for another aliphatic residue), or a residue present in ataxin-1 polypeptides or the AXH domain at that conserved position. Conversely, if a change is made to an IMX97018 amino acid sequence resulting in a substitution of the residue at that position in the alignment from one of the other Table 1 ataxin-1 polypeptide or AXH domain sequences, it is less likely that such an alteration will affect the function of the altered IMX97018 polypeptide. For example, the consensus residue at position 504 in Table 1 is lysine, and the AXH domain has an aspartate at that position. Substitution of aspartate or the chemically similar glutamate for lysine at that position is considered less likely to alter the function of the polypeptide than substitution of tryptophan or tyrosine etc. Embodiments of the invention include IMX97018 polypeptides and fragments of IMX97018 polypeptides, comprising altered amino acid sequences. Altered IMX97018 polypeptide sequences share at least 30%, or more preferably at least 40%, or more preferably at least 50%, or more preferably at least 55%, or more preferably at least 60%, or more preferably at least 65%, or more preferably at least 70%, or more preferably at least 75%, or more preferably at least 80%, or more preferably at least 85%, or more preferably at least 90%, or more preferably at least 95%, or more preferably at least 97.5%, or more preferably at least 99%, or most preferably at least 99.5% amino acid identity with one or more of the amino acid sequences shown in Table 1. When IMX97018 polypeptide variants according to the invention, such as allelic variants or IMX97018 polypeptides having deliberately engineered modifications, are analyzed using the GeneFold algorithm (Tripos Inc., St. Louis, Mo., see tripos.com/admin/LitCtr/genefold app.pdf), the top five scoring template structures will be ataxin-1 or AXH-domain-containing polypeptides. The score for these top five hits, using any of the three types of score reported by GeneFold (sequence only, sequence plus local conformation preferences plus burial terms, or sequence plus local conformation preferences plus burial terms plus secondary structure) preferably will be at least 500, more preferably at least 750, and most preferably 999.9.
TABLE 1
Alignment of IMX97018 amino acid sequence with those of ataxin-1
polypeptides
Protein
(SEQ ID NO)
1 50
IMX97018
MXpvhERsqE CLPPKKRdlP vTSedmgrtt scstnhtpss daseWsrgvv
(SEQ ID NO: 2)
Hs
MKsnqERsnE CLPPKKReiP aTSrsseeka ptlpsdnhrv egtaWlpg.n
ATX1(SEQ
ID NO: 3)
Mm
MKsnqERtnE CLPPKKReiP aTSrpseeka talpsdnhcv egvaWlps.t
ATX1(SEQ
ID NO: 4)
Rn
MKsnqERsnE CLPPKKReiP aTSrpseeka talpsdnhcv egvaWlps.t
ATX1(SEQ
ID NO: 5)
51 98
IMX97018
vagqsqaGaR vslgGdgaEa itGLtvdqyG m.LyKvavpp atfSrtglP.
(SEQ ID NO:
2)
Hs
pggrghgGgR hgpaGtsvE. .lGL...qqG igLhKalstg ldySPpsaPr
ATX1(SEQ
ID NO: 3)
Mm
pgirghgGgR hgsaGtsgE. .hGL....qG mgLIXalsag ldySPpsaPr
ATX1(SEQ
ID NO: 4)
Rn
pgsrghgGgR hgpaGtSgE. .hGL....qG mgLhKalsag ldySPpsaPr
ATX1(SEQ
ID NO: 5)
99 .147
IMX97018
SVvnmspLPp tfnvassliq hpGihypPIh YAqLpsTslQ FIG.SpYSlp
(SEQ ID NO:
2)
Hs
SVpvattLPa ayatpqp... ..GtpvsPvq YAhLphT.fQ FIGsSqYSgt
ATX1(SEQ
ID NO: 3)
Mm
SVptantLPt vypppqs... ..GtpvsPvq YAhLshT.fQ FIGsSqYSgp
ATX1(SEQ
ID NO: 4)
Rn
SVptantLPt vypppqs... ..GtpvsPvq YAhLShT.fQ FIGsSqYSgp
ATX1(SEQ
ID NO: 5)
148 190
IMX97018
YAvppnFlPS pLlsPsaNla TShlphfvpy AsllAeGATp PpQ~~~~~~~
(SEQ ID NO:
2)
Hs
YA...sFiPS qLipPtaNpv TS........ AvasAaGATt PsQrsqleay
ATX1(SEQ
ID NO: 3)
Mm
YA...gFiPS qLisPsgNpv TS........ AvasAaGATt PsQrsqleay
ATX1(SEQ
ID NO: 4)
Rn
YA...gFiPS qLisPpgNpv TS........ AvnsAnGATt PsQrsqleay
ATX1(SEQ
ID NO: 5)
191 197
IMX97018
~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~apspahs
(SEQ ID NO:
2)
Hs
stllanmgsl sqtpghkaeq qqqqqqqqqq qhqhqqqqqq qqqqqqqqqh
ATX1(SEQ
ID NO:3)
Mm
stllanmgsl sqapghkve. .......... .......... ....pppqqh
ATX1(SEQ
ID NO: 4)
Rn
stllanmgsl sqapgbkve. .......... .......... ....pppqqh
ATX1(SEQ
ID NO: 5)
198 236
1MX97018
fnkApsatsP sgqlPh.... ...HsStqPl d....lapgr mPiyyqmsrl
(SEQ ID NO:
2)
Hs
lsrApglitP gs.pPpaqqn qyvHiSssPq ntgrtasppa iPvhlhphqt
ATX1(SEQ
ID NO: 3)
Mm
lsrAaglvnP gsppPptqqn qyiHiSssPq ssgratsppp iPvhlhphqt
ATX1(SEQ
ID NO: 4)
Rn
lgrAaglvnP gs.pPptqqn qyiHiSssPq ssgrats.pp iPvhlhpbqt
ATX1(SEQ
ID NO: 5)
237 270
IMX97018
pagyTLhetP p.......ag aspvltPqEs .........Q sAleAaaang
(SEQ ID NO:
2)
Hs
miphTLtlgP psqvvmqyad sgshfvPrEa tkkaessrlQ qAiqAkevln
ATX1(SEQ
ID NO: 3)
Mm
miphTLtlgP ssqvvvqysd agghfvPrEs tkkaessrlQ qAmqAkevln
ATX1(SEQ
ID NO: 4)
Rn
miphTLtlgP ssqvvvqysd agghfvPrEs tkkaessrlQ qAmqAkevln
ATX1(SEQ
ID NO: 5)
271 313
IMX97018
GqrpreRnlv rreSe..aLd spnsKg.... .EgqglVpVv ecvvDgqlfS
(SEQ ID NO:
2)
Hs
GemeksRryg apsSadlgLg kaggKsvphp yEsrhvV.Vh pspsD...yS
ATX1(SEQ
ID NO: 3)
Mm
GemeksBryg assSvelsLg kassKsvphp yEsrhvV.Vh pspaD...yS
ATX1(SEQ
ID NO: 4)
Rn
GemeksRryg assSvelsLg ktssKsvphp yEsrhvV.Vh pspaD...yS
ATX1(SEQ
ID NO: 5)
314 361
IMX97018
gsqtp..Rve VaapahrgTP dtDLEvQrvv galasqdyrv vaaqRkeePS
(SEQ ID NO:
2)
Hs
srdpsgvRas VmvlpnsnTP aaDLEvQq.. .......... .athRcasPS
ATX1(SEQ
ID NO: 3)
Mm
srdtsgvRgs VmvlpnssTP saDLEaQq.. .......... .tthReasPS
ATX1(SEQ
ID NO: 4)
Rn
srdtsgvRgs VmvlpnssTP saDLEtQq.. .......... .athReaspS
ATX1(SEQ
ID NO: 5)
362 398
IMX97018
pLN....Lsh htPdHqg... ......egrg SArnPaeLae ksqArgFYpq
(SEQ ID NO:
2)
Hs
tLNdksgLhI gkPgHrsyal sphtviqtth SAseP..Lpv glpAtaFYag
ATX1(SEQ
ID NO: 3)
Mm
tLNdksgLap rkPgHrsyal sphtviqtth SAseP..Lpv glpAtaFYag
ATX1(SEQ
ID NO: 4)
Rn
tLNdksgLbl gkPgHrsyal sphtviqtth SAseP..Lpv glpAtaFYag
ATX1(SEQ
ID NO: 5)
399 434
IMX97018
shQePV..kh rplpkAmvvA ng...nLVpt GtdsgLIPVG S.........
(SEQ ID NO:
2)
Hs
t.QpPVigyl sgqqqAityA gslpqhLVip GtqplLiPVG Stdmeasgaa
ATX1(SEQ
ID NO: 3)
Mn
t.QpPVigyl sgqqqAityA gglpqhtVip GnqplLiPVG Spdmdmpgaa
ATX1(SEQ
ID NO: 4)
Rn
a.QpPVigyl ssqqqAityA gglpqhLVip GtqplLiPVQ Spdmdtpgaa
ATX1(SEQ
ID NO: 5)
Self-ass'n
435 451
IMX97018
eilVaSSld. .......... .......... .......... ..VQAratfP
(SEQ ID NO:
2)
Hs
paiVtSSpqf aavphtfvtt alpksenfnp ealvtqaayp amVQAqihIP
ATX1(SEQ
ID NO: 3)
Mm
saiVtSSpqf aavphtfvtt alpksenfnp ealvtqasyp amVQAqihIP
ATX1(SEQ
ID NO: 4)
Rn
saiVtSSpqf aavphtfvtt alpksenfhp ealvtqaayp amVQAqihlP
ATX1(SEQ
ID NO: 5)
Self-ass'n
RNA-binding
452 500
IMX97018
dkeptppPit ss.hLPshFM KGaIIQLAtG ELKrVEDLqT qDFvrSAEvS
(SEQ ID NO:
2)
Hs
vvqsvasPaa apptLPpyFM KGsIIQLAnG BLKkVEDLkT eDFiqSAEiS
ATX1(SEQ
ID NO: 3)
Mm
vvqsvasPtt asptLPpyFM KGsIIQLAnG ELKkVEDLkT eDFiqSAEiS
ATX1(SEQ
ID NO: 4)
Rn
vvqsvasPaa asptLPpyFM KGsIIQLAnG ELKkVEDLkT eDFiqSAEiS
ATX1(SEQ
ID NO: 5)
AXH (SEQ
tvPhcFM KGtrlcLAnG snKkVEDLrT eDFirSAgeS
ID NO: 6)
Self-ass'n
RNA-binding
501 550
IMX97018
ggLKIdSSTV vdIqeSqwPG fvmIhFvVGE qqskVSiEVp pEhPFFVyGQ
(SEQ ID NO:
2)
Ms
ndLKIdSSTV erIedShspG vaviqFaVGE hraqVSvEVI vEyPFFVfGQ
ATX1(SEQ
ID NO: 3)
Mm
ndLKIhSSTV erIeeShsPG vaviqFaVGE hraqVSvEVI vEyPFFVfGQ
ATX1(SEQ
ID NO: 4)
Rn
ndLKIdSSTV erIedShsPG vaviqFaVGE hraqVSvEVI vEyPFFVIGQ
ATX1(SEQ
ID NO: 5)
AXH(SEQ
ndedlqmSTV krIgsSglPs vvtltFdpGv edalltvEcq vEhPFFVkGk
ID NO: 6)
RNA-binding
551 600
IMX97018
GWSSCsPgRT tQLFsLPChr LqVGDVCISi sLqsLnsnSV sqascapPsq
(SEQ ID NO:
2)
Hs
GWSSCcPeRT sQLFdLPCsk LsVGDVCISJ tLknLkngSV kkgqpvdPas
ATX1(SEQ
ID NO: 3)
Mn
GWSSCcPeRT sQLFdLPCsk LsVGDVCISl tLknLkngsV kkgqpvdPas
ATX1(SEQ
ID NO: 4)
Rn
GWSSCcPeRT SQLFdLPCsk LsVGDVCISl tLknLkngSV kkgqpvdPas
ATX1(SEQ
ID NO: 5)
AXH (SEQ
GWSSCyPslT vQLygLPCce LqVGDVClSl thn
ID NO: 6)
RNA-binding
601 629
IMX97018
.......... LgppReR... pErtv.lGSr elcdseGksq .......Pag
(SEQ ID NO:
2)
Hs
vllkhskadg LagsRhRyae qEnginqGSa qmlsenGelk fpekmglPAa
ATX1(SBQ
ID NO: 3)
Mm
vllkqvktds LagsRhRyae qEnginqGSa qvlsenGelk fpekiglPAa
ATX1(SEQ
ID NO: 4)
Rn
allkhaktds LagsRhRyae qEnginqGSa qvlsenGelk fpekiglPAa
ATX1(SEQ
ID NO: 5)
RNA-binding
630 678
IMX97018
egsrvvEPSq Pcsgaqa.cW pAPsfqrysm qgeEaraaLl rPSfIPQEVK
(SEQ ID NO:
2)
Hs
pfltkiEPSk Paatrk.rrW sAPesrklek sedEppltLp kPSlIPQEVK
ATX1(SEQ
ID NO: 3)
Mm
pflskiEPSk PtatrkrrrW sAPetrklek sedEppltLp kPSlIPQEVK
ATX1(SEQ
ID NO: 4)
Rn
pfltkiEPSk Ptatrk.rrW sAPetrklek sedEppltLp kPSlIPQEVK
ATX1(SEQ
ID NO: 5)
RNA-binding
679 689
IMX97018
lsIEGRSNaQ K
SEQ ID NO:
2)
Hs
icIEGRSNvG K
ATX1(SEQ
ID NO: 3)
Mn
icIEGRSNvG K
ATX1(SEQ
ID NO: 4)
Rn
icIEGRSNvG K
ATX1(SEQ
ID NO: 5)
Expression of polynucleotides encoding IMX97018 polypeptides. An array of polynucleotides, including polynucleotide probes specific for IMX97018-encoding sequences, was contacted with human RNA samples prepared from a variety of tissues and cell types. Low levels of expression of IMX97018-encoding sequences were detected in all samples tested.
Example 2
Monoclonal Antibodies that Bind Polypeptides of the Invention
This example illustrates a method for preparing monoclonal antibodies that bind IMX97018 polypeptides. Other conventional techniques may be used, such as those described in U.S. Pat. No. 4,411,993. Suitable immunogens that may be employed in generating such antibodies include, but are not limited to, purified IMX97018 polypeptide, an immunogenic fragment thereof, and cells expressing high levels of IMX97018 polypeptide or an immunogenic fragment thereof. DNA encoding an IMX97018 polypeptide can also be used as an immunogen, for example, as reviewed by Pardoll and Beckerleg in Immunity 3: 165, 1995.
Rodents (BALB/c mice or Lewis rats, for example) are immunized with IMX97018 polypeptide immunogen emulsified in an adjuvant (such as complete or incomplete Freund's adjuvant, alum, or another adjuvant, such as Ribi adjuvant R700 (Ribi, Hamilton, Mont.)), and injected in amounts ranging from 10-100 micrograms subcutaneously or intraperitoneally. DNA may be given intradermally (Raz et al., 1994, Proc. Natl. Acad. Sci. USA 91: 9519) or intamuscularly (Wang et al., 1993, Proc. Natl. Acad. Sci. USA 90: 4156); saline has been found to be a suitable diluent for DNA-based antigens. Ten days to three weeks days later, the immunized animals are boosted with additional immunogen and periodically boosted thereafter on a weekly, biweekly or every third week immunization schedule.
Serum samples are periodically taken by retro-orbital bleeding or tail-tip excision to test for IMX97018 polypeptide antibodies by dot-blot assay, ELISA (enzyme-linked immunosorbent assay), immunoprecipitation, or other suitable assays, such as FACS analysis of inhibition of binding of IMX97018 polypeptide to an IMX97018 polypeptide binding partner. Following detection of an appropriate antibody titer, positive animals are provided one last intravenous injection of IMX97018 polypeptide in saline. Three to four days later, the animals are sacrificed, and spleen cells are harvested and fused to a murine myeloma cell line, e.g., NS1 or preferably P3X63Ag8.653 (ATCC CRL-1580). These cell fusions generate hybridoma cells, which are plated in multiple microtiter plates in a HAT (hypoxanthine, aminopterin and thymidine) selective medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
The hybridoma cells may be screened by ELISA for reactivity against purified IMX97018 polypeptide by adaptations of the techniques disclosed in Engvall et al., (Immunochem. 8: 871, 1971) and in U.S. Pat. No. 4,703,004. A preferred screening technique is the antibody capture technique described in Beckmann et al., (J. Immunol. 144: 4212, 1990). Positive hybridoma cells can be injected intraperitoneally into syngeneic rodents to produce ascites containing high concentrations (for example, greater than 1 milligram per milliliter) of anti-IMX97018 polypeptide monoclonal antibodies. Alternatively, hybridoma cells can be grown in vitro in flasks or roller bottles by various techniques. Monoclonal antibodies can be purified by ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can also be used, as can affinity chromatography based upon binding to IMX97018 polypeptide.
Example 3
Antisense Inhibition of IMX97018 Nucleic Acid Expression
In accordance with the present invention, a series of oligonucleotides are designed to target different regions of the IMX97018 mRNA molecule, using the nucleotide sequence of SEQ ID NO: 1 as the basis for the design of the oligonucleotides. The oligonucleotides are selected to be approximately 10, 12, 15, 18, or more preferably 20 nucleotide residues in length, and to have a predicted hybridization temperature that is at least 37 degrees C. Preferably, the oligonucleotides are selected so that some will hybridize toward the 5′ region of the mRNA molecule, others will hybridize to the coding region, and still others will hybridize to the 3′ region of the mRNA molecule. Methods such as those of Gray and Clark (U.S. Pat. Nos. 5,856,103 and 6,183,966) can be used to select oligonucleotides that form the most stable hybrid structures with target sequences, as such oligonucleotides are desirable for use as antisense inhibitors.
The oligonucleotides may be oligodeoxynucleotides, with phosphorothioate backbones (internucleoside linkages) throughout, or may have a variety of different types of internucleoside linkages. Generally, methods for the preparation, purification, and use of a variety of chemically modified oligonucleotides are described in U.S. Pat. No. 5,948,680. As specific examples, the following types of nucleoside phosphoramidites may be used in oligonucleotide synthesis: deoxy and 2′-alkoxy amidites; 2′-fluoro amidites such as 2′-fluorodeoxyadenosine amidites, 2′-fluorodeoxyguanosine, 2′-fluorouridine, and 2′-fluorodeoxycytidine; 2′-O-(2-methoxyethyl)-modified amidites such as 2,2′-anhydro[1-(beta-D-arabino-furanosyl)-5-methyluridine], 2′-O-methoxyethyl-5-methyluridine, 2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine, 3′-O-acetyl-2′-O-methoxy-ethyl-5′-O-dimethoxytrityl-5-methyluridine, 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine, 2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine, N4-benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine, and N4-benzoyl-2′-O-methoxyethyl-5′-O-di-methoxytrityl-5-methylcytidine-3′-amidite; 2′-O-(aminooxyethyl) nucleoside amidites and 2′-O-(dimethylaminooxyethyl) nucleoside amidites such as 2′-(dimethylaminooxyethoxy) nucleoside amidites, 5′-O-tert-butyldiphenylsilyl-O2-2′-anhydro-5-methyluridine, 5′-O-tert-butyl-diphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine, 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenyl-silyl-5-methyluridine, 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine, 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine, 2′-O-(dimethylaminooxy-ethyl)-5-methyluridine, 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine, and 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphor-amidite]; and 2′-(aminooxyethoxy) nucleoside amidites such as N2-isobutyryl-6-O-diphenyl-carbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diiso-propylphosphoramidite].
Modified oligonucleosides may also be used in oligonucleotide synthesis, for example methylenemethylimino-linked oligonucleosides, also called MMI-linked oligonucleosides; methylene-dimethylhydrazo-linked oligonucleosides, also called MDH-linked oligonucleosides; methylene-carbonylamino-linked oligonucleosides, also called amide-3-linked oligonucleosides; and methylene-aminocarbonyl-linked oligonucleosides, also called amide-4-linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages, which are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289. Formacetal- and thioformacetal-linked oligonucleosides may also be used and are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564; and ethylene oxide linked oligonucleosides may also be used and are prepared as described in U.S. Pat. No. 5,223,618. Peptide nucleic acids (PNAs) may be used as in the same manner as the oligonucleotides described above, and are prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23; and U.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262.
Chimeric oligonucleotides, oligonucleosides, or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”. Some examples of different types of chimeric oligonucleotides are: [2′-O-Me]-[2′-deoxy]-[2′-O-Me] chimeric phosphorothioate oligonucleotides, [2′-O-(2-methoxyethyl)]-[2′-deoxy]-[2′-O-(methoxyethyl)] chimeric phosphorothioate oligonucleotides, and [2′-O-(2-methoxy-ethyl)phosphodiester]-[2′deoxy phosphoro-thioate]-[2′-O-(2-methoxyethyl)phosphodiester] chimeric oligonucleotides, all of which may be prepared according to U.S. Pat. No. 5,948,680. In one preferred embodiment, chimeric oligonucleotides (“gapmers”) 18 nucleotides in length are utilized, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by four-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. Cytidine residues in the 2′-MOE wings are 5-methylcytidines. Other chimeric oligonucleotides, chimeric oligonucleosides, and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065.
Oligonucleotides are preferably synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a standard 96 well format. The concentration of oligonucleotide in each well is assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products is evaluated by capillary electrophoresis, and base and backbone composition is confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy.
The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. Cells are routinely maintained for up to 10 passages as recommended by the supplier. When cells reached 80% to 90% confluency, they are treated with oligonucleotide. For cells grown in 96-well plates, wells are washed once with 200 microliters OPTI-MEM-1 reduced-serum medium (Gibco BRL) and then treated with 130 microliters of OPTI-MEM-1 containing 3.75 g/mL LIPOFECTIN (Gibco BRL) and the desired oligonucleotide at a final concentration of 150 nM. After 4 hours of treatment, the medium is replaced with fresh medium. Cells are harvested 16 hours after oligonucleotide treatment. Preferably, the effect of several different oligonucleotides should be tested simultaneously, where the oligonucleotides hybridize to different portions of the target nucleic acid molecules, in order to identify the oligonucleotides producing the greatest degree of inhibition of expression of the target nucleic acid.
Antisense modulation of IMX97018 nucleic acid expression can be assayed in a variety of ways known in the art. For example, IMX97018 mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation and Northern blot analysis are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions. This fluorescence detection system allows high-throughput quantitation of PCR products. As opposed to standard PCR, in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., JOE or FAM, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular (six-second) intervals by laser optics built into the ABI PRISM 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples. Other methods of quantitative PCR analysis are also known in the art. IMX97018 protein levels can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA, or fluorescence-activated cell sorting (FACS). Antibodies directed to IMX97018 polypeptides can be prepared via conventional antibody generation methods such as those described herein. Immunoprecipitation methods, Western blot (immunoblot) analysis, and enzyme-linked immunosorbent assays (ELISA) are standard in the art (see, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, 10.8.1-10.8.21, and 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991).
All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
Sequences Presented in the Sequence Listing
SEQ ID NO
Type
Description
SEQ ID NO: 1
Nucleotide
IMX97018 coding sequence
SEQ ID NO: 2
Amino acid
IMX97018 amino acid sequence
SEQ ID NO: 3
Amino acid
Homo sapiens ataxin-1 polypeptide
(SWISSPROT accession # P54253)
SEQ ID NO: 4
Amino acid
Mus musculus ataxin-1 polypeptide
(SWISSPROT accession # P54254)
SEQ ID NO: 5
Amino acid
Rattus norvegicus ataxin-1 polypeptide
(GenBank accession # NP_036858)
SEQ ID NO: 6
Amino acid
AXH domain (ncbi.nlm.nih.gov/
Structure/cdd/cdd.shtml; smart00536)
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11592413
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immunex corporation
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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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424/141.1
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Mar 31st, 2022 02:17PM
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Mar 31st, 2022 02:17PM
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Amgen
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:amgn
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Amgen
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Jul 29th, 2008 12:00AM
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Jun 13th, 2006 12:00AM
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https://www.uspto.gov?id=US07405058-20080729
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Human TSLP polynucleotides
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The invention is directed to purified and isolated novel TSLP polypeptides, the nucleic acids encoding such polypeptides, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, fragmented peptides derived from these polypeptides, and the uses of the above.
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7405058
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1. An isolated nucleic acid molecule selected from the group consisting of:
(a) the DNA sequence of SEQ ID NO: 1; and
(b) an isolated nucleic acid molecule encoding a polypeptide having the amino acid sequence comprising at least 30 contiguous amino acids of the sequence of SEQ ID NO:2.
2. The nucleic acid molecule of claim 1 comprising the DNA sequence of SEQ ID NO:1.
3. A recombinant vector that directs the expression of the nucleic acid molecule of claim 1.
4. A host cell transfected or transduced with the vector of claim 3.
5. The host cell of claim 4, wherein the host cell is a bacteria.
6. The host cell of claim 4, wherein the host cell is a yeast cell.
7. The host cell of claim 4, wherein the host cell is a plant cell.
8. The host cell of claim 4, wherein the host cell is an animal cell.
9. A method for the production of a polypeptide encoded by the nucleic acid molecule of claim 1, said method comprising culturing a host cell comprising said nucleic acid molecule under conditions promoting expression, and recovering the polypeptide from the culture medium.
10. The method of claim 9, wherein the host cell is a bacteria.
11. The method of claim 9, wherein the host cell is a yeast cell.
12. The method of claim 9, wherein the host cell is a plant cell.
13. The method of claim 9, wherein the host cell is an animal cell.
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13
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CROSS REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. Ser. No. 10/376,406 filed Feb. 27, 2003, which is a divisional of U.S. Ser. No. 09/852,391 filed May 9, 2001, now issued U.S. Pat. No. 6,555,520, which is a continuation of PCT/US99/27069 filed Nov. 12, 1999 which claims the benefit of U.S. provisional application Ser. No. 60/108,452, filed Nov. 13, 1998, the entire disclosure of which is relied upon and incorporated by reference herein.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention is directed to purified and isolated novel human thymic stromal lymphopoietin (TSLP) polypeptides and fragments thereof, the nucleic acids encoding such polypeptides, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, fragmented peptides derived from these polypeptides, and uses thereof.
2. Description of Related Art
Although B cell development has been extensively studied, there still remain gaps in the pathway leading from hematopoeitic stem cells to mature B cells. It is recognized that cytokines influence and play a critical role in B cell development and growth. Known cytokines that influence B cell development include IL-2, IL-4, IL-5, IL-6, IL-7, IFN-gamma, and granulocyte-macrophage colony-stimulating factor (GM-CSF).
In recent years, a novel murine growth factor, designated thymic stromal lymphopoietin (TSLP), has been shown to play a role in B cell development and maturation. The cytokine activity of murine TSLP is very similar to that of IL-7, which is required during proliferation and survival of pre-B cells (Janeway et al., Immuno Biology, 2nd Ed. (1996)). Both of these cytokines have been shown to sustain NAG8/7 cells (Friend et al., Exp. Hematol., 22:321-328 (1994)) and support B lymphopoiesis. In addition, mature B lymphocytes fail to develop in the absence of either IL-7 or murine TSLP. Moreover, it has been shown that murine TSLP can replace IL-7 in sustaining B cell proliferative responses (Ray et al., Eur. J. Immunol., 26:10-16 (1996)). Thus, in the mouse system, TSLP has a significant function in B cell development.
Like IL-7, murine TSLP can also costimulate thymocytes and mature T cells (Friend et al., Exp. Hematol., 22:321-328 (1994)). Studies with IL-7 receptor (IL-7R) knockout mice indicate that IL-7, TSLP, or both play a crucial role in controlling the rearrangement of the T cell receptor-gamma (TCRγ) locus, presumably by mediating accessibility of the TCRγ genes to the VDJ recombinase (Candeias et al., Immunology Letters, 57:9-14 (1997)). Thus, murine TSLP also plays a significant role in T cell development.
Murine TSLP receptors and IL-7 receptors both use the IL-7R α-chain as part of their signaling complexes (Levin et al., J. Immunol., 162:677-683 (1999)). Despite the common IL-7R α-chain, however, IL-7 and TSLP appear to mediate their lymphopoietic effects through distinct mechanisms. IL-7 induces activation of Stat5 and the Janus family kinases Jak1 and Jak3, whereas murine TSLP induces activation of Stat5, but not any of the known Janus family kinases (Levin et al., J. Immunol., 162:677-683 (1999)).
Given the important function of murine TSLP and the significance of its role in B cell and T cell development and maturation in the mouse system, there is a need in the art to identify and isolate human TSLP and to study its role in human B cell and T cell development and maturation. In addition, in view of the continuing interest in lymphocyte development and the immune system, the discovery, identification, and roles of new proteins, such as human TSLP and its receptors, are at the forefront of modem molecular biology, biochemistry, and immunology. Despite the growing body of knowledge, there is still a need in the art for the identity and function of proteins involved in cellular and immune responses.
In another aspect, the identification of the primary structure, or sequence, of an unknown protein is the culmination of an arduous process of experimentation. In order to identify an unknown protein, the investigator can rely upon a comparison of the unknown protein to known peptides using a variety of techniques known to those skilled in the art. For instance, proteins are routinely analyzed using techniques such as electrophoresis, sedimentation, chromatography, sequencing and mass spectrometry.
In particular, comparison of an unknown protein to polypeptides of known molecular weight allows a determination of the apparent molecular weight of the unknown protein (T. D. Brock and M. T. Madigan, Biology of Microorganisms, pp. 76-77, Prentice Hall, 6d ed., (1991)). Protein molecular weight standards are commercially available to assist in the estimation of molecular weights of unknown protein (New England Biolabs Inc. Catalog:130-131 (1995)); (J. L. Hartley, U.S. Pat. No. 5,449,758). However, the molecular weight standards may not correspond closely enough in size to the unknown protein to allow an accurate estimation of apparent molecular weight. The difficulty in estimation of molecular weight is compounded in the case of proteins that are subjected to fragmentation by chemical or enzymatic means, modified by post-translational modification or processing, and/or associated with other proteins in non-covalent complexes.
In addition, the unique nature of the composition of a protein with regard to its specific amino acid constituents results in unique positioning of cleavage sites within the protein. Specific fragmentation of a protein by chemical or enzymatic cleavage results in a unique “peptide fingerprint” (D. W. Cleveland et al., J. Biol. Chem. 252:1102-1106 (1977); M. Brown et al., J. Gen. Virol. 50:309-316 (1980)). Consequently, cleavage at specific sites results in reproducible fragmentation of a given protein into peptides of precise molecular weights. Furthermore, these peptides possess unique charge characteristics that determine the isoelectric pH of the peptide. These unique characteristics can be exploited using a variety of electrophoretic and other techniques (T. D. Brock and M. T. Madigan, Biology of Microorganisms, pp. 76-77, Prentice Hall, 6d ed. (1991)).
Fragmentation of proteins is further employed for amino acid composition analysis and protein sequencing (P. Matsudiara, J. Biol. Chem., 262:10035-10038 (1987); C. Eckerskom et al., Electrophoresis, 9:830-838 (1988)), particularly the production of fragments from proteins with a “blocked” N-terminus. In addition, fragmented proteins can be used for immunization, for affinity selection (R. A. Brown, U.S. Pat. No. 5,151,412), for determination of modification sites (e.g. phosphorylation), for generation of active biological compounds (T. D. Brock and M. T. Madigan, Biology of Microorganisms, 300-301 (Prentice Hall, 6d ed., (1991)), and for differentiation of homologous proteins (M. Brown et al., J. Gen. Virol., 50:309-316 (1980)).
In addition, when a peptide fingerprint of an unknown protein is obtained, it can be compared to a database of known proteins to assist in the identification of the unknown protein using mass spectrometry (W. J. Henzel et al., Proc. Natl. Acad. Sci. USA 90:5011-5015 (1993); D. Fenyo et al., Electrophoresis, 19:998-1005 (1998)). A variety of computer software programs to facilitate these comparisons are accessible via the Internet, such as Protein Prospector (Internet site: prospector.uscf.edu), MultiIdent (Internet site: www.expasy.ch/sprot/multiident.html), PeptideSearch (Internet site: www.mann.embl-heiedelberg.de...deSearch/FR_PeptideSearch Form.html), and ProFound (Internet site: www.chait-sgi.rockefeller.edu/cgi-bin/prot-id-frag.html). These programs allow the user to specify the cleavage agent and the molecular weights of the fragmented peptides within a designated tolerance. The programs compare these molecular weights to protein molecular weight information stored in databases to assist in determining the identity of the unknown protein. Accurate information concerning the number of fragmented peptides and the precise molecular weight of those peptides is required for accurate identification. Therefore, increasing the accuracy in determining of the number of fragmented peptides and the precise molecular weight should result in enhanced likelihood of success in the identification of unknown proteins.
In addition, peptide digests of unknown proteins can be sequenced using tandem mass spectrometry (MS/MS) and the resulting sequence searched against databases (J. K. Eng, et al., J. Am. Soc. Mass Spec. 5:976-989 (1994); M. Mann and M. Wilm, Anal. Chem., 66:4390-4399 (1994); J. A. Taylor and R. S. Johnson, Rapid Comm. Mass Spec., 11: 1067-1075 (1997)). Searching programs that can be used in this process exist on the Internet, such as Lutefisk 97 (Internet site: www.lsbc.com:70/Lutefisk97.html), and the Protein Prospector, Peptide Search and ProFound programs described above. Therefore, adding the sequence of a gene and its predicted protein sequence and peptide fragments to a sequence database can aid in the identification of unknown proteins using tandem mass spectrometry.
Thus, there also exists a need in the art for polypeptides suitable for use in peptide fragmentation studies, for use in molecular weight measurements, and for use in protein sequencing using tandem mass spectrometry.
SUMMARY OF THE INVENTION
The invention aids in fulfilling these various needs in the art by providing isolated human TSLP nucleic acids and polypeptides encoded by these nucleic acids. Particular embodiments of the invention are directed to an isolated TSLP nucleic acid molecule comprising the DNA sequence of SEQ ID NO:1 and an isolated TSLP nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:2, as well as nucleic acid molecules complementary to these sequences. Both single-stranded and double-stranded RNA and DNA nucleic acid molecules are encompassed by the invention, as well as nucleic acid molecules that hybridize to a denatured, double-stranded DNA comprising all or a portion of SEQ ID NO:1. Also encompassed are isolated nucleic acid molecules that are derived by in vitro mutagenesis of the nucleic acid molecule comprising the sequence of SEQ ID NO:1, that are degenerate from the nucleic acid molecule comprising the sequence of SEQ ID NO:1, and that are allelic variants of DNA of the invention. The invention also encompasses recombinant vectors that direct the expression of these nucleic acid molecules and host cells transformed or transfected with these vectors.
In addition, the invention encompasses methods of using the nucleic acid noted above to identify nucleic acids encoding proteins having the ability to induce B lineage or T lineage cell proliferation; to identify human chromosome number 5; to map genes on human chromosome number 5; to identify genes associated with certain diseases, syndromes, or other human conditions associated with human chromosome number 5; and to study cell signaling and the immune system.
The invention also encompasses the use of sense or antisense oligonucleotides from the nucleic acid of SEQ ID NO:1 to inhibit the expression of the polynucleotide encoded by the TSLP gene.
The invention also encompasses isolated polypeptides and fragments thereof encoded by these nucleic acid molecules including soluble polypeptide portions of SEQ ID NO:2. The invention further encompasses methods for the production of these polypeptides, including culturing a host cell under conditions promoting expression and recovering the polypeptide from the culture medium. Especially, the expression of these polypeptides in bacteria, yeast, plant, insect, and animal cells is encompassed by the invention.
In general, the polypeptides of the invention can be used to study cellular processes such as immune regulation, cell proliferation, cell differentiation, cell death, cell migration, cell-to-cell interaction, and inflammatory responses. In addition, these polypeptides can be used to identify proteins associated with TSLP ligands and TSLP receptors.
In addition, the invention includes assays utilizing these polypeptides to screen for potential inhibitors of activity associated with polypeptide counter-structure molecules, and methods of using these polypeptides as therapeutic agents for the treatment of diseases mediated by TSLP polypeptide counter-structure molecules. Further, methods of using these polypeptides in the design of inhibitors thereof are also an aspect of the invention.
The invention further includes a method for using these polypeptides as molecular weight markers that allow the estimation of the molecular weight of a protein or a fragmented protein, as well as a method for the visualization of the molecular weight markers of the invention thereof using electrophoresis. The invention further encompasses methods for using the polypeptides of the invention as markers for determining the isoelectric point of an unknown protein, as well as controls for establishing the extent of fragmentation of a protein.
Further encompassed by this invention are kits to aid in these determinations.
Further encompassed by this invention is the use of the human TSLP nucleic acid sequences, predicted amino acid sequences of the polypeptide or fragments thereof, or a combination of the predicted amino acid sequences of the polypeptide and fragments thereof for use in searching an electronic database to aid in the identification of sample nucleic acids and/or proteins.
Isolated polyclonal or monoclonal antibodies that bind to these polypeptides are also encompassed by the invention, as well as the use of these antibodies to aid in purifying the TSLP polypeptide. In addition, the isolated antibodies can be used to establish an Enzyme-Linked Immunosorbent Assay (ELISA) to measure TSLP in samples such as serum.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 presents the nucleotide sequence of human TSLP DNA (SEQ ID NO:1), and
FIG. 2 presents the amino acid sequence of human TSLP (SEQ ID NO:2).
DETAILED DESCRIPTION OF THE INVENTION
The nucleic acid molecules encompassed in the invention include the following nucleotide sequence:
Name: TSLP
1
GCAGCCAGAA AGCTCTGGAG CATCAGGGAG ACTCCAACTT AAGGCAACAG
(SEQ ID NO:1)
51
CATGGGTGAA TAAGGGCTTC CTGTGGACTG GCAATGAGAG GCAAAACCTG
101
GTGCTTGAGC ACTGGCCCCT AAGGCAGGCC TTACAGATCT CTTACACTCG
151
TGGTGGGAAG AGTTTAGTGT GAAACTGGGG TGGAATTGGG TGTCCACGTA
201
TGTTCCCTTT TGCCTTACTA TATGTTCTGT CAGTTTCTTT CAGGAAAATC
251
TTCATCTTAC AACTTGTAGG GCTGGTGTTA ACTTACGACT TCACTAACTG
301
TGACTTTGAG AAGATTAAAG CAGCCTATCT CAGTACTATT TCTAAAGACC
351
TGATTACATA TATGAGTGGG ACCAAAAGTA CCGAGTTCAA CAACACCGTC
401
TCTTGTAGCA ATCGGCCACA TTGCCTTACT GAAATCCAGA GCCTAACCTT
451
CAATCCCACC GCCGGCTGCG CGTCGCTCGC CAAAGAAATG TTCGCCATGA
501
AAACTAAGGC TGCCTTAGCT ATCTGGTGCC CAGGCTATTC GGAAACTCAG
551
ATAAATGCTA CTCAGGCAAT GAAGAAGAGG AGAAAAAGGA AAGTCACAAC
601
CAATAAATGT CTGGAACAAG TGTCACAATT ACAAGGATTG TGGCGTCGCT
651
TCAATCGACC TTTACTGAAA CAACAGTAAA CCATCTTTAT TATGGTCATA
701
TTTCACAGCC CAAAATAAAT CATCTTTATT AAGTAAAAAA AAA
The amino acid sequence of the polypeptide encoded by the nucleotide sequence of the invention includes:
Name: TSLP (Polypeptide)
1
MFPFALLYVL SVSFRKIFIL QLVGLVLTYD FTNCDFEKIK AAYLSTISKD
(SEQ ID NO:2)
51
LITYMSGTKS TEFNNTVSCS NRPHCLTEIQ SLTFNPTAGC ASLAKEMFAM
101
KTKAALAIWC PGYSETQINA TQAMKKRRKR KVTTNKCLEQ VSQLQGLWRR
151
FNRPLLKQQ
The discovery of the nucleic acids of the invention enables the construction of expression vectors comprising nucleic acid sequences encoding polypeptides; host cells transfected or transformed with the expression vectors; isolated and purified biologically active polypeptides and fragments thereof; the use of the nucleic acids or oligonucleotides thereof as probes to identify nucleic acid encoding proteins having TSLP-like activity (e.g., inducing B lineage or T lineage cell proliferation), the use of the nucleic acids or oligonucleotides thereof to identify human chromosome number 5; the use of the nucleic acids or oligonucleotides thereof to map genes on human chromosome number 5; the use of the nucleic acid or oligonucleotides thereof to identify genes associated with certain diseases, syndromes or other human conditions associated with human chromosome number 5 and, in particular, with the q21-q22 region of chromosome number 5, including Gardner syndrome, adenomatous polyposis coli, hereditary desmoid disease, Turcot syndrome, and colorectal cancer; the use of single-stranded sense or antisense oligonucleotides from the nucleic acids to inhibit expression of polynucleotides encoded by the TSLP gene; the use of such polypeptides and soluble fragments to induce B lineage or T lineage cell proliferation; the use of such polypeptides and fragmented peptides as molecular weight markers; the use of such polypeptides and fragmented peptides as controls for peptide fragmentation, and kits comprising these reagents; the use of such polypeptides and fragments thereof to generate antibodies; and the use of the antibodies to purify TSLP polypeptides.
Nucleic Acid Molecules
In a particular embodiment, the invention relates to certain isolated nucleotide sequences that are free from contaminating endogenous material. A “nucleotide sequence” refers to a polynucleotide molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. The nucleic acid molecule has been derived from DNA or RNA isolated at least once in substantially pure form and in a quantity or concentration enabling identification, manipulation, and recovery of its component nucleotide sequences by standard biochemical methods (such as those outlined in (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd sed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)). Such sequences are preferably provided and/or constructed in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, that are typically present in eukaryotic genes. Sequences of non-translated DNA can be present 5′ or 3′ from an open reading frame, where the same do not interfere with manipulation or expression of the coding region.
Nucleic acid molecules of the invention include DNA in both single-stranded and double-stranded form, as well as the RNA complement thereof. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. Genomic DNA may be isolated by conventional techniques, e.g., using the cDNA of SEQ ID NO:1, or a suitable fragment thereof, as a probe.
The DNA molecules of the invention include full length genes as well as polynucleotides and fragments thereof. The full length gene may also include the N-terminal signal peptide. Other embodiments include DNA encoding a soluble form, e.g., encoding the extracellular domain of the protein, either with or without the signal peptide.
The nucleic acids of the invention are preferentially derived from human sources, but the invention includes those derived from non-human species, as well.
Preferred Sequences
The particularly preferred nucleotide sequence of the invention is SEQ ID NO:1, as set forth above. A cDNA clone having the nucleotide sequence of SEQ ID NO:1 was isolated as described in Example 1. The sequence of amino acids encoded by the DNA of SEQ ID NO:1 is shown in SEQ ID NO:2. This sequence identifies the TSLP polynucleotide as a member of a group of factors that influence the growth of B lineage and T lineage cells (Ray et al., Eur. J. Immunol, 26:10-16 (1996)); (Friend et al., Exp. Hematol., 22:321-328 (1994)).
Additional Sequences
Due to the known degeneracy of the genetic code, wherein more than one codon can encode the same amino acid, a DNA sequence can vary from that shown in SEQ ID NO:1, and still encode a polypeptide having the amino acid sequence of SEQ ID NO:2. Such variant DNA sequences can result from silent mutations (e.g., occurring during PCR amplification), or can be the product of deliberate mutagenesis of a native sequence.
The invention thus provides isolated DNA sequences encoding polypeptides of the invention, selected from: (a) DNA comprising the nucleotide sequence of SEQ ID NO:1; (b) DNA encoding the polypeptide of SEQ ID NO:2; (c) DNA capable of hybridization to a DNA of (a) or (b) under conditions of moderate stringency and which encodes polypeptides of the invention; (d) DNA capable of hybridization to a DNA of (a) or (b) under conditions of high stringency and which encodes polypeptides of the invention, and (e) DNA which is degenerate as a result of the genetic code to a DNA defined in (a), (b), (c), or (d) and which encode polypeptides of the invention. Of course, polypeptides encoded by such DNA sequences are encompassed by the invention.
As used herein, conditions of moderate stringency can be readily determined by those having ordinary skill in the art based on, for example, the length of the DNA. The basic conditions are set forth by (Sambrook et al. Molecular Cloning: A Laboratory Manual, 2ed. Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989)), and include use of a prewashing solution for the nitrocellulose filters 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of about 50% formamide, 6×SSC at about 42° C.(or other similar hybridization solution, such as Stark's solution, in about 50% formamide at about 42° C.), and washing conditions of about 60° C., 0.5×SSC, 0.1% SDS. Conditions of high stringency can also be readily determined by the skilled artisan based on, for example, the length of the DNA. Generally, such conditions are defined as hybridization conditions as above, and with washing at approximately 68° C., 0.2×SSC, 0.1% SDS. The skilled artisan will recognize that the temperature and wash solution salt concentration can be adjusted as necessary according to factors such as the length of the probe.
Also included as an embodiment of the invention is DNA encoding polypeptide fragments and polypeptides comprising inactivated N-glycosylation site(s), inactivated protease processing site(s), or conservative amino acid substitution(s), as described below.
In another embodiment, the nucleic acid molecules of the invention also comprise nucleotide sequences that are at least 80% identical to a native sequence. Also contemplated are embodiments in which a nucleic acid molecule comprises a sequence that is at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.9% identical to a native sequence.
The percent identity may be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two nucleic acid sequences can be determined by comparing sequence information using the GAP computer program, version 6.0 described by (Devereux et al., Nucl. Acids Res., 12:387 (1984)) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of (Gribskov and Burgess, Nucl. Acids Res., 14:6745 (1986)), as described by (Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979)); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. Other programs used by one skilled in the art of sequence comparison may also be used.
The invention also provides isolated nucleic acids useful in the production of polypeptides. Such polypeptides may be prepared by any of a number of conventional techniques. A DNA sequence encoding a human TSLP polypeptide, or desired fragment thereof may be subcloned into an expression vector for production of the polypeptide or fragment. The DNA sequence advantageously is fused to a sequence encoding a suitable leader or signal peptide. Alternatively, the desired fragment may be chemically synthesized using known techniques. DNA fragments also may be produced by restriction endonuclease digestion of a full length cloned DNA sequence, and isolated by electrophoresis on agarose gels. If necessary, oligonucleotides that reconstruct the 5′ or 3′ terminus to a desired point may be ligated to a DNA fragment generated by restriction enzyme digestion. Such oligonucleotides may additionally contain a restriction endonuclease cleavage site upstream of the desired coding sequence, and position an initiation codon (ATG) at the N-terminus of the coding sequence.
The well-known polymerase chain reaction (PCR) procedure also may be employed to isolate and amplify a DNA sequence encoding a desired protein fragment. Oligonucleotides that define the desired termini of the DNA fragment are employed as 5′ and 3′ primers. The oligonucleotides may additionally contain recognition sites for restriction endonucleases, to facilitate insertion of the amplified DNA fragment into an expression vector. PCR techniques are described in (Saiki et al., Science, 239:487 (1988)); (Wu et al., Recombinant DNA Methodology, eds., Academic Press, Inc., San Diego, pp. 189-196 (1989)); and (Innis et al., PCR Protocols: A Guide to Methods and Applications, eds., Academic Press, Inc. (1990)).
Polypeptides and Fragments Thereof
The invention encompasses polypeptides and fragments thereof in various forms, including those that are naturally occurring or produced through various techniques such as procedures involving recombinant DNA technology. Such forms include, but are not limited to, derivatives, variants, and oligomers, as well as fusion proteins or fragments thereof.
Polypeptides and Fragments Thereof
The polypeptides of the invention include full length proteins encoded by the nucleic acid sequences set forth above. Particularly preferred polypeptides comprise the amino acid sequence of SEQ ID NO:2 with particularly preferred fragments comprising amino acids 29 to 159 (the mature polypeptide sequence) of SEQ ID NO:2.
The polypeptide of SEQ ID NO:2 includes an N-terminal hydrophobic region that functions as a signal peptide. Computer analysis predicts that the signal peptide corresponds to residues 1 to 28 of SEQ ID NO:2 (although the next most likely computer-predicted signal peptide cleavage sites (in descending order) occur after amino acids 34 and 116 of SEQ ID NO:2). Cleavage of the signal peptide thus would yield a mature protein comprising amino acids 29 through 159 of SEQ ID NO:2.
The skilled artisan will recognize that the above-described boundaries of such regions of the polypeptide are approximate. To illustrate, the boundaries of the signal peptide (which may be predicted by using computer programs available for that purpose) may differ from those described above.
The polypeptides of the invention may be membrane bound or they may be secreted and thus soluble. Soluble polypeptides are capable of being secreted from the cells in which they are expressed. In general, soluble polypeptides may be identified (and distinguished from non-soluble membrane-bound counterparts) by separating intact cells which express the desired polypeptide from the culture medium, e.g., by centrifugation, and assaying the medium (supernatant) for the presence of the desired polypeptide. The presence of polypeptide in the medium indicates that the polypeptide was secreted from the cells and thus is a soluble form of the protein.
In one embodiment, the soluble polypeptides and fragments thereof comprise all or part of the extracellular domain, but lack the transmembrane region that would cause retention of the polypeptide on a cell membrane. A soluble polypeptide may include the cytoplasmic domain, or a portion thereof, as long as the polypeptide is secreted from the cell in which it is produced.
Other embodiments include soluble fragments having an N-terminus at amino acids 29 or 35 and a C-terminus at amino acid 159.
In general, the use of soluble forms is advantageous for certain applications. Purification of the polypeptides from recombinant host cells is facilitated, since the soluble polypeptides are secreted from the cells. Further, soluble polypeptides are generally more suitable for intravenous administration.
The invention also provides polypeptides and fragments of the extracellular domain that retain a desired biological activity. Particular embodiments are directed to polypeptide fragments that retain the ability to bind TSLP receptors. Such a fragment may be a soluble polypeptide, as described above. In another embodiment, the polypeptides and fragments advantageously include regions that are conserved among the family of proteins that influence the growth of B lineage or T lineage cells described above.
Also provided herein are polypeptide fragments comprising at least 20, or at least 30, contiguous amino acids of the sequence of SEQ ID NO:2. Fragments derived from the cytoplasmic domain find use in studies of signal transduction, and in regulating cellular processes associated with transduction of biological signals. Polypeptide fragments also may be employed as immunogens, in generating antibodies.
Variants
Naturally occurring variants as well as derived variants of the polypeptides and fragments are provided herein.
Variants may exhibit amino acid sequences that are at least 80% identical. Also contemplated are embodiments in which a polypeptide or fragment comprises an amino acid sequence that is at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.9% identical to the preferred polypeptide or fragment thereof. Percent identity may be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two protein sequences can be determined by comparing sequence information using the GAP computer program, based on the algorithm of (Needleman and Wunsch, J. Mol. Bio., 48:443 (1970)) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The preferred default parameters for the GAP program include: (1) a scoring matrix, blosum62, as described by (Henikoff and Henikoff Proc. Natl. Acad. Sci. USA, 89:10915 (1992)); (2) a gap weight of 12; (3) a gap length weight of 4; and (4) no penalty for end gaps. Other programs used by one skilled in the art of sequence comparison may also be used.
The variants of the invention include, for example, those that result from alternate mRNA splicing events or from proteolytic cleavage. Alternate splicing of mRNA may, for example, yield a truncated but biologically active protein, such as a naturally occurring soluble form of the protein. Variations attributable to proteolysis include, for example, differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the protein (generally from 1-5 terminal amino acids). Proteins in which differences in amino acid sequence are attributable to genetic polymorphism (allelic variation among individuals producing the protein) are also contemplated herein.
Additional variants within the scope of the invention include polypeptides that may be modified to create derivatives thereof by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives may be prepared by linking the chemical moieties to functional groups on amino acid side chains or at the N-terminus or C-terminus of a polypeptide. Conjugates comprising diagnostic (detectable) or therapeutic agents attached thereto are contemplated herein, as discussed in more detail below.
Other derivatives include covalent or aggregative conjugates of the polypeptides with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions. Examples of fusion proteins are discussed below in connection with oligomers. Further, fusion proteins can comprise peptides added to facilitate purification and identification. Such peptides include, for example, poly-His or the antigenic identification peptides described in U.S. Pat. No. 5,011,912 and in (Hopp et al., Bio/Technology, 6:1204 (1988)). One such peptide is the FLAG7 peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, (SEQ ID NO:3) which is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant protein. A murine hybridoma designated 4E11 produces a monoclonal antibody that binds the FLAG7 peptide in the presence of certain divalent metal cations, as described in U.S. Pat. No. 5,011,912, hereby incorporated by reference. The 4E11 hybridoma cell line has been deposited with the American Type Culture Collection under accession no. HB 9259. Monoclonal antibodies that bind the FLAG7 peptide are available from Eastman Kodak Co., Scientific Imaging Systems Division, New Haven, Conn.
Among the variant polypeptides provided herein are variants of native polypeptides that retain the native biological activity or the substantial equivalent thereof. One example is a variant that binds with essentially the same binding affinity as does the native form. Binding affinity can be measured by conventional procedures, e.g., as described in U.S. Pat. No. 5,512,457 and as set forth below.
Variants include polypeptides that are substantially homologous to the native form, but which have an amino acid sequence different from that of the native form because of one or more deletions, insertions or substitutions. Particular embodiments include, but are not limited to, polypeptides that comprise from one to ten deletions, insertions or substitutions of amino acid residues, when compared to a native sequence.
A given amino acid may be replaced, for example, by a residue having similar physiochemical characteristics. Examples of such conservative substitutions include substitution of one aliphatic residue for another, such as Ile, Val, Leu, or Ala for one another; substitutions of one polar residue for another, such as between Lys and Arg, Glu and Asp, or Gln and Asn; or substitutions of one aromatic residue for another, such as Phe, Trp, or Tyr for one another. Other conservative substitutions, e.g., involving substitutions of entire regions having similar hydrophobicity characteristics, are well known.
Similarly, the DNAs of the invention include variants that differ from a native DNA sequence because of one or more deletions, insertions or substitutions, but that encode a biologically active polypeptide.
The invention further includes polypeptides of the invention with or without associated native-pattern glycosylation. Polypeptides expressed in yeast or mammalian expression systems (e.g., COS-1 or COS-7 cells) can be similar to or significantly different from a native polypeptide in molecular weight and glycosylation pattern, depending upon the choice of expression system. Expression of polypeptides of the invention in bacterial expression systems, such as E. coli, provides non-glycosylated molecules. Further, a given preparation may include multiple differentially glycosylated species of the protein. Glycosyl groups can be removed through conventional methods, in particular those utilizing glycopeptidase. In general, glycosylated polypeptides of the invention can be incubated with a molar excess of glycopeptidase (Boehringer Mannheim).
Correspondingly, similar DNA constructs that encode various additions or substitutions of amino acid residues or sequences, or deletions of terminal or internal residues or sequences are encompassed by the invention. For example, N-glycosylation sites in the polypeptide extracellular domain can be modified to preclude glycosylation, allowing expression of a reduced carbohydrate analog in mammalian and yeast expression systems. N-glycosylation sites in eukaryotic polypeptides are characterized by an amino acid triplet Asn-X-Y, wherein X is any amino acid and Y is Ser or Thr. Appropriate substitutions, additions, or deletions to the nucleotide sequence encoding these triplets will result in prevention of attachment of carbohydrate residues at the Asn side chain. Alteration of a single nucleotide, chosen so that Asn is replaced by a different amino acid, for example, is sufficient to inactivate an N-glycosylation site. Alternatively, the Ser or Thr can by replaced with another amino acid, such as Ala. Known procedures for inactivating N-glycosylation sites in proteins include those described in U.S. Pat. No. 5,071,972 and EP 276,846, hereby incorporated by reference.
In another example of variants, sequences encoding Cys residues that are not essential for biological activity can be altered to cause the Cys residues to be deleted or replaced with other amino acids, preventing formation of incorrect intramolecular disulfide bridges upon folding or renaturation.
Other variants are prepared by modification of adjacent dibasic amino acid residues, to enhance expression in yeast systems in which KEX2 protease activity is present. EP 212,914 discloses the use of site-specific mutagenesis to inactivate KEX2 protease processing sites in a protein. KEX2 protease processing sites are inactivated by deleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of these adjacent basic residues. Lys-Lys pairings are considerably less susceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and preferred approach to inactivating KEX2 sites.
Oligomers
Encompassed by the invention are oligomers or fusion proteins that contain human TSLP polypeptides. Such oligomers may be in the form of covalently-linked or non-covalently-linked multimers, including dimers, trimers, or higher oligomers. As noted above, preferred polypeptides are soluble and thus these oligomers may comprise soluble polypeptides. In one aspect of the invention, the oligomers maintain the binding ability of the polypeptide components and provide therefor, bivalent, trivalent, etc., binding sites.
One embodiment of the invention is directed to oligomers comprising multiple polypeptides joined via covalent or non-covalent interactions between peptide moieties fused to the polypeptides. Such peptides may be peptide linkers (spacers), or peptides that have the property of promoting oligomerization. Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote oligomerization of the polypeptides attached thereto, as described in more detail below.
Immunoglobulin-based Oligomers
As one alternative, an oligomer is prepared using polypeptides derived from immunoglobulins. Preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., by (Ashkenazi et al., PNAS USA, 88:10535 (1991)); (Byrn et al., Nature, 344:677 (1990)); and (Hollenbaugh and Aruffo “Construction of Immunoglobulin Fusion Proteins”, in Current Protocols in Immunology, Suppl. 4, pp. 10.19.1-10.19.11 (1992)).
One embodiment of the present invention is directed to a dimer comprising two fusion proteins created by fusing a polypeptide of the invention to an Fc polypeptide derived from an antibody. A gene fusion encoding the polypeptide/Fc fusion protein is inserted into an appropriate expression vector. Polypeptide/Fc fusion proteins are expressed in host cells transformed with the recombinant expression vector, and allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between the Fc moieties to yield divalent molecules.
The term “Fc polypeptide” as used herein includes native and mutein forms of polypeptides made up of the Fc region of an antibody comprising all of the CH domains of the Fc region. Truncated forms of such polypeptides containing the hinge region that promotes dimerization are also included. Preferred polypeptides comprise an Fc polypeptide derived from a human IgG1 antibody.
One suitable Fc polypeptide, described in PCT application WO 93/10151 (hereby incorporated by reference), is a single chain polypeptide extending from the N-terminal hinge region to the native C-terminus of the Fc region of a human IgG1 antibody. Another useful Fc polypeptide is the Fc mutein described in U.S. Pat. No. 5,457,035 and in (Baum et al., EMBO J., 13:3992-4001 (1994)) incorporated herein by reference. The amino acid sequence of this mutein is identical to that of the native Fc sequence presented in WO 93/10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 has been changed from Gly to Ala. The mutein exhibits reduced affinity for Fc receptors.
The above-described fusion proteins comprising Fc moieties (and oligomers formed therefrom) offer the advantage of facile purification by affinity chromatography over Protein A or Protein G columns.
In other embodiments, the polypeptides of the invention may be substituted for the variable portion of an antibody heavy or light chain. If fusion proteins are made with both heavy and light chains of an antibody, it is possible to form an oligomer with as many as four TSLP extracellular regions.
Peptide-linker Based Oligomers
Alternatively, the oligomer is a fusion protein comprising multiple polypeptides, with or without peptide linkers (spacer peptides). Among the suitable peptide linkers are those described in U.S. Pat. Nos. 4,751,180 and 4,935,233, which are hereby incorporated by reference. A DNA sequence encoding a desired peptide linker may be inserted between, and in the same reading frame as, the DNA sequences of the invention, using any suitable conventional technique. For example, a chemically synthesized oligonucleotide encoding the linker may be ligated between the sequences. In particular embodiments, a fusion protein comprises from two to four soluble TSLP polypeptides, separated by peptide linkers.
Leucine-Zippers
Another method for preparing the oligomers of the invention involves use of a leucine zipper. Leucine zipper domains are peptides that promote oligomerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759 (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize.
The zipper domain (also referred to herein as an oligomerizing, or oligomer-forming, domain) comprises a repetitive heptad repeat, often with four or five leucine residues interspersed with other amino acids. Examples of zipper domains are those found in the yeast transcription factor GCN4 and a heat-stable DNA-binding protein found in rat liver (C/EBP; Landschulz et al., Science, 243:1681 (1989)). Two nuclear transforming proteins, fos and jun, also exhibit zipper domains, as does the gene product of the murine proto-oncogene, c-myc (Landschulz et al., Science, 240:1759 (1988)). The products of the nuclear oncogenesfos and jun comprise zipper domains that preferentially form heterodimer (O'Shea et al., Science, 245:646 (1989)), (Turner and Tjian, Science, 243:1689 (1989)). The zipper domain is necessary for biological activity (DNA binding) in these proteins.
The fusogenic proteins of several different viruses, including paramyxovirus, coronavirus, measles virus and many retroviruses, also possess zipper domains (Buckland and Wild, Nature, 338:547 (1989); (Britton, Nature, 353:394 (1991)); (Delwart and Mosialos, AIDS Research and Human Retroviruses, 6:703 (1990)). The zipper domains in these fusogenic viral proteins are near the transmembrane region of the proteins; it has been suggested that the zipper domains could contribute to the oligomeric structure of the fusogenic proteins. Oligomerization of fusogenic viral proteins is involved in fusion pore formation (Spruce et al, Proc. Natl. Acad. Sci. U.S.A. 88:3523 (1991)). Zipper domains have also been recently reported to play a role in oligomerization of heat-shock transcription factors (Rabindran et al., Science 259:230 (1993)).
Zipper domains fold as short, parallel coiled coils. (O'Shea et al., Science 254:539 (1991)). The general architecture of the parallel coiled coil has been well characterized, with a “knobs-into-holes” packing as proposed by (Crick, Acta Crystallogr., 6:689)). The dimer formed by a zipper domain is stabilized by the heptad repeat, designated (abcdefg)n according to the notation of (McLachlan and Stewart, J. Mol. Biol., 98:293 (1975)), in which residues a and d are generally hydrophobic residues, with d being a leucine, which line up on the same face of a helix. Oppositely-charged residues commonly occur at positions g and e. Thus, in a parallel coiled coil formed from two helical zipper domains, the “knobs” formed by the hydrophobic side chains of the first helix are packed into the “holes” formed between the side chains of the second helix.
The residues at position d (often leucine) contribute large hydrophobic stabilization energies, and are important for oligomer formation (Krystek et al., Int. J. Peptide Res., 38:229 (1991)). (Lovejoy et al., Science 259:1288 (1993)) recently reported the synthesis of a triple-stranded α-helical bundle in which the helices run up-up-down. Their studies confirmed that hydrophobic stabilization energy provides the main driving force for the formation of coiled coils from helical monomers. These studies also indicate that electrostatic interactions contribute to the stoichiometry and geometry of coiled coils. Further discussion of the structure of leucine zippers is found in (Harbury et al., Science, 262:1401 (26 Nov. 1993)).
Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT application WO 94/10308, and the leucine zipper derived from lung surfactant protein D (SPD) described in (Hoppe et al., FEBS Letters, 344:191 (1994)), hereby incorporated by reference. The use of a modified leucine zipper that allows for stable trimerization of a heterologous protein fused thereto is described in (Fanslow et al., Semin. Immunol., 6:267-278 (1994)). Recombinant fusion proteins comprising a soluble polypeptide fused to a leucine zipper peptide are expressed in suitable host cells, and the soluble oligomer that forms is recovered from the culture supernatant.
Certain leucine zipper moieties preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in (Hoppe et al., FEBS Letters, 344:191 (1994)) and in U.S. Pat. No. 5,716,805, hereby incorporated by reference in their entirety. This lung SPD-derived leucine zipper peptide comprises the amino acid sequence Pro Asp Val Ala Ser Leu Arg Gln Gln Val Glu Ala Leu Gln Gly Gln Val Gln His Leu Gln Ala Ala Phe Ser Gln Tyr (SEQ ID NO: 4).
Another example of a leucine zipper that promotes trimerization is a peptide comprising the amino acid sequence Arg Met Lys Gln Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu Arg, (SEQ ID NO: 5), as described in U.S. Pat. No. 5,716,805. In one alternative embodiment, an N-terminal Asp residue is added; in another, the peptide lacks the N-terminal Arg residue.
Fragments of the foregoing zipper peptides that retain the property of promoting oligomerization may be employed as well. Examples of such fragments include, but are not limited to, peptides lacking one or two of the N-terminal or C-terminal residues presented in the foregoing amino acid sequences. Leucine zippers may be derived from naturally occurring leucine zipper peptides, e.g., via conservative substitution(s) in the native amino acid sequence, wherein the peptide's ability to promote oligomerization is retained.
Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric oligomers. Alternatively, synthetic peptides that promote oligomerization may be employed. In particular embodiments, leucine residues in a leucine zipper moiety are replaced by isoleucine residues. Such peptides comprising isoleucine may be referred to as isoleucine zippers, but are encompassed by the term “leucine zippers” as employed herein.
Production of Polypeptides and Fragments Thereof
Expression, isolation and purification of the polypeptides and fragments of the invention may be accomplished by any suitable technique, including but not limited to the following:
Expression Systems
The present invention also provides recombinant cloning and expression vectors containing DNA, as well as host cell containing the recombinant vectors. Expression vectors comprising DNA may be used to prepare the polypeptides or fragments of the invention encoded by the DNA. A method for producing polypeptides comprises culturing host cells transformed with a recombinant expression vector encoding the polypeptide, under conditions that promote expression of the polypeptide, then recovering the expressed polypeptides from the culture. The skilled artisan will recognize that the procedure for purifying the expressed polypeptides will vary according to such factors as the type of host cells employed, and whether the polypeptide is membrane-bound or a soluble form that is secreted from the host cell.
Any suitable expression system may be employed. The vectors include a DNA encoding a polypeptide or fragment of the invention, operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such as those derived from a mammalian, microbial, viral, or insect gene. Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, an mRNA ribosomal binding site, and appropriate sequences which control transcription and translation initiation and termination. Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA sequence. Thus, a promoter nucleotide sequence is operably linked to a DNA sequence if the promoter nucleotide sequence controls the transcription of the DNA sequence. An origin of replication that confers the ability to replicate in the desired host cells, and a selection gene by which transformants are identified, are generally incorporated into the expression vector.
In addition, a sequence encoding an appropriate signal peptide (native or heterologous) can be incorporated into expression vectors. A DNA sequence for a signal peptide (secretory leader) may be fused in frame to the nucleic acid sequence of the invention so that the DNA is initially transcribed, and the mRNA translated, into a fusion protein comprising the signal peptide. A signal peptide that is functional in the intended host cells promotes extracellular secretion of the polypeptide. The signal peptide is cleaved from the polypeptide upon secretion of polypeptide from the cell.
The skilled artisan will also recognize that the position(s) at which the signal peptide is cleaved may differ from that predicted by computer program, and may vary according to such factors as the type of host cells employed in expressing a recombinant polypeptide. A protein preparation may include a mixture of protein molecules having different N-terminal amino acids, resulting from cleavage of the signal peptide at more than one site. Particular embodiments of mature proteins provided herein include, but are not limited to, proteins having the residue at position 16, 29, 35, 95, or 117 of SEQ ID NO:2 as the N-terminal amino acid.
Suitable host cells for expression of polypeptides include prokaryotes, yeast or higher eukaryotic cells. Mammalian or insect cells are generally preferred for use as host cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example, in (Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., (1985)). Cell-free translation systems could also be employed to produce polypeptides using RNAs derived from DNA constructs disclosed herein.
Prokaryotic Systems
Prokaryotes include gram-negative or gram-positive organisms. Suitable prokaryotic host cells for transformation include, for example, E. coli, Bacillus subtilis, Salmonella typhimurium, and various other species within the genera Pseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic host cell, such as E. coli, a polypeptide may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-terminal Met may be cleaved from the expressed recombinant polypeptide.
Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker genes. A phenotypic selectable marker gene is, for example, a gene encoding a protein that confers antibiotic resistance or that supplies an autotrophic requirement. Examples of useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids such as the cloning vector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells. An appropriate promoter and a DNA sequence are inserted into the pBR322 vector. Other commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA).
Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include β-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615 (1978); and (Goeddel et al., Nature 281:544 (1979)), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057 (1980); and EP-A-36776) and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412 (1982)). A particularly useful prokaryotic host cell expression system employs a phage λPL promoter and a cI857ts thermolabile repressor sequence. Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the λPL promoter include plasmid pHUB2 (resident in E. coli strain JMB9, ATCC 37092) and pPLc28 (resident in E. coli RR1, ATCC 53082).
Yeast Systems
Alternatively, the polypeptides may be expressed in yeast host cells, preferably from the Saccharomyces genus (e.g., S. cerevisiae). Other genera of yeast, such as Pichia or Kluyveromyces, may also be employed. Yeast vectors will often contain an origin of replication sequence from a 2μ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073 (1980)) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149 (1968)); and (Holland et al., Biochem. 17:4900 (1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phospho-glucose isomerase, and glucokinase. Other suitable vectors and promoters for use in yeast expression are further described in (Hitzeman, EPA-73,657). Another alternative is the glucose-repressible ADH2 promoter described by (Russell et al., J. Biol. Chem. 258:2674 (1982)) and (Beier et al., Nature 300:724 (1982)). Shuttle vectors replicable in both yeast and E. coli may be constructed by inserting DNA sequences from pBR322 for selection and replication in E. coli (Ampr gene and origin of replication) into the above-described yeast vectors.
The yeast α-factor leader sequence may be employed to direct secretion of the polypeptide. The α-factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. See, e.g., (Kurjan et al., Cell 30:933 (1982)) and (Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330 (1984)). Other leader sequences suitable for facilitating secretion of recombinant polypeptides from yeast hosts are known to those of skill in the art. A leader sequence may be modified near its 3′ end to contain one or more restriction sites. This will facilitate fusion of the leader sequence to the structural gene.
Yeast transformation protocols are known to those of skill in the art. One such protocol is described by (Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929 (1978)). The Hinnen et al. protocol selects for Trp+ transformants in a selective medium, wherein the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 mg/ml adenine and 20 mg/ml uracil.
Yeast host cells transformed by vectors containing an ADH2 promoter sequence may be grown for inducing expression in a “rich” medium. An example of a rich medium is one consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 mg/ml adenine and 80 mg/ml uracil. Derepression of the ADH2 promoter occurs when glucose is exhausted from the medium.
Mammalian or Insect Systems
Mammalian or insect host cell culture systems also may be employed to express recombinant polypeptides. Bacculovirus systems for production of heterologous proteins in insect cells are reviewed by (Luckow and Summers, Bio/Technology, 6:47 (1988)).
Established cell lines of mammalian origin also may be employed. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175 (1981)), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) as described by (McMahan et al., EMBO J., 10: 2821 (1991)).
Established methods for introducing DNA into mammalian cells have been described (Kaufman, R. J., Large Scale Mammalian Cell Culture, pp. 15-69 (1990)).
Additional protocols using commercially available reagents, such as Lipofectamine lipid reagent (Gibco/BRL) or Lipofectamine-Plus lipid reagent, can be used to transfect cells (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987)). In addition, electroporation can be used to transfect mammalian cells using conventional procedures, such as those in (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1-3, Cold Spring Harbor Laboratory Press (1989)). Selection of stable transformants can be performed using methods known in the art, such as, for example, resistance to cytotoxic drugs. (Kaufman et al., Meth. in Enzymology 185:487-511 (1990)), describes several selection schemes, such as dihydrofolate reductase (DHFR) resistance. A suitable host strain for DHFR selection can be CHO strain DX-B11, which is deficient in DHFR (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220 (1980)). A plasmid expressing the DHFR cDNA can be introduced into strain DX-B11, and only cells that contain the plasmid can grow in the appropriate selective media. Other examples of selectable markers that can be incorporated into an expression vector include cDNAs conferring resistance to antibiotics, such as G418 and hygromycin B. Cells harboring the vector can be selected on the basis of resistance to these compounds.
Transcriptional and translational control sequences for mammalian host cell expression vectors can be excised from viral genomes. Commonly used promoter sequences and enhancer sequences are derived from polyoma virus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites can be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell. Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment, which can also contain a viral origin of replication (Fiers et al., Nature 273:113 (1978)); (Kaufman, Meth. in Enzymology (1990)). Smaller or larger SV40 fragments can also be used, provided the approximately 250 bp sequence extending from the HindIII site toward the BglI site located in the SV40 viral origin of replication site is included.
Additional control sequences shown to improve expression of heterologous genes from mammalian expression vectors include such elements as the expression augmenting sequence element (EASE) derived from CHO cells (Morris et al., Animal Cell Technology, pp. 529-534 and PCT Application WO 97/25420 (1997)) and the tripartite leader (TPL) and VA gene RNAs from Adenovirus 2 (Gingeras et al., J. Biol. Chem. 257:13475-13491 (1982)). The internal ribosome entry site (IRES) sequences of viral origin allows dicistronic mRNAs to be translated efficiently (Oh and Sarnow, Current Opinion in Genetics and Development 3:295-300 (1993)); (Ramesh et al., Nucleic Acids Research 24:2697-2700 (1996)). Expression of a heterologous cDNA as part of a dicistronic mRNA followed by the gene for a selectable marker (e.g. DHFR) has been shown to improve transfectability of the host and expression of the heterologous cDNA (Kaufman, Meth. in Enzymology (1990)). Exemplary expression vectors that employ dicistronic mRNAs are pTR-DC/GFP described by (Mosser et al., Biotechniques 22:150-161 (1997)), and p2A5I described by (Morris et al., Animal Cell Technology, pp. 529-534 (1997)).
A useful high expression vector, pCAVNOT, has been described by (Mosley et al., Cell 59:335-348 (1989)). Other expression vectors for use in mammalian host cells can be constructed as disclosed by (Okayama and Berg, Mol. Cell. Biol. 3:280 (1983)). A useful system for stable high level expression of mammalian cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by (Cosman et al., Mol. Immunol. 23:935 (1986)). A useful high expression vector, PMLSV N1/N4, described by (Cosman et al., Nature 312:768 (1984)), has been deposited as ATCC 39890. Additional useful mammalian expression vectors are described in EP-A-0367566, and in WO 91/18982, incorporated by reference herein. In yet another alternative, the vectors can be derived from retroviruses.
Another useful expression vector, pFLAG7, can be used. FLAG7 technology is centered on the fusion of a low molecular weight (1 kD), hydrophilic, FLAG7 marker peptide to the N-terminus of a recombinant protein expressed by pFLAG7 expression vectors. pDC311 is another specialized vector used for expressing proteins in CHO cells. pDC311 is characterized by a bicistronic sequence containing the gene of interest and a dihydrofolate reductase (DHFR) gene with an internal ribosome binding site for DHFR translation, an expression augmenting sequence element (EASE), the human CMV promoter, a tripartite leader sequence, and a polyadenylation site.
Regarding signal peptides that may be employed, the native signal peptide may be replaced by a heterologous signal peptide or leader sequence, if desired. The choice of signal peptide or leader may depend on factors such as the type of host cells in which the recombinant polypeptide is to be produced. To illustrate, examples of heterologous signal peptides that are functional in mammalian host cells include the signal sequence for interleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195; the signal sequence for interleukin-2 receptor described in (Cosman et al., Nature 312:768 (1984)); the interleukin-4 receptor signal peptide described in EP 367,566; the type I interleukin-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; and the type II interleukin-1 receptor signal peptide described in EP 460,846.
Purification
The invention also includes methods of isolating and purifying the polypeptides and fragments thereof.
Isolation and Purification
The “isolated” polypeptides or fragments thereof encompassed by this invention are polypeptides or fragments that are not in an environment identical to an environment in which it or they can be found in nature. The “purified” polypeptides or fragments thereof encompassed by this invention are essentially free of association with other proteins or polypeptides, for example, as a purification product of recombinant expression systems such as those described above or as a purified product from a non-recombinant source such as naturally occurring cells and/or tissues.
In one preferred embodiment, the purification of recombinant polypeptides or fragments can be accomplished using fusions of polypeptides or fragments of the invention to another polypeptide to aid in the purification of polypeptides or fragments of the invention. Such fusion partners can include the poly-His or other antigenic identification peptides described above as well as the Fc moieties described previously.
With respect to any type of host cell, as is known to the skilled artisan, procedures for purifying a recombinant polypeptide or fragment will vary according to such factors as the type of host cells employed and whether or not the recombinant polypeptide or fragment is secreted into the culture medium.
In general, the recombinant polypeptide or fragment can be isolated from the host cells if not secreted, or from the medium or supernatant if soluble and secreted, followed by one or more concentration, salting-out, ion exchange, hydrophobic interaction, affinity purification or size exclusion chromatography steps. As to specific ways to accomplish these steps, the culture medium first can be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a purification matrix such as a gel filtration medium. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. In addition, a chromatofocusing step can be employed. Alternatively, a hydrophobic interaction chromatography step can be employed. Suitable matrices can be phenyl or octyl moieties bound to resins. In addition, affinity chromatography with a matrix which selectively binds the recombinant protein can be employed. Examples of such resins employed are lectin columns, dye columns, and metal-chelating columns. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, (e.g., silica gel or polymer resin having pendant methyl, octyl, octyldecyl or other aliphatic groups) can be employed to further purify the polypeptides. Some or all of the foregoing purification steps, in various combinations, are well known and can be employed to provide an isolated and purified recombinant protein.
It is also possible to utilize an affinity column comprising a polypeptide-binding protein of the invention, such as a monoclonal antibody generated against polypeptides of the invention, to affinity-purify expressed polypeptides. These polypeptides can be removed from an affinity column using conventional techniques, e.g., in a high salt elution buffer and then dialyzed into a lower salt buffer for use or by changing pH or other components depending on the affinity matrix utilized, or be competitively removed using the naturally occurring substrate of the affinity moiety, such as a polypeptide derived from the invention.
In this aspect of the invention, polypeptide-binding proteins, such as the anti-polypeptide antibodies of the invention or other proteins that may interact with the polypeptide of the invention, can be bound to a solid phase support such as a column chromatography matrix or a similar substrate suitable for identifying, separating, or purifying cells that express polypeptides of the invention on their surface. Adherence of polypeptide-binding proteins of the invention to a solid phase contacting surface can be accomplished by any means, for example, magnetic microspheres can be coated with these polypeptide-binding proteins and held in the incubation vessel through a magnetic field. Suspensions of cell mixtures are contacted with the solid phase that has such polypeptide-binding proteins thereon. Cells having polypeptides of the invention on their surface bind to the fixed polypeptide-binding protein and unbound cells then are washed away. This affinity-binding method is useful for purifying, screening, or separating such polypeptide-expressing cells from solution. Methods of releasing positively selected cells from the solid phase are known in the art and encompass, for example, the use of enzymes. Such enzymes are preferably non-toxic and non-injurious to the cells and are preferably directed to cleaving the cell-surface binding partner.
Alternatively, mixtures of cells suspected of containing polypeptide-expressing cells of the invention first can be incubated with a biotinylated polypeptide-binding protein of the invention. Incubation periods are typically at least one hour in duration to ensure sufficient binding to polypeptides of the invention. The resulting mixture then is passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin provides the binding of the polypeptide-binding cells to the beads. Use of avidin-coated beads is known in the art. See (Berenson, et al. J. Cell. Biochem., 10D:239 (1986)). Wash of unbound material and the release of the bound cells is performed using conventional methods.
The desired degree of purity depends on the intended use of the protein. A relatively high degree of purity is desired when the polypeptide is to be administered in vivo, for example. In such a case, the polypeptides are purified such that no protein bands corresponding to other proteins are detectable upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be recognized by one skilled in the pertinent field that multiple bands corresponding to the polypeptide may be visualized by SDS-PAGE, due to differential glycosylation, differential post-translational processing, and the like. Most preferably, the polypeptide of the invention is purified to substantial homogeneity, as indicated by a single protein band upon analysis by SDS-PAGE. The protein band may be visualized by silver staining, Coomassie blue staining, or (if the protein is radiolabeled) by autoradiography.
Assays
The purified polypeptides of the invention (including proteins, polypeptides, fragments, variants, oligomers, and other forms) may be tested for the ability to bind TSLP receptors in any suitable assay, such as a conventional binding assay. To illustrate, the polypeptide may be labeled with a detectable reagent (e.g., a radionuclide, chromophore, enzyme that catalyzes a colorimetric or fluorometric reaction, and the like). The labeled polypeptide is contacted with cells expressing TSLP receptors. The cells then are washed to remove unbound labeled polypeptide, and the presence of cell-bound label is determined by a suitable technique, chosen according to the nature of the label.
One example of a binding assay procedure is as follows. A recombinant expression vector containing TSLP cDNA is constructed by methods known in the art. The mouse TSLP receptor comprises an N-terminal extracellular domain, a transmembrane region, and a C-terminal cytoplasmic domain. CV1-EBNA-1 cells in 10 cm2 dishes are transfected with the recombinant expression vector. CV-1I/EBNA-1 cells (ATCC CRL 10478) constitutively express EBV nuclear antigen-1 driven from the CMV immediate-early enhancer/promoter. CV1-EBNA-1 was derived from the African Green Monkey kidney cell line CV-1 (ATCC CCL 70), as described by (McMahan et al., EMBO J. 10:2821 (1991)).
The transfected cells are cultured for 24 hours, and the cells in each dish then are split into a 24-well plate. After culturing an additional 48 hours, the transfected cells (about 4×104 cells/well) are washed with BM-NFDM, which is binding medium (RPMI 1640 containing 25 mg/ml bovine serum albumin, 2 mg/ml sodium azide, 20 mM Hepes pH 7.2) to which 50 mg/ml nonfat dry milk has been added. The cells then are incubated for 1 hour at 37° C.with various concentrations of, for example, a soluble polypeptide/Fc fusion protein made as set forth above. Cells then are washed and incubated with a constant saturating concentration of a 125I-mouse anti-human IgG in binding medium, with gentle agitation for 1 hour at 37° C. After extensive washing, cells are released via trypsinization.
The mouse anti-human IgG employed above is directed against the Fc region of human IgG and can be obtained from Jackson Immunoresearch Laboratories, Inc., West Grove, Pa. The antibody is radioiodinated using the standard chloramine-T method. The antibody will bind to the Fc portion of any polypeptide/Fc protein that has bound to the cells. In all assays, non-specific binding of 125I-antibody is assayed in the absence of the Fc fusion protein/Fc, as well as in the presence of the Fc fusion protein and a 200-fold molar excess of unlabeled mouse anti-human IgG antibody.
Cell-bound 125I-antibody is quantified on a Packard Autogamma counter. Affinity calculations (Scatchard, Ann. N.Y. Acad. Sci. 51:660 (1949)) are generated on RS/1 (BBN Software, Boston, Mass.) run on a Microvax computer.
Another type of suitable binding assay is a competitive binding assay. To illustrate, biological activity of a variant may be determined by assaying for the variant's ability to compete with the native protein for binding to TSLP receptors.
Competitive binding assays can be performed by conventional methodology. Reagents that may be employed in competitive binding assays include radiolabeled TSLP and intact cells expressing TSLP receptors (endogenous or recombinant) on the cell surface. For example, a radiolabeled soluble TSLP fragment can be used to compete with a soluble TSLP variant for binding to cell surface TSLP receptors. Instead of intact cells, one could substitute a soluble TSLP receptor/Fc fusion protein bound to a solid phase through the interaction of Protein A or Protein G (on the solid phase) with the Fc moiety. Chromatography columns that contain Protein A and Protein G include those available from Pharmacia Biotech, Inc., Piscataway, N.J.
Another type of competitive binding assay utilizes radiolabeled soluble TSLP receptor, such as a soluble TSLP receptor/Fc fusion protein, and intact cells expressing endogenous or recombinant TSLP receptor. The radiolabeled TSLP receptor can be used to compete with the membrane bound TSLP receptor for soluble TSLP. Qualitative results can be obtained by competitive autoradiographic plate binding assays, while Scatchard plots (Scatchard, Ann. N.Y. Acad. Sci. 51:660 (1949)) may be utilized to generate quantitative results.
Use of Human TSLP Nucleic Acid or Oligonucleotides
In addition to being used to express polypeptides as described above, the nucleic acids of the invention, including DNA, RNA, mRNA and oligonucleotides thereof can be used:
as probes to identify nucleic acid encoding proteins having the ability to induce B lineage or T lineage cell proliferation;
to identify human chromosome number 5;
to map genes on human chromosome number 5;
to identify genes associated with certain diseases, syndromes, or other conditions associated with human chromosome number 5;
as single-stranded sense or antisense oligonucleotides, to inhibit expression of polypeptide encoded by the TSLP gene;
to help detect defective genes in an individual; and
for gene therapy.
Probes
Among the uses of nucleic acids of the invention is the use of fragments as probes or primers. Such fragments generally comprise at least about 17 contiguous nucleotides of a DNA sequence. In other embodiments, a DNA fragment comprises at least 30, or at least 60, contiguous nucleotides of a DNA sequence.
Because homologs of SEQ ID NO:1, from other mammalian species, are contemplated herein, probes based on the human DNA sequence of SEQ ID NO:1 may be used to screen cDNA libraries derived from other mammalian species, using conventional cross-species hybridization techniques.
Using knowledge of the genetic code in combination with the amino acid sequences set forth above, sets of degenerate oligonucleotides can be prepared. Such oligonucleotides are useful as primers, e.g., in polymerase chain reactions (PCR), whereby DNA fragments are isolated and amplified.
Chromosome Mapping
All or a portion of the nucleic acids of SEQ ID NO:1, including oligonucleotides, can be used by those skilled in the art using well-known techniques to identify the human chromosome 5, and the specific locus thereof, that may contain the DNA of other TSLP family members. Useful techniques include, but are not limited to, using the sequence or portions, including oligonucleotides, as a probe in various well-known techniques such as radiation hybrid mapping (high resolution), in situ hybridization to chromosome spreads (moderate resolution), and Southern blot hybridization to hybrid cell lines containing individual human chromosomes (low resolution).
For example. chromosomes can be mapped by using PCR and radiation hybridization. PCR is performed using the Whitehead institute/MIT Center for Genome Research Genebridge4 panel of 93 radiation hybrids. Primers are used which lie within a putative exon, across an intron, or across an intron-exon fragment of the gene of interest and which amplify a product from human genomic DNA, but do not amplify, for example, control hamster genomic DNA. The results of the PCRs are converted into a data vector that is submitted to the Whitehead/MIT Radiation Mapping site on the internet. The data is scored and the chromosomal assignment and placement relative to known Sequence Tag Site (STS) markers on the radiation hybrid map is provided.
Identifying Associated Diseases
As set forth below, SEQ ID NO:1 has been mapped to the q21-q22 region of chromosome 5 by syntenic analysis of the murine gene. Thus, the nucleic acid of SEQ ID NO:1 or a fragment thereof can be used by one skilled in the art using well-known techniques to analyze abnormalities associated with human chromosome number 5 and, in particular, with the q21-q22 region of chromosome number 5, including Gardner syndrome, adenomatous polyposis coli, hereditary desmoid disease, Turcot syndrome, and colorectal cancer. This enables one to distinguish conditions in which this marker is rearranged or deleted. In addition, nucleotides of SEQ ID NO:1 or a fragment thereof can be used as a positional marker to map other genes of unknown location.
The DNA may be used in developing treatments for any disorder mediated (directly or indirectly) by defective or insufficient amounts of the genes corresponding to the nucleic acids of the invention. Disclosure herein of native nucleotide sequences permits the detection of defective genes, and the replacement thereof with normal genes. Defective genes may be detected in in vitro diagnostic assays, and by comparison of a native nucleotide sequence disclosed herein with that of a gene derived from a person suspected of harboring a defect in this gene.
Sense-Antisense
Other useful fragments of the nucleic acids include antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment of DNA (SEQ ID NO:1). Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 to about 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example, (Stein and Cohen, Cancer Res. 48:2659 (1988)) and (van der Krol et al., BioTechniques 6:958 (1988)).
Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block or inhibit protein expression by one of several means, including enhanced degradation of the mRNA by RNAseH, inhibition of splicing, premature termination of transcription or translation, or by other means. The antisense oligonucleotides thus may be used to block expression of proteins. Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as those described in WO91/06629) and wherein such sugar linkages are resistant to endogenous nucleases. Such oligonucleotides with resistant sugar linkages are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences.
Other examples of sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/10448, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine). Further still, intercalating agents, such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence.
Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, lipofection, CaPO4-mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus.
Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.
Alternatively, a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448. The sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.
Use of Human TSLP Polypeptides and Fragmented Polypeptides
Uses include, but are not limited to, the following:
Purifying proteins and measuring activity thereof
Delivery Agents
Therapeutic and Research Reagents
Molecular weight and Isoelectric focusing markers
Controls for peptide fragmentation
Identification of unknown proteins
Preparation of Antibodies
Purification Reagents
The polypeptide of the invention finds use as a protein purification reagent. For example, the polypeptides may be used to purify TSLP binding partners, such as human TSLP receptors. In particular embodiments, a polypeptide (in any form described herein that is capable of binding TSLP receptors) is attached to a solid support by conventional procedures. As one example, affinity chromatography columns containing functional groups that will react with functional groups on amino acid side chains of proteins are available (Pharmacia Biotech, Inc., Piscataway, N.J.). In an alternative, a TSLP polypeptide/Fc protein (as discussed above) is attached to Protein A- or Protein G-containing chromatography columns through interaction with the Fc moiety.
The polypeptide also finds use in purifying or identifying cells that express TSLP receptors on the cell surface. Polypeptides are bound to a solid phase such as a column chromatography matrix or a similar suitable substrate. For example, magnetic microspheres can be coated with the polypeptides and held in an incubation vessel through a magnetic field. Suspensions of cell mixtures containing TSLP receptor expressing cells are contacted with the solid phase having the polypeptides thereon. Cells expressing TSLP receptor on the cell surface bind to the fixed polypeptides, and unbound cells then are washed away.
Alternatively, the polypeptides can be conjugated to a detectable moiety, then incubated with cells to be tested for TSLP receptor expression. After incubation, unbound labeled matter is removed and the presence or absence of the detectable moiety on the cells is determined.
In a further alternative, mixtures of cells suspected of containing TSLP receptors are incubated with biotinylated polypeptides. Incubation periods are typically at least one hour in duration to ensure sufficient binding. The resulting mixture then is passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin provides binding of the desired cells to the beads. Procedures for using avidin-coated beads are known (see Berenson, et al. J. Cell. Biochem., 10D:239 (1986)). Washing to remove unbound material, and the release of the bound cells, are performed using conventional methods.
Measuring Activity
Polypeptides also find use in measuring the biological activity of TSLP receptors in terms of their binding affinity. The polypeptides thus may be employed by those conducting “quality assurance” studies, e.g., to monitor shelf life and stability of protein under different conditions. For example, the polypeptides may be employed in a binding affinity study to measure the biological activity of a TSLP receptor that has been stored at different temperatures, or produced in different cell types. The proteins also may be used to determine whether biological activity is retained after modification of a TSLP receptor (e.g., chemical modification, truncation, mutation, etc.). The binding affinity of the modified TSLP receptor is compared to that of an unmodified TSLP receptor to detect any adverse impact of the modifications on biological activity of TSLP receptors. The biological activity of a TSLP receptor thus can be ascertained before it is used in a research study, for example.
Delivery Agents
The polypeptides also find use as carriers for delivering agents attached thereto to cells bearing TSLP receptors. Cells expressing TSLP receptors include those identified in thymus, spleen, kidney, and bone marrow. The polypeptides thus can be used to deliver diagnostic or therapeutic agents to such cells (or to other cell types found to express TSLP receptors on the cell surface) in in vitro or in vivo procedures.
Detectable (diagnostic) and therapeutic agents that may be attached to a polypeptide include, but are not limited to, toxins, other cytotoxic agents, drugs, radionuclides, chromophores, enzymes that catalyze a colorimetric or fluorometric reaction, and the like, with the particular agent being chosen according to the intended application. Among the toxins are ricin, abrin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, ribosomal inactivating proteins, mycotoxins such as trichothecenes, and derivatives and fragments (e.g., single chains) thereof. Radionuclides suitable for diagnostic use include, but are not limited to, 123I, 131I, 99mTc, 111In, and 76Br. Examples of radionuclides suitable for therapeutic use are 131I, 211At, 77Br, 186Re, 188Re, 212Pb, 212Bi, 109Pd, 64Cu, and 67Cu.
Such agents may be attached to the polypeptide by any suitable conventional procedure. The polypeptide comprises functional groups on amino acid side chains that can be reacted with functional groups on a desired agent to form covalent bonds, for example. Alternatively, the protein or agent may be derivatized to generate or attach a desired reactive functional group. The derivatization may involve attachment of one of the bifunctional coupling reagents available for attaching various molecules to proteins (Pierce Chemical Company, Rockford, Ill.). A number of techniques for radiolabeling proteins are known. Radionuclide metals may be attached to polypeptides by using a suitable bifunctional chelating agent, for example.
Conjugates comprising polypeptides and a suitable diagnostic or therapeutic agent (preferably covalently linked) are thus prepared. The conjugates are administered or otherwise employed in an amount appropriate for the particular application.
Therapeutic Agents
Polypeptides of the invention may be used in developing treatments for any disorder mediated (directly or indirectly) by defective, or insufficient amounts of the polypeptides. These polypeptides may be administered to a mammal afflicted with such a disorder.
The polypeptides may also be employed in inhibiting the biological activity of TSLP receptors in in vitro or in vivo procedures. For example, a purified or modified polypeptide or a fragment thereof (e.g., modified TSLP polypeptides that bind the receptor but lack the ability to induce signaling) may be used to inhibit binding of endogenous TSLP to cell surface receptors. Biological effects that result from the binding of endogenous TSLP to receptors thus are inhibited.
In addition, TSLP receptor polypeptides may be administered to a mammal to treat a TSLP receptor-mediated disorder. Such TSLP receptor-mediated disorders include conditions caused (directly or indirectly) or exacerbated by TSLP receptors.
Compositions of the present invention may contain a polypeptide in any form described herein, such as native proteins, variants, derivatives, oligomers, and biologically active fragments. In particular embodiments, the composition comprises a soluble TSLP polypeptide or an oligomer comprising soluble TSLP polypeptides.
Compositions comprising an effective amount of a polypeptide of the present invention, in combination with other components such as a physiologically acceptable diluent, carrier, or excipient, are provided herein. The polypeptides can be formulated according to known methods used to prepare pharmaceutically useful compositions. They can be combined in admixture, either as the sole active material or with other known active materials suitable for a given indication, with pharmaceutically acceptable diluents (e.g., saline, Tris-HCl, acetate, and phosphate buffered solutions), preservatives (e.g., thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/or carriers. Suitable formulations for pharmaceutical compositions include those described in (Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Company, Easton, Pa. (1980)).
In addition, such compositions can be complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, etc., or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application.
The compositions of the invention can be administered in any suitable manner, e.g., topically, parenterally, or by inhalation. The term “parenteral” includes injection, e.g., by subcutaneous, intravenous, or intramuscular routes, also including localized administration, e.g., at a site of disease or injury. Sustained release from implants is also contemplated. One skilled in the pertinent art will recognize that suitable dosages will vary, depending upon such factors as the nature of the disorder to be treated, the patient's body weight, age, and general condition, and the route of administration. Preliminary doses can be determined according to animal tests, and the scaling of dosages for human administration is performed according to art-accepted practices.
Compositions comprising nucleic acids in physiologically acceptable formulations are also contemplated. DNA may be formulated for injection, for example.
Research Agents
Another use of the polypeptide of the present invention is as a research tool for studying the biological effects that result from inhibiting TSLP/TSLP receptor interactions on different cell types. Polypeptides also may be employed in in vitro assays for detecting TSLP or TSLP receptors or the interactions thereof.
Another embodiment of the invention relates to uses of human TSLP to study B cell or T cell signal transduction. Human TSLP and other cytokines play a central role in B cell and T cell development and immune responses, including transducing cellular signals, stimulating cells to secrete cytokines, and inducing B cell and T cell proliferation. As such, alterations in the expression and/or activation of TSLP can have profound effects on a plethora of cellular processes, including, but not limited to, activation or inhibition of cell specific responses and proliferation. Expression of cloned TSLP or of catalytically inactive mutants of TSLP has been used to identify the role a particular protein plays in mediating specific signaling events.
Cellular signaling often involves a molecular activation cascade, during which a receptor propagates a ligand-receptor mediated signal by specifically activating intracellular kinases which phosphorylate target substrates. These substrates can themselves be kinases which become activated following phosphorylation. Alternatively, they can be adaptor molecules that facilitate down stream signaling through protein-protein interaction following phosphorylation. Regardless of the nature of the substrate molecule(s), expressed catalytically active versions of the TSLP ligand receptors can be used to identify what substrate(s) were recognized and activated by the TSLP ligand receptor(s). As such, these novel TSLP receptors can be used as reagents to identify novel molecules involved in signal transduction pathways.
In addition, TSLP can be used by one skilled in the art using well-known techniques to stimulate B lineage or T lineage cell proliferation (Ray et al., Eur. J. Immunology 26, 10-16 (1996)) and (Namikawa et al., Blood 87:1881-1890 (1996)), to expression clone the human TSLP receptor (Sims et al., Science 241:585-589 (1988)), to clone a related protein (Kozlosky et. al., Cytokine 9:540-549 (1997)) and (Lyman et al., Blood 10:2795-2801 (1994)), and to ex vivo expand cells (Piacibello et al., Blood 89:2644-2653 (1997)).
Uses Thereof
Thus, the present invention encompasses methods of stimulating B- and T-lymphocyte proliferation, where the method comprises incubating lymphocytes with human TSLP. In a further embodiment, the method comprises incubating lymphocytes with human TSLP and at least one other cytokine in vivo or in vitro. Preferably, the cytokine is selected from the group of IL-7, Steel Factor, Stem Cell Factor, Mast Cell Growth Factor or flt3-Ligand. More preferably the cytokine is IL-7.
The present invention also encompasses methods of stimulating lymphocyte development or lymphopoiesis, where the method comprises incubating progenitor cells, such as bone marrow-derived mononuclear cells, with human TSLP in vivo or in vitro. In a further embodiment, the method comprises incubating lymphocytes with human TSLP and at least one other cytokine. Preferably, the cytokine is selected from the group of IL-7, Steel Factor, Stem Cell Factor, Mast Cell Growth Factor or flt3-Ligand. More preferably the cytokine is IL-7.
Molecular Weight and Isoelectric Point Markers
The polypeptides of the present invention can be subjected to fragmentation into smaller peptides by chemical and enzymatic means, and the peptide fragments so produced can be used in the analysis of other proteins or polypeptides. For example, such peptide fragments can be used as peptide molecular weight markers, peptide isoelectric point markers, or in the analysis of the degree of peptide fragmentation. Thus, the invention also includes these polypeptides and peptide fragments, as well as kits to aid in the determination of the apparent molecular weight and isoelectric point of an unknown protein and kits to assess the degree of fragmentation of an unknown protein.
Although all methods of fragmentation are encompassed by the invention, chemical fragmentation is a preferred embodiment, and includes the use of cyanogen bromide to cleave under neutral or acidic conditions such that specific cleavage occurs at methionine residues (E. Gross, Methods in Enz. 11:238-255 (1967)). This can further include additional steps, such as a carboxymethylation step to convert cysteine residues to an unreactive species.
Enzymatic fragmentation is another preferred embodiment, and includes the use of a protease such as Asparaginylendo-peptidase, Arginylendo-peptidase, Achromobacter protease I, Trypsin, Staphlococcus aureus V8 protease, Endoproteinase Asp-N, or Endoproteinase Lys-C under conventional conditions to result in cleavage at specific amino acid residues. Asparaginylendo-peptidase can cleave specifically on the carboxyl side of the asparagine residues present within the polypeptides of the invention. Arginylendo-peptidase can cleave specifically on the carboxyl side of the arginine residues present within these polypeptides. Achromobacter protease I can cleave specifically on the carboxyl side of the lysine residues present within the polypeptides (Sakiyama and Nakat, U.S. Pat. No. 5,248,599; T. Masaki et al., Biochim. Biophys. Acta 660:44-50 (1981); T. Masaki et al., Biochim. Biophys. Acta 660:51-55 (1981)). Trypsin can cleave specifically on the carboxyl side of the arginine and lysine residues present within polypeptides of the invention. Enzymatic fragmentation may also occur with a protease that cleaves at multiple amino acid residues. For example, Staphlococcus aureus V8 protease can cleave specifically on the carboxyl side of the aspartic and glutamic acid residues present within polypeptides (D. W. Cleveland, J. Biol. Chem. 3:1102-1106 (1977)). Endoproteinase Asp-N can cleave specifically on the amino side of the asparagine residues present within polypeptides. Endoproteinase Lys-C can cleave specifically on the carboxyl side of the lysine residues present within polypeptides of the invention. Other enzymatic and chemical treatments can likewise be used to specifically fragment these polypeptides into a unique set of specific peptides.
Of course, the peptides and fragments of the polypeptides of the invention can also be produced by conventional recombinant processes and synthetic processes well known in the art. With regard to recombinant processes, the polypeptides and peptide fragments encompassed by invention can have variable molecular weights, depending upon the host cell in which they are expressed. Glycosylation of polypeptides and peptide fragments of the invention in various cell types can result in variations of the molecular weight of these pieces, depending upon the extent of modification. The size of these pieces can be most heterogeneous with fragments of polypeptide derived from the extracellular portion of the polypeptide. Consistent polypeptides and peptide fragments can be obtained by using polypeptides derived entirely from the transmembrane and cytoplasmic regions, pretreating with N-glycanase to remove glycosylation, or expressing the polypeptides in bacterial hosts.
The molecular weight of these polypeptides can also be varied by fusing additional peptide sequences to both the amino and carboxyl terminal ends of polypeptides of the invention. Fusions of additional peptide sequences at the amino and carboxyl terminal ends of polypeptides of the invention can be used to enhance expression of these polypeptides or aid in the purification of the protein. In addition, fusions of additional peptide sequences at the amino and carboxyl terminal ends of polypeptides of the invention will alter some, but usually not all, of the fragmented peptides of the polypeptides generated by enzymatic or chemical treatment. Of course, mutations can be introduced into polypeptides of the invention using routine and known techniques of molecular biology. For example, a mutation can be designed so as to eliminate a site of proteolytic cleavage by a specific enzyme or a site of cleavage by a specific chemically induced fragmentation procedure. The elimination of the site will alter the peptide fingerprint of polypeptides of the invention upon fragmentation with the specific enzyme or chemical procedure.
The polypeptides and the resultant fragmented peptides can be analyzed by methods including sedimentation, electrophoresis, chromatography, and mass spectrometry to determine their molecular weights. Because the unique amino acid sequence of each piece specifies a molecular weight, these pieces can thereafter serve as molecular weight markers using such analysis techniques to assist in the determination of the molecular weight of an unknown protein, polypeptides or fragments thereof. The molecular weight markers of the invention serve particularly well as molecular weight markers for the estimation of the apparent molecular weight of proteins that have similar apparent molecular weights and, consequently, allow increased accuracy in the determination of apparent molecular weight of proteins.
When the invention relates to the use of fragmented peptide molecular weight markers, those markers are preferably at least 10 amino acids in size. More preferably, these fragmented peptide molecular weight markers are between 10 and 100 amino acids in size. Even more preferable are fragmented peptide molecular weight markers between and 50 amino acids in size and especially between 10 and 35 amino acids in size. Most preferable are fragmented peptide molecular weight markers between 10 and 20 amino acids in size.
Among the methods for determining molecular weight are sedimentation, gel electrophoresis, chromatography, and mass spectrometry. A particularly preferred embodiment is denaturing polyacrylamide gel electrophoresis (U. K. Laemmli, Nature 227:680-685 (1970)). Conventionally, the method uses two separate lanes of a gel containing sodium dodecyl sulfate and a concentration of acrylamide between 6-20%. The ability to simultaneously resolve the marker and the sample under identical conditions allows for increased accuracy. It is understood, of course, that many different techniques can be used for the determination of the molecular weight of an unknown protein using polypeptides of the invention, and that this embodiment in no way limits the scope of the invention.
Each unglycosylated polypeptide or fragment thereof has a pI that is intrinsically determined by its unique amino acid sequence (which pI can be estimated by the skilled artisan using any of the computer programs designed to predict pI values currently available, calculated using any well-known amino acid pKa table, or measured empirically). Therefore these polypeptides and fragments thereof can serve as specific markers to assist in the determination of the isoelectric point of an unknown protein, polypeptide, or fragmented peptide using techniques such as isoelectric focusing. These polypeptide or fragmented peptide markers serve particularly well for the estimation of apparent isoelectric points of unknown proteins that have apparent isoelectric points close to that of the polypeptide or fragmented peptide markers of the invention.
The technique of isoelectric focusing can be further combined with other techniques such as gel electrophoresis to simultaneously separate a protein on the basis of molecular weight and charge. The ability to simultaneously resolve these polypeptide or fragmented peptide markers and the unknown protein under identical conditions allows for increased accuracy in the determination of the apparent isoelectric point of the unknown protein. This is of particular interest in techniques, such as two dimensional electrophoresis (T. D. Brock and M. T. Madigan, Biology of Microorganisms 76-77, Prentice Hall, 6d ed. (1991)), where the nature of the procedure dictates that any markers should be resolved simultaneously with the unknown protein. In addition, with such methods, these polypeptides and fragmented peptides thereof can assist in the determination of both the isoelectric point and molecular weight of an unknown protein or fragmented peptide.
Polypeptides and fragmented peptides can be visualized using two different methods that allow a discrimination between the unknown protein and the molecular weight markers. In one embodiment, the polypeptide and fragmented peptide molecular weight markers of the invention can be visualized using antibodies generated against these markers and conventional immunoblotting techniques. This detection is performed under conventional conditions that do not result in the detection of the unknown protein. It is understood that it may not be possible to generate antibodies against all polypeptide fragments of the invention, since small peptides may not contain immunogenic epitopes. It is further understood that not all antibodies will work in this assay; however, those antibodies which are able to bind polypeptides and fragments of the invention can be readily determined using conventional techniques.
The unknown protein is also visualized by using a conventional staining procedure. The molar excess of unknown protein to polypeptide or fragmented peptide molecular weight markers of the invention is such that the conventional staining procedure predominantly detects the unknown protein. The level of these polypeptide or fragmented peptide molecular weight markers is such as to allow little or no detection of these markers by the conventional staining method. The preferred molar excess of unknown protein to polypeptide molecular weight markers of the invention is between 2 and 100,000 fold. More preferably, the preferred molar excess of unknown protein to these polypeptide molecular weight markers is between 10 and 10,000 fold and especially between 100 and 1,000 fold.
It is understood of course that many techniques can be used for the determination and detection of molecular weight and isoelectric point of an unknown protein, polypeptides, and fragmented peptides thereof using these polypeptide molecular weight markers and peptide fragments thereof and that these embodiments in no way limit the scope of the invention.
In another embodiment, the analysis of the progressive fragmentation of the polypeptides of the invention into specific peptides (D. W. Cleveland et al., J. Biol. Chem. 252:1102-1106 (1977)), such as by altering the time or temperature of the fragmentation reaction, can be used as a control for the extent of cleavage of an unknown protein. For example, cleavage of the same amount of polypeptide and unknown protein under identical conditions can allow for a direct comparison of the extent of fragmentation. Conditions that result in the complete fragmentation of the polypeptide can also result in complete fragmentation of the unknown protein.
As to the specific use of the polypeptides and fragmented peptides of the invention as molecular weight markers, the fragmentation of the polypeptide of SEQ ID NO:2 with cyanogen bromide generates a unique set of fragmented peptide molecular weight markers. The distribution of methionine residues determines the number of amino acids in each peptide and the unique amino acid composition of each peptide determines its molecular weight.
In addition, the preferred purified polypeptide of the invention (SEQ ID NO:2) has an observed molecular weight of approximately 21,000 Daltons.
Where an intact protein is used, the use of these polypeptide molecular weight markers allows increased accuracy in the determination of apparent molecular weight of proteins that have apparent molecular weights close to 21,000 Daltons. Where fragments are used, there is increased accuracy in determining molecular weight over the range of the molecular weights of the fragment.
Finally, as to the kits that are encompassed by the invention, the constituents of such kits can be varied, but typically contain the polypeptide and fragmented peptide molecular weight markers. Also, such kits can contain the polypeptides wherein a site necessary for fragmentation has been removed. Furthermore, the kits can contain reagents for the specific cleavage of the polypeptide and the unknown protein by chemical or enzymatic cleavage. Kits can further contain antibodies directed against polypeptides or fragments thereof of the invention.
Identification of Unknown Proteins
As set forth above, a polypeptide or peptide fingerprint can be entered into or compared to a database of known proteins to assist in the identification of the unknown protein using mass spectrometry (W. J. Henzel et al., Proc. Natl. Acad. Sci. USA 90:5011-5015 (1993); D. Fenyo et al., Electrophoresis 19:998-1005 (1998)). A variety of computer software programs to facilitate these comparisons are accessible via the Internet, such as Protein Prospector (Internet site: prospector.uscf.edu), MultiIdent (Internet site: www.expasy.ch/sprot/multiident.html), PeptideSearch (Internet site: www.mann.embl-heiedelberg.de...deSearch/FR_PeptideSearch Form.html), and ProFound (Internet site: www.chait-sgi.rockefeller.edu/cgi-bin/prot-id-frag.html). These programs allow the user to specify the cleavage agent and the molecular weights of the fragmented peptides within a designated tolerance. The programs compare these molecular weights to protein databases to assist in determining the identity of the unknown protein.
In addition, a polypeptide or peptide digest can be sequenced using tandem mass spectrometry (MS/MS) and the resulting sequence searched against databases (J. K. Eng, et al., J. Am. Soc. Mass Spec. 5:976-989 (1994); M. Mann and M. Wilm, Anal. Chem. 66:4390-4399 (1994); J. A. Taylor and R. S. Johnson, Rapid Comm. Mass Spec. 11: 1067-1075 (1997)). Searching programs that can be used in this process exist on the Internet, such as Lutefisk 97 (Internet site: www.lsbc.com:70/Lutefisk97.html), and the Protein Prospector, Peptide Search and ProFound programs described above. Therefore, adding the sequence of a gene and its predicted protein sequence and peptide fragments to a sequence database can aid in the identification of unknown proteins using tandem mass spectrometry.
Antibodies
Antibodies that are immunoreactive with the polypeptides of the invention are provided herein. Such antibodies specifically bind to the polypeptides via the antigen-binding sites of the antibody (as opposed to non-specific binding). Thus, the polypeptides, fragments, variants, fusion proteins, etc., as set forth above may be employed as “immunogens” in producing antibodies immunoreactive therewith. More specifically, the polypeptides, fragment, variants, fusion proteins, etc. contain antigenic determinants or epitopes that elicit the formation of antibodies.
These antigenic determinants or epitopes can be either linear or conformational (discontinuous). Linear epitopes are composed of a single section of amino acids of the polypeptide, while conformational or discontinuous epitopes are composed of amino acids sections from different regions of the polypeptide chain that are brought into close proximity upon protein folding (C. A. Janeway, Jr. and P. Travers, Immuno Biology 3:9, Garland Publishing Inc., 2nd ed. (1996)). Because folded proteins have complex surfaces, the number of epitopes available is quite numerous; however, due to the conformation of the protein and steric hinderances, the number of antibodies that actually bind to the epitopes is less than the number of available epitopes (C. A. Janeway, Jr. and P. Travers, Immuno Biology 2:14, Garland Publishing Inc., 2nd ed. (1996)). Epitopes may be identified by any of the methods known in the art.
Thus, one aspect of the present invention relates to the antigenic epitopes of the polypeptides of the invention. Such epitopes are useful for raising antibodies, in particular monoclonal antibodies, as described in more detail below. Additionally, epitopes from the polypeptides of the invention can be used as research reagents, in assays, and to purify specific binding antibodies from substances such as polyclonal sera or supernatants from cultured hybridomas. Such epitopes or variants thereof can be produced using techniques well known in the art such as solid-phase synthesis, chemical or enzymatic cleavage of a polypeptide, or using recombinant DNA technology.
As to the antibodies that can be elicited by the epitopes of the polypeptides of the invention, whether the epitopes have been isolated or remain part of the polypeptides, both polyclonal and monoclonal antibodies may be prepared by conventional techniques. See, for example, (Kennet et al., Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, eds., Plenum Press, New York (1980); and Harlow and Land, Antibodies: A Laboratory Manual, eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988)).
Hybridoma cell lines that produce monoclonal antibodies specific for the polypeptides of the invention are also contemplated herein. Such hybridomas may be produced and identified by conventional techniques. One method for producing such a hybridoma cell line comprises immunizing an animal with a polypeptide; harvesting spleen cells from the immunized animal; fusing said spleen cells to a myeloma cell line, thereby generating hybridoma cells; and identifying a hybridoma cell line that produces a monoclonal antibody that binds the polypeptide. The monoclonal antibodies may be recovered by conventional techniques.
The monoclonal antibodies of the present invention include chimeric antibodies, e.g., humanized versions of murine monoclonal antibodies. Such humanized antibodies may be prepared by known techniques and offer the advantage of reduced immunogenicity when the antibodies are administered to humans. In one embodiment, a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody. Alternatively, a humanized antibody fragment may comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in (Riechmann et al., Nature 332:323 (1988), Liu et al., PNAS 84:3439 (1987), Larrick et al., Bio/Technology 7:934 (1989), and Winter and Harris, TIPS 14:139 (May 1993)). Procedures to generate antibodies transgenically can be found in GB 2,272,440, U.S. Pat. Nos. 5,569,825 and 5,545,806 and related patents claiming priority therefrom, all of which are incorporated by reference herein.
Antigen-binding fragments of the antibodies, which may be produced by conventional techniques, are also encompassed by the present invention. Examples of such fragments include, but are not limited to, Fab and F(ab′)2 fragments. Antibody fragments and derivatives produced by genetic engineering techniques are also provided.
In one embodiment, the antibodies are specific for the polypeptides of the present invention and do not cross-react with other proteins. Screening procedures by which such antibodies may be identified are well known, and may involve immunoaffinity chromatography, for example.
Uses Thereof
The antibodies of the invention can be used in assays to detect the presence of the polypeptides or fragments of the invention, either in vitro or in vivo. The antibodies also may be employed in purifying polypeptides or fragments of the invention by immunoaffinity chromatography.
Those antibodies that additionally can block binding of the polypeptides of the invention to TSLP receptors may be used to inhibit a biological activity that results from such binding. Such blocking antibodies may be identified using any suitable assay procedure, such as by testing antibodies for the ability to inhibit binding of TSLP to certain cells expressing the TSLP receptors. Examples of such cells are the B and T lymphoid cell lines 70Z/3 and 7B9, respectively. Alternatively, blocking antibodies may be identified in assays for the ability to inhibit a biological effect that results from binding of TSLP to TSLP receptors on target cells. Antibodies may be assayed for the ability to inhibit TSLP-mediated lysis of cells expressing TSLP receptors, for example.
Such an antibody may be employed in an in vitro procedure, or administered in vivo to inhibit a biological activity mediated by the entity that generated the antibody. Disorders caused or exacerbated (directly or indirectly) by the interaction of TSLP with cell surface TSLP receptors thus may be treated. A therapeutic method involves in vivo administration of a blocking antibody to a mammal in an amount effective in inhibiting TSLP-mediated biological activity. Monoclonal antibodies are generally preferred for use in such therapeutic methods. In one embodiment, an antigen-binding antibody fragment is employed.
Antibodies may be screened for agonistic (i.e., ligand-mimicking) properties. Such antibodies, upon binding to cell surface TSLP receptors, induce biological effects (e.g., transduction of biological signals) similar to the biological effects induced when TSLP binds to cell surface TSLP receptors. Agonistic antibodies may be used to induce B lineage or T lineage cell proliferation.
Compositions comprising an antibody that is directed against human TSLP, and a physiologically acceptable diluent, excipient, or carrier, are provided herein. Suitable components of such compositions are as described above for compositions containing human TSLP proteins.
Also provided herein are conjugates comprising a detectable (e.g., diagnostic) or therapeutic agent, attached to the antibody. Examples of such agents are presented above. The conjugates find use in in vitro or in vivo procedures.
The following examples are provided to further illustrate particular embodiments of the invention, and are not to be construed as limiting the scope of the present invention.
EXAMPLE 1
Isolation of the Nucleic Acid
Human TSLP nucleic acid sequence was obtained by sequencing EST IMAGE clone 1407260, accession #AA889581. This sequence suggested, in comparison to the murine TSLP sequence, that the EST clone was a partial clone. A number of cDNA libraries were screened with internal primers to determine a source of cDNA that could be used to obtain the missing 3′ end of the TSLP cDNA clone. After 60 cycles of PCR using two internal primers of human TSLP sequence, the following cDNA libraries were positive for TSLP sequences: human testis, human foreskin fibroblasts, and fetal brain (weakly positive); while MoT, HS431, bone marrow, HPT4, HBT3, W126, Hut102, PBT, Sk Hep, human dermal fibroblast, Raji, human placenta, and KB libraries were all negative.
Using PCR on the human testis λgt10 library with an internal TSLP primer and a λgt10 vector primer, two clones (19E and 19F) with sequences identical to internal human TSLP sequences were isolated. Both clones had identical 5′ ends but different length 3′ ends. The coding as well as the non-coding sequences of clone 19E were identical to clone 19F; these clones differed in the length of the 3′ non-coding region, where clone 19F was about 34 bp longer than 19E. Therefore, sequences from 19F were used to complete the 3′ coding sequence of the human TSLP protein. This allowed for the identification of the C-terminal 15 amino acids not present in the EST. PCR was conducted according to conventional procedures.
EXAMPLE 2
Purification of TSLP Polypeptide
TSLP-specific ELISA:
Serial dilutions of TSLP-containing samples (in 50 mM NaHCO3, brought to pH 9 with NaOH) are coated onto Linbro/Titertek 96 well flat bottom E.I.A. microtitration plates (ICN Biomedicals Inc., Aurora, Ohio) at 100:1/well. After incubation at 4EC for 16 hours, the wells are washed six times with 200:1 PBS containing 0.05% Tween-20 (PBS-Tween). The wells are then incubated with FLAG7-TSLP receptor at 1 μg/ml in PBS-Tween with 5% fetal calf serum (FCS) for 90 minutes (100:1 per well), followed by washing as above. Next, each well is incubated with the anti-FLAG7 (monoclonal antibody M2 at 1 μg/ml in PBS-Tween containing 5% FCS for 90 minutes (100:1 per well), followed by washing as above. Subsequently, wells are incubated with a polyclonal goat anti-mIgG1-specific horseradish peroxidase-conjugated antibody (a 1:5000 dilution of the commercial stock in PBS-Tween containing 5% FCS) for 90 minutes (100:1 per well). The HRP-conjugated antibody is obtained from Southern Biotechnology Associates, Inc., Birmingham, Ala. Wells then are washed six times, as above.
For development of the ELISA, a substrate mix [100:1 per well of a 1:1 premix of the TMB Peroxidase Substrate and Peroxidase Solution B (Kirkegaard Perry Laboratories, Gaithersburg, Md.)] is added to the wells. After sufficient color reaction, the enzymatic reaction is terminated by addition of 2 N H2SO4 (50:1 per well). Color intensity (indicating TSLP-TSLP receptor binding) is determined by measuring extinction at 450 nm on a V Max plate reader (Molecular Devices, Sunnyvale, Calif.).
EXAMPLE 3
Amino Acid Sequence
The amino acid sequence of human TSLP was determined by translation of the complete human TSLP nucleotide sequence. The reading frame chosen was based on the homology of human TSLP with murine TSLP.
EXAMPLE 4
DNA and Amino Acid Sequences
The human TSLP nucleic acid sequence was determined by standard double stranded sequencing of the composite sequence of EST IMAGE clone 1407260, accession #AA889581, and the additional 3′ sequence from clone 19F.
The nucleotide sequence of the isolated human TSLP DNA and the amino acid sequence encoded thereby, are presented in SEQ ID NOs:1 and 2. The sequence of the entire human TSLP DNA fragment isolated by PCR corresponds to nucleotides 1 to 767 of SEQ ID NO:1, which encode amino acids 1 to 159 of SEQ ID NO:2.
The amino acid sequence in SEQ ID NO:2 bears significant similarity (49%) and identity (43%) to murine TSLP and weak homology to IL-7.
EXAMPLE 5
Monoclonal Antibodies That Bind TSLP
This example illustrates a method for preparing monoclonal antibodies that bind TSLP. Suitable immunogens that may be employed in generating such antibodies include, but are not limited to, purified human TSLP polypeptide or an immunogenic fragment thereof such as the extracellular domain, or fusion proteins containing human TSLP (e.g., a soluble TSLP/Fc fusion protein).
Purified human TSLP can be used to generate monoclonal antibodies immunoreactive therewith, using conventional techniques such as those described in U.S. Pat. No. 4,411,993. Briefly, mice are immunized with human TSLP immunogen emulsified in complete Freund's adjuvant, and injected in amounts ranging from 10-100 μg subcutaneously or intraperitoneally. Ten to twelve days later, the immunized animals are boosted with additional human TSLP emulsified in incomplete Freund's adjuvant. Mice are periodically boosted thereafter on a weekly to bi-weekly immunization schedule. Serum samples are periodically taken by retro-orbital bleeding or tail-tip excision to test for TSLP antibodies by dot blot assay, ELISA (Enzyme-Linked Immunosorbent Assay) or inhibition of TSLP receptor binding.
Following detection of an appropriate antibody titer, positive animals are provided one last intravenous injection of human TSLP in saline. Three to four days later, the animals are sacrificed, spleen cells harvested, and spleen cells are fused to a murine myeloma cell line, e.g., NS1 or preferably P3x63Ag8.653 (ATCC CRL 1580). Fusions generate hybridoma cells, which are plated in multiple microtiter plates in a HAT (hypoxanthine, aminopterin and thymidine) selective medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
The hybridoma cells are screened by ELISA for reactivity against purified TSLP by adaptations of the techniques disclosed in (Engvall et al., Immunochem. 8:871 (1971)) and in U.S. Pat. No. 4,703,004. A preferred screening technique is the antibody capture technique described in (Beckmann et al., J. Immunol. 144:4212 (1990)). Positive hybridoma cells can be injected intraperitoneally into syngeneic BALB/c mice to produce ascites containing high concentrations of anti-TSLP monoclonal antibodies. Alternatively, hybridoma cells can be grown in vitro in flasks or roller bottles by various techniques. Monoclonal antibodies produced in mouse ascites can be purified by ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to Protein A or Protein G can also be used, as can affinity chromatography based upon binding to TSLP.
EXAMPLE 6
Northern Blot Analysis
The tissue distribution of human TSLP mRNA was investigated by Northern blot analysis, as follows. An aliquot of a radiolabeled probe was added to two different human multiple tissue Northern blots (Clontech, Palo Alto, Calif.; Biochain, Palo Alto, Calif.). The blots were hybridized in 10× Denhardts, 50 mM Tris pH 7.5, 900 mM NaCl, 0.1% Na pyrophosphate, 1% SDS, 200 g/mL salmon sperm DNA. Hybridization was conducted overnight at 63EC in 50% formamide as previously described (March et al., Nature 315:641-647 (1985)). The blots then were washed with 2×SSC, 0.1% SDS at 68EC for 30 minutes.
A single transcript of 1.4 kilobases (kb) was present in heart, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testes, ovary, small intestine, colon. Negative tissues were brain, placenta, and peripheral blood leukocytes. The cells and tissues with the highest levels of TSLP mRNA are heart, liver, prostate, and testes, as shown by comparison to control probing with a β-actin-specific probe.
EXAMPLE 7
Binding Assay
Full length human TSLP can be expressed and tested for the ability to bind TSLP receptors. The binding assay can be conducted as follows.
A fusion protein comprising a leucine zipper peptide fused to the N-terminus of a soluble human TSLP polypeptide (LZ-TSLP) is employed in the assay. An expression construct is prepared, essentially as described for preparation of the FLAG7(TSLP) expression construct in (Wiley et al., Immunity, 3:673-682 (1995)); hereby incorporated by reference), except that DNA encoding the FLAG7 peptide was replaced with a sequence encoding a modified leucine zipper that allows for trimerization. The construct, in expression vector pDC409, encodes a leader sequence derived from human cytomegalovirus, followed by the leucine zipper moiety fused to the N-terminus of a soluble human TSLP polypeptide. The LZ-TSLP is expressed in CHO cells, and purified from the culture supernatant.
The expression vector designated pDC409 is a mammalian expression vector derived from the pDC406 vector described in (McMahan et al., EMBO J. 10:2821-2832 (1991)) hereby incorporated by reference). Features added to pDC409 (compared to pDC406) include additional unique restriction sites in the multiple cloning site (mcs); three stop codons (one in each reading frame) positioned downstream of the mcs; and a T7 polymerase promoter, downstream of the mcs, that facilitates sequencing of DNA inserted into the mcs.
For expression of full length human TSLP protein, the entire coding region (i.e., the DNA sequence presented in SEQ ID NO:1) is amplified by polymerase chain reaction (PCR). The template employed in the PCR is the cDNA clone isolated from a human testis cDNA library, as described in Example 1. The isolated and amplified DNA is inserted into the expression vector pDC409, to yield a construct designated pDC409-TSLP.
LZ-TSLP polypeptide is employed to test the ability to bind to host cells expressing recombinant or endogenous TSLP receptors, as discussed above. Cells expressing TSLP receptor are cultured in DMEM supplemented with 10% fetal bovine serum, penicillin, streptomycin, and glutamine. Cells are incubated with LZ-TSLP (5 mg/ml) for about 1 hour. Following incubation, the cells are washed to remove unbound LZ-TSLP and incubated with a biotinylated anti-LZ monoclonal antibody (5 mg/ml), and phycoerythrin-conjugated streptavidin (1:400), before analysis by fluorescence-activated cell scanning (FACS). The cytometric analysis was conducted on a FACscan (Beckton Dickinson, San Jose, Calif.).
The cells expressing TSLP receptors showed significantly enhanced binding of LZ-TSLP, compared to the control cells not expressing TSLP receptors.
EXAMPLE 8
Induction of T Cell Growth from Bone Marrow by TSLP and IL-7
Human TSLP, in combination with IL-7, induces the outgrowth of T cells from human bone marrow.
Human bone marrow-derived mononuclear cells (BM MNC) were isolated by centrifugation of whole bone marrow over Ficoll. BM MNC were cultured in McCoy's media supplemented with 10% fetal bovine serum, and amino acid and vitamin supplements, at a concentration ranging between 4.5-10×105 cells/ml in a total volume of 6 or 7 ml per flask (T25). Human TSLP (20 ng/ml) and other cytokines, i.e., IL-7, SLF (i.e., steel factor or stem cell factor, or mast cell growth factor), or flt3L, either alone or in combination, were added to the cultures at day 0. After 14 days and weekly thereafter, half the culture was removed for counting. Fresh media and cytokines were added to the cultures to return the total volume to 6 or 7 ml.
Harvested cells were also analyzed via flow cytometry fourteen days after culture and weekly thereafter, using antibodies specific for cell surface antigens. The antibodies used were specific for T cell antigens (i.e., the αβ T cell receptor, γδ T cell receptor, and CD3), B cell antigens (i.e., CD19 and surface IgM), Natural Killer cell antigens (i.e., CD56), monocyte antigens (i.e., CD14), and granulocyte antigens (i.e., CD15).
Addition of human TSLP and IL-7 to BM MNC cultures induced cellular growth as indicated in Table 1. At day 0, approximately 5% of BM MNC were T cells. After 2 weeks of culture with TSLP and IL-7, the cultures consisted of 70% CD3+ T cells. At day 21, 86% of the cells were CD3+ T cells. The cultures contained predominantly T cells until the termination of the experiment at day 42.
TABLE 1
Total Cell Yield (×105)
Day
Day
Day
Day
Day
Treatment
0
14
21
28
42
Cumulative
13.5
Media
6
1.1
0.4
0.9
8.4
TSLP
3.9
2.1
1
2.9
9.9
IL-7
4.2
7.4
4.4
4.6
20.6
IL-7 + TSLP
10.3
12.1
17.2
7.5
47.1
SLF
3.7
4.3
1.1
0.9
10
SLF + TSLP
5.4
6.9
1
1.6
14.9
flt3L
6.3
2.3
2.8
1.8
13.2
flt3L + TSLP
7.7
4.7
2.7
3.1
18.2
In another set of experiments, three separate batches of human TSLP tagged with His/FLAG7 (TSLP 7489, TSLP 7811, or TSLP 7812) were tested alone or in combination with IL-7 for the ability to affect cell survival and expansion. BM MNC cultures were obtained from two separate, fresh bone marrow samples and seeded at a concentration of either 5×105 cells/ml (Group 1) or 10×105 cells/ml (Group 2). His/FLAG7-tagged TSLP (20 mg/ml) and IL-7 were added to cultures as described above. TSLP combined with IL-7 resulted in expansion of BM MNC cultures as indicated in Table 2 (bone marrow sample 1) and Table 3 (bone marrow sample 2). By day 21, 80% of the expanded cell population consisted of CD4+ αβ+ or CD8+ αβ+ T cells. In four of the cultures treated with IL-7 and TSLP, cells expanded at such a rapid rate that an additional harvest was required at day 23 (Table 3). The cultures contained predominantly T cells until the termination of the experiments at 4-5 weeks.
TABLE 2
Total Cell Yield (×105)
Day
Day
Day
Day
Day
Treatment
0
14
21
28
35
Cumulative
Group 1
17.5
(5 × 105)
Media
4
1.3
1.4
ND*
6.7
IL-7
8.4
6.5
7.1
ND*
22
TSLP 7489
4.4
1.5
1.2
ND*
7.1
TSLP 7811
5.2
1.7
1.2
ND*
8.1
TSLP 7812
2.8
1.4
2.3
ND*
6.5
IL-7 + T7489
12.4
9.1
8.3
ND*
29.8
IL-7 + T7811
10.5
5.3
8.4
ND*
24.2
IL-7 + T7812
9.7
6.5
4.7
ND*
20.9
Group 2
35
(10 × 105)
Media
6.6
3.1
2.2
ND*
11.9
IL-7
14.8
10.1
3.7
ND*
32.3
TSLP 7489
11.5
3.3
2.9
ND*
17.7
TSLP 7811
13.3
2.8
3.1
ND*
19.2
TSLP 7812
13
3.2
2.6
ND*
18.8
IL-7 + T7489
25.6
17.7
8
10.9
62.2
IL-7 + T7811
18.8
16.8
10
15.7
61.3
IL-7 + T7812
22.4
13.5
10.4
11.6
57.9
*ND = not determined (culture exhausted)
TABLE 3
Total Cell Yield (×105)
Day
Day
Day
Day
Day
Day
Treatment
0
14
21
23
28
35
Cumulative
Group 1
17.5
(5 × 105)
Media
3.1
0.9
ND*
0.8
ND*
4.8
IL-7
3.8
8.9
ND*
8
ND*
20.7
TSLP 7489
3
1.1
ND*
0.8
ND*
4.9
TSLP 7811
2.6
1.3
ND*
ND*
ND*
3.9
TSLP 7812
3.8
1.2
ND*
0.9
ND*
5.9
IL-7 + T7489
8.9
80
39.4
18.2
21
167.5
IL-7 + T7811
6.2
12.5
ND*
16.7
14.3
49.7
IL-7 + T7812
7.1
14.5
ND*
11.1
11.6
44.3
Group 2
35
(10 × 105)
Media
6.6
1.9
ND*
1.8
ND*
10.3
IL-7
10.7
19
ND*
16.5
29.2
75.4
TSLP 7489
6.8
3.2
ND*
3.3
ND*
13.3
TSLP 7811
8.7
3.3
ND*
3.4
ND*
15.4
TSLP 7812
7.1
3.1
ND*
2.7
ND*
12.9
IL-7 + T7489
18.1
31.4
20
16.7
20.4
106.6
IL-7 + T7811
13.9
26.2
46.8
17.9
19.2
124
IL-7 + T7812
15.1
24.4
88.4
20.6
26.6
175.1
*ND = not determined (culture exhausted)
The specification is most thoroughly understood in light of the teachings of the references cited within the specification which are hereby incorporated by reference. The embodiments within the specification provide an illustration of embodiments of the invention and should not be construed to limit the scope of the invention. The skilled artisan readily recognizes that many other embodiments are encompassed by the invention.
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11452762
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immunex corporation
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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/ 69.1
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Mar 31st, 2022 02:17PM
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Mar 31st, 2022 02:17PM
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Amgen
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:amgn
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Amgen
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Feb 24th, 2009 12:00AM
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Oct 22nd, 2004 12:00AM
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https://www.uspto.gov?id=US07495086-20090224
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TWEAK receptor
|
The present invention provides the TWEAK receptor and methods for identifying and using agonists and antagonists of the TWEAK receptor. In particular, the invention provides methods of screening for agonists and antagonists and for treating diseases or conditions mediated by angiogenesis, such as solid tumors and vascular deficiencies of cardiac or peripheral tissue.
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7495086
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1. An isolated monoclonal antibody that binds specifically to a polypeptide consisting of residues 34 to 68 of SEQ ID NO:4, wherein said antibody inhibits angiogenesis.
2. The isolated monoclonal antibody of claim 1 wherein said antibody is conjugated to a radioisotope, a plant-derived toxin, a fungus-derived toxin, a bacterial-derived toxin, ricin A, or diphtheria toxin.
3. The isolated monoclonal antibody of claim 1, wherein said antibody is conjugated to a detectable marker.
4. The isolated monoclonal antibody of claim 3, wherein said detectable marker is a radioisotope, antigenic, or colorimetric.
5. The isolated monoclonal antibody of claim 1 or antigen binding fragment thereof, wherein said antibody is selected from the group consisting of:
a) an intact human antibody;
b) a human antibody fragment;
c) an intact chimeric antibody;
d) a chimeric antibody fragment;
e) an intact humanized antibody;
f) a humanized antibody fragment;
g) a Fab fragment;
h) an Fv fragment;
i) an F(ab')2 fragment; and
j) a single chain antibody.
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5
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REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. patent application Ser. No. 10/862,109, filed Jun. 4, 2004, which is a continuation of U.S. patent application Ser. No. 10/754,847, filed Jan. 8, 2004, which is a divisional of U.S. patent application Ser. No. 09/883,777, filed Jun. 18, 2001, now U.S. Pat. No. 6,727,225, which is a continuation-in-part of International Application Number PCT/US00/34755, filed 19 Dec. 2000, and is a continuation-in-part of U.S. patent application Ser. No. 09/742,454 , filed Dec. 19, 2000 now U.S. Pat. No. 6,824,773, , both of which claim the benefit of U.S. Provisional Application Ser. No. 60/172,878, filed 20 Dec. 1999, and U.S. Provisional Application Ser. No. 60/203,347, filed 10 May 2000. The above-identified applications are incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates to the discovery of the functional TWEAK receptor (TWEAKR) for the TWEAK protein. More particularly, the invention relates to the use of TWEAKR agonists and antagonists in methods of treatment, and to screening methods based on TWEAKR and the TWEAK-TWEAKR interaction.
BACKGROUND
Angiogenesis is a multi-step developmental process that results in the formation of new blood vessels off of existing vessels. This spatially and temporally regulated process involves loosening of matrix contacts and support cell interactions in the existing vessels by proteases, followed by coordinated movement, morphological alteration, and proliferation of the smooth muscle and endothelial cells of the existing vessel. The nascent cells then extend into the target tissue followed by cell-cell interactions in which the endothelial cells form tubes which the smooth muscle cells surround. In a coordinated fashion, extracellular matrix proteins of the vessel are secreted and peri-endothelial support cells are recruited to support and maintain structural integrity (see, e.g., Daniel et al., Ann. Rev. Physiol. 2000(62):649, 2000). Angiogenesis plays important roles in both normal and pathological physiology.
Under normal physiological conditions, angiogenesis is involved in fetal and embryonic development, wound healing, organ regeneration, and female reproductive remodeling processes including formation of the endometrium, corpus luteum, and placenta. Angiogenesis is stringently regulated under normal conditions, especially in adult animals, and perturbation of the regulatory controls can lead to pathological angiogenesis.
Pathological angiogenesis has been implicated in the manifestation and/or progression of inflammatory diseases, certain eye disorders, and cancer. In particular, several lines of evidence support the concept that angiogenesis is essential for the growth and persistence of solid tumors and their metastases (see, e.g., Folkman, N. Engl. J. Med. 285:1182, 1971; Folkman et al., Nature 339:58, 1989; Kim et al., Nature 362:841, 1993; Hori et al., Cancer Res., 51:6180, 1991). Angiogenesis inhibitors are therefore useful for the prevention (e.g., treatment of premalignant conditions), intervention (e.g., treatment of small tumors), and regression (e.g., treatment of large tumors) of cancers (see, e.g., Bergers et al., Science 284:808, 1999).
The TWEAK protein, which has also been called TREPA and Apo3L, is a member of the tumor necrosis factor (TNF) family and is expressed in a wide variety of human tissues (Chicheportiche et al., J. Biol. Chem., 272(51):32401, 1997; see also Wiley, PCT Publication No. WO 98/35061, 13 Aug. 1998). Like most TNF family members, TWEAK is a Type II membrane protein with an extracellular C-terminal domain. Although TWEAK was originally described as a weak inducer of apoptosis, this induction of cell death was later shown to be indirect (Schneider et al., Eur. J. Immunol. 29:1785, 1999).
Lynch et al. demonstrated that TWEAK directly induces endothelial cell proliferation and angiogenesis (J. Biol. Chem., 274(13):8455, 1999). Picomolar concentrations of recombinant soluble TWEAK induce proliferation in multiple endothelial cell lines and in aortic smooth muscle cells, and reduce the requirement for serum and growth factors in culture. Moreover, TWEAK induces a strong angiogenic response in a rat corneal pocket assay. Since TNF family members initiate biological responses by signaling through members of the TNF receptor family, there has been great interest in identifying and characterizing a TWEAKR.
Marsters et al. reported that TWEAK binds to and signals through a death-domain containing receptor known variously as DR3, Apo3, WSL-1, TRAMP, or LARD (Marsters et al., Current Biology 8(9):525, 1998). Schneider et al., however, showed that TWEAK binds to and signals in Kym-1 cells but that Kym-1 cells do not express the receptor DR3 (Schneider et al., Eur. J. Immunol. 29:1785, 1999). These results suggest the existence of a yet to be identified TWEAK receptor.
Because TWEAK induces angiogenesis in vivo, there is a particular need to identify the major functional TWEAKR. Once identified, TWEAKR may be used to screen for and develop TWEAKR agonists and antagonists for the modulation of angiogenesis and the treatment of human disease.
There is a need for additional compositions and methods of modulating angiogenesis for the prevention, abrogation, and mitigation of disease.
SUMMARY OF THE INVENTION
The present invention is based upon the identification and biological characterization of the major functional TWEAK receptor (TWEAKR). As described below, cDNA encoding the TWEAKR was molecularly cloned from a human endothelial cell expression library.
Although DNA and deduced amino acid sequences corresponding to the TWEAKR identified herein have been reported (see, e.g., Kato et al., PCT Publication No. WO 98/55508, 10 Dec. 1998 and Incyte, PCT Publication No. WO 99/61471, 2 Dec. 1999), it was not heretofore appreciated that these sequences encode a receptor for TWEAK or that the encoded polypeptide, fragments, agonists, or antagonists thereof can be used to modulating angiogenesis. Similarly, investigators have recently claimed methods of making and using TWEAKR antagonists to treat immunological disorders, but without identifying the major TWEAKR or its role in angiogenesis (Rennert, PCT Publication No. WO 00/42073, 20 Jul. 2000). These deficiencies have been addressed, as described herein, by identification of the major TWEAKR and characterization of its biological activities. The identification of TWEAKR has led to the development of compositions for the modulation of angiogenesis, and also provides screening tools for the identification of diagnostics and therapeutics.
In one aspect, the present invention provides a polypeptide consisting of: a) SEQ ID NO:23, b) SEQ ID NO:28, c) SEQ ID NO:29, d) SEQ ID NO:30, e) a plurality of sequences, wherein each sequence is independently selected from sequences a)-d), f) e) and one or more linker sequences, g) a sequence selected from the group consisting of a), b), c), d), e), and f), and further comprising a multimerization domain, or h) a sequence selected from the group consisting of a), b), c), d), e), f), and g), and further comprising at least one sequence of amino acids that is not identical to a subsequence of contiguous amino acids found within SEQ ID NO:4, wherein said polypeptide binds to human TWEAK. In one embodiment, said multimerization domain is selected from the group consisting of an Fc domain and a leucine zipper. In another embodiment, said polypeptide does not comprise a subsequence of contiguous amino acids that is identical to the subsequence of contiguous amino acids from residue 36 to residue 67 of SEQ ID NO:4. In another embodiment, said polypeptide consists of: a) SEQ ID NO:23, b) SEQ ID NO:28, c) SEQ ID NO:29, or d) SEQ ID NO:30.
In another aspect, the present invention provides a polynucleotide comprising a sequence encoding said polypeptide. In one embodiment, the invention provides a plasmid comprising said polynucleotide. In another embodiment, said plasmid is an expression vector.
In another aspect, the present invention provides a cell comprising said plasmid. In one embodiment, said cell comprises said expression vector. In another embodiment, the present invention provides a method of making a polypeptide, comprising incubating said cell under conditions that allow said expression vector to express said polypeptide.
In another aspect, the present invention provides a pharmaceutical composition comprising said polypeptide and an excipient or diluent.
In another aspect, the present invention provides a method of inhibiting binding of TWEAK to a TWEAK receptor in a subject in need of such treatment comprising administering to said subject an inhibition-effective amount of said polypeptide. In one embodiment, said subject is a human. In another embodiment, said subject has a disease or condition mediated by angiogenesis. In another embodiment, said disease or condition is characterized by ocular neovascularization. In another embodiment, said disease or condition is a malignant or metastatic condition. In another embodiment, said malignant or metastatic condition is a solid tumor. In another embodiment, said method further comprises treating the subject with radiation. In another embodiment, said method further comprises treating said subject with a second chemotherapeutic agent. In another embodiment, said second chemotherapeutic agent is selected from the group consisting of alkylating agents, antimetabolites, vinca alkaloids and other plant-derived chemotherapeutics, nitrosoureas, antitumor antibiotics, antitumor enzymes, topoisomerase inhibitors, platinum analogs, adrenocortical suppressants, hormones, hormone agonists, hormone antagonists, antibodies, immunotherapeutics, blood cell factors, radiotherapeutics, and biological response modifiers. In another embodiment, said second chemotherapeutic agent is selected from the group consisting of cisplatin, cyclophosphamide, mechloretamine, melphalan, bleomycin, carboplatin, fluorouracil, 5-fluorodeoxyuridine, methotrexate, taxol, asparaginase, vincristine, and vinblastine, lymphokines, cytokines, interleukins, interferons, alpha interferon, beta interferon, delta interferon, TNF, chlorambucil, busulfan, carmustine, lomustine, semustine, streptozocin, dacarbazine, cytarabine, mercaptopurine, thioguanine, vindesine, etoposide, teniposide, dactinomycin, daunorubicin, doxorubicin, plicamycin, mitomycin, L-asparaginase, hydroxyurea, methylhydrazine, mitotane, tamoxifen, and fluoxymesterone. In another embodiment, said second chemotherapeutic agent is selected from the group consisting of Flt3 ligand, CD40 ligand, interleukin-2, interleukin-12, 4-1BB ligand, anti-4-1BB antibodies, TNF antagonists and TNF receptor antagonists, TRAIL, CD148 agonists, VEGF antagonists, VEGF receptor antagonists, and Tek antagonists.
In another aspect, the present invention provides an isolated antibody that binds specifically to a mature form of the TWEAK receptor and to a polypeptide consisting of the sequence of SEQ ID NO:28. In one embodiment, said antibody binds specifically to a polypeptide consisting of the sequence of SEQ ID NO:29. In another embodiment, said antibody binds specifically to a polypeptide consisting of residues 28 to 80 of SEQ ID NO:4. In another embodiment, said antibody inhibits angiogenesis. In another embodiment, said antibody promotes angiogenesis. In another embodiment, said antibody is conjugated to a radioisotope, a plant-derived toxin, a fungus-derived toxin, a bacterial-derived toxin, ricin A, or diphtheria toxin. In another embodiment, said antibody is conjugated to a detectable marker. In another embodiment, said detectable marker is a radioisotope, antigenic, or colorimetric. In another embodiment, said antibody is selected from the group consisting of: a) an intact human antibody; b) a human antibody fragment; c) an intact chimeric antibody; d) a chimeric antibody fragment; e) an intact humanized antibody; f) a humanized antibody fragment; g) a Fab fragment; h) an Fv fragment; i) an F(ab′)2 fragment; and j) a single chain antibody.
In another aspect, the present invention provides a nucleic acid comprising a sequence encoding: a) a heavy chain of an antibody, b) a heavy chain variable region of an antibody, c) a light chain of an antibody, d) a light chain variable region of an antibody, e) a) and c), or f) b) and d), wherein each of said antibodies in a)-f) is said antibody. In one embodiment, the present invention provides a plasmid comprising said nucleic acid. In another embodiment, said plasmid is an expression vector.
In another aspect, the present invention provides a cell comprising said plasmid. In one embodiment, the invention provides a cell comprising said expression vector. In another embodiment, the present invention provides a method of making an antibody, or an antibody derivative, comprising incubating said cell under conditions that allow said expression vector to express said antibody or antibody derivative. In another embodiment, said cell is a mammalian cell. In another embodiment, said mammalian cell is a Chinese Hamster Ovary cell.
In another aspect, the present invention provides a method of inhibiting binding of TWEAK to a TWEAK receptor in a subject in need of such treatment comprising administering to said subject an inhibition-effective amount of said antibody. In one embodiment, said subject is a human. In another embodiment, said subject has a disease or condition mediated by angiogenesis. In another embodiment, said disease or condition is characterized by ocular neovascularization. In another embodiment, said disease or condition is a malignant or metastatic condition. In another embodiment, said malignant or metastatic condition is a solid tumor. In another embodiment, said method further comprises treating the subject with radiation. In another embodiment, said method further comprises treating the subject with a second chemotherapeutic agent. In another embodiment, said second chemotherapeutic agent is selected from the group consisting of alkylating agents, antimetabolites, vinca alkaloids and other plant-derived chemotherapeutics, nitrosoureas, antitumor antibiotics, antitumor enzymes, topoisomerase inhibitors, platinum analogs, adrenocortical suppressants, hormones, hormone agonists, hormone antagonists, antibodies, immunotherapeutics, blood cell factors, radiotherapeutics, and biological response modifiers. In another embodiment, said second chemotherapeutic agent is selected from the group consisting of cisplatin, cyclophosphamide, mechloretamine, melphalan, bleomycin, carboplatin, fluorouracil, 5-fluorodeoxyuridine, methotrexate, taxol, asparaginase, vincristine, and vinblastine, lymphokines, cytokines, interleukins, interferons, alpha interferon, beta interferon, delta interferon, TNF, chlorambucil, busulfan, carmustine, lomustine, semustine, streptozocin, dacarbazine, cytarabine, mercaptopurine, thioguanine, vindesine, etoposide, teniposide, dactinomycin, daunorubicin, doxorubicin, plicamycin, mitomycin, L-asparaginase, hydroxyurea, methylhydrazine, mitotane, tamoxifen, and fluoxymesterone. In another embodiment, said second chemotherapeutic agent is selected from the group consisting of Flt3 ligand, CD40 ligand, interleukin-2, interleukin-12, 4-1BB ligand, anti-4-1BB antibodies, TNF antagonists and TNF receptor antagonists, TRAIL, CD148 agonists, VEGF antagonists, VEGF receptor antagonists, and Tek antagonists. In another embodiment, the present invention provides a method of promoting angiogenesis in a subject in need of such treatment comprising administering to said subject an angiogenesis promoting-effective amount of said antibody. In another embodiment, said subject has an ischemic condition, and said method treats said ischemic condition. In another embodiment, said ischemic condition is ischemia of the heart, liver, or brain. In another embodiment, said subject has a wound, and said method treats said wound. In another embodiment, said subject has organ damage, and said method treats said organ damage. In another embodiment, said subject has coronary or peripheral atherosclerosis, and said method treats said coronary or peripheral atherosclerosis.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows a sequence alignment of the human and murine TWEAKR polypeptide sequences. The top sequence is the murine TWEAKR polypeptide (SEQ ID NO:5), and the bottom sequence is the human TWEAKR polypeptide (SEQ ID NO:4).
FIG. 1B shows the primary amino acid sequence of TweakR (SEQ ID NQ:4) showing major features. The leader sequence Is underlined. The arrow indicates the predicted site of cleavage of the leader seqaence. The region of TNF family receptor homology is shown in bold. The predicted transmembrane region is doubly underlined. The putative c TRAF binding motif in the cytoplasmic domain is boxed.
FIG. 2 shows the effect of TWEAKR-Fc on PMA-induced HRMEC wound closure.
FIG. 3 shows the effect of TWEAKR-Fc on EGF-induced HRMEC wound closure.
FIG. 4 shows the effect of human TWEAKR-Fc on TWEAK-induced (100 ng/ml) HUVEC proliferation.
FIG. 5 shows the effect of human TWEAKR-Fc on FGF-2-induced (10 ng/ml) HUVEC proliferation.
FIG. 6 collectively shows a scatchard analysis of TWEAK-TWEAKR interaction. CV-1 cells transfected with human full-length TWEAK mixed 1:30 with Raji cells and incubated with various concentrations of 125I-labeled TWEAKR-Fc. A) Shows scatchard representation of specific binding. B) Plot of competitive inhibition of unlabeled vs. 125I-labeled TWEAKR-Fc.
FIG. 7 collectively shows that human TWEAKR-Fc inhibits PMA- or EGF-stimulated endothelial cell migration in vitro. A, Shows that TWEAKR-Fc inhibited the PMA-stimulated migration rate to baseline at concentrations greater than or equal to 1.5 μg/ml, whereas huIgG at similar concentrations did not effect migration. Neither huIgG nor TweakR-Fc increased or decreased the basal migration rate when added to the cultures alone. B, Human TweakR-Fc inhibits EGF-induced endothelial cell migration. TweakR-Fc inhibited EGF-stimulated migration to basal levels at 5 μg/ml. Partial inhibition of EGF-induced migration was also observed at huTweakR/Fc concentrations of 500 ng/ml and 1.5 μg/ml.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to TWEAKR and methods for identifying and using agonists and antagonists of TWEAKR. The invention provides methods of screening for agonists and antagonists and for treating diseases or conditions mediated by angiogenesis.
Abbreviations and Terminology Used in the Specification
“4-1BB” and “4-1BB ligand” (4-1BB-L) are polypeptides described, inter alia, in U.S. Pat. No. 5,674,704, including soluble forms thereof.
“CD40 ligand” (CD40L) is a polypeptide described, inter alia, in U.S. Pat. No. 5,716,805, including soluble forms thereof.
“Flt3L” is Flt3 ligand, a polypeptide described, inter alia, in U.S. Pat. No. 5,554,512, including soluble forms thereof.
“RTKs” are receptor tyrosine kinases.
“Tek,” which has also been called Tie2 and ork, is an RTK that is predominantly expressed in vascular endothelium. The molecular cloning of human Tek (ork) has been described by Ziegler, U.S. Pat. No. 5,447,860. “Tek antagonists” are described, inter alia, in Cerretti et al., PCT Publication No. WO 00/75323, 14 Dec. 2000.
“TRAIL” is TNF-related apoptosis-inducing ligand, a type II transmembrane polypeptide in the TNF family described, inter alia, in U.S. Pat. No. 5,763,223, including soluble forms thereof.
“VEGF” is vascular endothelial growth factor, also known as VPF or vascular permeability factor.
Soluble TWEAKR Polypeptides
As described in the examples below, the native human TWEAKR cDNA has a sequence as set forth in SEQ ID NO:3, which encodes a 129 residue polypeptide (SEQ ID NO:4). Several distinct regions can be discerned within the TWEAKR polypeptides of the invention (see, e.g., FIG. 1). A leader sequence, also called a signal peptide, is present in these polypeptides. The leader sequence present in the full-length TWEAKR polypeptide of the invention is predicted to include amino acids 1-27 of SEQ ID NO:4. The signal peptide cleavage site for TWEAKR polypeptide can be predicted using a computer algorithm. However, one of skill in the art will recognize that the cleavage site of the signal sequence may vary depending upon a number of factors including the organism in which the polypeptide is expressed. Accordingly, the N-terminus of a mature form of a TWEAKR polypeptide of the invention may vary by about 2 to 5 amino acids. Thus, a mature form of a TWEAKR polypeptide of the invention may include at its N-terminus amino acid 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 of SEQ ID NO:4. Accordingly, a mature form of a TWEAKR polypeptide includes amino acids 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 to about amino acid 129 (or, in the case of a soluble polypeptide, an amino acid between 68 and 80) of SEQ ID NO:4. The extracellular region of a TWEAKR polypeptide is located at about amino acids 28 to 80 of SEQ ID NO:4. The transmembrane region for the TWEAKR polypeptide is located at about amino acids 81 to 100 of SEQ ID NO:4. The intracellular region is located at about amino acids 101 to 129 of SEQ ID NO:4. A putative TWEAKR sequence has also been reported by Kato et al., PCT Publication No. WO 98/55508, 10 Dec. 1998 and by Incyte, PCT Publication No. WO 99/61471, 2 Dec. 1999. As used herein, “TWEAKR” includes polypeptides having these sequences, and in particular comprising amino acids 28 to x1 of SEQ ID NO:4, wherein x1 is an amino acid from 68 to 80 of SEQ ID NO:4, as well as naturally occurring variants thereof.
The invention provides both full-length and mature forms of TWEAKR polypeptides. Full-length polypeptides are those having the complete primary amino acid sequence of the polypeptide as initially translated (see, e.g., SEQ ID NO:4). The amino acid sequences of full-length polypeptides can be obtained, for example, by translation of the complete open reading frame (“ORF”) of a cDNA molecule (see, e.g., SEQ ID NO:3). An example of a full length TWEAKR polypeptide of the invention comprises a sequence as set forth in SEQ ID NO:4 from amino acid 1 to amino acid 129. Such a full length polypeptide is contemplated to include, for example, the signal peptide comprising amino acid 1 to about amino acid 27 of SEQ ID NO:4.
The “mature form” of a polypeptide refers to a polypeptide that has undergone post-translational processing steps, if any, such as, for example, cleavage of the signal sequence or proteolytic cleavage to remove a prodomain. Multiple mature forms of a particular full-length polypeptide may be produced, for example, by imprecise cleavage of the signal sequence, or by differential regulation of proteases that cleave the polypeptide. The mature form(s) of such polypeptide may be obtained by expression, in a suitable mammalian cell or other host cell, of a polynucleotide that encodes the full-length polypeptide. The sequence of the mature form of the polypeptide may also be determinable from the amino acid sequence of the full-length form, through identification of signal sequences or protease cleavage sites (e.g., a protease cleavage site is predicted between the Gly-Glu residues at positions 27 and 28 of SEQ ID NO:4). An example of a mature form of a TWEAKR polypeptide of the invention comprises a sequence as set forth in SEQ ID NO:4 from about amino acid 28 to amino acid 129.
In another aspect of the invention, fragments of TWEAKR polypeptides are provided. Such fragments include, for example, the various domains identified above (e.g., the signal sequence domain, the extracellular domain, the transmembrane domain, and the cytoplasmic or intracellular domain). Such domains find use in recombinant DNA techniques (e.g., creation of fusion proteins and the like). Of particular interest is the extracellular domain of TWEAKR from about amino acid 28 to amino acid 68 to 80 of SEQ ID NO:4. The extracellular domain of TWEAKR comprises a soluble TWEAKR amino acid sequence. Also included in the invention are fragments of the extracellular domain that retain a biological activity of TWEAKR. For example, a biological activity associated with a TWEAKR extracellular domain or fragment thereof includes the ability to bind to TWEAK.
In one aspect of the invention, a soluble TWEAKR fragment is used as a TWEAKR antagonist to inhibit angiogenesis and/or to inhibit the binding of TWEAK ligand to TWEAKR. A TWEAKR fragment preferably comprises the extracellular domain of TWEAKR or a portion thereof as described herein such that the fragment comprises a soluble TWEAKR amino acid sequence. Accordingly, a TWEAKR antagonist includes, for example, a soluble portion of the TWEAKR molecule, preferably a portion of the extracellular domain of TWEAKR, either alone, fused, or conjugated to one or more other molecules or polypeptides (e.g., an Fc, leucine zipper polypeptide, or a peptide linker). For example, the invention provides compositions and fusion proteins that comprise at least one soluble TWEAKR polypeptide domain (e.g., the extracellular domain).
Soluble polypeptides are capable of being secreted from the cells in which they are expressed. The use of soluble forms of polypeptides is advantageous for certain applications. Purification of the polypeptides from recombinant host cells is facilitated since the polypeptides are secreted, and soluble proteins are generally suited for parenteral administration. A secreted soluble polypeptide may be identified (and distinguished from its non-soluble membrane-bound counterparts) by separating intact cells which express the desired polypeptide from the culture medium, e.g., by centrifugation, and assaying the medium (supernatant) for the presence of the desired polypeptide. The presence of the desired polypeptide in the medium indicates that the polypeptide was secreted from the cells and thus is a soluble form of the polypeptide. Soluble polypeptides may be prepared by any of a number of conventional techniques. A polynucleotide encoding a desired soluble polypeptide may be subcloned into an expression vector for production of the polypeptide, or the desired encoding polynucleotide or soluble polypeptide may be chemically synthesized. Examples of a nucleic acid molecule encoding a soluble TWEAKR polypeptide comprises about nucleotides 134 to 256, 134 to 262, 134 to 289, and 134 to 292 of SEQ ID NO:3. In one embodiment, D-amino acids are substituted for the naturally occurring L-amino acids. D-amino acids provide improved stability under in vivo conditions. In addition, due to the size of the extracellular domain or soluble polypeptide sequence of the invention it may be advantageous to synthesize the polypeptide using D-amino acids. It will be recognized that the polypeptide of the invention can be synthesized such that the polypeptide comprises a combination of L- and D-amino acids.
Soluble TWEAKR polypeptides comprise all or part of the TWEAKR extracellular domain, but generally lack the transmembrane domain that would cause retention of the polypeptide at the cell surface. Soluble polypeptides may include part of the transmembrane domain or all or part of the cytoplasmic domain so long as the polypeptide is secreted from the cell in which it is produced. Soluble TWEAKR polypeptides advantageously comprise a native or heterologous signal peptide when initially synthesized, to promote secretion from the cell, but the signal sequence is cleaved upon secretion. The term “TWEAKR extracellular domain” is intended to encompass all or part of the native TWEAKR extracellular domain, as well as related forms including but not limited to: (a) fragments, (b) variants, (c) derivatives, and (d) fusion polypeptides. The ability of these related forms to inhibit angiogenesis or other TWEAKR-mediated responses may be determined in vitro or in vivo, using methods such as those exemplified below or using other assays known in the art. Examples of soluble TWEAKR polypeptides are provided below. In some embodiments of the present invention a multimeric form of a soluble TWEAKR polypeptide (“soluble TWEAKR multimer”) is used as an antagonist to block the binding of TWEAK to TWEAKR, to inhibit angiogenesis or other TWEAKR-mediated responses.
Soluble TWEAKR multimers are covalently-linked or non-covalently-linked multimers, including dimers, trimers, or higher multimers. Multimers may be linked by disulfide bonds formed between cysteine residues on different soluble TWEAKR polypeptides. One embodiment of the invention is directed to multimers comprising multiple soluble TWEAKR polypeptides joined via covalent or non-covalent interactions between peptide moieties fused to the soluble TWEAKR polypeptides. Such peptides may be peptide linkers (spacers), or peptides that have the property of promoting multimerization. In one embodiment peptide linkers are fused to the C-terminal end of a first soluble TWEAKR molecule and the N-terminal end of a second soluble TWEAKR molecule. This structure may be repeated multiple times such that at least one, preferably 2, 3, 4, or more soluble TWEAKR polypeptides are linked to one another via peptide linkers at their respective termini. For example, a polypeptide of the invention comprises a sequence Z1-X-Z2, wherein Z1 and Z2 are each individually a polypeptide consisting of amino acid 28 to x1 of SEQ ID NO:4, wherein x1 is an amino acid from about 68 to 80 of SEQ ID NO:4 and X is a peptide linker. In another embodiment, the polypeptide comprises Z1-X-Z2(-X-Z)n, wherein ‘n’ is any integer, but is preferably 1 or 2. In a further embodiment, the peptide linkers should be of sufficient length to allow the soluble TWEAKR polypeptide to form bonds with adjacent soluble TWEAKR polypeptides. Examples of peptide linkers include—Gly-Gly—, GGGGS (SEQ ID NO:10) (GGGGS)n (SEQ ID NO:11), GKSSGSGSESKS (SEQ ID NO:12), GSTSGSGKSSEGKG (SEQ ID NO:13), GSTSGSGKSSEGSGSTKG (SEQ ID NO:14), GSTSGSGKPGSGEGSTKG (SEQ ID NO:15), or EGKSSGSGSESKEF (SEQ ID NO:16). Linking moieties are described, for example, in Huston et al., PNAS 85:5879-5883, 1988; Whitlow et al., Protein Engineering 6:989-995, 1993; and Newton et al., Biochemistry 35:545-553, 1996. Other suitable peptide linkers are those described in U.S. Pat. Nos. 4,751,180 and 4,935,233, which are hereby incorporated by reference. A polynucleotide encoding a desired peptide linker can be inserted between, and in the same reading frame as, a polynucleotide encoding a soluble TWEAKR polypeptide, using any suitable conventional technique. In particular embodiments, a fusion polypeptide comprises from two to four soluble TWEAKR polypeptides separated by peptide linkers.
In some embodiments, a soluble TWEAKR multimer is prepared using polypeptides derived from immunoglobulins. Preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., by Ashkenazi et al. (Proc. Natl. Acad. Sci. USA 88:10535, 1991); Byrn et al. (Nature 344:677, 1990); and Hollenbaugh and Aruffo (“Construction of Immunoglobulin Fusion Proteins”, in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11, 1992).
One preferred embodiment of the present invention is directed to a TWEAKR-Fc dimer comprising two fusion proteins created by fusing a soluble TWEAKR to an Fc polypeptide. A gene fusion encoding the TWEAKR-Fc fusion protein is inserted into an appropriate expression vector. TWEAKR-Fc fusion proteins are expressed in host cells transformed with the recombinant expression vector, and allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between the Fc moieties to yield divalent soluble TWEAKR. The term “Fc polypeptide” as used herein includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Truncated forms of such polypeptides containing the hinge region that promotes dimerization are also included.
One suitable Fc polypeptide, described in PCT application WO 93/10151, is a single chain polypeptide extending from the N-terminal hinge region to the native C-terminus of the Fc region of a human IgG1 antibody. Another useful Fc polypeptide is the Fc mutein described in U.S. Pat. No. 5,457,035 and by Baum et al., EMBO J. 13:3992, 1994. The amino acid sequence of this mutein is identical to that of the native Fc sequence presented in WO 93/10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 has been changed from Gly to Ala. The mutein exhibits reduced affinity for Fc receptors. Fusion polypeptides comprising Fc moieties, and multimers formed therefrom, offer an advantage of facile purification by affinity chromatography over Protein A or Protein G columns, and Fc fusion polypeptides may provide a longer in vivo half life, which is useful in therapeutic applications, than unmodified polypeptides.
In other embodiments, a soluble TWEAKR polypeptide may be substituted for the variable portion of an antibody heavy or light chain. If fusion proteins are made with both heavy and light chains of an antibody, it is possible to form a soluble TWEAKR multimer with as many as four soluble TWEAKR polypeptides.
Another method for preparing soluble TWEAKR multimers involves use of a leucine zipper domain. Leucine zipper domains are peptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, 1988), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric proteins are described in PCT application WO 94/10308, and the leucine zipper derived from lung surfactant protein D (SPD) described in Hoppe et al. FEBS Lett. 344:191, 1994. The use of a modified leucine zipper that allows for stable trimerization of a heterologous protein fused thereto is described in Fanslow et al., Semin. Immunol. 6:267, 1994. Recombinant fusion proteins comprising a soluble TWEAKR polypeptide fused to a leucine zipper peptide are expressed in suitable host cells, and the soluble TWEAKR multimer that forms is recovered from the culture supernatant.
For some applications, the soluble TWEAKR multimers of the present invention are believed to provide certain advantages over the use of monomeric forms. Fc fusion polypeptides, for example, typically exhibit an increased in vivo half-life as compared to an unmodified polypeptide.
The present invention encompasses the use of various forms of soluble TWEAKR multimers that retain the ability to inhibit angiogenesis or other TWEAKR-mediated responses. The term “soluble TWEAKR multimer” is intended to encompass multimers containing all or part of the native TWEAKR extracellular domain, as well as related forms including, but not limited to, multimers of: (a) fragments, (b) variants, (c) derivatives, and (d) fusion polypeptides of soluble TWEAKR. The ability of these related forms to inhibit angiogenesis or other TWEAKR-mediated responses may be determined in vitro or in vivo, using methods such as those exemplified in the examples or using other assays known in the art.
Among the soluble TWEAKR polypeptides and soluble TWEAKR multimers useful in practicing the present invention are TWEAKR variants that retain the ability to bind ligand (e.g., TWEAK) and/or inhibit angiogenesis or other TWEAKR-mediated responses. Such TWEAKR variants include polypeptides that are substantially homologous to native TWEAKR, but which have an amino acid sequence different from that of a native TWEAKR because of one or more deletions, insertions or substitutions. Particular embodiments include, but are not limited to, TWEAKR polypeptides that comprise from one to ten deletions, insertions or substitutions of amino acid residues, when compared to a native TWEAKR sequence. Included as variants of TWEAKR polypeptides are those variants that are naturally occurring, such as allelic forms and alternatively spliced forms, as well as variants that have been constructed by modifying the amino acid sequence of a TWEAKR polypeptide or the nucleotide sequence of a nucleic acid encoding a TWEAKR polypeptide.
Generally, substitutions for one or more amino acids present in the native polypeptide should be made conservatively. Examples of conservative substitutions include substitution of amino acids outside of the active domain(s), and substitution of amino acids that do not alter the secondary and/or tertiary structure of TWEAKR. Additional examples include substituting one aliphatic residue for another, such as Ile, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gln and Asn, or substitutions of one aromatic residue for another, such as Phe, Trp, or Tyr for one another. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are known in the art.
In some preferred embodiments, the TWEAKR variant is at least about 70% identical in amino acid sequence to the amino acid sequence of native TWEAKR; in some preferred embodiments, the TWEAKR variant is at least about 80% identical in amino acid sequence to the amino acid sequence of native TWEAKR. In some more preferred embodiments, the TWEAKR variant is at least about 90% identical in amino acid sequence to the amino acid sequence of native TWEAKR; in some more preferred embodiments, the TWEAKR variant is at least about 95% identical in amino acid sequence to the amino acid sequence of native TWEAKR. In some most preferred embodiments, the TWEAKR variant is at least about 98% identical in amino acid sequence to the amino acid sequence of native TWEAKR; in some most preferred embodiments, the TWEAKR variant is at least about 99% identical in amino acid sequence to the amino acid sequence of native TWEAKR. Percent identity, in the case of both polypeptides and nucleic acids, may be determined by visual inspection. Percent identity may also be determined using the alignment method of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970) as revised by Smith and Waterman (Adv. Appl. Math 2:482, 1981). Preferably, percent identity is determined by using a computer program, for example, the GAP computer program version 10.x available from the Genetics Computer Group (GCG; Madison, Wis., see also Devereux et al., Nucl. Acids Res. 12:387, 1984). The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979 for amino acids; (2) a penalty of 30 (amino acids) or 50 (nucleotides) for each gap and an additional 1 (amino acids) or 3 (nucleotides) penalty for each symbol in each gap; (3) no penalty for end gaps; and (4) no maximum penalty for long gaps. Other programs used by one skilled in the art of sequence comparison may also be used. For fragments of TWEAKR, the percent identity is calculated based on that portion of TWEAKR that is present in the fragment.
The present invention further encompasses the use of soluble TWEAKR polypeptides with or without associated native-pattern glycosylation. TWEAKR expressed in yeast or mammalian expression systems (e.g., COS-1 or COS-7 cells) may be similar to or significantly different from a native TWEAKR polypeptide in molecular weight and glycosylation pattern, depending upon the choice of expression system. Expression of TWEAKR polypeptides in bacterial expression systems, such as E. coli, provides non-glycosylated molecules. Different host cells may also process polypeptides differentially, resulting in heterogeneous mixtures of polypeptides with variable N- or C-termini.
The primary amino acid structure of soluble TWEAKR polypeptides may be modified to create derivatives by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives of TWEAKR may be prepared by linking particular functional groups to TWEAKR amino acid side chains or at the N-terminus or C-terminus of a TWEAKR polypeptide. In addition, TWEAKR can be complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, and the like, or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application.
Fusion polypeptides of soluble TWEAKR that are useful in practicing the invention also include covalent or aggregative conjugates of a TWEAKR polypeptide with other polypeptides added to provide novel polyfunctional entities.
TWEAKR Antibodies
One aspect of the present invention relates to the antigenic epitopes of the TWEAKR extracellular domain. Such epitopes are useful for raising antibodies, and in particular the blocking monoclonal antibodies described in more detail below. Such epitopes or variants thereof can be produced using techniques well known in the art such as solid-phase synthesis, chemical or enzymatic cleavage of a polypeptide, or using recombinant DNA technology.
The claimed invention encompasses compositions and uses of antibodies that are immunoreactive with TWEAKR polypeptides. Such antibodies “bind specifically” to TWEAKR polypeptides, meaning that they bind via antigen-binding sites of the antibody as compared to non-specific binding interactions. The terms “antibody” and “antibodies” are used herein in their broadest sense, and include, without limitation, intact monoclonal and polyclonal antibodies as well as fragments such as Fv, Fab, and F(ab′)2 fragments, single-chain antibodies such as scFv, and various chain combinations. The antibodies of the present invention are preferably humanized, and more preferably human. The antibodies may be prepared using a variety of well-known methods including, without limitation, immunization of animals having native or transgenic immune repertoires, phage display, hybridoma and recombinant cell culture, and transgenic plant and animal bioreactors.
Both polyclonal and monoclonal antibodies may be prepared by conventional techniques. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).
Hybridoma cell lines that produce monoclonal antibodies specific for the polypeptides of the invention are also contemplated herein. Such hybridomas may be produced and identified by conventional techniques. One method for producing such a hybridoma cell line comprises immunizing an animal with a polypeptide, harvesting spleen cells from the immunized animal, fusing said spleen cells to a myeloma cell line, thereby generating hybridoma cells, and identifying a hybridoma cell line that produces a monoclonal antibody that binds the polypeptide. The monoclonal antibodies produced by hybridomas may be recovered by conventional techniques.
The monoclonal antibodies of the present invention include chimeric antibodies, e.g., “humanized” versions of antibodies originally produced in mice or other non-human species. A humanized antibody is an engineered antibody that typically comprises the variable region of a non-human (e.g., murine) antibody, or at least complementarity determining regions (CDRs) thereof, and the remaining immunoglobulin portions derived from a human antibody. Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick et al. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS 14:139, May, 1993). Such humanized antibodies may be prepared by known techniques and offer the advantage of reduced immunogenicity when the antibodies are administered to humans.
Procedures that have been developed for generating human antibodies in non-human animals may be employed in producing antibodies of the present invention. The antibodies may be partially human or preferably completely human. For example, transgenic mice into which genetic material encoding one or more human immunoglobulin chains has been introduced may be employed. Such mice may be genetically altered in a variety of ways. The genetic manipulation may result in human immunoglobulin polypeptide chains replacing endogenous immunoglobulin chains in at least some, and preferably virtually all, antibodies produced by the animal upon immunization.
Mice in which one or more endogenous immunoglobulin genes have been inactivated by various means have been prepared and are commercially available from, for example, Medarex Inc. (Princeton, N.J.) and Abgenix Inc. (Fremont, Calif.). Human immunoglobulin genes have been introduced into the mice to replace the inactivated mouse genes. Antibodies produced in the animals incorporate human immunoglobulin polypeptide chains encoded by the human genetic material introduced into the animal. Examples of techniques for the production and use of such transgenic animals to make antibodies (which are sometimes called “transgenic antibodies”) are described in U.S. Pat. Nos. 5,814,318, 5,569,825, and 5,545,806, which are incorporated by reference herein.
Inhibitory Antisense, Ribozyme and Triple Helix Approaches
Modulation of angiogenesis in a tissue or group of cells may also be ameliorated by decreasing the level of TWEAKR gene expression and/or TWEAKR-ligand interaction by using TWEAKR or ligand gene sequences in conjunction with well-known antisense, gene “knock-out,” ribozyme and/or triple helix methods to decrease the level of TWEAKR or ligand gene expression. Among the compounds that may exhibit the ability to modulate the activity, expression or synthesis of the TWEAKR or a ligand gene, including the ability to modulate angiogenesis, are antisense, ribozyme, and triple helix molecules. Such molecules may be designed to reduce or inhibit either unimpaired, or if appropriate, mutant target gene activity. Techniques for the production and use of such molecules are known to those of skill in the art.
Recombinant Production of TWEAKR Polypeptides
TWEAKR polypeptides, including soluble TWEAKR polypeptides, fragments, and fusion polypeptides, used in the present invention may be prepared using a recombinant expression system. Host cells transformed with a recombinant expression vector or a polynucleotide encoding a TWEAKR polypeptide, soluble TWEAKR polypeptide, or fusion polypeptide (“recombinant host cells”) are cultured under conditions that promote expression of TWEAKR molecule and the TWEAKR molecule is recovered. TWEAKR polypeptides can also be produced in transgenic plants or animals, or by chemical synthesis.
TWEAKR Nucleic Acids
The invention encompasses nucleic acid molecules (i.e., polynucleotides) encoding a TWEAKR polypeptide used in the invention, including: (a) nucleic acids that encode residues from about 28 to x1 (x1 is residue 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80) of SEQ ID NO:4 and fragments thereof that bind TWEAK; (b) nucleic acids that are at least 70%, 80%, 90%, 95%, 98%, or 99% identical to a nucleic acid of (a), and which encode a polypeptide capable of binding TWEAK; and (c) nucleic acids that hybridize at moderate stringency to a nucleic acid of (a), and which encode a polypeptide capable of binding TWEAK.
Due to degeneracy of the genetic code, there can be considerable variation in nucleotide sequences encoding the same amino acid sequence. Included as embodiments of the invention are nucleic acid sequences capable of hybridizing under moderately stringent conditions (e.g., prewashing solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridization conditions of 50° C., 5×SSC, overnight) to the DNA sequences encoding TWEAKR. The skilled artisan can determine additional combinations of salt and temperature that constitute moderate hybridization stringency (see also, Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1982; and Ausubel, Current Protocols in Molecular Biology, Wiley and Sons, 1989 and later versions, which are incorporated herein by reference). Conditions of higher stringency include higher temperatures for hybridization and post-hybridization washes, and/or lower salt concentration. Percent identity of nucleic acids may be determined using the methods described above for polypeptides, e.g., by methods including visual inspection and/or the use of computer programs such as GAP.
Any suitable expression system may be employed for the production of recombinant TWEAKR. Recombinant expression vectors include nucleic acids (e.g., DNA or RNA) encoding a TWEAKR polypeptide operably linked to suitable transcriptional and translational regulatory nucleotide sequences, such as those derived from a mammalian, microbial, viral, or insect gene. A TWEAKR nucleic acid molecule and a regulatory sequence are operably linked when the regulatory sequence functionally relates to the TWEAKR nucleic acid molecule. Thus, a regulatory sequence such as a promoter is operably linked to a TWEAKR nucleic acid molecule if the promoter controls the transcription of the TWEAKR nucleic acid molecule. Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, an mRNA ribosomal binding site, internal ribosome entry sites (IRES), and appropriate sequences which control transcription and translation initiation and termination. A sequence encoding an appropriate signal peptide (native or heterologous) can be incorporated into expression vectors. A DNA sequence for a signal peptide (referred to by a variety of names including secretory leader, leader peptide, or leader) may be fused in frame to the TWEAKR sequence so that the TWEAKR polypeptide is initially translated as a fusion protein comprising the signal peptide. A signal peptide that is functional in the intended host cells promotes extracellular secretion of the TWEAKR polypeptide. The signal peptide is cleaved from the TWEAKR polypeptide upon secretion of TWEAKR from the cell.
Suitable host cells for expression of TWEAKR polypeptides include prokaryotes, yeast, and higher eukaryotic cells, including insect and mammalian cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, insect, and mammalian cellular hosts are described, for example, in Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985.
Prokaryotes include gram negative or gram positive organisms, for example, E. coli or Bacilli. Suitable prokaryotic host cells for transformation include, for example, E. coli, Bacillus subtilis, Salmonella typhimurium, and various other species within the genera Pseudomonas, Streptomyces , and Staphylococcus. In a prokaryotic host cell, such as E. coli, TWEAKR polypeptides may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-terminal Met may be cleaved from the expressed recombinant polypeptide.
Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker gene(s). A phenotypic selectable marker gene is, for example, a gene encoding a protein that confers antibiotic resistance or that supplies an autotrophic requirement. Examples of useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids such as the cloning vector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells. An appropriate promoter and a TWEAKR DNA sequence are inserted into the pBR322 vector. Other commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA).
Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include β-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615, 1978; Goeddel et al., Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, 1980; EP-A-36776) and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412, 1982). A particularly useful prokaryotic host cell expression system employs a phage λ PL promoter and a cI857ts thermolabile repressor sequence. Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the λ PL promoter include plasmid pHUB2 (resident in E. coli strain JMB9, ATCC 37092) and pPLc28 (resident in E. coli RR1, ATCC 53082).
The stability of TWEAKR lends itself to expression in prokaryotic systems. For example, TweakR ligand binding domain will spontaneously re-fold into an active conformation even after being reduced and boiled in SDS loading buffer.
TWEAKR polypeptides may also be expressed in yeast host cells, preferably from the Saccharomyces genus (e.g., S. cerevisiae). Other genera of yeast, such as Pichia or Kluyveromyces, may also be employed. Yeast vectors will often contain an origin of replication sequence from a 2μ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; Holland et al., Biochem. 17:4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phospho-glucose isomerase, and glucokinase. Other suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EPA-73,657. Another alternative is the glucose-repressible ADH2 promoter described by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature 300:724, 1982). Shuttle vectors replicable in both yeast and E. coli may be constructed by inserting DNA sequences from pBR322 for selection and replication in E. coli (Ampr gene and origin of replication) into the above-described yeast vectors.
The yeast α-factor leader sequence may be employed to direct secretion of recombinant polypeptides. The α-factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. See, e.g., Kurjan et al., Cell 30:933, 1982; Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330, 1984. Other leader sequences suitable for facilitating secretion of recombinant polypeptides from yeast hosts are known to those of skill in the art. A leader sequence may be modified near its 3′ end to contain one or more restriction sites. This will facilitate fusion of the leader sequence to the structural gene.
Yeast transformation protocols are known to those of skill in the art. One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp+ transformants in a selective medium, wherein the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 μg/ml adenine and 20 μg/ml uracil.
Yeast host cells transformed by vectors containing an ADH2 promoter sequence may be grown for inducing expression in a “rich” medium. An example of a rich medium is one consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/ml uracil. Derepression of the ADH2 promoter occurs when glucose is exhausted from the medium.
Insect host cell culture systems also may be employed to express recombinant TWEAKR polypeptides, including soluble TWEAKR polypeptides. Bacculovirus systems for production of heterologous polypeptides in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47, 1988.
Mammalian cells are typically used as host cells. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) as described by McMahan et al. (EMBO J. 10:2821, 1991). For the production of therapeutic polypeptides it is particularly advantageous to use a mammalian host cell line which has been adapted to grow in media that does not contain animal proteins.
Established methods for introducing DNA into mammalian cells have been described (Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990, pp. 15-69). Additional protocols using commercially available reagents, such as Lipofectamine (Gibco/BRL) or Lipofectamine-Plus, can be used to transfect cells (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987). In addition, electroporation can be used to transfect mammalian cells using conventional procedures, such as those in Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1-3, Cold Spring Harbor Laboratory Press, 1989. Selection of stable transformants can be performed using methods known in the art, such as, for example, resistance to cytotoxic drugs. Kaufman et al., Meth. in Enzymology 185:487, 1990, describes several selection schemes, such as dihydrofolate reductase (DHFR) resistance. A suitable host strain for DHFR selection can be CHO strain DX-B11, which is deficient in DHFR (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216, 1980). A plasmid expressing the DHFR cDNA can be introduced into strain DX-B11, and only cells that contain the plasmid can grow in the appropriate selective media. Other examples of selectable markers that can be incorporated into an expression vector include cDNAs conferring resistance to antibiotics, such as G418 and hygromycin B. Cells harboring the vector can be selected on the basis of resistance to these compounds.
Transcriptional and translational control sequences for mammalian host cell expression vectors can be excised from viral genomes. Commonly used promoter sequences and enhancer sequences are derived from polyoma virus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites can be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell. Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment, which can also contain a viral origin of replication (Fiers et al., Nature 273:113, 1978; Kaufman, Meth. in Enzymology, 1990). Smaller or larger SV40 fragments can also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the Bgl I site located in the SV40 viral origin of replication site is included.
Additional control sequences shown to improve expression of heterologous genes from mammalian expression vectors include such elements as the expression augmenting sequence element (EASE) derived from CHO cells (Morris et al., Animal Cell Technology, 1997, pp. 529-534) and the tripartite leader (TPL) and VA gene RNAs from Adenovirus 2 (Gingeras et al., J. Biol. Chem. 257:13475, 1982). The internal ribosome entry site (IRES) sequences of viral origin allows dicistronic mRNAs to be translated efficiently (Oh and Sarnow, Current Opinion in Genetics and Development 3:295, 1993; Ramesh et al., Nucleic Acids Research 24:2697, 1996). Expression of a heterologous cDNA as part of a dicistronic mRNA followed by the gene for a selectable marker (e.g. DHFR) has been shown to improve transfectability of the host and expression of the heterologous cDNA (Kaufman, Meth; in Enzymology, 1990). Exemplary expression vectors that employ dicistronic mRNAs are pTR-DC/GFP described by Mosser et al., Biotechniques 22:150, 1997, and p2A5I described by Morris et al., Animal Cell Technology, 1997, pp. 529-534.
A useful high expression vector, pCAVNOT, has been described by Mosley et al., Cell 59:335, 1989. Other expression vectors for use in mammalian host cells can be constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983). A useful system for stable high level expression of mammalian cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al. (Mol. Immunol. 23:935, 1986). A useful high expression vector, PMLSV N1/N4, described by Cosman et al., Nature 312:768, 1984, has been deposited as ATCC 39890. Additional useful mammalian expression vectors are known in the art.
Regarding signal peptides that may be employed in producing TWEAKR polypeptides, the native TWEAKR signal peptide may used or it may be replaced by a heterologous signal peptide or leader sequence, if desired. The choice of signal peptide or leader may depend on factors such as the type of host cells in which the recombinant TWEAKR is to be produced. Examples of heterologous signal peptides that are functional in mammalian host cells include the signal sequence for interleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195, the signal sequence for interleukin-2 receptor described in Cosman et al., Nature 312:768, 1984; the interleukin-4 receptor signal peptide described in EP 367,566; the type I interleukin-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; and the type II interleukin-1 receptor signal peptide described in EP 460,846.
Using the techniques of recombinant DNA including mutagenesis, directed evolution, and the polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 6,171,820 and 6,238,884), the skilled artisan can produce DNA sequences that encode TWEAKR polypeptides comprising various additions or substitutions of amino acid residues or sequences, or deletions of terminal or internal residues or sequences, including TWEAKR fragments, variants, derivatives, and fusion polypeptides.
Transgenic animals, including mice, goats, sheep, and pigs, transgenic plants, including tobacco, tomato, legumes, grasses, and grains, and transgenic algae may also be used as bioreactors for the production of TWEAKR polypeptides, including soluble TWEAKR polypeptides. In the case of transgenic animals, it is particularly advantageous to construct a chimeric DNA including a TWEAKR coding sequence operably linked to cis-acting regulatory sequences that promote expression of the soluble TWEAKR in milk and/or other body fluids (see, e.g., U.S. Pat. Nos. 5,843,705; 5,880,327). In the case of transgenic plants it is particularly advantageous to produce TWEAKR in a particular cell type, tissue, or organ (see, e.g., U.S. Pat. Nos. 5,639,947; 5,889,189).
The skilled artisan will recognize that the procedure for purifying expressed soluble TWEAKR polypeptides will vary according to the host system employed, and whether or not the recombinant polypeptide is secreted. Soluble TWEAKR polypeptides may be purified using methods known in the art, including one or more concentration, salting-out, ion exchange, hydrophobic interaction, affinity purification, HPLC, or size exclusion chromatography steps. Fusion polypeptides comprising Fc moieties (and multimers formed therefrom) offer the advantage of facile purification by affinity chromatography over Protein A or Protein G columns.
Methods of Treatment
Described below are methods and compositions employing the TWEAK receptor or ligand, or the genes encoding the TWEAK receptor or ligand, to promote or suppress angiogenesis in a subject, a target tissue, or a group of cells. The terms “treat,” “treating,” “treatment,” “therapy,” “therapeutic,” and the like are intended to include preventative therapy, prophylactic therapy, ameliorative therapy, and curative therapy. By “subject” is meant any mammal (e.g., bovine, equine, porcine, canine, feline, and primates), but preferably is a human.
The disclosed polypeptides, compositions, and methods are used to inhibit angiogenesis, modulate cell migration and/or proliferation, or other TWEAKR-mediated responses in a subject in need of such treatment. The term “TWEAKR-mediated response” includes any cellular, physiological, or other biological response that is caused at least in part by the binding of TWEAK ligand to TWEAKR, or which may be inhibited or suppressed, in whole or in part, by blocking TWEAK from binding to TWEAKR. The treatment is advantageously administered in order to prevent the onset or the recurrence of a disease or condition mediated by angiogenesis, or to treat a subject that has a disease or condition mediated by angiogenesis. Diseases and conditions mediated by angiogenesis include but are not limited to ocular disorders, malignant and metastatic conditions, and inflammatory diseases. In some instances stimulation of a TWEAK-TWEAKR response may be beneficial (e.g., during tissue or would repair). Accordingly, administration of TWEAK or TWEAKR in such tissue or cells may be used to promote wound repair.
Among the ocular disorders that can be treated according to the present invention are eye diseases characterized by ocular neovascularization including, but not limited to, diabetic retinopathy (a major complication of diabetes), retinopathy of prematurity (this devastating eye condition, that frequently leads to chronic vision problems and carries a high risk of blindness, is a severe complication during the care of premature infants), neovascular glaucoma, retinoblastoma, retrolental fibroplasia, rubeosis, uveitis, macular degeneration, and corneal graft neovascularization. Other eye inflammatory diseases, ocular tumors, and diseases associated with choroidal or iris neovascularization can also be treated according to the present invention.
The present invention can also be used to treat cell proliferative disorders, including malignant and metastatic conditions such as solid tumors. Solid tumors include both primary and metastatic sarcomas and carcinomas.
The present invention can also be used to treat inflammatory diseases including, but not limited to, arthritis, rheumatism, and psoriasis.
Other diseases and conditions that can be treated according to the present invention include benign tumors and preneoplastic conditions, myocardial angiogenesis, hemophilic joints, scleroderma, vascular adhesions, atherosclerotic plaque neovascularization, telangiectasia, and wound granulation.
Disease states that are angiogenic-dependent include coronary or peripheral atherosclerosis and ischemia of any tissue or organ, including the heart, liver, brain, and the like. These types of diseases can be treated by compositions that promote angiogenesis.
In addition to polypeptides comprising a fragment of TWEAKR extracellular domain, soluble TWEAKR multimers, and antibodies that bind to the TWEAKR extracellular domain, other forms of TWEAKR antagonists can also be administered to achieve a therapeutic effect. Examples of other forms of TWEAKR antagonists include other antibodies such as antibodies against TWEAK, antisense nucleic acids, ribozymes, muteins, aptamers, and small molecules directed against TWEAKR or against TWEAK.
The methods according to the present invention can be tested in in vivo animal models to confirm the desired prophylactic or therapeutic activity, as well as to determine the optimal therapeutic dosage, prior to administration to humans.
The amount of a particular TWEAKR antagonist that will be effective in a particular method of treatment depends upon age, type and severity of the condition to be treated, body weight, desired duration of treatment, method of administration, and other parameters. Effective dosages are determined by a physician or other qualified medical professional. Typical effective dosages are about 0.01 mg/kg to about 100 mg/kg body weight. In some preferred embodiments the dosage is about 0.1-50 mg/kg; in some preferred embodiments the dosage is about 0.5-10 mg/kg. The dosage for local administration is typically lower than for systemic administration. In some embodiments a single administration is sufficient; in some embodiments the TWEAKR antagonist is administered as multiple doses over one or more days.
The TWEAKR antagonists are typically administered in the form of a pharmaceutical composition comprising one or more pharmacologically acceptable carriers. Pharmaceutically acceptable carriers include diluents, fillers, adjuvants, excipients, and vehicles that are pharmaceutically acceptable for the route of administration, and may be aqueous or oleaginous suspensions formulated using suitable dispersing, wetting, and suspending agents.
Pharmaceutically acceptable carriers are generally sterile and free of pyrogenic agents, and may include water, oils, solvents, salts, sugars and other carbohydrates, emulsifying agents, buffering agents, antimicrobial agents, and chelating agents. The particular pharmaceutically acceptable carrier and the ratio of active compound to carrier are determined by the solubility and chemical properties of the composition, the mode of administration, and standard pharmaceutical practice.
The compositions as described herein may be contained in a vial, bottle, tube, syringe inhaler or other container for single or multiple administrations. Such containers may be made of glass or a polymer material such as polypropylene, polyethylene, or polyvinylchloride, for example. Preferred containers may include a seal or other closure system, such as a rubber stopper that may be penetrated by a needle in order to withdraw a single dose and then re-seal upon removal of the needle. All such containers for injectable liquids, lyophilized formulations, reconstituted lyophilized formulations or reconstitutable powders for injection known in the art or for the administration of aerosolized compositions are contemplated for use in the presently disclosed compositions and methods.
The TWEAKR antagonists are administered to the subject in a manner appropriate to the indication. Thus, for example, a TWEAKR antagonist, or a pharmaceutical composition thereof, may be administered by intravenous, transdermal, intradermal, intraperitoneal, intramuscular, intranasal, epidural, oral, topical, subcutaneous, intracavity, sustained release from implants, peristaltic routes, or by any other suitable technique. Parenteral administration is preferred.
In certain embodiments of the claimed invention, the treatment further comprises treating a subject with one or more additional agents such as additional chemotherapeutic agents. The additional chemotherapeutic agent(s) may be administered prior to, concurrently with, or following the administration of the TWEAKR antagonist. The use of more than one chemotherapeutic agent is particularly advantageous when the subject that is being treated has a solid tumor. In some embodiments of the claimed invention, the treatment further comprises treating the subject with radiation. Radiation, including brachytherapy and teletherapy, may be administered prior to, concurrently with, or following the administration of the second chemotherapeutic agent(s) and/or TWEAKR antagonist.
When the subject that is being treated has a solid tumor, the method preferably includes the administration of, in addition to a TWEAKR antagonist, one or more chemotherapeutic agents selected from the group consisting of alkylating agents, antimetabolites, vinca alkaloids and other plant-derived chemotherapeutics, nitrosoureas, antitumor antibiotics, antitumor enzymes, topoisomerase inhibitors, platinum analogs, adrenocortical suppressants, hormones, hormone agonists and antagonists, antibodies, immunotherapeutics, blood cell factors, radiotherapeutics, and biological response modifiers.
In some preferred embodiments the method includes administration of, in addition to a TWEAKR antagonist, one or more chemotherapeutic agents selected from the group consisting of cisplatin, cyclophosphamide, mechloretamine, melphalan, bleomycin, carboplatin, fluorouracil, 5-fluorodeoxyuridine, methotrexate, taxol, asparaginase, vincristine, and vinblastine, lymphokines and cytokines such as interleukins, interferons (including alpha, beta, or delta), and TNF, chlorambucil, busulfan, carmustine, lomustine, semustine, streptozocin, dacarbazine, cytarabine, mercaptopurine, thioguanine, vindesine, etoposide, teniposide, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, L-asparaginase, hydroxyurea, methylhydrazine, mitotane, tamoxifen, and fluoxymesterone.
In some preferred embodiments the method includes administration of, in addition to a TWEAKR antagonist, one or more chemotherapeutic agents, including various soluble forms thereof, selected from the group consisting of Flt3 ligand, CD40 ligand, interleukin-2, interleukin-12, 4-1BB ligand, anti-4-1BB antibodies, TNF antagonists and TNF receptor antagonists, TRAIL, VEGF antagonists, VEGF receptor (including VEGF-R1 and VEGF-R2, also known as Flt1 and Flk1 or KDR) antagonists, Tek antagonists, and CD148 (also referred to as DEP-1, ECRTP, and PTPRJ, see Takahashi et al., J. Am. Soc. Nephrol. 10:2135-45, 1999) agonists. In some preferred embodiments the TWEAKR antagonists of the invention are used as a component of, or in combination with, “metronomic therapy,” such as that described by Browder et al. and Klement et al. (Cancer Research 60:1878, 2000; J. Clin. Invest. 105(8):R15, 2000; see also Barinaga, Science 288:245, 2000).
The polypeptides, compositions, and methods of the present invention may be used as a first line treatment, for the treatment of residual disease following primary therapy, or as an adjunct to other therapies including chemotherapy, surgery, radiation, and other therapeutic methods known in the art.
When the nucleic acid sequences of the present invention are delivered according to the methods disclosed herein, it is advantageous to use a delivery mechanism so that the sequences will be incorporated into a cell for expression. Delivery systems that may advantageously be employed in the contemplated methods include the use of, for example, viral delivery systems such as retroviral and adenoviral vectors, as well as non-viral delivery systems. Such delivery systems are well known by those skilled in the art.
Methods of Screening
TWEAKR as described herein may be used in a variety of methods of screening to isolate, for example, TWEAKR agonists and antagonists. TWEAKR agonists are compounds that promote the biological activity of TWEAKR and TWEAKR antagonists are compounds that inhibit the biological activity of TWEAKR. Compounds identified via the following screening assays can be used in compositions and methods for modulating angiogenesis to treat a variety of disease states. The present invention provides methods of screening for compounds that (1) modulate TWEAKR or ligand gene expression in a target tissue or cell, (2) modulate the TWEAKR-ligand interaction to regulate angiogenesis; (3) bind to the TWEAKR or ligand to influence angiogenesis; or (4) interfere with or regulate the bound TWEAKR-ligand complex's influence on downstream events such as angiogenesis. Accordingly, the polypeptides, and fragments thereof, of the invention can be used to regulate, influence, and modulate (i.e., increase or decrease) a biological activity associated with interaction of TWEAK or TWEAKR with its cognate.
The present invention contemplates the use of assays that are designed to identify compounds that modulate the activity of a TWEAKR or ligand gene (e.g., modulate the level of TWEAKR or TWEAK gene expression and/or modulate the level of TWEAKR or TWEAK gene product activity). Assays may additionally be utilized that identify compounds that bind to TWEAKR or TWEAK gene regulatory sequences (e.g., promoter sequences; see e.g., Platt, J. Biol. Chem. 269, 28558-28562, 1994), and that may modulate the level of TWEAKR or TWEAK gene expression.
Such an assay may involve, for example, the use of a control system, in which transcription and translation of the TWEAKR or ligand gene occurs, in comparison to a system including a test agent suspected of influencing normal transcription or translation of a TWEAKR or ligand gene. For example, one could determine the rate of TWEAKR RNA produced by cardiac cells, and use this to determine if a test agent influences that rate. To assess the influence of a test agent suspected to influence this normal rate of transcription, one would first determine the rate of TWEAKR RNA production in a cardiac cell culture by, for example, Northern Blotting. One could then administer the test agent to a cardiac cell culture under otherwise identical conditions as the control culture. The rate of TWEAKR RNA in the culture treated with the test agent could be determined by, for example, Northern Blotting, and compared to the rate of TWEAKR RNA produced by the control culture cells. An increase in the TWEAKR RNA in the cells contacted with the test agent relative to control cells is indicative of a stimulator of TWEAKR gene transcription in cardiac cells, while a decrease is indicative of an inhibitor of TWEAKR gene transcription in cardiac cells.
There are a variety of other methods that can be used to determine the level of TWEAKR or ligand gene expression as well, and may further be used in assays to determine the influence of a test agent on the level of TWEAKR or ligand gene expression. For example; RNA from a cell type or tissue known, or suspected, to express the TWEAK receptor or ligand gene, such as cardiac tissue, may be isolated and tested utilizing hybridization or PCR techniques. The isolated cells can be derived from cell culture or from a subject. The analysis of cells taken from culture may be a necessary step in the assessment of cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the TWEAK receptor or ligand gene. Such analyses may reveal both quantitative and qualitative aspects of the expression pattern of the TWEAK receptor or ligand gene, including activation or inactivation of TWEAKR or ligand gene expression.
In one embodiment of such a detection scheme, a cDNA molecule is synthesized from an RNA molecule of interest (e.g., by reverse transcription of the RNA molecule into cDNA). A sequence within the cDNA is then used as the template for a nucleic acid amplification reaction, such as a PCR amplification reaction, or the like. The nucleic acid reagents used as synthesis initiation reagents (e.g., primers) in the reverse transcription and nucleic acid amplification steps of this method are chosen from among the TWEAKR or ligand gene nucleic acid segments described above. The preferred lengths of such nucleic acid reagents are at least 9-30 nucleotides. For detection of the amplified product, the nucleic acid amplification may be performed using radioactively or non-radioactively labeled nucleotides. Alternatively, enough amplified product may be made such that the product may be visualized by standard ethidium bromide staining or by utilizing any other suitable nucleic acid staining method.
Additionally, it is possible to perform such TWEAKR or ligand gene expression assays “in situ,” i.e., directly upon tissue sections (fixed and/or frozen) of subject tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. TWEAKR or ligand gene nucleic acid segments described above can be used as probes and/or primers for such in situ procedures (see, for example, Nuovo, G. J., 1992, “PCR In situ Hybridization: Protocols And Applications,” Raven Press, NY).
Compounds identified via assays such as those described herein may be useful, for example, in modulating angiogenesis influenced by TWEAKR or TWEAKR-ligand interaction. Such methods of stimulating or inhibiting TWEAK- or TWEAKR-influenced angiogenesis are discussed herein.
Alternatively, assay systems may be designed to identify compounds capable of binding the TWEAKR or ligand polypeptide of the invention and thereby influencing angiogenesis resulting from this interaction. Compounds identified may be useful, for example, in modulating the vascularization of target tissues or cells, may be utilized in screens for identifying compounds that disrupt normal TWEAKR-ligand interactions, or may in themselves disrupt such interactions.
The principle of the assays used to identify compounds that bind to the TWEAK receptor or ligand involves preparing a reaction mixture of the TWEAK receptor or ligand and the test agent under conditions and for a time sufficient to allow the two components to interact or bind, thus forming a complex that can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways. For example, one method to conduct such an assay screening for compounds that bind to the TWEAK receptor, would involve anchoring the TWEAK receptor or the test substance onto a solid phase and detecting TWEAKR/test agent complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the TWEAK receptor may be anchored onto a solid surface, and the test agent, which is not anchored, may be labeled, either directly or indirectly. Alternatively, these same methods could be used to screen for test agents that bind to the TWEAK ligand rather than receptor.
In practice, microtiter plates may conveniently be utilized as the solid phase. The anchored component may be immobilized by non-covalent or covalent attachments. Non-covalent attachment may be accomplished by simply coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the protein to be immobilized may be used to anchor the protein to the solid surface. The surfaces may be prepared in advance and stored.
In order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the previously non-immobilized component (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody).
Alternatively, a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for the TWEAK receptor or ligand or the test agent to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes.
Those compounds identified as binding agents for either the TWEAK receptor or the TWEAK ligand may further be assessed for their ability to interfere with TWEAKR-ligand interaction, as described below, and thereby suppress or promote angiogenesis resulting from TWEAKR-ligand interaction. Such compounds may then be used therapeutically to stimulate or inhibit angiogenesis.
The TWEAKR and ligand polypeptides of the present invention may also be used in a screening assay to identify compounds and small molecules which specifically interact with the disclosed TWEAK receptor or ligand to either inhibit (antagonize) or enhance (agonize) interaction between these molecules. Thus, for example, polypeptides of the invention may be used to identify antagonists and agonists from cells, cell-free preparations, chemical libraries, and natural product mixtures. The antagonists and agonists may be natural or modified substrates, ligands, enzymes, receptors, and the like, of the polypeptides of the instant invention, or may be structural or functional mimetics of the polypeptides. Potential antagonists of the TWEAKR-ligand interaction of the instant invention may include small molecules, polypeptides, peptides, peptidomimetics, and antibodies that bind to and occupy a binding site of the polypeptides, causing them to be unavailable to interact and therefore preventing their normal ability to modulate angiogenesis. Other potential antagonists are antisense molecules that may hybridize to mRNA in vivo and block translation of the mRNA into the polypeptides of the instant invention. Potential agonists include small molecules, polypeptides, peptides, peptidomimetics, and antibodies that bind to the instant TWEAKR and TWEAK polypeptides and influence angiogenesis as caused by the disclosed interactions of the TWEAKR and TWEAK polypeptides of the instant invention.
Small molecule agonists and antagonists are usually less than 10K molecular weight and may possess a number of physiochemical and pharmacological properties that enhance cell penetration, resist degradation and prolong their physiological half-lives. (Gibbs, “Pharmaceutical Research in Molecular Oncology,” Cell, Vol. 79, 1994). Antibodies, which include intact molecules as well as fragments such as Fab and F(ab′)2 fragments, may be used to bind to and inhibit the polypeptides of the instant invention by blocking the commencement of a signaling cascade. It is preferable that the antibodies are humanized, and more preferable that the antibodies are human. The antibodies of the present invention may be prepared by any of a variety of well-known methods. Alternatively, antibodies may bind to and activate the polypeptides of the instant by mimicking the interaction of a polypeptide of the invention with its cognate. One of skill in the art using the assay methods and techniques herein can determine whether an antibody is an antagonist or agonist.
Specific screening methods are known in the art and many are extensively incorporated in high throughput test systems so that large numbers of test agents can be screened within a short amount of time. The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, cell based assays, and the like. These assay formats are well known in the art. The screening assays of the present invention are amenable to screening of chemical libraries and are suitable for the identification of small molecule drug candidates, antibodies, peptides and other antagonists and agonists.
One embodiment of a method for identifying molecules which antagonize or inhibit TWEAKR-ligand interaction involves adding a candidate molecule to a medium which contains cells that express the polypeptides of the instant invention; changing the conditions of said medium so that, but for the presence of the candidate molecule, the polypeptides would interact; and observing the binding and inhibition of angiogenesis. Binding of the TWEAK receptor and ligand can be determined according to competitive binding assays outlined above, and well known in the art. The angiogenic effect of this binding can be determined via cell proliferation assays such as, for example, cell density assays, corneal pocket assays, or other cell proliferation assays that are also well-known in the art. The activity of the cells contacted with the candidate molecule may then be compared with the identical cells, which were not contacted, and agonists and antagonists of the TWEAK polypeptide interactions of the instant invention may be identified. The measurement of biological activity may be performed by a number of well-known methods such as measuring the amount of protein present (e.g. an Enzyme-Linked Immunosorbent Assay (ELISA)), production of cytokines (e.g., IL-8 and IL-6; see, e.g., Saas et al., Glia 32(1):102-7, 2000), or of the protein's activity. A decrease in biological stimulation or activation would indicate an antagonist. An increase would indicate an agonist.
Screening assays can further be designed to find molecules that mimic the biological activity resulting from the TWEAKR and/or TWEAK polypeptide interactions of the instant invention. Molecules which mimic the biological activity of a polypeptide may be useful for enhancing the biological activity of the polypeptide. To identify compounds for therapeutically active agents that mimic the biological activity of a polypeptide, it must first be determined whether a candidate molecule binds to the polypeptide. A binding candidate molecule is then added to a biological assay to determine its biological effects. The biological effects of the candidate molecule are then compared to those of the polypeptide.
Additionally, complex formation within reaction mixtures containing the test agent and normal TWEAKR or ligand gene protein may also be compared to complex formation within reaction mixtures containing the test agent and a mutant TWEAKR or ligand gene protein. This comparison may be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal TWEAKR or ligand gene proteins.
The assay for compounds that interfere with the interaction of the TWEAKR or ligand gene products and binding partners can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either TWEAKR or ligand gene product or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test agents that interfere with the interaction between the TWEAKR or ligand gene products and the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance; e.g., by adding the test substance to the reaction mixture prior to or simultaneously with the TWEAKR and ligand gene products. Alternatively, test agents that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test agent to the reaction mixture after complexes have been formed.
In a particular embodiment, the TWEAKR or ligand gene product can be prepared for immobilization using recombinant DNA techniques. For example, the TWEAKR or ligand coding region can be fused to a glutathione-S-transferase (GST) gene using a fusion vector, such as pGEX-5X-1, in such a manner that its binding activity is maintained in the resulting fusion protein. The interactive binding partner can be purified and used to raise a monoclonal antibody, using methods routinely practiced in the art. This antibody can be labeled with the radioactive isotope 125I, for example, by methods routinely practiced in the art. In a heterogeneous assay, e.g., the GST-TWEAKR or ligand fusion protein can be anchored to glutathione-agarose beads. The TWEAKR or ligand gene product can then be added in the presence or absence of the test agent in a manner that allows interaction and binding to occur. At the end of the reaction period, unbound material can be washed away, and the labeled monoclonal antibody can be added to the system and allowed to bind to the complexed components. The interaction between the TWEAKR and ligand gene products can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test agent will result in a decrease in measured radioactivity.
Alternatively, a GST-TWEAKR gene fusion protein and TWEAK ligand gene product (or vice versa) can be mixed together in liquid in the absence of the solid glutathione-agarose beads. The test agent can be added either during or after the species is allowed to interact. This mixture can then be added to the glutathione-agarose beads and unbound material is washed away. Again the extent of inhibition of the TWEAKR-ligand gene product interaction can be detected by adding the labeled antibody and measuring the radioactivity associated with the beads.
In another embodiment of the invention, these same techniques can be employed using peptide fragments that correspond to the binding domains of the TWEAKR and/or ligand protein, in place of one or both of the full-length proteins. Any number of methods routinely practiced in the art can be used to identify and isolate the binding sites. These methods include, but are not limited to, mutagenesis of the gene encoding one of the proteins and screening for disruption of binding in a co-immunoprecipitation assay. Compensating mutations in the gene encoding the second species in the complex can then be selected. Sequence analysis of the genes encoding the respective proteins will reveal the mutations that correspond to the region of the protein involved in interactive binding. Alternatively, one protein can be anchored to a solid surface using methods described above, and allowed to interact with and bind to its labeled binding partner, which has been treated with a proteolytic enzyme, such as trypsin. After washing, a short, labeled peptide comprising the binding domain may remain associated with the solid material, which can be isolated and identified by amino acid sequencing. Also, once the gene coding for the segments can be engineered to express peptide fragments of the protein, which can then be tested for binding activity and purified or synthesized.
As an example, and not by way of limitation, a TWEAKR or ligand gene product can be anchored to a solid material, as described above, by making a GST-TWEAKR or ligand fusion protein and allowing it to bind to glutathione agarose beads. The interactive binding partner obtained can be labeled with a radioactive isotope, such as 35S, and cleaved with a proteolytic enzyme such as trypsin. Cleavage products can then be added to the anchored GST-TWEAKR fusion protein or TWEAK ligand fusion protein and allowed to bind. After washing away unbound peptides, labeled bound material, representing the binding partner binding domain, can be eluted, purified, and analyzed for amino acid sequence by well-known methods. Peptides so identified can be produced synthetically or fused to appropriate facilitative proteins using recombinant DNA technology.
The TWEAKR-ligand interactions of the invention, in vivo, initiate a cascade of events that either stimulate or suppress angiogenesis in a target group of cells or tissues. Molecules, such as nucleic acid molecules, proteins, or small molecules may, in turn, influence this cascade. Compounds that disrupt the TWEAKR-ligand interaction may be useful in regulating angiogenesis.
The basic principle of the assay systems used to identify compounds that interfere with the angiogenic or anti-angiogenic effect of TWEAKR-ligand interaction involves preparing a reaction mixture containing the TWEAK receptor and ligand under conditions and for a time sufficient to allow the two to interact or bind, thus forming a complex. In order to test a compound for inhibitory activity of the effect of this interaction, the reaction mixture is prepared in the presence and absence of the test agent. The test agent may be initially included in the reaction mixture, or may be added at a time subsequent to the addition of the TWEAKR-ligand complex. Control reaction mixtures are incubated without the test agent or with a placebo. The inhibition or potentiation of any effect of the TWEAK complex on vascularization is then detected. Normal angiogenic response in the control reaction, but not in the reaction mixture containing the test agent, indicates that the compound interferes with the cascade of events initiated by the TWEAKR-ligand interaction. Enhanced angiogenesis in the test agents-containing culture indicates a stimulator of the TWEAKR-ligand complex effect.
In another embodiment, the techniques of rational drug design can be used to develop TWEAKR binding agents (e.g., agonist or antagonists of TWEAKR). The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact, e.g., substrates, binding agents, inhibitors, agonists, antagonists, and the like. The methods provided herein can be used to fashion or identify agents which are more active or stable forms of the polypeptide or which enhance or interfere with the function of a polypeptide in vivo (Hodgson J Biotechnology 9:19-21, 1991, incorporated herein by reference). In one approach, the three-dimensional structure of a TWEAKR polypeptide of the invention, a ligand or binding partner, or of a polypeptide-binding partner complex, is determined by x-ray crystallography, by nuclear magnetic resonance, or by computer homology modeling or, most typically, by a combination of these approaches. Both the shape and charges of the polypeptide are ascertained to elucidate the structure and to determine active site(s) or sites of interaction of the molecule. Relevant structural information is used to design analogous molecules, to identify efficient inhibitors, or to identify small molecules that may bind to a polypeptide of the invention. Useful examples of rational drug design may include molecules which have improved activity or stability as shown by Braxton and Wells (Biochemistry 31:7796-7801, 1992) or which act as inhibitors, agonists, or antagonists of native peptides as shown by Athauda et al. (J Biochem 113:742-746, 1993), incorporated herein by reference. The use of TWEAKR or TWEAK polypeptide structural information in molecular modeling software systems provides for the design of inhibitors or binding agents useful in modulating TWEAKR-TWEAK interactions or biological activity. A particular method of the invention comprises analyzing the three dimensional structure of TWEAK or TWEAKR polypeptides for likely binding/interaction sites of substrates or ligands, synthesizing a new molecule that incorporates a predictive reactive site, and assaying the new molecule as described further herein. Examples of algorithms, software, and methods for modeling substrates or binding agents based upon the three-dimensional structure of a protein are described in PCT publication WO107579A2, entitled “METHODS AND COMPOSITIONS FOR DETERMINING ENZYMATIC ACTIVITY,” the disclosure of which is incorporated herein.
EXAMPLES
The following examples are intended to illustrate particular embodiments and not to limit the scope of the invention.
Example 1
Identification of the TWEAK Receptor
Expression Cloning of TWEAKR cDNA
To clone TWEAKR cDNA, an expression vector encoding a growth hormone leader, a leucine zipper multimerization domain, and the C-terminal extracellular domain of human TWEAK (see Chicheportiche et al., J. Biol. Chem. 272(51):32401, 1997) was constructed. This expression vector, which was named pDC409-LZ-TWEAK, comprised the DNA sequence SEQ ID NO:1 and encoded the polypeptide SEQ ID NO:2. pDC409-LZ-TWEAK conditioned supernatants were produced by transient transfection into CV1-EBNA cells. These supernatants were incubated with magnetic beads coated with polyclonal goat anti-mouse antibody that had previously been incubated with a mouse monoclonal antibody against the leucine zipper. Control beads were produced by mixing the coated beads with supernatants from cells transfected with empty vector.
A monolayer of COS cells grown in a T175 flask was transfected with 15 μg of DNA pools of complexity of 100,000 from a human umbilical vein endothelial cell (HUVEC) cDNA expression library. After 2 days these cells were lifted from the flask, and incubated in 1.5 mls of binding media plus 5% non-fat dried milk for 3 hours at 4° C. on a rotator wheel. Cells were pre-cleared by adding control beads and rotated at 4° C. for an additional 45 minutes after which bead bound cells were removed with a magnet. Pre-clearing was repeated 2-3 times, then TWEAK coated beads were added to the cells and rotated 30 minutes at 4° C. Cells binding the TWEAK beads were separated by use of a magnet and washed 4× in phosphate buffered saline (PBS). Plasmid DNA was extracted from these cells by lysing in 0.1% SDS, and electroporating the supernatants in DH101B cells. Colonies were grown overnight on ampicilin selective media. Transformants were pooled and used as a source of plasmid DNA for a further round of panning. After 2 rounds of panning, positive clones were picked from the resulting pool based on their ability to bind TWEAK using a slide binding protocol. Slide binding was performed as described more fully below, with the exception that TWEAKR positive slides were detected by incubation with TWEAKR conditioned supernatants followed by incubation with 125I-labeled M15 anti-leucine zipper.
The human TWEAK receptor (also called TWEAKR) cDNA was determined to have the sequence SEQ ID NO:3, which encodes a 129 residue polypeptide (SEQ ID NO:4). Examination of the sequence predicts a polypeptide having an approximately 80 amino acid extracellular domain (residues 1-80 of SEQ ID NO:4, including the signal peptide, amino acids 1-27), an approximately 20 amino acid transmembrane domain (residues 81-100 of SEQ ID NO:4), and an approximately 29 amino acid intracellular domain (residues 101-129 of SEQ ID NO:4). TWEAKR is the smallest known TNF receptor family member. It has a single cysteine-rich repeat region in the extracellular domain, as compared to the 3-4 repeats of other TNF receptor family members. The TWEAKR polypeptide was previously described as a transmembrane protein encoded by a human liver cDNA clone (WO 98/55508, see also WO 99/61471), but had not been identified as the TWEAK receptor. A murine homolog, the FGF-inducible Fn14 (Meighan-Mantha et al., J. Biol. Chem. 274(46):33166, 1999), is approximately 82% identical to the human protein, as shown by the alignment in FIG. 1.
The newly identified TWEAK receptor was tested side by side with DR3 (which had been identified as the TWEAK receptor by Marsters et al., Current Biology 8:525, 1998) for the ability to bind to TWEAK.
TWEAKR Binds to TWEAK
Slides of COS cells were transfected with expression vectors containing TWEAKR, DR3, or vector without insert (control). After two days the cells were incubated with concentrated supernatants from CV-1 cells transfected with a vector encoding the leucine zipper TWEAK extracellular domain fusion protein. One hour later the cells were washed and probed with an 125I labeled antibody against the leucine-zipper domain. The slides were washed, fixed, and autoradiography was performed using x-ray film. The TWEAKR transfected cells bound significant amounts of TWEAK. TWEAK did not bind to the cells transfected with DR3 or the control cells. This experiment confirmed that the TWEAKR polypeptide identified in part A above, rather than DR3, is the major receptor for TWEAK. After discovery of the functional TWEAK receptor, other investigators also reported that DR3 is not the major receptor for TWEAK (Kaptein et al., FEBS Lett., 485(2-3):135, 2000). The TWEAK-TWEAKR binding interaction was further characterized by Scatchard analysis.
CV-1 cells were transfected with human full length TWEAK and mixed 1:30 with Raji cells, which do not express TWEAK. The cells were incubated with serial dilutions of 125-I labeled human TWEAKR-Fc for 2 hours at 4° C. Free and bound probe was separated by microfuging the samples through a phalate oil mixture in plastic tubes. Supernatants and pellets were gamma-counted. Scatchard analyses of TWEAK ligand binding the TWEAK receptor showed a binding affinity constant (Ka) of approximately 4.5×108 M−1.
The TWEAK Receptor is Strongly Expressed in Cardiac Tissue
To determine the expression pattern of the TWEAK receptor, Northern blot analyses were performed. Human multiple tissue northern blots were purchased from Clontech (Palo Alto, Calif.) and probed with 32P labeled random primed DNA from the TWEAKR coding region. The blots were washed and autoradiography was performed using x-ray film. Results showed that in the adult TWEAKR is strongly expressed in heart, placenta, and some skeletal muscle samples. Strong expression in heart tissue further supports the utility of TWEAKR in the diagnosis and treatment of cardiac disease. In contrast to the adult, the fetal tissues expressed TWEAKR more ubiquitously; TWEAKR transcripts were seen in the lung and liver.
Example 2
Preparation of TWEAKR Antagonists and Agonists
Because TWEAK induces angiogenesis, TWEAKR agonists (such as agonistic antibodies) may be used to promote angiogenesis and TWEAKR antagonists (such as soluble receptors and antagonistic antibodies) may be used to inhibit angiogenesis.
Recombinant Production of Soluble TWEAKR-Fc Fusion Polypeptides
To construct a nucleic acid encoding the TWEAKR extracellular domain fused to Fc, a nucleic acid encoding the N-terminal 79 amino acids from TWEAKR, including the leader (signal peptide), was joined to a nucleic acid encoding an Fc portion from human IgG1. Sequences for this construct are shown as SEQ ID NO:6 and 8 (nucleic acid) and SEQ ID NO:7 and 9 (amino acid). In SEQ ID NO:7 and 9, residues 1-27 are the predicted signal peptide (predicted to be cleaved upon secretion from the cell; the actual cleavage site was identified by N-terminal sequence analysis, see below), residues 28-79 and 28-70 of SEQ ID NO:7 and 9, respectively, are from the cysteine-rich TWEAKR extracellular domain, residues 80-81 and 71-72 of SEQ ID NO:7 and 9, respectively, are from a BglII cloning site, and the remainder is the Fc portion. Upon insertion into a mammalian expression vector, and expression in and secretion from a mammalian host cells, these construct produced a polypeptide designated TWEAKR-Fc (SEQ ID NO:7) and TWEAKR-FcΔ9 (SEQ ID NO:9). N-terminal sequence analysis determined that the secreted polypeptides designated TWEAKR-Fc and TWEAKR-FcΔ9 had an N-terminus corresponding to residue 28 (Glu) of SEQ ID NO:7 and 9, respectively. Anti-angiogenic activity of TWEAKR-Fc was demonstrated using assays such as those described in the following examples. An analogous Fc-fusion construct was prepared using the murine TWEAKR extracellular domain.
The extracellular domain of human TWEAKR was expressed in E. coli as a leucine zipper dimer fusion protein. A cDNA was constructed with the aid of PCR to place an initiator Met residue next to TWEAKR DNA encoding amino acids Glu28 to Trp79. In addition, cDNA sequences were added that encoded Flag and the leucine zipper dimer at the C-terminal end. The cDNA was then ligated into an E. coli expression vector. The vector was designed to express recombinant protein upon induction in E. coli. Several promoters or transcriptional control units can be used including the T7 promoter, the PL promoter, and the Tac promoter. A number of commercially available vectors are known in the art.
E. coli cells containing the TWEAKR-Flag-LeuZip2 were cultured and induced for expression. After several hours, E. coli cells were collected and lysed to release intracellular proteins. The E. coli lysate was fractionated on SDS-PAGE and Western blotted for the Flag antigen. A specific Flag reactive band was seen at approximately 12.5 kDa, the expected size of the TWEAKR-Flag-LeuZip2. Additional blots were probed with TWEAK and bands visualized with an anti-TWEAK antibody. The same 12.5 kDa band was visualized indicating the E. coli-expressed TWEAKR is able to bind its ligand.
Production of Antibodies that Bind the TWEAKR Extracellular Domain
BALB/c mice are immunized with TWEAKR extracellular domain and spleen cells are collected and used to prepare hybridomas using standard procedures. Hybridoma supernatants are screened, using ELISA, for the ability to bind TWEAKR. Positives are cloned two times, to insure monoclonality, then isotyped and reassayed for reactivity to TWEAKR. Antibodies and antibody derivatives are also prepared using transgenic mice that express human immunoglobulins and through the use of phage display. The resulting antibodies are tested in assays such as those described in the examples below, to characterize their ability to modulate the TWEAK-TWEAKR interaction, TWEAKR signaling, angiogenesis, and other downstream biological activities.
Agonistic antibodies are used to promote TWEAK-induced biological activities such as angiogenesis, and antagonistic antibodies are used to inhibit TWEAK-induced biological activities such as angiogenesis. For some applications, the activity of antagonistic antibodies is augmented by conjugation to a radioisotope, to a plant-, fungus-, or bacterial-derived cytotoxin such as ricin A or diptheria toxin, or to another chemical poison. And because of the restricted tissue distribution of TWEAKR, antibodies that bind to TWEAKR are particularly useful as targeting agents for imaging or delivering therapeutics to the vasculature. Antibodies that bind TWEAKR can be used, for example, to target a detectable label or chemotherapeutic to the mural cells (pericytes and vascular smooth muscle cells). Detectable labels may include radioisotopes, chemiluminescent and fluorescent compounds, and enzymes. These techniques are useful, for example, in the diagnosis, staging, and treatment of neoplasms.
Example 3
Activity of TWEAKR-Fc in a Wound Closure Assay
A planar endothelial cell migration (wound closure) assay was used to quantitate the inhibition of angiogenesis by TWEAKR-Fc in vitro. In this assay, endothelial cell migration is measured as the rate of closure of a circular wound in a cultured cell monolayer. The rate of wound closure is linear, and is dynamically regulated by agents that stimulate and inhibit angiogenesis in vivo.
Primary human renal microvascular endothelial cells (HRMEC) were isolated, cultured, and used at the third passage after thawing, as described in Martin et al., In vitro Cell Dev Biol 33:261, 1997. Replicate circular lesions, “wounds,” (600-800 micron diameter) were generated in confluent HRMEC monolayers using a silicon-tipped drill press. At the time of wounding the medium (Dulbecco's Modified eagle Medium (DMEM)+1% bovine serum albumin (BSA)) was supplemented with 20 ng/ml PMA (phorbol-12-myristate-13-acetate), EGF (4 ng/ml), and 0.150 to 5 μg/ml TWEAKR-Fc, or a combination of 40 ng/ml EGF and 0.150 to 5 μg/ml TWEAKR-Fc. As a control for TWEAKR-Fc indicated samples received 5 μg/ml IgG-Fc. The residual wound area was measured as a function of time (0-12 hours) using a microscope and image analysis software (Bioquant, Nashville, Tenn.). The relative migration rate was calculated for each agent and combination of agents by linear regression of residual wound area plotted over time. The results are shown in FIGS. 2-3.
Compared to huIgG or media+BSA, TWEAKR-Fc inhibited PMA-induced endothelial migration in a dose responsive manner, reducing the rate of migration to unstimulated levels at 1.5 to 5 μg/ml (FIG. 2). Neither huIgG nor TWEAKR-Fc inhibited basal (uninduced) migration. When HRMEC migration was induced by EGF, TWEAKR-Fc inhibited endothelial migration in a dose-dependent manner, reducing the rate of migration to unstimulated levels at 5 μg/ml (FIG. 3).
Example 4
Activity of TWEAKR-Fc in a Corneal Pocket Assay
A mouse corneal pocket assay was used to quantitate the inhibition of angiogenesis by TWEAKR-Fc in vivo. In this assay, agents to be tested for angiogenic or anti-angiogenic activity are immobilized in a slow release form in a hydron pellet, which is implanted into micropockets created in the corneal epithelium of anesthetized mice. Vascularization is measured as the appearance, density, and extent of vessel ingrowth from the vascularized corneal limbus into the normally avascular cornea.
Hydron pellets, as described in Kenyon et al., Invest Opthamol. & Visual Science 37:1625, 1996, incorporated sucralfate with basic fibroblast growth factor (bFGF) (90 ng/pellet), bFGF and IgG (14 μg/pellet, control), or bFGF and TWEAKR-Fc (14 μg). The pellets were surgically implanted into corneal stromal micropockets created by micro-dissection 1 mm medial to the lateral corneal limbus of 6-8 week old male C57BL mice. After five days, at the peak of neovascular response to bFGF, the corneas were photographed, using a Zeiss slit lamp, at an incipient angle of 35-50 degrees from the polar axis in the meridian containing the pellet. Images were digitized and processed by subtractive color filters (Adobe Photoshop 4.0) to delineate established microvessels by hemoglobin content. Image analysis software (Bioquant, Nashville, Tenn.) was used to calculate the fraction of the corneal image that was vascularized, the vessel density within the vascularized area, and the vessel density within the total cornea.
As shown in Table 1, TWEAKR-Fc (100 pmol) inhibited bFGF (3 pmol)-induced corneal angiogenesis, reducing the vascular density to 50% of that induced by FGF alone or FGF+IgG. In addition to reducing vascular area, local administration of TWEAKR-Fc significantly inhibited FGF induced vessel density (imaged on hemoglobin) by 70% compared to the vessel density in the presence of the control protein IgG-Fc.
TABLE 1
Effect of TWEAKR-Fc on FGF-induced Angiogenesis
in the Mouse Corneal Pocket Assay
Greater than 50% Reduction in
Number and Length of Vessels
Treatment
n/total n (%)
FGF alone
0/2 (0%)
FGF + IgG
0/2 (0%)
FGF + TWEAKR-Fc
6/9 (67%)
Example 5
Qualitative TRAF Binding to the TWEAK Receptor (TWEAKR) Cytoplasmic Domain
Members of the TRAF family are intra-cellular signaling molecules. Several members of the TRAF family are known to associate with members of the TNF receptor family in order to initiate a signaling cascade that activates the NF-kappa-B pathway, resulting in cell activation and proliferation. A qualitative in vitro binding assay was performed to test whether members of the TRAF family of intra-cellular signaling molecules bind to the cytoplasmic domain of TWEAKR and to learn, therefore, whether the small cytoplasmic domain of TWEAKR is capable of mediating a signal into the cell via the TRAF pathway.
A GST fusion vector consisting of the C-terminal 29 amino acids of TWEAKR fused to glutathione S-transferase was created by sub-cloning the appropriate insert into the pGEX-4T (Amersham Pharmacia Biotech) vector at the BamHI and NotI sites. The product from this vector was expressed in E. coli and bound to sepharose beads as described by Galibert et al., J. Biol. Chem. 273(51):34120, 1998. Similarly constructed beads coated with RANK cytoplasmic domain-GST fusion proteins were used as a positive control, and beads coated with GST alone were used as a negative control. 35S-methionine/cysteine labeled TRAF proteins were produced in reticulocyte lysates (TNT-coupled Reticulocyte Lysate Systems, Promega) according to the manufacturer's protocol. Reticulocyte lysates containing the labeled TRAF molecules were first pre-cleared using the control beads followed incubation with the indicated fusion protein coated beads in binding buffer (50 mM HEPES [pH 7.4], 250 mM NaCl, 0.25% (v/v) Nonidet P-40, 10% glycerol, 2 mM EDTA) at 4° C. for 2 hours. After washing 4× with binding buffer bound TRAF molecules eluted from the beads in SDS-loading buffer, separated by SDS-PAGE, dried and exposed to X-ray film.
Binding above background levels was seen with TRAFS 1,2 and 3. No binding above background levels was seen with TRAFS 4,5, and 6. The ability of TWEAKR to bind to TRAFs 1,2, and 3 demonstrates that TWEAKR is capable of inducing a signal to the cell via the TRAF pathway, and therefore transmitting a proliferative signal into the host cell. This experiment provides further evidence that TWEAKR is the functional receptor for TWEAK. It also illustrates a further means by which signaling can be inhibited: by disrupting the TRAF-TWEAKR interaction with a small molecule, or by use of a dominant negative variant of the TRAF molecule.
Example 6
Activity of TWEAKR-Fc in an Endothelial Cell Proliferation Assay
An endothelial cell proliferation assay was used to quantitate the inhibition of bFGF or TWEAK induced-proliferation by TWEAKR-Fc in vitro. In this assay, endothelial cell proliferation is measured after 4 days of cell growth in microtiter wells using a cell-labeling molecule called calcein AM. Esterases expressed by the cells cleave the calcein and cause it to fluoresce when excited at 485 nm. Uncleaved calcein does not fluoresce. The amount of fluorescence is directly related to the number of endothelial cells in the culture well. Endothelial cell proliferation is often regulated by agents that stimulate and/or inhibit angiogenesis in vivo.
Primary HUVEC (human umbilical vein endothelial cells) were obtained from a commercial source (Clonetics, Walkersville, Md.), cultured, and used at passage 2 to 7. Replicate cultures were set up by adding 3000 HUVEC to each microtiter well in endothelial cell basal media (EBM, an endothelial cell basal media that contains no growth factors or serum and is based on the media formulations developed by Dr. Richard Ham at the University of Colorado, Clonetics) plus 0.05% FBS (fetal bovine serum). At the time of culture initiation FGF-2 (fibroblast growth factor-2, 10 ng/ml) or human TWEAK (100 ng/ml) was added to the cultures in the presence of human IgG (huIgG, control) or human TWEAKR-Fc at concentrations ranging from 0.08 μg/ml to 20 μg/ml (0.25 to 20 μg/ml for TWEAK-induced and 0.08 to 6.7 μg/ml for FGF-2-induced). The HUVEC containing cultures were incubated for 4 days at 37° C., 5% CO2. On the fourth day of culture 4 μM calcein-AM was added to the cultures and 2 hours later the wells were evaluated for fluorescence. The results, expressed as the average fluorescence (485-530 nm) counts for replicate wells plus or minus the SEM, are shown in FIGS. 4 and 5.
TWEAKR-Fc specifically inhibited TWEAK-induced HUVEC proliferation in a dose-dependent manner when compared to huIgG, which did not effect TWEAK-induced proliferation (FIG. 4). In addition, TWEAKR-Fc inhibited the basal proliferation of HUVEC observed during culture in EBM plus 0.05% FBS, as compared to huIgG, which did not. Interestingly, TWEAKR-Fc also inhibited FGF-2 mediated HUVEC proliferation at concentrations of greater than 2 μg/ml, as compared to huIgG, which did not effect the FGF-2 induced HUVEC proliferative response (FIG. 5). These results show that TWEAKR-Fc inhibits HUVEC proliferation induced by the addition of exogenous recombinant human TWEAK. That TWEAKR-Fc partially inhibits serum-induced HUVEC-proliferation indicates HUVEC produce endogenous TWEAK that promotes growth/survival of the EC (endothelial cell) via the TWEAKR. TWEAKR-Fc attenuation of FGF-2 induced proliferation indicates that at least part of the EC response to FGF-2 is dependent on endogenous TWEAK/TWEAKR interaction.
In another set of experiments to examine the effects of TWEAKR on proliferation of HUVEC cells a construct was made that fused a synthetic FLAG octapeptide epitope onto the N-terminal extracellular domain of TWEAKR (FLAG-TWEAKR). The resulting protein was expressed by transient transfection in HUVEC and incubated with cross-linked anti-FLAG monoclonal antibody. Cross-linking the receptor in this manner avoids background from the endogenous TWEAKR expressed by HUVEC. Proliferation was measured by BrdU uptake. Lipid mediated transfection of HUVEC with FLAG-TWEAKR resulted in expression of recombinant FLAG-TWEAKR on the cell surface by 36 hours post transfection. In vitro culture of FLAG-TWEAKR expressing HUVEC with the complex of M2 anti-FLAG and goat anti-mouse IgG increased BrdU incorporation 3-fold over the level of BrdU incorporation observed by culturing FLAG-TWEAKR expression cells with goat anti-mouse IgG alone. Cultures of FLAG-TWEAKR expressing HUVEC with the complex of M2 anti-FLAG and goat anti-IgG increased BrdU incorporation 6-fold over the level of BrdU incorporation observed by culturing vector-only transfected HUVEC with the cross-linking complex. Incubation with the cross-linking complex did not alter BrdU incorporation in vector alone transfected HUVEC. As an additional control, cells transfected with the FLAG construct that were not exposed to anti-FLAG also showed decreased BrdU uptake relative those that were exposed to crosslinked anti-FLAG. This data provides additional evidence that despite the small size of TWEAKR, TWEAKR is capable of initiating a proliferative signal in human endothelial cells.
Example 7
Inhibition of Neovascularization by TWEAKR-Fc in a Murine Transplant Model
Survival of heterotopically transplanted cardiac tissue from one mouse donor to the ear skin of another genetically similar mouse requires adequate neovascularization by the transplanted heart and the surrounding tissue, to promote survival and energy for cardiac muscle function. Inadequate vasculature at the site of transplant causes excessive ischemia to the heart, tissue damage, and failure of the tissue to engraft. Agents that antagonize the angiopoietins and endothelial specific factors involved in endothelial cell migration and vessel formation can decrease angiogenesis at the site of transplant, thereby limiting graft tissue function and ultimately engraftment itself.
The following studies were carried out, utilizing a murine heterotopic cardiac isograft model, in order to demonstrate the antagonistic effects of TWEAKR-Fc on neovascularization. In all experiments, female BALB/c (≈12 weeks of age) recipients received neonatal heart grafts from donor mice of the same strain.
TWEAKR-Fc Dose Titration
In the described experiments, the donor heart tissue was engrafted into the left ear pinnae of the recipient on day 0 and the mice were divided into treatment groups. The control group received human IgG (Hu IgG, 400 μg/day) while the other treatment groups human TWEAKR-Fc at a dose of 400 μg/day or 150 μg/day. All treatments (proteins administered by intraperitoneal injection) began on day 0 and continued for four consecutive days. The functionality of the grafts was determined by monitoring visible pulsatile activity on days 7 and 12 post-engraftment. Table 2 shows the experimental results.
TABLE 2
Functional Heart Isoengraftment Following Dose Titration with
TweakR/Fc
Treatment
Day 7
Day 12
N =
Hu IgG
100*
100
3
400 μg
HuTWEAKR-Fc
100
100
5
150 μg
HuTWEAKR-Fc
40
80
5
400 μg
*all results are reported as percent of mice with pulsatile heart grafts
Administration of TWEAKR-Fc to isograft-bearing mice caused a significant, dose-dependent, delay in cardiac isoengraftment. Sixty percent of TWEAKR-Fc-treated mice at the 400 ug/day dose, failed to exhibit pulsatile activity on day 7 post transplant as compared to huIgG control, where no effect on isoengraftment was observed. At this dose, TWEAKkR-Fc administration caused permanent engraftment failure in one fifth of the mice compared to huIgG control where no effect on engraftment was observed. While a dose of 400 μg of huTWEAKR-Fc showed a significant anti-angiogenic effect, a 150 ug dose of TWEAKR-Fc did not show measurable activity in this model.
Example 8
Regulation of TWEAKR mRNA Expression I Vascular Smooth Muscle Cells (SMC)
Rat aortic SMC were serum-starved and then treated with FGF for various lengths of time. RNA was isolated and TWEAKR mRNA levels were examined by Northern Blot hybridization. A single TWEAKR transcript of ≈1.2 kb in size was detected in SMC. TWEAKR mRNA expression was transiently induced following FGF addition, with maximal levels detected at 2 hours post stimulation. Serum-starved SMC were also treated for 4 hours with various agents (e.g., phorbol ester, polypeptide growth factors, peptide hormones) and then a Northern blot was performed to determine whether TWEAKR gene expression could be induced by multiple distinct growth promoters. TWEAKR mRNA levels were significantly elevated following PMA, FBS, PDGF-BB, EGF, FGF or Ang II treatment of rat SMC. TGF-beta1, IGF-1 and alpha-thrombin treatment had only a slight stimulatory effect. These results indicate that WEAKR is a growth factor-regulated gene in vascular SMC.
Example 9
Chromosome Mapping
The gene corresponding to a TWEAKR polypeptide is mapped using PCR-based mapping strategies. Initial human chrornosomal assignments are made using TWEAKR-specific PCR primers and a BIOS Somatic Cell Hybrid PCkable DNA kit from BIOS Laboratories (New Haven, Conn.), following the manufacturer's instructions. More detailed mapping is performed using a Genebridge 4 Radiation Hybrid Panel (Research Genetics, Huntsville, Ala.; described in Walter et al., Nature Genetics 7:22-28, 1994). Data from this analysis is ten submitted electronically to the MIT Radiation Hybrid Mapper following the instructions contained therein. This analysis yields specific genetic marker names which, when submitted electronically to the NCBI Genemap browser, yield the specific chromosome interval.
Example 10
TWEAKR Stability
Ligand blots were generated by running either TweakR-Fc or RP-Fc as a control on a standard SDS-PAGE and blotted onto nitrocellulose. The separate samples were prepared by with and without the addition of reducing agent (DTT) and with and without heating at 100° C. to denature the proteins. This blot was probed with TWEAK-leucine zipper conditioned supernatants followed by 125I labeled M15 anti-leucine zipper. The results showed that all TweakR-FC samples strongly bound TWEAK while the RP-Fc samples did not. This shows that TweakR ligand binding domain will spontaneously re-fold into an active conformation even after begin reduced and boiled in SDS loading buffer.
Example 11
Treatment of Tumors with TWEAKR Antagonists
TWEAKR antagonists, including antibodies and TWEAKR-Fc, are tested in animal models of solid tumors. The effect of the TWEAKR antagonists is determined by measuring tumor frequency and tumor growth.
The relevant disclosures of publications cited herein are specifically incorporated by reference. The examples presented above are not intended to be exhaustive or to limit the scope of the invention. The skilled artisan will understand that variations and modifications and variations are possible in light of the above teachings, and such modifications and variations are intended to be within the scope of the invention.
Example 12
Identification of a TWEAK Binding Domain
The three-dimensional structure of TWEAKR was modeled on the crystal structure of the extracellular domain of the Tumor Necrosis Factor Receptor family member p55 (Naismith et al., Structure 4:1251-62, 1996; Research Collaboratory for Structural Bioinformatics Protein Data Bank Identification Number 1ext (Berman et al., Nucl Acids Res. 28:235-42, 2000)). The conserved extracellular TWEAKR cysteines best align with the cysteines in the second domain of p55. This resulted in the following alignment (conserved cysteines in bold and underlined):
TWEAKR
EQAPGTAP------CSRGS-SWSAD-LDK
p55
CMDCASCRARP-HSDF--CL-NDCPGPGQ
DTDCRECESGSFTASENHLRHCLSCSKCR
KEMGQVEISSCTV
TWEAKR
----GCAAAPPAPFRLLW
(SEQ ID NO:17)
p55
DRDTVCGCRK-NQYRHYW
(SEQ ID NO:18)
The structure was then modeled using the Modeler software package (Blundell, J Mol. Biol. 234:779-815, 1993; Fiser et al., Protein Science 9:1753-73, 2000; Marti-Renom et al., Ann Rev Biophys Biomol Struct. 29:291-325, 2000) from Accelrys Inc. (San Diego, Calif.). Conserved cysteine residues in the family were forced to align as shown above. The alignment and the resulting structure indicate that disulfide bonds form between residues Cys36-Cys49, Cys52-Cys64, and Cys55-Cys67. The presence of a disulfide bond between residues Cys36 and Cys49 was confirmed by LC/MS. The model also predicted that residues 36 to 68 help maintain secondary structural conformation. Thus, amino acid residues 36-68 of the human TWEAKR constitute a putative TWEAK binding domain.
The sequence of soluble huTWEAKR was aligned to domain 2 of DR5 where the domains of DR5 were defined as follows:
DR5 Domains
Domain 1:
(SEQ ID NO:20)
PQQKRSSPSEGLCPPGHHISEDGRDCI
Domain 2:
(SEQ ID NO:21)
SCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQ
Domain 3:
(SEQ ID NO:22)
CEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKESGD
The sequence of a soluble huTWEAKR fragment was superimposed on the three dimensional structure of DR5 in complex with its ligand TRAIL. The TWEAKR residues predicted to be within 4.5 angstroms of TRAIL ligand, and therefore predicted to be important for ligand binding, were determined and are shown below in bold, underlined font:
(SEQ ID NO:4)
MARGSLRRLL RLLVLGLWLA LLRSVAGEQA PGTAPCSRGS
SWSADLDKCM DCASCRARPH SDFCLGCAAA PPAPFRLLWP
ILGGALSLTF VLGLLSGFLV WRRCRRREKF TTPIEETGGE
GCPAVALIQ
Thus, TWEAK is predicted to bind to a polypeptide comprising the sequence:
CX12CXDCASCRAXPX4CX2C
(SEQ ID NO:30)
Example 13
Identification of a TWEAK Receptor Epitope Bound by Inhibiting and Inducing Antibodies
Antagonistic and agonistic functional anti-huTWEAKR antibodies and antibody derivatives were tested for binding to fragments of the TWEAKR extracellular domain by competitive inhibition of TWEAK binding. For dissection of the antigenic epitope of TWEAKR, various biotin conjugated TWEAKR peptides were synthesized. Each peptide was allowed to fold and form disulfide bonds. The peptides were analyzed by mass spectrometry and HPLC. To dissect the role of Cys residues important for TWEAK or anti-huTWEAKR antibody binding to the receptor, Cys residues were replaced with amino-butyric acid (“Abu”), whose methyl group mimics the hydrophobicity of the thiol group, as shown in the following sequences:
TWEAKR (34–68):
(SEQ ID NO:23)
APCSRGSSWSADLDKCMDCASCRARPHSDFCLGCA
TWEAKR (28–68)Δ4,6:
(SEQ ID NO:24)
EQAPGTAPCSRGSSWSADLDKCMDCAS[Abu]RARPHSDFCLG[Abu]A
TWEAKR (28–68)Δ3,5:
(SEQ ID NO:25)
EQAPGTAPCSRGSSWSADLDKCMD[Abu]ASCRARPHSDF[Abu]LGC
TWEAKR (50–66)b:
(SEQ ID NO:26)
MDCAS[Abu]RARPHSDFCLG (C3–C5)
TWEAKR (54–74):
(SEQ ID NO:27)
SCRARPHSDF[Abu]LGCAAAPPAP (C4–C6)
Anti-huTWEAKR antibody and TWEAK binding to the soluble biotin labeled TWEAKR.Fc and biotin-labeled TWEAKR peptides were compared using a plate based binding assay.
A significant number of anti-huTWEAKR antibodies bound equally well to huTWEAKR(28-70).Fc and to huTWEAKR(28-79).Fc. (where the numbers in parentheses indicate the range of huTWEAKR amino acid residues in the fragment, and the initiating methionine is considered the first residue). Competition assays showed that some anti-huTWEAKR antibodies bound to TWEAKR(28-68).
Two antibodies were identified that each either potently inhibited TWEAKR signaling in the presence of TWEAK, or potently induced TWEAKR signaling in the absence of TWEAK, depending of the type of antibody or derivative of the antibody that was used. Each of these antibodies bound to TWEAKR(34-68) but not to TWEAKR(28-68)A3,5 or TWEAKR(28-68)A4,6. Thus, residues within TWEAKR(34-68), including the third, fourth, fifth, and sixth conserved cysteine residues, are part of the epitope recognized by these antibodies. Examples of such potential sequences include:
CX12CX2CX2CX8CX2C
(SEQ ID NO:28)
CSRGX8KCMDCASCRAX1PX4CX2C
(SEQ ID NO:29)
Using methods known in the art or described herein, antibodies or antibody derivatives against a polypeptide consisting of or comprising one of these sequences, or a fragment or derivative thereof, are made. Optionally, the antibodies or antibody derivatives are further tested, using methods known in the art or taught herein, for the ability to inhibit TWEAKR signaling in the presence of TWEAK, to induce TWEAKR signaling in the absence of TWEAK, or to increase TWEAKR signaling in the presence of TWEAK.
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10971250
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immunex corporation
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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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530/388.1
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Mar 31st, 2022 02:17PM
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Mar 31st, 2022 02:17PM
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Amgen
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:amgn
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Amgen
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Jun 27th, 2006 12:00AM
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Feb 3rd, 2003 12:00AM
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https://www.uspto.gov?id=US07067475-20060627
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Tek antagonists
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The present invention provides Tek antagonists and methods of inhibiting angiogenesis in a mammal by administering Tek antagonists. The methods are particularly useful in treating diseases or conditions mediated by angiogenesis, such as solid tumors and diseases or conditions characterized by ocular neovascularization.
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7067475
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1. A method of inhibiting angiogenesis in a mammal in need of such treatment, comprising administering to the mammal an inhibition-effective amount of a polypeptide comprising a fragment of Tek extracellular domain, shown as residues 19–745 of SEQ ID NO:1, wherein the polypeptide lacks residues 473–745 of SEQ ID NO:1 containing fibronectin type III (FN III) motifs and wherein the polypeptide has a higher binding affinity for angiopoietin-1 or angiopoietin-2 or angiopoietin-4 than does a polypeptide comprising the full length Tek extracellular domain.
2. A method as claimed in claim 1, wherein said polypeptide is a multimer.
3. The method of claim 2 wherein the multimer is a dimer or trimer.
4. The method of claim 2 wherein said multimer comprises an Fc polypeptide or a leucine zipper.
5. The method of one of claims 2–4 wherein the Tek is human Tek.
6. The method of claim 5 wherein the Tek multimer comprises a polypeptide having a sequence selected from the group consisting of residues 23–704 of SEQ ID NO:2, and residues 23–472 of SEQ ID NO:2.
7. The method of one of claims 2–4 wherein the Tek multimer comprises a polypeptide having a sequence selected from the group consisting of residues 23–704 of SEQ ID NO:2, and residues 23–472 of SEQ ID NO:2.
8. A method of inhibiting angiogenesis in a mammal in need of such treatment, comprising administering to the mammal an inhibition-effective amount of a compound selected from the group consisting of:
(a) a polypeptide comprising a fragment of Tek extracellular domain, shown as residues 19–745 of SEQ ID NO:1, wherein the polypeptide lacks residues 473–745 of SEQ ID NO:1 containing fibronectin type III (FN III) motifs and wherein the polypeptide has a higher binding affinity for angiopoietin-1 or angiopoietin-2 or angiopoietin-4 than does a polypeptide comprising the full length Tek extracellular domain; and
(b) a multimer of the polypeptide described in (a).
9. The method of claim 8 wherein the mammal has a disease or condition mediated by angiogenesis.
10. The method of claim 9 wherein the disease or condition is characterized by ocular neovascularization.
11. The method of claim 9 wherein the disease or condition is a solid tumor.
12. The method of claim 8 wherein the method further comprises treating the mammal with a second chemotherapeutic agent.
13. The method of claim 12 wherein the second chemotherapeutic agent is selected from the group consisting of alkylating agents, antimetabolites, vinca alkaloids and other plant-derived chemotherapeutics, nitrosoureas, antitumor antibiotics, antitumor enzymes, topoisomerase inhibitors, platinum analogs, adrenocortical suppressants, hormones, hormone agonists, hormone antagonists, antibodies, immunotherapeutics, blood cell factors, radiotherapeutics, and biological response modifiers.
14. The method of claim 12 wherein the second chemotherapeutic agent is selected from the group consisting of cisplatin, cyclophosphamide, mechloretamine, melphalan, bleomycin, carboplatin, fluorouracil, 5-fluorodeoxyuridine, methotrexate, taxol, asparaginase, vincristine, and vinblastine, lymphokines and cytokines such as interleukins, interferons (including alpha., beta, or delta), and TNF, chlorambucil, busulfan, carmustine, lomustine, semustine, streptozocin, dacarbazine, cytarabine, mercaptopurine, thioguanine, vindesine, etoposide, teniposide, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, L-asparaginase, hydroxyurea, methylhydrazine, mitotane, tamoxifen, and fluoxymesterone.
15. The method of claim 12 wherein the second chemotherapeutic agent is selected from the group consisting of Flt3 ligand, CD40 ligand, interleukin-2, interleukin-12, 4-1 BB ligand, anti-4-1 BB antibodies, TNF antagonists and TNF receptor antagonists including TNFR/Fc, TWEAK antagonists and TWEAK-R antagonists including TWEAK-R/Fc, TRAIL, CD148 agonists, VEGF antagonists including anti-VEGF antibodies, and VEGF receptor antagonists.
16. The method of claim 8 wherein the method further comprises treating the mammal with radiation.
17. A method of inhibiting the binding of a Tek ligand to Tek in a mammal in need of such treatment, comprising administering to the mammal an inhibition-effective amount of compound selected from the group consisting of:
(a) a polypeptide comprising a fragment of Tek extracellular domain, shown as residues 19–745 of SEQ ID NO:1, wherein the polypeptide lacks residues 473–745 of SEQ ID NO:1 containing fibronectin type III (FN III) motifs and wherein the polypeptide has a higher binding affinity for angiopoietin-1 or angiopoietin-2 or angiopoietin-4 than does a polypeptide comprising the full length Tek extracellular domain; and
(b) a multimer of the polypeptide described in (a).
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17
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REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 09/733,764, filed Dec. 7, 2000 now U.S. Pat. No. 6,521,424 and incorporated herein by reference, which is a continuation-in-part of U.S. application Ser. No. 09/590,656, filed Jun. 7, 2000 now U.S. Pat. No. 6,413,932 and incorporated herein by reference, which claims the benefit of U.S. Provisional Application Ser. No. 60/137,889, filed 7 Jun. 1999 and incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates to Tek antagonists and to the use of Tek antagonists to inhibit angiogenesis or other Tek-mediated responses in a mammal.
BACKGROUND OF THE INVENTION
A. Angiogenesis
Angiogenesis, the generation of new blood vessels, is a spatially and temporally regulated process in which endothelial cells proliferate, migrate, and assemble into tubes, in response to endogenous positive and negative regulatory molecules. Angiogenesis plays important roles in both normal and pathological physiology.
Under normal physiological conditions, angiogenesis is involved in fetal and embryonic development, wound healing, organ regeneration, and female reproductive remodeling processes including formation of the endometrium, corpus luteum, and placenta. Angiogenesis is stringently regulated under normal conditions, especially in adult animals, and perturbation of the regulatory controls can lead to pathological angiogenesis.
Pathological angiogenesis has been implicated in the manifestation and/or progression of inflammatory diseases, certain eye disorders, and cancer. In particular, several lines of evidence support the concept that angiogenesis is essential for the growth and persistence of solid tumors and their metastases (see, e.g., Folkman, N. Engl. J. Med. 285:1182, 1971; Folkman et al., Nature 339:58, 1989; Kim et al., Nature 362:841, 1993; Hori et al., Cancer Res., 51:6180, 1991). Angiogenesis inhibitors are therefore being tested for the prevention (e.g., treatment of premalignant conditions), intervention (e.g., treatment of small tumors), and regression (e.g., treatment of large tumors) of cancers (see, e.g., Bergers et al., Science 284:808, 1999).
Although several anti-angiogenic agents are presently under development and testing as therapeutics, there is a need for additional methods of inhibiting angiogenesis for the prevention, abrogation, and mitigation of disease processes that are dependent on pathological angiogenesis.
B. Tek Polypeptides
The receptor tyrosine kinases (RTKs) are a large and evolutionarily conserved family of proteins involved in the transduction of extracellular signals to the cytoplasm. Among the RTKs believed to be involved in vascular morphogenesis and maintenance are the vascular endothelial growth factor (VEGF) receptors and Tek (see Hanahan, Science 277:48, 1997).
Tek, which has also been called Tie2 and ork, is an RTK that is predominantly expressed in vascular endothelium. The molecular cloning of human Tek (ork) has been described by Ziegler, U.S. Pat. No. 5,447,860. Four Tek ligands, angiopoietin-1, angiopoietin-2, angiopoietin-3, and angiopoietin-4 (Ang1, Ang2, Ang3, and Ang4), have been described (Davis et al., Cell 87:1161, 1996; Maisonpierre et al., Science 277:55, 1997; Valenzuela et al., Proc. Natl. Acad. Sci. USA 96:1904, 1999). These ligands have distinct expression patterns and activities with respect to Tek. “Tie ligand homologues” designated NL1, NL5, NL8, and NL4 are described in U.S. Pat. No. 6,057,435.
Tek knockout mice have defects in vascular development, and die during embryogenesis (see Dumont, Genes Dev. 8:1897, 1994; Sato, Nature 376:70, 1995), suggesting that Tek plays a role in the development of embryonic vasculature.
Lin et al. have described a soluble Tek (Tie2) inhibitor designated ExTek.6His, consisting of the entire extracellular portion of murine Tek fused to a six-histidine tag (J. Clin. Invest. 100(8):2072, 1997; WO 98/18914). ExTek.6His inhibited growth and tumor vascularization in a rat cutaneous window chamber model, and blocked angiogenesis stimulated by tumor cell conditioned media in a rat corneal micropocket assay. Peters et al. have also described a replication-defective adenoviral vector designated AdExTek, which expresses the murine Tek extracellular domain (Proc. Natl. Acad. Sci. USA 95:8829, 1998; WO 98/18914). AdExTek inhibited the growth and metastasis of a murine mammary carcinoma and a murine melanoma.
While ExTek.6His and AdExTek may prove useful as anti-angiogenic agents, there is a need for additional and improved Tek antagonists and additional and improved methods of inhibiting angiogenesis or other Tek-mediated responses using Tek antagonists.
SUMMARY OF THE INVENTION
The present invention provides Tek antagonists and methods of using Tek antagonists to inhibit angiogenesis or other Tek-mediated responses in a mammal in need of such treatment. The invention is based in part on the unexpected discovery that fragments of the Tek extracellular domain, lacking all or part of the region containing fibronectin type III (FNIII) motifs, can have a higher binding affinity for Tek ligands than polypeptides comprising full length Tek extracellular domain.
In some preferred embodiments the Tek antagonist is a polypeptide comprising a fragment of Tek extracellular domain, wherein the fragment lacks all or part of the region containing fibronectin type III (FNIII) motifs and wherein the polypeptide retains the ability to bind at least one Tek ligand. In preferred embodiments the fragment lacks at least residues 473–745 of the Tek extracellular domain; in more preferred embodiments the Tek ligand is angiopoietin-1, angiopoietin-2, or angiopoietin-4. In most preferred embodiments, the Tek antagonist is a polypeptide that has a higher binding affinity for a Tek ligand than does a polypeptide comprising full length Tek extracellular domain.
The invention also encompasses nucleic acids encoding polypeptides according to the invention, and polypeptides produced by expressing such a nucleic acid in a recombinant host cell under conditions that permit expression of the polypeptide.
In some preferred embodiments, the Tek antagonist is a soluble Tek multimer, preferably a dimer or trimer, and most preferably comprising an Fc polypeptide or a leucine zipper. The Tek is preferably human Tek. In some preferred embodiments the soluble Tek multimer comprises a fragment of Tek extracellular domain, wherein the fragment lacks all or part of the region containing fibronectin type III (FNIII) motifs and wherein the polypeptide retains the ability to bind at least one Tek ligand. In some preferred embodiments the soluble Tek multimer comprises residues 23–472 or 23–704 of SEQ ID NO:2.
The invention also encompasses antibodies or antibody fragments that bind specifically to a polypeptide according to the invention, and antibodies or antibody fragments that are capable of competitively inhibiting the binding of a Tek ligand to a polypeptide according to the invention. The antibodies are preferably selected from the group consisting of monoclonal antibodies, humanized antibodies, transgenic antibodies, and human antibodies.
The invention also provides methods of inhibiting angiogenesis or other Tek-mediated responses in a mammal in need of such treatment, comprising administering to the mammal an inhibition-effective amount of a Tek antagonist. The Tek antagonist is preferably a fragment of Tek extracellular domain, a soluble Tek multimer, or an antibody or antibody fragment. In some preferred embodiments the Tek antagonist is administered in a composition comprising a pharmaceutically acceptable carrier.
The soluble Tek multimer is preferably administered to a mammal that has a disease or condition mediated by angiogenesis, more preferably a solid tumor or a disease or condition characterized by ocular neovascularization.
In some embodiments the method further comprises treating the mammal with a second chemotherapeutic agent and or with radiation. The second chemotherapeutic agent may be selected from the group consisting of alkylating agents, antimetabolites, vinca alkaloids and other plant-derived chemotherapeutics, nitrosoureas, antitumor antibiotics, antitumor enzymes, topoisomerase inhibitors, platinum analogs, adrenocortical suppressants, hormones, hormone agonists, hormone antagonists, antibodies, immunotherapeutics, blood cell factors, radiotherapeutics, and biological response modifiers, and more preferably selected from the group consisting of cisplatin, cyclophosphamide, mechloretamine, melphalan, bleomycin, carboplatin, fluorouracil, 5-fluorodeoxyuridine, methotrexate, taxol, asparaginase, vincristine, and vinblastine, lymphokines and cytokines such as interleukins, interferons (including alpha, beta, or delta), and TNF, chlorambucil, busulfan, carmustine, lomustine, semustine, streptozocin, dacarbazine, cytarabine, mercaptopurine, thioguanine, vindesine, etoposide, teniposide, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, L-asparaginase, hydroxyurea, methylhydrazine, mitotane, tamoxifen, and fluoxymesterone, Flt3 ligand, CD40 ligand, interleukin-2, interleukin-12, 4-1BB ligand, anti-4-1BB antibodies, TNF antagonists and TNF receptor antagonists including TNFR/Fc, TWEAK antagonists and TWEAK-R antagonists including TWEAK-R/Fc, TRAIL, CD148 agonists, VEGF antagonists including anti-VEGF antibodies, and VEGF receptor antagonists.
The invention is further directed to a method of inhibiting the binding of a Tek ligand to Tek in a mammal in need of such treatment, comprising administering to the mammal an inhibition-effective amount of a Tek antagonist. The Tek antagonist is preferably a fragment of Tek extracellular domain, a soluble Tek multimer, or an antibody or antibody fragment.
The invention is also directed to the use of a Tek antagonist for the preparation of a medicament for inhibiting angiogenesis in a mammal in need of such treatment, or for inhibiting the binding of a Tek ligand to Tek in a mammal in need of such treatment. The Tek antagonist is preferably a fragment of Tek extracellular domain, a soluble Tek multimer, or an antibody or antibody fragment.
These and other aspects of the present invention will become evident upon reference to the following drawings and detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows inhibition by Tek472/Fc of endothelial cell migration in a wound closure assay.
FIG. 2 shows inhibition by Tek472/Fc of angiogenesis in a corneal pocket assay.
FIG. 3 shows tumor growth after treatment with Tek472/Fc, Flt3L, and combinations of Tek472/Fc and Flt3L, in mice with 87 fibrosarcoma tumors.
FIG. 4 shows tumor growth after treatment with Tek472/Fc, Flt3L, and combinations of Tek472/Fc and Flt3L in mice with B 10.2 fibrosarcoma tumors.
FIG. 5 shows the binding of Tek472/Fc and Tek745/Fc to human angiopoietin-2.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to Tek antagonists and methods of using Tek antagonists to inhibit angiogenesis or other Tek-mediated responses in a mammal. Tek antagonists are compounds or compositions that interfere with one or more biological activities of Tek, including ligand binding and signal transduction, and may be characterized using methods such as those exemplified below. Tek antagonists include fragments of the Tek extracellular domain, soluble Tek multimers, and Tek antibodies and antibody fragments. The molecular cloning of a cDNA encoding human Tek (ork, Tie2) is described in U.S. Pat. No. 5,447,860.
A. Abbreviations and Terminology Used in the Specification
“4-1BB” and “4-1BB ligand” (4-1BB-L) are polypeptides described, inter alia, in U.S. Pat. No. 5,674,704, including soluble forms thereof.
“bFGF” is basic fibroblast growth factor.
“BSA” is bovine serum albumin.
“CD40 ligand” (CD40L) is a polypeptide described, inter alia, in U.S. Pat. No. 5,716,805, including soluble forms thereof.
“CHO” is a Chinese hamster ovary cell line.
“DMEM” is Dulbecco's Modified eagle Medium, a commercially available cell culture medium.
“ELISA” is Enzyme-Linked Immunosorbent Assay.
“Flt3L” is Flt3 ligand, a polypeptide described, inter alia, in U.S. Pat. No. 5,554,512, including soluble forms thereof.
“HMVEC-d” are primary dermal human microvascular endothelial cells.
“HRMEC” are primary human renal microvascular endothelial cells.
“HUVEC” is a line of human umbilical vein endothelial cells.
“mAb” is a monoclonal antibody.
“MSA” is mouse serum albumin.
“PBS” is phosphate buffered saline.
“PE” is phycoerythrin.
“PMA” is phorbol 12-myristate-13-acetate.
“RTKs” are receptor tyrosine kinases.
“TNFR” is a tumor necrosis factor receptor, including soluble forms thereof. “TNFR/Fc” is a tumor necrosis factor receptor-Fc fusion polypeptide.
“TRAIL” is TNF-related apoptosis-inducing ligand, a type II transmembrane polypeptide in the TNF family described, inter alia, in U.S. Pat. No. 5,763,223, including soluble forms thereof.
“TWEAK” is TNF-weak effector of apoptosis, a type II transmembrane polypeptide in the TNF family described, inter alia, in Chicheportiche et al., J. Biol. Chem., 272(51):32401, 1997, including soluble forms thereof. “TWEAK-R” is the “TWEAK receptor,” which is described, inter alia, in U.S. Ser. Nos. 60/172,878 and 60/203,347 and Feng et al., Am. J. Pathol. 156(4):1253, 2000, including soluble forms thereof.
“VEGF” is vascular endothelial growth factor, also known as VPF or vascular permeability factor.
B. Soluble Tek Polypeptides
In one aspect of the present invention, a soluble Tek polypeptide is used as a Tek antagonist to inhibit angiogenesis or to inhibit the binding of a Tek ligand to Tek.
Soluble polypeptides are capable of being secreted from the cells in which they are expressed. The use of soluble forms of polypeptides is advantageous for certain applications. Purification of the polypeptides from recombinant host cells is facilitated since the polypeptides are secreted, and soluble proteins are generally suited for parenteral administration. A secreted soluble polypeptide may be identified (and distinguished from its non-soluble membrane-bound counterparts) by separating intact cells which express the desired polypeptide from the culture medium, e.g., by centrifugation, and assaying the medium (supernatant) for the presence of the desired polypeptide. The presence of the desired polypeptide in the medium indicates that the polypeptide was secreted from the cells and thus is a soluble form of the polypeptide. Soluble polypeptides may be prepared by any of a number of conventional techniques. A DNA sequence encoding a desired soluble polypeptide may be subcloned into an expression vector for production of the polypeptide, or the desired encoding DNA fragment may be chemically synthesized.
Soluble Tek polypeptides comprise all or part of the Tek extracellular domain, but generally lack the transmembrane domain that would cause retention of the polypeptide at the cell surface. Soluble polypeptides may include part of the transmembrane domain or all or part of the cytoplasmic domain as long as the polypeptide is secreted from the cell in which it is produced. Soluble Tek polypeptides advantageously comprise a native or heterologous signal peptide when initially synthesized, to promote secretion from the cell, but the signal sequence is cleaved upon secretion. The term “Tek extracellular domain” is intended to encompass all or part of the native Tek extracellular domain, as well as related forms including but not limited to: (a) fragments, (b) variants, (c) derivatives, and (d) fusion polypeptides. The ability of these related forms to inhibit angiogenesis or other Tek-mediated responses may be determined in vitro or in vivo, using methods such as those exemplified below or using other assays known in the art.
Examples of soluble Tek polypeptides are provided the examples below. As described in the examples, the Inventors unexpectedly discovered that certain fragments of the Tek extracellular domain bind Tek ligands better than the full length Tek extracellular domain, that these fragments can therefore be used as antagonists to block the binding of Tek ligands to Tek (for example, the Tek found on a cell surface), and that antibodies to these fragments can also be used as antagonists to block the binding of Tek ligands to Tek. In some embodiments of the present invention a multimeric form of a soluble Tek polypeptide (“soluble Tek multimer”) is used as an antagonist to block the binding of Tek ligands to Tek, to inhibit angiogenesis or other Tek-mediated responses.
C. Soluble Tek Multimers
Soluble Tek multimers are covalently-linked or non-covalently-linked multimers, including dimers, trimers, or higher multimers. Multimers may be linked by disulfide bonds formed between cysteine residues on different soluble Tek polypeptides. One embodiment of the invention is directed to multimers comprising multiple soluble Tek polypeptides joined via covalent or non-covalent interactions between peptide moieties fused to the soluble Tek polypeptides. Such peptides may be peptide linkers (spacers), or peptides that have the property of promoting multimerization. Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote multimerization of soluble Tek polypeptides attached thereto, as described in more detail below. In particular embodiments, the multimers comprise from two to four soluble Tek polypeptides.
In some embodiments, a soluble Tek multimer is prepared using polypeptides derived from immunoglobulins. Preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., by Ashkenazi et al. (Proc. Natl. Acad. Sci. USA 88:10535, 1991); Byrn et al. (Nature 344:677, 1990); and Hollenbaugh and Aruffo (“Construction of Immunoglobulin Fusion Proteins”, in Current Protocols in Immunology, Suppl. 4, pages 10.19.1–10.19.11, 1992).
One preferred embodiment of the present invention is directed to a Tek/Fc dimer comprising two fusion proteins created by fusing soluble Tek to an Fc polypeptide. A gene fusion encoding the Tek/Fc fusion protein is inserted into an appropriate expression vector. Tek/Fc fusion proteins are expressed in host cells transformed with the recombinant expression vector, and allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between the Fe moieties to yield divalent soluble Tek. The term “Fe polypeptide” as used herein includes native and mutein forms of polypeptides derived from the Fe region of an antibody. Truncated forms of such polypeptides containing the hinge region that promotes dimerization are also included.
One suitable Fe polypeptide, described in PCT application WO 93/10151, is a single chain polypeptide extending from the N-terminal hinge region to the native C-terminus of the Fc region of a human IgG1 antibody. Another useful Fe polypeptide is the Fe mutein described in U.S. Pat. No. 5,457,035 and by Baum et al., EMBO J. 13:3992, 1994. The amino acid sequence of this mutein is identical to that of the native Fe sequence presented in WO 93/10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 has been changed from Gly to Ala. The mutein exhibits reduced affinity for Fe receptors. Fusion polypeptides comprising Fe moieties, and multimers formed therefrom, offer an advantage of facile purification by affinity chromatography over Protein A or Protein G columns, and Fe fusion polypeptides may provide a longer in vivo half life, which is useful in therapeutic applications, than unmodified polypeptides.
In other embodiments, a soluble Tek polypeptide may be substituted for the variable portion of an antibody heavy or light chain. If fusion proteins are made with both heavy and light chains of an antibody, it is possible to form a soluble Tek multimer with as many as four soluble Tek polypeptides.
Alternatively, the soluble Tek multimer is a fusion protein comprising multiple soluble Tek polypeptides, with or without peptide linkers (spacers), or peptides that have the property of promoting multimerization. Among the suitable peptide linkers are those described in U.S. Pat. Nos. 4,751,180, 4,935,233, and 5,073,627. A DNA sequence encoding a desired peptide linker may be inserted between, and in the same reading frame as, the DNA sequences encoding Tek, using conventional techniques known in the art. For example, a chemically synthesized oligonucleotide encoding the linker may be ligated between sequences encoding soluble Tek. In particular embodiments, a fusion protein comprises from two to four soluble Tek polypeptides, separated by peptide linkers.
Another method for preparing soluble Tek multimers involves use of a leucine zipper domain. Leucine zipper domains are peptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, 1988), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric proteins are described in PCT application WO 94/10308, and the leucine zipper derived from lung surfactant protein D (SPD) described in Hoppe et al. FEBS Lett. 344:191, 1994. The use of a modified leucine zipper that allows for stable trimerization of a heterologous protein fused thereto is described in Fanslow et al., Semin. Immunol. 6:267, 1994. Recombinant fusion proteins comprising a soluble Tek polypeptide fused to a leucine zipper peptide are expressed in suitable host cells, and the soluble Tek multimer that forms is recovered from the culture supernatant.
For some applications, the soluble Tek multimers of the present invention are believed to provide certain advantages over the use of monomeric forms, including the advantage of mimicking the natural interaction between a ligand and a receptor tyrosine kinase (RTK). In general, a dimer ligand will bind and cause dimerization of the RTK (van der Geer et al., Ann. Rev. Cell Biol. 10:251, 1994). This high affinity binding causes transphosphorylation of the RTK and the beginning of the signal transduction process. The binding of a soluble Tek multimer may occur at higher affinity than will a soluble Tek monomer. Fc fusion polypeptides offer an additional advantage in that this form typically exhibits an increased in vivo half life as compared to an unmodified polypeptide.
The present invention encompasses the use of various forms of soluble Tek multimers that retain the ability to inhibit angiogenesis or other Tek-mediated responses. The term “soluble Tek multimer” is intended to encompass multimers containing all or part of the native Tek extracellular domain, as well as related forms including, but not limited to, multimers of: (a) fragments, (b) variants, (c) derivatives, and (d) fusion polypeptides of soluble Tek. The ability of these related forms to inhibit angiogenesis or other Tek-mediated responses may be determined in vitro or in vivo, using methods such as those exemplified in the examples or using other assays known in the art.
Among the soluble Tek polypeptides and soluble Tek multimers useful in practicing the present invention are Tek variants that retain the ability to bind ligand and/or inhibit angiogenesis or other Tek-mediated responses. Such Tek variants include polypeptides that are substantially homologous to native Tek, but which have an amino acid sequence different from that of a native Tek because of one or more deletions, insertions or substitutions. Particular embodiments include, but are not limited to, Tek polypeptides that comprise from one to ten deletions, insertions or substitutions of amino acid residues, when compared to a native Tek sequence. Included as variants of Tek polypeptides are those variants that are naturally occurring, such as allelic forms and alternatively spliced forms, as well as variants that have been constructed by modifying the amino acid sequence of a Tek polypeptide or the nucleotide sequence of a nucleic acid encoding a Tek polypeptide.
Generally, substitutions for one or more amino acids present in the native polypeptide should be made conservatively. Examples of conservative substitutions include substitution of amino acids outside of the active domain(s), and substitution of amino acids that do not alter the secondary and/or tertiary structure of Tek. Additional examples include substituting one aliphatic residue for another, such as Ile, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gln and Asn, or substitutions of one aromatic residue for another, such as Phe, Trp, or Tyr for one another. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are known in the art.
The native sequence of the full length Tek extracellular domain is set forth as residues 23–745 of SEQ ID NO:1. In some preferred embodiments the Tek variant is at least about 70% identical in amino acid sequence to the amino acid sequence of native Tek; in some preferred embodiments the Tek variant is at least about 80% identical in amino acid sequence to the amino acid sequence of native Tek. In some more preferred embodiments the Tek variant is at least about 90% identical in amino acid sequence to the amino acid sequence of native Tek; in some more preferred embodiments the Tek variant is at least about 95% identical in amino acid sequence to the amino acid sequence of native Tek. In some most preferred embodiments the Tek variant is at least about 98% identical in amino acid sequence to the amino acid sequence of native Tek; in some most preferred embodiments the Tek variant is at least about 99% identical in amino acid sequence to the amino acid sequence of native Tek. Percent identity, in the case of both polypeptides and nucleic acids, may be determined by visual inspection. Percent identity may also be determined using the alignment method of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970) as revised by Smith and Waterman (Adv. Appl. Math 2:482, 1981. Preferably, percent identity is determined by using a computer program, for example, the GAP computer program version 10.x available from the Genetics Computer Group (GCG; Madison, Wis., see also Devereux et al., Nucl. Acids Res. 12:387, 1984). The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353–358, 1979 for amino acids; (2) a penalty of 30 (amino acids) or 50 (nucleotides) for each gap and an additional 1 (amino acids) or 3 (nucleotides) penalty for each symbol in each gap; (3) no penalty for end gaps; and (4) no maximum penalty for long gaps. Other programs used by one skilled in the art of sequence comparison may also be used. For fragments of Tek, the percent identity is calculated based on that portion of Tek that is present in the fragment.
The present invention further encompasses the use of soluble Tek polypeptides with or without associated native-pattern glycosylation. Tek expressed in yeast or mammalian expression systems (e.g., COS-1 or COS-7 cells) may be similar to or significantly different from a native Tek polypeptide in molecular weight and glycosylation pattern, depending upon the choice of expression system. Expression of Tek polypeptides in bacterial expression systems, such as E. coli, provides non-glycosylated molecules. Different host cells may also process polypeptides differentially, resulting in heterogeneous mixtures of polypeptides with variable N- or C-termini.
The primary amino acid structure of soluble Tek polypeptides may be modified to create derivatives by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives of Tek may be prepared by linking particular functional groups to Tek amino acid side chains or at the N-terminus or C-terminus of a Tek polypeptide.
Fusion polypeptides of soluble Tek that are useful in practicing the invention also include covalent or aggregative conjugates of a Tek polypeptide with other polypeptides added to provide novel polyfunctional entities.
D. Recombinant Production of Tek Polypeptides
Tek polypeptides, including soluble Tek polypeptides, fragments, and fusion polypeptides, used in the present invention may be prepared using a recombinant expression system. Host cells transformed with a recombinant expression vector (“recombinant host cells”) encoding the Tek polypeptide are cultured under conditions that promote expression of Tek and the Tek is recovered. Tek polypeptides can also be produced in transgenic plants or animals, or by chemical synthesis.
The invention encompasses nucleic acid molecules encoding the Tek polypeptides used in the invention, including: (a) nucleic acids that encode residues 23–472 of SEQ ID NO:2 and fragments thereof that bind a Tek ligand; (b) nucleic acids that are at least 70%, 80%, 90%, 95%, 98%, or 99% identical to a nucleic acid of (a), and which encode a polypeptide capable of binding at least one Tek ligand; and (c) nucleic acids that hybridize at moderate stringency to a nucleic acid of (a), and which encode a polypeptide capable of binding at least one Tek ligand.
Due to degeneracy of the genetic code, there can be considerable variation in nucleotide sequences encoding the same amino acid sequence. Included as embodiments of the invention are nucleic acid sequences capable of hybridizing under moderately stringent conditions (e.g., prewashing solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridization conditions of 50° C., 5×SSC, overnight) to the DNA sequences encoding Tek. The skilled artisan can determine additional combinations of salt and temperature that constitute moderate hybridization stringency (see also, Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1982; and Ausubel, Current Protocols in Molecular Biology, Wiley and Sons, 1989 and later versions, which are incorporated herein by reference). Conditions of higher stringency include higher temperatures for hybridization and post-hybridization washes, and/or lower salt concentration. Percent identity of nucleic acids may be determined using the methods described above for polypeptides, i.e., by methods including visual inspection and the use of computer programs such as GAP.
Any suitable expression system may be employed for the production of recombinant Tek. Recombinant expression vectors include DNA encoding a Tek polypeptide operably linked to suitable transcriptional and translational regulatory nucleotide sequences, such as those derived from a mammalian, microbial, viral, or insect gene. Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the Tek DNA sequence. Thus, a promoter nucleotide sequence is operably linked to a Tek DNA sequence if the promoter nucleotide sequence controls the transcription of the Tek DNA sequence. Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, an mRNA ribosomal binding site, and appropriate sequences which control transcription and translation initiation and termination. A sequence encoding an appropriate signal peptide (native or heterologous) can be incorporated into expression vectors. A DNA sequence for a signal peptide (referred to by a variety of names including secretory leader, leader peptide, or leader) may be fused in frame to the Tek sequence so that the Tek polypeptide is initially translated as a fusion protein comprising the signal peptide. A signal peptide that is functional in the intended host cells promotes extracellular secretion of the Tek polypeptide. The signal peptide is cleaved from the Tek polypeptide upon secretion of Tek from the cell.
Suitable host cells for expression of Tek polypeptides include prokaryotes, yeast and higher eukaryotic cells, including insect and mammalian cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, insect, and mammalian cellular hosts are described, for example, in Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985.
Prokaryotes include gram negative or gram positive organisms, for example, E. coli or Bacilli. Suitable prokaryotic host cells for transformation include, for example, E. coli, Bacillus subtilis, Salmonella typhimurium, and various other species within the genera Pseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic host cell, such as E. coli, Tek polypeptides may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-terminal Met may be cleaved from the expressed recombinant polypeptide.
Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker gene(s). A phenotypic selectable marker gene is, for example, a gene encoding a protein that confers antibiotic resistance or that supplies an autotrophic requirement. Examples of useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids such as the cloning vector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells. An appropriate promoter and a Tek DNA sequence are inserted into the pBR322 vector. Other commercially available vectors include, for example, pKK223–3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA).
Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include β-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615, 1978; Goeddel et al., Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, 1980; EP-A-36776) and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412, 1982). A particularly useful prokaryotic host cell expression system employs a phage λ PL promoter and a cI857ts thermolabile repressor sequence. Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the λ PL promoter include plasmid pHUB2 (resident in E. coli strain JMB9, ATCC 37092) and pPLc28 (resident in E. coli RR1, ATCC 53082).
Tek polypeptides may also be expressed in yeast host cells, preferably from the Saccharomyces genus (e.g., S. cerevisiae). Other genera of yeast, such as Pichia or Kluyveromyces, may also be employed. Yeast vectors will often contain an origin of replication sequence from a 2μ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; Holland et al., Biochem. 17:4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phospho-glucose isomerase, and glucokinase. Other suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EPA-73,657. Another alternative is the glucose-repressible ADH2 promoter described by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature 300:724, 1982). Shuttle vectors replicable in both yeast and E. coli may be constructed by inserting DNA sequences from pBR322 for selection and replication in E. coli (Ampr gene and origin of replication) into the above-described yeast vectors.
The yeast α-factor leader sequence may be employed to direct secretion of recombinant polypeptides. The α-factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. See, e.g., Kurjan et al., Cell 30:933, 1982; Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330, 1984. Other leader sequences suitable for facilitating secretion of recombinant polypeptides from yeast hosts are known to those of skill in the art. A leader sequence may be modified near its 3′ end to contain one or more restriction sites. This will facilitate fusion of the leader sequence to the structural gene.
Yeast transformation protocols are known to those of skill in the art. One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp+ transformants in a selective medium, wherein the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 μg/ml adenine and 20 μg/ml uracil.
Yeast host cells transformed by vectors containing an ADH2 promoter sequence may be grown for inducing expression in a “rich” medium. An example of a rich medium is one consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/ml uracil. Derepression of the ADH2 promoter occurs when glucose is exhausted from the medium.
Insect host cell culture systems also may be employed to express recombinant Tek polypeptides, including soluble Tek polypeptides. Bacculovirus systems for production of heterologous polypeptides in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47, 1988.
Mammalian cells are particularly preferred for use as host cells. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) as described by McMahan et al. (EMBO J. 10: 2821, 1991). For the production of therapeutic polypeptides it is particularly advantageous to use a mammalian host cell line which has been adapted to grow in media that does not contain animal proteins.
Established methods for introducing DNA into mammalian cells have been described (Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990, pp. 15–69). Additional protocols using commercially available reagents, such as Lipofectamine (Gibco/BRL) or Lipofectamine-Plus, can be used to transfect cells (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987). In addition, electroporation can be used to transfect mammalian cells using conventional procedures, such as those in Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1–3, Cold Spring Harbor Laboratory Press, 1989). Selection of stable transformants can be performed using methods known in the art, such as, for example, resistance to cytotoxic drugs. Kaufman et al., Meth. in Enzymology 185:487, 1990, describes several selection schemes, such as dihydrofolate reductase (DHFR) resistance. A suitable host strain for DHFR selection can be CHO strain DX-B11, which is deficient in DHFR (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216, 1980). A plasmid expressing the DHFR cDNA can be introduced into strain DX-B11, and only cells that contain the plasmid can grow in the appropriate selective media. Other examples of selectable markers that can be incorporated into an expression vector include cDNAs conferring resistance to antibiotics, such as G418 and hygromycin B. Cells harboring the vector can be selected on the basis of resistance to these compounds.
Transcriptional and translational control sequences for mammalian host cell expression vectors can be excised from viral genomes. Commonly used promoter sequences and enhancer sequences are derived from polyoma virus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites can be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell. Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment, which can also contain a viral origin of replication (Fiers et al., Nature 273:113, 1978; Kaufman, Meth. in Enzymology, 1990). Smaller or larger SV40 fragments can also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the Bgl I site located in the SV40 viral origin of replication site is included.
Additional control sequences shown to improve expression of heterologous genes from mammalian expression vectors include such elements as the expression augmenting sequence element (EASE) derived from CHO cells (Morris et al., Animal Cell Technology, 1997, pp. 529–534) and the tripartite leader (TPL) and VA gene RNAs from Adenovirus 2 (Gingeras et al., J. Biol. Chem. 257:13475, 1982). The internal ribosome entry site (IRES) sequences of viral origin allows dicistronic mRNAs to be translated efficiently (Oh and Sarnow, Current Opinion in Genetics and Development 3:295, 1993; Ramesh et al., Nucleic Acids Research 24:2697, 1996). Expression of a heterologous cDNA as part of a dicistronic mRNA followed by the gene for a selectable marker (e.g. DHFR) has been shown to improve transfectability of the host and expression of the heterologous cDNA (Kaufman, Meth. in Enzymology, 1990). Exemplary expression vectors that employ dicistronic mRNAs are pTR-DC/GFP described by Mosser et al., Biotechniques 22:150, 1997, and p2A5I described by Morris et al., Animal Cell Technology, 1997, pp. 529–534.
A useful high expression vector, pCAVNOT, has been described by Mosley et al., Cell 59:335, 1989. Other expression vectors for use in mammalian host cells can be constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983). A useful system for stable high level expression of mammalian cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al. (Mol. Immunol. 23:935, 1986). A useful high expression vector, PMLSV N1/N4, described by Cosman et al., Nature 312:768, 1984, has been deposited as ATCC 39890. Additional useful mammalian expression vectors are known in the art.
Regarding signal peptides that may be employed in producing Tek polypeptides, the native Tek signal peptide may used or it may be replaced by a heterologous signal peptide or leader sequence, if desired. The choice of signal peptide or leader may depend on factors such as the type of host cells in which the recombinant Tek is to be produced. Examples of heterologous signal peptides that are functional in mammalian host cells include the signal sequence for interleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195, the signal sequence for interleukin-2 receptor described in Cosman et al., Nature 312:768 (1984); the interleukin-4 receptor signal peptide described in EP 367,566; the type I interleukin-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; and the type II interleukin-1 receptor signal peptide described in EP 460,846.
Using the techniques of recombinant DNA including mutagenesis and the polymerase chain reaction (PCR), the skilled artisan can produce DNA sequences that encode Tek polypeptides comprising various additions or substitutions of amino acid residues or sequences, or deletions of terminal or internal residues or sequences, including Tek fragments, variants, derivatives, and fusion polypeptides.
Transgenic animals, including mice, goats, sheep, and pigs, and transgenic plants, including tobacco, tomato, legumes, grasses, and grains, may also be used as bioreactors for the production of Tek polypeptides, including soluble Tek polypeptides. In the case of transgenic animals, it is particularly advantageous to construct a chimeric DNA including a Tek coding sequence operably linked to cis-acting regulatory sequences that promote expression of the soluble Tek in milk and/or other body fluids (see, e.g., U.S. Pat. No. 5,843,705; U.S. Pat. No. 5,880,327). In the case of transgenic plants it is particularly advantageous to produce Tek in a particular cell type, tissue, or organ (see, e.g., U.S. Pat. No. 5,639,947; U.S. Pat. No. 5,889,189).
The skilled artisan will recognize that the procedure for purifying expressed soluble Tek polypeptides will vary according to the host system employed, and whether or not the recombinant polypeptide is secreted. Soluble Tek polypeptides may be purified using methods known in the art, including one or more concentration, salting-out, ion exchange, hydrophobic interaction, affinity purification, HPLC, or size exclusion chromatography steps. Fusion polypeptides comprising Fc moieties (and multimers formed therefrom) offer the advantage of facile purification by affinity chromatography over Protein A or Protein G columns.
E. Tek Antibodies
One aspect of the present invention relates to the antigenic epitopes of the Tek extracellular domain. Such epitopes are useful for raising antibodies, and in particular the blocking monoclonal antibodies described in more detail below. Such epitopes or variants thereof can be produced using techniques well known in the art such as solid-phase synthesis, chemical or enzymatic cleavage of a polypeptide, or using recombinant DNA technology. As exemplified below, the Inventors have determined that the Tek extracellular domain comprises at least three epitopes, and that antibodies generated against a deleted form of the Tek extracellular domain can compete with Tek ligands for binding to Tek.
The claimed invention encompasses compositions and uses of antibodies that are immunoreactive with Tek polypeptides. Such antibodies “bind specifically” to Tek polypeptides, meaning that they bind via antigen-binding sites of the antibody as compared to non-specific binding interactions. The terms “antibody” and “antibodies” are used herein in their broadest sense, and include, without limitation, intact monoclonal and polyclonal antibodies as well as fragments such as Fv, Fab, and F(ab′)2 fragments, single-chain antibodies such as scFv, and various chain combinations. The antibodies of the present invention are preferably humanized, and more preferably human. The antibodies may be prepared using a variety of well-known methods including, without limitation, immunization of animals having native or transgenic immune repertoires, phage display, hybridoma and recombinant cell culture, and transgenic plant and animal bioreactors.
Both polyclonal and monoclonal antibodies may be prepared by conventional techniques. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).
Hybridoma cell lines that produce monoclonal antibodies specific for the polypeptides of the invention are also contemplated herein. Such hybridomas may be produced and identified by conventional techniques. One method for producing such a hybridoma cell line comprises immunizing an animal with a polypeptide, harvesting spleen cells from the immunized animal, fusing said spleen cells to a myeloma cell line, thereby generating hybridoma cells, and identifying a hybridoma cell line that produces a monoclonal antibody that binds the polypeptide. The monoclonal antibodies produced by hybridomas may be recovered by conventional techniques.
The monoclonal antibodies of the present invention include chimeric antibodies, e.g., “humanized” versions of antibodies originally produced in mice or other non-human species. A humanized antibody is an engineered antibody that typically comprises the variable region of a non-human (e.g., murine) antibody, or at least complementarity determining regions (CDRs) thereof, and the remaining immunoglobulin portions derived from a human antibody. Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick et al. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS 14:139, May, 1993). Such humanized antibodies may be prepared by known techniques and offer the advantage of reduced immunogenicity when the antibodies are administered to humans.
Procedures that have been developed for generating human antibodies in non-human animals may be employed in producing antibodies of the present invention. The antibodies may be partially human or preferably completely human. For example, transgenic mice into which genetic material encoding one or more human immunoglobulin chains has been introduced may be employed. Such mice may be genetically altered in a variety of ways. The genetic manipulation may result in human immunoglobulin polypeptide chains replacing endogenous immunoglobulin chains in at least some, and preferably virtually all, antibodies produced by the animal upon immunization.
Mice in which one or more endogenous immunoglobulin genes have been inactivated by various means have been prepared. Human immunoglobulin genes have been introduced into the mice to replace the inactivated mouse genes. Antibodies produced in the animals incorporate human immunoglobulin polypeptide chains encoded by the human genetic material introduced into the animal. Examples of techniques for the production and use of such transgenic animals to make antibodies (which are sometimes called “transgenic antibodies”) are described in U.S. Pat. Nos. 5,814,318, 5,569,825, and 5,545,806, which are incorporated by reference herein.
F. Therapeutic Methods
The disclosed polypeptides, compositions, and methods are used to inhibit angiogenesis or other Tek-mediated responses in a mammal in need of such treatment. The term “Tek-mediated response” includes any cellular, physiological, or other biological response that is caused at least in part by the binding of a Tek ligand to Tek, or which may be inhibited or suppressed, in whole or in part, by blocking a Tek ligand from binding to Tek. The treatment is advantageously administered in order to prevent the onset or the recurrence of a disease or condition mediated by angiogenesis, or to treat a mammal that has a disease or condition mediated by angiogenesis. Diseases and conditions mediated by angiogenesis include but are not limited to ocular disorders, malignant and metastatic conditions, and inflammatory diseases.
Among the ocular disorders that can be treated according to the present invention are eye diseases characterized by ocular neovascularization including, but not limited to, diabetic retinopathy (a major complication of diabetes), retinopathy of prematurity (this devastating eye condition, that frequently leads to chronic vision problems and carries a high risk of blindness, is a severe complication during the care of premature infants), neovascular glaucoma, retinoblastoma, retrolental fibroplasia, rubeosis, uveitis, macular degeneration, and corneal graft neovascularization. Other eye inflammatory diseases, ocular tumors, and diseases associated with choroidal or iris neovascularization can also be treated according to the present invention.
The present invention can also be used to treat malignant and metastatic conditions such as solid tumors. Solid tumors include both primary and metastatic sarcomas and carcinomas.
The present invention can also be used to treat inflammatory diseases including, but not limited to, arthritis, rheumatism, and psoriasis.
Other diseases and conditions that can be treated according to the present invention include benign tumors and preneoplastic conditions, myocardial angiogenesis, hemophilic joints, scleroderma, vascular adhesions, atherosclerotic plaque neovascularization, telangiectasia, and wound granulation.
In addition to polypeptides comprising a fragment of Tek extracellular domain, soluble Tek multimers, and antibodies that bind to the Tek extracellular domain, other forms of Tek antagonists can also be administered to achieve a therapeutic effect. Examples of other forms of Tek antagonists include other antibodies such as antibodies against a Tek ligand, antisense nucleic acids, ribozymes, muteins, aptamers, and small molecules directed against Tek or against one or more of the Tek ligands.
The methods according to the present invention can be. tested in in vivo animal models to confirm the desired prophylactic or therapeutic activity, as well as to determine the optimal therapeutic dosage, prior to administration to humans.
The amount of a particular Tek antagonist that will be effective in a particular method of treatment depends upon age, type and severity of the condition to be treated, body weight, desired duration of treatment, method of administration, and other parameters. Effective dosages are determined by a physician or other qualified medical professional. Typical effective dosages are about 0.01 mg/kg to about 100 mg/kg body weight. In some preferred embodiments the dosage is about 0.1–50 mg/kg; in some preferred embodiments the dosage is about 0.5–10 mg/kg. The dosage for local administration is typically lower than for systemic administration. In some embodiments a single administration is sufficient; in some embodiments the Tek antagonist is administered as multiple doses over one or more days.
The Tek antagonists are typically administered in the form of a pharmaceutical composition comprising one or more pharmacologically acceptable carriers. Pharmaceutically acceptable carriers include diluents, fillers, adjuvants, excipients, and vehicles which are pharmaceutically acceptable for the route of administration, and may be aqueous or oleaginous suspensions formulated using suitable dispersing, wetting, and suspending agents.
Pharmaceutically acceptable carriers are generally sterile and free of pyrogenic agents, and may include water, oils, solvents, salts, sugars and other carbohydrates, emulsifying agents, buffering agents, antimicrobial agents, and chelating agents. The particular pharmaceutically acceptable carrier and the ratio of active compound to carrier are determined by the solubility and chemical properties of the composition, the mode of administration, and standard pharmaceutical practice.
The Tek antagonists are administered to the patient in a manner appropriate to the indication. Thus, for example, a Tek antagonist, or a pharmaceutical composition thereof, may be administered by intravenous, transdermal, intradermal, intraperitoneal, intramuscular, intranasal, epidural, oral, topical, subcutaneous, intracavity, sustained release from implants, peristaltic routes, or by any other suitable technique. Parenteral administration is preferred.
In certain embodiments of the claimed invention, the treatment further comprises treating the mammal with one or more additional chemotherapeutic agents. The additional chemotherapeutic agent(s) may be administered prior to, concurrently with, or following the administration of the Tek antagonist. The use of more than one chemotherapeutic agent is particularly advantageous when the mammal that is being treated has a solid tumor. In some embodiments of the claimed invention, the treatment further comprises treating the mammal with radiation. Radiation, including brachytherapy and teletherapy, may be administered prior to, concurrently with, or following the administration of the second chemotherapeutic agent(s) and/or Tek antagonist.
When the mammal that is being treated has a solid tumor, the method preferably includes the administration of, in addition to a Tek antagonist, one or more chemotherapeutic agents selected from the group consisting of alkylating agents, antimetabolites, vinca alkaloids and other plant-derived chemotherapeutics, nitrosoureas, antitumor antibiotics, antitumor enzymes, topoisomerase inhibitors, platinum analogs, adrenocortical suppressants, hormones, hormone agonists and antagonists, antibodies, immunotherapeutics, blood cell factors, radiotherapeutics, and biological response modifiers.
In some preferred embodiments the method includes administration of, in addition to a Tek antagonist, one or more chemotherapeutic agents selected from the group consisting of cisplatin, cyclophosphamide, mechloretamine, melphalan, bleomycin, carboplatin, fluorouracil, 5-fluorodeoxyuridine, methotrexate, taxol, asparaginase, vincristine, and vinblastine, lymphokines and cytokines such as interleukins, interferons (including alpha, beta, or delta), and TNF, chlorambucil, busulfan, carmustine, lomustine, semustine, streptozocin, dacarbazine, cytarabine, mercaptopurine, thioguanine, vindesine, etoposide, teniposide, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, L-asparaginase, hydroxyurea, methylhydrazine, mitotane, tamoxifen, and fluoxymesterone.
In some preferred embodiments the method includes administration of, in addition to a Tek antagonist, one or more chemotherapeutic agents, including various soluble forms thereof, selected from the group consisting of Flt3 ligand, CD40 ligand, interleukin-2, interleukin-12, 4-1BB ligand, anti-4-1BB antibodies, TNF antagonists and TNF receptor antagonists including TNFR/Fc, TWEAK antagonists and TWEAK-R antagonists including TWEAK-R/Fc, TRAIL, VEGF antagonists including anti-VEGF antibodies, VEGF receptor (including VEGF-R1 and VEGF-R2, also known as Flt1 and Flk1 or KDR) antagonists, and CD148 (also referred to as DEP-1, ECRTP, and PTPRJ, see Takahashi et al., J. Am. Soc. Nephrol. 10:2135–45, 1999) agonists.
In some preferred embodiments the Tek antagonists of the invention are used as a component of, or in combination with, “metronomic therapy,” such as that described by Browder et al. and Klement et al. (Cancer Research 60:1878, 2000; J. Clin. Invest. 105(8):R15, 2000; see also Barinaga, Science 288:245, 2000).
The polypeptides, compositions, and methods of the present invention may be used as a first line treatment, for the treatment of residual disease following primary therapy, or as an adjunct to other therapies including chemotherapy, surgery, radiation, and other therapeutic methods known in the art.
EXAMPLES
The following examples are intended to illustrate particular embodiments and not to limit the scope of the invention.
Example 1
Recombinant Production of Soluble Tek/Fc Fusion Polypeptides
The molecular cloning of a cDNA encoding the human receptor tyrosine kinase (RTK) Tek (ork, Tie2) is described in U.S. Pat. No. 5,447,860. The Tek cDNA (deposited with the American Type Culture Collection under the terms of the Budapest Treaty on May 28, 1992, Accession No. ATCC 69003) encodes 1124 amino acids, including a signal peptide, an N-terminal extracellular domain, a transmembrane domain, and a C-terminal cytoplasmic domain. Based on sequence analysis, the signal peptide is predicted to encompass residues 1–18, the N-terminal extracellular domain is predicted to encompass residues 19–745, the transmembrane domain is predicted to encompass residues 746–772, and the C-terminal cytoplasmic domain is predicted to encompass residues 773–1124. The extracellular domain includes two immunoglobulin (Ig)-like loops, a region containing three EGF-like cysteine repeats (between residues 211–340), and a region containing three fibronectin type III (FNIII) motifs (between residues 440–733). Tek cDNA was used to construct recombinant expression vectors for the production of various Tek/Fc fusion polypeptides.
To construct a nucleic acid encoding the full length Tek extracellular domain fused to Fc, a nucleic acid encoding the N-terminal 745 amino acids from Tek, including the Tek leader (signal peptide) and extracellular domain, was fused to a nucleic acid encoding a 232 amino acid Fc portion from human IgG1. The amino acid sequence of the Tek/Fc fusion polypeptide encoded by this construct is shown as SEQ ID NO:1. In SEQ ID NO:1, residues 1–18 are the predicted signal peptide (predicted to be cleaved upon secretion from the cell; the actual cleavage site was identified by N-terminal sequence analysis, see below), residues 19–745 are the Tek extracellular domain, and residues 746–977 are the Fc portion. Upon insertion into a mammalian expression vector, and expression in and secretion from a mammalian host cell, this construct produced a polypeptide designated Tek745/Fc. Based on the predicted signal peptide cleavage site, the amino acid sequence of Tek745/Fc was predicted to be residues 19–977 of SEQ ID NO:1.
To construct a nucleic acid encoding a fragment of the Tek extracellular domain fused to Fc, a nucleic acid encoding the N-terminal 472 amino acids from Tek, including the Tek leader (signal peptide) and a deleted extracellular domain, was fused to a nucleic acid encoding a 232 amino acid Fc portion from human IgG1. The amino acid sequence of the Tek/Fc fusion polypeptide encoded by this construct is shown as SEQ ID NO:2. In SEQ ID NO:2, residues 1–18 are the predicted signal peptide (predicted to be cleaved upon secretion from the cell; the actual cleavage site was identified by N-terminal sequence analysis, see below), residues 19–472 are the fragment of the Tek extracellular domain, and residues 473–704 are the Fc portion. Upon insertion into a mammalian expression vector, and expression in and secretion from a mammalian host cell, this construct produced a polypeptide designated Tek472/Fc. Based on the predicted signal peptide cleavage site, the amino acid sequence of Tek472/Fc was predicted to be residues 19–704 of SEQ ID NO:2.
Nucleic acids encoding each of the Tek/Fc fusion polypeptides were inserted into mammalian expression vectors, and each vector was transfected into CHO cells. After amplification, stably transfected CHO cell lines were cultured under conditions promoting the expression and secretion of the recombinant fusion polypeptides and the Tek/Fc fusion polypeptides were recovered and isolated from the culture medium. N-terminal sequence analysis determined that the secreted polypeptide designated Tek745/Fc had an N-terminus corresponding to residue 23 (alanine) of SEQ ID NO:1. N-terminal sequence analysis determined that the secreted polypeptide designated Tek472/Fc had an N-terminus corresponding to residue 23 (alanine) of SEQ ID NO:2.
Anti-angiogenic activity of the Tek/Fc fusion polypeptides is demonstrated in the in vitro and in vivo systems described in Examples 2–6.
Example 2
Activity of Tek/Fc in a Wound Closure Assay
A planar endothelial cell migration (wound closure) assay was used to quantitate the inhibition of angiogenesis by Tek/Fc in vitro. In this assay, endothelial cell migration is measured as the rate of closure of a circular wound in a cultured cell monolayer. The rate of wound closure is linear, and is dynamically regulated by agents that stimulate and inhibit angiogenesis in vivo.
Primary human renal microvascular endothelial cells, HRMEC, were isolated, cultured, and used at the third passage after thawing, as described in Martin et al., In Vitro Cell Dev Biol 33:261, 1997. Replicate circular lesions, “wounds,” (600–800 micron diameter) were generated in confluent HRMEC monolayers using a silicon-tipped drill press. At the time of wounding the medium (DMEM+1% BSA) was supplemented with 20 ng/ml PMA (phorbol-12-myristate-13-acetate), 10 μg/ml Tek472/Fc, or combinations of 20 ng/ml PMA and 0.001–10 μg/ml Tek472/Fc. The residual wound area was measured as a function of time (0–12 hours) using a microscope and image analysis software (Bioquant, Nashville, Tenn.). The relative migration rate was calculated for each agent and combination of agents by linear regression of residual wound area plotted over time. The results are shown in FIG. 1. Tek472/Fc inhibited PMA-induced endothelial migration in a dose responsive manner, reducing the rate of migration to unstimulated levels at 10 μg/ml.
Example 3
Activity of Tek/Fc in a Corneal Pocket Assay
A mouse corneal pocket assay was used to quantitate the inhibition of angiogenesis by Tek/Fc in vivo. In this assay, agents to be tested for angiogenic or anti-angiogenic activity are immobilized in a slow release form in a hydron pellet, which is implanted into micropockets created in the corneal epithelium of anesthetized mice. Vascularization is measured as the appearance, density, and extent of vessel ingrowth from the vascularized corneal limbus into the normally avascular cornea.
Hydron pellets, as described in Kenyon et al., Invest Opthamol. & Visual Science 37:1625, 1996, incorporated sucralfate with bFGF (90 ng/pellet), bFGF and IgG (11 μg/pellet, control), or bFGF and Tek472/Fc (12.8 μg). The pellets were surgically implanted into corneal stromal micropockets created by micro-dissection 1 mm medial to the lateral corneal limbus of 6–8 week old male C57BL mice. After five days, at the peak of neovascular response to bFGF, the corneas were photographed, using a Zeiss slit lamp, at an incipient angle of 35–50° from the polar axis in the meridian containing the pellet. Images were digitized and processed by subtractive color filters (Adobe Photoshop 4.0) to delineate established microvessels by hemoglobin content. Image analysis software (Bioquant, Nashville, Tenn.) was used to calculate the fraction of the corneal image that was vascularized, the vessel density within the vascularized area, and the vessel density within the total cornea. The results are shown in FIG. 2. Tek472/Fc (50 pmol) inhibited bFGF (3 pmol)-induced corneal angiogenesis, reducing the vascular density to 30% of that induced by FGF alone.
Example 4
Inhibition of Neovascularization by Tek/Fc in a Murine Transplant Model
Survival of heterotopically transplanted cardiac tissue from one mouse donor to the ear skin of another genetically similar mouse requires adequate neovascularization by the transplanted heart and the surrounding tissue, to promote survival and energy for cardiac muscle function. Inadequate vasculature at the site of transplant causes excessive ischemia to the heart, tissue damage, and failure of the tissue to engraft. Agents that antagonize the angiopoietins and endothelial specific factors involved in endothelial cell migration and vessel formation can decrease angiogenesis at the site of transplant, thereby limiting graft tissue function and ultimately engraftment itself.
The following studies were carried out, utilizing a murine heterotopic cardiac isograft model, in order to demonstrate the antagonistic effects of Tek/Fc on neovascularization. In all experiments, female BALB/c (≈12 weeks of age) recipients received neonatal heart grafts from donor mice of the same strain.
A. Tek/Fc at 500 μg/Day Dose
In each of three experiments, the donor heart tissue was engrafted into the left ear pinnae of the recipient on day 0 and the mice were divided into two groups. The control group received human IgG (Hu IgG) while the other group received human Tek472/Fc, both intraperitoneally at 500 μg per day. All treatments began on day 0 and continued for five consecutive days. The functionality of the grafts was determined by monitoring visible pulsatile activity on days 7 and 14 post-engraftment.
Table 1 shows the cumulative results from the three experiments. All 8 mice receiving Hu IgG had functioning grafts on days 7 and 14, indicating 100% engraftment. The Tek472/Fc treated mice initially demonstrated no functional activity, indicative of diminished engraftment, with only 36% having functioning grafts at day 7. By day 14, ten days after cessation of Tek472/Fc treatment, 82% of the mice had functioning grafts.
TABLE 1
Functional Engraftment at Days 7 and 14
Total
Treatment
Day 7
Day 14
N = 8
Hu IgG
8/8 (100%)
8/8 (100%)
N = 11
Tek472/Fc
4/11 (36%)
9/11 (82%)
Histological studies on the transplanted hearts of mice receiving the Tek/Fc showed increased edema at the site of transplant, indicative of vascular leakage, and decreased host and donor tissue vasculature staining (Factor VIII) as compared to that observed in transplanted hearts from mice receiving the control protein IgG.
This experiment showed that treatment with Tek472/Fc severely compromised cardiac isograft function and prevented engraftment of tissue in 64% of mice at day 7 after a 5 day course of therapy.
B. Tek/Fc Dose Titration
Three different doses of Tek/Fc were tested in the cardiac isograft model described above. Each test group contained four female BALB/c mice. The control group received human IgG (Hu IgG), intraperitoneally, at 500 μg per day for five consecutive days. The Tek/Fc groups received human Tek472/Fc, intraperitoneally, at 90, 250, or 500 μg per day for five consecutive days. The functionality of the grafts was determined by monitoring visible pulsatile activity on post-engraftment days 7, 11, 14, 17, and 21. The results are shown in Table 2.
TABLE 2
Functional Engraftment Following Dose Titration with Tek
Treatment
Day 7
Day 11
Day 14
Day 17
Day 21
Hu IgG
100*
100
100
100
100
500 μg
Tek472/Fc
75
00
100
100
100
90 μg
Tek472/Fc
25
75
75
100
100
250 μg
Tek472/Fc
25
75
75
75
75
500 μg
*all results are reported as percent of mice with pulsatile heart grafts
A similar magnitude of cardiac isograft engraftment disruption was observed at both the 250 μg and 500 μg doses of Tek/Fc, as compared to Hu IgG control where no effect on engraftment was observed. A small, albeit significantly insignificant, reduction in engraftment was observed at the 90 μg dose.
C. Tek/Fc in Combination with a VEGF Antagonist
The anti-angiogenic activity of Tek/Fc in combination with an anti-murine VEGF monoclonal antibody was tested in the cardiac isograft model. The antibody, JH121 (Lab Vision Corporation, Fremont, Calif.), is an IgG1 that recognizes various isoforms of VEGF and neutralizes the bioactivity of VEGF. Each treatment group contained five mice. The mice were administered control protein (Hu IgG, 250 micrograms per day), anti-murine VEGF antibody (100 micrograms per day), Tek472/Fc (250 micrograms per day), or the combination of anti-murine VEGF antibody (100 micrograms per day) and Tek472/Fc (250 micrograms per day) intraperitoneally for five consecutive days starting at day 0, the day of the cardiac transplant. The effect of each treatment on cardiac isoengraftment/neovascularization was compared by determining functional engraftment on day 7 post transplant. The results are shown in Table 3.
TABLE 3
Functional Engraftment Following Treatment With
Tek/Fc and Anti-VEGF Antibody
Treatment
Engraftment at Day 7
Hu IgG
5/5 (100%)
250 μg
Anti-VEGF
2/5 (40%)
100 μg
Tek472/Fc
2/5 (40%)
250 μg
Anti-VEGF and
0/5 (0%)
Tek472/Fc
Both anti-VEGF and Tek/Fc were effective antiangiogenic agents, and treatment with the two agents in combination had a more pronounced biological effect than either agent administered alone. These results indicate that in combination lower doses of Tek antagonists and/or VEGF antagonists may be used to achieve significant biological antiangiogenic responses in vivo.
Example 5
Treatment of Tumors with Tek472/Fc
A. Tek/Fc Alone and in Combination with a Second Chemotherapeutic Agent
Tek472/Fc was administered alone, and in combination with Flt3L, to treat mice bearing 87 fibrosarcoma or B10.2 fibrosarcoma tumors. The B10.2 and 87 tumors are of the progressor phenotype, i.e. they grow progressively in normal mice. The B10.2 fibrosarcoma was induced by subcutaneous implantation of a paraffin pellet containing 5 mg of methylcholanthrene in C57BL mice (Lynch and Miller, Eur. J. Immunol., 21:1403, 1991). The 87 fibrosarcoma is a progressor variant of a tumor induced by chronic exposure of C3H/HeN mice to UVB irradiation. To innoculate tumors in mice for these experiments, 5×105 cells were injected (day 0) intradermally in the abdomen (see, also, Borges et al., J. Immunol. 163:1289, 1999, which is incorporated herein by reference).
The 87 fibrosarcoma tumors in C3H/HeN mice were treated with MSA (murine serum albumin, control), Tek/Fc (312 μg/day, days 4–19 after tumor cell injection), Flt3L (10 μg/day, days 1–19 after tumor cell injection), or a combination of Tek/Fc and Flt3L (Tek/Fc at 312 μg/day, days 4–19; Flt3L at 10 μg/day, days 1–19 after tumor cell injection). Each treatment group consisted of ten mice. Tumor frequency and tumor size were measured weekly for five weeks. The results are shown in FIG. 3. Mice treated with the combination of Tek/Fc and Flt3L showed the slowest tumor growth rates. In week 6 an additional animal in the Tek/Fc plus Flt3L group rejected the tumor, decreasing the tumor frequency to 68%. Based upon the results of this experiment, the combination of Tek/Fc and Flt3L was used to treat pre-existing B10.2 fibrosarcoma tumors.
The B10.2 fibrosarcoma tumors in C57BL/10 mice were treated with MSA (control), Tek/Fc (625 μg/day, days 7–32 after tumor cell injection), Flt3L (10 μg/day, days 7–26 after tumor cell injection combination of Tek/Fc and Flt3L (Tek/Fc at 312.5 or 625 μg/day, days 7–32; Flt3L at 10 μg/day, days 7–26 after tumor cell injection). Each treatment group consisted of ten mice. Tumor frequency and tumor size were measured weekly for six weeks. The results are shown in FIG. 4. Mice treated with both combinations of Tek/Fc and Flt3L showed reduced tumor growth rates; mice treated with 625 μg/day Tek/Fc in combination with Flt3L showed the slowest tumor growth rate.
B. Tek/Fc Alone and in Combination with Ionizing Radiation
3LL Lewis lung adenocarcinoma (1×105 cells) were innoculated in the foot pad of C57B1/6 mice. Mice with palpable tumors (2–3 weeks after innoculation, <5 mm diameter) were treated with either Hu IgG or Tek472/Fc (500 μg, intraperitoneally, per day) for 21 days. Initial tumor volume (Vo) was established for each mouse prior to the initiation of therapy. Radiation therapy (RT) was started on day 8 of Tek472/Fc and/or IgG treatment. The RT regimen comprised 6 Gy/day for 4 days/week to a total of 48 Gy. Tumor size was determined biweekly. Animals were sacrificed one week after the completion of Tek472/Fc treatment (day 28), and the mean relative or fractional tumor volume (Vf/Vo) was determined. Vf/Vo is equal to the final tumor volume of the primary footpad tumor in each mouse on day 28 divided by the initial tumor volume determined on the day of therapy initiation.
The results are shown in Tables 4 and 5. Administration of Tek/Fc, as a single agent, to mice bearing established 3LL tumors decreased tumor growth by almost 50% compared to Hu IgG treatment alone (p=0.035). Radiation therapy was also effective at slowing tumor growth in this 3LL model. When Tek/Fc treatment was combined with radiation therapy, tumor growth was inhibited significantly more than was the tumor growth after Tek/Fc treatment alone or radiation +IgG treatment (p<0.001).
TABLE 4
Summary of
Fractional Tumor Volume (Vf/V0) as a Function of Treatment
Treatment Group
Number (n)
Mean Vf/V0
S.D.
SEM
Control
5
62.51
34.88
15.59
IgG
4
42.19
11.92
5.33
RT
5
20.93
9.5
4.25
IgG + RT
15
14.98
5.77
1.49
Tek472/Fc
10
24.85
12.43
3.93
Tek472/Fc + RT*
14
6.76
3.29
0.88
*One way analysis of variance, p < 0.001
TABLE 5
Comparison Between Groups*
Treatment Group
p Value
Tek/Fc vs. Control (no treatment)
0.008
Tek/Fc vs. IgG
0.035
Tek/Fc vs. RT
0.548
Tek/Fc + RT vs. Tek/Fc
<0.001
Tek/Fc + RT vs. RT
<0.001
Tek/Fc + RT vs. IgG + RT
<0.001
*p values after t-test
Example 6
Binding of Tek/Fc Fusion Polypeptides to Angiopoietin
Both Tek745/Fc and Tek472/Fc were examined for the ability to bind the human Tek ligand angiopoietin 2 (Ang2), using a solid-phase plate binding assay based on time-resolved fluorescence. Comparison of binding to human Ang2 with the two different forms of soluble Tek/Fc revealed that Tek472/Fc bound significantly better (21-fold better) to Ang2 than did Tek745/Fc.
Low fluorescence 8×12 strip microtiter plate wells (Perkin-Wallac, Ackron, Ohio) were incubated with human Ang2 (R&D systems) at 500 ng/ml (100 μl) overnight at 2–8° C. The wells were then blocked by the addition of 100 μl of 1% BSA/PBS solution for 1 hour at room temperature. Following a 4×PBS-T (PBS-Tween 20 0.05%) wash, samples containing Tek745/Fc, Tek472/Fc, or TNFR/Fc (control/Fc) were titrated in diluent (1% BSA/PBS), in duplicate, beginning at 30 μg/ml in 3-fold dilutions. The samples were allowed to bind for 1 hour at room temperature with gentle agitation and then the unbound material was washed away 4×with PBS-T. Bound Tek/Fc was detected by adding goat anti human IgG-Europium conjugate (Perkin-Wallac), diluted to 100 ng/ml in assay buffer, to the wells and incubating for 30 minutes at room temperature. Unbound goat anti-human IgG-Europium was removed by a 4×PBS-T wash. Following the wash 150 μl of Enhancement solution (Perkin Wallac) was added to each well and the plate allowed to incubate at room temperature for a minimum of 5 minutes. Binding was determined by reading the fluorescence emitted from each well on a Victor II Multilabel counter equipped with software and light excitation/emission devices to measure Europium-derived fluorescence. The results, expressed as fluorescence counts, are shown in FIG. 5.
The TNFR/Fc control did not exhibit detectable binding (over that observed for background) to human Ang2. Both Tek472/Fc and Tek745/Fc bound to human Ang2 in a concentration dependent manner, but Tek472/Fc had a higher binding affinity. Tek472/Fc bound greater than 20-fold better than Tek745/Fc, based on mass concentration. Much higher concentrations of Tek745/Fc were required to achieve the same level of binding observed at lower concentrations of Tek472/Fc. The BC40K (the concentration of Tek/Fc required to achieve 40,000 fluorescence counts of huANG-2 binding) for Tek745/Fc was 20,596 ng/ml, compared to the BC40K for Tek472/Fc which was 994 ng/ml.
Example 7
Tek-Specific Blocking Monoclonal Antibodies
A. Antibodies to Tek472/Fc
Antibodies against “recombinant Tie2 extracellular domain-Fc fusion” have been described by Holmes et al., WO 00/18437. The present Inventors, in contrast, made antibodies against the deleted Tek extracellular domain fusion polypeptide Tek472/Fc. As shown in example 6, Tek472/Fc binds Tek ligand with higher affinity than does Tek745/Fc.
BALB/c mice were immunized with the Tek/Fc fusion polypeptide Tek472/Fc described in Example 1. Spleen cells were collected and used to prepare hybridomas using standard procedures. Hybridoma supernatants were screened, using ELISA, for the ability to bind (a) Tek472/Fc and (b) CV1 cells expressing human Tek. Positives were cloned two times, to insure monoclonality, then isotyped and reassayed for reactivity to Tek.
Three antibodies were chosen for further experiments: M530 (IgG2b isotype), M531 (IgG2b isotype), and M532 (IgG1 isotype). M530 and M531 appear to recognize the same epitope and M532 recognizes a second (different) epitope. M530 and M532 were therefore used as an antibody pair (e.g., for capture and detection) in various immunoassays. M530 was shown (by immunoprecipitation and by solid phase plate binding assays) to bind Tek745/Fc, Tek472/Fc, and to bind to naturally occurring Tek as expressed on the surface of human endothelial cells. The M530 antibody was further characterized in the binding and epitope mapping studies described in Example 8, below.
B. Additional Tek Antibodies
A workshop panel of putative endothelial cell-specific antibodies, which were not yet clustered, was obtained from the Human Leukocyte Differentiation Antigens (HLDA) Workshop. Some of the antibodies were generated by immunizing mice with human endothelial cells. One antibody in the panel was known to react with human Tek. These antibodies were further characterized in the binding and epitope mapping studies described in Example 8, below.
Example 8
Tek Antibody Binding to Tek and to Human Microvascular Endothelial Cells
A. Antibody Binding to Full Length Tek Extracellular Domain and to Endothelial Cells
Using a solid phase binding assay (time resolved fluorescence, as described in Example 6), the huTek monoclonal antibody M530 described in Example 7A and eight monoclonal antibodies described in Example 7B (endothelial cell-specific antibodies numbered WS#70098, #70099, #70100, #70101, #70104, #70108, #70112, and putative Tek-specific antibody #70637) bound specifically to the full length Tek extracellular fusion polypeptide Tek745/Fc. An IgG1 negative control mAb (MOPC21) did not bind to Tek745/Fc over background. Two other endothelial cell-specific workshop antibodies (WS#70110 and #70115 did not detectably bind to Tek745/Fc.
Using flow cytometry, the human Tek monoclonal antibodies M530, M531, and M532 described in Example 7A and eight monoclonal antibodies described in Example 7B (endothelial cell-specific antibodies numbered #70098, #70099, #70100, #70101, #70104, #70108, #70112, and Tek-specific antibody #70637) were shown to bind to naturally occurring Tek as expressed on human endothelial cells (both human microvascular endothelial cells from adult skin and HUVEC).
B. Antibody Binding to a Tek Extracellular Domain Lacking FN3 Motifs
The monoclonal antibody M530 described in Example 7A and seven monoclonal antibodies described in Example 7B (endothelial cell-specific antibodies numbered #70098, #70099, #70100, #70101, #70104, #70108, and #70112) bound specifically to the deleted Tek extracellular fusion polypeptide Tek472/Fc. Workshop antibodies #70637 (which bound to Tek745/Fc), #70110, and #70115 did not bind to Tek472/Fc.
C. Competitive Inhibition of Antibody Binding by Tek Ligands
Angiopoietin-1 (Ang1, Davis et al., Cell 87:1161, 1996) and Angiopoietin-2 (Ang2, Maisonpierre et al., Science 277:55, 1997) are two closely related Tek ligands. Both Ang1 and Ang2 bind with similar affinity to human Tek. The addition of a molar excess of Ang2 to EC cultures in the presence of Ang1 has been shown to inhibit Ang1 induced activation of Tek on endothelial cells via competition of Ang1 binding to endothelial cells (Maisonpierre et al., Science 277:55, 1997). A recombinant human angiopoietin-2 preparation was obtained from R&D Systems, Inc. (Minneapolis, Minn.). According to the manufacturer, the angiopoietin-2 preparation migrates as a 66 kDa protein in SDS-PAGE under both reducing and non-reducing conditions. Based on N-terminal amino acid sequencing, the preparation contains two peptides: a major polypeptide (75% of the total) having Asp68 as its N-terminus and a minor polypeptide (25% of the total) having Tyr19 as its N-terminus.
The ability of this Ang2 preparation to competitively inhibit the binding of Tek antibodies to Tek expressed on skin human microvascular endothelial cells was tested using flow cytometry.
Each mAb was added to 500,000 HMVEC-d at 5 μg/ml in 12×75 mm falcon tubes in duplicate and allowed to incubate for 15 minutes at 4° C. in binding medium. To one set of the duplicates, human Ang2 was added at 10 μg/ml (a five-fold molar excess) for an additional 30 minutes. The cells with the bound Tek mAb were then washed in 20 volumes of PBS-containing wash buffer. After the wash step, bound mouse mAb was detected by the addition of F(ab′2) sheep anti mouse IgG-PE fluorescent conjugate to the cells, followed by a 30 minute incubation at 4° C. and an additional 20 volume wash. Binding of the Tek mAb was measured by flow cytometric analysis on a single-laser FACSCAN (Becton Dickinson, Sunnyvale Calif.). The percent inhibition of antibody binding was calculated using the formula:
MFI(no Ang2)−MFI(+Ang2)/MFI(noAng2)×100.
The results are shown in Table 6.
TABLE 6
Inhibition of Tek Antibody Binding by Ang2
Monoclonal Antibody
Percent Inhibition of Antibody
5 μg/ml
Binding by Ang2
negative control (MOPC-21)
0
binding control (αvβ3)
6.4
M530
41.6
#70098
45.9
#70099
44.4
#70100
0
#70101
38.7
#70104
6.3
#70108
50.8
#70112
47.6
#70637
0
These results, inhibition of Tek antibody binding by Ang2, suggest that the M530, #70098, #70099, #70101, #70108, and #70112 antibodies bind at or near the Tek ligand binding site. The mAbs M530, WS#70099 and #70112 were also able to inhibit Ang2 binding (100 ng/ml) to recombinant human Tek472/Fc, by greater than 50% for mAb M530 and #70112 at concentrations of 10 μg/ml or greater and for mAb 70099 at concentrations of 3 μg/ml or greater.
In combination the binding results described in this example define at least three antibody epitopes in the human Tek extracellular domain, and exemplify the utility of preparing antibodies using a fragment of the Tek extracellular domain that lacks all or part of the region containing fibronectin type III (FNIII) motifs as an immunogen/target.
The relevant disclosures of publications cited herein are specifically incorporated by reference. The examples presented above are not intended to be exhaustive or to limit the scope of the invention. The skilled artisan will understand that variations and modifications and variations are possible in light of the above teachings, and such modifications and variations are intended to be within the scope of the invention.
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10357653
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immunex corporation
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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514/2
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Mar 31st, 2022 02:17PM
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Mar 31st, 2022 02:17PM
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Amgen
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:amgn
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Amgen
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Jul 17th, 2007 12:00AM
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Dec 19th, 2003 12:00AM
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https://www.uspto.gov?id=US07244822-20070717
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BTL-II proteins
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The invention provides isolated BTL-II proteins, nucleic acids, antibodies, antagonists, and agonists and methods of making and using the same. Diagnostic, screening, and therapeutic methods using the compositions of the invention are provided. For example, the compositions of the invention can be used for diagnosis and treatment of inflammatory bowel diseases and for enhancing a mucosal immune response to an antigen.
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7244822
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1. An isolated BTL-II protein comprising an amino acid sequence consisting of amino acids 29 to 457 of SEQ ID NO:4.
2. The protein of claim 1 comprising amino acids 29 to 482 of SEQ ID NO:4.
3. An isolated protein comprising a polypeptide consisting of an amino acid sequence at least 85% identical to amino acids 30 to 358 of SEQ ID NO:10, wherein the identity region of the amino acid sequence aligned with amino acids 30 to 358 of SEQ ID NO:10, is at least 150 amino acids,
wherein the amino acid sequence is at least 85% identical to amino acids 127 to 157 of SEQ ID NO:10, wherein the identity region of the amino acid sequence aligned with amino acids 127 to 157 of SEQ ID NO:10, is at least 20 amino acids long, and
wherein the protein can inhibit the proliferation of T cells induced by an anti-CD3 antibody.
4. The protein of claim 3, wherein the amino acid sequence is at least 90% identical to amino acids 30 to 358 of SEQ ID NO:10.
5. The protein of claim 3, wherein the amino acid sequence is at least 90% identical to amino acids 127 to 157 of SEQ ID NO:10.
6. An isolated protein comprising a polypeptide consisting of an amino acid sequence at least 85% identical to amino acids 32 to 358 of SEQ ID NO:10,
wherein the identity region of the amino acid sequence aligned with amino acids 32 to 358 of SEQ ID NO:10 is at least 275 amino acids, and
wherein the protein can inhibit the proliferation of T cells induced by an anti-CD3 antibody.
7. The protein of claim 6, wherein the amino acid sequence is at least 90% identical to amino acids 32 to 358 of SEQ ID NO:10.
8. An isolated protein comprising a first polypeptide consisting of a first amino acid sequence at least 85% identical to amino acids 32 to 358 of SEQ ID NO:10, wherein the identity region of the first amino acid sequence aligned to SEQ ID NO:10 is at least about 175 amino acids long,
wherein the first amino acid sequence is not more than 390 amino acids in length,
wherein the protein can inhibit the proliferation of T cells induced by an anti-CD3 antibody,
wherein the first amino acid sequence is not at least 85% identical to amino acids 148 to 232 of SEQ ID NO:4 with an identity region of the first amino acid sequence aligned to amino acids 148 to 232 of SEQ ID NO:4 of at least about 40 amino acids, and
wherein the protein does not comprise a second amino acid sequence that is at least 85% identical to amino acids 148 to 232 of SEQ ID NO:4 with an identity region of the second amino acid sequence aligned to amino acids 148 to 232 of SEQ ID NO:4 of at least about 40 amino acids.
9. The protein of claim 8, wherein the first amino acid sequence is not more than about 270 amino acids in length.
10. The protein of claim 8, wherein the first amino acid sequence is at least 90% identical to SEQ ID NO:10.
11. The protein of claim 1 further comprising another polypeptide.
12. The protein of claim 11, wherein the other polypeptide is an Fc region of an antibody.
13. The protein of claim 3 further comprising another polypeptide.
14. The protein of claim 13, wherein the other polypeptide is an Fc region of an antibody.
15. The protein of claim 8 further comprising another polypeptide.
16. The protein of claim 15, wherein the other polypeptide is an Fc region of an antibody.
17. An immunogenic fragment of SEQ ID NO:10,
wherein the immunogenic fragment is capable of eliciting antibodies that bind specifically to a protein consisting of amino acids 30–358 of SEQ ID NO:10,
wherein the immunogenic fragment is at least 10 amino acids long, and
wherein the immunogenic fragment spans a portion of SEQ ID NO:10 encode by the splice junction between nucleotides 427 and 428 of SEQ ID NO:9.
18. An immunogenic fragment of SEQ ID NO:10, wherein the immunogenic fragment spans positions 141 to 143 of SEQ ID NO:10,
wherein the immunogenic fragment is capable of eliciting antibodies that bind specifically to a protein consisting of amino acids 30 to 358 of SEQ ID NO:10, and
wherein the immunogenic fragment is at least 10 amino acids long.
19. An isolated protein comprising an amino acid sequence identical to amino acids 32 to 358 of SEQ ID NO:10.
20. The protein of claim 19 comprising amino acids 30 to 358 of SEQ ID NO:10.
21. The protein of claim 4, wherein the amino acid sequence is at least 94% identical to amino acids 30 to 358 of SEQ ID N:10.
22. The protein of claim 5, wherein the amino acid sequence is at least 94% identical to amino acids 127 to 157 of SEQ ID NO:10.
23. The protein of claim 7, wherein the amino acid sequence is at least about 96% identical to amino acids 32–358 of SEQ ID NO:10.
24. The protein of claim 10, wherein the first amino acid sequence is at least 94% identical to SEQ ID NO:10.
25. The protein of claim 6 further comprising another polypeptide.
26. The protein of claim 25, wherein the other polypeptide is an Fc region of an antibody.
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26
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This application claims benefit of U.S. Provisional Application No. 60/436,185, filed Dec. 23, 2002 and U.S. Provisional Application No. 60/525,298, filed Nov. 26, 2003.
FIELD OF THE INVENTION
The invention relates to butyrophilin-like proteins, specifically butyrophilin-like proteins of the B7 subfamily, which are known to modulate the function of immune effector cells such as, for example, B cells and/or T cells. Nucleic acids encoding such proteins, processes for producing such proteins, antibodies that bind to such proteins, pharmaceutical compositions containing such proteins or antibodies, methods of using such nucleic acids, proteins, and antibodies against such proteins are also included.
BACKGROUND
Modulation of an immune or inflammatory response can be a valuable tool in controlling various kinds of diseases including autoimmune diseases, diseases characterized by abnormal inflammation and/or immune response, and infections. In treating diseases characterized by abnormal inflammation and/or immune responses, such as inflammatory bowel diseases and autoimmune or inflammatory diseases, down-modulation of an immune response is desirable. In other situations, for example when vaccinating a patient to impart immunity to an infectious disease, stimulation of an immune response is desirable. In the vaccine setting, adjuvants that can heighten an immune response to a coadministered antigen can be valuable in providing long term protection against disease. Particularly lacking in the art are adjuvants capable of stimulating a mucosal immune response. A mucosal immune response, as opposed to a systemic immune response, is valuable because it can attack an infection at a very common point of entry, that is, at a mucosal surface. The present invention addresses these needs in the art by providing therapeutic agents to diagnose and treat diseases characterized by inappropriate and/or abnormal inflammation and/or immune responses and therapeutic agents that can act as adjuvants to stimulate an immune response, particularly a mucosal immune response.
SUMMARY
The invention encompasses isolated BTL-II proteins, nucleic acids, antibodies, BTL-II inhibitors and agonists, and methods for using these compositions.
An isolated BTL-II protein comprising an amino acid sequence consisting of amino acids x–y of SEQ ID NO:4, wherein x is any amino acid from position 1 to 35 of SEQ ID NO:4 and y is any amino acid from position 452–462 of SEQ ID NO:4 is provided. Such a BTL-II protein can comprise amino acids 30 to 453, 1 to 453, 29 to 457, 1 to 457, 1 to 482, and/or 29 to 482 of SEQ ID NO:4. The invention further provides an isolated BTL-II protein comprising a polypeptide consisting of an amino acid sequence at least 80%, optionally at least 85%, 90%, 92%, 94%, 96%, or 98%, identical to amino acids 127 to 157 of SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:18 or amino acids 126 to 156 of SEQ ID NO:16, wherein the identity region of the amino acid sequence aligned with amino acids 127 to 157 of SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:18 or amino acids 126 to 156 of SEQ ID NO:16 is at least 20, optionally at least 25, or 30, amino acids long and the polypeptide can bind to a cell surface receptor expressed on B cells or T cells and/or can inhibit the proliferation of T cells. Such an amino acid sequence can be at least 150 amino acids long and can be at least 80%, optionally at least 85%, 90%, 92%, 94%, 96%, 98%, 99%, or 99.5%, identical to amino acids 30 to 358 of SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18, wherein the identity region of the amino acid sequence aligned with amino acids 30 to 358 of SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18 is at least 150, optionally at least 200, 250, or 300, amino acids. Further, the amino acid sequence can be at least 90%, optionally at least 92%, 94%, 96%, 98%, 99%, or 99.5%, identical to amino acids 30 to 358 of SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18 and/or can comprise amino acids 30 to 358 of SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18.
In another embodient the invention encompasses an isolated BTL-II protein comprising a first polypeptide consisting of a first amino acid sequence at least 80%, optionally at least 85%, 90%, 92%, 94%, 96%, 98%, 99%, or 99.5%, identical to amino acids 30 to 358 of SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18, wherein the identity region of the first amino acid sequence aligned with amino acids 30 to 358 of SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18 is at least 150 amino acids, wherein the first polypeptide comprises a second polypeptide consisting of a second amino acid sequence at least 80%, optionally at least 85%, 90%, 92%, 94%, 96%, 98%, 99%, or 99.5%, identical to amino acids 127 to 157 of SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:18, or amino acids 126 to 156 of SEQ ID NO:16, wherein the identity region of the second amino acid sequence aligned with amino acids 127 to 157 of SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:18 or amino acids 126 to 156 of SEQ ID NO:16 is at least 20 amino acids long, and wherein the first polypeptide can inhibit the proliferation of T cells. The first amino acid sequence can be identical to amino acids 127 to 157 of SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:18, or amino acids 126 to 156 of SEQ ID NO:16,
Alternatively, the invention provides an isolated BTL-II protein comprising a polypeptide consisting of an amino acid sequence at least 80%, optionally at least 85%, 90%, 92%, 94%, 96%, 98%, 99%, or 99.5%, identical to amino acids 30 to 457 of SEQ ID NO:4, wherein the polypeptide comprises no more or less than 2 Ig-like domains, and wherein the polypeptide can inhibit the proliferation of T cells. The amino acid sequence can be at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to amino acids 30 to 247 of SEQ ID NO:8 or to amino acids 30 to 243 of SEQ ID NO:12. The amino acid sequence can be at least 90%, optionally at least 92%, 94%, 96%, 98%, 99%, 99.5%, or 100%, identical to amino acids 30 to 457 of SEQ ID NO:4.
Alternatively, a BTL-II protein of the invention can comprise a first polypeptide consisting of a first amino acid sequence at least 80%, optionally at least 85%, 90%, 92%, 94%, 96%, 98%, 99%, or 99.5%, identical to amino acids 247 to 452 SEQ ID NO:4 or to amino acids 248 to 447 of SEQ ID NO:6, wherein the first amino acid sequence does not comprise an amino acid sequence at least 80%, optionally at least 85%, 90%, 92%, 94%, 96%, 98%, 99%, or 99.5%, identical to amino acids 32 to 232 of SEQ ID NO:4 or to amino acid 27 to 232 of SEQ ID NO:6 with an identity region of the first amino acid sequence aligned with SEQ ID NO:4 of at least 25, optionally, at least 50, 75, 100, or 150, amino acids, and wherein the protein does not comprise a second polypeptide consisting of a second amino acid sequence at least 80%, optionally at least 85%, 90%, 92%, 94%, 96%, 98%, 99%, or 99.5%, identical to amino acids 32 to 232 of SEQ ID NO:4 or to amino acid 27 to 232 of SEQ ID NO:6 with an identity region of the second amino acid sequence aligned with SEQ ID NO:4 of at least 25, optionally, at least 50, 75, 100, or 150, amino acids, and wherein the first polypeptide can inhibit the proliferation of T cells.
In another embodiment, an isolated BTL-II protein of the invention can comprise a first polypeptide consisting of a first amino acid sequence at least 80%, optionally at least 85%, 90%, 92%, 94%, 96%, 98%, 99%, or 99.5%, identical to amino acids 32 to 242 of SEQ ID NO:8 or SEQ ID NO:12, wherein the identity region of the first amino acid sequence aligned with SEQ ID NO:8 or SEQ ID NO:12 is at least about 50, optionally at least about 75, 100, 150, or 200 amino acids long, wherein the first polypeptide comprises a second polypeptide consisting of an second amino acid sequence at least 80%, optionally at least 85%, 90%, 92%, 94%, 96%, 98%, 99%, or 99.5%, identical to amino acids 10 to 40 of SEQ ID NO:8 or SEQ ID NO:12, wherein the identity region of the second amino acid sequence aligned with SEQ ID NO:8 or SEQ ID NO:12 is at least about 20, optionally at least about 25 or 30, amino acids long, and wherein the first polypeptide can inhibit the proliferation of T cells.
In still another embodiment, the invention encompasses an isolated BTL-II protein comprising a polypeptide consisting of an amino acid sequence at least 80%, optionally at least 85%, 90%, 92%, 94%, 96%, 98%, 99%, or 99.5%, identical to amino acids 30 to 358 of SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18, wherein the identity region of the amino acid sequence aligned with amino acids 30 to 358 of SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18 is at least 250, optionally at least 275 or 300, amino acids, and wherein the polypeptide can inhibit the proliferation of T cells.
BTL-II proteins of the invention can comprise a polypeptide that can inhibit the proliferation of T cells that may be at most about 480 amino acids, about 380 amino acids, about 270 amino acids, or about 160 amino acids in length.
The invention further encompasses an isolated BTL-II protein comprising a first polypeptide consisting of a first amino acid sequence at least 80%, optionally at least 85%, 90%, 92%, 94%, 96%, 98%, 99%, or 99.5%, identical to amino acids 32 to 358 of SEQ ID NO:10, wherein the identity region of the first amino acid sequence aligned to SEQ ID NO:10 is at least about 175, optionally about 200, 250, 275 or 300, amino acids long, wherein the first amino acid sequence is not more than about 380, optionally not more than about 390, 270, or 170, amino acids in length, wherein the first polypeptide can inhibit the proliferation of T cells, wherein the first amino acid sequence is not at least 80% identical to amino acids 148 to 232 of SEQ ID NO:4 with an identity region of the first amino acid sequence aligned to amino acids 148 to 232 of SEQ ID NO:4 of at least about 20, 30, 40, 50, 60, or 75 amino acids, and wherein the BTL-II protein does not comprise a second amino acid sequence that is at least 80% identical to amino acids 148 to 232 of SEQ ID NO:4 with an identity region of the second amino acid sequence aligned to amino acids 148 to 232 of SEQ ID NO:4 of at least about 20, 30, 40, 50, 60, or 75 amino acids. Such a BTL-II protein may comprise amino acids 32 to 242 of SEQ ID NO:8 or SEQ ID NO:12.
In another embodiment, the invention comprises a BTL-II recombinant fusion protein comprising the BTL-II protein and a heterologous polypeptide, which can be an Fc region of an antibody or a leucine zipper. The invention also encompasses an immunogenic fragment of amino acids 29 to 457 SEQ ID NO:4 that is capable of eliciting antibodies that bind specifically to the fragment, that is at least 10 amino acids long, and that spans position 360 of SEQ ID NO:4. Immunogenic fragments of SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:16, and SEQ ID NO:18 at least 10 amino acids long are provided, wherein the immunogenic fragment spans position 141 to 143 of SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, or SEQ ID NO:16 and can elicit antibodies that bind specifically to the fragment. Alternatively, the immunogenic fragments can span position 142 of SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:18 or position 141 of SEQ ID NO:16.
In alternate embodiments, murine BTL-II proteins are provided. Specifically, the invention provides an isolated BTL-II protein comprising an amino acid sequence consisting of amino acids x–y of SEQ ID NO:6, wherein x is any amino acid from position 1 to 35 of SEQ ID NO:6 and y is any amino acid from position 450–460 of SEQ ID NO:6. Such a BTL-II protein can comprise amino acids 32–450, 29 to 456, and/or 29 to 514 of SEQ ID NO:6.
Other embodiments include isolated antibodies that bind specifically to a BTL-II protein consisting of amino acids 1–457 of SEQ ID NO:4, 1–456 of SEQ ID NO:6., 1–247 of SEQ ID NO:8, 1–363 of SEQ ID NO:10, 1–243 of SEQ ID NO:12, 1–359 of SEQ ID NO:14, 1–358 of SEQ ID NO:16, or 1–362 of SEQ ID NO:18. Such antibodies can be monoclonal antibodies, humanized antibodies, or human antibodies and may inhibit the binding of BTL-II to its receptor. The invention encompasses nucleic acids that encode such antibodies and cells that can produce such antibodies, which may be hybridoma cells or cells that have been genetically engineered to produce such an antibody. The invention further encompasses methods of producing antibodies by culturing such cells, which may secrete the antibody.
Other embodiments include BTL-II nucleic acids. The invention encompasses an isolated BTL-II nucleic acid comprising a polynucleotide consisting of nucleotides x to y of SEQ ID NO:3, wherein x is from nucleotide 1 to 105 and y is from nucleotide 1345 to 1375, or comprising the complement of the polynucleotide. Such a nucleic acid can comprise nucleotides 105 to 1345 or 1 to 1371 of SEQ ID NO:3. Further, nucleic acids encoding immunogenic fragments are provided, as are BTL-II nucleic acids encoding any of the BTL-II proteins described above.
The invention further provides a vector comprising any of the BTL-II nucleic acids described above or nucleic acids encoding anti-BTL-II antibodies and a host cell containing such a vector. Alternatively, the invention provides a host cell genetically engineered to express a BTL-II protein, an immunogenic fragment of BTL-II, or an antibody against BTL-II. Such host cells can be mammalian cells, including CHO cells. A method for producing a BTL-II protein, immunogenic fragment, or an anti-BTL-II antibody comprising culturing such a host cells under conditions allowing expression of the BTL-II protein, immunogenic fragment, or antibody is also encompassed by the invention. This method may further comprise isolating the BTL-II protein, immunogenic fragment, or antibody from the host cells or the medium. BTL-II proteins, immunogenic fragments, or antibodies produced by such methods are also contemplated. The invention further encompasses mammalian cells that produce antibodies against BTL-II, including hybridoma or myeloma cells and methods for making antibodies by culturing such cells.
Various therapeutic methods employing the compositions encompassed by the invention are also contemplated. The invention provides a method for reducing inflammation in the gut in a patient suffering from an inflammatory bowel disease, optionally either Crohn's disease or ulcerative colitis, comprising administering a therapeutically effective amount of a BTL-II protein, optionally a soluble BTL-II protein. A method for inducing an immune response, including a system and/or a mucosal immune response, against an antigen comprising administering a therapeutically effective amount of a BTL-II antagonist and the antigen is also provided. The BTL-II antagonist can be an antibody or a small molecule, and the antigen can be administered directly to a mucosal surface or can be administered systemically. Further provided is a method for diagnosing an inflammatory bowel disease or predicting the onset of an inflammatory bowel disease comprising assaying a tissue sample from the bowel of a patient to determine whether BTL-II mRNA or protein is overexpressed. The tissue can be assayed for BTL-II protein expression using an anti-BTL-II antibody. The invention further provides a method for dampening an immune response to an antigen, especially an auto-antigen in a patient suffering from an autoimmune or inflammatory disease, comprising co-administering the antigen and a soluble BTL-II protein. The antigen can be administered via a mucosal surface.
In further embodiments, the invention encompasses methods for inhibiting T cell proliferation and cytokine production. In one embodiment, the invention comprises a method for inhibiting T cell proliferation comprising contacting the T cells with a BTL-II polypeptide. The T cells can be human T cells and can be contacted with the BTL-II polypeptide in vivo. As an alternative to a BTL-II polypeptide, an agonistic antibody that binds to a BTL-II receptor expressed on T cells can be used, provided that it can inhibit the proliferation of T cells. In another embodiment, the invention includes a method for suppressing cytokine production by a T cell comprising contacting the T cell with a BTL-II polypeptide. The T cells can be human T cells and can be contacted with the BTL-II polypeptide in vivo. The cytokine can be, for example, interferon gamma (IFNγ), interleukin 2 (IL2), or interleukin 5 (IL5).
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the domain structures of selected members of the family of butyrophilin-like proteins. Most of the selected proteins are members of the B7 subfamily discussed below. The name of each protein is shown to the left of the diagram depicting its structure. The “Ig-like” domains are immunoglobulin-like domains as described below. The domains labeled “7” are heptad repeat regions as described below. The domains labeled “TM” are transmembrane domains. Open boxes are cytoplasmic domains not identified are part of a specific family of domains. Domains labeled “B30.2” are B30.2 domains as explained below. The BTL-II protein is depicted here as having four Ig-like domains, but forms of BTL-II also exist that have two or three Ig-like domains. B7-H3 is depicted with four Ig-like domains, and it also exists in a form containing only two Ig-like domains.
FIG. 2 is a diagram of the structure the human BTL-II gene and mRNA. The boxes indicate exons. The numbers below the horizontal lines below the boxes indicate the positions within SEQ ID NO:3 of the exons. The horizontal lines at the bottom of the figure denote the extent of SEQ ID NO:3 encoding the extracellular domain and the transmembrane and cytoplasmic domains (“TM/Cyto”) and forming the 3′ untranslated region (“3′ UTR”). Stop codons are denoted by a mark that could be described as a sunburst or a small explosion.
FIG. 3a is a diagram of the structure of a first category of splice variants of the human BTL-II mRNA. Symbols are the same as described for FIG. 2. The large Xs over exons 2 and 3 indicate that these exons are missing in this category of splice variants.
FIG. 3b is a representative sequence of a member of the first category of splice variants (bottom line, SEQ ID NO:7) aligned to a portion of SEQ ID NO:3 (top line). There are no mismatches other than the gap created by the missing exons 2 and 3.
FIG. 4a shows the structure of a second category of splice variants of the human BTL-II RNA. Symbols are the same as described for FIG. 2. The large X over exon 3 indicates that this exon is missing in this category of splice variants.
FIG. 4b shows a representative sequence of a member of the second category of splice variants (bottom line, SEQ ID NO:9) aligned to a portion of SEQ ID NO:3 (top line). There are no mismatches other than the gap created by the missing exon 3.
FIG. 5a shows the structure of a third category of splice variants of the human BTL-II mRNA. Symbols are the same as described for FIG. 3. The large Xs over exons 2 and 3 indicate that these exons are missing in this category of splice variants. The stars accompanied by numbers adjacent to the boxes indicate the positions of sequence polymorphisms present in this category of splice variants. The variations are present at the following positions within SEQ ID NO:3: variation 2 is at position 1050; variation 3 is at positions 1136 and 1140; variation 4 is at positions 1178 and 1179; and variation 5 is at position 1212; and variation 6 is at position 1242.
FIG. 5b shows a representative sequence of a member of the third category of splice variants (bottom line, SEQ ID NO:11) aligned to a portion of SEQ ID NO:3 (top line). Mismatched bases are indicated in boldface.
FIG. 6a shows the structure of a fourth category of splice variants of the human BTL-II mRNA. The large X over exon 3 indicates that this exon is missing in this category of splice variants. Sequence polymorphisms are indicated as in FIG. 5a, and variations 2 to 6 are as in FIG. 5a.
FIG. 6b shows a representative sequence of a member of the fourth category of splice variants (bottom line, SEQ ID NO:7) aligned to a portion of SEQ ID NO:3 (top line). Mismatched bases are indicated in boldface.
FIG. 7a shows the structure on a fifth category of splice variants of the human BTL-II mRNA. The large X over exon 3 indicates that this exon is missing in this category of splice variants. Sequence polymorphisms are indicated as in FIG. 5a, and variations 2 to 6 are as in FIG. 5a. Variation 1 is a deletion of nucleotides 78 to 80 of SEQ ID NO:3.
FIG. 7b shows a representative sequence of a member of the fifth category of splice variants (bottom line, SEQ ID NO:15) aligned to a portion of SEQ ID NO:3 (top line). Mismatched bases are indicated in boldface.
FIG. 8 is a diagram of the structure of the murine BTL-II gene and MRNA. Symbols are as in FIG. 5 except that the number refer to positions in SEQ ID NO:5.
FIG. 9a shows the structure of a first category of splice variants of the murine BTL-II MRNA. The large X over exon 3 indicates that this exon is missing in this category of splice variants.
FIG. 9b shows a representative sequence of a member of the first category of murine splice variants (bottom line, SEQ ID NO:17) aligned to SEQ ID NO:5 (top line). There are no mismatches other than the gap created by the missing exon 3.
FIG. 10 represents expression of BTL-II MRNA in colonic tissue of mdr1a −/− mice (which carry the mdr1a null mutation in an FVB background) when exhibiting no symptoms of inflammatory bowel disease (horizontal stripes) or when exhibiting symptoms of inflammatory bowel disease (checkerboard pattern) relative to expression of BTL-II MRNA in colonic tissue of non-symptomatic, wild type FVB mice. Measurements were done by hybridizing fluorescently-labeled cDNA to an Affymetrix chip containing an oligonucleotide complementary to BTL-II MRNA.
FIG. 11 is a graph showing the relative concentrations of BTL-II mRNA detected by a real time PCR assay of colon tissue from two different wild type mice of the FVB strain (FVB #1 and #2; diagonal lines) and from two mdr1a −/− mice showing symptoms of inflammatory bowel disease (Mdr1a −/− #1 and #2; checkerboard patterns).
FIG. 12a is a bar graph showing proliferation (as evidenced by uptake of 3H-thymidine) of purified human T cells cultured with the following proteins: anti-CD3ε antibody alone, ; anti-CD3ε antibody and BTL-II:Fc, ; anti-CD3ε antibody and B7RP-1:Fc, ; anti-CD3ε antibody, B7RP-1:Fc, and BTL-II:Fc, ; anti-CD3ε antibody and B7-2:Fc, ; and anti-CD3ε antibody, B7-2:Fc, and BTL-II:Fc, .
FIG. 12b is identical to FIG. 12a, except that a linear, rather than a logarithmic scale is used.
FIG. 13 is a bar graph showing proliferation of purified human T cells in response to a constant amount of anti-CD3ε antibody and a varying amount of BTL-II:Fc, as indicated.
FIG. 14 is a bar graph showing proliferation of murine T cells in response to ariti-CD3ε antibody alone, , anti-CD3ε antibody plus BTL-II:Fc, , or anti-CD3ε antibody plus a control protein consisting of a human Fc region, .
FIG. 15i is a bar graph showing proliferation of murine B cells in response to no added protein, , TALL-1 protein alone, , an anti-IgM antibody alone, , TALL-1 plus and anti-IgM antibody, , and TALL-1, anti-IgM antibody, and BTL-II:Fc, .
FIG. 16a shows proliferation of purified human T cells in response to various combinations of proteins indicated as in FIG. 12a.
FIG. 16b shows the relative interferon gamma (IFNγ) production in response to various combinations of proteins indicated as in FIG. 12a.
FIG. 16c shows the relative interleukin 2 (IL2) production in response to various combinations of proteins indicated as in FIG. 12a.
FIG. 16d shows the relative interleukin 5 (IL5) production in response to various combinations of proteins indicated as in FIG. 12a.
FIG. 17 is a bar graph showing the total number of dead cells in cultures of purified T cells cultured with various combinations of proteins, as indicated in FIG. 12a.
FIG. 18 shows FACS scans of cells transfected with either an empty vector (top line), or a vector containing cDNA encoding murine BTL-II (second line), murine B7RP-1 (third line), or murine CD80 (bottom line). The cells were stained with the following proteins: (1) the extracellular region of murine CTLA4 fused to a human Fc region of an IgG1 antibody (first column); (2) the extracellular region of murine CD28 fused to a human Fc region from an IgG1 antibody (second column); (3) the extracellular region of murine ICOS fused to a human Fc region from an IgG1 antibody (third column); and (4) the extracellular region of PD1 fused to a human Fc region of an IgG1 antibody (fourth column). The vertical axis of each scan (labeled “counts”) represents cell number and the horizontal axis (labeled “FL2-H”) represents fluorescence. The horizontal line labeled “M1” shows where the “gate” was set on the FACS machine. Cells encompassed in this gate are considered positive; all others are considered negative. The small lettering above each FACS scan indicates an individual sample number.
BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS
SEQ ID NO:1 is the nucleotide sequence of the human BTL-II cDNA from the National Center for Biotechnology Information (NCBI) entry with the accession number NM—019602.
SEQ ID NO:2 is the amino acid sequence of the of the human BTL-II protein predicted from the cDNA sequence of the NCBI entry with the accession number NM—019602.
SEQ ID NO:3 is the nucleotide sequence of a full length human BTL-II cDNA of the invention.
SEQ ID NO:4 is the amino acid sequence of the full length human BTL-II protein encoded by SEQ ID NO:3.
SEQ ID NO:5 is the nucleotide sequence of the full length murine BTL-II cDNA of the invention.
SEQ ID NO:6 is the amino acid sequence of the full length murine BTL-II protein encoded by SEQ ID NO:5.
SEQ ID NO:7 is the nucleotide sequence of the cDNA from a representative member of the first category of human BTL-II splice variants (FIG. 3a).
SEQ ID NO:8 is the amino acid sequence encoded by SEQ ID NO:7.
SEQ ID NO:9 is the nucleotide sequence of the cDNA from a representative member of the second category of human BTL-II splice variants (FIG. 4a).
SEQ ID NO:10 is the amino acid sequence encoded by SEQ ID NO:9.
SEQ ID NO:11 is a partial nucleotide sequence of the cDNA from a representative member of the third category of human BTL-II splice variants (FIG. 5a).
SEQ ID NO:12 is the amino acid sequence encoded by SEQ ID NO:11.
SEQ ID NO:13 is a partial nucleotide sequence of the cDNA from a representative member of the fourth category of human BTL-II splice variants (FIG. 6a).
SEQ ID NO:14 is the amino acid sequence encoded by SEQ ID NO:13.
SEQ ID NO:15 is a partial nucleotide sequence of the cDNA from a representative member of a fifth category of human BTL-II splice variants (FIG. 7a).
SEQ ID NO:16 is the amino acid sequence encoded by SEQ ID NO:15.
SEQ ID NO:17 is the nucleotide sequence of a representative member of a first category of murine BTL-II splice variants (FIG. 9a).
SEQ ID NO:18 is the amino acid sequence encoded by SEQ ID NO:17.
SEQ ID NO:19 is the nucleotide sequence encoding the BTL-II:Fc fusion protein described in Example 5.
SEQ ID NO:20 is the amino acid sequence of the BTL-II:Fc fusion protein described in Example 5.
DETAILED DESCRIPTION
The present invention provides BTL-II proteins and nucleic acids, including recombinant vectors encoding BTL-II proteins, anti-BTL-II antibodies, which can be agonists or antagonists, as well as methods for producing and using these molecules and pharmaceutical compositions containing them. BTL-II expression is restricted to a small number of tissue types. BTL-II is overexpressed in the gut prior to the onset of symptoms and during the symptomatic phase in a murine inflammatory bowel disease model system as illustrated in Example 4. BTL-II antibodies can therefore serve to diagnose or to predict the likelihood of the onset of an inflammatory bowel disease. In addition, the invention provides a number of allelic variants of the BTL-II nucleotide sequence (FIGS. 5a to 7a). These can find use in predicting susceptibility to inflammatory bowel disease. Further, since a soluble BTL-II protein can inhibit T cell proliferation and cytokine production (Examples 6–10), BTL-II proteins can find use in the treatment of autoimmune and inflammatory diseases.
Further, BTL-II is expressed in Peyer's patches, which are specialized structures known to play a role in immune sampling in the gut. BTL-II is preferentially expressed on are CD11c+ (low expressing) CD8+ B220+ dendritic cells (also called plasmacytoid dendritic cells) found in Peyer's patches as compared to other cells found in Peyer's patches, including other dendritic cells. Peyer's patch dendritic cells have been hypothesized to play a role in inducing tolerance at mucosal surfaces due to their influence on T cell differentiation. Weiner (2001), Nature Immunology 2 (8): 671–71; Weiner (2001), Immunol. Rev. 182: 207–14; Iwasaki and Kelsall (1999), American Journal of Physiology-Gastrointestinal and Liver Physiology 276 (5): G1074–78. Thus, anti-BTL-II antibodies can be used to identify CD11c+ (low expressing) CD8+ B220+ dendritic cells within Peyer's patches.
As explained below, BTL-II is within the B7 subfamily of butyrophilin-like proteins that play roles in regulating T and B cell-mediated responses. BTL-II may play a role in either dampening and/or promoting immune system-mediated inflammation, especially in the gut. Given the complexity of the immune system, a single cell surface protein may, in some cases, both stimulate or dampen an immune response by immune effector cells, depending on what receptors on the effector cells are available for the molecule to interact with. The B7-1 and B7-2 proteins discussed below are examples of immune-regulating cell surface proteins with both stimulatory and dampening effects on immune effector cells, in this case, T cells. Hence, BTL-II may play similar dual roles in vivo. However, the gut is, overall, highly tolerant to foreign antigens, as evidenced by its tolerance to food antigens and commensal microorganisms. It is therefore likely that BTL-II plays a role in dampening immune responses or inflammation in vivo in at least some situations. Thus, BTL-II antagonists, which can include antibodies, binding proteins selected in vitro, or small molecules, may serve to stimulate a mucosal immune response to an antigen. Further, soluble BTL-II proteins, or functionally equivalent anti-idiotypic antibodies, may dampen an immune response in the gut or in other mucosal surfaces in the body, such as the lungs.
An “antibody,” as used herein, can be a chimeric antibody, can be monomeric or single chain, dimeric, trimeric, tetrameric, or multimeric antibody, and can be a recombinant protein or a non-recombinant protein. A “domain” is part or all of a protein that can be distinguished by primary sequence motifs and/or tertiary structural characteristics. Programs designed to locate protein domains include, for example, Pfam (Bateman et al. (1999), Nucleic Acids Res. 27: 260–62; Bateman et al. (1999), Nucleic Acids Res. 28: 263–66), ProDom (Corpet et al. (1999), Nucleic Acids Res. 27: 263–67; Corpet et al. (1999), Nucleic Acids Res. 28: 267–69), Domo (Gracy and Argos (1998), Bioinformatics 14: 164–87), and SMART (Ponting et al. (1999), Nucleic Acids Res. 27: 229–32). Tertiary structure can be determined empirically, for example by X-ray crystallography, or can be predicted using computer software designed for such uses. For example, structural data can be accessed through the Entrez website of NCBI from the Molecular Modeling Database (Wang et al. (2000), Nucleic Acids Res. 28 (1): 243–45) or by the use of software such as DALI (Holm and Sander (1993), J. Mol. Biol. 233: 123–38). Immunoglobulin-like domains (Ig-like), for example, are distinguished mainly by their tertiary structure rather than by primary sequence homologies. See e.g. Bork et al. (1994), J. Mol. Biol. 242: 309–20; Hunkapiller and Hood (1989), Adv. Immunol. 44: 1–63; Williams and Barclay (1988), Ann. Rev. Immunol. 6: 381–405. However, IgV and IgC domains do contain a handful of highly conserved amino acids that occur at conserved positions within their primary amino acid sequence. See e.g. Kabat et al. (1991), Sequences of Proteins of Immunological Interest, U.S. Dept. of Health and Human Services, Public Health Service, National Institutes of Health, NIH Publication No. 91-3242. The presence of such highly conserved amino acids occurring in the proper spacing can indicate the presence of an IgC-like or IgV-like domain.
A nucleic acid “encodes” a protein, as meant herein, if the nucleic acid or its complement comprises the codons encoding the protein.
Cells have been “genetically engineered” to express a specific protein when recombinant nucleic acid sequences that allow expression of the protein have been introduced into the cells using methods of “genetic engineering,” such as viral infection, transfection, transformation, or electroporation. See e.g. Kaufman et al. (1990), Meth. Enzymol. 185: 487–511. This can include, for example, the introduction of nucleic acids encoding the protein into the cells or the introduction of regulatory sequences to enhance the expression of a host gene encoding the protein as described in U.S. Pat. No. 5,272,071 to Chappel. The methods of “genetic engineering” encompass numerous methods including, but not limited to, amplifying nucleic acids using polymerase chain reaction, assembling recombinant DNA molecules by cloning them in Escherichia coli, restriction enzyme digestion of nucleic acids, ligation of nucleic acids, in vitro synthesis of nucleic acids, and transfer of bases to the ends of nucleic acids, among numerous other methods that are well-known in the art. See e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1–3, Cold Spring Harbor Laboratory, 1989.
A “heterologous polypeptide” is any polypeptide that is at least 3 amino acids long that is not a BTL-II polypeptide as meant herein.
In connection with comparisons to determine sequence identity of polynucleotides or polypeptides, what is meant by an “identity region” is the portion of the polynucleotide or polypeptide that is matched, partially or exactly, with another polynucleotide or polypeptide by the computer program GAP (Devereux et al. (1984), Nucleic Acids Res. 12: 387–95) using the parameters stated below. For example, when a polypeptide of 20 amino acids is aligned with a considerably longer protein, the first 10 amino acids match the longer protein exactly, and the last 10 amino acids do not match the longer protein at all, the identity region is 10 amino acids. If, on the other hand, the first and last amino acids of the 20 amino acid polypeptide match the longer protein, and eight other matches are scattered between, the identity region is 20 amino acids long. However, long stretches in either aligned strand without identical or conservatively substituted amino acids or identical nucleotides of at least, for example, 20 amino acids or 60 nucleotides constitute an endpoint of an identity region, as meant herein.
“Ig-like” domains are immunoglobulin like domains and may be either IgV-like or IgC-like domains or be domains that can fold into an immunoglobulin structure but cannot be unambigously classified as either IgV-like or IgC-like.
“IgV-like” domains have amino acid sequences that can be folded into an immunoglobulin fold with the characteristics common to immunoglobulin variable region domains. See Bork et al., supra; Miller et al. (1991), Proc. Natl. Acad. Sci. USA 88: 4377–81; Williams and Barclay (1988), Ann. Rev. Immunol. 6: 381–405. One of skill in the art is aware that the presence of amino acids that are highly conserved in IgV domains at conserved positions can identify a domain as IgV-like.
“IgC-like” domains have amino acid sequences that can be folded into an immunoglobulin fold with the characteristics common to immunoglobulin constant region domains. See Bork et al., supra; Williams and Barclay, supra. One of skill in the art is aware that the presence of amino acids that are highly conserved in IgC domains at conserved positions can identify a domain as IgC-like.
“Inflammatory bowel diseases” include Crohn's disease, ulcerative colitis, ileitis, and any other disease characterized by chronic inflammation of the gastrointestinal tract.
A protein comprises “no more or less than 2 Ig-like domains” when it contains two Ig-like domains and does not contain all or some recognizable portion of another Ig-like domain. However, such a protein can contain other amino acid sequences that are not Ig-like domains and still contain “no more or less than 2 Ig-like domains.” Thus, the phrase “no more or less” refers only to Ig-like domains, not to other amino acid sequences that may be part of the protein.
When a polypeptide is said to be able to “inhibit the proliferation of T cells” or to be able to perform some other biological function, it is meant that a protein comprising the polypeptide can perform the function and that the addition of the polypeptide to at least some proteins that cannot perform the biological function enables these proteins to perform the function. In some cases, a polypeptide that can perform the biological function can do so without any additional sequences. In other cases, a polypeptide may require other sequences, for example, oligomerization sequences, to perform a biological function. In one scenario, a polypeptide may be able to effectively perform a particular biological function when it is linked to an Fc region or a leucine zipper or some other dimerizing domain, but not without the dimerizing domain. As meant herein, such a polypeptide can perform the biological function.
A “protein” is any polypeptide comprising at least 10 amino acids, optionally at least 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, and/or 300 amino acids.
“Recombinant,” as it applies to polypeptides or proteins, means that the production of the protein is dependent on at least one step in which nucleic acids, which may or may not encode the protein, are introduced into a cell in which they are not naturally found.
“Recombinant fusion proteins” are recombinant proteins comprising part or all of at least two proteins, which are not found fused together in nature, fused into a single polypeptide chain.
A “silent mutation” in a nucleic acid sequence is one that changes the sequence of the nucleic acid without changing the sequence of the protein encoded by the nucleic acid.
A “soluble” protein is one lacking a transmembrane domain or some other amino acid sequence, such as a GPI anchor sequence, that normally causes the protein to be embedded in or to associate with a membrane. Such proteins might typically comprise all or part of the extracellular region of a transmembrane protein.
For the purposes of the invention, two proteins or nucleic acids are “substantially similar” if they are at least 80%, optionally at least 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.7% identical to each other in amino acid or nucleotide sequence and maintain or alter in a desirable manner a biological activity of the unaltered protein. The percent identity of two amino acid or two nucleic acid sequences can be determined by visual inspection and mathematical calculation, or more preferably, the comparison is done by comparing sequence information using a computer program. An exemplary computer program is the Genetics Computer Group (GCG; Madison, Wis.) Wisconsin package version 10.0 program, GAP (Devereux et al. (1984), Nucleic Acids Res. 12: 387–95). The preferred default parameters for the GAP program includes: (1) The GCG implementation of a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted amino acid comparison matrix of Gribskov and Burgess, ((1986) Nucleic Acids Res. 14: 6745) as described in Atlas of Polypeptide Sequence and Structure, Schwartz and Dayhoff, eds., National Biomedical Research Foundation, pp. 353–358 (1979) or other comparable comparison matrices; (2) a penalty of 8 for each gap and an additional penalty of 2 for each symbol in each gap for amino acid sequences, or a penalty of 50 for each gap and an additional penalty of 3 for each symbol in each gap for nucleotide sequences; (3) no penalty for end gaps; and (4) no maximum penalty for long gaps. Other programs used by those skilled in the art of sequence comparison can also be used.
Butyrophilin-Like Proteins
Butyrophilin-like proteins reported to date share some structural features, and many are encoded within or adjoining the major histocompatability locus (MHC). See e.g. Henry et al. (1999), Immunology Today 20 (6): 285–88. Butyrophilin is a protein that constitutes 40% of the total protein associated with the fat globule of bovine milk, and a human homolog exists. Ruddy et al. (1997), Genome Research 7:441–56, citing Jack and Mather (1990), J. Biol. Chem. 265: 14481–86. Similarity at the amino acid sequence level among butyrophilin-like proteins can be low, but domain structure is somewhat conserved. All members comprise an amino terminal signal peptide followed by an Ig-like domain, usually reported to be an IgV-like domain. In many cases, this is followed by another Ig-like domain, usually reported to be an IgC-like domain, a transmembrane domain, and a cytoplasmic domain. The two Ig-like domains can be repeated immediately following their first occurrence, as in BTL-II or B7-H3. This domain structure is illustrated in FIG. 1. Most of the proteins diagrammed in FIG. 1 are members of the B7 subfamily of butyrophilin-like proteins; only three (human BTN2A1, human butyrophilin, and myelin oligodendrocyte glycoprotein) are not.
Ig-like domains can be very divergent in sequence and still retain one of a number of conserved folding patterns, which all include a common structural core comprising four β strands. Immunoglobulin constant and variable regions have a tertiary structure which is characterized by seven to nine antiparallel β strands forming a barrel-like shape. Bork et al. (1994), J. Mol. Biol. 242: 309–20. IgV- and IgC-like immunoglobulin domains each include a handful of distinct, highly conserved residues. Hunkapiller and Hood (1989), Adv. Immunol. 44: 1–63; Miller et al. (1991), Proc. Natl. Acad. Sci. USA 88: 4377–81; Williams and Barclay (1988), Ann. Rev. Immunol. 6: 381–405. Conserved residues are presumably important for structure or function. Such conserved residues are found in the Ig-like domains of BTL-II. For example, the first Ig-like domain of the human BTL-II protein (SEQ ID NO:4) contains such highly conserved residues characteristic of IgV-like domains in the appropriate locations at, for example, positions G43, C50, W65, L109, D118, G120, Y122 and C124. See Table 3. The third Ig-like domain also contains residues that correspond to residues conserved in IgV-like domains. The second and fourth Ig-like domains of human BTL-II contain contain residues that correspond to highly conserved residues in IgC-like domains. Table 3.
Transmembrane and cytoplasmic domains occur in butyrophilin-like proteins, regardless of whether they have a second Ig-like domain. The transmembrane domain may or may not be followed by one or more seven amino acid units reminiscent of the heptad repeats typical of an α-helical coiled coil motif. Heptad repeats may also occur at other positions in a butyrophilin-like protein. For a discussion of heptad repeats, see Miller et al. (1991), Proc. Nat. Acad. Sci. 88: 4377–81. Methods for predicting transmembrane domains are well known in the art. See e.g. Ikeda et al. (2002), In Silico Biol. 2 (1): 19–33; Tusnady and Simon (1998), J. Mol. Biol. 283 (2):489–506. Butyrophilin-like proteins can have a cytoplasmic domain. Such a cytoplasmic domain may or may not comprise a B30.2 domain. Sequences of various B30.2 domains are displayed, and putative functions of B30.2 domains are discussed by Henry et al. ((1998), Mol. Biol. Evol. 15 (12): 1696–1705. B30.2 domains are found in a variety of proteins, and the function of the B30.2 domain is unknown. A proposed ligand of the B30.2 domain is xanthine oxidase, which interacts with the cytoplasmic domain of butyrophilin. Mutations in B30.2 domains of two different B30.2-containing proteins have been correlated with two different diseases, although causal relationships between the mutations and the disease phenotypes have not been established. Henry et al., supra.
The B7 Subfamily of Butyrophilin-Like Proteins
BTL-II shares a domain structure with a number of butyrophilin-like immune regulatory proteins lacking the B30.2 domain that play roles in regulating immune effector cells, such as, for example, T cells, B cells and myeloid cells. This subfamily is referred to herein as “the B7 subfamily” of butyrophilin-like proteins. As meant herein, characteristics of B7 subfamily members include, without limitation, the following: (1) having one or more extracellular Ig-like domains; (2) having transmembrane and cytoplasmic domains; (3) lacking a B30.2 domain; (4) being expressed on antigen presenting cells; (5) undergoing regulation of expression during an activated immune response; and (6) modulating an immune response. No secreted, soluble B7 proteins, lacking a transmembrane domain, that modulate immune response have been reported to date. Most B7 proteins have two extracellular Ig-like domains, but some isoforms of B7-H3 have four. See FIG. 1. Human BTL-II can have from two to four extracellular Ig-like domains and has all of the characteristics of B7 family members listed above. We therefore consider it to be a member of the B7 subfamily of butyrophilin-like proteins. All other known members of the B7 subfamily interact with receptors on immune effector cells, such as B cells and/or T cells, which interaction serves as a signal to modulate an immune response. BTL-II is therefore predicted to interact with a receptor on an immune effector cell, such as a T cell, and thereby to modulate an immune response.
B7 family members play roles in modulating the activity of immune effector cells, especially T cells. Henry et al. (1999), Immunology Today 20 (6): 285–88; Sharpe and Freeman (2002), Nat. Rev. Immunol. 2: 116–26. The best characterized examples are, CD80 and CD86 (also called B7-1 and B7-2, respectively), which are expressed on antigen presenting cells and can promote T cell activation when they interact with CD28, which is constitutively expressed on the surface of T cells, or inhibit T cell activation when they interact with a CTLA-4, which is also expressed on the surface of T cells. CTLA-4 expression is not constitutive, but is rapidly upregulated following T cell activation. See e.g. Masteller et al. (2000), J. Immunol. 164: 5319–27; Hehner et al. (2000), J. Biol. Chem. 275 (24): 18160–71; Sharpe and Freeman (2002), Nat. Rev. Immunol. 2: 116–26. Moreover, it has been reported that numerous individual amino acid residues in both Ig-like domains of CD80 are important for binding to CTLA-4 and CD28. Peach et al. (1995), J. Biol. Chem. 270 (36): 21181–87. CD86 is constitutively expressed at low levels and is rapidly upregulated after activation, whereas CD80 is inducibly expressed later after activation. Sharpe and Freeman (2002), Nature Reviews Immunology 2: 116–26.
Another T cell regulatory molecule, referred to herein as B7RP-1, has a plethora of names, KIAA0653 (Ishikawa et al. (1998), DNA Res. 5: 169–76), B7h (Swallow et al. (1999), Immunity 11: 423–32), GL50 (Ling et al. (2000), J. Immunol. 164: 1653–57), B7RP-1 (Yoshinaga et al. (1999), Nature 402: 827–32), LICOS (Brodie et al. (2000), Curr. Biol. 10: 333–36), B7-H2 (Wang et al. (2000), Blood 96:2808–13), and ICOSL (Sharpe and Freeman, supra). B7RP-1 is expressed in peripheral lymphoid tissues, spleen, lymph nodes, lung, thymus, splenocytes, and B cells. Interaction of B7RP-1 with T cells can increase T cell proliferation and cytokine production. B7RP-1 signals T cells through the ICOS receptor, which is expressed on activated T cells. Yoshinaga et al. (1999), Nature 402: 827–32; Swallow et al. (1999), Immunity 11: 423–32. B7RP-1 is constitutively expressed in unstimulated B cell lines, and its expression can be induced in monocytes by interferon γ. Aicher et al. (2000), J. Immunol. 164: 4689–96. The development and regulatory function of regulatory T cells is dependent on the interaction between B7RP-1 and its receptor on T cells. Akbari et al. (2002), Nature Medicine 8 (9):1024–32.
Further, three other T cell regulatory molecules, PD-L1 (also called B7-H1), PD-L2 (also called B7-DC), and B7-H4 (also called B7S 1 and B7x) can inhibit T cell proliferation and cytokine production. PD-L1 and PD-L2 act through their common receptor, PD-1, which is expressed on T cells, B cells, and myeloid cells. Freeman et al. (2000), J. Exp. Med. 192 (7): 1027–34; Dong et al. (1999), Nature Medicine 5 (12): 1365–69; Latchman et al. (2001), Nature Immunology 2 (3): 261–68; Tamura et al. (2001), Blood 97 (6): 1809–16; and Tseng et al. (2001), J. Exp. Med. 193 (7): 839–45. Expression of PD-L1 and PD-L2 can be induced by interferon γ, a generally pro-inflammatory cytokine. Latchman et al., supra. B7-H4 is expressed on B cells, macrophages, and dendritic cells and likely acts through BTLA, an inhibitory receptor expressed on B and T cells. Watanabe et al. (2003), Nature Immunology 4 (7): 670–79; Zang et al. (2003), Proc. Natl. Acad. Sci. 100 (18): 10388–92; Sica et al. (2003), Immunity 18: 849–61; Prasad et al. (2003), Immunity 18: 863–73; Carreno and Collins (2003), Trends Immunol. 24 (10): 524–27.
Still another butyrophilin-like protein that plays a costimulatory role in stimulating T cells is B7-H3. Chapoval et al. (2001), Nature Immunol. 2 (3):269–74. Like the other B7 family members, B7-H3 has a signal sequence, extracellular Ig-like domains, a transmembrane domain, and a cytoplasmic domain. The human B7-H3 gene encodes isoforms with two or four extracellular Ig-like domains. Sun et al. (2002), J. Immunol. 168: 6294–97. Expression of B7-H3 can be induced on dendritic cells by inflammatory cytokines. B7-H3 can stimulate proliferation and the cytotoxic response of T cells. B7-H3 acts through a putative T cell receptor that is distinct from CD28, CTLA-4, ICOS, and PD-1. Chapoval et al, supra.
BTL-II Proteins
The existence of human and murine BTL-II proteins has been predicted from genomic sequence. Stammers et al. (2000), Immunogenetics 51: 373–82. However, these authors found no evidence of a transmembrane or a cytoplasmic domain in human or murine BTL-II proteins based on genome analysis and no evidence of a transcript connecting exons 1–4 (which encode a signal peptide, two Ig-like domains, and a heptad repeat region, respectively) with exons 5 and 6 (which encode another two Ig-like domains, respectively) in PCR experiments designed to detect murine BTL-II mRNAs. Stammers et al., supra. Based on these findings, BTL-II was not placed in the B7 subfamily of cell surface, immunomodulatory proteins. Later sequence submissions to public databases by these same authors predict a human BTL-II mRNA of 1368 nucleotides, which includes exons 5 and 6, encoding a protein of 455 amino acids, which lacks a transmembrane domain and a cytoplasmic domain (NCBI accession no. NM—019602, which discloses SEQ ID NO:1 (human BTL-II cDNA sequence) and SEQ ID NO:2 (human BTL-II protein sequence)).
The BTL-II nucleic acid and protein sequences of the invention differ from these sequences in a number of respects. The cDNA sequence encodes a protein comprising a signal sequence, an extracellular domain, a transmembrane domain, and a cytoplasrnic domain, unlike the previously reported BTL-II protein sequence, which contained no transmembrane or cytoplasmic domains. These characteristics, along with the expression pattern of BTL-II, place BTL-II within the B7 subfamily of butyrophilin-like proteins. Table 1 (below) highlights the differences between the BTL-II protein of the invention and the previously reported sequence. A BTL-II protein of the invention is shown on the top line (SEQ ID NO:4), and the BTL-II protein reported in NCBI accession no. NM—019602 is shown on the bottom line (SEQ ID NO:2). From this comparison, it is apparent that there are three mismatches between the two sequences (at positions 360, 454, and 455) and that SEQ ID NO:4 has 27 more amino acids, which constitute additional sequence in the extracellular domain as well as a transmembrane and a cytoplasmic domain. These sequences 99.3% identical according to the GAP program using the parameters recited above.
TABLE 1
Comparison of human BTL-II predicted protein sequences
Besides an overall similarity in domain structure as illustrated in FIG. 1, the B7 subfamily proteins have similarities at the primary sequence level. For example, when aligned pairwise using the computer program GAP, the human BTL-II protein sequence (SEQ ID NO:4) is similar to other B7 subfamily members as displayed in Table 2 below.
TABLE 2
Percent identity to
Percent similarity
human BTL-II
to human BTL-II
protein
protein
Human PD-L1 (NCBI
19%
28%
accession no. NP_054862)
Human PD-L2 (NCBI
19%
29%
accession no. NP_079515)
Human CD80 (NCBI
26%
33%
accession no. P33681)
Human CD86 (NCBI
23%
32%
accession no. P42081)
Murine BTL-II
62%
68%
One of skill in the art will realize that residues that are conserved in any of these alignments are more likely to play an essential role in the structure or function of human BTL-II than those that are not conserved.
In Table 3 (below), the human BTL-II amino acid sequence (SEQ ID NO:4, top line) is aligned with the murine BTL-II amino acid sequence (SEQ ID NO:6; bottom line). Identical amino acids are joined by a vertical line, and similar amino acids have one or two dots between them. The percent identity between these sequences as determined GAP (described above) is about 62%, and the percent similarity is about 68%. Residues found in IgV- or IgC-like domains or in the so-called “I set” of IgV-like immunoglobulin superfamily members or conservative substitutions of such residues are shown in boldface. Peach et al., supra; Harpaz and Chothia (1994), J. Mol. Biol. 238: 528–39. Such residues are likely to be structurally important and, thus, may have functional effects. The occurrence of a substantial number of such amino acids in the proper spacing can identify a sequence as IgV-like or IgC-like.
TABLE 3
One of skill in the art will appreciate that non-conserved residues are less likely to play a role in determining the overall tertiary structure of a BTL-II protein than conserved residues, since structure is more conserved in evolution than sequence. Bork et al. (1994), J. Mol. Biol. 242: 309–20. As used herein, “non-conserved residues” are amino acids within a BTL-II protein that are not conserved when the human and the murine BTL-II protein sequences are compared as in Table 3. In BTL-II proteins encoded by splice variants, such residues will occur a different numerical positions within the sequence. For example, the non-conserved residue at position 397 of SEQ ID NO:4 is the same non-conserved residue seen at position 187 of SEQ ID NO:8. One of skill in the art will appreciate that protein structure can affect protein function. Further, non-conserved amino acids are also less likely to play a direct role in BTL-II function. For example, residues 4, 6, 25, 26, 35, 36, and many others are neither identical nor similar. Thus, one of skill in the art would realize that alteration of such residues would be less likely to affect BTL-II protein function that would alteration of conserved or similar residues. Moreover, conservative substitutions are less likely to affect protein function that non-conservative substitutions. Examples of amino acid substitutions that are conservative substitutions, unlikely to affect biological activity, including the following: Ala for Ser, Val for Ile, Asp for Glu, Thr for Ser, Ala for Gly, Ala for Thr, Ser for Asn, Ala for Val, Ser for Gly, Tyr for Phe, Ala for Pro, Lys for Arg, Asp for Asn, Leu for Ile, Leu for Val, Ala for Glu, Asp for Gly, and these changes in the reverse. See e.g. Neurath et al., The Proteins, Academic Press, New York (1979). Further, an exchange of one amino acid within a group for another amino acid within the same group is a conservative substitution where the groups are the following: (1) alanine, valine, leucine, isoleucine, methionine, norleucine, and phenylalanine; (2) histidine, arginine, lysine, glutamine, and asparagine; (3) aspartate and glutamate; (4) serine, threonine, alanine, tyrosine, phenylalanine, tryptophan, and cysteine; (5) glycine, proline, and alanine;
Human BTL-II protein comprises several recognizable domains. First, is a signal sequence (encoded by exon 1), which extends from residue 1 of SEQ ID NO:4 to a second position from about residue 22 to 29 of SEQ ID NO:4. Next is an Ig-like domain (encoded by exon 2), which extends from residue x to residue y, where x is from residue 22 to 32 and y is from residue 138 to 148 of SEQ ID NO:4. Following this is another Ig-like domain (encoded by exon 3) extending from residue v to residue w, where residue v is from residue 138 to 148 and w is from residue 232 to 242 of SEQ ID NO:4. Next is a heptad repeat region (encoded by exon 4) from residue t to residue u, where t is from 234 to 239 and u is from residue 240 to 247 of SEQ ID NO:4. Another Ig-like region extends from residue r to residue s, where r is from residue 240 to 247 and s is from residue 354 to 364 of SEQ ID NO:4. A fourth Ig-like region extends from residue p to residue q, where p is from residue 355 to 365 and q is from residue 452 to 462 of SEQ ID NO:4. These first six domains of human BTL-II make up the extracellular region of human BTL-II. The signal sequence may, but need not, be cleaved from the rest of the protein upon secretion of the protein. A transmembrane domain extends from residue n to residue o, where n is from residue 454 to 462 and o is from residue 473 to 481 of SEQ ID NO:4. Finally, a cytoplasmic domain extends from residue k to residue m, where k is from residue 474 to 481 and m is at about residue 482 of SEQ ID NO:4.
Murine BTL-II protein comprises a similar set of domains. First is a signal sequence starting at residue 1 and ending at a position from about residue 20 to about residue 27 of SEQ ID NO:6. Second is an Ig-like domain extending from residue x to y, where x is from residue 21 to 27 and y is from residue 138 to 148 of SEQ ID NO:6. Third is another Ig-like domain from residue v to w, where v is from 139 to 148 and w is from 232 to 242 of SEQ ID NO:6. A heptad repeat region extends from residue t to u, where t is from 233 to 242 and u is from 238 to 248 of SEQ ID NO:6. Another Ig-like region extends from residue r to s, where r is from 239 to 248 and s is from 355 to 365 of SEQ ID NO:6. A final Ig-like region extends from residue p to q, where p is from 356 to 365 and q is from 447 to 457 of SEQ ID NO:6. These first six domains make up the extracellular region of murine BTL-II. A transmembrane domain extends from residue n to o, where n is from 448 to 459 and o is from 470 to 478 of SEQ ID NO:6. Finally, a cytoplasmic domain extends from residue k to m, where is from 471 to 478 and m is at about 514 of SEQ ID NO:6.
The instant invention encompasses secreted, soluble versions of BTL-II as well as versions comprising a transmembrane domain that can be expressed on a cell surface. The invention further includes BTL-II proteins encoded by the BTL-II nucleic acids described below. Recombinant versions of all of these proteins can be used to produce antibodies, in screening, and/or as therapeutic agents as described herein. For example, the invention encompasses BTL-II proteins comprising all or part of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and/or SEQ ID NO:18. BTL-II proteins of the invention include proteins that differ from SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18 by insertion, deletion, alteration, or substitution in the primary amino acid sequence. Such variant sequences are at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.7% identical to SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and/or SEQ ID NO:18, and contain no internal gaps of over 10 amino acids when aligned using GAP with the above-mentioned sequences. Examples of such sequences include the naturally-occurring human allelic variants of BTL-II shown in FIGS. 5b, 6b, and 7b. If such variant sequences contain amino substitutions when compared to SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and/or SEQ ID NO:18, these substitutions can be conservative amino acid substitutions. Further, variant BTL-II proteins may contain no more than 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 insertions, deletions, or substitutions of a single amino acid with respect to SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and/or SEQ ID NO:18.
The BTL-II proteins of the invention include proteins encoded by various splice variants of the human and mouse BTL-II mRNA. Sequences of such variant human BTL-II proteins are shown in SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16. The sequence of a variant mouse BTL-II protein is shown in SEQ ID NO:18. Human splice variants lack either exon 3 alone or lack both exons 2 and 3, and the murine splice variant disclosed lacks exon 3 only. See SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, and SEQ ID NO:17; FIGS. 3a, 4a, 5a, 6a, 7a, and 9a. Proteins encoded by these sequences and substantially similar sequences, where an alignment of the protein sequence with at least one of the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18 using GAP comprises no gaps longer than 10 amino acids, are encompassed by the invention. If these proteins contain amino acid substitutions relative to SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and/or SEQ ID NO:18, such substitutions are preferably conservative amino acid substitutions. BTL-II proteins of the invention can bind to a receptor on the surface of a T cell and can inhibit proliferation and/or cytokine production by T cells.
Further, the invention provides BTL-II proteins encoded by nucleic acids that span the splice junctions of exons 1 and 4 (SEQ ID NO:8 and SEQ ID NO:12) or exons 2 and 4 (SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, and SEQ ID NO:18) and substantially similar proteins that can bind to a receptor expressed on the surface of T cells and/or can inhibit proliferation and/or cytokine production by T cells. This specifically includes BTL-II proteins comprising a polypeptide consisting of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.7%, or 100% identical to amino acids 127 to 157 of SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:18 or amino acids 126 to 156 of SEQ ID NO:16. The identity region of the amino acid sequence aligned with amino acids 127 to 157 of SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:18 or amino acids 126 to 156 of SEQ ID NO:16 is preferably at least 20, 23, 25, 27, 30, 35, or 40. amino acids long. Such an amino acid sequence can be at least 150 amino acids long and can be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.7%, or 100% identical to amino acids 30 to 358 of SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18. The identity region of the amino acid sequence aligned with amino acids 30 to 358 of SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18 can be at least 50, 75, 100, 125, 150, 175, 200, or 300 amino acids.
The invention also provides BTL-II proteins comprising a polypeptide consisting of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.7%, or 100% identical to amino acids 30 to 457 of SEQ ID NO:4 that contains no more or less than 2 Ig-like domains and that can bind to a cell surface receptor expressed on B cells or T cells. The amino acid sequence can be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.7%, or 100% identical to amino acids 30 to 247 of SEQ ID NO:8 or to amino acids 30 to 243 of SEQ ID NO:12.
In further embodiments, the invention provides proteins encoded by nucleic acids comprising a polynucleotide that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.7%, or 100% identical to a polynucleotide consisting of nucleotides 34 to 124 of SEQ ID NO:11, where the identity region is at least 60,70, 80,90, or 100 nucleotides long. Mature BTL-II proteins encoded by such polynucleotides may, but need not, lack a signal sequence (which is present in the immature version of the protein) that is at least partially encoded by this polynucleotide. Such proteins can bind to a T cell and/or can inhibit proliferation and/or cytokinine production by the T cell.
BTL-II proteins may be glycosylated to varying degrees or not glycosylated. As an illustration, a BTL-II protein of the invention may comprise one or more N- or O-linked glycosylation sites in addition to those already found in a protein comprising SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and/or SEQ ID NO:18. Such a BTL-II protein can have a longer in vivo half life than an unaltered protein since it may have more sialic acid moieties attached to it. BTL-II proteins also include proteins comprising any one, any two, any three, or all four of the Ig-like domains of a human or murine BTL-II or substantially similar domains.
Variants
Polypeptides derived from any BTL-II protein by any type of alteration (for example, but not limited to, insertions, deletions, or substitutions of amino acids; changes in the state of glycosylation of the polypeptide; refolding or isomerization to change its three-dimensional structure or self-association state; and changes to its association with other polypeptides or molecules) are also BTL-II proteins as meant herein. The BTL-II proteins provided by the invention include polypeptides characterized by amino acid sequences substantially similar to those of the BTL-II proteins SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and/or SEQ ID NO:18 that can bind to a receptor expressed on the surface of T cells and/or can inhibit proliferation of and/or cytokine production of a T cell. The region of identity can start at position 30 or higher of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18. A GAP alignment of such a variant protein with at least one of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and/or SEQ ID NO:18 may have no internal gaps longer than 10 amino acids. The portion of the BTL-II protein that is substantially similar to SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18 can be at least 100, at least 125, at least 150, at least 175, at least 200, or at least 250 amino acids long. Modifications in such proteins can be naturally provided or deliberately engineered. For example, SEQ ID NO:11 (FIG. 6a) is an allelic or polymorphic variant of SEQ ID NO:9 (FIG. 4a) containing base changes at a handful of positions including at positions.
The invention provides BTL-II protein variants that contain single or multiple amino acid alterations, which can be insertions, deletions, or substitutions of a single amino acid, relative to the sequence of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18, wherein the BTL-II variant protein can inhibit T cell proliferation and/or cytokine production. The alterations can be conservative amino acid substitutions. One of skill in the art is guided as to what amino acids can be changed without affecting function by, for example, the alignment of human and mouse BTL-II proteins shown in Table 3. Amino acids that are identical or similar in human and mouse BTL-II are more likely to be important for function that those that are not. Further, amino acids that are highly conserved in IgV or IgC domains (shown in boldface in Table 3) are also likely to be functionally important. Such allelic or polymorphic variants can be valuable as diagnostic agents to determine a predisposition to disease. Human allelic variants with sequences varying from SEQ ID NO:3 are disclosed in FIGS. 5a, 5b, 6a, 6b, 7a, and 7b. For example, among normal people without symptoms of inflammatory bowel disease, a certain ratio can exist between the number having BTL-II genes encoding cDNAs with the sequence of SEQ ID NO:3 and the number having BTL-II genes encoding the allelic variant mutations labeled 3–6 in FIGS. 5a, 6a, and 7a. The ratio of the occurrence of these alleles may be altered among patients with symptoms of inflammatory bowel disease such that a greater proportion of these people have BTL-II genes with the allelic variant mutations labeled 3–6 in FIGS. 5a, 6a, and 7a. The existence of various allelic variations in a patient's tissues can be determined by, for example, PCR amplification of a segment of a DNA or RNA molecule spanning the polymorphic site. As mentioned above, conservative amino acid substitutions, particularly at sites not conserved between human and mouse BTL-II protein sequences, are more likely to preserve biological function that are non-conservative substitutions at conserved sites. One of skill in the art will also appreciate that substitutions that substantially upset the tertiary structure of a BTL-II protein as predicted by programs such as, for example, DALI (Holm and Sander (1993), J. Mol. Biol. 233: 123–38), are likely to also impair function.
Fragments
Included among BTL-II proteins are proteins comprising fragments of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18 or fragments that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.7% identical to these sequences. Preferably, such fragments can bind to a receptor expressed on the surface of a T cell and are at least about 50, 60, 70, 80, 90, 100, 150, or 200 amino acids long. Preferably, such fragments are soluble in aqueous solution and comprise part or all of the extracellular region of a BTL-II protein. The protein comprising such a fragment can be secreted. For example, BTL-II proteins comprising at least one of the domains of the human or murine BTL-II proteins described above or a substantially similar protein, wherein the domain has at least one of the biological properties of BTL-II proteins, are encompassed by the invention. For example, such proteins can inhibit proliferation and/or cytokine production of T cells.
Further, encompassed by the invention are the following altered versions the human and murine BTL-II proteins or substantially similar proteins: (1) versions of the human and murine BTL-II protein lacking the second Ig-like domain (see FIG. 1) encoded by exon 3; (2) versions of a human or murine BTL-II protein lacking the first and second Ig-like domains encoded by exons 2 and 3; (3) versions lacking third and fourth Ig-like domains encoded by exons 5 and 6; (4) versions lacking any two of the four Ig-like domains; and (5) versions lacking any one of the Ig-like domains.
Also encompassed within the invention are immunogenic fragments of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18 that are capable of eliciting antibodies that bind specifically to the fragment. Such fragments are preferably at least 10 amino acids long and preferably comprise contiguous amino acid residues from sequences mentioned above. Antibodies generated by immunizing animals with such fragments can be useful for prediction, diagnosis, and treatment of inflammatory bowel diseases, as discussed elsewhere in this application. Such fragments can span regions of these proteins encoded by splice junctions, which may have the advantage of generating antibodies specific for proteins encoded by splice variants or the full length protein. Alternatively, an antibody against a portion of BTL-II that is encoded by exon 3 would detect only full length BTL-II proteins, not proteins encoded by any other splice variants, all of which lack exon 3.
Recombinant Fusion Proteins
The invention further encompasses fusion proteins comprising at least one BTL-II polypeptide, which is one of the BTL-II proteins, variants, or fragments described above, and at least one other moiety. The other moiety can be a heterologous polypeptide, that is, a polypeptide other than a BTL-II polypeptide. The other moiety can also be a non-protein moiety such as, for example, a polyethylene glycol (PEG) moiety or a cytotoxic, cytostatic, luminescent, and/or radioactive moiety. Attachment of PEG has been shown to increase the in vivo half life of at least some proteins. Moreover, cytotoxic, cytostatic, luminescent, and/or radioactive moieties have been fused to antibodies for diagnostic or therapeutic purposes, for example, to locate, to inhibit proliferation of, or to kill cells to which the antibodies can bind. Similarly, BTL-II polypeptides fused to such moieties can be used to locate, to inhibit proliferation of, or to kill cells that BTL-II can bind to. Among such cytotoxic, cytostatic, luminescent, and/or radioactive moieties are, for example, maytansine derivatives (such as DM1), enterotoxins (such as a Staphlyococcal enterotoxin), iodine isotopes (such as iodine-125), technetium isotopes (such as Tc-99 m), cyanine fluorochromes (such as Cy5.5.18), ribosome-inactivating proteins (such as bouganin, gelonin, or saporin-S6), and calicheamicin, a cytotoxic substance that is part of a product marketed under the trademark MYLOTARG™ (Wyeth-Ayerst).
A variety of heterologous polypeptides can be fused to a BTL-II polypeptide for a variety of purposes such as, for example, to increase in vivo half life of the protein, to facilitate identification, isolation and/or purification of the protein, to increase the activity of the protein, and to promote oligomerization of the protein. Since some proteins of the B7 subfamily, such as, for example, CD80, bind to their receptors primarily in oligomeric form (dimeric in the case of CD80; see Collins et al. (2002), Immunity 17: 201–10), oligomerization can be very important to preserve the biological activity of a soluble protein.
Many heterologous polypeptides can facilitate identification and/or purification of recombinant fusion proteins of which they are a part. Examples include polyarginine, polyhistidine, or HAT™ (Clontech), which is a naturally-occurring sequence of non-adjacent histidine residues that possess a high affinity for immobilized metal ions. Proteins comprising these heterologous polypeptides can be purified by, for example, affinity chromatography using immobilized nickel or TALON™ resin (Clontech), which comprises immobilized cobalt ions. See e.g. Knol et al. (1996), J. Biol. Chem. 27 (26): 15358–15366. Heterologous polypeptides comprising polyarginine allow effective purification by ion exchange chromatography. Other useful heterologous polypeptides include, for example, the antigenic identification peptides described in U.S. Pat. No. 5,011,912 and in Hopp et al. (1988), Bio/Technology 6:1204. One such peptide is the FLAG® peptide, which is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant fusion protein. A murine hybridoma designated 4E11 produces a monoclonal antibody that binds the FLAG® peptide in the presence of certain divalent metal cations, as described in U.S. Pat. No. 5,011,912. The 4E11 hybridoma cell line has been deposited with the American Type Culture Collection under accession no. HB 9259. Monoclonal antibodies that bind the FLAG® peptide can be used as affinity reagents to recover a polypeptide purification reagent that comprises the FLAG® peptide. Other suitable protein tags and affinity reagents are: 1) those described in GST-Bind™ system (Novagen), which utilizes the affinity of glutathione-S-transferase fusion proteins for immobilized glutathione; 2) those described in the T7-Tag® affinity purification kit (Novagen), which utilizes the affinity of the amino terminal 11 amino acids of the T7 gene 10 protein for a monoclonal antibody; or 3) those described in the Strep-tag® system (Novagen), which utilizes the affinity of an engineered form of streptavidin for a protein tag. Some of the above-mentioned protein tags, as well as others, are described in Sassenfeld (1990), TIBTECH 8: 88–93, Brewer et al., in Purification and Analysis of Recombinant Proteins, pp.239–266, Seetharam and Sharma (eds.), Marcel Dekker, Inc. (1991), and Brewer and Sassenfeld, in Protein Purification Applications, pp. 91–111, Harris and Angal (eds.), Press, Inc., Oxford England (1990). Further, fusions of two or more of the tags described above, such as, for example, a fusion of a FLAG tag and a polyhistidine tag, can be fused to a BTL-II protein of the invention.
Recombinant fusion proteins comprising other heterologous polypeptides may have other kinds of unique advantages, such as, for example, a propensity to form dimers, trimers, or higher order multimers, an increased in vivo half-life, and/or an increased biological activity. Techniques for preparing fusion proteins are known, and are described, for example, in WO 99/31241 and in Cosman et al. ((2001). Immunity 14: 123–133). As an illustration, a heterologous polypeptide that comprises an Fc region of an IgG antibody, or a substantially similar protein, can be fused to a BTL-II polypeptide or fragment. An Fc region of an antibody is a polypeptide comprising CH2 and CH3 domains from an antibody of human or animal origin or immunoglobulin domains substantially similar to these. For discussion, see Hasemann and Capra, Immunoglobulins: Structure and Function, in William E. Paul, ed., Fundamental Immunology, Second Edition, 212–213 (1989). Truncated forms of Fc regions comprising the hinge region that promotes dimerization can also be used. Other portions of antibodies and other immunoglobulin isotypes can be used. Recombinant fusion proteins comprising Fc regions of IgG antibodies are likely to form dimers. Fusion proteins comprising various portions of antibody-derived proteins have been described by Ashkenazi et al. ((1991) Proc. Natl. Acad. Sci. USA 88:10535–39), Byrn et al. ((1990), Nature 344: 677–70), Hollenbaugh and Aruffo (in Current Protocols in Immunology, Suppl. 4, pp. 10.19.1–10.19.11 (1992)), Baum et al. ((1994), EMBO J. 13: 3992–4001) and in U.S. Pat. No. 5,457,035 and WO 93/10151. In some embodiments, an altered Fc region can have the advantage of having a lower affinity for Fc receptors compared to a wild type Fc region. This is an advantage because it may lessen the lysis of cells to which such recombinant fusion proteins bind by immune effector cells. Example 5 describes the production of a fusion protein containing the extracellular region of murine BTL-II fused to a human Fc region. The nucleic acid sequence encoding this protein and its amino acid sequence are disclosed in SEQ ID NO:19 and SEQ ID NO:20, respectively.
As another alternative, recombinant fusion proteins of the invention can comprise a heterologous polypeptide comprising a leucine zipper. Among known leucine zipper sequences are sequences that promote dimerization and sequences that promote trimerization. See e.g. Landschulz et al. (1988), Science 240: 1759–64. Leucine zippers comprise a repetitive heptad repeat, often with four or five leucine residues interspersed with other amino acids. Use and preparation of leucine zippers are well-known in the art.
Alternatively, a heterologous polypeptide forming part of a recombinant fusion protein can be one or more peptide linkers, connecting two or more BTL-II polypeptides. Generally, a peptide linker is a stretch of amino acids that serves to link plural identical, similar, or different polypeptides to form multimers and provides the flexibility or rigidity required for the desired function of the linked portions of the protein. Typically, a peptide linker is between about 1 and 30 amino acids in length. Examples of peptide linkers include, but are not limited to, -Gly-Gly-, GGGGS (SEQ ID NO:21), (GGGGS)n (SEQ ID NO:22), GKSSGSGSESKS (SEQ ID NO:23), GSTSGSGKSSEGKG (SEQ ID NO:24), GSTSGSGKSSEGSGSTKG (SEQ ID NO:25), GSTSGSGKSSEGKG (SEQ ID NO:26), GSTSGSGKPGSGEGSTKG (SEQ ID NO:27), or EGKSSGSGSESKEF (SEQ ID NO:28). Linking moieties are described, for example, in Huston, J. S., et al., Proc. Nat. Acad. Sci. 85: 5879–83 (1988), Whitlow, M., et al., Protein Engineering 6: 989–95 (1993), and Newton, D. L., et al., Biochemistry 35: 545–53 (1996). Other suitable peptide linkers are those described in U.S. Pat. Nos. 4,751,180 and 4,935,233.
Further, a recombinant fusion protein can comprise a BTL-II protein that lacks its normal signal sequence and has instead a heterologous signal sequence replacing it. The choice of a signal sequence depends on the type of host cells in which the recombinant protein is to be produced, and a heterologous signal sequence can replace the native signal sequence. Examples of signal sequences that are functional in mammalian host cells include the following: the signal sequence for interleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195; the signal sequence for interleukin-2 receptor described in Cosman et al. ((1984), Nature 312: 768); the interleukin-4 receptor signal peptide described in EP Patent No. 0 367 566; the type I interleukin-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; and the type II interleukin-1 receptor signal peptide described in EP Patent No. 0 460 846.
BTL-II Nucleic Acids
The invention encompasses isolated nucleic acids that encode the BTL-II proteins, fragments, or immunogenic fragments described above, including variants, fragments, recombinant fusion proteins, full-length proteins, soluble proteins, and secreted proteins. These nucleic acids are useful for, inter alia, producing recombinant proteins and detecting the presence of BTL-II nucleic acids in tissue samples, e.g. for diagnostic uses. Such nucleic acids can be genomic DNA or cDNA. The nucleic acid can comprise an uninterrupted open reading frame encoding a BTL-II protein of the invention. Nucleic acid molecules of the invention include DNA and RNA in both single-stranded and double-stranded form, as well as the corresponding complementary sequences. An “isolated nucleic acid” is a nucleic acid that has been separated from adjacent genetic sequences present in the genome of the organism from which the nucleic acid was isolated, in the case of nucleic acids isolated from naturally-occurring sources. In the case of nucleic acids synthesized chemically, such as oligonucleotides, or enzymatically from a template, such as polymerase chain reaction (PCR) products or cDNAs, it is understood that the nucleic acids resulting from such processes are isolated nucleic acids. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct.
Further, the invention encompasses fragments of a nucleic acid encoding a BTL-II protein that can serve (1) as probes for detecting BTL-II nucleic acids by a number of methods well known in the art, e.g. Southern and northern blotting, dot blotting, colony hybridizations, etc., (2) as polymerase chain reaction (PCR) primers to amplify BTL-II nucleic acids, or (3) as a means to regulate expression of BTL-II nucleic acids, e.g. through inhibition of expression with antisense nucleic acids (including peptide nucleic acids), ribozymes, triple helix-forming molecules, or interfering RNAs or DNAs that encode any of these RNAs. PCR primers can comprise, in addition to BTL-II nucleic acid sequences, other sequences such as restriction enzyme cleavage sites that facilitate the use of the amplified nucleic acid. PCR is described in the following references: Saiki et al. (1988), Science 239: 487–91; PCR Technology, Erlich, ed., Stockton Press, (1989). As explained below, PCR can be useful to detect overexpression of BTL-II mRNAs, and PCR primers can be taken from various parts of the gene and can also be selected to distinguish between different splice variants. Antisense RNAs (and DNAs encoding them), DNAs, or synthetic nucleotides and their use to regulate expression are well known in the art and are described in, e.g. Izant and Weintraub (1984), Cell 36 (4): 1007–15; Izant and Weintraub (1985), Science 229 (4711): 345–52; Harel-Bellan et al. (1988), J. Exp. Med. 168 (6): 2309–18; Sarin et al. (1988), Proc. Nat. Acad. Sci. USA 85 (20): 7448–51; Zon (1988), Pharm. Res. 5 (9): 539–49; Harel-Bellan et al. (1988), J. Immunol. 140 (7): 2431–35; Marcus-Sekura et al. (1987), Nucleic Acids Res. 15 (14): 5749–63; Gambari (2001), Curr. Pharm. Des. 7 (17): 1839–62; and Lemaitre et al. (1987), Proc. Natl. Acad. Sci. USA 84 (3): 648–52. Similarly, interfering RNAs (and DNAs encoding them) and their use to inhibit expression of selected genes are well known in the art and described in, e.g., Fjose et al. (2001), Biotechnol. Ann. Rev. 7: 31–57; Bosher and Labouesse (2000), Nature Cell Biol. 2: E31–E36. Further, ribozymes or DNAzymes can be targeted to cleave specific RNAs and thus used to inhibit gene expression as described in, e.g., Lewin and Hauswirth (2001), Trends Mol. Med. 7 (5): 221–28; Menke and Hobom (1997), Mol. Biotechnol. 8 (1): 17–33; Norris et al. (2000), Adv. Exp. Med. Biol. 465: 293–301; Sioud (2001), Curr. Mol. Med. 1 (5): 575–88; and Santiago and Khachigian (2001), J. Mol. Med. 79 (12): 695–706. Nucleic acids that can regulate BTL-II expression can find use in in vivo or in vitro studies of BTL-II function or as therapeutics, optionally as gene therapy agents.
The present invention also includes nucleic acids that hybridize under moderately stringent conditions, and more preferably highly stringent conditions, to nucleic acids encoding the BTL-II proteins described herein. Such nucleic acids include SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17. Preferably such nucleic acids encode proteins that can bind to a receptor on the surface of T cells and/or can inhibit T cell proliferation and/or cytokine production. Hybridization techniques are well known in the art and are described by Sambrook, J., E. F. Fritsch, and T. Maniatis (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, (1989)) and Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3–6.4 (1995)). Moderately stringent conditions include hybridization in about 50% formamide, 6×SSC at a temperature from about 42 to 55° C. and washing at about 60° C. in 0.5×SSC, 0.1% SDS. Highly stringent conditions are defined as hybridization conditions as above, but with washing at approximately 68° C. in 0.2×SSC, 0.1% SDS. SSPE (1×SSPE is 0.15 M NaCl, 10 mM NaH2PO4, and 1.26 mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is 0.15 M NaCi and 15 mM sodium citrate) in the hybridization and wash buffers; washes, preferably at least two, are performed for 15 minutes after hybridization is complete.
It should be understood that the wash temperature and wash salt concentration can be adjusted as necessary to achieve a desired degree of stringency by applying the basic principles that govern hybridization reactions and duplex stability, as known to those skilled in the art and described further below (see e.g., Sambrook et al., supra). When nucleic acids of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the nucleic acids and identifying the region or regions of optimal sequence complementarity. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5 to 10° C. less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm (degrees C)=2 (# of A+T bases)+4 (# of G+C bases). For hybrids above 18 base pairs in length, Tm (degrees C)=81.5+16.6 (log10[Na+])+0.41 (% G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1×SSC=0.165 M). Each such hybridizing nucleic acid has a length that is at least 15 nucleotides (or at least 18 nucleotides, or at least 20, or at least 25, or at least 30, or at least 40, or at least 50, or at least 100. Sambrook et al., supra.
BTL-II nucleic acids include nucleic acids comprising the following polynucleotides: (1) all or or a fragment of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17, wherein the fragment encodes a BTL-II protein that can bind to a receptor expressed on the surface of a T cell and/or inhibit proliferation and/or cytokine production of T cells; (2) sequences at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.7% identical to SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17 that are at least 100, 125, 150, 175, 200, 225, 250, 300, 400, 500, 600, 800, 1000. 1200, 1400, or 1600 nucleotides long and encode a BTL-II protein that can bind to a receptor expressed on the surface of T cells and/or inhibit proliferation and/or cytokine production of T cells; (3) fragments of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17 or substantially similar sequences that are useful for detecting or amplifying nucleic acids encoding the BTL-II proteins of the invention or for regulating the expression of BTL-II mRNAs and/or proteins; (4) nucleic acids comprising a polynucleotide that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.7% identical to a polynucleotide consisting of nucleotides 30 to 130 of SEQ ID NO:7 or SEQ ID NO:11 or to nucleotide 377–477 of SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17, wherein the region of identity is at least 60, 70, 80, 90, or 100 nucleotides long and the protein encoded by the nucleic acid can inhibit proliferation and/or cytokine production of T cells; and (5) nucleic acids that comprise at least 1, 2, 3, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 50, or 75 alteration(s) relative to SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17, wherein an alteration can be an insertion, deletion or substitution of a single nucleotide.
Antibodies that Bind Specifically to BTL-II Polypeptides
Antibodies that bind specifically to the BTL-II proteins of the invention, including variants, fragments, and recombinant fusion proteins, are encompassed by the invention. As used herein, specific binding of an epitope on a BTL-II protein by another protein (such as an antibody) means that the specifically-bound protein can be displaced from the molecule of BTL-II protein to which it is bound by another protein comprising the same epitope but not by another protein which does not comprise this epitope. Numerous competitive binding assays are known in the art. Epitopes may comprise only contiguous amino acids, but also may comprise non-contiguous amino acids that are brought into proximity by the tertiary folding of a BTL-II protein. Epitopes can be identified by methods known in the art. See e.g. Leinonen et al. (2002), Clin. Chem. 48 (12): 2208–16; Kroger et al. (2002), Biosens. Bioelectron. 17 (11–12): 937–44; Zhu et al. (2001), Biochem. Biophys. Res. Commun. 282 (4): 921–27. The invention also encompasses epitopes of the BTL-II proteins described herein that are useful for generating antibodies, which are referred to herein as immunogenic fragments. Immunogenic fragments are preferably at least 10 amino acids long and preferably comprise contiguous amino acids from SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18. Such epitopes can span regions of BTL-II proteins encoded by splice junctions, which may have the advantage of specific binding to proteins encoded by specific splice variants.
Antibodies can be polyclonal or monoclonal antibodies and can be produced by methods well-known in the art. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988); Kohler and Milstein (1980) Proc. Natl. Acad. Sci., USA, 77: 2197; Kozbor et al. (1984), J. Immunol. 133: 3001–3005 (describing the human B-cell hybridoma technique); Cole et al., Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77–96 (1985)(which describes EBV-hybridoma technique);. Kuby, Immunology, Second Edition, p. 162–64, W.H. Freeman and Co., New York (1994). Hybridoma cell lines that produce monoclonal antibodies specific for the BTL-II proteins of the invention are also contemplated herein. Such hybridomas can be produced and identified by conventional techniques. The hybridoma producing-the mAb of this invention can be cultivated in vitro or in vivo. Further, anti-BTL-II antibodies of the invention can be produced in other cultured cells, including, for example, Chinese hamster ovary (CHO), HeLa, VERO, BHK, Cos, MDCK, 293, 3T3, myeloma (e.g. NSO, NSI), or W138 cells, yeast cells, insect cells, and bacterial cells, including, for example, Eschericha coli. Such antibodies can be produced by introducing nucleic acids encoding the antibodies plus nucleic acids to enable expression of these nucleic acids into desired host cells. The antibodies can then be produced by culturing the cells into which these nucleic acids have been introduced. Monoclonal antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
Alternatively, antibodies can be single chain antibodies comprising a heavy and a light chain variable region-like domain and, optionally, also one or more constant region-like domain (U.S. Pat. No. 4,946,778; Bird et al. (1988), Science 242: 423–26; Huston et al. (1988), Proc. Natl. Acad. Sci. USA 85: 5879–83), dimeric or multivalent antibodies (see e.g. Lantto et al. (2002), J. Gen. Virol. 83: 2001–05; Hudson and Souriau (2001), Expert Opin. Biol. Ther. 1 (5): 845–55), tetrameric antibodies (see e.g. Janeway et al., Immunobiology: The Immune System in Health and Disease, Fifth Edition, Part II, Ch. 3, Garland Publishing (2001)), chimeric antibodies (Hudson and Souriau, supra; Boulianne et al. (1984), Nature 312:643–46; Morrison et al (1984), Proc. Natl. Acad. Sci. USA 81: 6851–55; Takeda et al. (1985), Nature 314: 452–54; Neuberger et al. (1985), Nature 314: 268–70), fully human antibodies produced in a different transgenic mammal (described in e.g., U.S. Pat. No. 6,150,584) or by in vitro selection (U.S. patent application No. 2002/0058033) or humanized antibodies (Morrison et al., supra; Takeda et al., supra; Boulianne et al., supra). Further, antibodies can be “matured” by in vitro selection schemes to yield an antibody with altered properties such as, for example, a higher affinity for the epitope to which it binds. See e.g. Jackson et al. (1995), J. Immunol. 154 (7): 3310–19; Pini and Bracci (2000), Curr. Protein Pept. Sci. 1 (2): 155–69; Ellmark et al. (2002), Mol. Immunol. 39 (5–6): 349; O'Connell et al. (2002), J. Mol. Biol. 321 (1): 49–56; Huls et al. (2001), Cancer Immunol. Immunother. 50: 163–71; Hudson and Souriau, supra; Adams and Schier (1999), J. Immunol. Methods 231 (1–2): 249–60; Schmitz et al. (2000), Placenta 21 Suppl. A: S106–12. Alternatively, fragments of an antibodies such as, for example, Fab fragments, F(ab′)2 fragments, or single chain Fv fragments (scFv's) that can bind specifically to a BTL-II protein of the invention are also encompassed by what is meant herein as an anti-BTL-II antibody. See Kuby, supra, pp.109–112 and Janeway et al., supra, for discussion of Fab and Fv fragments. The invention also encompasses anti-idiotypic antibodies that bind specifically to antibodies that bind specifically to BTL-II proteins and that mimic the effects of BTL-II proteins. Such anti-idiotypic antibodies find the same uses as BTL-II proteins. Methods for generating anti-idiotypic antibodies are well known in the art. See e.g. Kuby et al., supra, at 371–72. Various kinds of recombinant and non-recombinant bispecific antibodies that can bind specifically to a BTL-II protein of the invention and another epitope are also contemplated. Various kinds of bispecific antibodies and methods for making them are described in e.g. U.S. Pat. Nos. 4,474,893, 6,060,285, and 6,106,833.
The anti-BTL-II antibodies may be antagonistic antibodies that block a biological function of BTL-II, such as the binding of BTL-II to its receptor, or agonistic antibodies that promote a biological function of BTL-II or mimic the function of BTL-II. Agonistic antibodies can include agonistic anti-idiotypic antibodies that mimic the function of BTL-II protein. Assays for BTL-II function are described herein. Anti-BTL-II antibodies that block a biological function of BTL-II as determined in such assays are antagonistic antibodies as meant herein. Antagonistic antibodies may, for example, block the binding of BTL-II to its receptor. Anti-BTL-II antibodies that, when added to such assays, promote or enhance a biological function of BTL-II are agonistic antibodies as meant herein. An antagonistic anti-BTL-II antibody can be used, for example, as an adjuvant to enhance a mucosal immune response. Further, an agonistic antibody against a BTL-II receptor can be used to depress a mucosal immune response to treat, for example, a disease that is characterized by inappropriate inflammation of the gut, such as Crohn's disease or inflammatory bowel disease.
The antibodies of the invention can also be used in assays to detect the presence of the BTL-II proteins of the invention, either in vitro or in vivo. The antibodies also can be employed in purifying BTL-II proteins of the invention by immunoaffinity chromatography.
The invention encompasses nucleic acids encoding the antibodies of the invention and methods for producing the antibodies by introducing such nucleic acids into cells and culturing the cells containing the nucleic acids.
Agonists and Antagonists of BTL-II Polypeptides
The invention comprises agonists and antagonists of BTL-II and methods for screening for and using agonists and antagonists. Assays for BTL-II biological activity are described herein such as, for example, cell proliferation assays, cytokine secretion assays, binding assays, and genetic assays involving the over- or under-expression of BTL-II protein in vivo or in vitro or a complete absence of BTL-II expression in vivo or in vitro. Candidate molecules can be added to such assays to determine their effects on the biological activity of BTL-II proteins. BTL-II antagonists can, for example, block the interaction of BTL-II with its receptor, which is preferably expressed on B cell or T cells. Antagonists include antagonistic antibodies, and agonists include agonistic antibodies. In addition, other antibody-related molecules that can bind specifically to the BTL-II proteins of the invention, such as affibodies (Rönnmark et al. (2002), J. Immunol. Methods 261 (1–2): 199–211) and the biologically active peptides described in WO 00/24782 that can bind specifically to the BTL-II proteins of the invention and inhibit the biological activity of BTL-II proteins are encompassed by the invention. Further, BTL-II antagonists include the nucleic acids described above that are useful for modulating expression of BTL-II protein and/or mRNA, such as, for example, interfering RNAs (or DNAs that encode them) or antisense RNAs or DNAs.
Antagonists further include proteins that comprise amino acid sequences selected in vitro to bind to BTL-II or its receptor and that can, optionally, interfere with the interaction of BTL-II and its receptor. Alternatively, such proteins can be BTL-II agonists that promote or mimic the biological function of BTL-II. Proteins that bind to BTL-II or its receptor can be screened for their ability to interfere with the interaction of BTL-II with its receptor, or, alternatively, a selection can be designed to obtain such proteins directly.
Proteins may be selected by a number of methods such as, for example, phage display or display of the surface of a bacterium. See e.g. Parmley and Smith (1989), Adv. Exp. Med. Biol. 251: 215–218; Luzzago et al. (1995), Biotechnol. Annu. Rev. 1: 149–83; Lu et al. (1995), Biotechnology (NY) 13 (4): 366–372. In these methods, each member of a library of binding domains can be displayed on individual phage particles or bacterial cells, and bacteria or phage that bind to a protein of interest under chosen conditions can be selected. Nucleic acids encoding the selected binding domains can be obtained by growing the selected phage or bacteria and isolating nucleic acids from them.
Alternatively, a protein can be selected entirely in vitro. For example, each individual polypeptide in a library of potential binding domains can be attached to nucleic acids encoding it, and those that bind to the protein of interest under chosen conditions can be selected. Since the polypeptides are attached to nucleic acids encoding them, subsequent operations, such as amplifying, cloning, or sequencing nucleic acids encoding effective binding domains are facilitated. Various schemes for such selections are known in the art, including antibody-ribosome-mRNA particles, ribosome display, covalent RNA-peptide fusions, or covalent DNA-RNA-peptide fusions. He and Taussig (1997), Nucleic Acids. Res. 25 (24): 5132–5134; Hanes and Pluckthun (1997), Proc. Natl. Acad. Sci. 94: 4937–4942; Roberts and Szostak (1997), Proc. Natl. Acad. Sci. 94: 12297–12302; Lohse and Wright (2001), Curr. Opin. Drug Discov. Devel. 4 (2): 198–204; Kurz et al. (2000), Nucleic Acids Res. 28 (18): E83; Liu et al. (2000), Methods Enzymol. 318: 268–93; Nemoto et al. (1997), FEBS Lett. 414 (2): 405–08; U.S. Pat. No. 6,261,804; WO0032823; and WO0034784. Such proteins can be selected to be antagonists or agonists.
Assays for the Biological Activity of BTL-II Proteins
Various assays can be used to detect the biological activity of a BTL-II protein and to identify binding partners of BTL-II. BTL-II proteins, receptor(s) of BTL-II, and agonists and/or antagonists of either can be used in such assays.
Assays to Identify Binding Partners
A binding partner for BTL-II protein can be identified by first determining what type of cells a soluble BTL-II protein can bind to. Since the known receptors for members of the B7 subfamily of butyrophilin-like proteins are all expressed on T cells, either constitutively (CD28) or after activation (CTLA-4, ICOS, PD-1), T cells can be tested to determine whether BTL-II can bind to them. Because of the expression pattern of BTL-II, T cells isolated from normal and inflamed gut can be included in such tests. In addition, various subsets of T cells, including memory T cells, naive T cells, αβ T cells, γδ T cells, and T cells in various activation states, can be tested.
Such experiments can be conducted using methods well known in the art. For example, a BTL-II recombinant fusion protein comprising the extracellular domain of murine BTL-II plus an Fc region of an antibody can be used to bind to the cells being tested. A fluorescently-labeled antibody that can bind to the Fc region in the recombinant fusion protein can be added. After washing, the cells can be analyzed using a fluorescence activated cell sorting (FACS) device to determine whether BTL-II can bind to the cells. One such assay is described by Chapoval et al. )(2001), Nature Immunol. 2 (3): 269–74). Other methods known in the art can also be suitable to determine whether BTL-II binds to specific cell populations.
Further, known receptors of B7 subfamily members will be tested to determine whether BTL-II binds to any of these proteins. Recombinant fusion proteins comprising an extracellular domain of a receptor of a B7 sub-family member (such as, for example, CTLA-4, a receptor for B7-1 and B7-2) and an Fc region of an antibody can be made by methods similar to those used to make such BTL-II fusion proteins. Cells can be transfected with full length forms of BTL-II nucleic acids encoding BTL-II proteins including transmembrane and cytoplasmic domains that are expressed on the cell surface. The receptor:Fc fusion protein to be tested can be added to such cells along with a fluorescently-labeled antibody that can bind to the Fc region. After washing, the cells can be analyzed by FACS to determine whether the receptor:Fc fusion protein binds to the BTL-II protein expressed on the transfected cells. The reverse experiment can also done where soluble BTL-II:Fc fusion protein is used and the full length receptor protein is introduced and expressed via transfection. Such experiments can reveal binding interactions between BTL-II proteins and known receptors of other B7 subfamily members.
If BTL-II binds to at least one variety of T cells but does not bind to a known receptor, a variety of expression cloning or protein purification methods known in the art can be used to identify the receptor that BTL-II binds to. As an example, a radioactive slide binding cDNA expression cloning method can be used. Briefly, a cell source with the greatest binding to soluble BTL-II is identified, mRNA is isolated, and a cDNA library is built in a mammalian expression vector. Mammalian cells are transfected with pools of cDNAs on slides, and after an appropriate incubation to allow expression, soluble BTL-II:Fc fusion protein is bound to the cells. Specific binding to receptor bearing cells is detected by the following series of steps: binding of a radioactive anti-Fc reagent to the bound BTL-II:Fc protein; application of film emulsion to the slides; incubation to allow exposure; film development to deposit silver grains; and detection of the grains by microscope. The receptor-expressing clone is then isolated from the pool by sub-dividing the pool and iterative slide binding assays to identify the single receptor clone. Such methods are described in McMahon et al. (1991), EMBO J. 10: 2821–32.
When binding to cells is achieved, a variety of means, either through animal immunization or phage display technology, can be used to isolate antibodies that bind BTL-II and disrupt its binding to cells. Such antibodies can be used to antagonize the activity of endogenous BTL-II and therefore can be used in assays to determine the effects of lowered effective amounts of BTL-II on immune responses and in disease models, such as the inflammatory bowel disease models, for example, those described by Cooper et al. ((1993), Lab. Invest. 69 (2): 238–49) and Tokoi et al. ((1996), J. Gastroenterol. 31 (2): 182–88).
Assays to Determine BTL-II Function
B7 subfamily proteins have been shown to have activity in T cell “co-stimulatory” assays that involve activating T cells through their T cell receptors with varying doses of “antigen.” The activity of the B7 subfamily proteins is most evident at “sub-optimal” T cell receptor stimulation. See e.g. Latchman et al. (2001), Nature Immunol. 2 (3): 261–68. A “surrogate antigen,” such as tissue culture dish bound anti-CD3ε antibody or antigens presented by the MHC molecules on irradiated antigen presenting cells, can be used. A soluble BTL-II protein can be added to determine its effect on the T cells' response to the “antigen.” Various parameters can be measured to determine whether the T cells are being stimulated or suppressed. Cellular proliferation, cell surface receptor expression, and levels of expression of immunomodulatory molecules (such as, for example, interferon γ and/or IL-2) at the protein and/or MRNA level can be measured. Such methods are used and described in e.g., Fitch et al. in T Cell Subsets in Infectious and Autoimmune Diseases, John Wiley and Sons, pp. 68–85 (1995); Freeman et al. (2000), J. Exp. Med. 192 (7): 1027–34; Swallow et al. (1999), Immunity 11:423–32; Hutloff et al. (1999), Nature 397: 263–66; Yoshinaga et al. (1999), Nature 402: 827–32; Latchman et al., supra. Given the expression pattern of BTL-II, such assays can include T cells derived from mucosal tissues or the lymph nodes that drain such tissues. In addition, naive and memory T cells, CD4+ and CD8+ T cells, and T cells expressing T cell receptors other than αβ T cell receptors can be examined. From such experiments can reveal whether BTL-II can alter responses of a number of distinct kinds of T cells. Such experiments are described below and show that a soluble version of BTL-II can inhibit cell proliferation and suppress production of cytokines, including interferon gamma (IFNγ), interleukin 2 (IL2), and interleukin 5 (IL5). Examples 6–10; FIGS. 12–17.
Variations of this kind of experiment include examining the effect of including soluble forms of BTL-II proteins on the co-stimulatory response found with other soluble B7 subfamily proteins or various TNF family members that can be co-stimulatory. This will help to define whether BTL-II can alter co-stimulation seen with other molecules. Further variations can include use of non-irradiated antigen presentation cells of various sorts. This will help define the effects of addition of a soluble BTL-II protein on the function of antigen presenting cells. In still another variation, antibodies that block BTL-II binding to cells (see above) can also be introduced in such costimulation experiments to determine the effects of preventing binding.
In another kind of experiment, antigen presenting cells (such as, for example, dendritic cells or B cells from Peyer's patches or intestinal epithelial cells (IECs)) that express BTL-II can be combined with T cells (such as the various kinds listed above), and one or more of the parameters listed above can be measured. The results obtained with these cells can be compared to results obtained when interfering RNAs, or DNAs encoding them, designed to lower BTL-II expression are introduced into the antigen presenting cells.
T regulatory cells act to suppress autoimmune responses and help to do so, in part, by inducing differentiation or enhancing regulatory function of T regulatory (T reg) cells in inflammatory bowel disease model systems. T reg cells include, for example, Tr1 cells (Cong et al. (2002), J. Immunol. 169 (11): 6112–19; Groux et al. (1997), Nature 389: 737–42), Th3 cells, CD4+Cd25+ T reg cells (see e.g. Maloy and Powrie (2001), Nature Immunology 2 (9): 816–22), CD4+RbloCD25+ T reg cells, and CD8+ T reg cells (Allez et al. (2002), Gastroenterology 123: 1516–26), among others. Thus, assessing the effects of BTL-II upon T regulatory cell proliferation and function may be necessary to determine the precise role of BTL-II in vivo. For example, antigen specific proliferation of T cells and/or cytokine production by T cells in the presence of Tr1 cells can be measured. Such assays are described in Cong et al., supra. Soluble forms of BTL-II, blocking antibodies, or inhibition of BTL-II expression using interfering RNAs can be used to determine whether BTL-II plays a role in T regulatory cell or Tr1 cell proliferation, maintenance, or ability to suppress antigen activation of other T cells.
One T cell subset, Th3 cells, produce transforming growth factor β (TGF β) and have been implicated in oral tolerance to mucosal antigens. See e.g. Fukaura et al. (1996), J. Clin. Invest. 98 (1): 70–77; Inobe et al. (1998), Eur. J. Immunol. 28: 2780–90. Such cells can be generated from native T cells by extensive culture with antigen in the presence of TGF β. Soluble BTL-II proteins or blocking antibodies can be used to ask whether BTL-II can alter the proliferation or function of these cells by methods similar to those described above.
The effects of administering soluble BTL-II proteins or antagonistic antibodies in model T cell systems using simple, well-defined antigens, such as, for example, ovalbumin, or complex antigens, including bacteria or viruses, will be ascertained. T cells responses to such antigens (including proliferation and/or production of molecules such as interferon γ or IL-2) can be measured in the presence and absence of BTL-II proteins or antibodies.
Similarly effects in animal systems can be tested. Early focus will be on systems particularly relevant to mucosal immune response. This includes model systems in which antigen is fed to animals (see e.g. Yamashiro et al. (1994), Acta Paediatr. Jpn. 36: 550–56; Jain et al. (1996), Vaccine 14 (13):1291–97; Chen et al. (2002), Immunology 105: 171–80), inflammatory bowel disease systems such as in the dextran sulfate sodium-induced inflammatory bowel disease model (Cooper et al. (1993), Lab. Invest. 69 (2): 238–49; Tokoi et al. (1996), J. Gastroenterol. 31 (2): 182–88), the CD45RBhi/CD4+ T cell-induced wasting disease model (Morrissey et al. (1993), J. Exp. Med. 178: 237–44), and/or model asthma systems in which antigen is fed or instilled into the airways to provoke an immune response such as the murine ovalalbumin-induced asthma model (Brusselle et al. (1994), Clin. Exp. Allergy 24 (1): 73–80). In particular, the magnitude of the humoral or cell mediated response will be measured, and the type of immunomodulatory cytokines produced will be measured. Such experiments can reveal whether increased or decreased BTL-II can be of benefit in dampening responses to antigens, including autoantigens, or increasing response to alloantigens. Effects of BTL-II proteins or anti-BTL-II antibodies can also be ascertained in other models of inflammatory diseases.
Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Coligan et al. eds, Greene Publishing Associates and Wiley-Interscience (pp. 3.1–3.19: In vitro assays for mouse lymphocyte function; Chapter 7: Immunologic studies in humans); Takai et al. (1986), J. Immunol. 137: 3494–3500; Bertagnolli et al. (1990), J. Immunol. 145: 1706–1712; Bertagnolli, et al. (1992), J. Immunol. 149: 3778–3783; Bowman et al. (1994), J. Immunol. 152: 1756–1761.
Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Kruisbeek and Shevach (1994), Polyclonal T cell stimulation, in Current Protocols in Immunology, Coligan et al. eds. Vol 1 pp. 3.12.1–3.12.14, John Wiley and Sons, Toronto (1991); and Schreiber, (1994), Measurement of mouse and human interferon gamma in Current Protocols in Immunology, Coligan et al. eds. Vol 1 pp. 6.8.1–6.8.8, John Wiley and Sons, Toronto (1991).
Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Bottomly et al., 1991, Measurement of human and murine interleukin 2 and interleukin 4, in Current Protocols in Immunology, Coligan et al. eds. Vol 1 pp. 6.3.1–6.3.12, John Wiley and Sons, Toronto (1991); deVries et al. (1991), J. Exp. Med. 173: 1205–1211; Moreau et al. (1988), Nature 336:690–692; Greenberger et al. (1983), Proc Natl Acad Sci.USA 80: 2931–2938; Nordan, Measurement of mouse and human interleukin 6, in Current Protocols in Immunology, Coligan et al. eds. Vol 1 pp. 6.6.1–6.6.5, John Wiley and Sons, Toronto (1991); Smith et al., (1986), Proc Natl Acad Sci USA 83: 1857–1861; Bennett et al., 1991, Measurement of human interleukin 11, in Current Protocols in Immunology Coligan et al. eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto (1991); Ciarletta et al., Measurement of mouse and human Interleukin 9, in Current Protocols in Immunology Coligan et al. eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto (1991).
Assays for T-cell clone responses to antigens (which will identify, among others, polypeptides that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Coligan et al. eds, Greene Publishing Associates and Wiley-Interscience (Chapter 3: In vitro assays for mouse lymphocyte function; Chapter 6: Cytokines and their cellular receptors; Chapter 7: Immunologic studies in humans)(1991); Weinberger et al. (1980), Proc Natl Acad Sci USA 77: 6091–6095; Takai et al. (1986), J. Immunol. 137:3494–3500; Takai et al. (1988), J. Immunol. 140: 508–512.
Assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Coligan et al. eds, Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1–3.19; Chapter 7, Immunologic studies in Humans)(1991); Herrmann and Mescher (1981), Proc. Natl. Acad. Sci. USA 78: 2488–2492; Herrmann et al. (1982), J. Immunol. 128: 1968–1974; Handa et al. (1985), J. Immunol. 135:1564–1572; Takai et al. (1986), J. Immunol. 137: 3494–3500; Takai et al. (1988), J. Immunol. 140:508–512,; Brown et al. (1994), J. Immunol. 153: 3079–3092.
Assays for immunoglobulin responses and isotype switching by B cells (which will identify, among others, polypeptides that modulate T-cell dependent antibody responses and that affect Th1/Th2 profiles) include, without limitation, those described in: Maliszewski (1990), J. Immunol 144: 3028–3033; and Mond and Brunswick, Assays for B cell function: in vitro antibody production, in Current Protocols in Immunology Coligan et al. eds. Vol 1 pp. 3.8.1–3.8.16, John Wiley and Sons, Toronto (1994).
Mixed lymphocyte reaction (MLR) assays (which will identify, among others, polypeptides that generate predominantly Th1 and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Coligan et al. eds, Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1–3.19; Chapter 7, Immunologic studies in Humans); Takai et al. (1986); J. Immunol. 137:3494–3500; Takai et al. (1988), J. Immunol. 140: 508–512; Bertagnolli et al. (1992), J. Immunol. 149: 3778–3783.
Dendritic cell-dependent assays (which will identify, among others, polypeptides expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery and Adorini (1995), J. Immunol 154: 536–544; Inaba et al. (1991), J Exp Med 173: 549–559; Macatonia et al. (1995), J Immunol 154: 5071–5079; Porgador and Gilboa (1995), J Exp Med 182: 255–260; Nair et al. (1993), J. Virology 67:4062–4069; Huang et al. (1994), Science 264:961–965; Macatonia et al. (1989), J Exp Med 169:1255–1264; Bhardwaj et al. (1994), J. Clin. Invest. 94:797–807; and Inaba et al. (1990), J. Exp. Med. 172:631–640.
Assays for polypeptides that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al. (1994), Blood 84: 111–117; Fine et al. (1994), Cell Immunol 155: 111–122; Galy et al. (1995), Blood 85: 2770–2778; Toki et al. (1991), Proc Natl Acad Sci. USA 88: 7548–7551.
Assays for receptor-ligand activity include without limitation those described in: Current Protocols in Immunology Coligan et al. eds, Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of cellular adhesion under static conditions 7.28.1–7.28.22); Takai et al. (1987), Proc. Natl. Acad. Sci. USA 84: 6864–6868; Bierer et al. (1988), J. Exp. Med. 168:1145–1156; Rosenstein et al. (1989), J. Exp. Med. 169: 149–160; Stoltenborg et al. (1994), J. Immunol. Methods 175: 59–68; Stitt et al. (1995), Cell 80: 661–670.
Genetic Assay For Function Utilizing Transgenic Animals
Transgenic animals, preferably mice, that have multiple copies of the gene(s) corresponding to the BTL-II nucleic acids disclosed herein, preferably produced by transformation of cells with genetic constructs that are stably maintained within the transformed cells and their progeny, are provided. Transgenic animals that have modified genetic control regions that increase or reduce gene expression levels, or that change temporal or spatial patterns of gene expression, are also provided (see European Patent No. 0 649 464 B1). In addition, organisms are provided in which the BTL-II gene has been partially or completely inactivated, through insertion of extraneous sequences into the corresponding gene or through deletion of all or part of the corresponding gene. Partial or complete gene inactivation can be accomplished through insertion, preferably followed by imprecise excision, of transposable elements (Plasterk (1992), Bioessays 14 (9): 629–633; Zwaal et al. (1993), Proc. Natl. Acad. Sci. USA 90 (16): 7431–7435; Clark et al., (1994), Proc. Natl. Acad. Sci. USA 91 (2): 719–722), or through homologous recombination, preferably detected by positive/negative genetic selection strategies (Mansour et al. (1988), Nature 336: 348–352; U.S. Pat. Nos. 5,464,764; 5,487,992; 5,627,059; 5,631,153; 5,614,396; 5,616,491; and 5,679,523). As an alternative, expression of the BTL-II gene can be inhibited in a transgenic or non-transgenic animal by introduction of an interfering RNA, antisense RNA, or a ribozyme, which may be encoded by DNA introduced into a transgenic animal. The phenotypes of such organisms can elucidate the in vivo function(s) of the BTL-II gene. For example, an increased propensity (compared to wild type animals) in a transgenic animal that does not express BTL-II protein to exhibit symptoms of inflammatory bowel disease in response to feeding of dextran sulfate sodium can indicate that BTL-II normally dampens inflammation in the gut. Alternatively, transgenic animals can overexpress BTL-II. Phenotypes of such transgenic animals can also give clues as to the in vivo function of BTL-II proteins.
Uses of the Proteins, Antibodies, Antagonists, and Agonists of the Invention
The mucosal immune system operates within a set of specialized anatomical structures, the mucus membranes, and the immune response it generates has properties that distinguish it from an immune response generated in other anatomical compartments of the body. The mucus membranes are just one of several distinct anatomical compartments, also including the peripheral lymph nodes and spleen, the body cavities, i.e. the peritoneum and the pleura, and the skin, in which the immune system is active. Mucosal surfaces are found in the lungs, the gut, the eyes, the nose, the mouth, the throat, the uterus, and the vagina. A mucosal surface is a thin sheet or layer of pliable tissue serving as the covering or envelope of a bodily structure, such as the lining of a body cavity, a partition or septum, or a connection between two structures. Since mucosal surfaces are the route of entry for the vast majority of infectious agents, an adjuvant that can promote a mucosal immune response is particularly desirable. Janeway et al., Immunobiology: The Immune System in Health and Disease, 5th Edition, Part IV, Ch. 10, Garland Publishing, New York and London (2001). Infectious diseases that typically enter through mucosal surfaces, such as, for example, Neisseria gonorrhoeae, often generate only a weak mucosal immune response. Russell et al. (1999), 42 (1): 58–63.
The gut is unique in several ways. Lining the gut are a number of specialized forms of lymphoid tissue collectively known as gut-associated lymphoid tissue (GALT) including: tonsils and adenoids, which together form Waldyer's ring at the back of the mouth; Peyer's patches in the small intestine; the appendix; and solitary lymphoid follicles in the large intestine and rectum. Activation of a lymphocyte by an encounter with a foreign antigen in the gut can lead to the spread of an adaptive immune response to the antigen throughout the mucosal immune system. The activated lymphocyte can enter the lymphatic system and, from there, the bloodstream. The bloodstream can deliver activated lymphocytes to mucosal sites throughout the body, which can be recognized by the lymphocytes by means of molecules such as the mucosal adressin MAdCAM-1, which is expressed in mucosal tissue. Janeway et al., supra. The dominant antibody type produced by the gut is IgA, which, upon expression, is found primarily in the mucus layer overlying the gut epithelium. In addition, a number of distinct kinds of T cells are found in the gut. Janeway et al., supra.
Introduction of a foreign antigen into the gut usually leads to immunological tolerance but may lead to a specific immune response. The gut normally receives and tolerates (in an immunological sense) a vast array of foreign antigens, that is, food and the commensal microorganisms residing in the gut. The feeding of a specific foreign protein can lead to a state of specific unresponsiveness to that protein known as oral tolerance, such that later injection of the protein, even in the presence of an adjuvant, yields no antibody response. This phenomenon may involve the spleen and lymph nodes as well as the mucosal immune system. See Gutgemann et al. (1998), Immunity 8: 667–73. However, enteric pathogens, such as, for example, Salmonella, Yersinia, or Entamoeba histolytica, can elicit a local, or even a systemic, immune response. The factors controlling whether the introduction of a foreign antigen into the gut elicits an immune response are incompletely understood. Janeway et al., supra at Part V, Ch. 14.
Many vaccines employ adjuvants to enhance the immune response to the antigen. An adjuvant strengthens or broadens the specificity of an immune response to an antigen. The immune response may include an increase in antibody titer or an increase in the number of antigen-reactive T cells. Methods for measuring such parameters exist in the art. See e.g. Zigterman et al. (1988), J. Immunol. Methods 106 (1): 101–07. The mechanism(s) by which adjuvants enhance an immune response are incompletely understood, but their use can be essential. Few existing vaccines can elicit a robust mucosal immune response to the selected antigen.
The invention encompasses a method for promoting a systemic or mucosal immune response against an antigen comprising administering a therapeutically effective amount of an antagonist of BTL-II and the antigen. The antagonists of BTL-II that can be used to practice the invention include, antagonistic antibodies or in vitro-selected binding proteins that bind specifically to the extracellular region of BTL-II, and small molecules that can inhibit the biological activity of BTL-II. Optionally, the antagonist of BTL-II protein and the antigen can be administered directly to a mucosal surface, such as orally, nasally, vaginally, gastrically, or rectally or by inhalation. For example, nasal administration has been reported to be more effective than vaginal administration in inducing a durable immune response in at least one case. Russell (2002), Am. J. Reprod. Immunol. 47 (5): 265–68. Alternatively, the BTL-II antagonist and/or the antigen can be injected, for example, subcutaneously, intravenously, intramuscularly, intraarterially, or intraperitoneally. In some embodiments, a BTL-II antagonist, such as an antibody, can be injected and an antigen can be administered directly to a mucosal surface.
Appropriate antigens for practicing the invention include all or part of any infectious agent or agent that is similar to an infectious agent. Infectious agents can include live or killed viruses, bacteria, and infectious eukaryotes such as amoeba, flagellates, or helminths. An agent that is similar to such an infectious agent may, for example, be a virus that is analogous to a virus that can infect the mammal being vaccinated, but cannot, itself, infect the mammal being vaccinated. An example of this is the vaccinia virus (which can produce disease in cows but not people) used by Jenner to produce a vaccine against smallpox, a similar virus that produces disease in humans. Janeway et al., supra, Part V, Ch. 14. Table 4 indicates specific examples of antigens that could be used to practice the invention.
TABLE 4
Antigen
Category
Some Specific Examples of Representative Antigens
Viruses
Rotavirus; foot and mouth disease; influenza, including influenza A
and B; parainfluenza; Herpes species (Herpes simplex, Epstein-Barr
virus, chicken pox, pseudorabies, cytomegalovirus); rabies; polio;
hepatitis A; hepatitis B; hepatitis C; hepatitis E; measles; distemper;
Venezuelan equine encephalomyelitis; feline leukemia virus; reovirus;
respiratory syncytial virus; bovine respiratory syncytial virus; Lassa
fever virus; polyoma tumor virus; parvovirus; canine parvovirus;
papilloma virus; tick-borne encephalitis; rinderpest; human rhinovirus
species; enterovirus species; Mengo virus; paramyxovirus; avian
infectious bronchitis virus; HTLV 1; HIV-1; HIV-2; LCMV
(lymphocytic choriomeningitis virus); adenovirus; togavirus (rubella,
yellow fever, dengue fever); corona virus
Bacteria
Bordetella pertussis; Brucella abortis; Escherichia coli; Salmonella
species including Salmonella typhi; streptococci; Vibrio species (V.
cholera, V. parahaemolyticus); Shigella species; Pseudomonas species;
Brucella species; Mycobacteria species (tuberculosis, avium, BCG,
leprosy); pneumococci; staphlylococci; Enterobacter species;
Rochalimaia henselae; Pasterurella species (P. haemolytica, P.
multocida); Chlamydia species (C. trachomatis, C. psittaci,
Lymphogranuloma venereum); Syphilis (Treponema pallidum);
Haemophilus species; Mycoplasma species; Lyme disease (Borrelia
burgdorferi); Legionnaires' disease; Botulism (Colstridium botulinum);
Corynebacterium diphtheriae; Yersinia entercolitica
Ricketsial
Rocky mountain spotted fever; thyphus; Ehrlichia species
Infections
Parasites
Malaria (Plasmodium falciparum, P. vivax, P. malariae); schistosomes;
and
trypanosomes; Leishmania species; filarial nematodes; trichomoniasis;
Protozoa
sarcosporidiasis; Taenia species (T. saginata, T. solium); Toxoplasma
gondii; trichinelosis (Trichinella spiralis); coccidiosis (Eimeria
species) ; helminths including Ascarus species
Fungi
Cryptococcus neoformans; Candida albicans; Apergillus fumigatus;
coccidioidomycosis
Recombinant
Herpes simplex; Epstein-Barr virus; hepatitis B; pseudorabies;
Proteins
flavivirus (dengue, yellow fever); Neisseria gonorrhoeae; malaria:
circumsporozoite protein, merozoite protein; trypanosome surface
antigen protein; pertussis; alphaviruses; adenovirus
Proteins
Diphtheria toxoid; tetanus toxoid; meningococcal outer membrane
protein (OMP); streptococcal M protein; hepatitis B; influenza
hemagglutinin; cancer antigen; tumor antigens; toxins; exotoxins;
neurotoxins; cytokines and cytokine receptors; monokines and
monokine receptors
Synthetic
Malaria; influenza; foot and mouth disease virus; hepatitis B; hepatitis C
Peptides
Polysaccharides
Pneumococcal polysaccharide; Haemophilis influenza polyribosyl-
ribitolphosphate (PRP); Neisseria meningitides; Pseudomonas
aeruginosa; Klebsiella pneumoniae
Oligosaccharide
Pneumococcal
Alternatively, soluble BTL-II proteins can be used to promote tolerance to an antigen that is implicated in an autoimmune or inflammatory disease. For example, experimental autoimmune encephalomyelitis (EAE), a condition similar in many respects to multiple sclerosis, can be induced in rodents by injection of, for example, various epitopes of myelin basic protein or myelin oligodendrocyte glycoprotein (MOG). MOG-induced EAE can, in some cases, be ameliorated by prior feeding of small portions of MOG or butyrophilin. Stefferl et al. (2000), J. Immunol. 165: 2859–65. Soluble BTL-II proteins can be co-administered with an antigen known to be targeted in an autoimmune disease to promote tolerance to the antigen and thereby ameliorate the symptoms of the autoimmune disease. Optionally, the antigen can be administered directly to a mucosal surface, for example, nasally.
Inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, include chronic inflammation of the gastrointestinal tract, possibly because of an abnormally enhanced immune response to antigens of normal gut flora. Both diseases likely have at least some genetic basis since occurrences tend to cluster in families and can be associated with some genetic markers. For example, mice that do not express the multiple drug resistance gene (mdr1a) spontaneously develop colitis. Panwala et al. (1998), J. Immunol. 161: 5733–44. The occurrence of both Crohn's disease and ulcerative colitis is likely also influenced by environmental factors because increased occurrence is observed among urbanized populations. Also, such diseases do not occur in the absence of normal gut flora.
Crohn's disease is involves an abnormal inflammation of any portion of the alimentary tract from the mouth to the anus, although in most patients abnormal inflammation is confined to the ileocolic, small-intestinal, and colonic-anorectal regions. Typically, the inflammation is discontinuous. Common symptoms include abdominal pain, anorexia, weight loss, fever, diarrhea, fullness and/or tenderness in the right lower quadrant of the abdomen, constipation, vomiting, and perianal discomfort and discharge. Other possible symptoms include peripheral arthritis, growth retardation, episcleritis, aphthous stomatitis, erythema nodosum, pyoderma gangrenosum, kidney stones, impaired urinary dilution and alkalinization, malabsorption, and gallstones, among others. See e.g. Strober et al., Medical Immunology, 10th Edition, Section III, Ch. 35 (2001); Merck Manual of Diagnosis and Therapy, 17th Edition, Section 3, Ch. 31 (1999). Macrophages isolated from patients with Crohn's disease produce increased amounts of IL-12, IFNγ, TNFα, and other inflammatory cytokines.
Ulcerative colitis, though it is sometimes hard to distinguish from Crohn's disease, is distinct from Crohn's disease in several respects. First, it is generally limited to the colon while Crohn's disease may occur throughout the alimentary tract. Second, ulcerative colitis mainly involves inflammation only of the superficial layers of the bowel, unlike Crohn's disease in which the inflammation can penetrate all way through the wall of the bowel or other location in the alimentary tract. Finally, ulcerative colitis typically involves a continuous area of inflammation, rather than the discontinuous sites of inflammation typical of Crohn's disease. Like Crohn's disease, ulcerative colitis is found primarily in urban areas. Also, genetic factors likely play a role in ulcerative colitis since there is a familial aggregation of cases. Autoantibodies are observed in ulcerative colitis patients more often than Crohn's disease patients. The autoantibodies are often directed to colonic epithelial cell components. Among the most common are antineutrophil cytoplasmic antibodies with specificities for catalase, α-enolase, and lactoferrin. In some cases such antibodies cross react with colonic microorganisms.
Symptoms of ulcerative colitis are variable. They may include diarrhea, tenesmus, abdominal cramps, blood and mucus in the stool, fever, and rectal bleeding. Toxic megacolon, a potentially life-threatening condition in which the colon is dilated beyond about 6 centimeters and may lose its muscular tone and/or perforate, may also occur. Other symptoms that may accompany ulcerative colitis include peripheral arthritis, ankylosing spondylitis, sacroiliitis, anterior uveitis, erythema nodosum, pyoderma gangrenosum, episcleritis, autoimmune hepatitis, primary sclerosing cholangitis, cirrhosis, and retarded growth and development in children.
Antibodies or in vitro-selected binding proteins that bind specifically to BTL-II proteins can be used to diagnose or predict the onset of inflammatory bowel disease. As illustrated in Example 4 below, BTL-II is overexpressed in the gut prior to the onset of symptoms and during the symptomatic phase in a mouse model of inflammatory bowel disease. Thus, overexpression of BTL-II can indicate the existence of inflammatory bowel disease and can predict the its onset. Anti-BTL-II antibodies can be used to detect overexpression of BTL-II by assaying a tissue sample from the bowel of a patient using an ELISA assay or other immune-based assays known in the art. See e.g. Reen (1994), Enzyme-Linked Immunosorbent Assay (ELISA), in Basic Protein and Peptide Protocols, Methods Mol. Biol. 32: 461–466. Overexpression can also be detected by nucleic acid-based methods for measuring BTL-II mRNA expression such as, for example, reverse transcription plus PCR (RT-PCR), among other mRNA expression assays known in the art. See e.g. Murphy et al. (1990), Biochemistry 29 (45): 10351–56.
In another embodiment, soluble BTL-II proteins of the invention can be used to treat an inflammatory bowel disease. A soluble BTL-II protein can bind to a specific receptor expressed on a B cell or a T cell, thereby enabling the downregulation of an immune response. Such a downregulation can, for example, prevent the activation of a macrophage or a B cell by a CD4+ T cell or prevent activation of a T cell by an antigen. Alternatively, such a downregulation can cause a T cell to become anergic when it encounters an antigen to which its T cell receptor can specifically bind.
Antibodies or in vitro-selected binding proteins that bind specifically to BTL-II also find use as diagnostic reagents to identify patients with inflammatory bowel disease or at risk of developing inflammatory bowel disease. Since BTL-II is overexpressed prior to the onset of and during inflammatory bowel disease symptoms in a mouse model system (Example 4), an abnormally high level of BTL-II expression can indicate the presence of an inflammatory disease in the gut or a high risk of developing an inflammatory disease in the gut.
In addition, soluble BTL-II proteins can be useful in situations where down-modulation of an immune response is desired, such as transplantation (Manilay et al., 1998, Curr. Opin. Immunol. 10:532–538), graft versus host disease, graft rejection, autoimmune or inflammatory disease, gene therapy (Hackett et al., 2000, Curr. Opin. Mol. Therap. 2: 376–382), and the like. For example, a soluble BTL-II protein can be administered prior to, at approximately the same time (or either shortly before or shortly after), or concurrently with administration of a gene therapy vector to a mammal, transplantation, or as otherwise appropriate for the desired immuno-suppression. Also appropriate for such a treatment is an anti-idiotypic antibody that mimics the function of BTL-II.
An agonistic BTL-II antibody, a soluble BTL-II protein, or an anti-idiotypic antibody can be administered to a patient suffering from an autoimmune or inflammatory disease in order to decrease the number of detectable autoantibodies, to decrease the activation of immune effector cells, and/or to decrease or eliminate the symptoms of the disease. Autoimmune and inflammatory diseases include all conditions in which the patient's own tissues are subject to deleterious effects caused by the patient's immune system. Such effects can be mediated by autoantibodies and/or by the activation of immune effector cells, among other possibilities. Although the causes of autoimmune and inflammatory diseases are usually unclear, a correlation between the existence of various kinds of infections and various autoimmune diseases has been established in some cases and is a recurring subject of discussion in the scientific literature. See e.g. Corapcioglu et al. (2002), Thyroid 12: 613–17;Sewell et al. (2002), Immunol. Lett. 82: 101–10; Rose (1998), Semin. Immunol. 10 (1): 5–13; Matsiota-Bernard (1996), Clin. Exp. Immunol. 104: 228–35; and McMurray and Elbourne (1997), Semin. Arthritis Rheum. 26: 690–701.
One of skill in the art will appreciate that symptoms of autoimmune and inflammatory diseases are extremely diverse and can depend on what tissues are targeted by the patient's immune system. Autoimmune and inflammatory diseases can be organ-specific or systemic. Autoimmune and inflammatory diseases include, for example, arthritis, Addison's disease, insulin-dependent diabetes mellitus (type I diabetes mellitus), asthma, polyglandular endocrinopathy syndromes, systemic lupus erythematosus, chronic active hepatitis, various forms of thyroiditis (including Hashimoto's thyroiditis, transient thyroiditis syndromes, and Grave's disease), lymphocytic adenohypophysitis, premature ovarian failure, idiopathic phyoparathyroidism, pernicious anemia, glomerulonephritis, autoimmune neutropenia, Goodpasture's syndrome, multiple sclerosis, vitiligo, myasthenia gravis, rheumatoid arthritis, scleroderma, primary Sjogren's syndrome, polymyositis, autoimmune hemolytic anemia, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), psoriasis, psoriatic arthritis, dermatitis, autoimmune thrombocytopenic purpura, pemphigus vulgaris, acute rheumatic fever, mixed essential cryoglobulinemia, and warm autoimmune hemolytic anemia, among many others.
Vectors and Host Cells
The present invention also provides vectors containing the nucliec acids of the invention, as well as host cells transformed with such vectors. Any of the nucleic acids of the invention may be contained in a vector, which generally includes a selectable marker and an origin of replication, for propagation in a host. The vectors further include suitable transcriptional or translational regulatory sequences, such as those derived from a mammalian, microbial, viral, or insect genes, operably linked to the BTL-II nucleic acid. Examples of such regulatory sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences that control transcription and translation. Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA encoding the target protein. Thus, a promoter nucleotide sequence is operably linked to a BTL-II nucleic sequence if the promoter nucleotide sequence directs the transcription of the BTL-II sequence.
Selection of suitable vectors for the cloning of BTL-II nucleic acids encoding the target BTL-II proteins of this invention will depend upon the host cell in which the vector will be transformed, and, where applicable, the host cell from which the target polypeptide is to be expressed. Suitable host cells for expression of BTL-II proteins include prokaryotes, yeast, insect, and higher eukaryotic cells, each of which is discussed below.
The BTL-II proteins to be expressed in such host cells may also be fusion proteins that include regions from heterologous proteins. As discussed above, such regions may be included to allow, for example, secretion, improved stability, facilitated purification, targeting, or oligomerization of the BTL-II protein. For example, a nucleic acid sequence encoding an appropriate signal sequence can be incorporated into an expression vector. A nucleic acid sequence encoding a signal sequence (secretory leader) may be fused in-frame to a BTL-II sequence so that BTL-II is translated as a fusion protein comprising the signal peptide. A signal peptide can be functional in the intended host cell can promote extracellular secretion of the BTL-II protein. A heterologous signal peptide can replace the native signal sequence. Examples of signal peptides that are functional in mammalian host cells include the signal sequence for interleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195, the signal sequence for interleukin-2 receptor described in Cosman et al. ((1984), Nature 312: 768); the interleukin-4 receptor signal peptide described in EP Patent No. 0 367 566; the type I interleukin-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; the type II interleukin-1 receptor signal peptide described in EP Patent No. 0 460 846; the signal sequence of human IgK (which is METDTLLLWVLLLWVPGSTG); SEQ ID NO:29); and the signal sequence of human growth hormone (which is MATGSRTSLLLAFGLLCLPWLQEGSA); SEQ ID NO:30). Preferably, the signal sequence will be cleaved from the BTL-II protein upon secretion of the BTL-II protein from the cell. Other signal sequences that can be used in practicing the invention include the yeast α-factor and the honeybee melatin leader in Sf9 insect cells. Brake (1989), Biotechnology 13: 269–280; Homa et al. (1995), Protein Exp. Purif. 6141–148; Reavy et zal. (2000), Protein Exp. Purif. 6: 221–228.
Suitable host cells for expression of the proteins of the invention include prokaryotes, yeast, and higher eukaryotic cells. Suitable prokaryotic hosts to be used for the expression of these polypeptides include bacteria of the genera Escherichia, Bacillus, and Salmonella, as well as members of the genera Pseudomonas, Streptomyces, and Staphylococcus. For expression in prokaryotic cells, for example, in E. coli, the polynucleotide molecule encoding BTL-II protein preferably includes an N-terminal methionine residue to facilitate expression of the recombinant polypeptide. The N-terminal Met may optionally be cleaved from the expressed polypeptide.
Expression vectors for use in cellular hosts generally comprise one or more phenotypic selectable marker genes. Such genes encode, for example, a protein that confers antibiotic resistance or that supplies an auxotrophic requirement. A wide variety of such vectors are readily available from commercial sources. Examples include pGEM vectors (Promega), pSPORT vectors, and pPROEX vectors (InVitrogen, Life Technologies, Carlsbad, Calif.), Bluescript vectors (Stratagene), and pQE vectors (Qiagen).
BTL-II can also be expressed in yeast host cells from genera including Saccharomyces, Pichia, and Kluveromyces. Preferred yeast hosts are S. cerevisiae and P. pastoris. Yeast vectors will often contain an origin of replication sequence from a 2μ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Vectors replicable in both yeast and E. coli (termed shuttle vectors) may also be used. In addition to the above-mentioned features of yeast vectors, a shuttle vector will also include sequences for replication and selection in E. coli. Direct secretion of the target polypeptides expressed in yeast hosts may be accomplished by the inclusion of nucleotide sequence encoding the yeast α-factor leader sequence at the 5′ end of the BTL-II-encoding nucleotide sequence. Brake (1989), Biotechnology 13: 269–280.
Insect host cell culture systems can also be used for the expression of BTL-II proteins. The proteins of the invention are preferably expressed using a baculovirus expression system, as described, for example, in the review by Luckow and Summers ((1988), BioTechnology 6: 47).
BTL-II proteins of the invention can be expressed in mammalian host cells. Non-limiting examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (Gluzman et al. (1981), Cell 23: 175–182), Chinese hamster ovary (CHO) cells (Puck et al. (1958), PNAS USA 60: 1275–1281), CV-1 (Fischer et al. (1970), Int. J. Cancer 5: 21–27) and human cervical carcinoma cells (HELA) (ATCC CCL 2).
The choice of a suitable expression vector for expression of BTL-II proteins of the invention will depend upon the specific mammalian host cell to be used. Examples of suitable expression vectors include pcDNA3.1/Hygro+ (Invitrogen), pDC409 (McMahan et al. (1991), EMBO J. 10: 2821–2832), and pSVL (Pharmacia Biotech). Expression vectors for use in mammalian host cells can include transcriptional and translational control sequences derived from viral genomes. Commonly used promoter sequences and enhancer sequences that can be used to express BTL-II include, but are not limited to, those derived from human cytomegalovirus (CMV), Adenovirus 2, Polyoma virus, and Simian virus 40 (SV40). Methods for the construction of mammalian expression vectors are disclosed, for example, in Okayama and Berg ((1982) Mol. Cell. Biol. 2:161–170), Cosman et al. ((1986) Mol. Immunol. 23:935–941), Cosman et al. ((1984) Nature 312: 768–771), EP-A-0367566, and WO 91/18982.
Modification of a BTL-II nucleic acid molecule to facilitate insertion into a particular vector (for example, by modifiying restriction sites), ease of use in a particular expression system or host (for example, using preferred host codons), and the like, are known and are contemplated for use in the invention. Genetic engineering methods for the production of BTL-II proteins include the expression of the polynucleotide molecules in cell free expression systems, in cellular hosts, in tissues, and in animal models, according to known methods.
Therapeutic Methods
“Treatment” of any disease mentioned herein encompasses an alleviation of at least one symptom of the disease, a reduction in the severity of the disease, or the delay or prevention of disease progression to more serious symptoms that may, in some cases, accompany the disease or to at least one other disease. Treatment need not mean that the disease is totally cured. A useful therapeutic agent needs only to reduce the severity of a disease, reduce the severity of symptom(s) associated with the disease or its treatment, or delay the onset of more serious symptoms or a more serious disease that can occur with some frequency following the treated condition. For example, if the disease is an inflammatory bowel disease, a therapeutic agent may reduce the number of distinct sites of inflammation in the gut, the total extent of the gut affected, reduce pain and/or swelling, reduce symptoms such as diarrhea, constipation, or vomiting, and/or prevent perforation of the gut. A patient's condition can be assessed by standard techniques such as an x-ray performed following a barium enema or enteroclysis, endoscopy, colonoscopy, and/or a biopsy. Suitable procedures vary according to the patient's condition and symptoms.
The invention encompasses a method of treating inflammatory diseases, including autoimmune diseases, graft versus host disease, and inflammatory bowel diseases, using an amount of a BTL-II protein or antibody for a time sufficient to induce a sustained improvement over baseline of an indicator that reflects the severity of a particular disorder or the severity of symptoms caused by the disorder or to delay or prevent the onset of a more serious disease that follows the treated condition in some or all cases. The treatments of the invention may be used before, after, or during other treatments for the disorder in question that are commonly used, or they may be used without other treatments. For example, Crohn's disease and ulcerative colitis are commonly treated with sulfasalazine, 5-aminosalicylic acid, or cortico-steroids. These treatments may be used before, during, or after the treatments of the invention.
Any of the above-described therapeutic agents can be administered in the form of a composition, that is, with one or more additional components such as a physiologically acceptable carrier, excipient, or diluent. For example, a composition may comprise a soluble BTL-II protein as described herein plus a buffer, an antioxidant such as ascorbic acid, a low molecular weight polypeptide (such as those having less than 10 amino acids), a protein, amino acids, carbohydrates such as glucose, sucrose, or dextrins, chelating agent such as EDTA, glutathione, and/or other stabilizers, excipients, and/or preservatives. The composition may be formulated as a liquid or a lyophilizate. Further examples of components that may be employed in pharmaceutical formulations are presented in Remington's Pharmaceutical Sciences, 16th Ed., Mack Publishing Company, Easton, Pa., (1980).
Compositions comprising therapeutic molecules described above can be administered by any appropriate means including, but not limited to, parenteral, topical, oral, nasal, vaginal, rectal, or pulmonary (by inhalation) administration. If injected, the composition(s) can be administered intra-articularly, intravenously, intraarterially, intramuscularly, intraperitoneally, or subcutaneously by bolus injection or continuous infusion. Localized administration, that is, at the site of disease, is contemplated, as are transdermal delivery and sustained release from implants, skin patches, or suppositories. Delivery by inhalation includes, for example, nasal or oral inhalation, use of a nebulizer, inhalation in aerosol form, and the like. Administration via a suppository inserted into a body cavity can be accomplished, for example, by inserting a solid form of the composition in a chosen body cavity and allowing it to dissolve. In the case of soluble BTL-II proteins or agonists to treat an inflammatory bowel disease, administration via a rectal suppository may be particularly appropriate since it localizes the therapeutic appropriately. Other alternatives include eyedrops, oral preparations such as pills, lozenges, syrups, and chewing gum, and topical preparations such as lotions, gels, sprays, and ointments. In most cases, therapeutic molecules that are polypeptides can be administered topically or by injection or inhalation.
The therapeutic molecules described above can be administered at any dosage, frequency, and duration that can be effective to treat the condition being treated. The dosage depends on the molecular nature of the therapeutic molecule and the nature of the disorder being treated. Treatment may be continued as long as necessary to achieve the desired results. Therapeutic molecules of the invention can be administered as a single dosage or as a series of dosages given periodically, including multiple times per day, daily, every other day, twice a week, three times per week, weekly, every other week, and monthly dosages, among other possible dosage regimens. The periodicity of treatment may or may not be constant throughout the duration of the treatment. For example, treatment may initially occur at weekly intervals and later occur every other week. Treatments having durations of days, weeks, months, or years are encompassed by the invention. Treatment may be discontinued and then restarted. Maintenance doses may be administered after an initial treatment.
Dosage may be measured as milligrams per kilogram of body weight (mg/kg) or as milligrams per square meter of skin surface (mg/m2) or as a fixed dose, irrespective of height or weight. All of these are standard dosage units in the art. A person's skin surface area is calculated from her height and weight using a standard formula.
The invention has been described with reference to specific examples. These examples are not meant to limit the invention in any way. It is understood for purposes of this disclosure, that various changes and modifications may be made to the invention that are well within the scope of the invention. Numerous other changes may be made which will readily suggest themselves to those skilled in the art and which are encompassed in the spirit of the invention disclosed herein and as defined in the appended claims.
This specification contains numerous citations to patents, patent applications, and publications. Each is hereby incorporated by reference for all purposes.
EXAMPLE 1
Isolation of Human BTL-II cDNAs
RNA was isolated from several sources, human colon tissue samples from patients with Crohn's disease or ulcerative colitis, the human colon cancer cell line Caco-2 (American Type Culture Collection (ATCC) No. HTB-37), and a colon epithelial cell line called T84 (ATCC No. CCL-248). The RNA was reverse transcribed and amplified by PCR using primers that were designed on the basis of the nucleic acid sequence disclosed in NCBI accession no. NM—019602. This yielded an upstream portion of the sequences of full length BTL-II (SEQ ID NO:3) and the splice variant sequences disclosed in SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:15. Isolation of a cDNA containing the 3′ end of the BTL-II mRNA was accomplished using 3′ RACE (Rapid Amplification of cDNA Ends), i.e., essentially the protocols of Frohman et al. ((1988), Proc. Natl. Acad. Sci. USA 85 (23): 8998–9002). From the analysis of many variants of the BTL-II cDNA by 3′ RACE revealed no variants that encoded soluble proteins lacking a transmembrane domain. As shown in FIGS. 5a, 6a, and 7a, many of the variants contained sequence polymorphisms (or allelic variations) at a number of sites in the BTL-II sequence.
EXAMPLE 2
Isolation of Murine BTL-II cDNAs
Murine BTL-II cDNAs were isolated as follows. RNA was isolated from murine colon and small intestine using a kit the isolation and purification of RNA (the RNEASY® kit; Qiagen) and treated with DNAse I (Ambion), according to recommendations of the manufacturer, to eliminate residual chromosomal DNA. Purified RNA was transcribed into cDNA, using a reaction mixture containing isolated RNA in 10 mM Tris-HCl, pH 8.3, 50 mM KCL, 5 mM MgCl2, 1 mM of each dNTP, 2.5 μM random hexamer primers, 1 U/μl RNAse inhibitor, and 2.5 U/□1 MuLV Reverse Transcriptase (PE Biosystems). The reaction mixture was incubated for 10 minutes at 25° C., followed by 30 minutes at 48° C., followed by 5 minutes at 95° C. PCR amplification reactions for the BTL-II gene were performed in a final volume of 100 μl containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 200 μM of each dNTP, and 2.5U ampliTaq DNA polymerase (Perkin Elmer) and 25 pmol of both the upstream (5′-TTACTGAGAGAGGGAAACGGGCTGTTTTCTCC; SEQ ID NO:31) and downstream (5′-GGACTTCATTGGTGACTGATGCCATCCAC; SEQ ID NO:32) primers. The amplification reactions were carried out for 35 cycles of 40 seconds at 94° C., 40 seconds at 55° C., and 40 seconds at 72° C. The amplification products were analyzed on a 2% agarose gel, visualized by ethidium bromide, and sequenced. By visual estimation, splice variants lacking exon 3 (FIG. 9a) were more abundant than variants containing exons 1 to 8 (FIG. 8).
EXAMPLE 3
Expression of BTL-II in Cells and Tissues from Various Sources
Expression of BTL-II MRNA was measured using real time PCR essentially according to the protocols of Heid et al. ((1996), Genome Res. 6 (10): 986–94) and using standard reverse transcription followed by PCR (RT-PCR; see e.g. Fuqua et al. (1990), Biotechniques 9 (2): 206–11). All cells types tested were of human origin except the CD11c+ CD8+ B220+ (or plasmacytoid) dendritic cells from Peyer's patches which were from mouse. Cells in which BTL-II expression was detected were the following: human B cells, unstimulated or stimulated with killed Staphylococcus aureus, CD40 ligand, and interleukin 4; normal human bronchial epithelial cells (NHBE cells; see Lechner et al. (1983), Cancer Res. 43 (12 pt. 1): 5915–21) stimulated with interferon γ; Calu-3 cells, a lung epithelial cell line (ATCC No. HTB-55), unstimulated; T84 cells, a human colon epithelial cell line, unstimulated or stimulated with interferon γ; Caco-2 cells, a human colon cancer cell line, unstimulated; CD11c+ (low expressing) CD8+ B220+ cells from murine Peyer's patches, which are predominantly dendritic cells; and murine peripheral blood leukocytes. Expression, on an absolute scale, was low in most cells tested. Expression of BTL-II mRNA was not detected in dendritic cells resulting from in vitro treatment of human peripheral blood monocytes to induce differentiation into a dendritic cell type. However, BTL-II expression was detected in CD123+ plasmacytoid dendritic cells purified from human blood purified from human peripheral blood. BTL-II MRNA was highly enriched in the CD11c+ (low expressing) CD8+ B220+ subset of murine dendritic cells from Peyer's patches. Expression of BTL-II mRNA was detected in a number of murine tissue types by similar methods including spleen, lymph node, stomach, mesenteric lymph nodes, bone marrow, small intestine, cecum, lung, large intestine, Peyer's patch, and thymus. The highest levels of expression were detected in small intestine, Peyer's patch, and cecum tissue.
EXAMPLE 4
Expression of BTL-II in a Murine Model for Inflammatory Bowel Disease
Mdr1a −/− mice can be a model system for the study of chronic inflammatory bowel disease. Panwala et al. (1998), J. Immunol. 161: 5733–44. The murine multiple drug resistance gene, mdr1a, encodes a 170 kDa transmembrane protein that is expressed in many tissues including intestinal epithelial cells and lymphoid cells. Mice deficient in mdr1a are susceptible to developing severe spontaneous intestinal inflammation characterized by dysregulated epithelial cell growth and massive leukocyte infiltration into the lamina propria of the large intestine. Treating mdr1a −/− mice with oral antibiotics prevents both the development of disease and resolves active inflammation. Lymphoid cells isolated from mice with active colitis demonstrate enhanced reactivity to intestinal bacterial antigens. Although mdr1a is expressed by both epithelial cells and leukocytes, the development of colitis correlates with lack of mdr1a expression on epithelial cells.
The mdr1a −/− mice used were in an FVB genetic background. Typically, approximately 20% of a group of mdr1a −/− mice spontaneously develop colitis at 18 to 20 weeks of age, while the remaining mice in the mdr1a −/− colony remain healthy and do not develop colitis. The percentage of animals that develop disease is dependent on the cleanliness of the animal facility.
Initially, RNA was prepared from gut tissue and, after reverse transcription incorporating a fluorescent label into the resulting cDNA, used to hybridize to an array of probes on a custom-prepared Affymetrix chip containing an oligonucleotide designed to detect BTL-II mRNA based on the sequence published by Stammers et al. ((2000), Immunogenetics 51: 373–82). Overexpression of BTL-II mRNA was detected in mdr1a −/− mice (relative to wild type individuals of the FVB strain) both before the onset of inflammatory bowel disease symptoms and during the symptoms. A sample of such data is shown in FIG. 10. These data show that BTL-II MRNA is expressed to a greater extent in mdr1a −/− mice than in wild type FVB mice. Moreover, even higher expression of BTL-II MRNA accompanies the onset of symptoms of inflammatory bowel disease.
These results were confirmed by analysis of the same RNA using a real time PCR technique (Heid et al. (1996), Genome Res. 6 (10): 986–94). This analysis showed overexpression of BTL-II MRNA by two symptomatic mdr1a −/− mice relative to two healthy wild type FVB mice. This data is diagrammed in FIG. 11. The two parental mice are represented by the two bars marked with diagonal stripes towards the left of FIG. 11, and the two symptomatic mdr1a −/− are represented by the two bars marked with checkered patterns towards the right of FIG. 11. These data indicate that there is approximately a 2 to 5 fold difference in expression between the wild type mice and the symptomatic mdr1a −/− mice. Thus, higher levels of BTL-II mRNA are expressed in mice with symptoms of inflammatory bowel disease than in wild type mice with no symptoms.
EXAMPLE 5
Construction of a BTL-II:Fc Fusion Protein
A soluble BTL-II protein consisting of the extracellular region of murine BTL-II fused to a human Fc region (BTL-II:Fc) was produced in the following way. A fusion cDNA construct encoding BTL-II:Fc was prepared by fusing nucleic acids encoding the extracellular region of murine BTL-II to nucleic acids encoding a human IgG1 (in-frame). To produce the BTL-II:Fc protein, mammalian cells were transfected with the fusion cDNA construct using the LIPOFECTAMINE™ 2000 transfection method (Invitrogen, Carlsbad, Calif., USA). BTL-II:Fc protein-containing supernatants were harvested 6–7 days post transfection, and the BTL-II:Fc protein was purified by Protein A column chromatography. The nucleic acid sequence encoding the BTL-II:Fc protein and the BTL-II:Fc amino acid sequence are found in SEQ ID NO:19 and SEQ ID NO:20, respectively.
EXAMPLE 6
Suppression of Human T Cell Proliferation by BTL-II:Fc
The following experiment tests whether a soluble form of BTL-II can suppress T cell proliferation in vitro in response to a monoclonal anti-CD3ε antibody with or without other costimulatory molecules.
BTL-II:Fc was made as described in Example 5. A 96 well, U bottom microtiter plate was coated with varying concentrations of anti-CD3ε antibody with or without one or more other proteins. FIG. 12 indicates what proteins were used in each sample as follows: anti-CD3ε antibody alone, ; anti-CD3ε antibody and BTL-II:Fc, ; anti-CD3ε antibody and B7RP-1:Fc, ; anti-CD3ε antibody, B7RP-1:Fc, and BTL-II:Fc, ; anti-CD3ε antibody and B7-2:Fc, ; anti-CD3ε antibody, B7-2:Fc, and BTL-II:Fc, . B7RP-1:Fc consists of the extracellular region of B7RP-1 (also known as B7h) fused to an Fc region of an antibody, and B7-2:Fc consists of the extracellular region of B7-2 fused to an Fc region of an antibody. Both of these proteins are members of the B7 family of proteins known to modulate T cell response to antigens. These fusion proteins can be purchased from commercial vendors such as, for example, R & D Systems (Minneapolis, Minn., USA), or can be isolated as described in Example 5 for the essentially as described in Example 5, although it also is available from R & D Systems under the name B7-H2/Fc. The microtiter plate wells were coated by adding 100 μl of phosphate buffered saline (PBS) containing the concentration of anti-CD3ε antibody indicated in FIG. 12 with or without one or more other proteins, that is BTL-II:Fc (10 μg/ml), B7RP1 :Fc (10 μg/ml), and/or B7-2:Fc (2 μg/ml). Plates were incubated at 4° C. overnight and then washed twice with PBS. Human T cells were purified from human peripheral blood mononuclear cells using a CD4+ T cell isolation kit from Miltenyi Biotec (Bergisch Gladbach, Germany), which functions by magnetically labeling and depleting peripheral blood cells other than CD4+ T cells, resulting in relatively pure population of untouched CD4+ T cells. About 1×105 purified T cells were added to each well in a volume of 200 μl of culture medium (RPMI with 10% fetal bovine serum). The cells were incubated for a total of 72 hours. At 64 hours, 1 μCi of 3H-thymidine was added to each well. At the end of the 72 hours, unincorporated thymidine was removed by using an automatic cell harvester (obtained from Tomtec, Hamden, Conn., USA), which deposits the cells onto a filter and washes away the culture medium. The filters with the cells on them were then counted in a scintillation counter to determine how much radioactivity the cells had incorporated. The results are shown in FIGS. 12a and 12b, which differ only in that FIG. 12a has a logarithmic scale and FIG. 12b has a linear scale.
The results indicate that BTL-II:Fc can suppress the T cell proliferation induced by anti-CD3ε antibody. FIG. 12a and 12b. In FIG. 12a, samples containing BTL-II plus anti-CD3ε antibody () proliferate less than samples containing only anti-CD3ε antibody () at the higher antibody concentrations tested. Moreover, it is clear that both B7-2 () and B7RP-1 () stimulate anti-CD3ε antibody-induced T cell proliferation. The concentration of anti-CD3ε antibody required to see an effect on T cell proliferation by adding B7-2:Fc is lower than that required to see a similar effect by adding B7RP-1:Fc. Further, BTL-II can also suppress this increased T cell proliferation in response to the addition of B7-2:Fc () and B7RP-1 :Fc (). The effect of BTL-II:Fc on proliferation induced by B7-2:Fc plus anti-CD3ε antibody is evident only at the lowest concentration of anti-CD3ε antibody tested, whereas the effects of BTL-II:Fc on proliferation induced by B7RP-1 :Fc are more apparent at the three higher concentrations of anti-CD3ε antibody tested. Thus, the observed effects depend on the concentration of anti-CD3ε antibody
FIG. 13 shows that the suppression of human T cell proliferation by BTL-II:Fc is also dependent on BTL-II:Fc concentration. The experiment was performed as described above except that the anti-CD3ε antibody concentration remained constant at 0.5 μg/ml. The BTL-II:Fc concentration varied from 0 to 10 μg/ml, as indicated in FIG. 13. The results shown in FIG. 13 indicate that suppression of anti-CD3ε-induced T cell proliferation is dependent on the concentration of BTL-II:Fc.
EXAMPLE 7
BTL-II:Fc Suppresses Murine T Cell Proliferation
This experiment was done to determine whether BTL-II:Fc can suppress the proliferation of murine, as well as human, T cells. A T cell proliferation assay was performed as essentially described in Example 6 except that murine T cells purified using magnetic microbeads purchased from Miltneyi Biotec GmbH (Bergisch Gladbach, Germany) were used instead of human T cells. A protein consisting of a human Fc region, which was made in transfected CHO cells, served as a control. The wells were coated with either anti-CD3ε antibody alone () at the concentration indicated in FIG. 14 or with anti-CD3ε antibody plus either BTL-II:Fc () at 10 μg/ml or the protein consisting of a human Fc region () at 10 μg/ml. The results indicate that BTL-II:Fc, but not a protein consisting of a human Fc region alone, can suppress murine T cell proliferation in response to anti-CD3ε antibody. FIG. 14.
EXAMPLE 8
BTL-II:Fc Does Not Supress Murine B Cell Proliferation
The following experiment was designed to determine whether human BTL-II:Fc can inhibit proliferation of murine B cells induced by TALL-1 and an IgM antibody. The experiment was performed essentially as described by Khare et al. ((2000), Proc. Natl. Acad. Sci. 97 (7): 3370–75). Briefly, murine B cells were purified from spleens by first purifying lymphocytes by density gradient centrifugation and then passing the lymphocytes over a B cell column, which removes monocytes/macrophages and CD4+ and CD8+ cells (Cedarlane, Westbury, N.Y.). About 1×105 purified B cells in MEM plus 10% heat inactivated fetal calf serum were incubated for 4 days at 37° C. in a 96 well microtiter plate with or without goat F(ab′)2 anti-mouse IgM (2 μg/ml), human TALL-1 (10 μg/ml), and/or BTL-II:Fc (10 μg/ml). 3H-thymidine (1 μCi) was added during the last 8 hours of incubation. Cells were harvested as described in Example 6 at the end of 4 days and counted in a scintillation counter. The markings in FIG. 15 indicate the following combinations of cells and proteins: B cells alone, (This is the leftmost bar on the graph shown in FIG. 15, but there is no detectable signal); B cells plus TALL-1, ; B cells plus anti-IgM antibody, ; B cells plus TALL-1 and anti-IgM antibody, ; and B cells plus TALL-1, anti-IgM antibody, and BTL-II: Fc, . The results indicate that BTL-II:Fc has no effect on the proliferation of B cells induced by TALL-I and anti-IgM antibody. FIG. 15. Therefore, BTL-II appears to inhibit proliferation of T cells, but not of B cells.
EXAMPLE 9
Effects of BTL-II:Fc on Cytokine Production by T Cells
The following experiment was aimed at determining whether BTL-II:Fc has effects on cytokine production by T cells. T cells were purified and microtiter plate wells were coated with proteins as described in Example 6. About 1×105 cells were added to each well in a volume of 200 μl of medium (RPMI with 10% fetal bovine serum). Cells were incubated for 64 hours at 37° C. Then 150 μl was removed to determine cytokine concentration, and 1 μi of 3H-thymidine was added to the remaining 50 μl in each well. The microtiter plate was then allowed to incubate for an additional 8 hours at 37° C. and then washed and counted as described in Example 6 to ascertain differences in proliferation (shown in FIG. 16a). Markings to indicate what proteins were used to coat the well are as in FIGS. 12a and 12b (explained in Example 6). Concentrations of interferon gamma (IFNγ), interleukin 2 (IL2), and interleukin 5 (IL5) were determined using an electrochemical-based immunoassay system for simultaneous detection of multiple cytokines sold by Meso Scale Discovery (MSD, Gaithersburg, Md., USA, which affiliated with IGEN International, Inc.). The principles and operation of this kind of cytokine detection system are explained, in e.g., Sennikov et al. (2003), J. Immunol. Methods 275: 81–88. The units for the amounts of cytokines are taken from the readings generated by the MSD machine. The actual concentrations of cytokines in the medium cannot be determined from this data without comparison to a standard curve generated with a protein of known concentration, which did not accompany this particular experiment. However, comparisons between readings within a single experiment can provide relative amounts of cytokines present in control versus experimental samples.
The results are shown in FIGS. 16a–16d. FIG. 16a indicates that BTL-II:Fc inhibited proliferation in response to anti-CD3ε antibody or anti-CD3ε antibody plus B7RP-1:Fc, but not in response to anti-CD3ε antibody plus B7-2:Fc at the concentrations of anti-CD3ε antibody used. However, as noted in Example 6 (FIGS. 12a and 12b), at lower anti-CD3ε antibody concentrations, i.e., 0.1 μg/ml, cell proliferation in response to anti-CD3ε antibody plus B7-2:Fc is inhibited by BTL-II:Fc. Production of IFNγ, IL2, and IL5 was low in wells coated with anti-CD3ε antibody alone, and addition of BTL-II:Fc did not decrease it significantly further. FIGS. 16b–16d. Increased IFNγ production in response to the addition of either B7RP-1:Fc or B7-2:Fc to anti-CD3ε antibody was inhibited by BTL-II:Fc. FIG. 16b. In addition, increased IL2 and IL5 production in response to the addition of B7-2:Fc to anti-CD3ε antibody was inhibited by BTL-II:Fc. FIGS. 16c and 16d. At the concentrations tested, the addition of B7RP-1:Fc to anti-CD3ε antibody did not appreciably increase IL2 or IL5 production. FIGS. 16c and 16d. These results indicate that BTL-II:Fc can inhibit production of at least some cytokines in response to a combination of anti-CD3ε antibody plus either B7-2:Fc or B7RP-1:Fc.
EXAMPLE 10
Treatment of T Cells With BTL-II:Fc Does Not Result in Massive Cell Death
The following experiment was done to determine whether inhibition of T cell proliferation by BTL-II:Fc involved massive cell death. A T cell proliferation assay was performed essentially as described in Example 6, except that the cells were not labeled with 3H-thymidine. Cells were counted in a hemocytometer after 72 hours of culture, and cell viability was determined by trypan blue staining. Proteins used to coat the microtiter plate wells are indicated in FIG. 17 as explained in Example 6 and shown in FIGS. 12a and 12b. The anti-CD3ε antibody was used at a concentration of 2 μg/ml. The results indicate that the number of dead cells in each well falls within a range between about 0.5×104 and 0.75×104 cells, regardless of the proteins used to coat the wells. FIG. 17. Therefore, the differences in cell proliferation observed in the presence of BTL-II:Fc do not reflect a substantial toxic effect of BTL-II:Fc.
EXAMPLE 11
Murine BTL-II Does Not Bind to Murine CTLA4, CD28, ICOS, or PD-1
The following set of experiments addresses the question of whether BTL-II binds to one of several known binding partners of other B7 proteins. For example, B7RP-1 is known to bind to ICOS (Yoshinaga et al. (1999), Nature 402: 827–32), CD80 (also called B7-1) is known to bind to CTLA4 and CD28, as does CD86 (also called B7-2; see e.g. Sharpe and Freeman (2002), Nature Reviews Immunology 2: 116–26), and PD-L1 and PD-L2 are known to bind to PD-1 (Latchman et al. (2001), Nature Immunology 2 (3): 261–68).
The experiment was done as follows. First, fusion proteins comprising the extracellular region of a protein known to bind a B7 protein plus the Fc region of a human IgG antibody were obtained or isolated. Fusion proteins comprising an Fc region of a human antibody plus the extracellular region of either murine CD28 (mCD28-huFC) or murine PD-1 (PD1-huFC) were purchased from R & D Systems (Minneapolis, Minn., USA). The other two B7 binding proteins (CTLA4-huFC and ICOS-huFC) are also available from R & D Systems, but were made as follows. A cDNA encoding the extracellular region of murine CTLA4 and another encoding the extracellular region of murine ICOS were each fused to cDNA encoding the Fc region of a human IgG antibody in a vector appropriate for expression in mammalian cells. Each of these constructs was used to transfect cells, and the fusion proteins were purified from the culture medium of the transfected cells by Protein A chromatography.
Full length versions of each of three murine cDNAs (encoding BTL-II, B7RP-1, CD80) were inserted into a vector appropriate for expression. About 10 μg of each of these constructs, along with an empty vector, were used separately to transfect 293 cells. Two days post transfection, about one million cells from each of the four transfections were stained with each of the fusion proteins described above. Bound protein was detected using a fluorescently labeled antibody against the human IgG Fc region. The stained cells were analyzed by FACS. The results are shown in FIG. 18.
As expected, cells transfected with the empty vector (top line of FIG. 18) did not stain with any of the four fusion proteins. Cells transfected with murine BTL-II (second line of FIG. 18) behaved similarly, indicating that none of the fusion proteins bind to BTL-II. As expected, cells transfected with murine B7RP-1 stained with ICOS-huFC, but not with any of the other fusion proteins. Also as expected, cells transfected with murine CD80 stained with CTLA4-huFC or mCD28-huFC, but not with ICOS-huFC or PD1-huFC. These results indicate that BTL-II fails to bind to four proteins, CTLA-4, PD-1, ICOS, and CD28, each of which is known to bind to at least one protein in the B7 family.
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10742682
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immunex corporation
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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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530/350
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Mar 31st, 2022 02:17PM
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Mar 31st, 2022 02:17PM
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Amgen
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:amgn
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Amgen
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Oct 29th, 2013 12:00AM
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Mar 19th, 2012 12:00AM
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https://www.uspto.gov?id=US08569456-20131029
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Receptor activator of NF-kappaB
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Isolated receptors, DNAs encoding such receptors, and pharmaceutical compositions made therefrom, are disclosed. The isolated receptors can be used to regulate an immune response. The receptors are also useful in screening for inhibitors thereof.
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8569456
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1. An isolated RANK polypeptide comprising the amino acid sequence of amino acids 33 through 196 of SEQ ID NO:6 or a fragment thereof, wherein the polypeptide or fragment can bind a RANKL polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:13.
2. An isolated RANK polypeptide, wherein the amino acid sequence of the polypeptide is at least 80% identical to the amino acid sequence as set forth in SEQ ID NO:6, and wherein the RANK polypeptide can bind a RANKL polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:13.
3. The RANK polypeptide of claim 1, further comprising a peptide selected from the group consisting of an immunoglobulin Fc domain, an immunoglobulin Fc mutein, a FLAG™ tag, a peptide comprising at least about 6 His residues, a leucine zipper, and combinations thereof
4. The RANK protein of claim 2, further comprising a peptide selected from the group consisting of an immunoglobulin Fc domain, an immunoglobulin Fc mutein, a FLAG™ tag, a peptide comprising at least about 6 His residues, a leucine zipper, and combinations thereof.
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4
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CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 13/092,718 filed Apr. 22, 2011 (now allowed), which is a continuation of U.S. patent application Ser. No. 11/881,911 filed Jul. 30, 2007 (now U.S. Pat. No. 7,932,375), which is a divisional of U.S. patent application Ser. No. 10/405,878 filed Apr. 1, 2003 (now U.S. Pat. No. 7,262,274), which is a continuation of U.S. patent application Ser. No. 09/871,291 filed May 30, 2001 (now U.S. Pat. No. 6,562,948), which is a divisional of U.S. patent application Ser. No. 09/577,800 filed May 24, 2000 (now U.S. Pat. No. 6,479,635), which is a continuation of U.S. patent application Ser. No. 09/466,496 filed Dec. 17, 1999 (now U.S. Pat. No. 6,528,482), which is a continuation of U.S. patent application Ser. No. 08/996,139 filed Dec. 22, 1997 (now U.S. Pat. No. 6,017,729), which claims the benefit of U.S. provisional application No. 60/064,671 filed Oct. 14, 1997, U.S. provisional application No. 60/077,181 filed Mar. 7, 1997, and U.S. provisional application No. 60/059,978, filed Dec. 23, 1996.
REFERENCE TO THE SEQUENCE LISTING
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 2851-US-CNT5Seq_List_ST25.txt, created Mar. 19, 2012, which is 76 KB in size. The information in the electronic format of the Sequence Listing is incorporated hereby reference in its entirety.
TECHNICAL FIELD OF THE INVENTION
The present invention relates generally to the field of cytokine receptors, and more specifically to cytokine receptor/ligand pairs having immunoregulatory activity.
BACKGROUND OF THE INVENTION
Efficient functioning of the immune system requires a fine balance between cell proliferation and differentiation and cell death, to ensure that the immune system is capable of reacting to foreign, but not self antigens. Integral to the process of regulating the immune and inflammatory response are various members of the Tumor Necrosis Factor (TNF) Receptor/Nerve Growth Factor Receptor superfamily (Smith et al., Science 248:1019; 1990). This family of receptors includes two different TNF receptors (Type I and Type II; Smith et al., supra; and Schall et al., Cell 61:361, 1990), nerve growth factor receptor (Johnson et al., Cell 47:545, 1986), B cell antigen CD40 (Stamenkovic et al., EMBO J. 8:1403, 1989), CD27 (Camerini et al., J. Immunol. 147:3165, 1991), CD30 (Durkop et al., Cell 68:421, 1992), T cell antigen OX40 (Mallett et al., EMBO J. 9:1063, 1990), human Fas antigen (Itoh et al., Cell 66:233, 1991), murine 4-1BB receptor (Kwon et al., Proc. Natl. Acad. Sci. USA 86:1963, 1989) and a receptor referred to as Apoptosis-Inducing Receptor (AIR; U.S. Ser. No. 08/720,864, filed Oct. 4, 1996).
CD40 is a receptor present on B lymphocytes, epithelial cells and some carcinoma cell lines that interacts with a ligand found on activated T cells, CD40L (U.S. Ser. No. 08/249,189, filed May 24, 1994). The interaction of this ligand/receptor pair is essential for both the cellular and humoral immune response. Signal transduction via CD40 is mediated through the association of the cytoplasmic domain of this molecule with members of the TNF receptor-associated factors (TRAFs; Baker and Reddy, Oncogene 12:1, 1996). It has recently been found that mice that are defective in TRAF3 expression due to a targeted disruption in the gene encoding TRAF3 appear normal at birth but develop progressive hypoglycemia and depletion of peripheral white cells, and die by about ten days of age (Xu et al., Immunity 5:407, 1996). The immune responses of chimeric mice reconstituted with TRAF3−/− fetal liver cells resemble those of CD40-deficient mice, although TRAF3−/− B cells appear to be functionally normal.
The critical role of TRAF3 in signal transduction may be in its interaction with one of the other members of the TNF receptor superfamily, for example, CD30 or CD27, which are present on T cells. Alternatively, there may be other, as yet unidentified members of this family of receptors that interact with TRAF3 and play an important role in postnatal development as well as in the development of a competent immune system. Identifying additional members of the TNF receptor superfamily would provide an additional means of regulating the immune and inflammatory response, as well as potentially providing further insight into post-natal development in mammals.
SUMMARY OF THE INVENTION
The present invention provides a novel receptor, referred to as RANK (for receptor activator of NF-κB), that is a member of the TNF receptor superfamily. RANK is a Type I transmembrane protein having 616 amino acid residues that interacts with TRAF3. Triggering of RANK by over-expression, co-expression of RANK and membrane bound RANK ligand (RANKL), and with addition of soluble RANKL or agonistic antibodies to RANK results in the upregulation of the transcription factor NF-κB, a ubiquitous transcription factor that is most extensively utilized in cells of the immune system.
Soluble forms of the receptor can be prepared and used to interfere with signal transduction through membrane-bound RANK, and hence upregulation of NF-κB; accordingly, pharmaceutical compositions comprising soluble forms of the novel receptor are also provided. Inhibition of NF-κB by RANK antagonists may be useful in ameliorating negative effects of an inflammatory response that result from triggering of RANK, for example in treating toxic shock or sepsis, graft-versus-host reactions, or acute inflammatory reactions. Soluble forms of the receptor will also be useful in vitro to screen for agonists or antagonists of RANK activity.
The cytoplasmic domain of RANK will be useful in developing assays for inhibitors of signal transduction, for example, for screening for molecules that inhibit interaction of RANK with TRAF2 or TRAF3. Deleted forms and fusion proteins comprising the novel receptor are also disclosed.
The present invention also identifies a counterstructure, or ligand, for RANK, referred to as RANKL. RANKL is a Type 2 transmembrane protein with an intracellular domain of less than about 50 amino acids, a transmembrane domain and an extracellular domain of from about 240 to 250 amino acids. Similar to other members of the TNF family to which it belongs, RANKL has a ‘spacer’ region between the transmembrane domain and the receptor binding domain that is not necessary for receptor binding. Accordingly, soluble forms of RANKL can comprise the entire extracellular domain or fragments thereof that include the receptor binding region.
These and other aspects of the present invention will become evident upon reference to the following detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 demonstrates the influence of RANK.Fc and hRANKL on activated T cell growth. Human peripheral blood T cells were cultured as described in Example 12; viable T cell recovery was determined by triplicate trypan blue countings.
FIG. 2 demonstrates that RANKL enhances DC allo-stimulatory capacity. Allogeneic T cells were incubated with varying numbers of irradiated DC cultured as described in Example 13. The cultures were pulsed with [3H]-thymidine and the cells harvested onto glass fiber sheets for counting. Values represent the mean±standard deviation (SD) of triplicate cultures.
DETAILED DESCRIPTION OF THE INVENTION
A novel partial cDNA insert with a predicted open reading frame having some similarity to CD40 was identified in a database containing sequence information from cDNAs generated from human bone marrow-derived dendritic cells (DC). The insert was used to hybridize to colony blots generated from a DC cDNA library containing full-length cDNAs. Several colony hybridizations were performed, and two clones (SEQ ID NOs:1 and 3) were isolated. SEQ ID NO:5 shows the nucleotide and amino acid sequence of a predicted full-length protein based on alignment of the overlapping sequences of SEQ ID NOs:1 and 3.
RANK is a member of the TNF receptor superfamily; it most closely resembles CD40 in the extracellular region. Similar to CD40, RANK associates with TRAF2 and TRAF3 (as determined by co-immunoprecipitation assays substantially as described by Rothe et al., Cell 83:1243, 1995). TRAFs are critically important in the regulation of the immune and inflammatory response. Through their association with various members of the TNF receptor superfamily, a signal is transduced to a cell. That signal results in the proliferation, differentiation or apoptosis of the cell, depending on which receptor(s) is/are triggered and which TRAF(s) associate with the receptor(s); different signals can be transduced to a cell via coordination of various signaling events. Thus, a signal transduced through one member of this family may be proliferative, differentiative or apoptotic, depending on other signals being transduced to the cell, and/or the state of differentiation of the cell. Such exquisite regulation of this proliferative/apoptotic pathway is necessary to develop and maintain protection against pathogens; imbalances can result in autoimmune disease.
RANK is expressed on epithelial cells, some B cell lines, and on activated T cells. However, its expression on activated T cells is late, about four days after activation. This time course of expression coincides with the expression of Fas, a known agent of apoptosis. RANK may act as an anti-apoptotic signal, rescuing cells that express RANK from apoptosis as CD40 is known to do. Alternatively, RANK may confirm an apoptotic signal under the appropriate circumstances, again similar to CD40. RANK and its ligand are likely to play an integral role in regulation of the immune and inflammatory response.
Moreover, the post-natal lethality of mice having a targeted disruption of the TRAF3 gene demonstrates the importance of this molecule not only in the immune response but in development. The isolation of RANK, as a protein that associates with TRAF3, and its ligand will allow further definition of this signaling pathway, and development of diagnostic and therapeutic modalities for use in the area of autoimmune and/or inflammatory disease.
DNAs, Proteins and Analogs
The present invention provides isolated RANK polypeptides and analogs (or muteins) thereof having an activity exhibited by the native molecule (i.e, RANK muteins that bind specifically to a RANK ligand expressed on cells or immobilized on a surface or to RANK-specific antibodies; soluble forms thereof that inhibit RANK ligand-induced signaling through RANK). Such proteins are substantially free of contaminating endogenous materials and, optionally, without associated native-pattern glycosylation. Derivatives of RANK within the scope of the invention also include various structural forms of the primary proteins which retain biological activity. Due to the presence of ionizable amino and carboxyl groups, for example, a RANK protein may be in the form of acidic or basic salts, or may be in neutral form. Individual amino acid residues may also be modified by oxidation or reduction. The primary amino acid structure may be modified by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like, or by creating amino acid sequence mutants. Covalent derivatives are prepared by linking particular functional groups to amino acid side chains or at the N- or C-termini.
Derivatives of RANK may also be obtained by the action of cross-linking agents, such as M-maleimidobenzoyl succinimide ester and N-hydroxysuccinimide, at cysteine and lysine residues. The inventive proteins may also be covalently bound through reactive side groups to various insoluble substrates, such as cyanogen bromide-activated, bisoxirane-activated, carbonyldiimidazole-activated or tosyl-activated agarose structures, or by adsorbing to polyolefin surfaces (with or without glutaraldehyde cross-linking). Once bound to a substrate, the proteins may be used to selectively bind (for purposes of assay or purification) antibodies raised against the proteins or against other proteins which are similar to RANK or RANKL, as well as other proteins that bind RANK or RANKL or homologs thereof.
Soluble forms of RANK are also within the scope of the invention. The nucleotide and predicted amino acid sequence of the RANK is shown in SEQ ID NOs:1 through 6. Computer analysis indicated that the protein has an N-terminal signal peptide; the predicted cleavage site follows residue 24. Those skilled in the art will recognize that the actual cleavage site may be different than that predicted by computer analysis. Thus, the N-terminal amino acid of the cleaved peptide is expected to be within about five amino acids on either side of the predicted, preferred cleavage site following residue 24. Moreover a soluble form beginning with amino acid 33 was prepared; this soluble form bound RANKL. The signal peptide is predicted to be followed by a 188 amino acid extracellular domain, a 21 amino acid transmembrane domain, and a 383 amino acid cytoplasmic tail.
Soluble RANK comprises the signal peptide and the extracellular domain (residues 1 to 213 of SEQ ID NO:6) or a fragment thereof. Alternatively, a different signal peptide can be substituted for the native leader, beginning with residue 1 and continuing through a residue selected from the group consisting of amino acids 24 through 33 (inclusive) of SEQ ID NO:6. Moreover, fragments of the extracellular domain will also provide soluble forms of RANK. Fragments can be prepared using known techniques to isolate a desired portion of the extracellular region, and can be prepared, for example, by comparing the extracellular region with those of other members of the TNFR family and selecting forms similar to those prepared for other family members. Alternatively, unique restriction sites or PCR techniques that are known in the art can be used to prepare numerous truncated forms which can be expressed and analyzed for activity.
Fragments can be prepared using known techniques to isolate a desired portion of the extracellular region, and can be prepared, for example, by comparing the extracellular region with those of other members of the TNFR family (of which RANK is a member) and selecting forms similar to those prepared for other family members. Alternatively, unique restriction sites or PCR techniques that are known in the art can be used to prepare numerous truncated forms which can be expressed and analyzed for activity.
Other derivatives of the RANK proteins within the scope of this invention include covalent or aggregative conjugates of the proteins or their fragments with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions. For example, the conjugated peptide may be a signal (or leader) polypeptide sequence at the N-terminal region of the protein which co-translationally or post-translationally directs transfer of the protein from its site of synthesis to its site of function inside or outside of the cell membrane or wall (e.g., the yeast α-factor leader).
Protein fusions can comprise peptides added to facilitate purification or identification of RANK proteins and homologs (e.g., poly-His). The amino acid sequence of the inventive proteins can also be linked to an identification peptide such as that described by Hopp et al., Bio/Technology 6:1204 (1988). Such a highly antigenic peptide provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant protein. The sequence of Hopp et al. is also specifically cleaved by bovine mucosal enterokinase, allowing removal of the peptide from the purified protein. Fusion proteins capped with such peptides may also be resistant to intracellular degradation in E. coli.
Fusion proteins further comprise the amino acid sequence of a RANK linked to an immunoglobulin Fc region. An exemplary Fc region is a human IgG1 having a nucleotide an amino acid sequence set forth in SEQ ID NO:8. Fragments of an Fc region may also be used, as can Fc muteins. For example, certain residues within the hinge region of an Fc region are critical for high affinity binding to FcγRI. Canfield and Morrison (J. Exp. Med. 173:1483; 1991) reported that Leu(234) and Leu(235) were critical to high affinity binding of IgG3 to FcγRI present on U937 cells. Similar results were obtained by Lund et al. (J. Immunol. 147:2657, 1991; Molecular Immunol. 29:53, 1991). Such mutations, alone or in combination, can be made in an IgG1 Fc region to decrease the affinity of IgG1 for FcR. Depending on the portion of the Fc region used, a fusion protein may be expressed as a dimer, through formation of interchain disulfide bonds. If the fusion proteins are made with both heavy and light chains of an antibody, it is possible to form a protein oligomer with as many as four RANK regions.
In another embodiment, RANK proteins further comprise an oligomerizing peptide such as a leucine zipper domain. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, 1988). Leucine zipper domain is a term used to refer to a conserved peptide domain present in these (and other) proteins, which is responsible for dimerization of the proteins. The leucine zipper domain (also referred to herein as an oligomerizing, or oligomer-forming, domain) comprises a repetitive heptad repeat, with four or five leucine residues interspersed with other amino acids. Examples of leucine zipper domains are those found in the yeast transcription factor GCN4 and a heat-stable DNA-binding protein found in rat liver (C/EBP; Landschulz et al., Science 243:1681, 1989). Two nuclear transforming proteins, fos and jun, also exhibit leucine zipper domains, as does the gene product of the murine proto-oncogene, c-myc (Landschulz et al., Science 240:1759, 1988). The products of the nuclear oncogenes fos and jun comprise leucine zipper domains preferentially form a heterodimer (O'Shea et al., Science 245:646, 1989; Turner and Tjian, Science 243:1689, 1989). The leucine zipper domain is necessary for biological activity (DNA binding) in these proteins.
The fusogenic proteins of several different viruses, including paramyxovirus, coronavirus, measles virus and many retroviruses, also possess leucine zipper domains (Buckland and Wild, Nature 338:547, 1989; Britton, Nature 353:394, 1991; Delwart and Mosialos, AIDS Research and Human Retroviruses 6:703, 1990). The leucine zipper domains in these fusogenic viral proteins are near the transmembrane region of the proteins; it has been suggested that the leucine zipper domains could contribute to the oligomeric structure of the fusogenic proteins. Oligomerization of fusogenic viral proteins is involved in fusion pore formation (Spruce et al, Proc. Natl. Acad. Sci. U.S.A. 88:3523, 1991). Leucine zipper domains have also been recently reported to play a role in oligomerization of heat-shock transcription factors (Rabindran et al., Science 259:230, 1993).
Leucine zipper domains fold as short, parallel coiled coils. (O'Shea et al., Science 254:539; 1991) The general architecture of the parallel coiled coil has been well characterized, with a “knobs-into-holes” packing as proposed by Crick in 1953 (Acta Crystallogr. 6:689). The dimer formed by a leucine zipper domain is stabilized by the heptad repeat, designated (abcdefg)n according to the notation of McLachlan and Stewart (J. Mol. Biol. 98:293; 1975), in which residues a and d are generally hydrophobic residues, with d being a leucine, which line up on the same face of a helix. Oppositely-charged residues commonly occur at positions g and e. Thus, in a parallel coiled coil formed from two helical leucine zipper domains, the “knobs” formed by the hydrophobic side chains of the first helix are packed into the “holes” formed between the side chains of the second helix.
The leucine residues at position d contribute large hydrophobic stabilization energies, and are important for dimer formation (Krystek et al., Int. J. Peptide Res. 38:229, 1991). Lovejoy et al. recently reported the synthesis of a triple-stranded α-helical bundle in which the helices run up-up-down (Science 259:1288, 1993). Their studies confirmed that hydrophobic stabilization energy provides the main driving force for the formation of coiled coils from helical monomers. These studies also indicate that electrostatic interactions contribute to the stoichiometry and geometry of coiled coils.
Several studies have indicated that conservative amino acids may be substituted for individual leucine residues with minimal decrease in the ability to dimerize; multiple changes, however, usually result in loss of this ability (Landschulz et al., Science 243:1681, 1989; Turner and Tjian, Science 243:1689, 1989; Hu et al., Science 250:1400, 1990). van Heekeren et al. reported that a number of different amino residues can be substituted for the leucine residues in the leucine zipper domain of GCN4, and further found that some GCN4 proteins containing two leucine substitutions were weakly active (Nucl. Acids Res. 20:3721, 1992). Mutation of the first and second heptadic leucines of the leucine zipper domain of the measles virus fusion protein (MVF) did not affect syncytium formation (a measure of virally-induced cell fusion); however, mutation of all four leucine residues prevented fusion completely (Buckland et al., J. Gen. Virol. 73:1703, 1992). None of the mutations affected the ability of MVF to form a tetramer.
Amino acid substitutions in the a and d residues of a synthetic peptide representing the GCN4 leucine zipper domain have been found to change the oligomerization properties of the leucine zipper domain (Alber, Sixth Symposium of the Protein Society, San Diego, Calif.). When all residues at position a are changed to isoleucine, the leucine zipper still forms a parallel dimer. When, in addition to this change, all leucine residues at position d are also changed to isoleucine, the resultant peptide spontaneously forms a trimeric parallel coiled coil in solution. Substituting all amino acids at position d with isoleucine and at position a with leucine results in a peptide that tetramerizes. Peptides containing these substitutions are still referred to as leucine zipper domains.
Also included within the scope of the invention are fragments or derivatives of the intracellular domain of RANK. Such fragments are prepared by any of the herein-mentioned techniques, and include peptides that are identical to the cytoplasmic domain of RANK as shown in SEQ ID NO:6, or of murine RANK as shown in SEQ ID NO:15, and those that comprise a portion of the cytoplasmic region. All techniques used in preparing soluble forms may also be used in preparing fragments or analogs of the cytoplasmic domain (i.e., RT-PCR techniques or use of selected restriction enzymes to prepare truncations). DNAs encoding all or a fragment of the intracytoplasmic domain will be useful in identifying other proteins that are associated with RANK signaling, for example using the immunoprecipitation techniques described herein, or another technique such as a yeast two-hybrid system (Rothe et al., supra).
The present invention also includes RANK with or without associated native-pattern glycosylation. Proteins expressed in yeast or mammalian expression systems, e.g., COS-7 cells, may be similar or slightly different in molecular weight and glycosylation pattern than the native molecules, depending upon the expression system. Expression of DNAs encoding the inventive proteins in bacteria such as E. coli provides non-glycosylated molecules. Functional mutant analogs of RANK protein having inactivated N-glycosylation sites can be produced by oligonucleotide synthesis and ligation or by site-specific mutagenesis techniques. These analog proteins can be produced in a homogeneous, reduced-carbohydrate form in good yield using yeast expression systems. N-glycosylation sites in eukaryotic proteins are characterized by the amino acid triplet Asn-A1-Z, where A1 is any amino acid except Pro, and Z is Ser or Thr. In this sequence, asparagine provides a side chain amino group for covalent attachment of carbohydrate. Such a site can be eliminated by substituting another amino acid for Asn or for residue Z, deleting Asn or Z, or inserting a non-Z amino acid between A1 and Z, or an amino acid other than Asn between Asn and A1.
RANK protein derivatives may also be obtained by mutations of the native RANK or subunits thereof. A RANK mutated protein, as referred to herein, is a polypeptide homologous to a native RANK protein, respectively, but which has an amino acid sequence different from the native protein because of one or a plurality of deletions, insertions or substitutions. The effect of any mutation made in a DNA encoding a mutated peptide may be easily determined by analyzing the ability of the mutated peptide to bind its counterstructure in a specific manner. Moreover, activity of RANK analogs, muteins or derivatives can be determined by any of the assays described herein (for example, inhibition of the ability of RANK to activate transcription).
Analogs of the inventive proteins may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues can be deleted or replaced with other amino acids to prevent formation of incorrect intramolecular disulfide bridges upon renaturation. Other approaches to mutagenesis involve modification of adjacent dibasic amino acid residues to enhance expression in yeast systems in which KEX2 protease activity is present.
When a deletion or insertion strategy is adopted, the potential effect of the deletion or insertion on biological activity should be considered. Subunits of the inventive proteins may be constructed by deleting terminal or internal residues or sequences. Soluble forms of RANK can be readily prepared and tested for their ability to inhibit RANK-induced NF-κB activation. Polypeptides corresponding to the cytoplasmic regions, and fragments thereof (for example, a death domain) can be prepared by similar techniques. Additional guidance as to the types of mutations that can be made is provided by a comparison of the sequence of RANK to proteins that have similar structures, as well as by performing structural analysis of the inventive RANK proteins.
Generally, substitutions should be made conservatively; i.e., the most preferred substitute amino acids are those which do not affect the biological activity of RANK (i.e., ability of the inventive proteins to bind antibodies to the corresponding native protein in substantially equivalent a manner, the ability to bind the counterstructure in substantially the same manner as the native protein, the ability to transduce a RANK signal, or ability to induce NF-κB activation upon overexpression in transient transfection systems, for example). Examples of conservative substitutions include substitution of amino acids outside of the binding domain(s) (either ligand/receptor or antibody binding areas for the extracellular domain, or regions that interact with other, intracellular proteins for the cytoplasmic domain), and substitution of amino acids that do not alter the secondary and/or tertiary structure of the native protein. Additional examples include substituting one aliphatic residue for another, such as Ile, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gln and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known.
Mutations in nucleotide sequences constructed for expression of analog proteins or fragments thereof must, of course, preserve the reading frame phase of the coding sequences and preferably will not create complementary regions that could hybridize to produce secondary mRNA structures such as loops or hairpins which would adversely affect translation of the mRNA.
Not all mutations in the nucleotide sequence which encodes a RANK protein or fragments thereof will be expressed in the final product, for example, nucleotide substitutions may be made to enhance expression, primarily to avoid secondary structure loops in the transcribed mRNA (see EPA 75,444A, incorporated herein by reference), or to provide codons that are more readily translated by the selected host, e.g., the well-known E. coli preference codons for E. coli expression.
Although a mutation site may be predetermined, it is not necessary that the nature of the mutation per se be predetermined. For example, in order to select for optimum characteristics of mutants, random mutagenesis may be conducted and the expressed mutated proteins screened for the desired activity. Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.
Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required. Exemplary methods of making the alterations set forth above are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462 disclose suitable techniques, and are incorporated by reference herein.
Other embodiments of the inventive proteins include RANK polypeptides encoded by DNAs capable of hybridizing to the DNA of SEQ ID NO:5 under moderately stringent conditions (prewashing solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridization conditions of 50° C., 5×SSC, overnight) to the DNA sequences encoding RANK, or more preferably under stringent conditions (for example, hybridization in 6×SSC at 63° C. overnight; washing in 3×SSC at 55° C.), and other sequences which are degenerate to those which encode the RANK. In one embodiment, RANK polypeptides are at least about 70% identical in amino acid sequence to the amino acid sequence of native RANK protein as set forth in SEQ ID NO:6. In a preferred embodiment, RANK polypeptides are at least about 80% identical in amino acid sequence to the native form of RANK; most preferred polypeptides are those that are at least about 90% identical to native RANK.
Percent identity may be determined using a computer program, for example, the GAP computer program described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). For fragments derived from the RANK protein, the identity is calculated based on that portion of the RANK protein that is present in the fragment
The biological activity of RANK analogs or muteins can be determined by testing the ability of the analogs or muteins to inhibit activation of transcription, for example as described in the Examples herein. Alternatively, suitable assays, for example, an enzyme immunoassay or a dot blot, employing an antibody that binds native RANK, or a soluble form of RANKL, can be used to assess the activity of RANK analogs or muteins, as can assays that employ cells expressing RANKL. Suitable assays also include, for example, signal transduction assays and methods that evaluate the ability of the cytoplasmic region of RANK to associate with other intracellular proteins (i.e., TRAFs 2 and 3) involved in signal transduction will also be useful to assess the activity of RANK analogs or muteins. Such methods are well known in the art.
Fragments of the RANK nucleotide sequences are also useful. In one embodiment, such fragments comprise at least about 17 consecutive nucleotides, preferably at least about 25 nucleotides, more preferably at least 30 consecutive nucleotides, of the RANK DNA disclosed herein. DNA and RNA complements of such fragments are provided herein, along with both single-stranded and double-stranded forms of the RANK DNA of SEQ ID NO:5, and those encoding the aforementioned polypeptides. A fragment of RANK DNA generally comprises at least about 17 nucleotides, preferably from about 17 to about 30 nucleotides. Such nucleic acid fragments (for example, a probe corresponding to the extracellular domain of RANK) are used as a probe or as primers in a polymerase chain reaction (PCR).
The probes also find use in detecting the presence of RANK nucleic acids in in vitro assays and in such procedures as Northern and Southern blots. Cell types expressing RANK can be identified as well. Such procedures are well known, and the skilled artisan can choose a probe of suitable length, depending on the particular intended application. For PCR, 5′ and 3′ primers corresponding to the termini of a desired RANK DNA sequence are employed to amplify that sequence, using conventional techniques.
Other useful fragments of the RANK nucleic acids are antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target RANK mRNA (sense) or RANK DNA (antisense) sequences. The ability to create an antisense or a sense oligonucleotide, based upon a cDNA sequence for a given protein is described in, for example, Stein and Cohen, Cancer Res. 48:2659, 1988 and van der Krol et al., BioTechniques 6:958, 1988.
Uses of DNAs, Proteins and Analogs
The RANK DNAs, proteins and analogs described herein will have numerous uses, including the preparation of pharmaceutical compositions. For example, soluble forms of RANK will be useful as antagonists of RANK-mediated NF-κB activation, as well as to inhibit transduction of a signal via RANK. RANK compositions (both protein and DNAs) will also be useful in development of both agonistic and antagonistic antibodies to RANK. The inventive DNAs are useful for the expression of recombinant proteins, and as probes for analysis (either quantitative or qualitative) of the presence or distribution of RANK transcripts.
The inventive proteins will also be useful in preparing kits that are used to detect soluble RANK or RANKL, or monitor RANK-related activity, for example, in patient specimens. RANK proteins will also find uses in monitoring RANK-related activity in other samples or compositions, as is necessary when screening for antagonists or mimetics of this activity (for example, peptides or small molecules that inhibit or mimic, respectively, the interaction). A variety of assay formats are useful in such kits, including (but not limited to) ELISA, dot blot, solid phase binding assays (such as those using a biosensor), rapid format assays and bioassays.
The purified RANK according to the invention will facilitate the discovery of inhibitors of RANK, and thus, inhibitors of an inflammatory response (via inhibition of NF-κB activation). The use of a purified RANK polypeptide in the screening for potential inhibitors is important and can virtually eliminate the possibility of interfering reactions with contaminants. Such a screening assay can utilize either the extracellular domain of RANK, the intracellular domain, or a fragment of either of these polypeptides. Detecting the inhibiting activity of a molecule would typically involve use of a soluble form of RANK derived from the extracellular domain in a screening assay to detect molecules capable of binding RANK and inhibiting binding of, for example, an agonistic antibody or RANKL, or using a polypeptide derived from the intracellular domain in an assay to detect inhibition of the interaction of RANK and other, intracellular proteins involved in signal transduction.
Moreover, in vitro systems can be used to ascertain the ability of molecules to antagonize or agonize RANK activity. Included in such methods are uses of RANK chimeras, for example, a chimera of the RANK intracellular domain and an extracellular domain derived from a protein having a known ligand. The effects on signal transduction of various molecule can then be monitored by utilizing the known ligand to transduce a signal.
In addition, RANK polypeptides can also be used for structure-based design of RANK-inhibitors. Such structure-based design is also known as “rational drug design.”The RANK polypeptides can be three-dimensionally analyzed by, for example, X-ray crystallography, nuclear magnetic resonance or homology modeling, all of which are well-known methods. The use of RANK structural information in molecular modeling software systems to assist in inhibitor design is also encompassed by the invention. Such computer-assisted modeling and drug design may utilize information such as chemical conformational analysis, electrostatic potential of the molecules, protein folding, etc. A particular method of the invention comprises analyzing the three dimensional structure of RANK for likely binding sites of substrates, synthesizing a new molecule that incorporates a predictive reactive site, and assaying the new molecule as described above.
Expression of Recombinant RANK
The proteins of the present invention are preferably produced by recombinant DNA methods by inserting a DNA sequence encoding RANK protein or an analog thereof into a recombinant expression vector and expressing the DNA sequence in a recombinant expression system under conditions promoting expression. DNA sequences encoding the proteins provided by this invention can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being inserted in a recombinant expression vector and expressed in a recombinant transcriptional unit.
Recombinant expression vectors include synthetic or cDNA-derived DNA fragments encoding RANK, or homologs, muteins or bioequivalent analogs thereof, operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral or insect genes. Such regulatory elements include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation, as described in detail below. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may additionally be incorporated.
DNA regions are operably linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous and, in the case of secretory leaders, contiguous and in reading frame. DNA sequences encoding RANK, or homologs or analogs thereof which are to be expressed in a microorganism will preferably contain no introns that could prematurely terminate transcription of DNA into mRNA.
Useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA). These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed. E. coli is typically transformed using derivatives of pBR322, a plasmid derived from an E. coli species (Bolivar et al., Gene 2:95, 1977). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells.
Promoters commonly used in recombinant microbial expression vectors include the β-lactamase (penicillinase) and lactose promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544, 1979), the tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, 1980; and EPA 36,776) and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412, 1982). A particularly useful bacterial expression system employs the phage λ PL promoter and cI857ts thermolabile repressor. Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the λ PL promoter include plasmid pHUB2, resident in E. coli strain JMB9 (ATCC 37092) and pPLc28, resident in E. coli RR1 (ATCC 53082).
Suitable promoter sequences in yeast vectors include the promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al., EPA 73,657.
Preferred yeast vectors can be assembled using DNA sequences from pBR322 for selection and replication in E. coli (Ampr gene and origin of replication) and yeast DNA sequences including a glucose-repressible ADH2 promoter and α-factor secretion leader. The ADH2 promoter has been described by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature 300:724, 1982). The yeast α-factor leader, which directs secretion of heterologous proteins, can be inserted between the promoter and the structural gene to be expressed. See, e.g., Kurjan et al., Cell 30:933, 1982; and Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330, 1984. The leader sequence may be modified to contain, near its 3′ end, one or more useful restriction sites to facilitate fusion of the leader sequence to foreign genes.
The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells may be provided by viral sources. For example, commonly used promoters and enhancers are derived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites may be used to provide the other genetic elements required for expression of a heterologous DNA sequence. The early and late promoters are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40 fragments may also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the BglI site located in the viral origin of replication is included. Further, viral genomic promoter, control and/or signal sequences may be utilized, provided such control sequences are compatible with the host cell chosen. Exemplary vectors can be constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983).
A useful system for stable high level expression of mammalian receptor cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al. (Mol. Immunol. 23:935, 1986). A preferred eukaryotic vector for expression of RANK DNA is referred to as pDC406 (McMahan et al., EMBO J. 10:2821, 1991), and includes regulatory sequences derived from SV40, human immunodeficiency virus (HIV), and Epstein-Barr virus (EBV). Other preferred vectors include pDC409 and pDC410, which are derived from pDC406. pDC410 was derived from pDC406 by substituting the EBV origin of replication with sequences encoding the SV40 large T antigen. pDC409 differs from pDC406 in that a Bgl II restriction site outside of the multiple cloning site has been deleted, making the Bgl II site within the multiple cloning site unique.
A useful cell line that allows for episomal replication of expression vectors, such as pDC406 and pDC409, which contain the EBV origin of replication, is CV-1/EBNA (ATCC CRL 10478). The CV-1/EBNA cell line was derived by transfection of the CV-1 cell line with a gene encoding Epstein-Barr virus nuclear antigen-1 (EBNA-1) and constitutively express EBNA-1 driven from human CMV immediate-early enhancer/promoter.
Host Cells
Transformed host cells are cells which have been transformed or transfected with expression vectors constructed using recombinant DNA techniques and which contain sequences encoding the proteins of the present invention. Transformed host cells may express the desired protein (RANK, or homologs or analogs thereof), but host cells transformed for purposes of cloning or amplifying the inventive DNA do not need to express the protein. Expressed proteins will preferably be secreted into the culture supernatant, depending on the DNA selected, but may be deposited in the cell membrane.
Suitable host cells for expression of proteins include prokaryotes, yeast or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram negative or gram positive organisms, for example E. coli or Bacillus spp. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems could also be employed to produce proteins using RNAs derived from the DNA constructs disclosed herein. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985), the relevant disclosure of which is hereby incorporated by reference.
Prokaryotic expression hosts may be used for expression of RANK, or homologs or analogs thereof that do not require extensive proteolytic and disulfide processing. Prokaryotic expression vectors generally comprise one or more phenotypic selectable markers, for example a gene encoding proteins conferring antibiotic resistance or supplying an autotrophic requirement, and an origin of replication recognized by the host to ensure amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
Recombinant RANK may also be expressed in yeast hosts, preferably from the Saccharomyces species, such as S. cerevisiae. Yeast of other genera, such as Pichia or Kluyveromyces may also be employed. Yeast vectors will generally contain an origin of replication from the 2μ yeast plasmid or an autonomously replicating sequence (ARS), promoter, DNA encoding the protein, sequences for polyadenylation and transcription termination and a selection gene. Preferably, yeast vectors will include an origin of replication and selectable marker permitting transformation of both yeast and E. coli, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae trp1 gene, which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, and a promoter derived from a highly expressed yeast gene to induce transcription of a structural sequence downstream. The presence of the trp1 lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
Suitable yeast transformation protocols are known to those of skill in the art; an exemplary technique is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929, 1978, selecting for Trp+ transformants in a selective medium consisting of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 μg/ml adenine and 20 μg/ml uracil. Host strains transformed by vectors comprising the ADH2 promoter may be grown for expression in a rich medium consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/ml uracil. Derepression of the ADH2 promoter occurs upon exhaustion of medium glucose. Crude yeast supernatants are harvested by filtration and held at 4° C. prior to further purification.
Various mammalian or insect cell culture systems can be employed to express recombinant protein. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988). Examples of suitable mammalian host cell lines include the COS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175, 1981), and other cell lines capable of expressing an appropriate vector including, for example, CV-1/EBNA (ATCC CRL 10478), L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell lines. Mammalian expression vectors may comprise nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5′ or 3′ flanking nontranscribed sequences, and 5′ or 3′ nontranslated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
Purification of Recombinant RANK
Purified RANK, and homologs or analogs thereof are prepared by culturing suitable host/vector systems to express the recombinant translation products of the DNAs of the present invention, which are then purified from culture media or cell extracts. For example, supernatants from systems which secrete recombinant protein into culture media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
Following the concentration step, the concentrate can be applied to a suitable purification matrix. For example, a suitable affinity matrix can comprise a counter structure protein or lectin or antibody molecule bound to a suitable support. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred. Gel filtration chromatography also provides a means of purifying the inventive proteins.
Affinity chromatography is a particularly preferred method of purifying RANK and homologs thereof. For example, a RANK expressed as a fusion protein comprising an immunoglobulin Fc region can be purified using Protein A or Protein G affinity chromatography. Moreover, a RANK protein comprising an oligomerizing zipper domain may be purified on a resin comprising an antibody specific to the oligomerizing zipper domain. Monoclonal antibodies against the RANK protein may also be useful in affinity chromatography purification, by utilizing methods that are well-known in the art. A ligand may also be used to prepare an affinity matrix for affinity purification of RANK.
Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify a RANK composition. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant protein.
Recombinant protein produced in bacterial culture is usually isolated by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange or size exclusion chromatography steps. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. Microbial cells employed in expression of recombinant protein can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
Fermentation of yeast which express the inventive protein as a secreted protein greatly simplifies purification. Secreted recombinant protein resulting from a large-scale fermentation can be purified by methods analogous to those disclosed by Urdal et al. (J. Chromatog. 296:171, 1984). This reference describes two sequential, reversed-phase HPLC steps for purification of recombinant human GM-CSF on a preparative HPLC column.
Protein synthesized in recombinant culture is characterized by the presence of cell components, including proteins, in amounts and of a character which depend upon the purification steps taken to recover the inventive protein from the culture. These components ordinarily will be of yeast, prokaryotic or non-human higher eukaryotic origin and preferably are present in innocuous contaminant quantities, on the order of less than about 1 percent by weight. Further, recombinant cell culture enables the production of the inventive proteins free of other proteins which may be normally associated with the proteins as they are found in nature in the species of origin.
Uses and Administration of RANK Compositions
The present invention provides methods of using therapeutic compositions comprising an effective amount of a protein and a suitable diluent and carrier, and methods for regulating an immune or inflammatory response. The use of RANK in conjunction with soluble cytokine receptors or cytokines, or other immunoregulatory molecules is also contemplated.
For therapeutic use, purified protein is administered to a patient, preferably a human, for treatment in a manner appropriate to the indication. Thus, for example, RANK protein compositions administered to regulate immune function can be given by bolus injection, continuous infusion, sustained release from implants, or other suitable technique. Typically, a therapeutic agent will be administered in the form of a composition comprising purified RANK, in conjunction with physiologically acceptable carriers, excipients or diluents. Such carriers will be nontoxic to recipients at the dosages and concentrations employed.
Ordinarily, the preparation of such protein compositions entails combining the inventive protein with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with conspecific serum albumin are exemplary appropriate diluents. Preferably, product is formulated as a lyophilizate using appropriate excipient solutions (e.g., sucrose) as diluents. Appropriate dosages can be determined in trials. The amount and frequency of administration will depend, of course, on such factors as the nature and severity of the indication being treated, the desired response, the condition of the patient, and so forth.
Soluble forms of RANK and other RANK antagonists such as antagonistic monoclonal antibodies can be administered for the purpose of inhibiting RANK-induced induction of NF-κB activity. NF-κB is a transcription factor that is utilized extensively by cells of the immune system, and plays a role in the inflammatory response. Thus, inhibitors of RANK signalling will be useful in treating conditions in which signalling through RANK has given rise to negative consequences, for example, toxic or septic shock, or graft-versus-host reactions. They may also be useful in interfering with the role of NF-κB in cellular transformation. Tumor cells are more responsive to radiation when their NF-κB is blocked; thus, soluble RANK (or other antagonists of RANK signalling) will be useful as an adjunct therapy for disease characterized by neoplastic cells that express RANK.
The following examples are offered by way of illustration, and not by way of limitation. Those skilled in the art will recognize that variations of the invention embodied in the examples can be made, especially in light of the teachings of the various references cited herein, the disclosures of which are incorporated by reference.
EXAMPLE 1
The example describes the identification and isolation of a DNA encoding a novel member of the TNF receptor superfamily. A partial cDNA insert with a predicted open reading frame having some similarity to CD40 (a cell-surface antigen present on the surface of both normal and neoplastic human B cells that has been shown to play an important role in B-cell proliferation and differentiation; Stamenkovic et al., EMBO J. 8:1403, 1989), was identified in a database containing sequence information from cDNAs generated from human bone marrow-derived dendritic cells (DC). The insert was excised from the vector by restriction endonuclease digestion, gel purified. labeled with 32P, and used to hybridize to colony blots generated from a DC cDNA library containing larger cDNA inserts using high stringency hybridization and washing techniques (hybridization in 5×SSC, 50% formamide at 42° C. overnight, washing in 0.5×SSC at 63° C.); other suitable high stringency conditions are disclosed in Sambrook et al. in Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; 1989), 9.52-9.55. Initial experiments yielded a clone referred to as 9D-8A (SEQ ID NO:1); subsequent analysis indicated that this clone contained all but the extreme 5′ end of a novel cDNA, with predicted intron sequence at the extreme 5′ end (nucleotides 1-92 of SEQ ID NO:1). Additional colony hybridizations were performed, and a second clone was isolated. The second clone, referred to as 9D-15C (SEQ ID NO:3), contained the 5′ end without intron interruption but not the full 3′ end. SEQ ID NO:5 shows the nucleotide and amino acid sequence of a predicted full-length protein based on alignment of the overlapping sequences of SEQ ID NOs:1 and 3.
The encoded protein was designated RANK, for receptor activator of NF-κB. The cDNA encodes a predicted Type 1 transmembrane protein having 616 amino acid residues, with a predicted 24 amino acid signal sequence (the computer predicted cleavage site is after Leu24), a 188 amino acid extracellular domain, a 21 amino acid transmembrane domain, and a 383 amino acid cytoplasmic tail. The extracellular region of RANK displayed significant amino acid homology (38.5% identity, 52.3% similarity) to CD40. A cloning vector (pBluescriptSK−) containing human RANK sequence, designated pBluescript:huRANK (in E. coli DH10B), was deposited with the American Type Culture Collection, Manassas, Va. (ATCC) on Dec. 20, 1996, under terms of the Budapest Treaty, and given accession number 98285.
EXAMPLE 2
This example describes construction of a RANK DNA construct to express a RANK/Fc fusion protein. A soluble form of RANK fused to the Fc region of human IgG1 was constructed in the mammalian expression vector pDC409 (U.S. Ser. No. 08/571,579). This expression vector encodes the leader sequence of the Cytomegalovirus (CMV) open reading frame R27080 (SEQ ID NO:9), followed by amino acids 33-213 of RANK, followed by a mutated form of the constant domain of human IgG1 that exhibits reduced affinity for Fc receptors (SEQ ID NO:8; for the fusion protein, the Fc portion of the construct consisted of Arg3 through Lys232). An alternative expression vector encompassing amino acids 1-213 of RANK (using the native leader sequence) followed by the IgG1 mutein was also prepared. Both expression vectors were found to induce high levels of expression of the RANK/Fc fusion protein in transfected cells.
To obtain RANK/Fc protein, a RANK/Fc expression plasmid is transfected into CV-1/EBNA cells, and supernatants are collected for about one week. The RANK/Fc fusion protein is purified by means well-known in the art for purification of Fc fusion proteins, for example, by protein A sepharose column chromatography according to manufacturer's recommendations (i.e., Pharmacia, Uppsala, Sweden). SDS-polyacrylamide gel electrophoresis analysis indicted that the purified RANK/Fc protein migrated with a molecular weight of ˜55 kDa in the presence of a reducing agent, and at a molecular weight of ˜110 kDa in the absence of a reducing agent.
N-terminal amino acid sequencing of the purified protein made using the CMV 827080 leader showed 60% cleavage after Ala20, 20% cleavage after Pro22 and 20% cleavage after Arg28 (which is the Furin cleavage site; amino acid residues are relative to SEQ ID NO:9); N-terminal amino acid analysis of the fusion protein expressed with the native leader showed cleavage predominantly after Gln25 (80% after Gln25 and 20% after Arg23; amino acid residues are relative to SEQ ID NO:6, full-length RANK). Both fusion proteins were able to bind a ligand for RANK is a specific manner (i.e., they bound to the surface of various cell lines such as a murine thymoma cell line, EL4), indicating that the presence of additional amino acids at the N-terminus of RANK does not interfere with its ability to bind RANKL. Moreover, the construct comprising the CMV leader encoded RANK beginning at amino acid 33; thus, a RANK peptide having an N-terminus at an amino acid between Arg23 and Pro33, inclusive, is expected to be able to bind a ligand for RANK in a specific manner.
Other members of the TNF receptor superfamily have a region of amino acids between the transmembrane domain and the ligand binding domain that is referred to as a ‘spacer’ region, which is not necessary for ligand binding. In RANK, the amino acids between 196 and 213 are predicted to form such a spacer region. Accordingly, a soluble form of RANK that terminates with an amino acid in this region is expected to retain the ability to bind a ligand for RANK in a specific manner. Preferred C-terminal amino acids for soluble RANK peptides are selected from the group consisting of amino acids 213 and 196 of SEQ ID NO:6, although other amino acids in the spacer region may be utilized as a C-terminus.
EXAMPLE 3
This example illustrates the preparation of monoclonal antibodies against RANK. Preparations of purified recombinant RANK, for example, or transfected cells expressing high levels of RANK, are employed to generate monoclonal antibodies against RANK using conventional techniques, such as those disclosed in U.S. Pat. No. 4,411,993. DNA encoding RANK can also be used as an immunogen, for example, as reviewed by Pardoll and Beckerleg in Immunity 3:165, 1995. Such antibodies are likely to be useful in interfering with RANK-induced signaling (antagonistic or blocking antibodies) or in inducing a signal by cross-linking RANK (agonistic antibodies), as components of diagnostic or research assays for RANK or RANK activity, or in affinity purification of RANK.
To immunize rodents, RANK immunogen is emulsified in an adjuvant (such as complete or incomplete Freund's adjuvant, alum, or another adjuvant, such as Ribi adjuvant R700 (Ribi, Hamilton, Mont.), and injected in amounts ranging from 10-100 μg subcutaneously into a selected rodent, for example, BALB/c mice or Lewis rats. DNA may be given intradermally (Raz et al., Proc. Natl. Acad. Sci. USA 91:9519, 1994) or intamuscularly (Wang et al., Proc. Natl. Acad. Sci. USA 90:4156, 1993); saline has been found to be a suitable diluent for DNA-based antigens. Ten days to three weeks days later, the immunized animals are boosted with additional immunogen and periodically boosted thereafter on a weekly, biweekly or every third week immunization schedule.
Serum samples are periodically taken by retro-orbital bleeding or tail-tip excision for testing by dot-blot assay (antibody sandwich), ELISA (enzyme-linked immunosorbent assay), immunoprecipitation, or other suitable assays, including FACS analysis. Following detection of an appropriate antibody titer, positive animals are given an intravenous injection of antigen in saline. Three to four days later, the animals are sacrificed, splenocytes harvested, and fused to a murine myeloma cell line (e.g., NS1 or preferably Ag 8.653 [ATCC CRL 1580]). Hybridoma cell lines generated by this procedure are plated in multiple microtiter plates in a selective medium (for example, one containing hypoxanthine, aminopterin, and thymidine, or HAT) to inhibit proliferation of non-fused cells, myeloma-myeloma hybrids, and splenocyte-splenocyte hybrids.
Hybridoma clones thus generated can be screened by ELISA for reactivity with RANK, for example, by adaptations of the techniques disclosed by Engvall et al., Immunochem. 8:871 (1971) and in U.S. Pat. No. 4,703,004. A preferred screening technique is the antibody capture technique described by Beckman et al., J. Immunol. 144:4212 (1990). Positive clones are then injected into the peritoneal cavities of syngeneic rodents to produce ascites containing high concentrations (>1 mg/ml) of anti-RANK monoclonal antibody. The resulting monoclonal antibody can be purified by ammonium sulfate precipitation followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can also be used, as can affinity chromatography based upon binding to RANK protein.
Monoclonal antibodies were generated using RANK/Fc fusion protein as the immunogen. These reagents were screened to confirm reactivity against the RANK protein. Using the methods described herein to monitor the activity of the mAbs, both blocking (i.e., antibodies that bind RANK and inhibit binding of a ligand to RANK) and non-blocking (i.e., antibodies that bind RANK and do not inhibit ligand binding) were isolated.
EXAMPLE 4
This example illustrates the induction of NF-κB activity by RANK in 293/EBNA cells (cell line was derived by transfection of the 293 cell line with a gene encoding Epstein-Barr virus nuclear antigen-1 (EBNA-1) that constitutively express EBNA-1 driven from human CMV immediate-early enhancer/promoter). Activation of NF-κB activity was measured in 293/EBNA cells essentially as described by Yao et al. (Immunity 3:811, 1995). Nuclear extracts were prepared and analyzed for NF-κB activity by a gel retardation assay using a 25 base pair oligonucleotide spanning the NF-κB binding sites. Two million cells were seeded into 10 cm dishes two days prior to DNA transfection and cultured in DMEM-F12 media containing 2.5% FBS (fetal bovine serum). DNA transfections were performed as described herein for the IL-8 promoter/reporter assays.
Nuclear extracts were prepared by solubilization of isolated nuclei with 400 mM NaCl (Yao et al., supra). Oligonucleotides containing an NF-κB binding site were annealed and endlabeled with 32P using T4 DNA polynucleotide kinase. Mobility shift reactions contained 10 μg of nuclear extract, 4 μg of poly(dI-dC) and 15,000 cpm labeled double-stranded oligonucleotide and incubated at room temperature for 20 minutes. Resulting protein-DNA complexes were resolved on a 6% native polyacrylamide gel in 0.25× Tris-borate-EDTA buffer.
Overexpression of RANK resulted in induction of NF-κB activity as shown by an appropriate shift in the mobility of the radioactive probe on the gel. Similar results were observed when RANK was triggered by a ligand that binds RANK and transduces a signal to cells expressing the receptor (i.e., by co-transfecting cells with human RANK and murine RANKL DNA; see Example 7 below), and would be expected to occur when triggering is done with agonistic antibodies.
EXAMPLE 5
This example describes a gene promoter/reporter system based on the human Interleukin-8 (IL-8) promoter used to analyze the activation of gene transcription in vivo. The induction of human IL-8 gene transcription by the cytokines Interleukin-1 (IL-1) or tumor necrosis factor-alpha (TNF-α) is known to be dependent upon intact NF-κB and NF-IL-6 transcription factor binding sites. Fusion of the cytokine-responsive IL-8 promoter with a cDNA encoding the murine IL-4 receptor (mIL-4R) allows measurement of promoter activation by detection of the heterologous reporter protein (mIL-4R) on the cell surface of transfected cells.
Human kidney epithelial cells (293/EBNA) are transfected (via the DEAE/DEXTRAN method) with plasmids encoding: 1). the reporter/promoter construct (referred to as pIL-8rep), and 2). the cDNA(s) of interest. DNA concentrations are always kept constant by the addition of empty vector DNA. The 293/EBNA cells are plated at a density of 2.5×104 cells/ml (3 ml/well) in a 6 well plate and incubated for two days prior to transfection. Two days after transfection, the mIL-4 receptor is detected by a radioimmunoassay (RIA) described below.
In one such experiment, the 293/EBNA cells were co-transfected with DNA encoding RANK and with DNA encoding RANKL (see Example 7 below). Co-expression of this receptor and its counterstructure by cells results in activation of the signaling process of RANK. For such co-transfection studies, the DNA concentration/well for the DEAE transfection were as follows: 40 ng of pIL-8rep [pBluescriptSK-vector (Stratagene)]; 0.4 ng CD40 (DNA encoding CD40, a control receptor; pCDM8 vector); 0.4 ng RANK (DNA encoding RANK; pDC409 vector), and either 1-50 ng CD40L (DNA encoding the ligand for CD40, which acts as a positive control when co-transfected with CD40 and as a negative control when co-transfected with RANK; in pDC304) or RANKL (DNA encoding a ligand for RANK; in pDC406). Similar experiments can be done using soluble RANKL or agonistic antibodies to RANK to trigger cells transfected with RANK.
For the mIL-4R-specific RIA, a monoclonal antibody reactive with mIL-4R is labeled with 125I via a Chloramine T conjugation method; the resulting specific activity is typically 1.5×1016 cpm/nmol. After 48 hours, transfected cells are washed once with media (DMEM/F12 5% FBS). Non-specific binding sites are blocked by the addition of pre-warmed binding media containing 5% non-fat dry milk and incubation at 37° C./5% CO2 in a tissue culture incubator for one hour. The blocking media is decanted and binding buffer containing 125I anti-mIL-4R (clone M1; rat IgG1) is added to the cells and incubated with rocking at room temperature for 1 hour. After incubation of the cells with the radio-labeled antibody, cells are washed extensively with binding buffer (2×) and twice with phosphate-buffered saline (PBS). Cells are lysed in 1 ml of 0.5M NaOH, and total radioactivity is measured with a gamma counter.
Using this assay, 293/EBNA co-transfected with DNAs encoding RANK demonstrated transcriptional activation, as shown by detection of muIL-4R on the cell surface. Overexpression of RANK resulted in transcription of muIL-4R, as did triggering of the RANK by RANKL. Similar results are observed when RANK is triggered by agonistic antibodies.
EXAMPLE 6
This example illustrates the association of RANK with TRAF proteins. Interaction of RANK with cytoplasmic TRAF proteins was demonstrated by co-immunoprecipitation assays essentially as described by Hsu et al. (Cell 84:299; 1996). Briefly, 293/EBNA cells were co-transfected with plasmids that direct the synthesis of RANK and epitope-tagged (FLAG®; SEQ ID NO:7) TRAF2 or TRAF3. Two days after transfection, surface proteins were labeled with biotin-ester, and cells were lysed in a buffer containing 0.5% NP-40. RANK and proteins associated with this receptor were immunoprecipitated with anti-RANK, washed extensively, resolved by electrophoretic separation on a 6-10% SDS polyacrylamide gel and electrophoretically transferred to a nitrocellulose membrane for Western blotting. The association of TRAF2 and TRAF3 proteins with RANK was visualized by probing the membrane with an antibody that specifically recognizes the FLAG® epitope. TRAFs 2 and 3 did not immunopreciptitate with anti-RANK in the absence of RANK expression.
EXAMPLE 7
This example describes isolation of a ligand for RANK, referred to as RANKL, by direct expression cloning. The ligand was cloned essentially as described in U.S. Ser. No. 08/249,189, filed May 24, 1994 (the relevant disclosure of which is incorporated by reference herein), for CD40L. Briefly, a library was prepared from a clone of a mouse thymoma cell line EL-4 (ATCC TIB 39), called EL-40.5, derived by sorting five times with biotinylated CD40/Fc fusion protein in a FACS (fluorescence activated cell sorter). The cDNA library was made using standard methodology; the plasmid DNA was isolated and transfected into sub-confluent CV1-EBNA cells using a DEAE-dextran method. Transfectants were screened by slide autoradiography for expression of RANKL using a two-step binding method with RANK/Fc fusion protein as prepared in Example 2 followed by radioiodinated goat anti-human IgG antibody.
A clone encoding a protein that specifically bound RANK was isolated and sequenced; the clone was referred to as 11H. An expression vector containing murine RANKL sequence, designated pDC406:muRANK-L (in E. coli DH10B), was deposited with the American Type Culture Collection, Manassas, Va. (ATCC) on Dec. 20, 1996, under terms of the Budapest Treaty, and given accession number 98284. The nucleotide sequence and predicted amino acid sequence of this clone are illustrated in SEQ ID NO:10. This clone did not contain an initiator methionine; additional, full-length clones were obtained from a 7B9 library (prepared substantially as described in U.S. Pat. No. 5,599,905, issued Feb. 4, 1997); the 5′ region was found to be identical to that of human RANKL as shown in SEQ ID NO: 12, amino acids 1 through 22, except for substitution of a Gly for a Thr at residue 9.
This ligand is useful for assessing the ability of RANK to bind RANKL by a number of different assays. For example, transfected cells expressing RANKL can be used in a FACS assay (or similar assay) to evaluate the ability of soluble RANK to bind RANKL. Moreover, soluble forms of RANKL can be prepared and used in assays that are known in the art (i.e., ELISA or BIAcore assays essentially as described in U.S. Ser. No. 08/249,189, filed May 24, 1994). RANKL is also useful in affinity purification of RANK, and as a reagent in methods to measure the levels of RANK in a sample. Soluble RANKL is also useful in inducing NF-κB activation and thus protecting cells that express RANK from apoptosis.
EXAMPLE 8
This example describes the isolation of a human RANK ligand (RANKL) using a PCR-based technique. Murine RANK ligand-specific oligonucleotide primers were used in PCR reactions using human cell line-derived first strand cDNAs as templates. Primers corresponded to nucleotides 478-497 and to the complement of nucleotides 858-878 of murine RANK ligand (SEQ ID NO:10). An amplified band approximately 400 bp in length from one reaction using the human epidermoid cell line KB (ATCC CCL-17) was gel purified, and its nucleotide sequence determined; the sequence was 85% identical to the corresponding region of murine RANK ligand, confirming that the fragment was from human RANKL.
To obtain full-length human RANKL cDNAs, two human RANKL-specific oligonucleotides derived from the KB PCR product nucleotide sequence were radiolabeled and used as hybridization probes to screen a human PBL cDNA library prepared in lambda gt10 (Stratagene, La Jolla, Calif.), substantially as described in U.S. Pat. No. 5,599,905, issued Feb. 4, 1997. Several positive hybridizing plaques were identified and purified, their inserts subcloned into pBluescript SK− (Stratagene, La Jolla, Calif.), and their nucleotide sequence determined One isolate, PBL3, was found to encode most of the predicted human RANKL, but appeared to be missing approximately 200 bp of 5′ coding region. A second isolate, PBL5 was found to encode much of the predicted human RANKL, including the entire 5′ end and an additional 200 bp of 5′ untranslated sequence.
The 5′ end of PBL5 and the 3′ end of PBL3 were ligated together to form a full length cDNA encoding human RANKL. The nucleotide and predicted amino acid sequence of the full-length human RANK ligand is shown in SEQ ID NO:12. Human RANK ligand shares 83% nucleotide and 84% amino acid identity with murine RANK ligand. A plasmid vector containing human RANKL sequence, designated pBluescript:huRANK-L (in E. coli DH10B), was deposited with the American Type Culture Collection, Manassas, Va. (ATCC) on Mar. 11, 1997 under terms of the Budapest Treaty, and given accession number 98354.
Murine and human RANKL are Type 2 transmembrane proteins. Murine RANKL contains a predicted 48 amino acid intracellular domain, 21 amino acid transmembrane domain and 247 amino acid extracellular domain. Human RANKL contains a predicted 47 amino acid intracellular domain, 21 amino acid transmembrane domain and 249 amino acid extracellular domain.
EXAMPLE 9
This example describes the chromosomal mapping of human RANK using PCR-based mapping strategies. Initial human chromosomal assignments were made using RANK and RANKL-specific PCR primers and a BIOS Somatic Cell Hybrid PCRable DNA kit from BIOS Laboratories (New Haven, Conn.), following the manufacturer's instructions. RANK mapped to human chromosome 18; RANK ligand mapped to human chromosome 13. More detailed mapping was performed using a radiation hybrid mapping panel Genebridge 4 Radiation Hybrid Panel (Research Genetics, Huntsville, Ala.; described in Walter, M A et al., Nature Genetics 7:22-28, 1994). Data from this analysis was then submitted electronically to the MIT Radiation Hybrid Mapper following the instructions contained therein. This analysis yielded specific genetic marker names which, when submitted electronically to the NCBI Entrez browser, yielded the specific map locations. RANK mapped to chromosome 18q22.1, and RANKL mapped to chromosome 13q14.
EXAMPLE 10
This example illustrates the preparation of monoclonal antibodies against RANKL. Preparations of purified recombinant RANKL, for example, or transfixed cells expressing high levels of RANKL, are employed to generate monoclonal antibodies against RANKL using conventional techniques, such as those disclosed in U.S. Pat. No. 4,411,993. DNA encoding RANKL can also be used as an immunogen, for example, as reviewed by Pardoll and Beckerleg in Immunity 3:165, 1995. Such antibodies are likely to be useful in interfering with RANKL signaling (antagonistic or blocking antibodies), as components of diagnostic or research assays for RANKL or RANKL activity, or in affinity purification of RANKL.
To immunize rodents, RANKL immunogen is emulsified in an adjuvant (such as complete or incomplete Freund's adjuvant, alum, or another adjuvant, such as Ribi adjuvant R700 (Ribi, Hamilton, Mont.), and injected in amounts ranging from 10-100 μg subcutaneously into a selected rodent, for example, BALB/c mice or Lewis rats. DNA may be given intradermally (Raz et al., Proc. Natl. Acad. Sci. USA 91:9519, 1994) or intamuscularly (Wang et al., Proc. Natl. Acad. Sci. USA 90:4156, 1993); saline has been found to be a suitable diluent for DNA-based antigens. Ten days to three weeks days later, the immunized animals are boosted with additional immunogen and periodically boosted thereafter on a weekly, biweekly or every third week immunization schedule.
Serum samples are periodically taken by retro-orbital bleeding or tail-tip excision for testing by dot-blot assay (antibody sandwich), ELISA (enzyme-linked immunosorbent assay), immunoprecipitation, or other suitable assays, including FACS analysis. Following detection of an appropriate antibody titer, positive animals are given an intravenous injection of antigen in saline. Three to four days later, the animals are sacrificed, splenocytes harvested, and fused to a murine myeloma cell line (e.g., NS1 or preferably Ag 8.653 [ATCC CRL 1580]). Hybridoma cell lines generated by this procedure are plated in multiple microtiter plates in a selective medium (for example, one containing hypoxanthine, aminopterin, and thymidine, or HAT) to inhibit proliferation of non-fused cells, myeloma-myeloma hybrids, and splenocyte-splenocyte hybrids.
Hybridoma clones thus generated can be screened by ELISA for reactivity with RANKL, for example, by adaptations of the techniques disclosed by Engvall et al., Immunochem. 8:871 (1971) and in U.S. Pat. No. 4,703,004. A preferred screening technique is the antibody capture technique described by Beckman et al., J. Immunol. 144:4212 (1990). Positive clones are then injected into the peritoneal cavities of syngeneic rodents to produce ascites containing high concentrations (>1 mg/ml) of anti-RANK monoclonal antibody. The resulting monoclonal antibody can be purified by ammonium sulfate precipitation followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can also be used, as can affinity chromatography based upon binding to RANKL protein. Using the methods described herein to monitor the activity of the mAbs, both blocking (i.e., antibodies that bind RANKL and inhibit binding to RANK) and non-blocking (i.e., antibodies that bind RANKL and do not inhibit binding) are isolated.
EXAMPLE 11
This example demonstrates that RANK expression can be up-regulated. Human peripheral blood T cells were purified by flow cytometry sorting or by negative selection using antibody coated beads, and activated with anti-CD3 (OKT3, Dako) coated plates or phytohemagglutinin in the presence or absence of various cytokines, including Interleukin-4 (IL-4), Transforming Growth Factor-β (TGF-β) and other commercially available cytokines (IL1-α, IL-2, IL-3, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IFN-γ, TNF-α). Expression of RANK was evaluated by FACS in a time course experiment for day 2 to day 8, using a mouse monoclonal antibody mAb144 (prepared as described in Example 3), as shown in the table below. Results are expressed as ‘+’ to ‘++++’referring to the relative increase in intensity of staining with anti-RANK. Double labeling experiments using both anti-RANK and anti-CD8 or anti-CD4 antibodies were also performed.
TABLE 1
Upregulation of RANK by Cytokines
Cytokine (concentration)
Results:
IL-4 (50 ng/ml)
+
TGF-β (5 ng/ml)
+ to ++
IL-4 (50 ng/ml) + TGF-β (5 ng/ml)
++++
IL1-α (10 ng/ml)
−
IL-2 (20 ng/ml)
−
IL-3 (25 ng/ml)
−
IL-7 (20 ng/ml)
−
IL-8 (10 ng/ml)
−
IL-10 (50 ng/ml)
−
IL-12 (10 ng/ml)
−
IL-15 (10 ng/ml)
−
IFN-γ (100 U/ml)
−
Of the cytokines tested, IL-4 and TGF-β increased the level of RANK expression on both CD8+cytotoxic and CD4+helper T cells from day 4 to day 8. The combination of IL-4 and TGF-β acted synergistically to upregulate expression of this receptor on activated T cells. This particular combination of cytokines is secreted by suppresser T cells, and is believed to be important in the generation of tolerance (reviewed in Mitchison and Sieper, Z. Rheumatol. 54:141, 1995), implicating the interaction of RANK in regulation of an immune response towards either tolerance or induction of an active immune response.
EXAMPLE 12
This example illustrates the influence of RANK.Fc and hRANKL on activated T cell growth. The addition of TGFβ to anti-CD3 activated human peripheral blood T lymphocytes induces proliferation arrest and ultimately death of most lymphocytes within the first few days of culture. We tested the effect of RANK:RANKL interactions on TGFβ-treated T cells by adding RANK.Fc or soluble human RANKL to T cell cultures.
Human peripheral blood T cells (7×105 PBT) were cultured for six days on anti-CD3 (OKT3, 5 μg/ml) and anti-Flag (M1, 5 μg/ml) coated 24 well plates in the presence of TGFβ (1 ng/ml) and IL-4 (10 ng/ml), with or without recombinant FLAG-tagged soluble hRANKL (1 μg/ml) or RANK.Fc (10 μg/ml). Viable T cell recovery was determined by triplicate trypan blue countings.
The addition of RANK.Fc significantly reduced the number of viable T cells recovered after six days, whereas soluble RANKL greatly increased the recovery of viable T cells (FIG. 1). Thus, endogenous or exogenous RANKL enhances the number of viable T cells generated in the presence of TGFβ. TGFβ, along with IL-4, has been implicated in immune response regulation when secreted by the TH3/regulatory T cell subset. These T cells are believed to mediate bystander suppression of effector T cells. Accordingly, RANK and its ligand may act in an auto/paracrine fashion to influence T cell tolerance. Moreover, TGFβ is known to play a role in the evasion of the immune system effected by certain pathogenic or opportunistic organisms. In addition to playing a role in the development of tolerance, RANK may also play a role in immune system evasion by pathogens.
EXAMPLE 13
This example illustrates the influence of the interaction of RANK on CD11a+ dendritic cells (DC). Functionally mature dendritic cells (DC) were generated in vitro from CD34+ bone marrow (BM) progenitors. Briefly, human BM cells from normal healthy volunteers were density fractionated using Ficoll medium and CD34+cells immunoaffinity isolated using an anti-CD34 matrix column (Ceprate, CellPro). The CD34+ BM cells were then cultured in human GM-CSF (20 ng/ml), human IL-4 (20 ng/ml), human TNF-α (20 ng/ml), human CHO-derived Flt3L (FL; 100 ng/ml) in Super McCoy's medium supplemented with 10% fetal calf serum in a fully humidified 37° C. incubator (5% CO2) for 14 days. CD1a+, HLA-DR+ DC were then sorted using a FACStar Plus™, and used for biological evaluation of RANK
On human CD1a+ DC derived from CD34+bone marrow cells, only a subset (20-30%) of CD1a+ DC expressed RANK at the cell surface as assessed by flow cytometric analysis. However, addition of CD40L to the DC cultures resulted in RANK surface expression on the majority of CD1a+ DC. CD40L has been shown to activate DC by enhancing in vitro cluster formation, inducing DC morphological changes and upregulating HLA-DR, CD54, CD58, CD80 and CD86 expression
Addition of RANKL to DC cultures significantly increased the degree of DC aggregation and cluster formation above control cultures, similar to the effects seen with CD40L. Sorted human CD11a+ DC were cultured in a cytokine cocktail (GM-CSF, IL-4, TNF-α and FL), in cocktail plus CD40L (1 μg/ml), in cocktail plus RANKL (1 μg/ml), or in cocktail plus heat inactivated (ΔH) RANKL (1 μg/ml) in 24-well flat bottomed culture plates in 1 ml culture media for 48-72 hours and then photographed using an inversion microscope. An increase in DC aggregation and cluster formation above control cultures was not evident when heat inactivated RANKL was used, indicating that this effect was dependent on biologically active protein. However, initial phenotypic analysis of adhesion molecule expression indicated that RANKL-induced clustering was not due to increased levels of CD2, CD11a, CD54 or CD58.
The addition of RANKL to CD1a+ DC enhanced their allo-stimulatory capacity in a mixed lymphocyte reaction (MLR) by at least 3- to 10-fold, comparable to CD40L-cultured DC (FIG. 2). Allogeneic T cells (1×105) were incubated with varying numbers of irradiated (2000 rad) DC cultured as indicated above in 96-well round bottomed culture plates in 0.2 ml culture medium for four days. The cultures were pulsed with 0.5 mCi [3H]-thymidine for eight hours and the cells harvested onto glass fiber sheets for counting on a gas phase β counter. The background counts for either T cells or DC cultured alone were <100 cpm. Values represent the mean±SD of triplicate cultures. Heat inactivated RANKL had no effect. DC allo-stimulatory activity was not further enhanced when RANKL and CD40L were used in combination, possibly due to DC functional capacity having reached a maximal level with either cytokine alone. Neither RANKL nor CD40L enhanced the in vitro growth of DC over the three day culture period. Unlike CD40L, RANKL did not significantly increase the levels of HLA-DR expression nor the expression of CD80 or CD86.
RANKL can enhance DC cluster formation and functional capacity without modulating known molecules involved in cell adhesion (CD18, CD54), antigen presentation (HLA-DR) or costimulation (CD86), all of which are regulated by CD40/CD40L signaling. The lack of an effect on the expression of these molecules suggests that RANKL may regulate DC function via an alternate pathway(s) distinct from CD40/CD40L. Given that CD40L regulates RANK surface expression on in vitro-generated DC and that CD40L is upregulated on activated T cells during DC-T cell interactions, RANK and its ligand may form an important part of the activation cascade that is induced during DC-mediated T cell expansion. Furthermore, culture of DC in RANKL results in decreased levels of CD1b/c expression, and increased levels of CD83. Both of these molecules are similarly modulated during DC maturation by CD40L (Caux et al. J. Exp. Med. 180:1263; 1994), indicating that RANKL induces DC maturation.
Dendritic cells are referred to as “professional” antigen presenting cells, and have a high capacity for sensitizing MHC-restricted T cells. There is growing interest in using dendritic cells ex vivo as tumor or infectious disease vaccine adjuvants (see, for example, Romani, et al., J. Exp. Med., 180:83, 1994). Therefore, an agent such as RANKL that induces DC maturation and enhances the ability of dendritic cells to stimulate an immune response is likely to be useful in immunotherapy of various diseases.
EXAMPLE 14
This example describes the isolation of the murine homolog of RANK, referred to as muRANK. MuRANK was isolated by a combination of cross-species PCR and colony hybridization. The conservation of Cys residues in the Cys-rich pseudorepeats of the extracellular domains of TNFR superfamily member proteins was exploited to design human RANK-based PCR primers to be used on murine first strand cDNAs from various sources. Both the sense upstream primer and the antisense downstream primer were designed to have their 3′ ends terminate within Cys residues.
The upstream sense primer encoded nucleotides 272-295 of SEQ ID NO:5 (region encoding amino acids 79-86); the downstream antisense primer encoded the complement of nucleotides 409-427 (region encoding amino acids 124-130). Standard PCR reactions were set up and run, using these primers and first strand cDNAs from various murine cell line or tissue sources. Thirty reaction cycles of 94° C. for 30 seconds, 50° C. for 30 seconds, and 72° C. for 20 seconds were run. PCR products were analyzed by electrophoresis, and specific bands were seen in several samples. The band from one sample was gel purified and DNA sequencing revealed that the sequence between the primers was approximately 85% identical to the corresponding human RANK nucleotide sequence.
A plasmid based cDNA library prepared from the murine fetal liver epithelium line FLE18 (one of the cell lines identified as positive in the PCR screen) was screened for full-length RANK cDNAs using murine RANK-specific oligonucleotide probes derived from the murine RANK sequence determined from sequencing the PCR product. Two cDNAs, one encoding the 5′ end and one encoding the 3′ end of full-length murine RANK (based on sequence comparison with the full-length human RANK) were recombined to generate a full-length murine RANK cDNA. The nucleotide and amino acid sequence of muRANK are shown in SEQ ID Nos:14 and 15.
The cDNA encodes a predicted Type 1 transmembrane protein having 625 amino acid residues, with a predicted 30 amino acid signal sequence, a 184 amino acid extracellular domain, a 21 amino acid transmembrane domain, and a 390 amino acid cytoplasmic tail. The extracellular region of muRANK displayed significant amino acid homology (69.7% identity, 80.8% similarity) to huRANK. Those of skill in the art will recognize that the actual cleavage site can be different from that predicted by computer; accordingly, the N-terminal of RANK may be from amino acid 25 to amino acid 35.
Other members of the TNF receptor superfamily have a region of amino acids between the transmembrane domain and the ligand binding domain that is referred to as a ‘spacer’ region, which is not necessary for ligand binding. In muRANK, the amino acids between 197 and 214 are predicted to form such a spacer region. Accordingly, a soluble form of RANK that terminates with an amino acid in this region is expected to retain the ability to bind a ligand for RANK in a specific manner. Preferred C-terminal amino acids for soluble RANK peptides are selected from the group consisting of amino acids 214, and 197 of SEQ ID NO:14, although other amino acids in the spacer region may be utilized as a C-terminus.
EXAMPLE 15
This example illustrates the preparation of several different soluble forms of RANK and RANKL. Standard techniques of restriction enzyme cutting and ligation, in combination with PCR-based isolation of fragments for which no convenient restriction sites existed, were used. When PCR was utilized, PCR products were sequenced to ascertain whether any mutations had been introduced; no such mutations were found.
In addition to the huRANK/Fc described in Example 2, another RANK/Fc fusion protein was prepared by ligating DNA encoding amino acids 1-213 of SEQ ID NO:6, to DNA encoding amino acids 3-232 of the Fc mutein described previously (SEQ ID NO:8). A similar construct was prepared for murine RANK, ligating DNA encoding amino acids 1-213 of full-length murine RANK (SEQ ID NO:15) to DNA encoding amino acids 3-232 of the Fc mutein (SEQ ID NO:8).
A soluble, tagged, poly-His version of huRANKL was prepared by ligating DNA encoding the leader peptide from the immunoglobulin kappa chain (SEQ ID NO:16) to DNA encoding a short version of the FLAG™ tag (SEQ ID NO:17), followed by codons encoding Gly Ser, then a poly-His tag (SEQ ID NO:18), followed by codons encoding Gly Thr Ser, and DNA encoding amino acids 138-317 of SEQ ID NO:13. A soluble, poly-His tagged version of murine RANKL was prepared by ligating DNA encoding the CMV leader (SEQ ID NO:9) to codons encoding Arg Thr Ser, followed by DNA encoding poly-His (SEQ ID NO:18) followed by DNA encoding amino acids 119-294 of SEQ ID NO:11.
A soluble, oligomeric form of huRANKL was prepared by ligating DNA encoding the CMV leader (SEQ ID NO:9) to a codon encoding Asp followed by DNA ending a trimer-former “leucine” zipper (SEQ ID NO:19), then by codons encoding Thr Arg Ser followed by amino acids 138-317 of SEQ ID NO:13.
These and other constructs are prepared by routine experimentation. The various DNAs are then inserted into a suitable expression vector, and expressed. Particularly preferred expression vectors are those which can be used in mammalian cells. For example, pDC409 and pDC304, described herein, are useful for transient expression. For stable transfection, the use of CHO cells is preferred; several useful vectors are described in U.S. Ser. No. 08/785,150, now allowed, for example, one of the 2A5-3 λ-derived expression vectors discussed therein.
EXAMPLE 16
This example demonstrates that RANKL expression can be up-regulated on murine T cells. Cells were obtained from mesenteric lymph nodes of C57BL/6 mice, and activated with anti-CD3 coated plates, Concanavalin A (ConA) or phorbol myristate acetate in combination with ionomycin (anti-CD3: 500A2; Immunex Corporation, Seattle Wash.; ConA, PMA, ionomycin, Sigma, St. Louis, Mo.) substantially as described herein, and cultured from about 2 to 5 days. Expression of RANKL was evaluated in a three color analysis by FACS, using antibodies to the T cell markers CD4, CD8 and CD45RB, and RANK/Fc, prepared as described herein.
RANKL was not expressed on unstimulated murine T cells. T cells stimulated with either anti-CD3, ConA, or PMA/ionomycin, showed differential expression of RANKL: CD4+/CD45RBLo and CD4+/CD45RBHi cells were positive for RANKL, but CD8+cells were not. RANKL was not observed on B cells, similar to results observed with human cells.
EXAMPLE 17
This example illustrates the effects of murine RANKL on cell proliferation and activation. Various cells or cell lines representative of cells that play a role in an immune response (murine spleen, thymus and lymphnode) were evaluated by culturing them under conditions promoting their viability, in the presence or absence of RANKL. RANKL did not stimulate any of the tested cells to proliferate. One cell line, a macrophage cell line referred to as RAW 264.7 (ATCC accession number TIB 71) exhibited some signs of activation.
RAW cells constitutively produce small amounts of TNF-α. Incubation with either human or murine RANKL enhanced production of TNF-α by these cells in a dose dependent manner. The results were not due to contamination of RANKL preparations with endotoxin, since boiling RANKL for 10 minutes abrogated TNF-α production, whereas a similar treatment of purified endotoxin (LPS) did not affect the ability of the LPS to stimulate TNF-α production. Despite the fact that RANKL activated the macrophage cell line RAW T64.7 for TNF-α production, neither human RANKL nor murine RANKL stimulated nitric oxide production by these cells.
EXAMPLE 18
This example illustrates the effects of murine RANKL on growth and development of the thymus in fetal mice. Pregnant mice were injected with 1 mg of RANK/Fc or vehicle control protein (murine serum albumin; MSA) on days 13, 16 and 19 of gestation. After birth, the neonates continued to be injected with RANK/Fc intraperitoneally (IP) on a daily basis, beginning at a dose of 1 μg, and doubling the dose about every four days, for a final dosage of 4 μg. Neonates were taken at days 1, 8 and 15 post birth, their thymuses and spleens harvested and examined for size, cellularity and phenotypic composition.
A slight reduction in thymic size at day 1 was observed in the neonates born to the female injected with RANK/Fc; a similar decrease in size was not observed in the control neonates. At day 8, thymic size and cellularity were reduced by about 50% in the RANK/Fc-treated animals as compared to MSA treated mice. Phenotypic analysis demonstrated that the relative proportions of different T cell populations in the thymus were the same in the RANK/Fc mice as the control mice, indicating that the decreased cellularity was due to a global depression in the number of thymic T cells as opposed to a decrease in a specific population(s). The RANK/Fc-treated neonates were not significantly different from the control neonates at day 15 with respect to either size, cellularity or phenotype of thymic cells. No significant differences were observed in spleen size, cellularity or composition at any of the time points evaluated. The difference in cellularity on day 8 and not on day 15 may suggest that RANK/Fc may assert its effect early in thymic development.
EXAMPLE 19
This example demonstrates that the C-terminal region of the cytoplasmic domain of RANK is important for binding of several different TRAF proteins. RANK contains at least two recognizable PXQX(X)T motifs that are likely TRAF docking sites. Accordingly, the importance of various regions of the cytoplasmic domain of RANK for TRAF binding was evaluated. A RANK/GST fusion protein was prepared substantially as described in Smith and Johnson, Gene 67:31 (1988), and used in the preparation of various truncations as described below.
Comparison of the nucleotide sequence of murine and human RANK indicated that there were several conserved regions that could be important for TRAF binding. Accordingly, a PCR-based technique was developed to facilitate preparation of various C-terminal truncations that would retain the conserved regions. PCR primers were designed to introduce a stop codon and restriction enzyme site at selected points, yielding the truncations described in Table 2 below. Sequencing confirmed that no undesired mutations had been introduced in the constructs.
Radio-labeled (35S-Met, Cys) TRAF proteins were prepared by in vitro translation using a commercially available reticulocyte lysate kit according to manufacturer's instructions (Promega). Truncated GST fusion proteins were purified substantially as described in Smith and Johnson (supra). Briefly, E. coli were transfected with an expression vector encoding a fusion protein, and induced to express the protein. The bacteria were lysed, insoluble material removed, and the fusion protein isolated by precipitation with glutathione-coated beads (Sepahrose 4B, Pharmacia, Uppsala Sweden)
The beads were washed, and incubated with various radiolabeled TRAF proteins. After incubation and wash steps, the fusion protein/TRAF complexes were removed from the beads by boiling in 0.1% SDS+β-mercaptoethanol, and loaded onto 12% SDS gels (Novex). The gels were subjected to autoradiography, and the presence or absence of radiolabeled material recorded. The results are shown in Table 2 below.
TABLE 2
Binding of Various TRAF Proteins
to the Cytoplasmic Domain of RANK
C terminal
E206-
E206-
E206-
E206-
Full
Truncations:
S339
Y421
M476
G544
length
TRAF1
−
−
−
−
++
TRAF2
−
−
−
−
++
TRAF3
−
−
−
−
++
TRAF4
−
−
−
−
−
TRAF5
−
−
−
−
+
TRAF6
−
+
+
+
++
These results indicate that TRAF1, TRAF2, TRAF3, TRAF 5 and TRAF6 bind to the most distal portion of the RANK cytoplasmic domain (between amino-acid G544 and A616). TRAF6 also has a binding site between S339 and Y421. In this experiment, TRAF5 also bound the cytoplasmic domain of RANK.
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13424179
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immunex corporation
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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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530/350
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Mar 31st, 2022 02:17PM
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Mar 31st, 2022 02:17PM
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Amgen
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:amgn
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Amgen
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Mar 13th, 2007 12:00AM
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Oct 15th, 2004 12:00AM
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https://www.uspto.gov?id=US07189521-20070313
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Claudin polypeptides, polynucleotides, and methods of making and use thereof
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This invention relates to new members of the human Claudin polypeptide family, to methods of making such polypeptides, and to methods of using them to treat Claudin-associated conditions and to identify agents that alter Claudin polypeptide activities.
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7189521
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1. A method for identifying an agent that modulates the polypeptide having SEQ ID NO: 6, comprising mixing a test agent with the said polypeptide and determining whether there is a change in the activity of the polypeptide in the presence of the test agent relative to the activity of the polypeptide in the absence of the test agent, wherein the change of the activity of the polypeptide comprises an assessment of transcription and/or translation of skin differentiation marker, thereby identifying an agent that modulates polypeptide activity.
2. The method of claim 1, wherein the test agent is selected from the group consisting of small molecules, peptides, and antibodies.
3. The method of claim 1, wherein the skin differentiation marker is selected from the group consisting of:
a) filaggrin,
b) profilaggrin,
c) involucrin, and
d) keratin markers.
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3
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This application is a continuation of International Application Serial No. PCT/US03/16052, filed May 20, 2003; which designates the United States and claims the benefit under 35 U.S.C. 119(e) of U.S. provisional application Ser. No. 60/382,040, filed May 20, 2002; all of which are incorporated in their entirety by reference herein.
FIELD OF THE INVENTION
This invention relates to novel human and murine polypeptides of the Claudin polypeptide family, and to methods of making and using them.
BACKGROUND
Tight junctions, which are also called “zona occludens”, form a regulated, semipermeable barrier in the intercellular spaces within sheets of epithelial or endothelial cells. The properly regulated formation of tight junctions is an important aspect of the normal development of tissues such as the skin, and maintenance of these junctions may assist in suppressing the formation and spread of tumors. Inadequate or improperly regulated epithelial or endothelial barrier function contributes to the initiation, maintenance, and exacerbation of inflammation in tissues such as the gut, lungs, and the like. Tight junctions also form a “fence” separating the apical and basolateral regions of these cells' membranes, allowing the establishment of different physiological environments on the opposite sides of a cell sheet, such as the different physiological environments required for transport of materials across the intestinal epithelium. It has also been proposed that tight junctions contain aqueous pores, with paracellular transport between the cells of an epithelial or endothelial sheet occurring through these pores. In order to develop more effective treatments for conditions involving disruption of epithelial or endothelial barrier function or unregulated transport across the epithelium or endothelium, such as inflammatory bowel disease or skin disorders such as psoriasis or contact dermatitis, the identification of proteins that play a role in tight junctions is critical in understanding and treating such diseases and disorders.
SUMMARY OF THE INVENTION
The invention is based upon the discovery of a new member of the Claudin polypeptide family, Claudin-23.
The invention provides a substantially purified or isolated polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of: (a) an amino acid sequence as set forth in SEQ ID NO:6; (b) a Claudin-23 fragment, wherein the Claudin-23 fragment consists of an extracellular loop of a Claudin-23 polypeptide; (c) a Claudin-23 fragment, wherein the Clauldin-23 fragment consists of a cytoplasmic domain of a Claudin-23 polypeptide; (d) a Claudin-23 fragment, wherein the Claudin-23 fragment consists of at least 20 contiguous amino acid of SEQ ID NO:6 or 8 and has Claudin polypeptide activity; and (e) a Claudin-23 polypeptide variant, wherein the Claudin-23 variant is at least 80%, at least 90%, at least 95%, at least 97.5%, at least 99% or at least 99.5% identical to SEQ ID NO:6 or 8 and wherein the Claudin-23 variant has Claudin-23 polypeptide activity. In one embodiment, the polypeptides of the invention consist essentially of specified portions of SEQ ID NO:6 or 8, such as amino acids 31 through 76 of SEQ ID NO:6 or amino acids 138 through 159 of SEQ ID NO:6, or variants thereof, and do not comprise any of the polypeptides disclosed in WO 02/099062, WO 03/008553, or WO 03/023013. In further embodiments, the polypeptides of the invention consist essentially of the amino acid sequence of a fragment of a Claudin-23 polypeptide, wherein the fragment consists of, in N-to-C order, a first transmembrane domain, a first extracellular loop, a second transmembrane domain, an intracellular loop, a third transmembrane domain, a second extracellular loop, and a fourth transmembrane domain of a Claudin-23 polypeptide of a Claudin-23 polypeptide. In such embodiments, each of the first and third transmembrane domains can be independently selected from the group consisting of amino acids 5 through 27 of SEQ ID NO:6 and amino acids 112 through 134 of SEQ ID NO:6, or the corresponding portions of SEQ ID NO:8, or variants thereof; and each of the second and fourth transmembrane domains can be independently selected from the group consisting of amino acids 77 through 99 of SEQ ID NO:6 and amino acids 160 through 182 of SEQ ID NO:6, or the corresponding portions of SEQ ID NO:8, or variants thereof.
The invention also provides an isolated polynucleotide selected from the group consisting of a polynucleotide comprising, consisting essentially of, or consisting of: (a) SEQ ID NO:5; (b) SEQ ID NO:5, wherein T can also be U; (c) a polynucleotide that encode an amino acid sequence as set forth in SEQ ID NO:6 or 8; (d) a fragment of (a), (b), or (c) that is at least 15 consecutive bases in length and that selectively hybridize to DNA which encodes a polypeptide of SEQ ID NO:6 or 8; and (e) a fragment of SEQ ID NO:5, wherein the fragment encodes a polypeptide having Claudin-23 polypeptide activity. In another aspect the invention provides an isolated polynucleotide that hybridizes under moderate to high stringency conditions to a polynucleotide consisting of a sequence as set forth in SEQ ID NO:5. In yet a further embodiment, the isolated polynucleotide comprises a Claudin-23 polynucleotide variant, wherein the variant shares nucleotide sequence identity with a nucleotide sequence as set forth by SEQ ID NO:5, wherein the percent nucleotide sequence identity is selected from the group consisting of: at least 90%, at least 95%, at least 97.5%, at least 99%, and at least 99.5%. In one embodiment, the polynucleotides of the invention consist essentially of specified portions of SEQ ID NO:5, or variants thereof, and do not comprise any of the polynucleotides disclosed in WO 02/099062, WO 03/008553, or WO 03/023013.
The invention also provides an expression vector comprising a polynucleotide of the invention, as well as a recombinant host cell transfected or transformed with the expression vector or a polynucleotide of the invention.
The invention further provides a process for producing a polypeptide, comprising culturing a recombinant host cell of the invention under conditions promoting expression of the polypeptide from the polynucleotide. In a further embodiment, the process further includes purifying said polypeptide.
The invention also provides a substantially purified antibody that binds to a polypeptide consisting of a sequence as set forth in SEQ ID NO:6 or 8. The antibody may be monoclonal, polyclonal, human, or humanized.
The invention provides a method of designing an inhibitor of a Claudin-23 polypeptide of the invention. The method includes determining a three-dimensional structure of the polypeptide, analyzing the three-dimensional structure for likely binding sites of a ligand or substrate, synthesizing a molecule is predicted to interact with the binding site, substrate, or ligand, and determining the polypeptide-inhibiting activity of the molecule.
The invention provides a method for identifying an agent that modulates Claudin-23 polypeptide activity comprising mixing a test agent with a Claudin-23 polypeptide and determining Claudin-23 polypeptide activity in the presence and absence of the test agent, wherein a difference in Claudin-23 polypeptide activity in the presence of the test agent relative to that in the absence of the test agent is indicative of an agent that modulates Claudin-23 polypeptide activity. In this manner both inhibitors (antagonists) and activators (agonists) of Claudin-23 polypeptide activity may be identified. In one embodiment of these methods of the invention, the determination of Claudin-23 polypeptide activity comprises an assessment of transcription and/or translation of skin differentiation markers such as, but not limited to, filaggrin, profilaggrin, involucrin, and keratin markers (such as K1, K2, K2e, K2p, K4, K5, K6, K8, K9, K10, K13, K14, K16, K17, K18, K19, and the like) by conventional techniques, for example the use of differentiation marker-specific probes.
In another aspect of the invention, a method is provided for identifying peptide agonists and antagonists of the cytokine polypeptides of the invention, the method comprising selecting at least one peptide that binds to a polypeptide of the invention, wherein the peptide is selected in a process comprising one or more techniques selected from yeast-based screening, rational design, protein structural analysis, screening of a phage display library, an E. coli display library, a ribosomal library, an RNA-peptide library, and a chemical peptide library. In further aspects of the invention, the peptide is selected from a plurality of randomized peptides.
Also provided by the invention is a method for increasing tight junction formation activity or epithelial or endothelial barrier function activity in a cell or subject comprising providing a Claudin-23 polypeptide of the invention, or an agonist thereof, to the cell or subject. In one embodiment of the invention, the agonist is an agonistic antibody or a peptide.
The invention further provides a method for decreasing tight junction formation activity or epithelial or endothelial barrier function activity in a cell or subject comprising providing an antagonist of a Claudin-23 polypeptide to the cell or subject. In one embodiment, the antagonist is an antibody or a soluble Claudin-23 domain.
The invention provides a method for treating an epithelial or endothelial barrier function condition in a subject comprising administering a Claudin-23 polypeptide, or an agonist thereof, to the subject. In one embodiment, the epithelial or endothelial barrier function condition is a disorder of cells derived from keratinocytes of the epithelium or of the hair follicle. In another embodiment, the epithelial or endothelial barrier function condition is selected from the group consisting of inflammation, asthma, allergy, metastasis of cancer cells, ion transport disorders such as magnesium transport defects in the kidney, psoriasis and other inflammatory dermatoses, hyperproliferative skin disorder, hair loss, and inflammatory bowel disease.
DETAILED DESCRIPTION OF THE INVENTION
The Claudin polypeptides are a related group of “tetraspan” polypeptides, polypeptides having four membrane-spanning or transmembrane domains that are associated with cellular tight junctions. Claudin family polypeptides are expressed in epithelial cells and/or endothelial cells throughout development, with individual members of the Claudin polypeptide family being expressed in different tissues. The physiological functions associated with a particular Claudin polypeptide are related to the functions performed by the particular tissue(s) in which it is expressed.
Because of their roles in tight junction formation, epithelial and endothelial barrier function, ion transport, and viral protein, enterotoxin, or allergen binding, Claudin polypeptides are associated with conditions involving unregulated or improperly regulated transport across the epithelium or endothelium such as inflammation, asthma, allergy, metastasis of cancer cells, and ion transport disorders such as magnesium transport defects in the kidney. In addition, because a Claudin polypeptide expressed in neural cells has been shown to be required for formation of the myelin sheath in oligodendrocytes, Claudin polypeptides are associated with demyelination conditions such as multiple sclerosis (MS), autoimmune encephalomyelitis, optic neuritis, progressive multifocal leukoencephalopathy (PML), and the like.
Characteristics and activities of the Claudin polypeptide family are described further in the following references:. Fujitaab K et al., 2000, Clostridium perfringens enterotoxin binds to the second extracellular loop of claudin-3, a tight junction integral membrane protein, FEBS Lett. 476: 258–261; Kinugasa T et al., 2000, Claudins regulate the intestinal barrier in response to immune mediators, Gastroenterology 118: 1001–1011; Tsukita S and Furuse M, 2000, Pores in the wall: claudins constitute tight junction strands containing aqueous pores, J Cell Biol. 149: 13–16; Bronstein J M et al., 2000, Involvement of OSP/claudin-11 in oligodendrocyte membrane interactions: role in biology and disease, J Neurosci Res. 59: 706–711; Itoh M et al., 1999, Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins, J Cell Biol. 147: 1351–1363; Furuse M et al., 1999, Manner of interaction of heterogeneous claudin species within and between tight junction strands, J Cell Biol. 147: 891–903; Morita K et al., 1999, Endothelial claudin: claudin-5/TMVCF constitutes tight junction strands in endothelial cells, J Cell Biol. 147: 185–194; Kubota K et al., 1999, Ca(2+)-independent cell-adhesion activity of claudins, a family of integral membrane proteins localized at tight junctions, Curr Biol. 9: 1035–1038; Wan H et al., 1999, Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions, J Clin Invest. 104: 123–133; Simon D B et al., 1999, Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorption, Science 285: 103–106; Morita K et at., 1999, Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands, Proc Natl Acad Sci USA. 96: 511–516; Furuse M et al., 1998, A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts, J Cell Biol. 143: 391–401; Furuse M et al., 1998, Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin, J Cell Biol. 141: 1539–1550; all of which are incorporated by reference herein.
The invention provides polypeptides that are members of the Claudin polypeptide family, human Claudin-23 and murine Claudin-23. Typical structural elements common to members of the Claudin polypeptide family include a non-cleaved signal peptide sequence, four membrane-spanning domains, two extracellular loops formed by the membrane-spanning domains, and a cytoplasmic tail at the C-terminus of the polypeptide. Both the N-terminus and the C-terminus of the polypeptide are intracellular. The two extracellular loop domains of Claudin polypeptides are located between the first and second transmembrane domains and between the third and fourth transmembrane domains of the polypeptide, respectively. The extracellular loop domains of Claudin polypeptides may contribute to tight junction formation, which is an important aspect of both the barrier function and the ion transport function of Claudin polypeptides, and/or act as a receptor for viral proteins, enterotoxins, or allergens. The tight junction formation activities of the Claudin polypeptide family are believed to occur through homotypic interactions with the extracellular loops of the same Claudin polypeptide expressed on neighboring epithelial or endothelial cells, or heterotypic interactions with the extracellular loops of other Claudin family members or other non-Claudin polypeptides. In addition, there is evidence that the biological effects of Claudin polypeptides involve a requirement for Claudin polypeptides, and particularly their most N-terminal extracellular domain, in the processing of matrix metalloproteinases to their active form (Miyamori et al., 2001, J Biol Chem 276: 2804–28211). The short region between the second and third transmembrane domains of the polypeptide is intracellular. The cytoplasmic tail domain of Claudin polypeptides extends from the fourth transmembrane domain to the C-terminus of the polypeptide. The cytoplasmic tail domain is thought to be involved in interactions with other tight-junction-associated proteins such as the ZO (zona occludens) family of proteins. These interactive activities of Claudin polypeptides are thought to involve PDZ-domain-containing polypeptides, with a PDZ domain binding to the C-terminal residues of the cytoplasmic tail domain of a Claudin polypeptide; association of PDZ-containing polypeptides may then result in oligomerization of Claudin polypeptides.
The amino acid sequences of human Claudin-23 (SEQ ID NO:6) and murine Claudin-23 (SEQ ID NO:8) contain the structural features of Claudin polypeptides. Human Claudin-23 contains a first transmembrane (TM) domain from about amino acid 5 to about 27 of SEQ ID NO:6. Consistent with the other Claudin family members the first transmembrane domain is inserted into the cell membrane with the very N-terminal end of the Claudin polypeptide (in this case, including amino acids from about 1 to 4 of SEQ ID NO:6) located inside the cell. Human Claudin-23 is also predicted to have a second TM domain comprising from about amino acids 77 to 99 of SEQ ID NO:6, a third TM domain comprising from about amino acids 112 to 134 of SEQ ID NO:6, and a fourth TM domain comprising from about amino acids 160 to 182 of SEQ ID NO:6. Hidden Markov Model (HMM) analysis predicts similar transmembrane domains: from about amino acids 5 through 27 of SEQ ID NO:6; from about amino acids 78 through 100 of SEQ ID NO:6; from about amino acids 112 through 134 of SEQ ID NO:6; and from about amino acids 161 through 183 of SEQ ID NO:6. These predicted locations for the Human Claudin-23 TM domains also correspond well with those identified by Morita et al. (1999, Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands, Proc Natl Acad Sci USA. 96: 511–516) for other members of the Claudin polypeptide family. Based on the alignments with other family members and by reference to FIG. 1 of Morita et al., the predicted locations for the four TM domains of Human Claudin-23 place the first extracellular loop of Human Claudin-23 as beginning approximately around amino acid 28 to amino acid 31 of SEQ ID NO:6 and extending to approximately amino acid 76 of SEQ ID NO:6, and the second extracellular loop of Human Claudin-23 as beginning approximately around amino acid 135 to amino acid 138 of SEQ ID NO:6 and extending to approximately amino acid 159 of SEQ ID NO:6. The intracellular sequence between the second and third TM domains begins at approximately amino acid 100 to 103 of SEQ ID NO:6 and extends to approximately amino acid 111 of SEQ ID NO:6. The cytoplasmic tail domain of Human Claudin-23 begins approximately around amino acid 182 to amino acid 184 (e.g., about amino acid 183) of SEQ ID NO:6 and extends to the predicted C-terminus of SEQ ID NO:6 at amino acid 292.
A Hidden Markov Model (HMM) analysis of murine Claudin-23 (SEQ ID NO:8) predicts the following transmembrane domains: from about amino acids 5 through 27 of SEQ ID NO:8; from about amino acids 79 through 101 of SEQ ID NO:8; from about amino acids 113 through 135 of SEQ ID NO:8; and amino acids 161 through 183 of SEQ ID NO:8. These predicted locations for the four TM domains of murine Claudin-23 place the first extracellular loop of murine Claudin-23 as beginning approximately around amino acid 28 to amino acid 31 of SEQ ID NO:8 and extending to approximately amino acid 78 of SEQ ID NO:8, and the second extracellular loop of murine Claudin-23 as beginning approximately around amino acid 136 to amino acid 138 of SEQ ID NO:8 and extending to approximately amino acid 160 of SEQ ID NO:8. The intracellular sequence between the second and third TM domains begins at approximately amino acid 102 to 105 of SEQ ID NO:8 and extends to approximately amino acid 112 of SEQ ID NO:8. The cytoplasmic tail domain of murine Claudin-23 begins approximately around amino acid 183 to amino acid 185 (e.g., about amino acid 184) of SEQ ID NO:8 and extends to the predicted C-terminus of SEQ ID NO:8 at amino acid 296.
The skilled artisan will recognize that the boundaries of these regions of these polypeptides are approximate and that the precise boundaries of such domains, as for example the boundaries of the transmembrane domains, may differ in 1–5 amino acids from those predicted herein for human and murine claudin-23.
The most C-terminal residues of the cytoplasmic tail domains of Claudin polypeptides are believed to be involved with interaction with PDZ-domain-containing proteins, such that substitutions of those residues are likely be associated with an altered PDZ domain recognition pattern or binding function, or with a lack of that function, for the polypeptide. Human Claudin-23 has an -Asp-Ser-Asp-Leu-COOH amino acid sequence at its C-terminus. Although this does not match exactly the C-terminal amino acid sequences of other Claudin family polypeptides, it is consistent in most respects with the consensus requirements for “Group 1” polypeptides that interact with PDZ domains (Cowburn D, 1997, Curr Opin Struct Biol 7: 835–838; which is incorporated by reference herein): Val/Ile/Leu/Met as the C-terminal residue, with preference for Thr/Ser/Tyr at the -2 position and Glu at the −3 position. Human Claudin-23 has Leu as the C-terminal residue and Asp, having an acidic side chain like Glu, at the −4 position. Human Claudin-23 may interact with PDZ-domain-containing polypeptides, although they may interact with different subsets of PDZ domains than other Claudin family members, or they may exhibit different kinetics or affinity in their interactions with PDZ-domain-containing polypeptides.
Biological Activities and Functions of Claudin Polypeptides of the Invention
As used herein, “Claudin polypeptides of the invention” includes human Claudin-23 (SEQ ID NO:6) and species homologues such as murine Claudin-23 (SEQ ID NO:8), and variants and fragments of these Claudin polypeptides and their species homologues. Claudin polypeptides of the invention have biological activities and functions that are consistent with those of the other Claudin family polypeptides. Polypeptides of the Claudin family are expressed in cell types including epithelial and endothelial cells throughout development. Typical biological activities or functions associated with this family of polypeptides are tight junction formation, epithelial or endothelial barrier function, ion transport, viral protein binding, homotypic or heterotypic binding, and binding PDZ domain binding. Polypeptides having tight junction formation activity bind to other tight-junction-associated molecules to form tight junction structures that regulate epithelial or endothelial barrier function and paracellular transport. The tight junction formation activity is associated with the extracellular loops and, at least under certain conditions, with the cytoplasmic tail domain of Claudin polypeptides. Thus, for uses requiring tight junction formation activity, human Claudin-23 polypeptides include those having the extracellular loop domains and exhibiting tight junction formation activities such as epithelial or endothelial barrier function, paracellular ion transport, or viral protein binding. Claudin polypeptides of the invention further include oligomers or fusion polypeptides comprising at least one extracellular loop or cytoplasmic tail domain of one or more Claudin polypeptides of the invention, and fragments of any of these polypeptides that have tight junction formation activity. The tight junction formation activity of human Claudin-23 and other Claudin family polypeptides may be determined, for example, by introducing Claudin polypeptides into cells that do not normally form tight junctions, such a L fibroblasts, along with occludin or any other polypeptide that the Claudin polypeptide needs to interact with in the formation of tight junctions, then visualizing the resulting tight junction structures by electron microscopy or immunofluorescence methods (see for example Furuse M et al., 1998, A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts, J Cell Biol. 143: 391–401). Alternatively, the paracellular ion transport activity of human Claudin-23 and other Claudin family polypeptides may be assayed by electrophysiology or through the use of luminescent ion indicator molecules such as aequorin, preferably in micellular preparations from cells expressing Claudin polypeptides.
Claudin polypeptides such as human Claudin-23 have homotypic binding, heterotypic binding, viral protein binding, and/or enterotoxin binding activity; each of these binding activities is associated with the extracellular loop domains of Claudin polypeptides. Thus, for uses requiring homotypic binding, heterotypic binding, viral protein binding, and/or enterotoxin binding activity, Claudin polypeptides of the invention will include those having at least one extracellular loop domain and exhibiting at least one such binding activity. Claudin polypeptides also have PDZ domain-binding activity associated with the cytoplasmic tail domains of Claudin polypeptides. Thus, for uses requiring PDZ domain-binding activity, Claudin polypeptides of the invention will include those having a cytoplasmic tail domain and exhibiting PDZ domain-binding activity. Claudin polypeptides of the invention further include oligomers or fusion polypeptides comprising at least one extracellular loop domain and/or cytoplasmic tail domain of one or more Claudin polypeptides of the invention, and fragments of any of these polypeptides that have homotypic binding, heterotypic binding, viral protein binding, enterotoxin binding, and/or PDZ domain binding activity. The binding activity or activities of human Claudin-23 and species homologues and other Claudin family polypeptides may be determined, for example, in a yeast two-hybrid assay, or in an in vitro assay that measures binding between a Claudin polypeptide and one of its homotypic, heterotypic, viral protein, enterotoxin, and/or PDZ-domain-containing binding partners, where either the Claudin polypeptide or its binding partner is labeled with a radioactive, fluorescent, or bioluminescent protein such that binding can be detected.
The term “human Claudin polypeptide activity,” as used herein, includes any one or more of the following: tight junction formation, epithelial or endothelial barrier function, and ion transport activity; homotypic binding, heterotypic binding, viral protein binding, enterotoxin binding, and PDZ domain binding activity; as well as the ex vivo and in vivo activities of Claudin polypeptides of the invention. The degree to which Claudin polypeptides of the invention and fragments and other derivatives of these polypeptides exhibit these activities can be determined by standard assay methods. Exemplary assays are disclosed herein; those of skill in the art will appreciate that other, similar types of assays can be used to measure the biological activities of Claudin polypeptides of the invention and other Claudin family members.
One aspect of the biological activity of Claudin polypeptides including human Claudin-23 is the ability of members of this polypeptide family to bind particular binding partners such homotypic and heterotypic polypeptides, viral proteins, enterotoxins, and PDZ-domain-containing polypeptides, with the extracellular loop domains binding, for example, to homotypic polypeptides, and the cytoplasmic tail domain binding to PDZ-domain-containing polypeptides. The term “binding partner,” as used herein, includes ligands, receptors, substrates, antibodies, other Claudin polypeptides, the same human Claudin-23 polypeptide (in the case of homotypic interactions), and any other molecule that interacts with a human Claudin-23 polypeptide through contact or proximity between particular portions of the binding partner and the human Claudin-23 polypeptide. Binding partners for Claudin polypeptides of the invention are also expressed by epithelial and endothelial cells, as Claudin polypeptides expressed in epithelial cells bind to molecules on neighboring epithelial cells to form tight junctions, and Claudin polypeptides expressed in endothelial cells bind to molecules on neighboring endothelial cells. Therefore, the interactions between Claudin polypeptides of the invention and their binding partners are involved in mediating interactions between adjacent epithelial cells, and interactions between adjacent endothelial cells. Because the extracellular loop domains of Claudin polypeptides of the invention bind to homotypic or heterotypic polypeptides, a derivative polypeptide comprising one or more extracellular loop domains when expressed as a separate fragment from the rest of a human Claudin-23 polypeptide, or as a soluble polypeptide, fused for example to an immunoglobulin Fc domain, is expected to disrupt the binding of Claudin polypeptides of the invention to its binding partners. By binding to one or more binding partners, the separate extracellular loop domain(s) polypeptide likely prevents binding by the native human Claudin-23 polypeptide(s), and so acts in a dominant negative fashion to inhibit the biological activities mediated via binding of Claudin polypeptides of the invention to homotypic or heterotypic polypeptides. The biological activities and partner-binding properties of human Claudin-23 and other Claudin family polypeptides may be assayed by standard methods and by those assays described herein.
Polypeptides of the Claudin family such as human Claudin-23 are involved in epithelial or endothelial barrier function and transport diseases or conditions, that share as a common feature abnormal tight junction formation or improperly regulated tight junction function (i.e. abnormal epithelial or endothelial barrier function) in their etiology. More specifically, the following conditions involving epithelial or endothelial barrier function and/or binding to Claudin polypeptides are those that are known or are likely to involve the biological activities of Claudin polypeptides: inflammation (e.g., psoriasis and other inflammatory dermatoses), asthma, allergy, cell proliferative disorders (e.g., hyperproliferative skin disorders including skin cancer), metastasis of cancer cells, ion transport disorders such as magnesium transport defects in the kidney, inflammatory bowel disease, and exposure to Clostridium perfringens enterotoxin (CPE). In addition, because a Claudin polypeptide expressed in neural cells has been shown to be required for formation of the myelin sheath in oligodendrocytes, Claudin polypeptides are associated with demyelination conditions such as multiple sclerosis (MS), autoimmune encephalomyelitis, optic neuritis, and progressive multifocal leukoencephalopathy (PML). Also, diseases that are promoted by one or more of the conditions above may involve Claudin polypeptides, directly or indirectly. For example, susceptibility to sudden infant death syndrome (SIDS) has been associated with exposure to CPE. As described in Example 1 below, Claudin-23 has been shown to be downregulated in mutant mice having defects of skin development, specifically differentation of keratinocyte-derived cells of the epithelium and the hair follicle. Therefore, Claudin-23 is involved in conditions and disorders affecting the skin epithelium and/or the hair follicle, for example conditions and disorders in which skin epithelial barrier function is abnormal or misregulated. Blocking or inhibiting the interactions between Claudin polypeptides of the invention and their substrates, ligands, receptors, binding partners, and or other interacting polypeptides is an aspect of the invention and provides methods for treating or ameliorating these diseases and conditions through the use of inhibitors of human Claudin-23 activity. Examples of such inhibitors or antagonists are described in more detail below. For certain conditions involving a defect in epithelial or endothelial barrier function or ion transport associated with too little human Claudin-23 activity, methods of treating or ameliorating these conditions comprise increasing the amount or activity of Claudin polypeptides of the invention by providing isolated Claudin polypeptides of the invention or active fragments or fusion polypeptides thereof, or by providing compounds (agonists) that activate endogenous or exogenous Claudin polypeptides of the invention. Additional uses for Claudin polypeptides of the invention and agonists and antagonists thereof include diagnostic reagents for epithelial or endothelial transport diseases; research reagents for investigation of occludin or ZO family polypeptides and the formation of tight junctions; purification, processing, and preservation of occludin or ZO polypeptides or of epithelial or endothelial cells; or as a carrier or targeting molecule for the delivery of therapeutic agents, particularly in view of the role of Claudins in the tight junctions of the blood-brain barrier (Kniesel U and Wolburg H. 2000, Cell Mol Neurobiol. 20: 57–76, which is incorporated by reference herein).
In one embodiment, a Claudin-23 polypeptide or polynucleotide plays a role as a tumor suppressor. For example, where there is a decrease in the amount or activity of Claudin-23 in a subject. the invention also provides methods of treating such a disorder characterized by a decrease in Claudin-23, and of preventing, reducing the risk of, ameliorating, or treating tumor formation or metastasis, comprising administering to the subject a therapeutically effective amount of a pharmaceutically acceptable solution containing an agonist of Claudin-23. The term “agonist,” as used herein, refers to an agent that causes a change in Claudin-23 that increases a biological activity associated with Claudin-23. An agonist includes molecules that (1) increase the bioavailability of a Claudin-23 polypeptide, and/or (2) increase the expression of a Claudin-23 polynucleotide, and/or (3) simulate a biological activity of a Claudin-23 gene product. Such a molecule can include a polynucleotide, polypeptide, peptidomimetic, or small molecule. In one embodiment, a vector or cell comprising a recombinant polynucleotide encoding a Claudin-23 polypeptide is administered to the subject such that the polynucleotide is expressed thereby increasing the bioavailability of a Claudin-23 polypeptide. Such vectors may be administered to a subject in vivo through, for example, intravenous administration, or via ex vivo transfection of a subject's wherein the cells are infused into the subject. Such cells are typically homologous cells derived from tissue or serum of the subject, or they may include heterologous cells.
As demonstrated in the Examples below, Claudin-23 is found to be expressed in a number of inflammatory cells including dendritic cells and thus may play a role in inflammation as an immune system modulator. Accordingly, agonists and antagonists of Claudin-23 can be used to modulate the immune system.
Claudin Polypeptides of the Invention
A human Claudin-23 polypeptide is a polypeptide that (a) has a sequence as set forth in SEQ ID NO:6; (b) shares a sufficient degree of amino acid identity or similarity to a Claudin-23 polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:6 or 7; (c) is identified by those of skill in the art as a polypeptide likely to share particular structural domains with a Claudin-23 polypeptide of SEQ ID NO:6 or 7; (d) has biological activities in common with a Claudin polypeptide; and/or (e) binds to antibodies that also specifically bind to a Claudin-23 polypeptide having a sequence as set forth in SEQ ID NO:6 or 7. Claudin polypeptides of the invention may be isolated from naturally occurring sources, or be recombinantly produced such that a recombinant Claudin polypeptide has the same structure as naturally occurring Claudin polypeptides, or may be produced to have structures that differ from naturally occurring Claudin polypeptides. Polypeptides derived from any human Claudin-23 polypeptide by any type of alteration (for example, but not limited to, insertions, deletions, or substitutions of, for example, 1–10 or more amino acids; changes in the state of glycosylation of the polypeptide; refolding or isomerization to change its three-dimensional structure or self-association state; and changes to its association with other polypeptides or molecules) are also Claudin polypeptides of the invention. Therefore, the polypeptides provided by the invention include polypeptides characterized by amino acid sequences similar to those of the Claudin polypeptides of the invention described herein, but into which modifications are naturally provided or deliberately engineered. A polypeptide that shares biological activities in common with Claudin polypeptides of the invention is a polypeptide having Claudin-23 activity. Examples of biological activities exhibited by members of the Claudin polypeptide family include, without limitation, tight junction formation, epithelial or endothelial barrier function, ion transport, homotypic or heterotypic binding, viral protein binding, and enterotoxin binding.
“An isolated polypeptide consisting essentially of an amino acid sequence” means that the polypeptide may have, in addition to said amino acid sequence, additional material covalently linked to either or both ends of the polypeptide, said additional material between 1 and 10,000 additional amino acids covalently linked to either end, each end, or both ends of polypeptide; or between 1 and 1,000 additional amino acids covalently linked to either end, each end, or both ends of the polypeptide; or between 1 and 100 additional amino acids covalently linked to either end, each end, or both ends of the polypeptide. Covalent linkage of additional amino acids to either end, each end, or both ends of the polypeptide according to the invention results in a novel combined amino acid sequence that is neither naturally occurring nor disclosed in the art.
The invention provides both full-length and mature forms of Claudin polypeptides of the invention. Full-length polypeptides are those having the complete primary amino acid sequence of the polypeptide as initially translated. The amino acid sequences of full-length polypeptides can be obtained, for example, by translation of the complete open reading frame (“ORF”) of a cDNA molecule. Several full-length polypeptides may be encoded by a single genetic locus if multiple mRNA forms are produced from that locus by alternative splicing or by the use of multiple translation initiation sites. The “mature form” of a polypeptide refers to a polypeptide that has undergone post-translational processing steps such as cleavage of the signal sequence or proteolytic cleavage to remove a prodomain. Multiple mature forms of a particular full-length polypeptide may be produced, for example by cleavage of the signal sequence at multiple sites, or by differential regulation of proteases that cleave the polypeptide. The mature form(s) of such polypeptide may be obtained by expression, in a suitable mammalian cell or other host cell, of a polynucleotide molecule that encodes the full-length polypeptide. The sequence of the mature form of the polypeptide may also be determinable from the amino acid sequence of the full-length form, through identification of signal sequences or protease cleavage sites. The Claudin polypeptides of the invention also include those that result from post-transcriptional or post-translational processing events such as alternate mRNA processing which can yield a truncated but biologically active polypeptide, for example, a naturally occurring soluble form of the polypeptide. Also encompassed within the invention are variations attributable to proteolysis such as differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the polypeptide (generally from 1 to 5 terminal amino acids).
The invention further includes Claudin polypeptides of the invention with or without associated native-pattern glycosylation. Polypeptides expressed in yeast or mammalian expression systems (e.g., COS-1 or CHO cells) can be similar to or significantly different from a native polypeptide in molecular weight and glycosylation pattern, depending upon the choice of expression system. Expression of polypeptides of the invention in bacterial expression systems, such as E. coli, provides non-glycosylated molecules. Further, a given preparation can include multiple differentially glycosylated species of the polypeptide. Glycosyl groups can be removed through conventional methods, in particular those utilizing glycopeptidase. In general, glycosylated polypeptides of the invention can be incubated with a molar excess of glycopeptidase (Boehringer Mannheim).
Species homologues of Claudin polypeptides of the invention (e.g., the Claudin-23 human and murine forms) and of polynucleotides encoding them are also provided by the invention. As used herein, a “species homologue” is a polypeptide or polynucleotide with a different species of origin from that of a given polypeptide or polynucleotide, but with significant sequence similarity to the given polypeptide or polynucleotide, as determined by those of skill in the art. Species homologues may be isolated and identified by making suitable probes or primers from polynucleotides encoding the amino acid sequences provided herein and screening a suitable nucleic acid source from the desired species. The invention also encompasses allelic variants of Claudin polypeptides of the invention and polynucleotides encoding them; that is, naturally-occurring alternative forms of such polypeptides and polynucleotides in which differences in amino acid or nucleotide sequence are attributable to genetic polymorphism (allelic variation among individuals within a population).
Fragments of the Claudin polypeptides of the invention may be in linear form or cyclized using known methods, for example, as described in H. U. Saragovi, et al., Bio/Technology 10, 773–778 (1992) and in R. S. McDowell, et al., J. Amer. Chem. Soc. 114 9245–9253 (1992), both of which are incorporated by reference herein. Polypeptides and polypeptide fragments of the invention, and polynucleotides encoding them, include polypeptides and polynucleotides with amino acid or nucleotide sequence lengths that are at least 25% (e.g., at least 50%, or at least 60%, or at least 70%, or at least 80%) of the length of a Claudin-23 polypeptide and have at least 60% sequence identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, or a 99%, or at least 99.5%) with a Claudin-23 polypeptide or encoding polynucleotide, where sequence identity is determined by comparing the amino acid sequences of the polypeptides when aligned so as to maximize overlap and identity while minimizing sequence gaps. Also included in the invention are polypeptides and polypeptide fragments, and polynucleotides encoding them, that contain or encode a segment typically comprising at least 8, or at least 10, or at least 15, or at least 20, or at least 30, or at least 40 contiguous amino acids. Such polypeptides and polypeptide fragments may also contain a segment that shares at least 70% sequence identity (or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 99%, or at least 99.5%) with any such segment of any of the Claudin polypeptides of the invention, where sequence identity is determined by comparing the amino acid sequences of the polypeptides when aligned so as to maximize overlap and identity while minimizing sequence gaps. The percent identity can be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two amino acid or two polynucleotide sequences can be determined by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The typical default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Polypeptide Sequence and Structure, National Biomedical Research Foundation, pp. 353–358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. Other programs used by those skilled in the art of sequence comparison may also be used, such as, for example, the BLASTN program version 2.0.9, available for use via the National Library of Medicine website ncbi.nlm.nih.gov/gorf/wblast2.cgi, or the UW-BLAST 2.0 algorithm. Standard default parameter settings for UW-BLAST 2.0 are described at the following Internet webpage: blast.wustl.edu/blast/README.html#References. In addition, the BLAST algorithm uses the BLOSUM64 amino acid scoring matrix, and optional parameters that may be used are as follows: (A) inclusion of a filter to mask segments of the query sequence that have low compositional complexity (as determined by the SEG program of Wootton & Federhen (Computers and Chemistry, 1993); also see Wootton J C and Federhen S. 1996, Analysis of compositionally biased regions in sequence databases, Methods Enzymol. 266: 554–71) or segments consisting of short-periodicity internal repeats (as determined by the XNU program of Claverie & States (Computers and Chemistry, 1993)), and (B) a statistical significance threshold for reporting matches against database sequences, or E-score (the expected probability of matches being found merely by chance, according to the stochastic model of Karlin and Altschul (1990); if the statistical significance ascribed to a match is greater than this E-score threshold, the match will not be reported.); typical E-score threshold values are 0.5, or 0.25, 0.1, 0.05, 0.01, 0.001, 0.0001, 1e-5, 1e-10, 1e-15, 1e-20, 1e-25, 1e-30, 1e-40, 1e-50, 1e-75, or 1e-100.
The invention also provides for soluble forms of Claudin polypeptides of the invention comprising certain fragments or domains of these polypeptides, and particularly those comprising the extracellular domain or one or more fragments of the extracellular domain. Soluble polypeptides are polypeptides that are capable of being secreted from the cells in which they are expressed. In such forms part or all of the intracellular and transmembrane domains of the polypeptide are deleted such that the polypeptide is fully secreted from the cell in which it is expressed. The intracellular and transmembrane domains of polypeptides of the invention can be identified in accordance with known techniques for determination of such domains from sequence information. Soluble Claudin polypeptides of the invention also include those polypeptides which include part of the transmembrane region, provided that the soluble Claudin-23 polypeptide is capable of being secreted from a cell, and which typically retains a human Claudin-23 activity. Soluble Claudin polypeptides of the invention further include oligomers or fusion polypeptides comprising the extracellular portion of at least one Claudin-23 polypeptide, and fragments that have Claudin-23 activity. A secreted soluble polypeptide may be identified (and distinguished from its non-soluble membrane-bound counterparts) by separating intact cells which express the desired polypeptide from the culture medium, e.g., by centrifugation, and assaying the medium (supernatant) for the presence of the desired polypeptide. The presence of the desired polypeptide in the medium indicates that the polypeptide was secreted from the cells and thus is a soluble form of the polypeptide. The use of soluble forms of Claudin polypeptides of the invention is advantageous for many applications. Purification of the polypeptides from recombinant host cells is facilitated, since the soluble polypeptides are secreted from the cells. Moreover, soluble polypeptides are generally more suitable than membrane-bound forms for parenteral administration and for many enzymatic procedures.
In another aspect of the invention, polypeptides comprise various combinations of Claudin-23 polypeptide domains, such as the cytoplasmic tail domain and the extracellular loop domain or a cytoplasmic tail and a cytoplasmic loop domain. Accordingly, polypeptides of the invention and polynucleotides encoding them include those comprising or encoding two or more copies of a domain such as the cytoplasmic tail domain, two or more copies of a domain such as the extracellular loop domain, or at least one copy of each domain, and these domains may be presented in any order within such polypeptides.
Further modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques. Modifications of interest in the polypeptide sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid. For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule, an alteration which may involve preventing formation of incorrect intramolecular disulfide bridges upon folding or renaturation. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Pat. No. 4,518,584). As another example, N-glycosylation sites in the polypeptide extracellular domain can be modified to preclude glycosylation, allowing expression of a reduced carbohydrate analog in mammalian and yeast expression systems. N-glycosylation sites in eukaryotic polypeptides are characterized by an amino acid triplet Asn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr. Appropriate substitutions, additions, or deletions to the nucleotide sequence encoding these triplets will result in prevention of attachment of carbohydrate residues at the Asn side chain. Alteration of a single nucleotide, chosen so that Asn is replaced by a different amino acid, for example, is sufficient to inactivate an N-glycosylation site. Alternatively, the Ser or Thr can by replaced with another amino acid, such as Ala. Known procedures for inactivating N-glycosylation sites in polypeptides include those described in U.S. Pat. Nos. 5,071,972 and EP 276,846, hereby incorporated by reference. Additional variants within the scope of the invention include polypeptides that can be modified to create derivatives thereof by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives can be prepared by linking the chemical moieties to functional groups on amino acid side chains or at the N-terminus or C-terminus of a polypeptide. Conjugates comprising diagnostic (detectable) or therapeutic agents attached thereto are contemplated herein. Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the polypeptide or a substantial equivalent thereof. One example is a variant that binds with essentially the same binding affinity as does the native form. Binding affinity can be measured by conventional procedures, e.g., as described in U.S. Pat. No. 5,512,457 and as set forth herein.
Other derivatives include covalent or aggregative conjugates of the polypeptides with other polypeptides or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions. Examples of fusion polypeptides are discussed below in connection with oligomers. Further, fusion polypeptides can comprise peptides added to facilitate purification and identification. Such peptides include, for example, poly-His or the antigenic identification peptides described in U.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988. One such peptide is the FLAG® peptide, which is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay And facile purification of expressed recombinant polypeptide. A murine hybridoma designated 4E11 produces a monoclonal antibody that binds the FLAG® peptide in the presence of certain divalent metal cations, as described in U.S. Pat. No. 5,011,912, hereby incorporated by reference. The 4E11 hybridoma cell line has been deposited with the American Type Culture Collection under accession no. HB 9259. Monoclonal antibodies that bind the FLAG® peptide are available from Eastman Kodak Co., Scientific Imaging Systems Division, New Haven, Conn.
Encompassed by the invention are oligomers or fusion polypeptides that contain a Claudin-23 polypeptide, one or more fragments of Claudin polypeptides of the invention, or any of the derivative or variant forms of Claudin polypeptides of the invention as disclosed herein. In particular embodiments, the oligomers comprise soluble Claudin polypeptides of the invention. Oligomers can be in the form of covalently linked or non-covalently-linked multimers, including dimers, trimers, or higher oligomers. In one aspect of the invention, the oligomers maintain the binding ability of the polypeptide components and provide therefor, bivalent, trivalent, etc., binding sites. In an alternative embodiment the invention is directed to oligomers comprising multiple Claudin polypeptides of the invention joined via covalent or non-covalent interactions between peptide moieties fused to the polypeptides, such peptides having the property of promoting oligomerization. Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote oligomerization of the polypeptides attached thereto, as described in more detail below.
In embodiments where variants of the Claudin polypeptides of the invention are constructed to include a membrane-spanning domain, they will form a membrane-spanning polypeptide. Membrane-spanning Claudin polypeptides of the invention can be fused with extracellular domains of receptor polypeptides for which the ligand is known. Such fusion polypeptides can then be manipulated to control the intracellular signaling pathways triggered by the membrane-spanning Claudin-23 polypeptide. Claudin polypeptides of the invention that span the cell membrane can also be fused with agonists or antagonists of cell-surface receptors, or cellular adhesion molecules to further modulate Claudin-23 intracellular effects. In another aspect of the invention, interleukins can be situated between Claudin-23 polypeptide fragment and other fusion polypeptide domains.
Immunoglobulin-based Oligomers. The polypeptides of the invention or fragments thereof may be fused to molecules such as immunoglobulins for many purposes, including increasing the valency of polypeptide binding sites. For example, fragments of a Claudin-23 polypeptide may be (a) fused directly or through a linker peptide to the Fc portion of an immunoglobulin, or (b) fused directly or through a linker peptide to another Claudin-23 polypeptide. For a bivalent form of the polypeptide, such a fusion could be to the Fc portion of an IgG molecule. Other immunoglobulin isotypes may also be used to generate such fusions. For example, a polypeptide-IgM fusion would generate a decavalent form of the polypeptide of the invention. The term “Fc polypeptide” as used herein includes native and mutein forms of polypeptides made up of the Fc region of an antibody comprising any or all of the CH domains of the Fc region. Truncated forms of such polypeptides containing the hinge region that promotes dimerization are also included. Useful Fc polypeptides comprise an Fc polypeptide derived from a human IgG1 antibody. As one alternative, an oligomer is prepared using polypeptides derived from immunoglobulins. Preparation of fusion polypeptides comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., by Ashkenazi et al. (PNAS USA 88:10535, 1991); Byrn et al. (Nature 344:677, 1990); and Hollenbaugh and Aruffo (“Construction of Immunoglobulin Fusion Polypeptides”, in Current Protocols in Immunology, Suppl. 4, pages 10.19.1–10.19.11, 1992). Methods for preparation and use of immunoglobulin-based oligomers are well known in the art. One embodiment of the invention is directed to a dimer comprising two fusion polypeptides created by fusing a polypeptide of the invention to an Fc polypeptide derived from an antibody. A gene fusion encoding the polypeptide/Fc fusion polypeptide is inserted into an appropriate expression vector. Polypeptide/Fc fusion polypeptides are expressed in host cells transformed with the recombinant expression vector, and allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between the Fc moieties to yield divalent molecules. One suitable Fc polypeptide, described in PCT application WO 93/10151 (hereby incorporated by reference), is a single chain polypeptide extending from the N-terminal hinge region to the native C-terminus of the Fc region of a human IgG1 antibody. Another useful Fc polypeptide is the Fe mutein described in U.S. Pat. No. 5,457,035 and in Baum et al., (EMBO J. 13:3992–4001, 1994) incorporated herein by reference. The amino acid sequence of this mutein is identical to that of the native Fc sequence presented in WO 93/10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 has been changed from Gly to Ala. The mutein exhibits reduced affinity for Fe receptors. The above-described fusion polypeptides comprising Fe moieties (and oligomers formed therefrom) offer the advantage of facile purification by affinity chromatography over Polypeptide A or Polypeptide G columns. In other embodiments, the polypeptides of the invention can be substituted for the variable portion of an antibody heavy or light chain. If fusion polypeptides are made with both heavy and light chains of an antibody, it is possible to form an oligomer with as many as four Claudin-23 extracellular regions.
Peptide-linker Based Oligomers. Alternatively, the oligomer is a fusion polypeptide comprising multiple Claudin polypeptides of the invention, with or without peptide linkers (spacer peptides). Among the suitable peptide linkers are those described in U.S. Pat. Nos. 4,751,180 and 4,935,233, which are hereby incorporated by reference. An oligonucleotide sequence encoding a desired peptide linker can be inserted between, and in the same reading frame as a Claudin polynucleotide of the invention, using any suitable conventional technique. For example, a chemically synthesized oligonucleotide encoding a peptide linker can be ligated between the sequences. In particular embodiments, a fusion polypeptide comprises from two to four soluble Claudin polypeptides of the invention, separated by peptide linkers. Suitable peptide linkers, their combination with other polypeptides, and their use are well known by those skilled in the art
Leucine-Zippers. Another method for preparing the oligomers of the invention involves use of a leucine zipper. Leucine zipper domains are peptides that promote oligomerization of the polypeptides in which they are found. Leucine zippers were originally identified in several DNA-binding polypeptides (Landschulz et al., Science 240:1759, 1988), and have since been found in a variety of different polypeptides. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. The zipper domain (also referred to herein as an oligomerizing, or oligomer-forming, domain) comprises a repetitive heptad repeat, often with four or five leucine residues interspersed with other amino acids. Use of leucine zippers and preparation of oligomers using leucine zippers are well known in the art.
Other fragments and derivatives of the sequences of polypeptides which would be expected to retain polypeptide activity in whole or in part and may thus be useful for screening or other immunological methodologies may also be made by those skilled in the art given the disclosures herein. Such modifications are encompassed by the invention.
Polynucleotides Encoding Claudin Polypeptides of the Invention
Encompassed within the invention are polynucleotides encoding Claudin polypeptides of the invention. These polynucleotides can be identified in several ways, including isolation of genomic or cDNA molecules from a suitable source. Nucleotide sequences corresponding to the amino acid sequences described herein, to be used as probes or primers for the isolation of polynucleotides or as query sequences for database searches, can be obtained by “back-translation” from the amino acid sequences, or by identification of regions of amino acid identity with polypeptides for which the coding DNA sequence has been identified. The well-known polymerase chain reaction (PCR) procedure can be employed to isolate and amplify a DNA sequence encoding a human Claudin-23 polypeptide or a desired combination of human Claudin-23 polypeptide fragments. Oligonucleotides that define the desired termini of the combination of DNA fragments are employed as 5′ and 3′ primers. The oligonucleotides can additionally contain recognition sites for restriction endonucleases, to facilitate insertion of the amplified combination of DNA fragments into an expression vector. PCR techniques are described in Saiki et al., Science 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds., Academic. Press, Inc., San Diego (1989), pp. 189–196; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., Academic Press, Inc. (1990).
Polynucleotide molecules of the invention include DNA and RNA in both single-stranded and double-stranded form, as well as the corresponding complementary sequences. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. The polynucleotide molecules of the invention include full-length genes or cDNA molecules as well as a combination of fragments thereof. The polynucleotides of the invention can be derived from human sources, but the invention includes those derived from non-human species, as well.
“An isolated polynucleotide consisting essentially of a Claudin-23 polynucleotide” means that the polynucleotide may have, in addition to a specified Claudin-23 polynucleotide, additional material covalently linked to either or both ends of the polynucleotide molecule, said additional material being in one embodiment between 1 and 100,000 additional nucleotides; or between 1 and 10,000 additional nucleotides covalently linked to either end, each end, or both ends of the polynucleotide molecule; or between 10 and 1,000 additional nucleotides covalently linked to either end, each end, or both ends of the polynucleotide molecule; wherein the Claudin-23 polynucleotide encodes a Claudin-23 polypeptide or a fragment or variant thereof. An isolated polynucleotide consisting essentially of a Claudin-23 polynucleotide may be an expression vector or other construct comprising said Claudin-23 polynucleotide.
An “isolated polynucleotide” is a polynucleotide that has been separated from adjacent genetic sequences present in the genome of the organism from which the polynucleotide was isolated, in the case of polynucleotides isolated from naturally occurring sources. In the case of polynucleotides synthesized enzymatically from a template or chemically, such as PCR products, cDNA molecules, or oligonucleotides for example, it is understood that the polynucleotides resulting from such processes are isolated polynucleotides. An isolated polynucleotide refers to a polynucleotide in the form of a separate fragment or as a component of a larger polynucleotide construct. In one embodiment, the invention relates to certain isolated polynucleotides that are substantially free from contaminating endogenous material. The polynucleotide has preferably been derived from DNA or RNA isolated at least once in substantially pure form and in a quantity or concentration enabling identification, manipulation, and recovery of its component nucleotide sequences by standard biochemical methods (such as those outlined in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)). Such sequences are typically provided and/or constructed in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, that are typically present in eukaryotic genes. Sequences of non-translated DNA can be present 5′ or 3′ from an open reading frame, where the same do not interfere with manipulation or expression of the coding region.
The invention also includes polynucleotides that hybridize under moderately stringent conditions, or under highly stringent conditions, to polynucleotides encoding Claudin polypeptides of the invention. The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by Sambrook, et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11; and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3–6.4, incorporated herein by reference), and can be readily determined by those having ordinary skill in the art based on, for example, the length and/or base composition of the DNA. One way of achieving moderately stringent conditions involves the use of a prewashing solution containing 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50% formamide, 6×SSC, and a hybridization temperature of about 55 degrees C. (or other similar hybridization solutions, such as one containing about 50% formamide, with a hybridization temperature of about 42 degrees C.), and washing conditions of about 60 degrees C., in 0.5×SSC, 0.1% SDS. Generally, highly stringent conditions are defined as hybridization conditions as above, but with washing at approximately 68degrees C., 0.2×SSC, 0.1% SDS. SSPE (1×SSPE is 0.15M NaCl, 10 mM NaH2 PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is 0.15M NaCl and 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete. It should be understood that the wash temperature and wash salt concentration can be adjusted as necessary to achieve a desired degree of stringency by applying the basic principles that govern hybridization reactions and duplex stability, as known to those skilled in the art and described further below (see, e.g., Sambrook et al., 1989). When hybridizing a polynucleotide to a target polynucleotide of unknown sequence, the hybrid length is assumed to be that of the hybridizing polynucleotide. When polynucleotides of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5 to 10 degrees C. less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm (degrees C.)=2(# of A+T bases)+4(# of #G+C bases). For hybrids above 18 base pairs in length, Tm (degrees C.)=81.5+16.6(log10 [Na+])+0.41(% G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1×SSC=0.165M). Typically, each such hybridizing polynucleotide has a length that is at least 15, 18, 20, 25, 30, 40, or more typically 50 nucleotides, or at least 25% (e.g., at least 50%, or at least 60%, or at least 70%, or at least 80%) of the length of the polynucleotide of the invention to which it hybridizes, and has at least 60% sequence identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 99%, or at leas 99.5%) with the polynucleotide of the invention to which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing nucleic acids when aligned so as to maximize overlap and identity while minimizing sequence gaps as described in more detail above.
The invention also provides genes corresponding to the polynucleotide sequences disclosed herein. “Corresponding genes” are the regions of the genome that are transcribed to produce the mRNAs from which cDNA polynucleotide sequences are derived and may include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes may therefore include but are not limited to coding sequences, 5′ and 3′ untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements. The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials. An “isolated gene” is a gene that has been separated from the adjacent coding sequences, if any, present in the genome of the organism from which the gene was isolated.
Methods for Making and Purifying Claudin Polypeptides of the Invention
Methods for making Claudin polypeptides of the invention are described below. Expression, isolation, and purification of the polypeptides and fragments of the invention can be accomplished by any suitable technique, including but not limited to the following methods. The isolated nucleic acid of the invention can be operably linked to an expression control sequence such as the pDC409 vector (Giri et al., 1990, EMBO J. 13: 2821) or the derivative pDC412 vector (Wiley et al., 1995, Immunity 3: 673). The pDC400 series vectors are useful for transient mammalian expression systems, such as CV-1 or 293 cells. Alternatively, the isolated nucleic acid of the invention can be linked to expression vectors such as pDC312, pDC316, or pDC317 vectors. The pDC300 series vectors all contain the SV40 origin of replication, the CMV promoter, the adenovirus tripartite leader, and the SV40 polyA and termination signals, and are useful for stable mammalian expression systems, such as CHO cells or their derivatives. Other expression control sequences and cloning technologies can also be used to produce the polypeptide recombinantly, such as the pMT2 or pED expression vectors (Kaufman et al., 1991, Nucleic Acids Res 19: 4485–4490; and Pouwels et al., 1985, Cloning Vectors: A Laboratory Manual, Elsevier, N.Y.) and the GATEWAY Vectors (Life Technologies; Rockville, Md.). The isolated nucleic acid of the invention, flanked by attB sequences, can be recombined through an integrase reaction with a GATEWAY vector such as pDONR201 containing attP sequences, providing an entry vector for the GATEWAY system containing the isolated nucleic acid of the invention. This entry vector can be further recombined with other suitably prepared expression control sequences, such as those of the pDC400 and pDC300 series described above. Many suitable expression control sequences are known in the art. General methods of expressing recombinant polypeptides are also described in Kaufman, 1990, Methods in Enzymology 185, 537–566. As used herein “operably linked” means that a polynucleotide of the invention and an expression control sequence are situated within a construct, vector, or cell in such a way that a polypeptide encoded by a polynucleotide is expressed when appropriate molecules (such as polymerases) are present. As one embodiment of the invention, at least one expression control sequence is operably linked to a polynucleotide of the invention in a recombinant host cell or progeny thereof, the polynucleotide and/or expression control sequence having been introduced into the host cell by transformation or transfection, for example, or by any other suitable method. As another embodiment of the invention, at least one expression control sequence is integrated into the genome of a recombinant host cell such that it is operably linked to a polynucleotide sequence encoding a polypeptide of the invention. In a further embodiment of the invention, at least one expression control sequence is operably linked to a polynucleotide of the invention through the action of a trans-acting factor such as a transcription factor, either in vitro or in a recombinant host cell.
In addition, a sequence encoding an appropriate signal peptide (native or heterologous) can be incorporated into expression vectors. The choice of signal peptide or leader can depend on factors such as the type of host cells in which the recombinant polypeptide is to be produced. To illustrate, examples of heterologous signal peptides that are functional in mammalian host cells include the signal sequence for interleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195; the signal sequence for interleukin-2 receptor described in Cosman et al., Nature 312:768 (1984); the interleukin-4 receptor signal peptide described in EP 367,566; the type I interleukin-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; and the type II interleukin-1 receptor signal peptide described in EP 460,846. A DNA sequence for a signal peptide (secretory leader) can be fused in frame to a polynucleotide of the invention so that the DNA is initially transcribed, and the mRNA translated, into a fusion polypeptide comprising the signal peptide. A signal peptide that is functional in the intended host cells promotes extracellular secretion of the polypeptide. The signal peptide is cleaved from the polypeptide upon secretion of polypeptide from the cell. The skilled artisan will also recognize that the position(s) at which the signal peptide is cleaved can differ from that predicted by computer program, and can vary according to such factors as the type of host cells employed in expressing a recombinant polypeptide. A polypeptide preparation can include a mixture of polypeptide molecules having different N-terminal amino acids, resulting from cleavage of the signal peptide at more than one site.
Established methods for introducing DNA into mammalian cells have been described (Kaufman, 1990, Large Scale Mammalian Cell Culture, pp. 15–69). Additional protocols using commercially available reagents, such as Lipofectamine lipid reagent (Gibco/BRL) or Lipofectamine-Plus lipid reagent, can be used to transfect cells (Felgner et al., 1987, Proc. Natl. Acad Sci. USA 84:7413–7417). In addition, electroporation can be used to transfect mammalian cells using conventional procedures, such as those in Sambrook et al., (Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1–3, Cold Spring Harbor Laboratory Press, 1989). Selection of stable transformants can be performed using methods known in the art such as, for example, resistance to cytotoxic drugs. Kaufman et al., Meth. in Enzymology 185:487–511, 1990, describes several selection schemes, such as dihydrofolate reductase (DHFR) resistance. A suitable strain for DHFR selection can be CHO strain DX-B11, which is deficient in DHFR (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216–4220, 1980). A plasmid expressing the DHFR cDNA can be introduced into strain DX-B11, and only cells that contain the plasmid can grow in the appropriate selective media. Other examples of selectable markers that can be incorporated into an expression vector include cDNAs conferring resistance to antibiotics, such as G418 and hygromycin B. Cells harboring the vector can be selected on the basis of resistance to these compounds.
Alternatively, gene products can be obtained via homologous recombination, or “gene targeting,” techniques. Such techniques employ the introduction of exogenous transcription control elements (such as the CMV promoter or the like) in a particular predetermined site on the genome, to induce expression of the endogenous polynucleotide sequence of interest. The location of integration into a host chromosome or genome can be easily determined by one of skill in the art, given the known location and sequence of the gene. In one embodiment, the invention also contemplates the introduction of exogenous transcriptional control elements in conjunction with an amplifiable gene, to produce increased amounts of the gene product, again, without the need for isolation of the gene itself from the host cell. The practice of homologous recombination or gene targeting is explained by Schimke, et al.” Amplification of Genes in Somatic Mammalian cells,” Methods in Enzymology 151:85–104 (1987), as well as by Capecchi, et al., “The New Mouse Genetics: Altering the Genome by Gene Targeting,” TIG 5:70–76 (1989).
A number of types of cells may act as suitable host cells for expression of a polypeptide. Mammalian host cells include, for example, the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) as described by McMahan et al. (EMBO J. 10:2821, 1991), human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HL-60, U937, HaK or Jurkat cells. Alternatively, it may be possible to produce a polypeptide in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous polypeptides. Potentially suitable bacterial strains include Eseherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous polypeptides. If the polypeptide is made in yeast or bacteria, it may be necessary to modify the polypeptide produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional polypeptide. Such covalent attachments may be accomplished using known chemical or enzymatic methods. The polypeptide may also be produced by operably linking an isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MAXBAC® Baculovirus expression system), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), and Luckow and Summers, Bio/Technology 6:47 (1988), incorporated herein by reference. As used herein, an insect cell capable of expressing a polynucleotide of the invention is “transformed.” Cell-free translation systems could also be employed to produce polypeptides using RNAs derived from polynucleotide constructs disclosed herein. A host cell that comprises an isolated polynucleotide of the invention, typically operably linked to at least one expression control sequence, is a “recombinant host cell”.
A polypeptide of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant polypeptide. The resulting expressed polypeptide may then be purified from such culture (e.g., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The purification of a polypeptide may also include an affinity column containing agents which will bind to the polypeptide; one or more colunm steps over such affinity resins as concanavalin A-agarose, HEPARIN-TOYOPEARL® (hydrophilic polymer gel) or Cibacrom blue 3GA SEPHAROSE® (agarose beads); one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography. Alternatively, a polypeptide of the invention may also be expressed in a form that will facilitate purification. For example, it may be expressed as a fusion polypeptide, such as those of maltose binding polypeptide (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion polypeptides are commercially available from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway, N.J.) and InVitrogen, respectively. A polypeptide can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope. One such epitope (“Flag”) is commercially available from Kodak (New Haven, Conn.). Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the polypeptide. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant polypeptide. A polypeptide thus purified is substantially free of other mammalian polypeptides and is defined in accordance with the invention as a “purified polypeptide”; such purified polypeptides of the invention include purified antibodies that bind to Claudin polypeptides of the invention, fragments, variants, binding partner, and the like. A polypeptide of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a polynucleotide encoding the polypeptide.
It is also possible to utilize an affinity column comprising a polypeptide-binding polypeptide of the invention, such as a monoclonal antibody generated against polypeptides of the invention, to affinity-purify expressed polypeptides. These polypeptides can be removed from an affinity column using conventional techniques, e.g., in a high salt elution buffer and then dialyzed into a lower salt buffer for use or by changing pH or other components depending on the affinity matrix utilized, or be competitively removed using the naturally occurring substrate of the affinity moiety, such as a polypeptide derived from the invention. In this aspect of the invention, polypeptide-binding polypeptides, such as the anti-polypeptide antibodies of the invention or other polypeptides that can interact with a polypeptide of the invention, can be bound to a solid phase support such as a column chromatography matrix or a similar substrate suitable for identifying, separating, or purifying cells that express polypeptides of the invention on their surface. Adherence of polypeptide-binding polypeptides of the invention to a solid phase contacting surface can be accomplished by any number of techniques, for example, magnetic microspheres can be coated with these polypeptide-binding polypeptides and held in the incubation vessel through a magnetic field. Suspensions of cell mixtures are contacted with the solid phase that has such polypeptide-binding polypeptides thereon. Cells having polypeptides of the invention on their surface bind to the fixed polypeptide-binding polypeptide and unbound cells then are washed away. This affinity-binding method is useful for purifying, screening, or separating such polypeptide-expressing cells from solution. Methods of releasing positively selected cells from the solid phase are known in the art and encompass, for example, the use of enzymes. Such enzymes are preferably non-toxic and non-injurious to the cells and are directed to cleaving the cell-surface binding partner. Alternatively, mixtures of cells suspected of containing .polypeptide-expressing cells of the invention can first be incubated with a biotinylated polypeptide-binding polypeptide of the invention. Incubation periods are typically at least one hour in duration to ensure sufficient binding to polypeptides of the invention. The resulting mixture then is passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin provides the binding of the polypeptide-binding cells to the beads. Use of avidin-coated beads is known in the art (see, e.g., Berenson, et al. J. Cell. Biochem., 10D:239, 1986). Wash of unbound material and the release of the bound cells is performed using conventional methods
A polypeptide may also be produced by known conventional chemical synthesis. Methods for constructing polypeptides of the invention by synthetic means are known to those skilled in the art. The synthetically constructed polypeptides, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with native polypeptides may possess biological properties in common therewith, including polypeptide activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified polypeptides in screening of therapeutic compounds and in immunological processes for the development of antibodies.
The desired degree of purity depends on the intended use of a polypeptide. A relatively high degree of purity is desired when a polypeptide is to be administered in vivo, for example. In such a case, polypeptides are purified such that no polypeptide bands corresponding to other polypeptides are detectable upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be recognized by one skilled in the pertinent field that multiple bands corresponding to the polypeptide can be visualized by SDS-PAGE, due to differential glycosylation, differential post-translational processing, and the like. A polypeptide of the invention is purified to substantial homogeneity, as indicated by a single polypeptide band upon analysis by SDS-PAGE. The polypeptide band can be visualized by silver staining, Coomassie blue staining, or (if the polypeptide is radiolabeled) by autoradiography.
Antagonists and Agonists of Claudin Polypeptides of the Invention
Any method that neutralizes Claudin polypeptides of the invention or inhibits expression of a Claudin-23 gene (either transcription or translation) can be used to reduce the biological activities of Claudin polypeptides of the invention. In particular embodiments, antagonists inhibit the binding of at least one Claudin-23 polypeptide to binding partners expressed on cells, thereby inhibiting biological activities induced by the binding of those Claudin polypeptides of the invention to the cells. In certain other embodiments of the invention, antagonists can be designed to reduce the level of endogenous Claudin-23 gene expression, e.g., using well-known antisense or ribozyme approaches to inhibit or prevent translation of Claudin-23 mRNA transcripts; triple helix approaches to inhibit transcription of Claudin-23 genes; or targeted homologous recombination to inactivate or “knock out” a Claudin-23 gene or their endogenous promoters or enhancer elements. Such antisense, ribozyme, and triple helix antagonists may be designed to reduce or inhibit either unimpaired, or if appropriate, mutant Claudin-23 gene activity. Techniques for the production and use of such molecules are well known to those of skill in the art.
Antisense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing polypeptide translation. Antisense approaches involve the design of oligonucleotides (either DNA or RNA) that are complementary to a Claudin-23 mRNA. The antisense oligonucleotides will bind to the complementary target gene mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required. A sequence “complementary” to a portion of a polynucleotide, as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the polynucleotide, forming a stable duplex (or triplex, as appropriate). In the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Oligonucleotides that are complementary to the 5′ end of the message, e.g., the 5′ untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, oligonucleotides complementary to either the 5′- or 3′- non-translated, non-coding regions of a Claudin-23 gene transcript could be used in an antisense approach to inhibit translation of endogenous Claudin-23 mRNA. Oligonucleotides complementary to the 5′ untranslated region of the mRNA should include the complement of the AUG start codon. Antisense nucleic acids should be at least six nucleotides in length, and typically range 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. The oligonucleotides 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, and the like. 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. U.S.A. 86:6553–6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648–652; PCT Publication No. WO88/09810, published Dec. 15, 1988), or hybridization-triggered cleavage agents or intercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5:539–549). The antisense molecules should be delivered to cells that express a human Claudin-23 transcript in vivo. A number of methods have been developed for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue or cell derivation site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically. However, it is often difficult to achieve intracellular concentrations of the antisense molecule sufficient to suppress translation of endogenous mRNAs. Therefore one approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter. The use of such a construct to transfect target cells in a subject will result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous Claudin-23 gene transcripts and thereby prevent translation of the Claudin-23 mRNA. For example, a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA. Such a vector can remain episomal or become chromosomally integrated, so 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 mammalian cells.
Ribozyme molecules designed to catalytically cleave Claudin-23 mRNA transcripts can also be used to prevent translation of Claudin-23 mRNA thereby inhibiting expression of Claudin polypeptides of the invention (see, e.g., PCT International Publication WO90/11364, published Oct. 4, 1990; U.S. Pat. No. 5,824,519). The ribozymes that can be used in the invention include hammerhead ribozymes (Haseloff and Gerlach, 1988, Nature, 334:585–591), RNA endoribonucleases (hereinafter “Cech-type ribozymes”) such as the one which occurs naturally in Tetrahymena Thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (International Patent Application No. WO 88/04300; Been and Cech, 1986, Cell, 47:207–216). As in the antisense approach, the ribozymes can be composed of modified oligonucleotides (e.g. for improved stability, targeting, and the like) and should be delivered to cells which express the human Claudin-23 polypeptide in vivo. A typical method of delivery involves using a DNA construct coding for the ribozyme under the control of a strong constitutive pol II or pol III promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous Claudin-23 messages and inhibit translation. Because ribozymes, unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
Alternatively, endogenous Claudin-23 gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the target gene (e.g., the target gene's promoter and/or enhancers) to form triple helical structures that prevent transcription of a Claudin-23 gene (see generally, Helene, 1991, Anticancer Drug Des., 6(6):569–584; Helene, et al., 1992, Ann. N.Y. Acad. Sci., 660, 27–36; and Maher, 1992, Bioassays 14(12):807–815).
Antisense nucleic acids, ribozyme, and triple helix molecules of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art such as, for example, solid phase phosphoramidite chemical synthesis. Oligonucleotides 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, and the like). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al., 1988, Nucl. Acids Res. 16:3209. Methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448–7451). Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase-promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
Endogenous target gene expression can also be reduced by inactivating or “knocking out” the target gene or its promoter using targeted homologous recombination (e.g., see Smithies, et al., 1985, Nature 317:230–234; Thomas and Capecchi, 1987, Cell 51:503–512; Thompson, et al., 1989, Cell 5:313–321; each of which is incorporated by reference herein in its entirety). For example, a mutant, non-functional target gene (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous target gene (either the coding regions or regulatory regions of the target gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express the target gene in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the target gene. Such approaches are particularly suited in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive target gene (see, e.g., Thomas and Capecchi, 1987 and Thompson, 1989, supra), or in model organisms such as Caenorhabditis elegans where the “RNA interference” (“RNAi”) technique (Grishok A, Tabara H, and Mello C C, 2000, Genetic requirements for inheritance of RNAi in C. elegans, Science 287 (5462): 2494–2497), or the introduction of transgenes (Dernburg A F, Zalevsky J, Colaiacovo M P, and Villeneuve A M, 2000, Transgene-mediated cosuppression in the C. elegans germ line, Genes Dev. 14 (13): 1578–1583) are used to inhibit the expression of specific target genes. However this approach can be adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors.
Organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein are provided. The desired change in gene expression can be achieved through the use of antisense nucleic acids or ribozymes that bind and/or cleave the mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol. Sci. 15(7):250–254; Lavarosky et al., 1997, Biochem. Mol. Med. 62(1): 11–22; and Hampel, 1998, Prog. Nucleic Acid Res. Mol. Biol. 58:1–39; all of which are incorporated by reference herein). Transgenic animals that have multiple copies of the gene(s) corresponding to the polynucleotide sequences disclosed herein, produced by transformation of cells with genetic constructs that are stably maintained within the transformed cells and their progeny, are provided. Transgenic animals that have modified genetic control regions that increase or reduce gene expression levels, or that change temporal or spatial patterns of gene expression, are also provided (see, e.g., European Patent No. 0 649 464 B1, incorporated by reference herein). In addition, organisms are provided in which the gene(s) corresponding to the polynucleotide sequences disclosed herein have been partially or completely inactivated, through insertion of extraneous sequences into the corresponding gene(s) or through deletion of all or part of the corresponding gene(s). Partial or complete gene inactivation can be accomplished through insertion, followed by imprecise excision, of transposable elements (Plasterk, 1992, Bioessays 14(9):629–633; Zwaal et al., 1993, Proc. Natl. Acad. Sci. USA 90(16):7431–7435; Clark et al., 1994, Proc. Natl. Acad. Sci. USA 91(2):719–722; all of which are incorporated by reference herein), or through homologous recombination which can be detected by positive/negative genetic selection strategies (Mansour et al., 1988, Nature 336:348–352; U.S. Pat. Nos. 5,464,764; 5,487,992; 5,627,059; 5,631,153; 5,614,396; 5,616,491; and 5,679,523; all of which are incorporated by reference herein). These organisms with altered gene expression are eukaryotes and typically are mammals. Such organisms are useful for the development of non-human models for the study of disorders involving the corresponding gene(s), and for the development of assay systems for the identification of molecules that interact with the polypeptide product(s) of the corresponding gene(s).
The Claudin polypeptides of the invention themselves can also be employed in inhibiting a biological activity of Claudin-23 in in vitro or in vivo procedures. Encompassed within the invention are extracellular loop domains of Claudin polypeptides of the invention that act as “dominant negative” inhibitors of native Claudin-23 polypeptide function when expressed as fragments or as components of fusion polypeptides. For example, a purified polypeptide domain of the invention can be used to inhibit binding of Claudin polypeptides of the invention to endogenous binding-partners. Such use would effectively block Claudin-23 polypeptide interactions and inhibit Claudin-23 polypeptide activities. In still another aspect of the invention, a soluble form of a Claudin-23 binding partner, which is expressed on epithelial and/or endothelial cells, is used to bind to and competitively inhibit activation of an endogenous Claudin-23 polypeptide. Furthermore, antibodies which bind to Claudin polypeptides of the invention can inhibit Claudin-23 activity and act as antagonists, or as agonists. For example, antibodies that specifically recognize one or more epitopes of Claudin polypeptides of the invention, or epitopes of conserved variants of Claudin polypeptides of the invention, or peptide fragments of a Claudin-23 polypeptide can be used in the invention to inhibit Claudin-23 activity (antagonistic antibodies). Agonistic antibodies bind to Claudin polypeptides of the invention or binding partners and increase Claudin-23 polypeptide activity by causing constitutive intracellular signaling (or “ligand mimicking”), or by preventing the binding of a native inhibitor of Claudin-23 polypeptide activity. Antibodies which bind to Claudin-23 polypeptides include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), human (also called “fully human”) antibodies, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′)2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. Alternatively, purified and modified Claudin polypeptides of the invention can be administered to modulate interactions between Claudin polypeptides of the invention and Claudin-23 binding partners that are not membrane-bound. Such an approach will allow an alternative method for the modification of human Claudin-23-influenced bioactivity.
Polypeptides of the invention may be used to identify antagonists and agonists from cells, cell-free preparations, chemical libraries, and natural product mixtures. The antagonists and agonists may be natural or modified substrates, ligands, enzymes, receptors, etc. of the polypeptides of the instant invention, or may be structural or functional mimetics of the polypeptides. Potential antagonists of the instant invention may include small molecules, peptides and antibodies that bind to and occupy a binding site of the inventive polypeptides or a binding partner thereof, causing them to be unavailable to bind to their natural binding partners and therefore preventing normal biological activity. Potential agonists include small molecules, peptides and antibodies which bind to the instant polypeptides or binding partners thereof, and elicit the same or enhanced biologic effects as those caused by the binding of the polypeptides of the instant invention. Peptide agonists and antagonists of the polypeptides of the invention can be identified and utilized according to known methods (see, for example, WO 00/24782 and WO 01/83525, which are incorporated by reference herein).
An approach to development of therapeutic agents is peptide library screening. The interaction of a protein ligand with its receptor often takes place at a relatively large interface. However, as demonstrated for human growth hormone and its receptor, only a few key residues at the interface contribute to most of the binding energy (Clackson et al., 1995. Science 267: 383–386). The bulk of the protein ligand merely displays the binding epitopes in the right topology or serves functions unrelated to binding. Thus, molecules of only “peptide” length (2 to 90 amino acids) can bind to the receptor protein or binding partner of even a large protein ligand such as a polypeptide of the invention. Such peptides may mimic the bioactivity of the large protein ligand (“peptide agonists”) or, through competitive binding, inhibit the bioactivity of the large protein ligand (“peptide antagonists”). Exemplary peptide agonists and antagonists of polypeptides of the invention may comprise a domain of a naturally occurring molecule or may comprise randomized sequences. The term “randomized” as used to refer to peptide sequences refers to fully random sequences (e.g., selected by phage display methods or RNA-peptide screening) and sequences in which one or more residues of a naturally occurring molecule is replaced by an amino acid residue not appearing in that position in the naturally occurring molecule. Phage display peptide libraries have emerged as a powerful method in identifying such peptide agonists and antagonists. See, for example, Scott et al., 1990, Science 249: 386; Devlin et al., 1990, Science 249: 404; U.S. Pat. Nos. 5,223,409; 5,733,731; 5,498,530; 5,432,018; 5,338,665; 5,922,545; WO 96/40987; and WO 98/15833 (each of which is incorporated by reference in its entirety). In such libraries, random peptide sequences are displayed by fusion with coat proteins of filamentous phage. Typically, the displayed peptides are affinity-eluted against an antibody-immobilized extracellular domain of a receptor. The retained phages may be enriched by successive rounds of affinity purification and repropagation. The best binding peptides may be sequenced to identify key residues within one or more structurally related families of peptides. The peptide sequences may also suggest which residues may be safely replaced by alanine scanning or by mutagenesis at the DNA level. Mutagenesis libraries may be created and screened to further optimize the sequence of the best binders (Lowman, 1997, Ann. Rev. Biophys. Biomol. Struct. 26: 401–424). Another biological approach to screening soluble peptide mixtures uses yeast for expression and secretion (Smith et al., 1993, Mol. Pharmacol. 43: 741–748) to search for peptides with favorable therapeutic properties. Hereinafter, this and related methods are referred to as “yeast-based screening.” A peptide library can also be fused to the carboxyl terminus of the lac repressor and expressed in E. coli. Another E. coli-based method allows display on the cell's outer membrane by fusion with a peptidoglycan-associated lipoprotein (PAL). Hereinafter, these and related methods are collectively referred to as “E. coli display.” In another method, translation of random RNA is halted prior to ribosome release, resulting in a library of polypeptides with their associated RNA still attached. Hereinafter, this and related methods are collectively referred to as “ribosome display.” Other methods employ peptides linked to RNA; for example, PROfusion technology, Phylos, Inc. (see, for example, Roberts and Szostak, 1997. Proc. Natl. Acad. Sci. USA 94: 12297–12303). Hereinafter, this and related methods are collectively referred to as “RNA-peptide screening.” Chemically derived peptide libraries have been developed in which peptides are immobilized on stable, non-biological materials, such as polyethylene rods or solvent-permeable resins. Another chemically derived peptide library uses photolithography to scan peptides immobilized on glass slides. Hereinafter, these and related methods are collectively referred to as “chemical-peptide screening.” Chemical-peptide screening may be advantageous in that it allows use of D-amino acids and other unnatural analogues, as well as non-peptide elements. Both biological and chemical methods are reviewed in Wells and Lowman, 1992, Curr. Opin. Biotechnol. 3: 355–362.
In the case of known bioactive peptides, rational design of peptide ligands with favorable therapeutic properties can be completed. In such an approach, one makes stepwise changes to a peptide sequence and determines the effect of the substitution upon bioactivity or a predictive biophysical property of the peptide (e.g., solution structure). Hereinafter, these techniques are collectively referred to as “rational design.” In one such technique, one makes a series of peptides in which one replaces a single residue at a time with alanine. This technique is commonly referred to as an “alanine walk” or an “alanine scan.” When two residues (contiguous or spaced apart) are replaced, it is referred to as a “double alanine walk.” The resultant amino acid substitutions can be used alone or in combination to result in a new peptide entity with favorable therapeutic properties. Structural analysis of protein-protein interaction may also be used to suggest peptides that mimic the binding activity of large protein ligands. In such an analysis, the crystal structure may suggest the identity and relative orientation of critical residues of the large protein ligand, from which a peptide may be designed (see, e.g., Takasaki et al., 1997, Nature Biotech. 15: 1266–1270). Hereinafter, these and related methods are referred to as “protein structural analysis.” These analytical methods may also be used to investigate the interaction between a receptor protein and peptides selected by phage display, which may suggest further modification of the peptides to increase binding affinity.
Peptide agonists and antagonists of polypeptides of the invention may be covalently linked to a vehicle molecule. The term “vehicle” refers to a molecule that prevents degradation and/or increases half-life, reduces toxicity, reduces immunogenicity, or increases biological activity of a therapeutic protein. Exemplary vehicles include an Fc domain or a linear polymer (e.g., polyethylene glycol (PEG), polylysine, dextran, etc.); a branched-chain polymer (see, for example, U.S. Pat. Nos. 4,289,872; 5,229,490; WO 93/21259); a lipid; a cholesterol group (such as a steroid); a carbohydrate or oligosaccharide (e.g., dextran); or any natural or synthetic protein, polypeptide or peptide that binds to a salvage receptor.
Antibodies to Claudin Polypeptides
Antibodies that are immunoreactive with the polypeptides of the invention are provided herein. Such antibodies specifically bind to the polypeptides via the antigen-binding sites of the antibody (as opposed to non-specific binding). In the invention, specifically binding antibodies are those that will specifically recognize and bind with Claudin polypeptides of the invention, homologues, and variants, but not with other molecules. In one embodiment, the antibodies are specific for the polypeptides of the invention and do not cross-react with other polypeptides. In this manner, the Claudin polypeptides of the invention, fragments, variants, fusion polypeptides, and the like, as set forth above, can be employed as “immunogens” in producing antibodies immunoreactive therewith.
More specifically, the polypeptides, fragment, variants, fusion polypeptides, and the like contain antigenic determinants or epitopes that elicit the formation of antibodies. These antigenic determinants or epitopes can be either linear or conformational (discontinuous). Linear epitopes are composed of a single section of amino acids of the polypeptide, while conformational or discontinuous epitopes are composed of amino acids sections from different regions of the polypeptide chain that are brought into close proximity upon polypeptide folding (C. A. Janeway, Jr. and P. Travers, Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed. 1996)). Because folded polypeptides have complex surfaces, the number of epitopes available is quite numerous; however, due to the conformation of the polypeptide and steric hinderances, the number of antibodies that actually bind to the epitopes is less than the number of available epitopes (C. A. Janeway, Jr. and P. Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nd ed. 1996)). Epitopes can be identified by any of the methods known in the art. Thus, one aspect of the invention relates to the antigenic epitopes of the polypeptides of the invention. Such epitopes are useful for raising antibodies, in particular monoclonal antibodies, as described in more detail below. Additionally, epitopes from the polypeptides of the invention can be used as research reagents, in assays, and to purify specific binding antibodies from substances such as polyclonal sera or supernatants from cultured hybridomas. Such epitopes or variants thereof can be produced using techniques well known in the art such as solid-phase synthesis, chemical or enzymatic cleavage of a polypeptide, or using recombinant DNA technology.
As to the antibodies that can be elicited by the epitopes of the polypeptides of the invention, whether the epitopes have been isolated or remain part of the polypeptides, both polyclonal and monoclonal antibodies can be prepared by conventional techniques. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988); Kohler and Milstein, (U.S. Pat. No. 4,376,110); the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026–2030); and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77–96). Hybridoma cell lines that produce monoclonal antibodies specific for the polypeptides of the invention are also contemplated herein. Such hybridomas can be produced and identified by conventional techniques. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the most common method of production. One method for producing such a hybridoma cell line comprises immunizing an animal with a polypeptide; harvesting spleen cells from the immunized animal; fusing said spleen cells to a myeloma cell line, thereby generating hybridoma cells; and identifying a hybridoma cell line that produces a monoclonal antibody that binds the polypeptide. For the production of antibodies, various host animals may be immunized by injection with one or more of the following: a Claudin-23 polypeptide, a fragment of a Claudin-23 polypeptide, a functional equivalent of a Claudin-23 polypeptide, or a mutant form of a Claudin-23 polypeptide. Such host animals may include, but are not limited to rabbits, mice and rats. Various adjuvants may be used to increase the immunological response, depending on the host species, including, but 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 hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. The monoclonal antibodies can be recovered by conventional techniques. Such monoclonal antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclass thereof.
In addition, techniques developed for the production of “chimeric antibodies” (Takeda et al., 1985, Nature, 314:452–454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a porcine mAb and a human immunoglobulin constant region. The monoclonal antibodies of the invention also include humanized versions of murine monoclonal antibodies. Such humanized antibodies can be prepared by known techniques and offer the advantage of reduced immunogenicity when the antibodies are administered to humans. In one embodiment, a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen-binding site thereof) and a constant region derived from a human antibody. Alternatively, a humanized antibody fragment can comprise the antigen-binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick et al. (Bio/Technology 7:934, 1989). and Winter and Harris (TIPS 14:139, Can, 1993). Procedures to generate antibodies transgenically can be found in GB 2,272,440U.S. Pat. No. 5,569,825 and 5,545,806 and related patents claiming priority therefrom, all of which are incorporated by reference herein. Preferably, for use in humans, the antibodies are human or humanized; techniques for creating such human or humanized antibodies are also well known and are commercially available from, for example, Medarex Inc. (Princeton, N.J.) and Abgenix Inc. (Fremont, Calif.).
Antigen-binding antibody fragments that recognize specific epitopes may be generated by known techniques. For example, such fragments include, but are not limited to: the F(ab′)2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the (ab′)2 fragments. Alternatively, Fab expression libraries may be constructed (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423–426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879–5883; and Ward et al., 1989, Nature 334:544–546) can also be adapted to produce single chain antibodies against Claudin-23 gene products. 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 polypeptide. In addition, antibodies to a Claudin-23 polypeptide can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” a Claudin-23 polypeptide and that may bind to a Claudin-23 polypeptide using techniques well known to those skilled in the art (see, e.g., Greenspan & Bona, 1993, FASEB J 7(5):437–444; and Nissinoff, 1991, J. Immunol. 147(8):2429–2438).
Screening procedures by which such antibodies can be identified are well known, and can involve immunoaffinity chromatography, for example. Antibodies can be screened for agonistic (i.e., ligand-mimicking) properties. Such antibodies, upon binding to cell surface Claudin-23, induce biological effects (e.g., transduction of biological signals) similar to the biological effects induced when a Claudin-23 binding partner binds to a cell surface Claudin-23. Agonistic antibodies can be used to induce Claudin-23-mediated activities, such as epithelial barrier formation, stimulatory pathways, or intercellular communication. Those antibodies that can block binding of the Claudin polypeptides of the invention to binding partners for Claudin-23 can be used to inhibit Claudin-23-mediated epithelial barrier formation, intercellular communication, or co-stimulation that results from such binding. Such blocking antibodies can be identified using any suitable assay procedure, such as by testing antibodies for the ability to inhibit binding of Claudin-23 to certain cells expressing a Claudin-23 binding partner. Alternatively, blocking antibodies can be identified in assays for the ability to inhibit a biological effect that results from binding of a Claudin-23 to target cells, such as epithelial barrier formation, using assays described herein. Such an antibody can be employed in an in vitro procedure, or administered in vivo to inhibit a biological activity mediated by the entity that generated the antibody. Disorders caused or exacerbated (directly or indirectly) by the interaction of Claudin-23 with cell surface binding partner receptor thus can be treated. A therapeutic method involves in vivo administration of a blocking antibody to a mammal in an amount effective in inhibiting Claudin-23 binding partner-mediated biological activity. Human or humanized antibodies can be used in such therapeutic methods. In one embodiment, an antigen-binding antibody fragment is employed. Compositions comprising an antibody that is directed against Claudin-23, and a physiologically acceptable diluent, excipient, or carrier, are provided herein. Suitable components of such compositions are as described below for compositions containing Claudin polypeptides of the invention.
Also provided herein are conjugates comprising a detectable (e.g., diagnostic) or a therapeutic agent, attached to the antibody. Examples of such agents are presented above. The conjugates find use in in vitro or in vivo procedures. The antibodies of the invention can also be used in assays to detect the presence of the polypeptides or fragments of the invention, either in vitro or in vivo. The antibodies also can be employed in purifying polypeptides or fragments of the invention by immunoaffinity chromatography.
Rational Design of Compounds that Interact with Claudin Polypeptides
The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact, e.g., inhibitors, agonists, antagonists, and the like. Any of these examples can be used to fashion drugs which are more active or stable forms of a polypeptide or which enhance or interfere with the function of a polypeptide in vivo (Hodgson J., 1991, Biotechnology 9:19–21, incorporated herein by reference). In one approach, the three-dimensional structure of a polypeptide of interest, or of a polypeptide-inhibitor complex, is determined by x-ray crystallography, by nuclear magnetic resonance, or by computer homology modeling or, most typically, by a combination of these approaches. Both the shape and charges of the polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the polypeptide. Less often, useful information regarding the structure of a polypeptide may be gained by modeling based on the structure of homologous polypeptides. In both cases, relevant structural information is used to design analogous Claudin-like molecules, to identify efficient inhibitors, or to identify small molecules that may bind a Claudin of the invention. Useful examples of rational drug design may include molecules which have improved activity or stability as shown by Braxton S and Wells J A (1992, Biochemistry 31:7796–7801) or which act as inhibitors, agonists, or antagonists of native peptides as shown by Athauda S B et al. (1993, J Biochem 113:742–746), incorporated herein by reference. The use of Claudin-23 polypeptide structural information in molecular modeling software systems to assist in inhibitor design and inhibitor-Claudin-23 polypeptide interaction is also encompassed by the invention. A particular method of the invention comprises analyzing the three-dimensional structure of Claudin polypeptides of the invention for likely binding sites of substrates, synthesizing a new molecule that incorporates a predictive reactive site, and assaying the new molecule as described further herein.
It is also possible to isolate a target-specific antibody, selected by functional assay, as described further herein, and then to solve its crystal structure. This approach, in principle, yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass polypeptide crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides would then act as the pharmacore.
Assays of Activities of Claudin Polypeptides of the Invention
The purified Claudin-23 polypeptides of the invention (including polypeptides, fragments, variants, oligomers, and other forms) are useful in a variety of assays. For example, a Claudin-23 molecule of the invention can be used to identify agonists and/or antagonists of Claudin-23 polypeptides of the invention, which can be used to modulate Claudin biological activities such as tight junction formation or endothelial or epithelial barrier formation. In one embodiment, the Claudin-23 polypeptides of the invention are used in binding assays to identify molecules (binding partners) that bind to Claudin-23, and then these molecules are tested in additional assays for modulation of Claudin-23 polypeptide activity as described herein. In one embodiment of these methods of the invention, the determination of Claudin-23 polypeptide activity comprises an assessment of transcription and/or translation of skin differentiation markers such as, but not limited to, filaggrin, profilaggrin, involucrin, and keratin markers (such as K1, K2, K2e, K2p, K4, K5, K6, K8, K9, K10, K13, K14, K16, K17, K18, K19, and the like) by conventional techniques, for example the use of differentiation marker-specific probes.
Assays to Identify Binding Partners. Claudin-23 polypeptides and fragments thereof can be used to identify binding partners. For example, they can be tested for the ability to bind a candidate binding partner in any suitable assay, such as a conventional binding assay. To illustrate, a Claudin-23 polypeptide can be labeled with a detectable reagent (e.g., a radionuclide, chromophore, enzyme that catalyzes a colorimetric or fluorometric reaction, and the like). The labeled polypeptide is contacted with cells expressing the candidate binding partner. The cells then are washed to remove unbound labeled polypeptide, and the presence of cell-bound label is determined by a suitable technique, chosen according to the nature of the label.
One example of a binding assay procedure is as follows. A recombinant expression vector containing the candidate binding partner cDNA is constructed. CV1-EBNA-1 cells in 10 cm2 dishes are transfected with this recombinant expression vector. CV-1/EBNA-1 cells (ATCC CRL 10478) constitutively express EBV nuclear antigen-1 driven from the CMV Immediate-early enhancer/promoter. CV1-EBNA-1 was derived from the African Green Monkey kidney cell line CV-1 (ATCC CCL 70), as described by McMahan et al., (EMBO J. 10:2821, 1991). The transfected cells are cultured for 24 hours, and the cells in each dish then are split into a 24-well plate. After culturing an additional 48 hours, the transfected cells (about 4×104 cells/well) are washed with BM-NFDM, which is binding medium (RPMI 1640 containing 25 mg/ml bovine serum albumin, 2 mg/ml sodium azide, 20 mM Hepes pH 7.2) to which 50 mg/ml nonfat dry milk has been added. The cells then are incubated for 1 hour at 37° C. with various concentrations of, for example, a soluble polypeptide/Fc fusion polypeptide made as set forth above. Cells then are washed and incubated with a constant saturating concentration of a 125I-mouse anti-human IgG in binding medium, with gentle agitation for 1 hour at 37° C. After extensive washing, cells are released via trypsinization. The mouse anti-human IgG employed above is directed against the Fc region of human IgG and can be obtained from Jackson Immunoresearch Laboratories, Inc., West Grove, Pa. The antibody is radioiodinated using the standard chloramine-T method. The antibody will bind to the Fc portion of any polypeptide/Fc polypeptide that has bound to the cells. In all assays, non-specific binding of 125I-antibody is assayed in the absence of the Fc fusion polypeptide/Fc, as well as in the presence of the Fc fusion polypeptide and a 200-fold molar excess of unlabeled mouse anti-human IgG antibody. Cell-bound 125I-antibody is quantified on a Packard Autogamma counter. Affinity calculations (Scatchard, Ann. N. Y. Acad. Sci. 51:660, 1949) are generated on RS/1 (BBN Software, Boston, Mass.) run on a Microvax computer. Binding can also be detected using methods that are well suited for high-throughput screening procedures such as scintillation proximity assays (Udenfriend et al., 1985, Proc Natl Acad Sci USA 82: 8672–8676), homogeneous time-resolved fluorescence methods (Park et al., 1999, Anal Biochem 269: 94–104), fluorescence resonance energy transfer (FRET) methods (Clegg R M, 1995, Curr Opin Biotechnol 6: 103–110), or methods that measure any changes in surface plasmon resonance when a bound polypeptide is exposed to a potential binding partner, such methods using, for example, a biosensor such as that supplied by Biacore AB (Uppsala, Sweden).
Compounds that can be assayed for binding to Claudin polypeptides of the invention include but are not limited to small organic molecules, such as those that are commercially available—often as part of large combinatorial chemistry compound ‘libraries’—from companies such as Sigma-Aldrich (St. Louis, Mass.), Arqule (Woburn, Ma.), Enzymed (Iowa City, Iowa), Maybridge Chemical Co.(Trevillett, Cornwall, UK), MDS Panlabs (Bothell, Wash.), Pharmacopeia (Princeton, N.J.), and Trega (San Diego, Calif.). Small organic molecules for screening using these assays are usually less than 10K molecular weight and can possess a number of physicochemical and pharmacological properties which enhance cell penetration, resist degradation, and/or prolong their physiological half-lives (Gibbs, J., 1994, Pharmaceutical Research in Molecular Oncology, Cell 79(2): 193–198). Compounds including natural products, inorganic chemicals, and biologically active materials such as proteins and toxins can also be assayed using these methods for the ability to bind to Claudin-23 polypeptides of the invention.
Specific screening methods are known in the art and along with integrated robotic systems and collections of chemical compounds/natural products are extensively incorporated in high throughput screening so that large numbers of test compounds can be tested for antagonist or agonist activity within a short amount of time. These methods include homogeneous assay formats such as fluorescence resonance energy transfer, fluorescence polarization, time-resolved fluorescence resonance energy transfer, scintillation proximity assays, reporter gene assays, fluorescence quenched enzyme substrate, chromogenic enzyme substrate and electrochemiluminescence, as well as more traditional heterogeneous assay formats such as enzyme-linked immunosorbant assays (ELISA) or radioimmunoassays. Homogeneous assays are “mix and read” assays that are very amenable to robotic application, whereas heterogeneous assays require separation of bound analyte from free by more complex unit operations such as filtration, centrifugation or washing. These assays are utilized to detect a wide variety of specific biomolecular interactions and the inhibition thereof by small organic molecules, including protein-protein, receptor-ligand, enzyme-substrate, etc. These assay methods and techniques are well known in the art and are described more fully in the following: High Throughput Screening: The Discovery of Bioactive Substances, John P. Devlin (ed.), Marcel Dekker, New York, 1997, ISBN: 0-8247-0067-8; and the internet sites of lab-robotics.org and sbsonline.org. The screening assays of the present invention are amenable to high throughput screening of chemical libraries and are suitable for the identification of small molecule drug candidates, antibodies, peptides, and other antagonists and/or agonists.
Yeast Two-Hybrid or “Interaction Trap ” Assays. Where a Claudin-23 polypeptide binds or potentially binds to another polypeptide (such as, for example, in a receptor-ligand interaction), the polynucleotide encoding a Claudin-23 polypeptide can also be used in interaction trap assays (such as, for example, described in Gyuris et al., 1993, Cell 75:791–803) to identify polynucleotides encoding the other polypeptide with which binding occurs or to identify inhibitors of the binding interaction. Polypeptides involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
Competitive Binding Assays. Another type of suitable binding assay is a competitive binding assay. To illustrate, biological activity of a variant can be determined by assaying for the variant's ability to compete with the native polypeptide for binding to the candidate binding partner. Competitive binding assays can be performed by conventional methodology. Reagents that can be employed in competitive binding assays include radiolabeled Claudin-23 and intact cells expressing Claudin-23 (endogenous or recombinant) on the cell surface. For example, a radiolabeled soluble Claudin-23 fragment can be used to compete with a soluble Claudin-23 variant for binding to cell surface receptors. Instead of intact cells, one could substitute a soluble binding partner/Fc fusion polypeptide bound to a solid phase through the interaction of Polypeptide A or Polypeptide G (on the solid phase) with the Fc moiety. Chromatography columns that contain Polypeptide A and Polypeptide G include those available from Pharmacia Biotech, Inc., Piscataway, N.J.
Assays to Identify Modulators of Intercellular Communication or Cell Activity. The influence of Claudin polypeptides of the invention on intercellular communication or cell activity can be manipulated to control these activities in target cells. For example, the disclosed Claudin polypeptides of the invention, polynucleotides encoding the disclosed Claudin polypeptides of the invention, or agonists or antagonists of such polypeptides can be administered to a cell or group of cells to induce, enhance, suppress, or arrest cellular communication or activity in the target cells. Identification of Claudin polypeptides of the invention, agonists or antagonists that can be used in this manner can be carried out via a variety of assays known to those skilled in the art. Included in such assays are those that evaluate the ability of a Claudin-23 polypeptide to influence intercellular communication or cell activity. Such an assay would involve, for example, the analysis of cell interaction in the presence of a Claudin-23 polypeptide. In such an assay, one would determine a rate of communication or cell stimulation in the presence of a Claudin-23 polypeptide and then determine if such communication or cell stimulation is altered in the presence of a candidate agonist or antagonist or another Claudin-23 polypeptide. Exemplary assays for this aspect of the invention include cytokine secretion assays, T-cell co-stimulation assays, and mixed lymphocyte reactions involving antigen presenting cells and T cells. These assays are well known to those skilled in the art.
In another aspect, the invention provides a method of detecting the ability of a test compound to affect the intercellular communication or co-stimulatory activity of a cell. In this aspect, the method comprises: (1) contacting a first group of target cells with a test compound including a Claudin-23 binding partner polypeptide or fragment thereof under conditions appropriate to the particular assay being used; (2) measuring the net rate of intercellular communication or co-stimulation among the target cells; and (3) observing the net rate of intercellular communication or co-stimulation among control cells containing a Claudin-23 binding partner polypeptide or fragment thereof, in the absence of a test compound, under otherwise identical conditions as the first group of cells. In this embodiment, the net rate of intercellular communication or co-stimulation in the control cells is compared to that of the cells treated with both a Claudin-23 molecule as well as a test compound. The comparison will provide a difference in the net rate of intercellular communication or co-stimulation such that an effector of intercellular communication or co-stimulation can be identified. The test compound can function as an effector by either activating or up-regulating, or by inhibiting or down-regulating intercellular communication or co-stimulation, and can be detected through this method.
Cell Proliferation, Cell Death, Cell Differentiation, and Cell Adhesion Assays. A polypeptide of the invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations. Many polypeptide factors discovered to date, including all known cytokines, have exhibited activity in one or more factor dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of a polypeptide of the invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK. The activity of a Claudin-23 polypeptide of the invention may, among other means, be measured by the following methods:
Assays for cell movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by Coligan et al., Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1–6.12.28; Taub et al., J. Clin. Invest. 95:1370–1376, 1995; Lind et al., APMIS 103:140–146, 1995; Muller et al., Eur. J. Immunol. 25:1744–1748; Gruber et al. J. Immunol. 152:5860–5867, 1994; Johnston et al., J. Immunol. 153:1762–1768, 1994.
Assays for cadherin adhesive and invasive suppressor activity include, without limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809–18817, 1995; Miyaki et al. Oncogene 11: 2547–2552, 1995; Ozawa et al. Cell 63:1033–1038, 1990.
Diagnostic and Other Uses of Claudin Polypeptides of the Invention and Polynucleotides
The polynucleotides encoding the Claudin polypeptides of the invention can be used for numerous diagnostic or other useful purposes. The polynucleotides of the invention can be used to express recombinant polypeptide for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding polypeptide is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in subjects to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to “subtract-out” known sequences in the process of discovering other novel nucleic acid molecules; for selecting and making oligomers for attachment to a “gene chip” or other support, including for examination of expression patterns; to raise anti-polypeptide antibodies using DNA immunization techniques; as an antigen to raise anti-DNA antibodies or elicit another immune response, and for gene therapy. Uses of Claudin polypeptides of the invention and fragmented polypeptides include, but are not limited to, the following: purifying polypeptides and measuring the activity thereof; delivery agents; therapeutic and research reagents; molecular weight and isoelectric focusing markers; controls for peptide fragmentation; identification of unknown polypeptides; and preparation of antibodies. Any or all polynucleotides suitable for these uses are capable of being developed into reagent grade or kit format for commercialization as products. Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without limitation “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.
Probes and Primers. Among the uses of a disclosed Claudin-23 polynucleotide, and combinations of fragments thereof, is the use of fragments as probes or primers. Such fragments generally comprise at least about 17 contiguous nucleotides of a DNA sequence. In other embodiments, a DNA fragment comprises at least 30, or at least 60, contiguous nucleotides of a DNA sequence. The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by Sambrook et al., 1989 and are described in detail above. Using knowledge of the genetic code in combination with the amino acid sequences set forth above, sets of degenerate oligonucleolides can be prepared. Such oligonucleotides are useful as primers, e.g., in polymerase chain reactions (PCR), whereby DNA fragments are isolated and amplified. In certain embodiments, degenerate primers can be used as probes for non-human genetic libraries. Such libraries include, but are not limited to, cDNA libraries, genomic libraries, and electronic EST (express sequence tag) or DNA libraries. Homologous sequences identified by this method can then be used as probes to identify Claudin-23 molecules from other species.
Diagnostics and Gene Therapy. The polynucleotides encoding Claudin polypeptides of the invention, and the disclosed fragments and combinations of these polynucleotides can be used by one skilled in the art using well-known techniques to analyze abnormalities associated with the genes corresponding to these polypeptides. This enables one to distinguish conditions in which this marker is rearranged or deleted. In addition, polynucleotides of the invention or a fragment thereof can be used as a positional marker to map other genes of unknown location. The DNA can be used in developing treatments for any disorder mediated (directly or indirectly) by defective, or insufficient amounts of, the genes corresponding to the polynucleotides of the invention. Disclosure herein of native nucleotide sequences permits the detection of defective genes, and the replacement thereof with normal genes. Defective genes can be detected in in vitro diagnostic assays, and by comparison of a native nucleotide sequence disclosed herein with that of a gene derived from a person suspected of harboring a defect in this gene.
Methods of Screening for Binding Partners. The Claudin polypeptides of the invention each can be used as reagents in methods to screen for or identify binding partners. For example, the Claudin polypeptides of the invention can be attached to a solid support material and may bind to their binding partners in a manner similar to affinity chromatography. In particular embodiments, a polypeptide is attached to a solid support by conventional procedures. As one example, chromatography columns containing functional groups that will react with functional groups on amino acid side chains of polypeptides are available (Pharmacia Biotech, Inc., Piscataway, N.J.). In an alternative, a polypeptide/Fc polypeptide (as discussed above) is attached to Polypeptide A- or Polypeptide G-containing chromatography columns through interaction with the Fc moiety. The Claudin polypeptides of the invention also find use in identifying cells that express a binding partner on the cell surface. Polypeptides are bound to a solid phase such as a column chromatography matrix or a similar suitable substrate. For example, magnetic microspheres can be coated with the polypeptides and held in an incubation vessel through a magnetic field. Suspensions of cell mixtures containing potential binding-partner-expressing cells are contacted with the solid phase having the polypeptides thereon. Cells expressing the binding partner on the cell surface bind to the fixed polypeptides, and unbound cells are washed away. Alternatively, Claudin polypeptides of the invention can be conjugated to a detectable moiety, then incubated with cells to be tested for binding partner expression. After incubation, unbound-labeled matter is removed and the presence or absence of the detectable moiety on the cells is determined. In a further alternative, mixtures of cells suspected of expressing the binding partner are incubated with biotinylated polypeptides. Incubation periods are typically at least one hour in duration to ensure sufficient binding. The resulting mixture is then passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin provides binding of the desired cells to the beads. Procedures for using avidin-coated beads are known (see Berenson, et al., J. Cell. Biochem., 10D:239, 1986). Washing to remove unbound material, and the release of the bound cells, are performed using conventional methods. In some instances, the above methods for screening for or identifying binding partners may also be used or modified to isolate or purify such binding partner molecules or cells expressing them.
Measuring Biological Activity. Polypeptides also find use in measuring the biological activity of Claudin-23-binding polypeptides in terms of their binding affinity. The polypeptides thus can be employed by those conducting “quality assurance” studies, e.g., to monitor shelf life and stability of polypeptide under different conditions. For example, the polypeptides can be employed in a binding affinity study to measure the biological activity of a binding partner polypeptide that has been stored at different temperatures, or produced in different cell types. The polypeptides also can be used to determine whether biological activity is retained after modification of a binding partner polypeptide (e.g., chemical modification, truncation, mutation, etc.). The binding affinity of the modified polypeptide is compared to that of an unmodified binding polypeptide to detect any adverse impact of the modifications on biological activity of the binding polypeptide. The biological activity of a binding polypeptide thus can be ascertained before it is used in a research study, for example.
Carriers and Delivery Agents. The polypeptides also find use as carriers for delivering agents attached thereto to cells bearing identified binding partners. The polypeptides thus can be used to deliver diagnostic or therapeutic agents to such cells (or to other cell types found to express binding partners on the cell surface) in in vitro or in vivo procedures. Detectable (diagnostic) and therapeutic agents that can be attached to a polypeptide include, but are not limited to, toxins, other cytotoxic agents, drugs, radionuclides, chromophores, enzymes that catalyze a colorimetric or fluorometric reaction, and the like, with the particular agent being chosen according to the intended application. Among the toxins are ricin, abrin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, ribosomal inactivating polypeptides, mycotoxins such as trichothecenes, and derivatives and fragments (e.g., single chains) thereof. Radionuclides suitable for diagnostic use include, but are not limited to, 123I, 131I, 99mTc, 111In, and 76Br. Examples of radionuclides suitable for therapeutic use are 131I, 211At, 77Br, 186Re, 188Re, 212Pb, 212Bi, 109Pd, 64Cu, and 67Cu. Such agents can be attached to the polypeptide by any suitable conventional procedure. The polypeptide comprises functional groups on amino acid side chains that can be reacted with functional groups on a desired agent to form covalent bonds, for example. Alternatively, the polypeptide or agent can be derivatized to generate or attach a desired reactive functional group. The derivatization can involve attachment of one of the bifunctional coupling reagents available for attaching various molecules to polypeptides (Pierce Chemical Company, Rockford, Ill.). A number of techniques for radiolabeling polypeptides are known. Radionuclide metals can be attached to polypeptides by using a suitable bifunctional chelating agent, for example. Conjugates comprising polypeptides and a suitable diagnostic or therapeutic agent (preferably covalently linked) are thus prepared. The conjugates are administered or otherwise employed in an amount appropriate for the particular application.
Cancer Diagnostics. Where Claudin-23 is down-regulated in cancer conditions detecting the presence of Claudin-23 polypeptide or polynucleotides can be used in cancer prognosis and diagnosis. The invention provides a method of diagnosing a cell proliferative disorder (e.g., a tumor) in a subject, comprising obtaining a fluid sample (e.g., blood, serum, urine, saliva, bile, lymph fluid, or spinal fluid) or a tissue biopsy (e.g., lymph, hepatic, or spleen tissue) or both fluid and biopsy samples from a subject and detecting a change in expression of Claudin-23. For example, methods include detecting down-regulation of expression of a Claudin-23 compared to a control sample. The method of detection may be any number of methods known in the art including radioimmunoassays, ELIZAs, Western blots, Northern or Southern Blots, polynucleotide amplification techniques, and the like.
Treating Diseases with Claudin Polypeptides, Agonists, and Antagonists Thereof
It is anticipated that the Claudin-23 polypeptides, fragments, variants, antagonists, agonists, antibodies, and binding partners of the invention will be useful for treating medical conditions and diseases including, but not limited to, conditions involving epithelial or endothelial barrier function or ion transport as described herein. The therapeutic molecule or molecules to be used will depend on the etiology of the condition to be treated and the biological pathways involved, and variants, fragments, and binding partners of Claudin polypeptides of the invention may have effects similar to or different from Claudin polypeptides of the invention. For example, an antagonist of the tight junction formation activity of Claudin polypeptides of the invention may be selected for treatment of conditions involving tight junction formation, but a particular fragment of a given Claudin-23 polypeptide may also act as an effective dominant negative antagonist of that activity. In the following paragraphs “Claudin-23 antagonists” or “antagonists of Claudin-23” refers to fragments of Claudin-23 polypeptides of the invention having a dominant negative effect on Claudin-23 polypeptide activity, polynucleotides such as antisense polynucleotides or silencing RNAs that decrease levels of Claudin-23 polypeptide expression, antagonistic antibodies, binding partners, and other Claudin-23 antagonists of the invention that function as antagonists of Claudin-23 polypeptide activity. In the following paragraphs “Claudin-23 agonists” or “agonists of Claudin-23” refers to Claudin-23 polypeptides of the invention having Claudin-23 polypeptide activity, polynucleotides that increase levels of Claudin-23 polypeptide expression, soluble forms, fragments, variants, antibodies, binding partners, and other Claudin-23 agonists of the invention that function as agonists of Claudin-23 polypeptide activity. It is understood that a specific molecule or molecules can be selected from those provided as embodiments of the invention by individuals of skill in the art, according to the biological and therapeutic considerations described herein. In one aspect, the invention entails administering compositions comprising a Claudin-23 polynucleotide and/or a Claudin-23 polypeptide and/or an agonist thereof to cells in vitro, to cells ex vivo, to cells in vivo, and/or to a multicellular organism. In still another aspect of the invention, the compositions comprise administering a Claudin-23-encoding polynucleotide for expression of a Claudin-23 polypeptide in a host organism for treatment of disease or disorder. Particularly useful in this regard is expression in a human subject for treatment of a dysfunction associated with aberrant (e.g., decreased) endogenous activity of a human Claudin-23 polypeptide. Furthermore, the invention encompasses the administration to cells and/or organisms of compounds found to increase the endogenous activity of Claudin polypeptides of the invention. One example of compounds that increases Claudin-23 polypeptide activity are agonistic antibodies, such as human or humanized antibodies, that bind to Claudin polypeptides of the invention or binding partners and increase Claudin-23 polypeptide activity by causing constitutive intracellular signaling (or “ligand mimicking”), or by preventing the binding of a native inhibitor of Claudin-23 polypeptide activity.
The invention encompasses the use of agonists of Claudin-23 activity to treat or ameliorate the symptoms of a disease for which increased Claudin-23 activity is beneficial. Such diseases include, but are not limited to, skin-related diseases as described in more detail below; inflammatory diseases (such as inflammatory bowel disease, inflammatory eye disease, herpetic stromal keratitis, and inflammatory eye disease associated with smoking and macular degeneration); allergies, including allergic rhinitis, contact dermatitis, atopic dermatitis and asthma; cell proliferative disorders including neoplasms/cancers and metastasis of cancer cells; ion transport disorders such as magnesium transport defects in the kidney; exposure to Clostridium perfringens enterotoxin (CPE); sudden infant death syndrome (SIDS); multiple sclerosis (MS); autoimmune encephalomyelitis; optic neuritis; progressive multifocal leukoencephalopathy (PML); and demyelinating neuropathy.
The disclosed Claudin-23 polypeptides and agonists thereof, including compositions and combination therapies described herein, are useful in medicines and methods of treatment involving disorders of the epithelium, such as disorders of the skin and/or of the mucous membranes. Such disorders include differentiative and proliferative disorders of the epithelium; hyperplastic growth of epithelium; acantholytic diseases, including Darier's disease, keratosis follicularis and pemphigus vulgaris; paraneoplastic pemphigus; aphthous stomatitis; bullous pemphigoid; epidermolysis bullosa, including bullous congenital icthyosiform erythroderma and Dowling-Meara type; pachyonychia congenita; hyperkeratosis, including epidermolytic hyperkeratosis; icthyosis, including icthyosis bullosa of Siemens and icthyosis vulgaris; palmoplantar keratoderma, including epidermolytic and non-epidermolytic palmoplantar keratoderma; pachyonychia congenita, including Jadassohn-Lewandowsky type; white sponge nevus; tricho-dento-osseous syndrome; tooth agenesis; autosomal dominant craniosyntosis, including Boston type; Papillon-Lefevre syndrome; Haim-Munk syndrome; prebubertal periodontis; burns; eczema; erythema, including erythema multiforme and erythema multiforme bullosum (Stevens-Johnson syndrome); inflammatory skin disease, including psoriasis, leukocutoclastic vasculitis, allergic contact dermatitis, pemphigus vulgaris, erythema multifome; lupus erythematosus; lichen planus; linear IgA bullous disease (chronic bullous dermatosis of childhood); loss of skin elasticity; fragility of the epidermis; ulcerations, including chronic ulcerations, diabetes-associated ulcerations, aphthous stomatitis, and mucosal surface ulcers; neutrophilic dermatitis (Sweet's syndrome); pityriasis rubra pilaris; pyoderma gangrenosum; acne; acne rosacea; alopecia areata; and toxic epidermal necrolysis; Kaposi's sarcoma; and erythema nodosum leprosum.
Agonists of Claudin-23 can be used to induce hair growth in patients in need thereof, for example, to treat alopecia, including but not limited to alopecia areata, male pattern baldness, and/or alopecia capitis totalis. Antagonists of Claudin-23 can be used to prevent unwanted growth of hair.
Conditions of the gastrointestinal system also are treatable with Claudin-23 agonists of the invention, compositions or combination therapies, including coeliac disease. In addition, the compounds, compositions and combination therapies of the invention are used to treat Crohn's disease; ulcerative colitis; and ulcers, including gastric and duodenal ulcers.
Also provided herein are methods for using Claudin-23 agonists of the invention, compositions or combination therapies to treat various hematologic and oncologic disorders. For example, Claudin-23 agonists of the invention are used to treat various forms of cancer, including acute myelogenous leukemia, Epstein-Barr virus-positive nasopharyngeal carcinoma, glioma, colon, stomach, prostate, renal cell, cervical and ovarian cancers, lung cancer (SCLC and NSCLC), including cancer-associated cachexia, fatigue, asthenia, paraneoplastic syndrome of cachexia and hypercalcemia. Additional diseases treatable with the subject Claudin-23 agonists of the invention, compositions or combination therapies are solid tumors, including sarcoma, osteosarcoma, and carcinomas such as adenocarcinoma (for example, breast cancer) and squamous cell carcinoma. In addition, the subject compounds, compositions or combination therapies are useful for treating leukemia, including acute myelogenous leukemia, chronic or acute lymphoblastic leukemia and hairy cell leukemia. Other malignancies with invasive metastatic potential can be treated with the Claudin-23 agonist compounds, compositions and combination therapies, including multiple myeloma.
Administration of Claudin Polypeptides, Antagonists, or Agonists Thereof
This invention provides compounds, compositions, and methods for treating a subject, such as a mammalian or a human subject, who is suffering from a medical disorder, and in particular a human Claudin-23-mediated disorder. Such human Claudin-23-mediated disorders include conditions caused (directly or indirectly) or exacerbated by binding between human Claudin-23 and a binding partner. For purposes of this disclosure, the terms “illness,” “disease,” “medical condition,” “abnormal condition” and the like are used interchangeably with the term “medical disorder.” The terms “treat”, “treating”, and “treatment” used herein includes curative, preventative (e.g., prophylactic) and palliative or ameliorative treatment. For such therapeutic uses, Claudin polypeptides of the invention and fragments, Claudin-23 polynucleotides encoding the Claudin polypeptides of the invention, and/or agonists or antagonists of a Claudin-23 polypeptide such as antibodies can be administered to the subject in need through well-known means. Compositions of the invention can contain a polypeptide in any form described herein, such as native polypeptides, variants, derivatives, oligomers, and biologically active fragments. In particular embodiments, the composition comprises a soluble polypeptide or an oligomer comprising soluble Claudin polypeptides of the invention.
Therapeutically Effective Amount. In practicing the method of treatment or use of the invention, a therapeutically effective amount of a therapeutic agent of the invention is administered to a subject having a condition to be treated, preferably to treat or ameliorate diseases associated with the activity of a human Claudin-23 polypeptide. “Therapeutic agent” includes, without limitation, any of the Claudin polypeptides of the invention, fragments, and variants; polynucleotides encoding the Claudin polypeptides of the invention, fragments, and variants; agonists or antagonists of the Claudin polypeptides of the invention such as antibodies; Claudin-23 polypeptide binding partners; complexes formed from the Claudin polypeptides of the invention, fragments, variants, and binding partners, and the like. As used herein, the term “therapeutically effective amount” means the total amount of each therapeutic agent or other active component of the pharmaceutical composition or method that is sufficient to show a meaningful subject benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual therapeutic agent or active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. As used herein, the phrase “administering a therapeutically effective amount” of a therapeutic agent means that the subject is treated with said therapeutic agent in an amount and for a time sufficient to induce an improvement, and preferably a sustained improvement, in at least one indicator that reflects the severity of the disorder. An improvement is considered “sustained” if the subject exhibits the improvement on at least two occasions separated by one or more weeks. The degree of improvement is determined based on signs or symptoms, and determinations may also employ questionnaires that are administered to the subject, such as quality-of-life questionnaires. Various indicators that reflect the extent of the subject's illness may be assessed for determining whether the amount and time of the treatment is sufficient. The baseline value for the chosen indicator or indicators is established by examination of the subject prior to administration of the first dose of the therapeutic agent. Typically, the baseline examination is done within about 60 days of administering the first dose. If the therapeutic agent is being administered to treat acute symptoms, the first dose is administered as soon as practically possible after the injury has occurred. Improvement is induced by administering a therapeutic agent of the invention until the subject manifests an improvement over baseline for the chosen indicator or indicators. In treating chronic conditions, this degree of improvement is obtained by repeatedly administering this medicament over a period of at least a month or more, e.g., for one, two, or three months or longer, or indefinitely. A period of one to six weeks, or even a single dose, often is sufficient for treating acute conditions. For injuries or acute conditions, a single dose may be sufficient. Although the extent of the subject's illness after treatment may appear improved according to one or more indicators, treatment may be continued indefinitely at the same level or at a reduced dose or frequency. Once treatment has been reduced or discontinued, it later may be resumed at the original level if symptoms should reappear.
Dosing. One skilled in the pertinent art will recognize that suitable dosages will vary, depending upon such factors as the nature and severity of the disorder to be treated, the subject's body weight, age, general condition, and prior illnesses and/or treatments, and the route of administration. Preliminary doses can be determined according to animal tests, and the scaling of dosages for human administration is performed according to art-accepted practices such as standard dosing trials. For example, the therapeutically effective dose can be estimated initially from cell culture assays. The dosage will depend on the specific activity of the compound and can be readily determined by routine experimentation. 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, while minimizing toxicities. Such information can be used to more accurately determine useful doses in humans. Ultimately, the attending physician will decide the amount of a therapeutic agent of the invention with which to treat each individual subject. Initially, the attending physician will administer low doses of a therapeutic agent of the invention and observe the subject's response. Larger doses of a therapeutic agent of the invention may be administered until the optimal therapeutic effect is obtained for the subject, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the invention should contain about 0.01 ng to about 100 mg (or about 0.1 ng to about 10 mg, or about 0.1 microgram to about 1 mg) of a therapeutic agent of the invention per kg body weight. In one embodiment of the invention, Claudin polypeptides of the invention or antagonists are administered one time per week to treat the various medical disorders disclosed herein, in another embodiment is administered at least two times per week, and in another embodiment is administered at least three times per week. If injected, the effective amount of a therapeutic agent of the invention per adult dose ranges from 1–20 mg/m2, and in one embodiment is about 5–12 mg/m2. Alternatively, a flat dose may be administered, whose amount may range from 5–100 mg/dose. Exemplary dose ranges for a flat dose to be administered by subcutaneous injection are 5–25 mg/dose, 25–50 mg/dose and 50–100 mg/dose. In one embodiment of the invention, the various indications described below are treated by administering a preparation acceptable for injection containing a therapeutic agent of the invention at 25 mg/dose, or alternatively, containing 50 mg per dose. The 25 mg or 50 mg dose may be administered repeatedly, particularly for chronic conditions. If a route of administration other than injection is used, the dose is appropriately adjusted in accord with standard medical practices. In many instances, an improvement in a subject's condition will be obtained by injecting a dose of about 25 mg of a therapeutic agent of the invention one to three times per week over a period of at least three weeks, or a dose of 50 mg of a therapeutic agent of the invention one or two times per week for at least three weeks, though treatment for longer periods may be necessary to induce the desired degree of improvement. For incurable chronic conditions, the regimen may be continued indefinitely, with adjustments being made to dose and frequency if such are deemed necessary by the subject's physician. The foregoing doses are examples for an adult subject who is a person who is 18 years of age or older. For pediatric subjects (age 4–17), a suitable regimen involves the subcutaneous injection of 0.4 mg/kg, up to a maximum dose of 25 mg of a therapeutic agent of the invention, administered by subcutaneous injection one or more times per week. If an antibody against a Claudin-23 polypeptide is used as a Claudin-23 polypeptide antagonist, a typical dose range is 0.1 to 20 mg/kg, and in one embodiment is 1–10 mg/kg. Another dose range for an anti-Claudin-23 polypeptide antibody is 0.75 to 7.5 mg/kg of body weight. Humanized antibodies are antibodies in which only the antigen-binding portion of the antibody molecule is derived from a non-human source. Such antibodies may be injected or administered intravenously.
Formulations. Compositions comprising an effective amount of a Claudin-23 polypeptide of the invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources), in combination with other components such as a physiologically acceptable diluent, carrier, or excipient, are provided herein. The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). Formulations suitable for administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The polypeptides can be formulated according to known methods used to prepare pharmaceutically useful compositions. They can be combined in admixture, either as the sole active material or with other known active materials suitable for a given indication, with pharmaceutically acceptable diluents (e.g., saline, Tris-HCl, acetate, and phosphate buffered solutions), preservatives (e.g., thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/or carriers. Suitable formulations for pharmaceutical compositions include those described in Remington's Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Company, Easton, Pa. In addition, such compositions can be complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, and the like, or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application, so that the characteristics of the carrier will depend on the selected route of administration. In one embodiment of the invention, sustained-release forms of Claudin polypeptides of the invention are used. Sustained-release forms suitable for use in the disclosed methods include, but are not limited to, Claudin polypeptides of the invention that are encapsulated in a slowly-dissolving biocompatible polymer (such as the alginate microparticles described in U.S. Pat. No. 6,036,978), admixed with such a polymer (including topically applied hydrogels), and or encased in a biocompatible semi-permeable implant.
Combinations of Therapeutic Compounds. A Claudin-23 polypeptide of the invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other polypeptides. As a result, pharmaceutical compositions of the invention may comprise a polypeptide of the invention in such multimeric or complexed form. The pharmaceutical composition of the invention may be in the form of a complex of the polypeptide(s) of invention along with polypeptide or peptide antigens. The invention further includes the administration of Claudin polypeptides of the invention or antagonists concurrently with one or more other drugs that are administered to the same subject in combination with the Claudin polypeptides of the invention or antagonists, each drug being administered according to a regimen suitable for that medicament. “Concurrent administration” encompasses simultaneous or sequential treatment with the components of the combination, as well as regimens in which the drugs are alternated, or wherein one component is administered long-term and the other(s) are administered intermittently. Components may be administered in the same or in separate compositions, and by the same or different routes of administration. Examples of components that may be included in the pharmaceutical composition of the invention are: cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-17, IL-18, IFN, G-CSF, thrombopoietin, stem cell factor, and erythropoietin. The pharmaceutical composition may further contain other agents which either enhance the activity of the polypeptide or compliment its activity or use in treatment. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with polypeptide of the invention, or to minimize side effects. Conversely, a Claudin-23 polypeptide or antagonist of the invention may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent. Additional examples of drugs to be administered concurrently include, but are not limited to, antivirals, antibiotics, analgesics, corticosteroids, antagonists of inflammatory cytokines, non-steroidal anti-inflammatories, pentoxifylline, thalidomide, and disease-modifying antirheumatic drugs (DMARDs) such as azathioprine, cyclophosphamide, cyclosporine, hydroxychloroquine sulfate, methotrexate, leflunomide, minocycline, penicillamine, sulfasalazine and gold compounds such as oral gold, gold sodium thiomalate, and aurothioglucose. Additionally, Claudin polypeptides of the invention or antagonists may be combined with a second Claudin-23 polypeptide/antagonist, including an antibody against a Claudin-23 polypeptide, or a Claudin-23 polypeptide-derived peptide that acts as a competitive inhibitor of a native Claudin-23 polypeptide.
Routes of Administration. Any efficacious route of administration may be used to therapeutically administer Claudin polypeptides of the invention or antagonists thereof, including those compositions comprising polynucleotides. Parenteral administration includes injection, for example, via intra-articular, intravenous, intramuscular, intralesional, intraperitoneal or subcutaneous routes by bolus injection or by continuous infusion, and also includes localized administration, e.g., at a site of disease or injury. Other suitable means of administration include sustained release from implants; aerosol inhalation and/or insufflation; eyedrops; vaginal or rectal suppositories; buccal preparations; oral preparations, including pills, syrups, lozenges or chewing gum; and topical preparations such as lotions, gels, sprays, ointments or other suitable techniques. Alternatively, polypeptideaceous Claudin-23 molecules of the invention or antagonists may be administered by implanting cultured cells that express a polypeptide, for example, by implanting cells that express Claudin polypeptides of the invention or antagonists. Cells may also be cultured ex vivo in the presence of polypeptides of the invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes. In another embodiment, the subject's own cells are induced to produce Claudin polypeptides of the invention or antagonists by transfection in vivo or ex vivo with a DNA that encodes Claudin polypeptides of the invention or antagonists. This DNA can be introduced into the subject's cells, for example, by injecting naked DNA or liposome-encapsulated DNA that encodes Claudin polypeptides of the invention or antagonists, or by other means of transfection. Polynucleotides of the invention may also be administered to subjects by other known methods for introduction of polynucleotide into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA). When Claudin polypeptides of the invention or antagonists are administered in combination with one or more other biologically active compounds, these may be administered by the same or by different routes, and may be administered simultaneously, separately or sequentially.
Oral Administration. When a therapeutically effective amount of polypeptide of the invention is administered orally, polypeptide of the invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% a polypeptide of the invention, and typically from about 25 to 90% polypeptide of the invention. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of polypeptide of the invention, and typically from about 1 to 50% a polypeptide of the invention.
Intravenous Administration. When a therapeutically effective amount of polypeptide of the invention is administered by intravenous, cutaneous or subcutaneous injection, polypeptide of the invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable polypeptide solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to polypeptide of the invention, 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 invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art. The duration of intravenous therapy using the pharmaceutical composition of the invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual subject. It is contemplated that the duration of each application of the polypeptide of the invention will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the invention.
Bone and Tissue Administration. For compositions of the invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage. Topical administration may be suitable for wound healing and tissue repair. Therapeutically useful agents other than a polypeptide of the invention, which may also optionally be included in the composition, as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention. Typically for bone and/or cartilage formation, the composition would include a matrix capable of delivering the polypeptide-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body. Such matrices may be formed of materials presently in use for other implanted medical applications. The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions will define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. Other potential materials are biodegradable and biologically well defined, such as bone or dermal collagen. Further matrices are comprised of pure polypeptides or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability. One embodiment is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns. In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the polypeptide compositions from disassociating from the matrix. A typical family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most common being cationic salts of carboxymethylcellulose (CMC). Other sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). The amount of sequestering agent useful herein is 0.5–20 wt %, typically 1–10 wt % based on total formulation weight, which represents the amount necessary to prevent desorbtion of the polypeptide from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the polypeptide the opportunity to assist the osteogenic activity of the progenitor cells. In further compositions, polypeptides of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin-like growth factor (IGF). The therapeutic compositions are also presently valuable for veterinary applications. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired subjects for such treatment with polypeptides of the invention. The dosage regimen of a polypeptide-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the polypeptides, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the subject's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other polypeptides in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.
Veterinary Uses. In addition to human subjects, Claudin polypeptides of the invention and antagonists are useful in the treatment of disease conditions in non-human animals, such as pets (dogs, cats, birds, primates, etc.), domestic farm animals (horses cattle, sheep, pigs, birds, etc.), or any animal that suffers from a TNFα-mediated inflammatory or arthritic condition. In such instances, an appropriate dose may be determined according to the animal's body weight. For example, a dose of 0.2–1 mg/kg may be used. Alternatively, the dose is determined according to the animal's surface area, an exemplary dose ranging from 0.1–20 mg/m2, and in one embodiment, from 5–12 mg/m2. For small animals, such as dogs or cats, a suitable dose is 0.4 mg/kg. In one embodiment, Claudin polypeptides of the invention or antagonists (preferably constructed from genes derived from the same species as the subject), is administered by injection or other suitable route one or more times per week until the animal's condition is improved, or it may be administered indefinitely.
Manufacture of Medicaments. The invention also relates to the use Claudin polypeptides of the invention, fragments, and variants; polynucleotides encoding the Claudin polypeptides of the invention, fragments, and variants; agonists or antagonists of the Claudin polypeptides of the invention such as antibodies; Claudin-23 polypeptide binding partners; complexes formed from the Claudin polypeptides of the invention, fragments, variants, and binding partners, etc, in the manufacture of a medicament for the prevention or therapeutic treatment of each medical disorder disclosed herein.
EXAMPLES
The following examples are intended to illustrate particular embodiments and not to limit the scope of the invention.
Example 1
Identification of Claudin-23
Mice deficient in RIP4 expression were analyzed (Holland et al., 2002, Current Biology 12: 1424–1428, which is incorporated by reference in its entirety herein). The protein kinase RIP4 is sometimes referred to as Death-Associated Kinase containing Ankyrin Repeats (DAKAR), Feldspar, protein kinase C-associated kinase or PKK (Chen et al., 2001, J Biol Chem 276: 21737–21744), or DIK (Bähr et al., 2000, J Biol Chem 275: 36350–36357). The phenotype observed for RIP4-deficient embryos shows some similarities to that reported for IKKalpha knockout animals (Takeda, K., et al., Science, 284:313. 1999; Hu, Y., et al., Science, 284:316, 1999; Hu, Y., et al., Nature, 410:710, 2001), but is distinct in certain respects. Unlike IKKalpha which has a role in the inflammation-related NFkB signaling pathway, it is suspected that the role of RIP4 in skin development is in regulating morphogenetic events, particularly keratinocyte proliferation and differentiation, not in regulating inflammatory responses. RIP4-deficient mice show variety of defects in cells derived from the keratinocyte lineage: fusion of all external orifices and of the esophagus; fusion of the interdigital epithelium; poor development of vibrissae; and replacement of the cornified layers of the skin by a thick layer of flattened, parakeratotic cells. RIP4 appears to act cell-autonomously within the keratinocyte cell lineage, because RIP4-deficient skin fails to differentiate when grafted onto a normal host.
RNA preparations were prepared from the skin of wild-type mouse embryos and also from the skin of embryos deficient in RIP4, using the RNeasy kit (Qiagen) followed by treatment with DNAse I (Ambion Inc.; Austin, Tex.) to eliminate residual chromosomal DNA contaminations. The RNA was labeled and hybridized to Affymetrix (Santa Clara, Calif.) U74Av2 chips according to the manufacturer's protocol. RESOLVER software (Rosetta Inpharmatics, a subsidiary of Merck & Co.; Whitehouse Station, N.J.) was used to analyze the data from the DNA array chips. RESOLVER analysis of the U74Av2 chips revealed a 4–8 fold down-regulation of a polynucleotide defined as IC2a-34134_at in RIP4-deficient mouse embryos. The down-regulated polynucleotide was homologous to a Genbank database entry, AK009330, for a putative mouse gene (SEQ ID NO:7). The down-regulated polynucleotide (SEQ ID NO:7) was used in a TBLASTN search of human genomic DNA sequences. The search revealed a similar human nucleic acid molecule, which is referred to herein as a human Claudin-23 polynucleotide (SEQ ID NO:5), present as a single exon in chromosome 8. The predicted polypeptide sequence of human Claudin-23 is provided in SEQ ID NO:6. Accordingly, the murine sequence first identified as down-regulated in RIP4-deficient mice is a homolog of human Claudin-23 and is referred to herein as a murine Claudin-23. Human Claudin-23 is notable for possession of the four characteristic transmembrane domains of Claudin polypeptides, the first of which spans from about amino acid 5 to about 27 of SEQ ID NO:6. This is consistent with other Claudin family members in that the first transmembrane domain is inserted into the cell membrane with the very N-terminal end of the Claudin polypeptide located inside the cell. Human Claudin-23's second TM domain comprises from about amino acids 77 to 99 of SEQ ID NO:6, a third TM domain comprises from about amino acids 112 to 134 of SEQ ID NO:6, and a fourth TM domain comprises from about amino acids 160 to 182 of SEQ ID NO:6. Based on the alignments with other family members and by reference to FIG. 1 of Morita et al. These predicted locations for the four TM domains of Human Claudin-23 places the first extracellular loop of Human Claudin-23 as beginning approximately around amino acid 28 to amino acid 31 of SEQ ID NO:6 and extending to approximately amino acid 76 of SEQ ID NO:6, and the second extracellular loop of Human Claudin-23 as beginning approximately around amino acid 135 to amino acid 138 of SEQ ID NO:6 and extending to approximately amino acid 159 of SEQ ID NO:6. The intracellular sequence between the second and third TM domains begins at approximately amino acid 100 to 103 of SEQ ID NO:6 and extends to approximately amino acid 111 of SEQ ID NO:6. The cytoplasmic tail domain of Human Claudin-23 begins approximately around amino acid 182 to amino acid 184 (e.g., about amino acid 183) of SEQ ID NO:6 and extends to the predicted C-terminus of SEQ ID NO:6 at amino acid 292.
The amino acid sequence of human Claudin-23 (SEQ ID NO:6) was compared with the amino acid sequences of other Claudin family members such as Claudin-1 (SEQ ID NO:1), Claudin4 (SEQ ID NO:2), Claudin-6 (SEQ ID NO:3), and Claudin-7 (SEQ ID NO:4), as shown in Table 1 below. This comparison used the GCG “pretty” multiple sequence alignment program, with amino acid similarity scoring matrix=blosum62, gap creation penalty=8, and gap extension penalty=2. The alignment of these sequences shown in Table 1 shows capitalized consensus residues that are identical among at least four of the amino acid sequences in the alignment, and the numbering of positions in the alignment is that of each residue's position in the human Claudin-23 amino acid sequence (SEQ ID NO:6). Embodiments of the invention include Claudin polypeptides and fragments of Claudin polypeptides comprising altered amino acid sequences. Altered Claudin-23 polypeptide sequences share at least 30%. or at least 40%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97.5%, or at least 99%, or at least 99.5% amino acid identity with a Claudin amino acid sequence shown in Table 1.
Amino acid substitutions and other alterations (deletions, insertions, and the like) to the Claudin polypeptides of the invention are predicted to be more likely to alter or disrupt Claudin polypeptide activities if they result in changes to the capitalized residues shown in Table 1, and particularly if those changes do not substitute a residue present in another Claudin polypeptide at that conserved position. Conversely, if a change is made to a Claudin amino acid sequence resulting in substitution of one or more Table 1 consensus sequence residues for the Claudin polypeptide residue at that conserved position, it is less likely that such an alteration will affect Claudin polypeptide function. For example, the consensus residue at position 40 in Table 1 is isoleucine in four of the five Claudin molecules, whereas human Claudin-23 has a proline at this position. Substitution of isoleucine or a chemically similar residues such as valine or one of the aliphatic amino acids at that position is considered less likely to alter the function of a Claudin-23 polypeptide than substitution of charged residues such as lysine or arginine. In addition to the substitution of isoleucine for proline at position 40 of SEQ ID NO:6 (symbolized as 40P—>I), the following additional variants of the amino acid sequence of SEQ ID NO:6 are expected to retain Claudin-23 polypeptide activity, based on substitution of an amino acid present in other Claudins at the corresponding position: 16C—>L; 18L—>W; 72F—>L; 78L—>Q; 121L—>F; and 176 S—>L:
Further types of variations that can be made to the Claudin-23 amino acid sequences of SEQ ID NO:6 and SEQ ID NO:8, such that the variants are expected to retain Claudin-23 polypeptide activity, are conservative changes to residues throughout the polypeptide. Conservative changes are those that substitute for a given amino acid an amino acid of a similar chemical type; amino acids can be grouped into similar chemical types as follows: aliphatic amino acids (alanine, glycine, isoleucine, leucine, proline, valine); aromatic amino acids (phenylalanine, tryptophan, tyrosine); amino acids with hydroxyl side chains (hydroxyproline, serine, threonine); sulfur-containing amino acids (cysteine, methionine); amino acids with amide side chains (asparagine, glutamine); amino acids with acidic side chains (aspartic acid, glutamic acid); and amino acids with basic side chains (arginine, histidine, lysine). Examples of conservative substitutions in SEQ ID NO:6 are: 37L—>G; 41V—>I; 46Y—>W; 58S—>T; 160L—>I; and 164Y—>W.
TABLE 1
Protein (SEQ ID NO)
1
49
hClaudin4
(2)
MAsmGlQvmGiaLAvLGWlavmlccAlPmWrvtafiGsnIVtsQtiweGL
hClaudin6
(3)
MAsaGmQiLGvvLtlLGWvnglvscAlPmWkvtafiGnsIVvaQvvweGL
hClaudin1
(1)
MAnaGlQlLGfiLAfLGWigaivstAlPqWriysyaGdnIVtaQamyeGL
hClaudin7
(4)
MAnsGlQlLGfsmAlLGWvglvactAiPqWqmssyaGdnIitaQamykGL
hClaudin23
(6)
MrtpvvmtLGmvLApcGlllnltgtlaPgWrlvkgflnqpVdve.lyqGL
Consensus
MA--G-Q-LG--LA-LGW-------A-P-W------G--IV--Q----GL
50
99
hClaudin4
(2)
WMnCVvQSTGqmqCKvyDSlLaL.pqdLQAaRALviisiivaalgvllsv
hClaudin6
(3)
WMsCVvQSTGqmqCKvyDSlLaL.pqdLQAaRALcViallvalfgllvyl
hClaudin1
(1)
WMsCVsQSTGqiqCKvfDSlLnL.sstLQAtRALmVvgillgviaifvat
hClaudin7
(4)
WMdCVtQSTGmmsCKmyDSvLaL.ssaLQAtRALmVvslvlgflamfvat
hClaudin23
(6)
WdmCreQSsrereCgqtDqwgyfeaqpvlvaRALmVtslaatvlglllas
Consensus
WM-CV-QSTG---CK--DS-L-L----LQA-RAL-V--------------
100
144
hClaudin4
(2)
vGgKCtnCl.eDesaKaktmivaGvvFllAGLmvivpvsWtaHniiqdFY
hClaudin6
(3)
aGaKCttCv.eekdsKarlvltsGivFvisGvltLipvcWtaHavirdFY
hClaudin1
(1)
vGmKCmkCledDevqKmrmavigGaiFllAGLaiLvataWygnrivqeFY
hClaudin7
(4)
mGmKCtrCggdDkvkKariamggGiiFivAGLaaLvacsWygHqivtdFY
hClaudin23
(6)
lGvrC....wqDepnfv.laglsGvvlfvAGLlgLipvsWynHflgdrdv
Consensus
-G-KC--C---D---K-------G--F--AGL--L----W--H-----FY
145
194
hClaudin4
(2)
nPlvasgqkrEmGasLyvGWAaSgLlLLGGgLLcCn.CP...prtdkpYs
hClaudin6
(3)
nPlvaeaqkrElGasLylGWAaSgLlLLGGgLLcCt.CPsggsqgpshYm
hClaudin1
(1)
dPmtpvnaryEfGqaLftGWAaasLcLLGGaLLcCs.CP....rkttsYp
hClaudin7
(4)
nPliptnikyEfGpaifiGWAgSaLviLGGaLLsCs.CP..gneskagYr
hClaudin23
(6)
lPapaspvtvqvsysLvlGylgScLlLLGGfsLalsfaPwcdercrrrrk
Consensus
-P--------E-G--L--GWA-S-L-LLGG-LL-C--CP---------Y-
195
244
hClaudin4
(2)
akysa...arsaaas nYv-------------------------------
hClaudin6
(3)
arystsapaisrgps eYptknyv--------------------------
hClaudin1
(1)
tprpypkpapssg.k.dYv-------------------------------
hClaudin7
(4)
aprsypk..snss.k.eYv-------------------------------
hClaudin23
(6)
gpsagprrssvstiqvewpepdlapaikyysdgqhrpppaqhrkpkpkpk
Concensus
-----------------Y--------------------------------
245
292
hClaudin23
(6)
vgfpmprprpkaytnsvdvldgegwesqdapscsthpcdsslpcdsdl
Additional types of variations that can be made to the Claudin-23 amino acid sequences of SEQ ID NO:6 and SEQ ID NO:8 are changes to the transmembrane domains of these polypeptides. Substitutions that preserve the hydrophobic nature of these transmembrane domains by substituting for transmembrane residues uncharged amino acids, and particularly aliphatic or aromatic amino acids, are expected to result in variants that retain Claudin-23 polypeptide activity. Insertions of 1 through about 10 amino acids, or deletions of 1 through about 8 amino acids, for each transmembrane domain, where such insertions or deletions preserve the hydrophobic nature of these transmembrane domains, are also within the scope of the invention. The overall topological structure of such Claudin-23 variants can be predicted using a program such as the TMHMM (TransMembrane Hidden Markov Model) application available on the internet from the Center for Biological Sequence Analysis of the Technical University of Denmark (cbs.dtu.dk/services/TMHMM). Examples of such insertions or deletions that retain the four transmembrane structure of Claudin-23 are: insertion of five leucine residues between amino acids 15 and 16 of SEQ ID NO:6; insertion of ten leucine residues between amino acids 15 and 16 of SEQ ID NO:6; deletion of amino acids 19 through 23 of SEQ ID NO:6; and deletion of amino acid 16 and amino acids 19 through 25 of SEQ ID NO:6. For the previous examples of Claudin-23 transmembrane domain variants, TMHMM analysis clearly indicated the presence of four transmembrane domains having the same overall topology as the Claudin-23 polypeptide of SEQ ID NO:6. Further, TMHMM analysis can indicate when a variant of a Claudin-23 polypeptide is not expected to retain the four transmembrane structure: for example, deletion of amino acids 11 through 12, 16, and 19 through 25 of SEQ ID NO:6 produced a variant that was predicted by TMHMM analysis to lack the most N-terminal transmembrane domain.
Polynucleotide sequences encoding human Claudin-23 map to human chromosome 8p23.1. Polynucleotides encoding Claudin polypeptides of the invention can be used to analyze genetic abnormalities associated with these chromosomal regions, for example, enabling one of skill in the art to identify subjects in which chromosomal regions comprising Claudin-encoding sequences are rearranged or deleted. There is also substantial utility in polynucleotides that can be used to confirm or to eliminate a particular genetic locus as a genetic factor for a kindred presenting with a hereditary disease. Human genetic disorders that have been mapped to the same chromosomal region as Claudin-23 include Keratolytic Winter Erythema (KWE) and Diamond-Blackfan Anemia 2 (DBA2). Claudin-23 polynucleotides are useful for more precisely mapping these genetic disorders within the p23 region of chromosome 8. Also, Claudin-23 is a candidate for being the gene implicated in the skin disorder KWE, because changes in Claudin-23 expression are associated with the skin disorders observed in RIP4-deficient mice as described above. Further, KWE is an autosomal dominant disorder; if Claudin-23 was the gene responsible for KWE, then KWE could be caused by dominant-negative forms of Claudin-23, with loss-of-function mutations of Claudin-23 being recessive lethal.
Example 2
Expression of Human Claudin-23 Transcripts and Proteins
The expression of murine Claudin-23 in different tissues was detected using RT-PCR. PCR was carried out as follows: 5′ (sense) oligo sequence was AAG AGG CTA CGC AGG ATG CGG ACG CC (SEQ ID NO:9) and the 3′ (antisense) oligo was CTG TCT ACA GGT CGG AGT CAC AGG GCA (SEQ ID NO:10) were incubated according to standard protocols with dNTP's, Clontech Mouse multiple tissue cDNAs (heart, brain, spleen, lung, liver, skeletal muscle, kidney, testis, E7, E11, E15, and E17), and Applied Biosystems Amplitaq as the polymerase. PCR cycling parameters were: denature at 95 degrees C. for 5 minutes followed by 35 cycles of (a) denaturation at 95 degrees C. for 1 minute; (b) annealing at 65 degrees C. for 1 minute; and (c) primer extension at 72 degrees C. for 2 minutes.
Claudin-23 transcripts were detected in the following murine tissues: brain, lung, and testis. Claudin-23 was also detected in embryos at stages E15 and E17. In addition, as described above, the skin showed differing rates of expression between normal and RIP4-deficient mice. In addition, human Claudin-23 was detected in Dendritic cells (DC) by standard RT-PCR. Human Claudin-23 was detected in sorted CD1b/c+ DC RNA from a normal Flt3-L treated donor. Claudin-23 was also weakly detected in CD8+ T cells and CD4+ T cells.
Example 3
Monoclonal Antibodies That Bind Polypeptides of the Invention
This example illustrates a method for preparing monoclonal antibodies that bind Claudin-23 polypeptides of the invention. Other conventional techniques may be used, such as those described in U.S. Pat. No. 4,411,993. Suitable immunogens that may be employed in generating such antibodies include, but are not limited to, purified Claudin-23 polypeptide of the invention, an immunogenic fragment thereof, and cells expressing high levels of said Claudin-23 polypeptide or an immunogenic fragment thereof. DNA encoding a Claudin-23 polypeptide of the invention can also be used as an immunogen, for example, as reviewed by Pardoll and Beckerleg in Immunity 3: 165, 1995.
Rodents (BALB/c mice or Lewis rats, for example) are immunized with Claudin-23 polypeptide immunogen emulsified in an adjuvant (such as complete or incomplete Freund's adjuvant, alum, or another adjuvant, such as Ribi adjuvant R700 (Ribi, Hamilton, and injected in amounts ranging from 10–100 micrograms subcutaneously or intraperitoneally. DNA may be given intradermally (Raz et al., 1994, Proc. Nail. Acad. Sci. USA 91: 9519) or intamuscularly (Wang et al., 1993, Proc. Natl. Acad. Sci. USA 90: 4156); saline has been found to be a suitable diluent for DNA-based antigens. Ten days to three weeks days later, the immunized animals are boosted with additional immunogen and periodically boosted thereafter on a weekly, biweekly or every third week immunization schedule.
Serum samples are periodically taken by retro-orbital bleeding or tail-tip excision to test for Claudin-23 polypeptide-specific antibodies by dot-blot assay, ELISA (enzyme-linked immunosorbent assay), immunoprecipitation, or other suitable assays, such as FACS analysis of inhibition of binding of Claudin-23 polypeptide of the invention to a Claudin-23 polypeptide binding partner. Following detection of an appropriate antibody titer, positive animals are provided one last intravenous injection of Claudin-23 polypeptide of the invention in saline. Three to four days later, the animals are sacrificed, and spleen cells are harvested and fused to a murine myeloma cell line, e.g., NS1 or preferably P3X63Ag8.653 (ATCC CRL-1580). These cell fusions generate hybridoma cells, which are plated in multiple microtiter plates in a HAT (hypoxanthine, aminopterin and thymidine) selective medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
The hybridoma cells may be screened by ELISA for reactivity against purified Claudin-23 polypeptide of the invention by adaptations of the techniques disclosed in Engvall et al., (Immunochem. 8: 871, 1971) and in U.S. Pat. No. 4,703,004. A preferred screening technique is the antibody capture technique described in Beckmann et al., (J. Immunol. 144: 4212, 1990). Positive hybridoma cells can be injected intraperitoneally into syngeneic rodents to produce ascites containing high concentrations (for example, greater than 1 milligram per milliliter) of anti-Claudin-23 polypeptide monoclonal antibodies. Alternatively, hybridoma cells can be grown in vitro in flasks or roller bottles by various techniques. Monoclonal antibodies can be purified by ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can also be used, as can affinity chromatography based upon binding to the Claudin-23 polypeptide of the invention.
All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
Sequences Presented in the Sequence Listing
SEQ ID NO
Sequence Type
Description
SEQ ID NO: 1
Amino acid
Human Claudin-1
SEQ ID NO: 2
Amino acid
Human Claudin-4
SEQ ID NO: 3
Amino acid
Human Claudin-6
SEQ ID NO: 4
Amino acid
Human Claudin-7
SEQ ID NO: 5
Nucleotide
Human Claudin-23
SEQ ID NO: 6
Amino acid
Human Claudin-23
SEQ ID NO: 7
Nucleotide
Murine Claudin-23
(GenBank AK009330)
SEQ ID NO: 8
Amino acid
Murine Claudin-23
SEQ ID NO: 9
Nucleotide
Claudin-23 ‘sense’
oligonucleotide primer
SEQ ID NO: 10
Nucleotide
Claudin-23 ‘antisense’
oligonucleotide primer
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10965972
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immunex corporation
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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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Mar 31st, 2022 02:17PM
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Mar 31st, 2022 02:17PM
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Amgen
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:amgn
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Amgen
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Mar 13th, 2012 12:00AM
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Nov 6th, 2009 12:00AM
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https://www.uspto.gov?id=US08133976-20120313
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Methods of use of the TACI/TACI-L interaction
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The invention discloses a novel interaction between a TNF receptor (TACI) and its interacting ligand (TACI-L). Also disclosed are methods of screening candidate molecules to determine potential antagonists and agonists of the TACI/TACI-L interaction. The use of the antagonists and agonists as therapeutics to treat autoimmune diseases, inflammation, and to inhibit graft vs. host rejections is further disclosed.
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8133976
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1. A method of inhibiting cellular activation through the binding of TACI (SEQ ID NO:2) to TACI-L (SEQ ID NO:4), comprising
(a) exposing a cell expressing a TACI-L polypeptide of SEQ ID NO:4 to a TACI-Fc conjugate comprising amino acids 2-166 of SEQ ID NO:2 conjugated to a human Fc domain;
(b) allowing the TACI-Fc conjugate to bind the TACI-L polypeptide; and
(c) inhibiting the binding of an endogenous TACI polypeptide comprising SEQ ID NO:2 to the TACI-L polypeptide and thereby inhibiting the activation of the cell.
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1
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 12/220,483, filed Jul. 23, 2008, which is a continuation of U.S. patent application Ser. No. 09/302,863 , filed Apr. 30, 1999, which is incorporated herein by reference.
FIELD OF INVENTION
This invention relates generally to the interaction between the transmembrane activator and CAML-interactor (TACI) protein and its ligand, TACI ligand (TACI-L), use of the interaction in screening assays thereof, and related kits.
BACKGROUND OF THE INVENTION
Cellular change is often triggered by the binding of an extrinsic element, such as a ligand, to the extracellular domain of a cell surface membrane receptor protein. This binding can result in cellular changes by activating and controlling intracellular signaling cascades and cell regulatory mechanisms. As such, understanding the initial binding interaction between the ligand and its receptor protein can be of great interest to the scientific community. A greater understanding of this interaction would enable one skilled in the art to modulate the resulting signaling cascade governed by the ligand/receptor interaction by selecting agents for co-stimulation or inhibition of the binding of the ligand to its receptor.
The tumor necrosis factor (TNF) receptor family is a class of mammalian signaling molecules that play an important role in protection against infection and immune inflammatory responses such as cellular signal transduction, stimulation of cells to secrete cytokines, cytotoxic T cell proliferation, general cellular proliferation, lymph node formation, bone formation, and bone degradation. TNF-mediated cellular signaling often involves a molecular activation cascade, during which a receptor triggers a ligand-receptor mediated signal. Alterations in TNF activation can have profound effects on a multitude of cellular processes, such as the activation or inhibition of cell-specific responses, cell proliferation, inflammatory reactions, and cell death.
The interactions between TNF ligands and receptors may result in one-directional signaling (the interaction of the TNF receptor/ligand triggers a signaling cascade in the receptor only) or may result in bi-directional or reverse signaling. In the instances of bi-directional or reverse signaling, the interaction would not only activate the signaling cascade of the TNF receptor but would also trigger a signaling cascade in a cell bearing the TNF ligand. (S. Wiley et al., Jour. of Immun., 3235-39 (1996).) Thus, understanding the interaction between a TNF receptor and ligand may result in therapeutic treatments involving the inhibition or enhancement of either one or both of the TNF receptor activity or TNF ligand activity.
One member of the TNF receptor family is the transmembrane activator and CAML-interactor (TACI), a cell surface protein. The TACI protein has been isolated and is described in WO 98/39361. When activated, TACI stimulates the influx of calcium in lymphocytes and initiates the activation of a transcription factor through a combination of a Ca2+-dependent pathway and a Ca2+-independent pathway. Functions of TACI include controlling the response of lymphocytes to cancer and to foreign antigens in infections, graft rejection, and graft-vs.-host disease (GVHD). Furthermore, activation of lymphocyte signaling allows the positive selection of functional lymphocytes and negative selection against self-reactive clones. (WO 98/39361 at 15.)
TACI modulated signals are often activated by a extracellular ligand/receptor interaction, which then triggers an intracellular protein/protein interaction. One of the intracellular proteins which bind with the TACI protein has been identified. TACI interacts with the calcium-signal modulating cyclophilin ligand (CAML), a protein associated with the calcium pathway in lymphocytes. According to WO 98/39361, after the binding of the extracellular domain of TACI to an extracellular ligand, the cytoplasmic domain of TACI binds CAML, initiating a Ca2+-dependent activation pathway, which includes the activation of the transcription factors, NF-AT, AP-1 and NFkB, a factor implicated in the actions of other members of the TNF-receptor family. The regions for the interaction between TACI and CAML were defined as the cytoplasmic COOH-terminal 126 amino acids of TACI and the NH2-terminal 201 amino acids of CAML. CAML's ability to act as a signaling intermediate was verified by the inhibition of TACI-induced activation of the transcription factor when blocked by a dominant-negative mutant. (Von Büllow, G. et al., Science, Vol. 278, p.138-141 (1997).)
Although this interaction between the cytoplasmic domain of TACI and CAML has been identified, little is known about the extracellular ligand with which TACI interacts to initiate the intracellular cascades. Given the important role TACI plays in signal transduction and given the potential therapies that may arise from the manipulation of the signaling cascades, there is a need in the art for the identification and understanding of the interaction of TACI with its signaling ligand. Further, there is a need for the development of assays and therapeutic methods using the interaction between TACI and its signaling ligand.
Another TNF protein that has been recently discovered is a ligand that has been designated Neutrokine α, which is described in WO 98/18921. Identical nucleotide and polypeptide sequences have also been disclosed as “TL5” in EP 0869180A1 and as “63954” in WO 98/27114. As a member of the TNF family, Neutrokine α polypeptides were described as useful in the treatment of tumor and tumor metastasis, infections by bacteria, virus and other parasites, immunodeficiencies, inflammatory disease, lymphadenopathy, autoimmune diseases, and GVHD. Neutrokine α was also described as useful to mediate cell activation and proliferation. Further, Neutrokine α polypeptides were described as primary mediators of immune regulation and inflammatory response. (WO 98/18921 at 11; EP 0869180A1 at 3.)
As Neutrokine α polypeptides may inhibit immune cell functions, the ligand was described as also having a variety of anti-inflammatory activities. (WO 98/18921 at 49.) Specifically, it was said that Neutrokine α polypeptides could be used as an anti-neovascularizing agent to treat solid tumors and for other non-cancer indications in which blood vessel proliferation is not wanted. (Id.) The polypeptides could also be employed to enhance host defenses against resistant chronic and acute infections and to inhibit T-cell proliferation by the inhibition of IL-2 biosynthesis. Finally, Neutrokine α polypeptides could also be used to stimulate wound healing and to treat other fibrotic disorders. (Id.)
As such activities may be modulated by the Neutrokine α polypeptides, knowledge of how the ligand functions would be of significant interest to the scientific community. WO 98/18921, EP 0869180A1 and WO 98/27114, however, fail to identify specific receptors with which Neutrokine α polypeptides bind. Identification of the related TNF receptor would allow those skilled in the art to identify antagonists which may then be used in therapies to treat the disorders associated with the Neutrokine α polypeptides. Thus, there is a need to greater understand this TNF ligand, identify the receptors with which it interacts, and determine how the interaction functions.
SUMMARY OF THE INVENTION
This invention aids in fulfilling these needs in the art by identifying a novel interaction between the extracellular domain of TACI and the Neutrokine α polypeptide (hereinafter referred to as TACI ligand (TACI-L)), and uses thereof. Specifically, the invention encompasses the identification of a novel interaction between TACI (SEQ. ID. NO.: 2) and TACI-L (SEQ. ID. NO.: 4).
The present invention provides a screening method for identifying molecules that enhance or inhibit the TACI/TACI-L interaction, or that prevent or inhibit dissociation of a complex formed by TACI and TACI-L. This screening method involves contacting a mixture of cells which express TACI and cells which express TACI-L with a candidate molecule, measuring cellular responses, and detecting the ability of the candidate molecule to inhibit or enhance the interaction between TACI and TACI-L or inhibit the dissociation of the complex formed by TACI and TACI-L. Successful inhibition indicates that the candidate molecule is an antagonist. Increased activation of TACI or TACI-L indicates that the candidate molecule is an agonist. The candidate molecules are preferably small molecules, antibodies or peptides.
In a further aspect of the present invention, a solid phase method may be used to identify small molecules which inhibit the interaction between TACI and TACI-L. Using this method, TACI may be bound and is placed in a mixture with labeled TACI-L. After contact, the amount of signal is measured. Diminished levels of signal indicate that the candidate molecule inhibited the interaction between TACI and TACI-L.
In a still further aspect, the present invention provides a screening method for identifying molecules which mimic the biological activity of the TACI/TACI-L interaction. This screening method involves adding a candidate molecule that binds to TACI or TACI-L to a biological assay and comparing the biological effect of the candidate molecule to the biological effect of TACI/TACI-L complex.
In yet a further aspect, the invention provides for a therapeutic use of agonists and antagonists of the TACI/TACI-L complex in the treatment of diseases modulated by the complex.
In still a further aspect, the invention provides for the antagonists and agonists of the TACI/TACI-L complex.
Finally, the invention relates to a kit to aid in the above determinations and uses.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the nucleotide (SEQ. ID. NO.:1) (FIG. 1a) and deduced amino acid (SEQ. ID. NO.:2)(FIG. 1b) sequences of the TACI protein.
FIG. 2 shows the nucleotide (SEQ. ID. NO.:3) (FIG. 2a) and deduced amino acid (SEQ. ID. NO.:4)(FIG. 2b) sequences of the TACI-L protein.
FIG. 3 shows the amino acid sequence of a polypeptide (SEQ. ID. NO.:5), in which a CMV leader followed by a leucine zipper motif is fused to the N-terminal region of the amino acid sequence of TACI-L.
FIG. 4 shows the results of a plate binding assay capturing TACI-L in which the ligand is diluted 1:2. FIG. 4a demonstrates the results of the assay and shows the complete saturation of the receptor binding sites. FIG. 4b, the Scatchard plot corresponding to FIG. 4a, demonstrates the actual number of sites that were bound. From these results, an affinity constant of 1.53×10−9 can be generated.
FIG. 5 shows the results of a plate binding assay capturing TACI-L in which the ligand is diluted 1:5. FIG. 5a demonstrates the results of the assay and shows the complete saturation of the receptor binding sites. FIG. 5b, the Scatchard plot corresponding to FIG. 5a, demonstrates the actual number of sites that were bound. From these results, an affinity constant of 2.2×10−9 can be generated.
FIG. 6 shows the results of a plate binding assay capturing HuTACI/Fc. FIG. 6a graphs the complete saturation of the receptor binding sites. FIG. 6b, the Scatchard graph which corresponds to FIG. 6a, demonstrates the actual number of sites that were bound. The Scatchard plot of FIG. 6b demonstrates a curvilinear binding, with a low affinity constant of 5.7×10−10 and a high affinity constant of 1.0×10−10.
DETAILED DESCRIPTION OF THE INVENTION
The terms “TACI” and “TACI protein” are used interchangeably to define the TNF receptor disclosed by WO 98/39361. TACI comprises an extracellular domain, a transmembrane domain, and a cytoplasmic domain.
“Fragments” of TACI encompass truncated amino acid sequences of the TACI protein that retain the biological ability to bind to TACI-L. An example of such a fragment is the extracellular domain. Such fragments are identified in WO 98/39361, which is incorporated in this application in its entirety. “Soluble TACI” includes truncated proteins that lack a functional transmembrane domain of the protein but retain the biological activity of binding to TACI-L. The soluble, extracellular domain can be used to inhibit cellular activation.
“Homologous analogs” of TACI include isolated nucleic acids of the TACI protein that are at least about 75% identical to SEQ.ID.NO.:1 and retain the biological activity of binding to TACI-L. Also contemplated by the term are embodiments in which a nucleic acid molecule comprises a sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.9% identical to SEQ.ID.NO.:1 and retain the biological activity of binding to TACI-L. Further included are nucleic acids which are at least 85% similar, at least 95% similar, or at least 99% similar to nucleic acids that encode the amino acids of the TACI protein, as described in SEQ. ID. NO.:2, and that maintain a binding affinity to TACI-L. Still further included are all substantially homologous analogs and allelic variations.
The percent identity and percent similar may be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two nucleic acid molecules can be determined by comparing their sequences using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities), and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. Other programs used by one skilled in the art of sequence comparison may also be used.
The terms “TACI-L” and “TACI ligand” are used interchangeably to define the member of the TNF ligand family disclosed by WO 98/18921. TACI-L is also disclosed as “TL5” in EP 0869180A1 and as “63954” in WO 98/27114. The full-length TACI-L comprises an extracellular domain, a transmembrane domain, and a cytoplasmic domain. Although the exact location of the extracellular, transmembrane, and cytoplasmic domains may differ slightly due to different analytical criteria for identifying the functional domains, the range of amino acids 1 to 46 generally represents the intracellular domain; amino acids 47 to 72 represent the transmembrane domain, and amino acids 73-285, the extracellular domain.
“Fragments” of TACI-L encompass truncated amino acids of the TACI-L protein that retain the biological ability to bind to TACI. An example of such a fragment is the extracellular domain of TACI-L, which binds TACI. Another example of a TACI-L fragment is amino acids 123-285 of the extracellular domain of the TACI ligand.
“Soluble TACI-L” includes truncated proteins that lack a functional transmembrane domain of the protein but retain the biological activity of binding to TACI. The soluble, extracellular domain can be used to inhibit cellular activation.
“Homologous analogs” of TACI-L include isolated nucleic acids of the TACI-L protein that are at least about 75% identical to SEQ.ID.NO.:3 and retain the biological ability to bind to TACI. Also contemplated by the term are embodiments in which a nucleic acid molecule comprises a sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.9% identical to SEQ.ID.NO.:3 and retain the biological ability to bind to TACI. Further included are nucleic acids which are at least 85% similar, at least 95% similar, or at least 99% similar to nucleic acids that encode the amino acids of TACI-L, as described in SEQ. ID. NO.:4 and that maintain a binding affinity to TACI. Still further included are all substantially homologous analogs and allelic variations.
Sequences are substantially homologous when at least 50% (preferably 60%, more preferably 65%, more preferably 75%, more preferably 85%, and most preferably 99%) of the nucleotides match over the defined length of the DNA sequences. Sequences which are substantially homologous can be identified by comparing the sequences using software known in the art or by the well-known Southern hybridization experiment. Substantially homologous analogs and allelic variations must maintain the same biological activity as the protein they are homologous to (e.g. bind to the same receptor or ligand).
The terms “TACI/TACI-L complex” or “TACI/TACI-L interaction” are used interchangeably and refer to the protein unit formed by the binding interaction of TACI to TACI-L.
The term “TACI/TACI-L fragment complex” includes the protein units formed in which at least one binding partner is either a fragment of TACI or TACI-L (e.g. the binding interaction of a TACI fragment to TACI-L, TACI to a TACI-L fragment, or a TACI fragment to a TACI-L fragment) or a homologous analog of TACI or TACI-L. The TACI/TACI-L fragment complex has the same biological activity, effects, and uses as the TACI/TACI-L complex, as described below. The term “biological activity” includes the binding of TACI to TACI-L or fragments thereof.
The term “biological effects” includes any cellular changes or effects which result from a protein-protein interaction or the interaction of a protein with an agonist or antagonist. Examples of a biological effect of the TACI/TACI-L complex include an increase or decrease in Ca2+ ions resulting from a protein-protein interaction or the activation of the transcription factors, NF-AT, AP-1 and NFKB.
The TACI/TACI-L interaction is a protein-protein interaction. Protein-protein interactions can be observed and measured in binding assays using a variety of detection methodologies that include, but are not limited to, surface plasmon resonance (Biacore), radioimmune based assays, and fluorescence polarization binding assays. When performed in the presence of a test compound, the ability of the test compound to modulate (e.g. enhance or inhibit) the protein-protein binding affinity is measured. In one embodiment of the instant invention, the binding interaction between TACI and TACI-L occurs between the extracellular domain of the TACI protein and amino acids 123-285 of the extracellular domain of the TACI ligand.
The discovery of the interaction between TACI and TACI-L is described in detail in Examples 1-3. Briefly, a ligand expression construct was transfected into cells. The cells were incubated with TACI:Fc, bound with an antibody of TACI:Fc, and followed by a detecting agent. A soluble form of TACI-L was used in verifying the interaction and was produced by fusing a CMV leader sequence followed by a leucine zipper motif to the polypeptide. Other useful leader sequences include IgKappa and Growth Hormone. PCR was used to amplify the cDNA sequence which encodes the extracellular domain (amino acids 123-285) of TACI-L by using the restriction sites of specific oligonucleotides. CMV and leucine zipper sequences can be obtained by methods well known in the art, such as by PCR or by enzymatic digestion of previously cloned sequences. These fragments are ligated and inserted into the appropriate expression vector. (Smith et al., Cell, Vol. 73, 1349-1360.)
The interaction between TACI and TACI-L was further characterized by plate binding assays, as described in Examples 4 and 5. Plate binding assays were conducted capturing either the TACI protein or the TACI ligand. In each instance, a high affinity constant was obtained, demonstrating the close binding interaction between TACI and TACI-L.
The discovery and understanding of the interaction between the extracellular region of TACI and TACI-L can be used to determine potential agonists or antagonists and to further develop understanding of which cell types TACI-L acts upon. Assays may utilize the interaction between TACI-L and TACI to screen for potential inhibitors (antagonists) or enhancers (agonists) of activity associated with TACI-L molecules and identify candidate molecules which may serve as therapeutically active agents that enhance, inhibit or modulate the TACI/TACI-L complex. Potential antagonists to the TACI/TACI-L interaction may include small molecules, peptides, and antibodies that bind to and occupy the binding site of either TACI or TACI-L, causing them to be unavailable to bind to each other and therefore preventing normal biological activity. Other potential antagonists are antisense molecules which may hybridize to mRNA in vivo and block translation of the mRNA into the TACI-L protein. Potential agonists include small molecules, peptides and antibodies which bind to TACI or TACI-L and elicit the same or enhanced biological effects as those caused by the binding of TACI to TACI-L.
Small molecules are usually less than 10K molecular weight and possess a number of physiochemical and pharmacological properties to enhance cell penetration, resist degradation and prolong their physiological half-lives. (Gibbs, J., Pharmaceutical Research in Molecular Oncology, Cell, Vol. 79 (1994).) Antibodies, which include intact molecules as well as fragments such as Fab and F(ab′)2 fragments, may be used to bind to and inhibit the TACI/TACI-L complex by blocking the commencement of the signaling cascade. Such activity by the antibodies could be useful in the treatment of Acute Respiratory Disease Syndrome (ARDS). (WO 98/18921 at 57.) It is preferable that the antibodies are humanized, and more preferable that the antibodies are human. The antibodies of the present invention may be prepared by any of a variety of well-known methods.
Antagonists may be employed to inhibit (antagonize) the interaction between TACI and TACI-L for therapeutic purposes to treat tumor and tumor metastasis and to combat various autoimmune diseases that may be modulated by the TACI/TACI-L complex, e.g. multiple sclerosis and diabetes, as well as other disorders, such as viral infection, rheumatoid arthritis, graft rejection, and IgE-mediated allergic reactions. A further disorder that may be treated by antagonists of the TACI/TACI-L interaction is inflammation mediated by the interaction. In general, the interaction may be used to study cellular processes associated with TNF-receptors such as immune regulation, cell proliferation, cell death, and inflammatory responses.
Specific screening methods are known in the art and many are extensively incorporated in high throughput test systems so that large numbers of test compounds can be screened within a short amount of time. The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, cell based assays, etc. These assay formats are well known in the art. The screening assays of the present invention are amenable to screening of chemical libraries and are suitable for the identification of small molecule drug candidates, antibodies, peptides.
A particular example of an assay for the identification of potential TACI antagonists is a competitive assay which combines TACI-L and a candidate molecule with TACI under the appropriate conditions for a competitive assay. Either TACI or TACI-L can be labeled so that the binding may be measured and the effectiveness of the antagonist judged. The label allows for detection by direct or indirect means. Direct means include, but are not limited to luminescence, radioactivity, optical or electron density. Indirect means include but are not limited to an enzyme or epitope tag.
By observing the effect that candidate molecules have on TACI/TACI-L complexes in various binding assays, on TACI/TACI-L mediated activity in functional tests, and in cell based screens, molecules that are potential therapeutics because they can modulate the TACI/TACI-L-binding interaction are identified. Such molecules either mimic the biological activity of the TACI/TACI-L complex, prevent the formation of the TACI/TACI-L complex or inhibit dissociation of the TACI/TACI-L complex already formed. Molecules preventing the interaction of TACI and TACI-L may be useful when enhancement of the immune system is desired. Antagonists of the dissociation of the TACI/TACI-L complex may be useful as immunosuppressants or antiinflammatory agents.
Molecules which inhibit or prevent the dissociation of the TACI/TACI-L complex can be identified by forming the complex in the absence of a candidate molecule, then adding the candidate molecule to the mixture, and changing the conditions so that, but for the presence of the candidate molecule, TACI would be released from the complex. The concentration of the free or bound TACI could then be measured and the dissociation constant of the complex could be determined and compared to a control.
Another method by which molecules which inhibit the interaction between TACI and TACI-L can be identified is the solid phase method, in which TACI is bound and placed in a medium with labeled TACI-L. After contact with a candidate molecule, the amount of signal produced by the interaction between TACI and TACI-L is measured. Diminished levels of signal, in comparison to a control, indicate that the candidate molecule inhibited the interaction between TACI and TACI-L. In a further embodiment of this method, TACI-L could be bound and TACI labeled.
Screening assays can further be designed to find molecules that mimic the biological activity of the TACI/TACI-L complex. Molecules which mimic the biological activity of the TACI/TACI-L complex may be useful for enhancing the interaction. To identify compounds for therapeutically active agents that mimic the biological activity of the TACI/TACI-L complex, it must first be determined whether a candidate molecule binds to TACI or TACI-L. A binding candidate molecule is added to a biological assay to determine its biological effects. The biological effects of the candidate molecule are then compared to the those of the TACI/TACI-L complex.
Thus, the present invention encompasses methods of screening candidate molecules for their ability to modulate TACI/TACI-L complexes and their ability to modulate activities mediated by TACI/TACI-L complexes. By observing the effect that the candidate molecule has on the known binding characteristics of TACI, TACI-L or fragments thereof, compounds that inhibit or enhance TACI/TACI-L binding can be identified. Typical candidate molecules are small molecules, antibodies, or peptides and may be part of extensive small molecule libraries developed for use in screening methods. In this context, the identification of small molecules which may interact with the TACI protein or the TACI ligand can be used to develop drugs that modulate the activation pathway and may allow physicians to treat distinct immune conditions without the negative side effects present in current therapies. For such therapeutic uses, the agonists or antagonists of the TACI/TACI-L complex identified can be administered through well-known means, including parenterally (subcutaneous, intramuscular, intravenous, intradermal, etc. injection) and with a suitable carrier. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation instonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
Generally, the conditions for an assay are conditions under which TACI and TACI-L would normally interact. In other words, for an assay to identify the inhibitor of the TACI//TACI-L interaction, the conditions would be such that, but for the candidate molecule, TACI and TACI-L would bind.
The following examples are offered by way of illustration, and not by way of limitation. Those skilled in the art will recognize that variations of the invention embodied in the examples can be made, especially in light of the teachings of the various references cited herein, the disclosures of which are incorporated by reference in their entirety.
EXAMPLE 1
Generation of TCI-FC
This Example describes a method of generating TACI-Fc. The cDNA sequence encoding the extracellular domain of TACI (amino acids 2-166) was amplified by PCR using a sense primer (5′-ataaccggtagtggcctgggccggagcaggcgag-3′) (SEQ. ID. NO. 6) and an antisense primer (5′-ataagatctgggctcgctgtagaccagggccacctgatc) (SEQ. ID. NO. 7). The amplified PCR fragment was digested with the appropriate restriction enzyme and then ligated into the mammalian expression vector pDC409, in-frame with the Ig kappa leader sequence at the 5′ end and with the Fc portion of human IgG1 at the 3′ end. The plasmid was transfected transiently in CV1/EBNA cells and the soluble protein TACI-FC was purified on a protein G-sepharose column. Protein concentration was determined by BCA analysis. Purity was assessed by SDS-PAGE analysis which, under reducing conditions, showed a single band at 42kDa.
EXAMPLE 2
Ligand Screening by Slide Binding Assay
This Example describes the method of a slide binding assay and demonstrates that the TACI-Fc protein interacted only with TACI-L. The purified TACI-Fc was used to screen against a cDNA panel containing known members of the ligand family (4-1BBL, CD40L, OX40L, CD27L, CD30L, RANKL, LT-alpha, LT-beta, LIGHT, TWEAK, FasL, TRAIL, proTNF and TACI-L). TACI-Fc was then bound to the slides by adding 2μg of the DNA encoding the members of the ligand family to a sterile tube and adding 75 μM choloroquine in transfection/growth medium to a final volume of 175 μl. 25 μl of DEAE-dextran (4mg/ml in PBS) was then added to the DNA solution and mixed.
The growth medium was aspirated from the slides and replaced with 3 ml of 75 μM choloroquine in the transfection/growth medium, followed by the addition of the DNA/DEAE-dextran mixture to the cells. The slides were rocked side-to-side and back-and-forth to distribute the precipitated DNA evenly. The slides were incubated at 37° C. for 4.5 hours.
The medium was aspirated and 3 ml 10% DMSO was added in the transfection/growth medium. After a 5 minute incubation period at room temperature, the medium was aspirated again and replaced with 3 ml fresh transfection/growth medium. The cells were then incubated at 37° C. for 2 days to allow for expression of the transfected cDNAs.
To screen for positive pools expressing the cell-bound protein, slides were incubated with TACI:Fc and then with a radioiodinated protein probe (labeled goat anti-human Fc F(ab′)2) for 30 minutes at room temperature. The probe solution is then removed by aspiration and washed to remove the non-specifically bound probe. Finally, the slides were fixed by incubating each slide with 1 ml 2.5% glutaraldehyde in PBS for 30 minutes at room temperature to retain specifically bound label. The slides were then washed twice with 1 ml PBS and air-dried.
The dried slides were dipped in liquid photographic emulsion that has been warmed to 42° C., dried at room temperature and exposed for 2 days at room temperature before developing. The slides were examined at 25× magnification under bright-field illumination to detect cell types upon which the ligand is acting. TACI-Fc protein was found to bind only to cells transfected with the TACI-L. The ability of the TACI-FC to bind to CV1 expressing the TACI-L was also demonstrated by the well-known methods of flow cytometry.
EXAMPLE 3
Immunoprecipitation of Membrane—Associated TACI with the TACI-Ligand
This Example demonstrates the interaction between the TACI protein and TACI-L. CV1 cells were transfected with soluble TACI-L plasmid and the two day supernatant was harvested. CV1 cells were transfected with membrane associated TACI and metabolically labeled with 35S-CYS-MET two days post-transfection (labeled cell lysate). Supernatant containing TACI-L was used in immunoprecipitation experiments with labeled cell lysate. A specific band at 45 kDa which was consistent with the predicted size of TACI was obtained, as shown in FIG. 1. Thus, the interaction between the TACI protein and TACI-L was confirmed.
EXAMPLE 4
Plate Binding Assay Capturing TACI-L
This example further characterizes the interaction between TACI and TACI-L by conducting a plate binding assay and demonstrates the high affinity between the proteins. Equilibrium binding isotherms were determined in 96-well microtiter plates that had been coated with TACI-L COS expressed supernatants, captured through Leucine Zipper M15 antibody. Plates were incubated with 5 μg/ml LZ M15 in PBS for 4 hr at 4° C. After being washed 3 times with PBS, the plates were incubated with a 1:2 or 1:5 dilution of the COS expressing TACI supernatant in PBS/0.05% Tween 20 for 12 hours at 4° C. The plates were then washed for an additional 3 times with PBS and nonspecific binding sites were blocked with 300 μl/well of a binding media (RPMI 1640, 2.5% BSA, 20MM HEPES, 0.02% sodium azide pH 7.2) and 2.5% non-fat dried milk. The plates were incubated for 1 hour at room temperature and washed 3 times with PBS.
HuTACI/Fc was diluted to 2 μg/ml to the first well, and serial dilutions were performed against the binding media. Incubation occurred for 2 hours at 4° C. Plates were then washed 3 times with PBS. A final incubation occurred for 30 minutes at room temperature with 125 ng/ml 125-I goat anti-human F(ab′)2. The goat anti-human F(ab′)2 was labeled with 125-I using solid phase chloramine T analog (Iodogen; Pierce Chemical, Rockford, Ill.) to a specific radioactivity of 8.73e14 cpm/mmol. Nonspecific binding was determined in the presence of 1000-fold excess of unlabeled goat anti-human F(ab′)2. Plates were washed 3 times in PBS and the specifically bound ligand was released with 50 mM citrate (pH 3.0) and then gamma counted. Data was processed as described (Dower et al., 1984).
FIG. 4a demonstrates the results of the assay using 1:2 dilution and shows the complete saturation of the receptor binding sites. FIG. 4b, the Scatchard graph corresponding to FIG. 4a, demonstrates the actual number of sites that were actually bound. From these results, the affinity constant of 1.53×10−9 can be generated.
FIG. 5a demonstrates the results of the assay using 1:5 dilution and shows the complete saturation of the receptor binding sites. FIG. 5b, the Scatchard graph corresponding to FIG. 5a, demonstrates the actual number of sites that were actually bound. From these results, the affinity constant of 2.2×10−9 is shown.
EXAMPLE 5
Plate Binding Assay Capturing HuTACI/FC
This example also characterizes the interaction between TACI and TACI-L by use of a plate binding assay and further demonstrates the high affinity between the proteins. Equilibrium binding isotherms were determined in 96-well microtiter plates that had been coated with HuTACI/Fc, captured through goat anti-human Fc polyclonal antibody. Plates were incubated with 5 μg/ml goat anti-human FC in PBS for 4 hours at 4° C. After being washed 3 times with PBS, the plates were incubated with 0.1 μg/ml Fc chimera in PBS/0.05% Tween 20 for 12 hours at 4° C. and then washed for an additional 3 times with PBS. Nonspecific binding sites were blocked with 300 μl/well of a binding media (RPMI 1640, 2.5% BSA, 20MM HEPES, 0.02% sodium azide pH 7.2) and 2.5% non-fat dried milk. The plates were incubated for 1 hour at room temperature and then washed 3 times with PBS. TACI-L was expressed in COS cells and concentrated 10-fold.
TACI-L supernatant was diluted 1:10 to the first well, and serial dilutions were performed against the binding media. Incubation occurred for 2 hours at 4° C. Plates were then washed 3 times with PBS. A final incubation occurred for 30 minutes at room temperature with 125-I Leucine Zipper M15. Leucine Zipper M15 (LZM15) was labeled with 125-I using solid phase chloramine T analog (Iodogen; Pierce Chemical, Rockford, Ill.) to a specific radioactivity of 8.73e14 cpm/mmol. Nonspecific binding was determined in the presence of 1000-fold excess of unlabeled LZM15. Plates were washed 3 times in PBS and specifically bound ligand was released with 50 mM citrate (pH 3.0) and then gamma counted. Data was processed as described (Dower et al., 1984).
FIG. 6a demonstrates the complete saturation of the receptor binding sites. FIG. 6b, the Scatchard graph which corresponds to FIG. 6a, demonstrates the actual number of sites that were actually bound. The Scatchard graph of FIG. 6b demonstrates a curvilinear binding, with a low affinity constant of 5.7×10−10 and a high affinity constant of 1.0×10−10. FIG. 6b demonstrates that the majority of the binding occurred at an affinity constant between 2−3×10−9.
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12614109
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immunex corporation
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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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Mar 31st, 2022 02:17PM
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Mar 31st, 2022 02:17PM
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Amgen
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:amgn
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Amgen
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Sep 7th, 2010 12:00AM
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Jun 11th, 2008 12:00AM
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https://www.uspto.gov?id=US07790684-20100907
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Method of inhibiting osteoclast activity
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Methods for inhibiting osteoclastogenesis by administering a soluble RANK polypeptide are disclosed. Such methods can be used to treat a variety of different cancers, including bone cancer, multiple myeloma, melanoma, breast cancer, squamous cell carcinoma, lung cancer, prostate cancer, hematologic cancers, head and neck cancer and renal cancer.
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7790684
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1. A method of inhibiting RANKL-induced osteoclastogenesis in a patient in need thereof, comprising administering to said patient a soluble RANK polypeptide composition comprising a soluble RANK polypeptide, wherein
said patient suffers from a condition selected from the group consisting of bone cancer, multiple myeloma, melanoma and breast cancer,
the soluble RANK polypeptide comprises amino acids 33-196 of SEQ ID NO:2 and is capable of binding to a RANKL polypeptide that consists of amino acids 1-317 of SEQ ID NO:8, and
said composition is administered in an amount sufficient to inhibit RANKL-induced osteoclastogenesis in said patient.
2. The method of claim 1, wherein the soluble RANK polypeptide further comprises a polypeptide selected from the group consisting of an immunoglobulin Fc domain, an immunoglobulin Fc mutein, a FLAG™ tag, a peptide comprising at least 6 His residues, a leucine zipper, and combinations thereof.
3. The method of claim 1, wherein the soluble RANK polypeptide comprises amino acids 33-213 of SEQ ID NO:2.
4. A method of inhibiting RANKL-induced osteoclastogenesis in a patient in need thereof, said method comprising administering to said patient a composition comprising a recombinant soluble RANK polypeptide, wherein
said patient suffers from a condition selected from the group consisting of squamous cell carcinoma, lung cancer, prostate cancer, hematologic cancer, head and neck cancer and renal cancer,
the soluble RANK polypeptide comprises amino acids 33-196 of SEQ ID NO:2 and is capable of binding to a RANKL polypeptide that consists of amino acids 1-317 of SEQ ID NO:8, and
said composition is administered in an amount sufficient to inhibit RANKL-induced osteoclastogenesis in said patient.
5. The method of claim 4, wherein the soluble RANK polypeptide comprises amino acids 33-213 of SEQ ID NO:2.
6. The method of claim 4, wherein the soluble RANK polypeptide further comprises one or more polypeptides selected from the group consisting of an immunoglobulin Fc domain, an immunoglobulin Fc mutein, a FLAG™ tag, a peptide comprising at least 6 His residues and a leucine zipper.
7. A method according to claim 2, wherein the further polypeptide is selected from the group consisting of an immunoglobulin Fc domain comprising the amino acid sequence as shown in SEQ ID NO:3 and a leucine zipper comprising the amino acid sequence as shown in SEQ ID NO:6.
8. A method according to claim 6, wherein the further polypeptide is selected from the group consisting of an immunoglobulin Fc domain having the amino acid sequence as shown in SEQ ID NO:3 and a leucine zipper having the amino acid sequence as shown in SEQ ID NO:6.
9. A method according to claim 7, wherein the soluble RANK polypeptide consists of amino acids 33-213 of SEQ ID NO:2 fused with the amino acid sequence as shown in SEQ ID NO:3.
10. A method according to claim 8, wherein the soluble RANK polypeptide consists of amino acids 33-213 of SEQ ID NO:2 fused with the amino acid sequence as shown in SEQ ID NO:3.
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10
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CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 09/705,985 filed Nov. 3, 2000, which is incorporated herein in its entirety, which is a continuation of International patent application No. PCT/US99/10588 filed May 13, 1999, which claims the benefit of U.S. provisional patent applications 60/110,836 filed Dec. 3, 1998 and 60/085,487 filed May 14, 1998, and is a continuation-in-part of U.S. patent application Ser. No. 11/881,911 filed Jul. 30, 2007, which is a divisional of U.S. patent application Ser. No. 10/405,878 filed Apr. 1, 2003 (now U.S. Pat. No. 7,262,274), which is a continuation of U.S. patent application Ser. No. 09/871,291 filed May 30, 2001 (now U.S. Pat. No. 6,562,948), which is a divisional of U.S. patent application Ser. No. 09/577,800 filed May 24, 2000 (now U.S. Pat. No. 6,479,635), which is a continuation of U.S. patent application Ser. No. 09/466,496 filed Dec. 17, 1999 (now U.S. Pat. No. 6,528,482), which is a continuation of U.S. patent application Ser. No. 08/996,139 filed Dec. 22, 1997 (now U.S. Pat. No. 6,017,729), which claims the benefit of U.S. provisional application No. 60/064,671 filed Oct. 14, 1997, U.S. provisional application No. 60/077,181 filed Mar. 7, 1997, and U.S. provisional application No. 60/059,978, filed Dec. 23, 1996.
TECHNICAL FIELD OF THE INVENTION
The present invention relates generally to the field of cytokine receptors, and more specifically to cytokine receptor/ligand pairs having osteoclast regulatory activity.
BACKGROUND OF THE INVENTION
RANK (Receptor Activator of NF-κB) and its ligand (RANKL) are a recently-described receptor/ligand pair that play an important role in an immune response. The cloning of RANK and RANKL is described in U.S. Ser. No. 08/996,139 and U.S. Ser. No. 08/995,659, respectively. It has recently been found that RANKL binds to a protein referred to as osteoprotegerin (OPG), a member of the Tumor Necrosis Factor Receptor (TNFR) family. Yasuda et al. (Proc. Natl. Acad. Sci. 95:3597; 1998) expression cloned a ligand for OPG, which they referred to as osteoclastogenesis inhibitory factor. Their work was repeated by Lacey et al. (Cell 93:165; 1998). In both cases, the ligand they cloned turned out to be identical to RANKL.
In osteoclastogenesis, the interaction of an osteoblast or stromal cell with an osteoclast precursor leads to the differentiation of the precursor into an osteoclast. OPG was known to inhibit this differentiation. A model has been proposed in which RANKL on the osteoblast or stromal cell surface interacts with a specific receptor on an osteoclast progenitor surface, signaling a differentiation event. OPG effectively blocks the interaction of RANKL with a receptor on osteoclast progenitors in vitro, and has been shown to ameliorate the effects of ovariectomy on bone-loss in mice. However, OPG is also known to bind other ligands in the TNF family, which may have a deleterious effect on the activities of such ligands in vivo. Moreover, the presence of other ligands that bind OPG in vivo may require high dosages of OPG to be administered in order to have sufficient soluble OPG available to inhibit osteoclastogenesis.
Accordingly, there is a need in the art to identify soluble factors that specifically bind RANKL and inhibit the ability of RANKL to induce osteoclastogenesis without reacting with other ligands.
SUMMARY OF THE INVENTION
The present invention provides processes associated with the use of a novel receptor, referred to as RANK (for receptor activator of NF-κB), that is a member of the TNF receptor superfamily. RANK is a Type I transmembrane protein having 616 amino acid residues, comprising an extracellular domain, transmembrane region and cytoplasmic domain. RANK interacts with various TNF Receptor Associated Factors (TRAFs); triggering of RANK results in the upregulation of the transcription factor NF-κB, a ubiquitous transcription factor that is most extensively utilized in cells of the immune system.
Soluble forms of the receptor can be prepared and used to interfere with signal transduction through membrane-bound RANK. Inhibition of RANKL-mediated signal transduction will be useful in ameliorating the effects of osteoclastogenesis and osteoclast activity in disease conditions in which there is excess bone break down. Examples of such conditions include osteoporosis, Paget's disease, cancers that may metastasize to bone and induce bone breakdown (i.e., multiple myeloma, breast cancer, some melanomas; see also Mundy, C. Cancer Suppl. 80:1546; 1997), and cancers that do not necessarily metastasize to bone, but result in hypercalcemia and bone loss (e.g. squamous cell carcinomas).
Soluble forms of RANK comprise the extracellular domain of RANK or a fragment thereof that binds RANKL. Fusion proteins of RANK may be made to allow preparation of soluble RANK. Examples of such fusion proteins include a RANK/Fc fusion protein, a fusion protein of a zipper moiety (i.e., a leucine zipper), and various tags that are known in the art. Other antagonists of the interaction of RANK and RANKL (i.e., antibodies to RANKL, small molecules) will also be useful in the inventive methods. These and other aspects of the present invention will become evident upon reference to the following detailed description of the invention.
DETAILED DESCRIPTION OF THE INVENTION
A novel partial cDNA insert with a predicted open reading frame having some similarity to CD40 was identified and was used to hybridize to colony blots generated from a dendritic cell (DC) cDNA library containing full-length cDNAs. SEQ ID NO:1 shows the nucleotide and amino acid sequence of a predicted full-length protein.
RANK is a member of the TNF receptor superfamily; it most closely resembles CD40 in the extracellular region. RANK is expressed on epithelial cells, some B cell lines, and on activated T cells. However, its expression on activated T cells is late, about four days after activation. This time course of expression coincides with the expression of Fas, a known agent of apoptosis. RANK may act as an anti-apoptotic signal, rescuing cells that express RANK from apoptosis as CD40 is known to do. Alternatively, RANK may confirm an apoptotic signal under the appropriate circumstances, again similar to CD40. RANK and its ligand are likely to play an integral role in regulation of the immune and inflammatory response. The isolation of a DNA encoding RANK is described in U.S. Ser. No. 08/996,139, filed Dec. 22, 1997, the disclosure of which is incorporated by reference herein. U.S. Ser. No. 08/996,139 describes several forms of RANK that are useful in the present invention.
Soluble RANK comprises the signal peptide and the extracellular domain (residues 1 to 213 of SEQ ID NO:2) or a fragment thereof. Alternatively, a different signal peptide can be substituted for the native leader, beginning with residue 1 and continuing through a residue selected from the group consisting of amino acids 24 through 33 (inclusive) of SEQ ID NO:2. Other members of the TNF receptor superfamily have a region of amino acids between the transmembrane domain and the ligand binding domain that is referred to as a ‘spacer’ region, which is not necessary for ligand binding. In RANK, the amino acids between 196 and 213 are predicted to form such a spacer region. Accordingly, a soluble form of RANK that terminates with an amino acid in this region is expected to retain the ability to bind a ligand for RANK in a specific manner. Preferred C-terminal amino acids for soluble RANK peptides are selected from the group consisting of amino acids 213 and 196 of SEQ ID NO:2, although other amino acids in the spacer region may be utilized as a C-terminus. In muRANK, the amino acids between 197 and 214 are predicted to form such a spacer region. Accordingly, a soluble form of RANK that terminates with an amino acid in this region is expected to retain the ability to bind a ligand for RANK in a specific manner. Preferred C-terminal amino acids for soluble RANK peptides are selected from the group consisting of amino acids 214, and 197 of SEQ ID NO:5, although other amino acids in the spacer region may be utilized as a C-terminus. Moreover, fragments of the extracellular domain will also provide soluble forms of RANK.
Fragments can be prepared using known techniques to isolate a desired portion of the extracellular region, and can be prepared, for example, by comparing the extracellular region with those of other members of the TNFR family (of which RANK is a member) and selecting forms similar to those prepared for other family members. Alternatively, unique restriction sites or PCR techniques that are known in the art can be used to prepare numerous truncated forms which can be expressed and analyzed for activity.
Other derivatives of the RANK proteins within the scope of this invention include covalent or aggregative conjugates of the proteins or their fragments with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions. For example, the conjugated peptide may be a signal (or leader) polypeptide sequence at the N-terminal region of the protein which co-translationally or post-translationally directs transfer of the protein from its site of synthesis to its site of function inside or outside of the cell membrane or wall (e.g., the yeast α-factor leader).
Protein fusions can comprise peptides added to facilitate purification or identification of RANK proteins and homologs (e.g., poly-His). The amino acid sequence of the inventive proteins can also be linked to an identification peptide such as that described by Hopp et al., Bio/Technology 6:1204 (1988; FLAG™). Such a highly antigenic peptide provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant protein. The sequence of Hopp et al. is also specifically cleaved by bovine mucosal enterokinase, allowing removal of the peptide from the purified protein.
Fusion proteins further comprise the amino acid sequence of a RANK linked to an immunoglobulin Fc region. An exemplary Fc region is a human IgG1 having an amino acid sequence set forth in SEQ ID NO:3. Fragments of an Fc region may also be used, as can Fc muteins. For example, certain residues within the hinge region of an Fc region are critical for high affinity binding to FcγRI. Canfield and Morrison (J. Exp. Med. 173:1483; 1991) reported that Leu(234) and Leu(235) were critical to high affinity binding of IgG3 to FcγRI present on U937 cells. Similar results were obtained by Lund et al. (J. Immunol. 147:2657, 1991; Molecular Immunol. 29:53, 1991). Such mutations, alone or in combination, can be made in an IgG1 Fc region to decrease the affinity of IgG1 for FcR. Depending on the portion of the Fc region used, a fusion protein may be expressed as a dimer, through formation of interchain disulfide bonds. If the fusion proteins are made with both heavy and light chains of an antibody, it is possible to form a protein oligomer with as many as four RANK regions.
In another embodiment, RANK proteins further comprise an oligomerizing peptide such as a zipper domain. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, 1988). Zipper domain is a term used to refer to a conserved peptide domain present in these (and other) proteins, which is responsible for multimerization of the proteins. The zipper domain comprises a repetitive heptad repeat, with four or five leucine, isoleucine or valine residues interspersed with other amino acids. Examples of zipper domains are those found in the yeast transcription factor GCN4 and a heat-stable DNA-binding protein found in rat liver (C/EBP; Landschulz et al., Science 243:1681, 1989). Two nuclear transforming proteins, fos and jun, also exhibit zipper domains, as does the gene product of the murine proto-oncogene, c-myc (Landschulz et al., Science 240:1759, 1988). The products of the nuclear oncogenes fos and jun comprise zipper domains that preferentially form a heterodimer (O'Shea et al., Science 245:646, 1989; Turner and Tjian, Science 243:1689, 1989). A preferred zipper moiety is that of SEQ ID NO:6 or a fragment thereof. This and other zippers are disclosed in U.S. Pat. No. 5,716,805.
Other embodiments of useful proteins include RANK polypeptides encoded by DNAs capable of hybridizing to the DNA of SEQ ID NO:1 under moderately stringent conditions (prewashing solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridization conditions of 50° C., 5×SSC, overnight) to the DNA sequences encoding RANK, or more preferably under stringent conditions (for example, hybridization in 6×SSC at 63° C. overnight; washing in 3×SSC at 55° C.), and other sequences which are degenerate to those which encode the RANK. In one embodiment, RANK polypeptides are at least about 70% identical in amino acid sequence to the amino acid sequence of native RANK protein as set forth in SEQ ID NO:2 for human RANK and NO:5 for murine RANK. In a preferred embodiment, RANK polypeptides are at least about 80% identical in amino acid sequence to the native form of RANK; most preferred polypeptides are those that are at least about 90% identical to native RANK.
Percent identity may be determined using a computer program, for example, the GAP computer program described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). For fragments derived from the RANK protein, the identity is calculated based on that portion of the RANK protein that is present in the fragment.
The biological activity of RANK analogs or muteins can be determined by testing the ability of the analogs or muteins to bind RANKL (SEQ ID NOS:7 and 8, for example as described in the Examples herein. Suitable assays include, for example, an enzyme immunoassay or a dot blot, and assays that employ cells expressing RANKL. Suitable assays also include, for example, inhibition assays, wherein soluble RANK is used to inhibit the interaction of RANKL with membrane-bound or solid-phase associated RANK (i.e., signal transduction assays). Such methods are well known in the art.
RANKL and RANK are important factors in osteoclastogenesis. RANK is expressed on osteoclasts and interacts with RANK ligand (RANKL) to mediate the formation of osteoclast-like (OCL) multinucleated cells. This was shown by treating mouse bone marrow preparations with M-CSF (CSF-1) and soluble RANKL for 7 days in culture. No additional osteoclastogenic hormones or factors were necessary for the generation of the multinucleated cells. Neither M-CSF nor RANKL alone led to the formation of OCL. The multinucleated cells expressed tartrate resistant acid phosphatase and were positive for [125] calcitonin binding. The tyrosine kinase c-src was highly expressed in multinucleated OCL and a subset of mononuclear cells as demonstrated by immunofluorescence microscopy. (See Example 2).
Purification of Recombinant RANK
Purified RANK, and homologs or analogs thereof are prepared by culturing suitable host/vector systems to express the recombinant translation products of the DNAs of the present invention, which are then purified from culture media or cell extracts. For example, supernatants from systems which secrete recombinant protein into culture media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
Following the concentration step, the concentrate can be applied to a suitable purification matrix. For example, a suitable affinity matrix can comprise a counter structure protein or lectin or antibody molecule bound to a suitable support. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred. Gel filtration chromatography also provides a means of purifying the inventive proteins.
Affinity chromatography is a particularly preferred method of purifying RANK and homologs thereof. For example, a RANK expressed as a fusion protein comprising an immunoglobulin Fc region can be purified using Protein A or Protein G affinity chromatography. Moreover, a RANK protein comprising an oligomerizing zipper domain may be purified on a resin comprising an antibody specific to the oligomerizing zipper domain. Monoclonal antibodies against the RANK protein may also be useful in affinity chromatography purification, by utilizing methods that are well-known in the art. A ligand may also be used to prepare an affinity matrix for affinity purification of RANK.
Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify a RANK composition. Suitable methods include those analogous to the method disclosed by Urdal et al. (J. Chromatog. 296:171, 1984). Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant protein.
Recombinant protein produced in bacterial culture is usually isolated by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange or size exclusion chromatography steps. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. Microbial cells employed in expression of recombinant protein can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Fermentation of yeast which express the inventive protein as a secreted protein greatly simplifies purification.
Protein synthesized in recombinant culture is characterized by the presence of cell components, including proteins, in amounts and of a character which depend upon the purification steps taken to recover the inventive protein from the culture. These components ordinarily will be of yeast, prokaryotic or non-human higher eukaryotic origin and preferably are present in innocuous contaminant quantities, on the order of less than about 1 percent by weight. Further, recombinant cell culture enables the production of the inventive proteins free of other proteins which may be normally associated with the proteins as they are found in nature in the species of origin.
Uses and Administration of RANK Compositions
The present invention provides methods of using therapeutic compositions comprising a protein and a suitable diluent and carrier. These methods involve the use of therapeutic compositions of RANK or soluble fragments of RANK for regulating an immune or inflammatory response. Further included within the present invention are methods for regulating osteoclast activity by administering therapeutic compositions of RANK or soluble RANK fragments to an individual in amounts sufficient to decrease excess bone resorption. Typically, the individual is inflicted with excess bone resorption and suffers from the effects of hypercalcemia, has symptoms of hypercalcemia, or is suffering a disease that involves excessive bone resorption. In addition to regulating osteoclast activity, the methods described herein are applicable to inhibiting osteoclast activity, regulating osteoclast generation and inhibiting osteoclast generation in individuals inflicted with excess bone resorption. In connection with the methods described herein, the present invention contemplates the use of RANK in conjunction with soluble cytokine receptors or cytokines, or other osteoclast/osteoblast regulatory molecules.
Soluble forms of RANK and other RANK antagonists such as antagonistic monoclonal antibodies can be administered for the purpose of inhibiting RANK-induced induction of NF-κB activity. NF-κB is a transcription factor that is utilized extensively by cells of the immune system, and plays a role in the inflammatory response. Thus, inhibitors of RANK signalling will be useful in treating conditions in which signalling through RANK has given rise to negative consequences, for example, toxic or septic shock, or graft-versus-host reactions. They may also be useful in interfering with the role of NF-κB in cellular transformation. Tumor cells are more responsive to radiation when their NF-κB is blocked; thus, soluble RANK (or other antagonists of RANK signalling) will be useful as an adjunct therapy for disease characterized by neoplastic cells that express RANK.
In connection with the methods described herein, RANK ligand (RANKL) on osteoblasts or stromal cells is known to interact with RANK on osteoclast progenitor surfaces signaling an event that leads to the differentiation of osteoclast precursors into osteoclasts. (See Example 2 below.) Thus, RANK, and in particular soluble forms of RANK, is useful for the inhibition of the RANKL-mediated signal transduction that leads to the differentiation of osteoclast precursors into osteoclasts. Soluble forms of RANK are also useful for the regulation and inhibition of osteoclast activity, e.g. bone resorption. By interfering with osteoclast differentiation, soluble forms of RANK are useful in the amelioration of the effects of osteoclastogenesis in disease conditions in which there is excess bone break down. Such disease conditions include Paget's disease, osteoporosis, and cancer. Many cancers metastasize to bone and induce bone breakdown by locally disrupting normal bone remodeling. Such cancers can be associated with enhanced numbers of osteoclasts and enhanced amount of osteoclastic bone resorption resulting in hypercalcemia. These cancers include, but are not limited to, breast cancer, multiple myeloma, melanomas, lung cancer, prostrate, hematologic, head and neck, and renal. (See Guise et al. Endocrine Reviews, 19(1):18-54, 1998.) Soluble forms of RANK can be administered to such cancer patients to disrupt the osteoclast differentiation pathway and result in fewer numbers of osteoclast, less bone resorption, and relief from the negative effects of hypercalcemia.
Other cancers do not metastasize to bone, but are known to act systemically on bone to disrupt bone remodeling and result in hypercalcemia. (See Guise et al. Endocrine Reviews, 19(1):18-54, 1998.) In accordance with this invention, RANKL has been found on the surface of certain squamous cells that do not metastasize to bone but are associated with hypercalcemia. (See Example 3 below) Squamous cells that are associated with hypercalcemia also express M-CSF (CSF-1), a cytokine that, together with RANKL, stimulates the proliferation and differentiation of osteoclast precursors to osteoclasts. In accordance with the present invention, it has been discovered that M-CSF directly upregulates RANK on surfaces of osteoclast precursors. When squamous cells release excessive amounts of CSF-1, increased expression of RANK occurs on the surfaces of osteoclast precursors. Thus, there is a higher probability that RANK will interact with RANKL on osteoblasts or stromal cells to produce increased numbers of osteoclasts, resulting in an enhanced amount of bone break down and hypercalcemia.
In addition to the ameliorating the effects of cancers that metastasize to bone, the present invention provides methods for ameliorating the systemic effects, e.g. hypercalcemia, of cancers that are associated with excess osteoclast activity (e.g. squamous cell carcinomas). Such methods include administering soluble forms of RANK in amounts sufficient to interfere with the RANK/RANKL signal transduction that leads to the differentiation of osteoclast precursors into osteoclasts. Fewer osteoclasts lead to reduced bone resorption and relief from the negative effects of hypercalcemia.
For therapeutic use, purified protein is administered to an individual, preferably a human, for treatment in a manner appropriate to the indication. Thus, for example, RANK protein compositions administered to regulate osteoclast function can be given by bolus injection, continuous infusion, sustained release from implants, or other suitable technique. Typically, a therapeutic agent will be administered in the form of a composition comprising purified RANK, in conjunction with physiologically acceptable carriers, excipients or diluents. Such carriers will be nontoxic to recipients at the dosages and concentrations employed.
Ordinarily, the preparation of such protein compositions entails combining the inventive protein with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with conspecific serum albumin are exemplary appropriate diluents. Preferably, product is formulated as a lyophilizate using appropriate excipient solutions (e.g., sucrose) as diluents. Appropriate dosages can be determined in trials. The amount and frequency of administration will depend, of course, on such factors as the nature and severity of the indication being treated, the desired response, the condition of the patient, and so forth.
Soluble forms of RANK and other RANK antagonists such as antagonistic monoclonal antibodies can be administered for the purpose of inhibiting RANK-induced osteoclastogenesis. It is desirable to inhibit osteoclastogenesis in various disease states in which excess bone loss occurs. Examples include osteoporosis, Pagett's disease, and various cancers. Various animal models of these diseases are known in the art; accordingly, it is a matter of routine experimentation to determine optimal dosages and routes of administration of soluble RANK, first in an animal model and then in human clinical trials.
The following examples are offered by way of illustration, and not by way of limitation. Those skilled in the art will recognize that variations of the invention embodied in the examples can be made, especially in light of the teachings of the various references cited herein, the disclosures of which are incorporated by reference.
EXAMPLE 1
This example describes a plate binding assay useful in comparing the ability of various ligands to bind receptors. The assay is performed essentially as described in Smith et al., Virology 236:316 (1997). Briefly, 96-well microtiter plates are coated with an antibody to human Fc (i.e., polyclonal goat anti human Fc). Receptor/Fc fusion proteins are then added, and after incubation, the plates are washed. Serial dilutions of the ligands are then added. The ligands may be directly labeled (i.e., with 1251), or a detecting reagent that is radioactively labeled may be used. After incubation, the plates are washed, specifically bound ligands are released, and the amount of ligand bound quantified.
Using this method, RANK/Fc and OPG/Fc were bound to 96-well plates. In an indirect method, a RANKL/zipper fusion is detected using a labeled antibody to the zipper moiety. It was found that human OPG/Fc binds mRANKL at 0.05 nM, and human RANK/Fc binds mRANKL at 0.1 nM. These values indicate similar binding affinities of OPG and RANK for RANKL, confirming the utility of RANK as an inhibitor of osteoclast activity in a manner similar to OPG.
EXAMPLE 2
The following describes the formation of osteoclast like cells from bone marrow cell cultures using a soluble RANKL in the form of soluble RANKL/leucine zipper fusion protein (RANKL LZ).
Using RANKL LZ at 1 μg/ml, osteoclasts were generated from murine bone marrow (BM) in the presence of CSF-1. These osteoclasts are formed by the fusion of macrophage-like cells and are characterized by their TRAP (tartrate-resistant acid phosphatase) positivity. No TRAP+ cells were seen in cultures containing CSF-1 alone or in cultures containing CSF-1 and TRAIL LZ (a control for the soluble RANKL LZ). Even though human and monkey bone marrow contains more contaminating fibroblasts than murine bone marrow, osteoclasts were generated from murine and monkey bone marrow with the combination of CSF-1 and soluble RANKL LZ. In a dose-response study using murine bone marrow and suboptimal amounts of CSF-1 (40 ng/ml), the effects of soluble RANKL LZ plateaued at about 100 ng/ml.
The effect of soluble RANKL LZ on proliferation of cells was studied in the same cultures using Alamar Blue. After 5 days, the proliferative response was lower in cultures containing CSF-1 and RANKL LZ than in those containing CSF-1 alone. The supports the observation that soluble RANKL LZ is inducing osteoclast differentiation. When CSF-1 and RANKL LZ are washed out of murine BM cultures at day 7 or 8, cells do not survive if they are recultured in medium or in RANKL LZ alone. In contrast, cells do survive if recultured in CSF-1. When RANKL LZ was added to these cultures there was no added benefit. Thus, the combination of CSF-1 and RANKL are required for the generation of osteoclast. Additionally, once formed, CSF-1 is sufficient to maintain their survival in culture.
Finally, using human bone marrow, soluble anti-human RANK mAb and immobilized anti-human RANK mAb were compared to RANKL LZ for the generation of osteoclasts in the presence of CSF-1. Immobilized M331 and RANKL LZ were found to be equally effective for osteoclast generation while soluble M331 was superior to both immobilized antibody and RANKL LZ. This confirms that the osteoclast differentiating activity of RANKL is mediated through RANK rather than via an alternative receptor.
Since osteoclasts cannot readily be harvested and analyzed by flow cytometry, 125I-labeled calcitonin binding assays were used to identify osteoclasts (the calcitonin receptor is considered to be an osteoclast-specific marker). Osteoclasts generated from murine BM cultured with CSF-1 and RANKL LZ for 9 days showed binding of radiolabeled calcitonin confirming their osteoclast identity.
EXAMPLE 3
In order to determine RANKL expression by either of two different squamous cell carcinomas, standard Western blot and RT-PCR studies were performed on MH-85 and OKK cells. One of these carcinoma cells, the MH-85 cells, is associated with hypercalcemia.
The results confirmed that MH-85 and OKK squamous cells express RANKL. MH-85 cells, in addition to being linked with hypercalcemia in patients inflicted with this carcinoma, also express M-CSF (CSF-1). It was also determined that CSF-1 upregulates RANK expression on osteoclast precursors. The enhanced amount of CSF-1 in MH-85 type squamous cell cancer patients can lead to an upregulation of RANK and increased RANK interaction with RANKL. Signals transduced by RANK and RANKL interaction result in increased numbers of mature osteoclasts and bone breakdown. Since soluble forms of RANK can inhibit the RANK/RANKL interaction, administering a soluble form of RANK (e.g. the extracellular region of RANK fused to an Fc) to a squamous cell cancer patient provides relief from adverse effects of this cancer, including hypercalcemia.
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12137397
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immunex corporation
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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514/12
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Mar 31st, 2022 02:17PM
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Mar 31st, 2022 02:17PM
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Amgen
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Health Care
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Pharmaceuticals & Biotechnology
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