CYTOKINE RECEPTOR MODULATORS, METHOD OF IDENTIFYING SAME, AND METHOD OF MODULATING CYTOKINE RECEPTORS ACTIVITY WITH SAME

Abstract

ates to a method for identifying a non-competitive peptide, which inhibits the activity of a cytokine receptor. This method includes the steps of selecting a candidate peptide containing from about 7 to about 20 amino acids derived from a flexible region of a cytokine receptor, and determining the ability of the peptide to inhibit or promote the oligomerization and/or activation of the receptor by measuring an activity of the receptor in the absence or the presence of the candidate peptide, wherein the non-competitive peptide is selected when the activity of the receptor is measurably lower in the presence of the peptide as compared to in the absence of the peptide so identified. This invention also provides agonists of cytokine receptor activity. Pharmaceutical compositions that comprise the identified peptides are disclosed. Also disclosed are methods for treating patients with a disease or condition associated with abnormal cytokine receptor mediated function or activity such as inflammatory, autoimmune and vascular diseases.

Description

[0001]This application claims the benefit of U.S. Provisional Application No. 60/420,679, filed Oct. 24, 2002, and U.S. Provisional Application No. 60/423,530, filed Nov. 5, 2002. The entire text of the above provisional applications are specifically incorporated by reference.

FIELD OF THE INVENTION

[0002]The present invention relates to cytokine receptor agonists and antagonists, to a method of identifying same, and to a method of modulating cytokine receptors activity with same. More specifically, the present invention is concerned with extracellular, non-competitive cytokine receptor modulators, a method of identifying same, their identification and their uses. More particularly, the present invention is concerned with extracellular, non-competitive cytokine receptor antagonists, a method of identifying same, their identification and their therapeutic uses.

BACKGROUND OF THE INVENTION

[0003]Cytokines are generic terms for designating biologically active hormone-like proteins (interleukins, interferons, tumor necrosis factor, growth factors) that mediate their effects through a superfamily of receptors. Cytokines and their receptors constitute a powerful control network by which cells signal and coordinate cell proliferation and differentiation, cell death and survival. Cytokines are low molecular weight peptides having very potent biological activity. Their mechanism of action is generally autocrine and paracrine and act by ultimately regulating gene expression.

[0004]Cytokines and their receptors are thus implicated in major diseases. They regulate hematopoiesis, immunity and development of the nervous system. Most of all, they contribute to the development of afflictions such as cancer, inflammatory and autoimmune reactions, asthma, allergy, thrombosis, vascular diseases and septic shock by influencing aberrant or overexpressing genes leading to diseases. Cytokines and growth factors mediate tightly regulated biological effects in order to ensure proper control and functioning of the immune system. Therefore, cytokines are also involved in pathological conditions such as inflammation (e.g. rhumatoid arthritis) and tissue degeneration. Diseases which may develop or progress as a result of defects in cytokine or growth factor mediated cell signaling have a high prevalence in the population and are associated with significant morbidity and/or mortality. For these reasons cytokine receptors are important therapeutic target.

[0005]The treatments available for these pathologies are currently limited. They often result in high toxicity and secondary effects. The demand in the medical world for safer and more targeted therapies is therefore considerable.

[0006]The current approaches in the field of cytokines antagonists include the development of soluble receptors, monoclonal antibodies directed against cytokines, mimetics of cytokines, antisense techniques and kinases inhibitors. Few of these strategies have been successful in drug development, however. Nevertheless, certain antibodies targeting the ligand and the receptor, natural soluble receptor inhibitors (eg. IL1ra), and decoy soluble receptors have displayed interesting results. For instance, Trastuzumab (Herceptin, Roche) a monoclonal antibody which binds the HER-2/neu protein tyrosine kinase, and ZD1839 (Iressa, Astra-Zeneca), a small molecule which binds the EGF receptor are either in clinical trials or available for the treatment of certain diseases.

[0007]Non competitive antagonists of cytokines have also been described. In international application no. WO 93/14781 published in 1993, Fox describes the use of non-competitive peptides targeting intracellular domains of EGF. Intracellular domains are difficult to reach by peptides because of the barrier that the cell membrane constitutes.

[0008]Antagonists of the prior art are thus either competitive (e.g. soluble receptors, antibodies, cytokine mimetics), not very selective (e.g. tyrosine kinase inhibitors), costly to produce or difficult to apply in vivo (e.g. antisense). Because the ligand exceeds by far the concentration of the receptor, the concentration of competitive antagonists needed to inhibit the receptor is often substantial.

[0009]There is therefore a need for non-competitive, selective, extracellular and simple to identify, select and produce antagonists of cytokines.

[0010]The present invention seeks to meet these needs and other needs.

[0011]The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

[0012]The present invention thus concerns non-competitive and selective extracellular cytokine receptor modulators, and methods of selecting and of using same.

[0013]The peptides, derivatives and peptidomimetics thereof of the present invention are derived from selected cytokine receptor flexible regions. Cytokine receptor agonists or antagonists of the present invention possess a unique mechanism and site of action for inhibiting cytokine receptors activity. They are peptides strategically positioned on at least one of an extracellular flexible region including juxtamembranous regions, flexible regions between domains of the cytokine receptor, and oligomerization site, that are important for the appropriate conformation of the receptor which enables signaling. In one embodiment the flexible region is required for proper oligomerization to occur. In such an embodiment, appropriate conformation of the receptor is needed to allow adequate positioning of the protein chains to enable oligomerization of the receptor and its resulting activation.

[0014]Cytokine receptors subfragments or peptides of this invention may promote or stabilize a particular conformation of the cytokine receptor which results in inhibition or activation of the receptor activity. In particular, the antagonists of this invention do not necessarily interfere directly with the oligomerization site. They may, for example, exert their antagonistic activity by directly or indirectly preventing the oligomerization of the complementary protein chains (of homodimers as well as heterodimers receptors) of the extracellular domain of the cytokine receptor. This process effectively prevents activation of the intracellular receptor domains responsible for cytokine enzymatic function. Subsequent cell transduction events leading to overexpression of the ligand and/or cell bound receptors responsible in part for disease expression are thereby prevented.

[0015]In the alternative, one can use cytokine receptors subfragment peptides or derivatives to promote or stabilize the active cytokine receptor structure capable signal transduction. Such peptides are considered agonists of the present invention. Cytokine receptor modulators of the present invention possess a number of advantages over the prior art.

[0016]Because they have extracellular targets, unlike certain known drug candidates which target intracellular regions of the cytokine receptors, the antagonists of the present invention do not necessitate a prior permeabilization or other disruption of cell membranes to gain access to the target in order to produce a pharmacological response.

[0017]Because they are non competitive, a smaller amount of the antagonists of the present invention is necessary to inhibit the receptor that they target, as compared to competitive inhibitors.

[0018]As peptides, the antagonists of the present invention are advantageously simple to synthesize.

[0019]In order to provide a clear and consistent understanding of terms used in the present description, a number of definitions are provided herein below.

[0020]In view of the importance of the function of cytokine receptors in numerous pathway and conditions in animals, the present invention has broad impact on the screening, identification, validation and treatment of conditions or diseases associated with abnormal functioning of these cytokine receptors.

[0021]Unless defined otherwise, the scientific and technological terms and nomenclature used herein have the same meaning as commonly understood by a person of ordinary skill to which this invention pertains. Generally, the procedures for cell cultures, infection, molecular biology methods and the like are common methods used in the art. Such standard techniques can be found in reference manuals such as for example Sambrook et al. (1989, Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Laboratories) and Ausubel et al. (1994, Current Protocols in Molecular Biology, Wiley, New York).

Cytokine Receptors

[0022]The term "cytokine" refers herein to any cytokine including growth factor. Similarly, the term "cytokine receptors" refers herein to any cytokine receptor including growth factor receptors. The cytokine receptors comprise a number of families including 1-tyrosine kinases receptors, such as vascular endothelial growth factor receptors (VEGFR), PDGFR, IGF-1R, FGFR, EGFR; 2-type I receptors, such as Interleukins-2, 3, 4, 5, 7, 9 and 15; 3-type II receptors, such as interleukins 10, IFNαR, IFNβR, IFNR; 4-TGFβ; 5-chemokines; and 6-NGF/TNF; 6-interleukins-1 types I and II. The present invention encompasses peptidic agonists or antagonists directed at any cytokine.

[0023]The method of identifying cytokines antagonists of the present invention is based on the localization of flexible extracellular regions, including regions between domains, long loops between two f chains, as well as juxtamembranous regions of the receptor, which are important for the appropriate conformation and/or oligomerization of the subunits of the receptor and/or its resulting activation. These regions can be determined using for example crystallography data, model structures, data bases, sequence alignments and the like. For example, the targeted regions were established herein based on crystal structure data provided by crystallography for IL-1R and IGF-1R and on published model structure for IL-4R. Databases such as Swiss-Prot and NCBI as well as sequences alignments with CLUSTALW and MOTIFSCAN enabled a comparison between many regions constituting the receptors domains and their structural similarities with flexible regions of the vascular endothelial growth factor receptor (VEGFR). It should be noted that the flexible regions of the present invention need not be directly involved in oligomerization. Indeed, regions which facilitate oligomerization or regions that are implicated in conformational changes needed for receptor signaling are also within the scope of the present invention. The same principle apply to the identification of cytokine agonists.

[0024]The terminology "juxtamembranous region of a receptor" refers herein to an extracellular region of the receptor located in the vicinity of the cellular membrane. More particularly in a region which spans a length of up to about 20 amino acids.

[0025]The terminology "flexible region of a receptor" refers herein to any region of the receptor that possesses sufficient flexibility to enable this region to bend, extend, twist or otherwise change its conformation and by which conformational change alone or in combination with other conformational changes of other flexible regions, receptor's activity is induced or facilitated. It includes juxtamembranous regions, oligomerization regions including those having secondary structures such as α helix, β sheet, loops, β turns, and flexible regions between domains of the receptor or in long loops between two β chains.

Peptides Preparation

[0026]The peptides of this invention, including the analogs and other modified variants, may generally be synthesized according to the FMOC protocol in an organic phase with protective groups. They can be purified with a yield of 70% with HPLC on a C18 column and eluted with an acetonitrile gradient of 10-60%. Their molecular weight can then be verified by mass spectrometry.

[0027]The peptides of the invention may also be prepared according to the solid phase synthetic method. For example, the solid phase synthesis is well known and is a common method for preparation of peptides, as are a variety of modifications of that technique [Merrifield (1964), J. Am. Chem. Soc., 85: 2149; Stewart and Young (1984), Solid Phase Peptide Synthesis, Pierce Chemical Company, Rockford, Ill.; Bodansky and Bodanszky (1984), The Practice of Peptide Synthesis, Springer-Verlag, New York; Atherton and Sheppard (1989), Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, New York].

[0028]Alternatively, peptides of this invention may be prepared in recombinant systems using polynucleotide sequences encoding the peptides. It is understood that a peptide of this invention may contain more than one of the above described modifications within the same peptide. Also included in this invention are pharmaceutically acceptable salt complexes of the peptides of this invention or their derivatives.

Peptides

[0029]Peptides of the present invention may therefore be constituted solely of L-amino acid sequences identical to amino acid sequences of flexible regions of an animal cytokine receptor and preferably of the human cytokines receptor that they target (subfragment peptides) and any mutated peptide that can be generated.

[0030]While subfragment peptides are effective in inhibiting wild-type cytokines in vitro, their effectiveness in vivo might be compromised by the presence of proteases. Serum proteases have specific substrate requirements. The substrate must have both L-amino acids and peptide bonds for cleavage. Furthermore, exopeptidases, which represent the most prominent component of the protease activity in serum, usually act on the first peptide bond of the peptide and require a free N-terminus (Power, et al. (1993), Pharmaceutical Res., 10:1268-1273). In light of this, it is often advantageous to utilize modified versions of subfragment peptides. The modified peptides retain the structural characteristics of the original L-amino acid peptides that confer biological activity with regard to cytokines, but are advantageously not readily susceptible to cleavage by proteases and/or exopeptidases.

[0031]Therefore, in specific modes, the peptides of this invention are not subfragment peptides, although their amino acid sequence is derived from the linear sequence of the human cytokine receptors or the corresponding sequences of non-human cytokine receptors and able to inhibit a cytokine receptor's activation (e.g. oligomerization) and more particularly the activation of a human cytokine receptor. Particularly, suitable non-human cytokine receptors sources include mouse, rat, quail and horse. It is thus apparent that multiple systems can provide suitable peptides and derivatives from which the cytokine receptor antagonists of the present invention can be derived.

[0032]The term "peptides" as referred to herein therefore includes cytokine receptor subfragment peptides, D-peptides and other modified forms of the peptides, so long as the modification does not alter ability to modulate cytokine receptor activity. All agonists and antagonists peptides of this invention share the ability to modulate the activity of specific cytokines receptors. Non-limiting examples of modifications include N-terminal acetylation, glycosylation, and biotinylation. Particular modified versions of the subfragment peptides according to the present invention are further described below. Although the peptides of the present invention encompass any peptide derived from the flexible regions of cytokines receptors, preferred peptides of the present invention are chosen so as to be specific to a particular receptor isoform (e.g. VEGFR-2) to ensure that their spatial conformation is complementary to the flexible region that they target. This latter characteristic is obtained by choosing where the peptide will be cut according to the properties afforded by each amino-acid in the remaining sequence (e.g. if the peptide has to follow the specific curve of the domain targeted).

[0033]The term "peptides derived from a flexible region" refers herein to peptides of 5 to about 20 amino acids that have been generated to correspond to segments of 5 to 20 contiguous amino acids located anywhere in the flexible regions and that may have been subjected to further modification or functional derivation as described herein. Preferably, the peptides derived from a flexible region is a peptide of at least 7 amino acids.

[0034]D-amino acid peptides can have modifications at the N-terminal amino-acid and at the C-terminal amino-acid. The presence of an N-terminal or C-terminal D-amino acid increases the serum stability of a peptide which otherwise contains L-amino acids, because exopeptidases acting on these residues cannot utilize a D-amino acid as a substrate (Powell, et al. (1993)). Cyclic peptides have no free N- or C-termini. Thus, they are not susceptible to proteolysis by exopeptidases, although they are of course susceptible to endopeptidases, which do not cleave at peptide termini. Thus, the amino acid sequences of the peptides with N-terminal or C-terminal D-amino acids and of the cyclic peptides are usually identical to the sequences of the subfragment peptides to which they correspond, except for the presence of an N-terminal or C-terminal D-amino acid residue, or their circular structure, respectively.

[0035]Substitution of unnatural amino acids for natural amino acids in a subsequence of the subfragment of cytokine receptor peptide can also confer resistance to proteolysis. Such a substitution can, for instance, confer resistance to proteolysis by exopeptidases acting on the N-terminus. Such substitutions have been described (Coller, et al. (1993), J. Biol. Chem., 268:20741-20743, incorporated herein by reference) and these substitutions do not affect biological activity. Furthermore, the synthesis of peptides with unnatural amino acids is routine and known in the art (see, for example, Coller, et al. (1993), supra).

[0036]An other effective approach to confer resistance to peptidases acting on the N-terminal or C-terminal residues of a peptide is to add chemical groups at the peptide termini, such that the modified peptide is no longer a substrate for the peptidase. One such chemical modification is glycosylation of the peptides at either or both termini. Certain chemical modifications, in particular N-terminal glycosylation, have been shown to increase the stability of peptides in human serum [Powell et al. (1993), supra]. Other chemical modifications which enhance serum stability include, but are not limited to, the addition of an N-terminal alkyl group, consisting of a lower alkyl of from 1 to 20 carbons, such as an acetyl group, and/or the addition of a C-terminal amide or substituted amide group. In particular the present invention includes modified peptides consisting of subfragment peptides bearing an N-terminal acetyl group and a C-terminal amide group.

[0037]Longer peptide sequences which result from the addition of extra amino acid residues to the peptides of the invention are encompassed in the present invention since they should have the same biological activity (inhibit oligomerization of cytokines) as the peptides described above. While peptides having a substantial number of additional amino acids are not excluded, it will be recognized that some large polypeptides may assume a configuration that masks the effective sequence, thereby preventing binding to cytokines. These derivatives will act as competitive antagonists and are thereby excluded from the invention. Thus, while the present invention encompasses peptides or derivatives having an extension, such longer peptides should be selected as not destroying the modulating activity of the peptide or derivative.

[0038]The present invention also encompasses peptides constituted of the sequences of two peptides having separately the property of inhibiting the activation (e.g. oligomerization) of a particular cytokine receptor, but not being contiguous within the flexibility regions. These peptides can also be described as having a sequence corresponding to the particular cytokine receptor with an internal deletion.

[0039]In another embodiment of this invention the peptides are reverse-D peptides corresponding to the amino acid sequence of the cytokine. The term "reverse-D peptide" refers herein to peptides containing D-amino acids, arranged in a reverse sequence relative to a peptide containing L-amino acids. Thus, the C-terminal residue of an L-amino acid peptide becomes N-terminal for the D-amino acid peptide, and so forth. For example, the sequence of the reverse-D peptide corresponding to subfragment peptide SEQ ID NO: 1 is: GVLIIIELNTKEQA. Reverse-D peptides retain the same tertiary conformation, and therefore the same activity, as the L-amino acid peptides, but are more stable to enzymatic degradation in vitro and in vivo, and thus have greater therapeutic efficacy than the original peptide (Brady and Dodson (1994), Nature, 368: 692-693; Jameson et al. (1994), Nature, 368: 744-746).

[0040]As used herein, the designation "functional derivative" denotes, in the context of a functional derivative of an amino acid sequence, a molecule that retains a biological activity (either function or structural) that is substantially similar to that of the original sequence. This functional derivative or equivalent may be a natural derivative or may be prepared synthetically. Such derivatives include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved (e.g. it acts as a non-competitive inhibitor or agonist of a cytokine receptor). The substituting amino acid generally has chemico-physical properties which are similar to that of the substituted amino acid. The similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophylicity and the like. The term "functional derivatives" is intended to include "segments", "variants", "analogs" or "chemical derivatives" of the subject matter of the present invention.

[0041]Thus, the term "variant" refers herein to a protein which is substantially similar in structure and biological activity to the protein or nucleic acid of the present invention.

[0042]The functional derivatives of the present invention can be synthesized chemically or produced through recombinant DNA technology. All these methods are well known in the art.

[0043]While peptides of specific embodiments of the present invention are preferably derived from human cytokines receptors, the invention should not be so limited. Indeed, in view of the significant conservation of flexible regions of these genes throughout evolution, sequences from different species, as discussed above and preferably mammalian species, could be used in the assays of the present invention. For instance, non-limiting examples for the VEGFR protein are the quail, mouse, rat and horse VEGFR protein sequences which show 70%, 82% and 82% similarity, respectively with the human VEGFR protein sequence. Similarly, the IL-1R mouse, rat and horse protein sequences show a 68%, 67% and 77% sequence similarity, respectively. Also, the IL-4R mouse and horse protein sequences show a 48% and 59% sequence similarity, respectively (as calculated by Blast®).

[0044]For administration to humans, the prescribing medical professional will ultimately determine the appropriate form and dosage for a given patient, and this can be expected to vary according to the chosen therapeutic regimen (e.g. peptides, variants, mimetics), the response and condition of the patient as well as the severity of the disease.

[0045]Composition within the scope of the present invention should contain the active agent (e.g. peptide) in an amount effective to achieve the desired therapeutic effect while avoiding adverse side effects. Typically, the nucleic acids in accordance with the present invention can be administered to mammals (e.g. humans) in doses ranging from 0.005 to 1 mg per kg of body weight per day of the mammal which is treated. Pharmaceutically acceptable preparations and salts of the active agent are within the scope of the present invention and are well known in the art (Remington's Pharmaceutical Science, 16th Ed., Mack Ed.). For the administration of polypeptides, antagonists, agonists and the like, the amount administered should be chosen so as to avoid adverse side effects. The dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient. Typically, 0.001 to 50 mg/kg/day will be administered to the mammal.

Assays to Identify Peptides of the Present Invention

[0046]Preferred methods for testing the ability of candidate compounds to inhibit the various cytokine receptors activity are presented herein. It will be understood that the invention is not so limited. Indeed, often assays well known in the art can be used in order to identify non-competitive, extracellular agonists or antagonists of the present invention.

[0047]As used herein, "cytokine receptor activity or activation" refers to any detectable biological activity of these proteins. This includes any physiological function attributable to a cytokine receptor such as any standard biochemical measurement of these receptors, conformational changes, phosphorylation status, any downstream effect of the receptor's signaling such as protein phosphorylation, kinase effect or any other feature of the protein that can be measured with techniques known in the art. Measuring the effect of a candidate peptide on its ability to modulate the oligomerization of the receptor is measuring a cytokine receptor's activity according to this invention. Broadly intra- or inter-molecular binding of the receptor in the absence vs the presence of the peptide of the invention is yet another example of a biological activity according to the invention.

[0048]The assays of this invention employ either a natural or recombinant cytokine receptor. A cell fraction or cell free screening assays for inhibitors of cytokine receptor activity can use in situ purified, or purified recombinant cytokine receptor. Cell-based assays can employ cells which express cytokine receptor naturally, or which contain recombinant cytokine receptor. In all cases, the biological activity of cytokine receptor can be directly or indirectly measured; thus inhibitors or activators of cytokine receptor activity can be identified. The inhibitors or activators themselves may be further modified by standard combinatorial chemistry techniques to provide improved analogs of the originally identified compounds.

[0049]It shall be understood that the "in vivo" experimental model can also be used to carry out an "in vitro" assay.

In Vitro Assays

[0050]In one embodiment, candidate peptides are tested for their ability to activate or inhibit cytokine receptor's ability to modulate cellular proliferation with the incorporated triated thymidine method. In yet other preferred embodiments, candidate peptides are tested for their ability to inhibit a particular cytokine receptors ability to modulate cellular proliferation, using for example, the assays described in Baker F. L. et al. (1995) Cell Prolif. 28(1):1-15; Cheviron N. et al. (1996) Cell Prolif. 29(8):43746; Hu Z. W. et al. (1999) J: Pharmacol. Exp. Ther. 290(1):28-37; and Elliott K. et al. (1999) Oncogene 18(24):3564-73.

[0051]In another preferred embodiment, candidate peptides are tested for their ability to modulate the phosphorylation state of cytokine protein or portion thereof, or an upstream or downstream target protein, using for example an in vitro kinase assay. Briefly, a cytokine receptor target molecule (e.g. an immunoprecipitated receptor from a cell line expressing such a molecule), can be incubated with radioactive ATP, e.g., [gamma-³²P]-ATP, in a buffer containing MgCl² and MnCl², e.g., 10 mM MgCl² and 5 mM MnCl². Following the incubation, the immunoprecipitated receptor target molecule, can be separated by SDS-polyacrylamide gel electrophoresis under reducing conditions, transferred to a membrane, e.g., a PVDF membrane, and autoradiographed. The appearance of detectable bands on the autoradiograph indicates that the receptor substrate has been phosphorylated. Phosphoaminoacid analysis of the phosphorylated substrate can also be performed in order to determine which residues on the receptor substrate are phosphorylated. Briefly, the radiophosphorylated protein band can be excised from the SDS gel and subjected to partial acid hydrolysis. The products can then be separated by one-dimensional electrophoresis and analyzed on, for example, a phosphoimager and compared to ninhydrin-stained phosphoaminoacid standards. Assays such as those described in, for example, Tamaskovic R. et al. (1999) Biol. Chem. 380(5):569-78.

[0052]In other embodiments, candidate peptides targeting IL-1R are tested with PGE₂ levels, IL-6, collagenase expression in chondrocytes and RPE; candidate peptides targeting IGF-1R are tested with Akt in Du145 and PC12; candidate peptides targeting IL-4R are tested with Akt in Thelper and PAEC and with VCAM-1 expression in PAEC.

In Vivo Assays

[0053]The assays described above may be used as initial or primary screens to detect promising lead compounds for further development. Lead peptides will be further assessed in additional, different screens. Therefore, this invention also includes secondary cytokine receptors screens which may involve various assays utilizing mammalian cell lines expressing these receptors or other assays.

[0054]Tertiary screens may involve the study of the identified inhibitors in animal models for clinical symptoms. Accordingly, it is within the scope of this invention to further use an agent (peptide or peptidomimetic) identified as described herein in an appropriate animal model such as a rat or a mouse. For example, a peptide can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatment (e.g. treatments of different types of disorders associated with a deregulation or malfunction of a cytokine receptor), as described herein. Preferred such experiments include collagen-induced arthritis in rat, acute septic shock in rat, tumor growth in immunosuppressed mouse, sensitization of the airways in newborn mice and any other known animal model including transgenics.

Assays to Identify Peptidomimetics

[0055]Non-peptidyl compounds generated to replicate the backbone geometry and pharmacophore display (peptidomimetics) of the peptides identified by the methods of the present invention often possess attributes of greater metabolic stability, higher potency, longer duration of action and better bioavailability.

[0056]The peptidomimetics compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the `one-bead one-compound` library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des. 12: 145, 1997). Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994), J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem, Int. Ed Engl. 33:2059; and ibid 2061; and in Gallop et al. (1994). Med. Chem. 37:1233. Libraries of compounds may be presented in solution (e.g. Houghten (1992) Biotechniques 13:412-421) or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria or spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990); Science 249:386-390). Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) supra; Erb et al. (1994) supra; Zuckermann et al. (1994) supra; Cho et al. (1993) supra; Carrell et al. (1994) supra, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

[0057]In one embodiment, the peptidomimetics compounds of the present invention are preferably obtained with the following three phase process. 1) Scanning the peptides of the present invention to identify regions of secondary structure necessary for recognition and activity toward the cytokine receptor; 2) use conformationally constrained dipeptide surrogates to refine the backbone geometry and provide organic platforms corresponding to these surrogates; 3) Use the best organic platforms to display organic pharmocophores in libraries of candidates designed to mimic the desired activity of the native peptide. In more details the three phases are as follows.

[0058]In phase 1, the peptide leads are scanned and their structure abridged to identify the requirements for their activity. A series of peptide analogs of the original are synthesized. In phase 2, the best peptide analogs are investigated using the conformationally constrained dipeptide surrogates. Indolizidin-2-one, indolizidin-9-one and quinolizidinone amino acids (I²aa, I⁹aa and Qaa respectively)) are used as platforms for studying backbone geometry of the best peptide candidates. These platforms are introduced at specific regions of the peptide in order to orient the pharmacophores in different directions. Biological evaluation of these analogs identifies improved leads that mimic the geometric requirements for activity. In phase 3, the platforms from the most active leads are used to display organic surrogates of the pharmacophores responsible for activity of the native peptide. The pharmacophores and scaffolds are combined in a parallel synthesis format.

[0059]In summary, based on the disclosure herein, those skilled in the art can develop peptides and peptidomimetics screening assays which are useful for identifying compounds which are useful for inhibiting cytokine receptors. Compounds so identified might also be shown to activate these receptors. The assays of this invention may be developed for low-throughput, high-throughput, or ultra-high throughput screening formats. Of course, assays of the present invention include assays which are amenable to automation.

[0060]More specifically, in accordance with one embodiment, the present invention, there is provided a method for identifying a non-competitive peptide which inhibits the oligomerization of a cytokine receptor, the method comprising the steps of selecting a candidate peptide containing from about 7 to about 20 amino acids derived from a flexible region of the receptor, and determining the ability of the peptide to inhibit the oligomerization of the receptor by measuring an activity of the receptor in the presence of a compound known to activate the receptor and in the absence or the presence of the candidate peptide, wherein the non-competitive peptide is selected when the activity of the receptor is measurably lower in the presence of the peptide as compared to in the absence thereof.

[0061]There is also provided a non-competitive extracellular cytokine receptor antagonist wherein the antagonist is a peptide containing from about 7 to about 20 amino-acids derived from a flexible region of the cytokine receptor.

[0062]The present invention also provides methods of treating diseases or conditions associated with a abnormal activity of a cytokine receptor comprising administration of a suitable amount of peptide or derivative of the invention.

[0063]The present invention also relates to pharmaceutical compositions comprising a modulating amount a or cytokine receptor subfragment peptide or derivative of the present invention, together with a suitable pharmacological carrier.

[0064]The terms "inhibiting," "reducing" or "prevention," or any variation of these terms, when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result.

[0065]The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0066]It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.

[0067]Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0068]The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

[0069]As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0070]Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0071]In the appended drawings:

[0072]FIG. 1 schematically illustrates the position of VEGFR antagonists on the receptor according to specific embodiments of the present invention;

[0073]FIG. 2 graphically illustrates in panel A results of proliferation assay in porcine microvascular endothelial cells in presence of VEGF (2 ng/ml) and peptides 2.1, 2.2, 2.3 (10 μM). In panel B are graphically illustrated dose-response of peptides in pulmonary arterial endothelial cells (PAEC) in presence of VEGF (2 ng/ml) and increasing doses of peptides. In panel C is graphically illustrated the effect of intravitreally injected peptides (10 μM [estimated final intraocular concentration]) of the present invention on neovascularization in rat retinas exposed to hyperoxic conditions;

[0074]FIG. 3 shows the sequence of the human VEGFR-2 (Flk-1). Boxed or underlined sequences represent the identified flexible region of VEGFR;

[0075]FIG. 4 shows the sequence of human Interleukin-1 receptor (IL-1R-alpha). Boxed or underlined sequences represent the identified flexible region of IL-1R-alpha;

[0076]FIG. 5 shows the sequence of human Interleukin-1 receptor accessory protein (IL-1 RacP). Boxed or underlined sequences represent the identified flexible region of IL-1 RacP;

[0077]FIG. 6 shows the sequence of human Insulin-like growth factor I receptor (IGF-1R). Boxed or underlined sequences represent the identified flexible region of; IGF-1R;

[0078]FIG. 7 shows the sequence of the human alpha chain of the Interleukin 4 receptor (IL-4R). Boxed or underlined sequences represent the identified flexible region of; (IL-4R).

[0079]FIG. 8 graphically illustrates results of proliferation assays in carcinome A549 cells in presence of IGF-1 (10 ng/ml-Panel A) (1 ng/ml-Panel B) and various concentrations of the peptides APG-201, APG-202 and APG-204;

[0080]FIG. 9 graphically illustrates results of proliferation assays in carcinome A549 cells in presence of IL-1 (10 ng/ml-Panel A) (1 ng/ml-Panel B) and various concentrations of the peptides API-101, API-103 and API-106;

[0081]FIG. 10 graphically illustrates results of proliferation assays in carcinome A549 cells in presence of IL-4 (1 ng/ml) and various concentrations of the peptides API-401, API-402, API-403, API-404 and API-405;

[0082]FIG. 11 shows an alignment of the human IL-1R sequence with corresponding mouse, rat and horse sequences;

[0083]FIG. 12 shows an alignment of the human IL-4R sequence with corresponding mouse and horse sequences; and

[0084]FIG. 13 shows an alignment of the human VEGFR2 sequence with corresponding mouse, rat and quail sequences.

DESCRIPTION OF SPECIFIC EMBODIMENTS

[0085]Table 1 presents the localization of flexible regions of various representative members of the cytokine receptors families along with exemplary peptide sequences derived from these regions and chosen for their specificity to the particular member they target. As explained above, many peptides can be derived from the targeted regions of the present invention and the peptides described hereinbelow are only exemplary.

TABLE-US-00001 TABLE 1 Amino acids involved in the oligomerization and stability of receptors of representative members of various cytokine receptors LOCALISATION OF THE SEQUENCE CYTOKINES FROM THE RECEPTOR SPECIFIC REGIONS STARTING TYPES RECEPTORS TARGETED METHIONINE PEPTIDE SEQUENCES Tyrosine VEGFR2 Juxtamembranous Aa 745-770 AQEKTNLEIIILVG (2.1) Kinase (Flk-1) SEQ ID NO. 1 receptor Ig3-Ig4 Aa 320-350 EATVGERVRL (2.2) SEQ ID NO. 2 Ig-4 dimerization Aa 350-400 LPLESNHTLK (2.3) domain SEQ ID NO. 3 Ig-4-Ig-5 Aa 400-440 SPVDSYQYGTT; SEQ ID NO. 4 VILTNPISKE; SEQ ID NO. 5 Ig-5-Ig 6 Aa 481-565 NKVGRGERVI; SEQ ID NO. 6 MPPTEQESV SEQ ID NO. 7 Ig-6-Ig-7 Aa 640-685 RKTKKRHCV; SEQ ID NO. 8 TVLERVAPT; SEQ ID NO. 9 TSIGESIEV SEQ ID NO. 10 IGF-1R On chain α: Juxtamembranous Aa 725-740 SIFVPRPERK; SEQ ID NO. 11 NFLHNSIFV; SEQ ID NO. 12 Cyst rich domain-L2 Aa 320-335 EGPCPKVCE; SEQ ID NO. 13 L2-FbnIII-1 Aa 487-527 ESDVLHFTST; SEQ ID NO. 14 FbnIII-1-FbnIII2a Aa 595-620 RTNASVPSI; SEQ ID NO. 15 FbnIII-2a-Insert Aa 660-690 IRKYADGTI; domain SEQ ID NO. 16 On chain β: Insert domain- Aa 780-799 ENFIHLIIA; FbnIII2b SEQ ID NO. 17 AKTGYENFIH; SEQ ID NO. 18 FbnIII2b-FbnIII3 Aa 820-840 KERTVISNLR; SEQ ID NO. 19 Juxtamembranous Aa 917-947 FVFARTMPA; SEQ ID NO. 30 EGFR Juxtamembranous Aa 640-650 NGPKIPSIAT; SEQ ID NO. 31 Loop L2-S2 Aa 495-515 ATGQVCHAL; (flexible) SEQ ID NO. 32 Loop S1-L2 (Hinge) Aa 335-345 RKVCNGIGIGE; SEQ ID NO. 33 Type I: IL-4R Juxtamembranous Aa 210-240 WHNSYREPF; Chainγc SEQ ID NO. 34 YREPFEQHLL SEQ ID NO. 35 Hinge zone D2 Aa 125-216 SDTLLLTWS; SEQ ID NO. 36 IYNVTYLE; SEQ ID NO. 37 IAASTLKSGIS; SEQ ID NO. 38 Loop D1-D2 Aa 112-125 KPSEHVKPR; SEQ ID NO. 39 Single GHR Juxtamembranous Aa 250-270 FTCEEDFYFPW; chain flexible region Aa 160-240 SEQ ID NO. 40 (D1-D2) SVDEIVQPD; SEQ ID NO. 41 MDPIDTTSVPVY; SEQ ID NO. 42 IL-1R IL-1R Juxtamembranous Aa 320-341 IDAAYIQLIYPV; SEQ ID NO. 43 LIYPVTNFQKHM SEQ ID NO. 44 Between Ig-like Aa 209-240 LEENKPTRPV; domain 2 and 3 SEQ ID NO. 45 (Hinge) NKPTRPVIVS SEQ ID NO. 46 Ig-like 2 loop e2-f2 Aa 181-200 VAEKHRGNYT; (pas int.ligand) SEQ ID NO. 47 WNGSVIDED SEQ ID NO. 48 IL-1RacP Juxtamembranous Aa 330-370 VPAPRYTVEL; SEQ ID NO. 49 APRYTVELA; SEQ ID NO. 50 Hinge regions: Loop Ig-1-2: Aa 115-160 VQKDSCFNSPM; SEQ ID NO. 51 MKLPVHKLY SEQ ID NO. 52 Loop Ig-2-3 Aa 170-266 VGSPKNAVPPV; SEQ ID NO. 53 VTYPENGRTF; SEQ ID NO. 54 IHSPNDHVVY; SEQ ID NO. 55 dimerization region Aa 200-215; LISNNGNYT; 275-295; SEQ ID NO. 56 300-315 VWWTIDGKKPD; SEQ ID NO. 57 WTIDGKKPDDI; SEQ ID NO. 58 HSRTEDETRTQ SEQ ID NO. 59

[0086]Cytokines receptors modulators according to specific embodiments of the present invention will now be described as well as the procedure to identify them and to test their efficiency in vitro and/or in vivo by the following non-limiting examples.

Example 1

VEGFR

Identification of VEGFR2 Antagonists

[0087]VEGF is a proliferating agent for endothelial cells. Its receptor (VEGFR) is present at the plasma membrane of endothelial cells as a monomer and its homodimerization is necessary for generating autophosphorylation via its intrinsic tyrosine kinase domain.

[0088]The method of identifying VEGFR antagonists of the present invention is based on the localization of extracellular flexible regions including regions between domains and juxtamembranous regions of the receptor that are important for the appropriate conformation and oligomerization of the subunits of the receptor and its resulting activation. These regions were established based on crystal structure data provided by crystallography. The antagonists able to bind to these regions block the signal transduction by interfering with the oligomerization. The regions so identified appear in green in FIG. 3. One of those regions is located under the IG-like 3 domain where ligand binding is located, namely between residues 320 and 350. The ligand binding location also appears in FIG. 1. A second region was identified in the oligomerization domain of two subunits of Ig-like 4, namely between residues 350 and 400. A third region was identified located at the juncture of the receptor with the cellular membrane, namely between residues 745 and 770. This region is important for the dimer stability. These regions do not interfere with the ligand binding so that any antagonist (peptide, small molecule) targeting these regions is not a competitor for the ligand binding sites (non-competitive antagonist) and prevents or limits the oligomerization required for the autophosphorylation of the receptor. Three D-peptides of up to 12 amino-acids (designated 2.1, 2.2 and 2.3) were derived from the amino-acid sequence of these regions and tested as antagonists. As mentioned earlier, D-peptides are preferred over subfragment peptides (of course subfragments could also be rendered protease resistant by well known means) because they are less likely degradable by various proteases. These particular peptides were selected among all those that could have been derived from the identified flexible regions of interest because of their specificity to VEGFR-flk-1: sequences alignments were performed with other receptors from VEGFR's family (PDGFR, Flt-1) showing the specificity of the selected three peptides. Of course, such alignments enable a selection of other specific peptides or alternatively of more general antagonists. It should be understood that the principles related to positioning discussed herein in relation to VEGFR can be applied to other types of cytokine receptors sharing similar morphologies.

[0089]The location of the three peptides appear in FIG. 1, the ligand binding region appears in red, the oligomerization domain per se appears in green and the tyrosine kinase domain appears in purple.

[0090]In FIG. 3, the domains of the VEGFR isoform VEGFR-2 are identified with arrows pointing at the start of each domain. The regions where antagonists of the present invention may bind to prevent the oligomerization and/or activation of the receptor are boxed or underlined. The underlined sequences denote the regions between domains while the boxed sequences denote the juxtamembranous regions. The regions from where peptides 2.1, 2.2 and 2.3 are derived are identified in italic and are underlined. The sequences that the peptides target according to the invention appear underlined and boxed.

Characterization of Peptides In Vitro

[0091]To determine the efficient and non cytotoxic concentration of VEGF to use in the assay, a dose-response curve of VEGF was generated in two types of cells, namely microvascular endothelial cells and pulmonary artery endothelial cells (PAEC) that had been transfected with the Flk-1 gene. The proliferation was then measured in those two types of cells in the presence of peptides 2.1, 2.2 and 2.3 and of VEGF (2 ng/ml) pursuant to the incorporated tritiated thymidine method. The cells were preincubated at 37° C. with the different peptides at different concentrations. They were incubated with VEGF (2 ng/ml) for 24 hours. The cells were contacted with ³H-Thymidine for 24 hours, washed and lysed. The radioactivity was measured with a scintillation counter.

[0092]As may be seen in panels A and B of FIG. 2, the peptides 2.1, and 2.2 completely abrogated VEGF induced proliferation in microvascular endothelial cells, and in PAEC with an EC₅₀ of 9 μM, respectively. In addition, using these PAEC transfected with the cDNAs for either of the VEGFR isoforms Flk-1 and Flt, the selectivity of the peptides was demonstrated as they were shown to be ineffective in modulating biological functions in the VEGFR Flt isoform-containing cells (data not shown).

Characterization of Peptides In Vivo

Ischemic Retinopathy Model

[0093]The efficiency of the selected peptides was verified in vivo in a ischemic retinopathy model, a phenomena highly dependant on VEGF activation. Rat pups were exposed to 80% O₂ followed by a period of normoxia (21% O₂). The peptides were injected at a final concentration of 10 μM in the vitreous body. The retinas were then retrieved, colored with the ADPase method and mounted on slides. Photographs of the retinas were taken with a microscope linked to a computer and the vascular density was evaluated with the Image prosoftware. As illustrated in FIG. 2, panel C, the results of this experiment demonstrated that all peptides tested prevented induced neovascularization in vivo. Peptide 2.2 was shown to be the most effective inhibitor of neovascularization. Specific peptides of the present invention were shown to prevent effects generated by activation of Flk-1 with VEGF by interfering with flexible regions of Flk-1 receptor.

Example 2

Insulin-Like Growth Factor-1 Receptor (IGF-1R)

[0094]IGF-1 is a small peptide and a member of a family of insulin related peptides. It consists of 70 amino acids and has structural similarity with insulin. IGF-1 is secreted by many tissues (cartilage, bone, epithelium, endothelium) but mostly by the liver to act on other tissues in an endocrine fashion. It exerts its actions by binding to IGF-1R upon which it sends a mitogenic signal. It can also protect cells from apoptosis, promote proliferation, regulate cell adhesion and motility and differentiation. The receptor itself is expressed in most cell types except in the hepatocytes. Because of its growth inducing functions, IGF-1R is also very much involved in malignant transformation or differentiation in various types of cancer such as glioblastomas, neuroblastomas, prostate, breast and ovarian cancer.

[0095]IGF-1 plays a critical role in cell growth, survival and metastatic differentiation. IGF-1R is a transmembrane tyrosine kinase protein, which is widely expressed. Increased IGF-1 R expression is observed in a number of tumour types, and epidemiological data implicates it in cancers such as those of the prostate and breast. Recent progress has been made on its 3-dimensional structure.

Design of Peptides for IGF-1

[0096]The approach described in Example 1 is used to generate antagonists to IGF-1R. The precise localization of these regions is described in Table 1 above along with exemplary sequences of subfragment peptides or modified peptides targeting one of these regions and presenting specificity to IGF-1R. Three D-peptides (designated APG201, APG202 and APG204) were then derived from the amino-acid sequence of these regions to act as antagonists. The sequences of these peptides antagonists are as follows: APG-201 SLFVPRPERK; APG-202 ESDVLHFTST; APG-204 LRKYADGTL. They generally correspond to the subfragment peptides having sequences SEQ ID NOs: 11, 14 and 16, respectively, except where the subfragment peptide contained an isoleucine. Similarly, In that case, this amino-acid was replaced by leucine in the synthesized peptide for economic reasons.

Characterization of Peptides In Vitro and In Vivo

[0097]The affinity is determined using binding studies on cells expressing and overexpressing IGF-1R. The selectivity is tested by performing bioassays on cells expressing receptors from the same family as IGF-1R and the specificity is tested against receptors of another family of cytokine.

[0098]The proliferation of IGF-1 was measured in A549 carcinoma cells in the presence of peptides APG201; APG202 and APG204 and of IGF-1 (10 ng/ml-Panel A) and (1 ng/ml-Panel B) pursuant to the incorporated tritiated thymidine method. The cells were preincubated at 37° C. with the different peptides at different concentrations, namely 10^-7, 10^-6 and 10^-5M. They were then incubated with IGF-1 (10 ng/ml or 1 ng/ml) for 24 hours. The cells were then contacted with ³H-Thymidine for 24 hours, washed and lysed. The radioactivity was then measured with a scintillation counter.

[0099]As may be seen in panels A and B of FIG. 8, the peptides completely abrogated IGF-1 induced proliferation in A549 carcinoma cells with an EC₅₀ of 10^-8M for APG-202 and 204; and of 10^-6M for APG-201.

[0100]Further in vitro testing of the antagonists are conducted as described in Table 2.

[0101]In vivo experiments are described in Table 5.

TABLE-US-00002 TABLE 2 In vitro bioassays for IGF-1R antagonist screening Cells Type Bioassay Method Du145 Prostate cancer Proliferation ³H-Thymidine cell line Akt incorporation phosphorylation Western Blot PC12 Pheochromo- Same as above Same as above cytoma cell line

Example 3

Interleukin 4 (IL-4)

[0102]IL-4 is a key cytokine involved in the development of allergic inflammation and allergy. It is generated early on in the process of inflammation in asthma. In allergy it is associated with the production of IgE immunoglobulins by B lymphocytes and will also up-regulate the expression of the IgE receptor on cell surface of B-lymphocytes, basophils and mast cells. In asthma it induces the expression of vascular cell adhesion molecule (VCAM-1) on vascular endothelium. This effect leads to direct migration chemotaxis of T lymphocytes, monocytes, basophils and eosinophils to the inflammatory site on pulmonary vascular endothelial cells. IL-4 inhibits eosinophil apoptosis and promotes eosinophilic inflammation by augmenting their presence in part by increasing expression of eotaxin. Another essential biological effect of IL-4 is Th2 differentiation and proliferation; in this process IL-4 diminishes T lymphocyte apoptosis. The II-4 receptor is a cell-surface protein consisting of an α subunit coupled to a γ subunit for signal transduction; its activation requires oligomerization.

[0103]Although IL-4R and IL-13R share a similar IL-4Rα chain, the two receptors exhibit distinct functions; moreover, the main receptor present on TH2 cells is that of IL-4, which for the most part consists of the IL-4Rα and IL-4γc chains. Nevertheless, the identification of modulators of IL-4R activity. Derived from the IL-4Rα are expected to also modulate IL-13R activity.

Design of Peptides for IL-4R

[0104]The approach described in Example 1 is used to generate antagonists to IL-4R. The precise localization of these regions is described in Table 1 above along with exemplary sequences of subfragment peptides or modified peptides targeting one of these regions and presenting specificity to IL-4R

Characterization of Peptides In Vitro and In Vivo

[0105]The affinity is determined using binding studies on cells expressing and overexpressing IL-4R. The selectivity is tested by performing bioassays on cells expressing receptors from the same family as IL-4R and the specificity is tested against receptors of another family of cytokine.

[0106]The proliferation of IL-4 was measured in A549 carcinoma cells in the presence of peptides API-401, API-402, API-403, API-404 and API-405 and of IL-4 (1 ng/ml) pursuant to the incorporated tritiated thymidine method. The cells were preincubated at 37° C. with the different peptides. They were then incubated with IL-4 (1 ng/ml) for 24 hours. The cells were then contacted with ³H-Thymidine for 24 hours, washed and lysed. The radioactivity was then measured with a scintillation counter. The sequences of peptides antagonists used are as follows: API-401 YREPFEQHLL, API-402 SDTLLLTWS; API-403 LYNVTYLE; API-404 LAASTLKSGLS; and API-405 KPSEHVKPR. They generally correspond to the subfragment peptides having sequences SEQ ID NOs: 35, 36, 37, 38 and 39, respectively except where the subfragment peptide contained an isoleucine. In that case, this amino-acid was replaced by leucine in the synthesized peptide as mentioned previously.

[0107]As may be seen in FIG. 10, four out of five peptides prevented IL-4 from stopping proliferation in A549 carcinoma cells.

[0108]Further In vitro testing of the antagonists is conducted, as described in Table 3. In vivo experiments are described in Table 5.

TABLE-US-00003 TABLE 3 In vitro bioassays for IL-4R antagonist screening Cells Type Bioassay Method T helper T helper cells Proliferation ³H-Thymidine Akt incorporation phosphorylation Western Blot PAEC Human pulmonary VCAM-1 expression Western blot artery endothelial cells

Example 4

Interleukin-1

[0109]Interleukin-1 (IL-1) plays a primary upstream role in the regulation of inflammation by stimulating generation of other inflammatory mediators and by enhancing the process of inflammation directly. Along with TNF, IL-1 is considered as a prototype for inflammatory cytokines. The effects of IL-1 are not limited to inflammation and this cytokine plays a role in bone formation and remodeling, insulin secretion and fever induction. IL-1 is also a major player in acute and chronic inflammation (e.g. septic shock, inflammatory bowel diseases, osteoarthritis, or rhumatoid arthritis), Alzheimer's disease and a number of autoimmune diseases. Monocytes are predominant sources of IL-1 but many other cell types express the protein: non-limiting examples include fibroblasts, endothelial cells, smooth muscle cells, osteoclasts, astrocytes, epithelial cells T-cells, B-cells and numerous cancer cells.

[0110]The interleukin-1 family of proteins consists of distinct but structurally related molecules: IL-1α, IL-1β and IL-18 which elicit a biologic response and IL-1ra, a naturally produced receptor antagonist. IL-1α is the predominant form in mice, IL-1β is predominant in human but both exert their effect through the same receptor. In addition, IL-1 induces the production of other inflammatory mediators like IL-6 and prostaglandin PGE₂ (induces COX-2 and PGE synthase expression) and induces proliferation and activation of numerous cell types.

[0111]As a major pro-inflammatory cytokine, IL-1 is a potentially powerful target for therapeutic interventions in diseases associated with articular cartilage injury such as in arthritis. Osteoarthritis and rhumatoid arthritis are only second to heart diseases for causing work disabilities in USA and their prevalence increase dramatically with age. Approximately 60 millions of American >40 years of age are at risk. In 1997, direct medical and disability costs for arthritis were approximately $75B (US). Other important disorders for which IL-1 contributes significantly include ulcerative colitis and Crohn's disease, which are also major causes of absenteeism in USA, and other types of auto-immune diseases.

[0112]Two distinct receptors of IL-1 have been cloned and characterized: IL-1R which generates the biological effects of IL-1, and IL-1RII which is a natural antagonist. In addition, a receptor accessory protein (IL-1RAcP), which is the putative signal-transducing subunit of the receptor complex has been identified. IL-1R type I is found mainly on T cells, keratinocytes, fibroblasts, chondrocytes, synoviocytes and epithelial cells. In order to generate a biological effect, IL-1R has to bind to IL-1 and subsequently to IL-1 RacP which is necessary for signal transduction. The extracellular portion of IL-1R contains 3 Ig-like domains that bind IL-1. Of note, according to studies involving antibodies directed against extracellular portions of IL-1 RacP, the latter does not interact with the cytokine and could therefore also be an excellent target for non-competitive peptidomimetic design.

Design of Peptides for IL-1R

[0113]The regions of the IL-1 receptor complex which were targeted are the third domain of IL-1R containing a flexible region and interacts with the accessory protein but not with the ligand. The equivalent domain on IL-1 RacP, is the juxtamembranous regions of IL-1R and IL-1AcP and the regions between the second and third extracellular domains of IL-1 RacP. The precise localization of these regions is described in Table 1 above along with exemplary sequences of subfragment peptides or modified peptides targeting one of these regions and presenting specificity to IL-1R.

Characterization of Peptides In Vitro and In Vivo

[0114]The affinity of the subfragment peptides or derivative is determined using binding studies on cells expressing or overexpressing IL-1R. The selectivity is tested by performing bioassays on cells expressing receptors from the same family as IL-1R (e.g. IL-18R) and the specificity is tested against receptors of another family of cytokine.

[0115]The proliferation effect of IL-1 was measured in A549 carcinoma cells in the presence of peptides API101; API103 and API106 and of IL-1 (10 ng/ml-Panel A) and (1 ng/ml-Panel B) pursuant to the incorporated tritiated thymidine method. The cells were preincubated at 37° C. with the different peptides at different concentrations, namely 10^-6, 10^-5 and 10⁴M. They were then incubated with IL-1 (10 ng/ml or 1 ng/ml) for 24 hours. The cells were then contacted with ³H-Thymidine for 24 hours, washed and lysed. The radioactivity was then measured with a scintillation counter. The sequences of the peptides antagonists used are as follows: API-101 APRYTVELA, API-103 MKLPVHKLY; and API-106 VGSPKNAVPPV. They generally correspond to the subfragment peptides having sequences SEQ ID NO: 50, NO: 52 and NO: 53, respectively except where the subfragment peptide contained an isoleucine. In that case, this isoleucine was replaced by a conservative leucine in the synthesized peptide for economic reasons.

[0116]As may be seen in panels A and B of FIG. 9, the peptides completely abrogated IL-1 induced proliferation in A549 carcinoma cells with an EC₅₀ of 10^-6M for API-101 and 103; and of 10^-5M for API-106.

[0117]The goal of the next experiment was to verify if the identified peptides can reverse the physiological actions of the natural cytokine in vivo either by injecting them through the jugular or directly in the stomach (to verify the stability of the peptide through the digestive tractus). 300 g Sprague-Dawley rats were anesthetized with isoflurane (2.54%). IL-1β was injected through the jugular. Blood was taken from the carotid for further analyses before and after (10 minutes) every injection. Peptides were then injected either directly in the stomach with a catheter or in the jugular at the concentration desired. Arterial blood pressure and other physiological characteristics were monitored at all time.

[0118]A severe hypotension induced by IL-1 was observed when administrated to the rats by either ways mentioned above. The following peptides constitute examples of antagonists that were able to prevent hypotension:

[0119]API-101.10 (target: juxtamembranous portion of the accessory protein of IL-1R, derivative of API-101): [0120]1) When administrated by jugular injection after IL-1β injection (5 ug/kg) it prevented hypotension by 95% at a concentration of 10^-8M. This demonstrated that the peptide has an hypotensor effect in vivo in animals by reversing the effect of IL-1β (data not shown). [0121]2) When administrated directly into the stomach, the peptide at a concentration of 10^-5M, reduced IL-1β induced hypotension by 60%. This result demonstrated that oral administration of the 101.10 peptide still maintained a major effect on IL-1β induced hypotension. (Data not Shown)

[0122]In another experiment, vasomotricity variation of piglets pial vessels was studied to further evaluate the particular effect of cytokine receptor subfragments on the vasodilatator effect of IL-1β. Brains were dissected from Yorkshire piglets. Slices of brain exposing the pial vessels were pinned to a wax base of a 20 ml bath containing Krebs buffer (pH 7.4) equilibrated with 95% O₂-5% CO₂ and maintained at 37° C. Microvessels were visualized and recorded using a video camera mounted on a dissecting microscope. Vascular diameter was measured using a digital image analyzer and the images were recorded before and after topical application of constricting agent U46619 at 10^-7M. After stabilization of the vasomotricity, IL-1β was added until stabilization of vasodilatation. Peptides were then injected at different concentrations from 10^-10 to 10^-5M. Reversal of vasodilatation (i.e. vasoconstriction) was visualized and measured as previously mentioned. IL-1β induced vasodilatation in the microvasculature of the piglet brain was observed. Examples of the inhibitory activity of cytokine subfragment peptides are given below: [0123]1) API-101 and 101.10 (Juxtamembranous part of accessory protein) could prevent the vasodilation induced by IL-1β (75 ng/ml) with an IC₅₀ of 182 nM (API-101) and 10.8 nM (API-101.10). The range of concentrations of the peptide administered was from 10^-10 to 10^-5M (data not shown) [0124]2) API-108 (hinge Ig-3 region of accessory protein) could prevent vasodilatation with an IC₅₀ of 1.9 nM (data not shown). The range of concentrations of the peptide administered was from 10^-10 to 10^-5M.

[0125]These results demonstrate that targeting of two flexible regions of one component of the receptor we could prevent IL-1β activity at a very low IC₅₀ and therefore with a very high efficiency.

[0126]Another way of assessing the effect of cytokine receptor subfragments on IL-1R activity in vivo is by measuring PGE₂ levels in rat blood serum. Rat blood samples were collected from in vivo experiments (e.g. Protocol for IL-1 induced hypotension) and centrifuged at maximum speed for 15 minutes. The serum was then passed through a Waters column in order to isolate the lipidic part. Samples were evaporated and PGE₂ quantities were determined with an RIA assay using a commercial kit (Cederlane).

[0127]If the cytokine receptor subfragment peptides can prevent hypotention in vivo they should be able to prevent also the synthesis of PGE₂. The prostaglandin was therefore measured in serum of rats used for experiments mentioned above (e.g. Arterial Blood Pressure variation measurement). An example of results obtained with a particular cytokine receptor subfragment peptide is described below: [0128]1) API-101.10 could prevent PGE₂ synthesis in vivo by 80% when the peptide was injected in the jugular. The same results were obtained when the peptide was injected directly in the stomach (data not shown).

[0129]These experiments demonstrate that the identified peptides derived from different flexible regions of a cytokine receptor (in this particular example, receptor IL-1R/IL-1 RacP) are efficient and very potent in vitro and an vivo at reversing various biological effects of IL-1β.

[0130]From these experiments the efficiency and specificity of the method used to select particular cytokine subfragment peptides to modulate cytokine receptor activity is clearly demonstrated. Furthermore, the particular experiments presented above (with the IL-1R/IL-1RacP receptors) serves as a complete example of how one can select a particular cytokine receptor subfragment peptide (derivitize and/or protect it if desired), test its modulating activity in vitro and than its efficiency and potency in vivo. It also demonstrate that the modulating activities demonstrated in vitro are translatable to the in vivo situation.

[0131]The stability and selectivity of the peptides in vitro and in vivo is further verified with the tests described in Table 4, and Table 5 below, respectively.

TABLE-US-00004 TABLE 4 In vitro bioassays for IL-1R antagonist screening Cells Type Bioassay Method Chondrocytes Human PGE₂ levels RIA kit chondrocytes IL-6 RIA kit Proliferation ³H-Thymidine Collagenase incorporation expression Western Blot RPE Human retinal Same as above Same as above pigment epithelial cells Thymocytes EL4-Mouse Proliferation ³H-Thymidine thymocytes-High incorporation IL1R expression Fibroblasts Human F7100 Proliferation ³H-Thymidine incorporation

[0132]Table 5 summarizes the nature of the in vivo experiments performed with various peptides of the present invention. They are presented in more details below.

TABLE-US-00005 TABLE 5 In vivo experiments to assess efficacy and specificity of antagonists against IL-1R, IGF-1R and IL-4R Target Animal model Method Treatment Parameters IL-1R Collagen-induced arthritis in s.c. injections Following onset of Destruction of rat of type II arthritis, cartilage collagen in continuous assessed by incomplete delivery of the histological Freund's drug via osmotic staining and adjuvant pump digital imaging Arterial blood pressure Injection of 10 minutes Blood pressure variation measurement in IL-1b in following IL-1b, variation rats jgular injection of peptide measurements antagonist in jugular or stomach. Vasomotricity experiment on Topical Following U46619 Vascular piglet pial vessels application of induced diameters U46619 vasodilatation, agent as application of vasoconstrictor peptide antagonist than, IL- in microvessels 1b as a vasodilatator PGE₂ levels in rat blood Injection of injection of peptide PGE₂ levels serum IL-1b in antagonist in jugular jugular or stomach and measurement of PGE₂ levels by RIA kit Acute septic shock in rat LPS-induced Preceding i.v. Blood pressure, septic shock bolus of LPS the body temperature animal will receive and cardiac an i.v. bolus of the rhythm will be antagonist monitored during the whole experiment (60 min) IGF-IR Tumor growth in s.c. injection Continuous Tumor size immunosuppressed mouse of tumoral delivery of the monitoring (nude mouse) cell line antagonist with osmotic pump after latency to obtain solid tumor IL-4R Sensitization of the airways Exposure of i.p. injection of IgE and TNF-γ in newborn mice the animals receptor dosage ovalbumin antagonist (i.p. injection and aerosolized)

Acute Septic Shock in Rats

[0133]The efficiency of the peptides is also verified with the acute septic shock in Sprague-Dawley rat. Sprague-Dawley (160-180 gm) rats (Charles River) are anesthesed with a solution 9:1 xelazine/ketamine at a concentration of 1 mg/Kg. A tracheotomy is performed so as to maintain ventilation with a tube linked to a respirator. A cannula is inserted into the right carotide artery to enable monitoring of the systemic arterial with a Stratham pressure transducer linked to a multichannel Gould apparatus. The right jugular vein is cannuled to enable drug administration. The animal is placed under radial heat to maintain a constant normal temperature. The septic shock is obtained by systemic injection of a lipopolysaccharide bolus (LPS) (1 mg/kg: Sigma). A decrease of about 30 mm Hg is observed after ˜5 minutes.

Collagen-Induced Arthritis Protocol in Lewis Rat

[0134]Type II Collagen (CII) that has been isolated and purified from bovine articulary is obtained from Sigma. CII (2 mg/ml) is dissolved over night at 4° C. with agitation in 0.01 M acetic acid. The solution is then emulsified in an incomplete Freund's adjuvant (CII: ICFA, Difco Laboratories, Detroit, Mich.). Lewis female rats (Charles River) of 140-180 gm and of 8 week old are immunised with 0.5 ml of the emulsion (0.5 mg CII) with many intradermal injections in the back and one or two injections in the tail base. The animals are then reinjected 7 days later in the tail base with 0.2 ml (0.2 mg CII) so as to obtain an acute inflammatory reaction. A different time points during the experiment (1 to 24 days) animals are sacrificed and knuckle joints samples are taken to be fixed and coated so as to enable cryosections of 6-7 μm. A double coloration of Goldner and toluidine blue is performed on slides to measure the importance of the articular inflammation. Digitalised images are taken and analysed with the Image Pro Plus® 4.1 software.

Tumor Growth in Immunosuppressed Mouse (Nude Mouse)

[0135]The colon Colo 205® carcinoma cell line is obtained from the American Type Culture Collection (ATCC: Rockville, Md.). Cells are maintained in a RPMI-1640 culture- and grown in 100 mm Petri at 37° C. in a humidified atmosphere controlled to maintain 5% CO₂ and 95% air. The medium is supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin.

[0136]2.5×10⁶ carcinoma colon Colo 205® cells in 100 μl de PBS are injected subcutaneously in the back (needle 25 G:BD, NJ) in 6 weeks old immunodeficient female mice (Balb/c, nu/nu: Charles River). Treatment begins 5 days after injection of tumorous cells measuring ˜0.5×0.5 cm. the tumour volume is measured every two days according to the following formula: length×width×height, with a vernier caliper. 14 days after the beginning of treatments, animals are sacrificed and tumours are sampled to be weighted and measured in volume. Specimens are then fixed in a 10% formalin buffer for 24H and then transferred in 70% ethanol. Tumours are then coated with paraffin and sections are cut for immunohistochemistry purposes. The general morphology is evaluated with a hematoxyline/eosin coloration.

[0137]Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims. In particular, although the flexible regions of all cytokines have not all been described herein nor have all peptidic extracellular non competitive modulators encompassed by the present invention targeting these regions have been described, in light of the procedure described above for screening peptides and identifying peptides of the present invention, a person of ordinary skill in the art would be able to rapidly develop peptidic modulators of cytokine receptor by selecting peptides of 5 to 20 amino acid derived from known flexible regions of cytokines.

REFERENCES

[0138]Christine Piossek et al. "Vascular Endothelial Growth Factor (VEGF) Receptor II-derived Peptides Inhibit VEGF" The Journal of Biological Chemistry vol. 274, No. 9, Feb. 26, 1999, pp. 5612-5619. [0139]Daren C. W. Tan et al "A small peptide derived from fit-1 (VEGFR-1) functions as an angiogenic inhibitor" FEBS Letters 494 (2001) 150-156. [0140]Guy Vigers et al. "X-Ray Crystal Structure of a Small Antagonist Peptide Bound to Interleukin-1 Receptor Type 1" The Journal of Biological Chemistry vol. 275, No. 47, Nov. 24, 2000, pp. 36927-36933. [0141]Do-Young Yoon et al. "Antibodies to domains II and III of the IL-1 Receptor Accessory Protein Inhibit IL-1β Activity But Not Binding: Regulation of IL-1 Responses Is Via Type I Receptor, Not the Accessory Protein" Journal of Immunology, 1998.

Sequence CWU 1

65114PRTArtificialsynthetic peptide 1Gly Val Leu Ile Ile Ile Glu Leu Asn Thr Lys Glu Gln Ala1 5 10210PRTArtificialsynthetic peptide 2Glu Ala Thr Val Gly Glu Arg Val Arg Leu1 5 10310PRTArtificialsynthetic peptide 3Leu Pro Leu Glu Ser Asn His Thr Leu Lys1 5 10411PRTArtificialsynthetic peptide 4Ser Pro Val Asp Ser Tyr Gln Tyr Gly Thr Thr1 5 10510PRTArtificialsynthetic peptide 5Val Ile Leu Thr Asn Pro Ile Ser Lys Glu1 5 10610PRTArtificialsynthetic peptide 6Asn Lys Val Gly Arg Gly Glu Arg Val Ile1 5 1079PRTArtificialsynthetic peptide 7Met Pro Pro Thr Glu Gln Glu Ser Val1 589PRTArtificialsynthetic peptide 8Arg Lys Thr Lys Lys Arg His Cys Val1 599PRTArtificialsynthetic peptide 9Thr Val Leu Glu Arg Val Ala Pro Thr1 5109PRTArtificialsynthetic peptide 10Thr Ser Ile Gly Glu Ser Ile Glu Val1 51110PRTArtificialsynthetic peptide 11Ser Ile Phe Val Pro Arg Pro Glu Arg Lys1 5 10129PRTArtificialsynthetic peptide 12Asn Phe Leu His Asn Ser Ile Phe Val1 5139PRTArtificialsynthetic peptide 13Glu Gly Pro Cys Pro Lys Val Cys Glu1 51410PRTArtificialsynthetic peptide 14Glu Ser Asp Val Leu His Phe Thr Ser Thr1 5 10159PRTArtificialsynthetic peptide 15Arg Thr Asn Ala Ser Val Pro Ser Ile1 5169PRTArtificialsynthetic peptide 16Ile Arg Lys Tyr Ala Asp Gly Thr Ile1 5179PRTArtificialsynthetic peptide 17Glu Asn Phe Ile His Leu Ile Ile Ala1 51810PRTArtificialsynthetic peptide 18Ala Lys Thr Gly Tyr Glu Asn Phe Ile His1 5 101910PRTArtificialsynthetic peptide 19Lys Glu Arg Thr Val Ile Ser Asn Leu Arg1 5 10201356PRTHomo sapiens 20Met Gln Ser Lys Val Leu Leu Ala Val Ala Leu Trp Leu Cys Val Glu1 5 10 15Thr Arg Ala Ala Ser Val Gly Leu Pro Ser Val Ser Leu Asp Leu Pro20 25 30Arg Leu Ser Ile Gln Lys Asp Ile Leu Thr Ile Lys Ala Asn Thr Thr35 40 45Leu Gln Ile Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro50 55 60Asn Asn Gln Ser Gly Ser Glu Gln Arg Val Glu Val Thr Glu Cys Ser65 70 75 80Asp Gly Leu Phe Cys Lys Thr Leu Thr Ile Pro Lys Val Ile Gly Asn85 90 95Asp Thr Gly Ala Tyr Lys Cys Phe Tyr Arg Glu Thr Asp Leu Ala Ser100 105 110Val Ile Tyr Val Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser115 120 125Val Ser Asp Gln His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys130 135 140Thr Val Val Ile Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser145 150 155 160Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg165 170 175Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile180 185 190Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser195 200 205Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr210 215 220Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu225 230 235 240Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile245 250 255Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu260 265 270Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe275 280 285Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu290 295 300Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr305 310 315 320Phe Val Arg Val His Glu Lys Pro Phe Val Ala Phe Gly Ser Gly Met325 330 335Glu Ser Leu Val Glu Ala Thr Val Gly Glu Arg Val Arg Ile Pro Ala340 345 350Lys Tyr Leu Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr Lys Asn Gly355 360 365Ile Pro Leu Glu Ser Asn His Thr Ile Lys Ala Gly His Val Leu Thr370 375 380Ile Met Glu Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr Val Ile Leu385 390 395 400Thr Asn Pro Ile Ser Lys Glu Lys Gln Ser His Val Val Ser Leu Val405 410 415Val Tyr Val Pro Pro Gln Ile Gly Glu Lys Ser Leu Ile Ser Pro Val420 425 430Asp Ser Tyr Gln Tyr Gly Thr Thr Gln Thr Leu Thr Cys Thr Val Tyr435 440 445Ala Ile Pro Pro Pro His His Ile His Trp Tyr Trp Gln Leu Glu Glu450 455 460Glu Cys Ala Asn Glu Pro Ser Gln Ala Val Ser Val Thr Asn Pro Tyr465 470 475 480Pro Cys Glu Glu Trp Arg Ser Val Glu Asp Phe Gln Gly Gly Asn Lys485 490 495Ile Glu Val Asn Lys Asn Gln Phe Ala Leu Ile Glu Gly Lys Asn Lys500 505 510Thr Val Ser Thr Leu Val Ile Gln Ala Ala Asn Val Ser Ala Leu Tyr515 520 525Lys Cys Glu Ala Val Asn Lys Val Gly Arg Gly Glu Arg Val Ile Ser530 535 540Phe His Val Thr Arg Gly Pro Glu Ile Thr Leu Gln Pro Asp Met Gln545 550 555 560Pro Thr Glu Gln Glu Ser Val Ser Leu Trp Cys Thr Ala Asp Arg Ser565 570 575Thr Phe Glu Asn Leu Thr Trp Tyr Lys Leu Gly Pro Gln Pro Leu Pro580 585 590Ile His Val Gly Glu Leu Pro Thr Pro Val Cys Lys Asn Leu Asp Thr595 600 605Leu Trp Lys Leu Asn Ala Thr Met Phe Ser Asn Ser Thr Asn Asp Ile610 615 620Leu Ile Met Glu Leu Lys Asn Ala Ser Leu Gln Asp Gln Gly Asp Tyr625 630 635 640Val Cys Leu Ala Gln Asp Arg Lys Thr Lys Lys Arg His Cys Val Val645 650 655Arg Gln Leu Thr Val Leu Glu Arg Val Ala Pro Thr Ile Thr Gly Asn660 665 670Leu Glu Asn Gln Thr Thr Ser Ile Gly Glu Ser Ile Glu Val Ser Cys675 680 685Thr Ala Ser Gly Asn Pro Pro Pro Gln Ile Met Trp Phe Lys Asp Asn690 695 700Glu Thr Leu Val Glu Asp Ser Gly Ile Val Leu Lys Asp Gly Asn Arg705 710 715 720Asn Leu Thr Ile Arg Arg Val Arg Lys Glu Asp Glu Gly Leu Tyr Thr725 730 735Cys Gln Ala Cys Ser Val Leu Gly Cys Ala Lys Val Glu Ala Phe Phe740 745 750Ile Ile Glu Gly Ala Gln Glu Lys Thr Asn Leu Glu Ile Ile Ile Leu755 760 765Val Gly Thr Ala Val Ile Ala Met Phe Phe Trp Leu Leu Leu Val Ile770 775 780Ile Leu Arg Thr Val Lys Arg Ala Asn Gly Gly Glu Leu Lys Thr Gly785 790 795 800Tyr Leu Ser Ile Val Met Asp Pro Asp Glu Leu Pro Leu Asp Glu His805 810 815Cys Glu Arg Leu Pro Tyr Asp Ala Ser Lys Trp Glu Phe Pro Arg Asp820 825 830Arg Leu Lys Leu Gly Lys Pro Leu Gly Arg Gly Ala Phe Gly Gln Val835 840 845Ile Glu Ala Asp Ala Phe Gly Ile Asp Lys Thr Ala Thr Cys Arg Thr850 855 860Val Ala Val Lys Met Leu Lys Glu Gly Ala Thr His Ser Glu His Arg865 870 875 880Ala Leu Met Ser Glu Leu Lys Ile Leu Ile His Ile Gly His His Leu885 890 895Asn Val Val Asn Leu Leu Gly Ala Cys Thr Lys Pro Gly Gly Pro Leu900 905 910Met Val Ile Val Glu Phe Cys Lys Phe Gly Asn Leu Ser Thr Tyr Leu915 920 925Arg Ser Lys Arg Asn Glu Phe Val Pro Tyr Lys Thr Lys Gly Ala Arg930 935 940Phe Arg Gln Gly Lys Asp Tyr Val Gly Ala Ile Pro Val Asp Leu Lys945 950 955 960Arg Arg Leu Asp Ser Ile Thr Ser Ser Gln Ser Ser Ala Ser Ser Gly965 970 975Phe Val Glu Glu Lys Ser Leu Ser Asp Val Glu Glu Glu Glu Ala Pro980 985 990Glu Asp Leu Tyr Lys Asp Phe Leu Thr Leu Glu His Leu Ile Cys Tyr995 1000 1005Ser Phe Gln Val Ala Lys Gly Met Glu Phe Leu Ala Ser Arg Lys1010 1015 1020Cys Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu Ser Glu1025 1030 1035Lys Asn Val Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile1040 1045 1050Tyr Lys Asp Pro Asp Tyr Val Arg Lys Gly Asp Ala Arg Leu Pro1055 1060 1065Leu Lys Trp Met Ala Pro Glu Thr Ile Phe Asp Arg Val Tyr Thr1070 1075 1080Ile Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile1085 1090 1095Phe Ser Leu Gly Ala Ser Pro Tyr Pro Gly Val Lys Ile Asp Glu1100 1105 1110Glu Phe Cys Arg Arg Leu Lys Glu Gly Thr Arg Met Arg Ala Pro1115 1120 1125Asp Tyr Thr Thr Pro Glu Met Tyr Gln Thr Met Leu Asp Cys Trp1130 1135 1140His Gly Glu Pro Ser Gln Arg Pro Thr Phe Ser Glu Leu Val Glu1145 1150 1155His Leu Gly Asn Leu Leu Gln Ala Asn Ala Gln Gln Asp Gly Lys1160 1165 1170Asp Tyr Ile Val Leu Pro Ile Ser Glu Thr Leu Ser Met Glu Glu1175 1180 1185Asp Ser Gly Leu Ser Leu Pro Thr Ser Pro Val Ser Cys Met Glu1190 1195 1200Glu Glu Glu Val Cys Asp Pro Lys Phe His Tyr Asp Asn Thr Ala1205 1210 1215Gly Ile Ser Gln Tyr Leu Gln Asn Ser Lys Arg Lys Ser Arg Pro1220 1225 1230Val Ser Val Lys Thr Phe Glu Asp Ile Pro Leu Glu Glu Pro Glu1235 1240 1245Val Lys Val Ile Pro Asp Asp Asn Gln Thr Asp Ser Gly Met Val1250 1255 1260Leu Ala Ser Glu Glu Leu Lys Thr Leu Glu Asp Arg Thr Lys Leu1265 1270 1275Ser Pro Ser Phe Gly Gly Met Val Pro Ser Lys Ser Arg Glu Ser1280 1285 1290Val Ala Ser Glu Gly Ser Asn Gln Thr Ser Gly Tyr Gln Ser Gly1295 1300 1305Tyr His Ser Asp Asp Thr Asp Thr Thr Val Tyr Ser Ser Glu Glu1310 1315 1320Ala Glu Leu Leu Lys Leu Ile Glu Ile Gly Val Gln Thr Gly Ser1325 1330 1335Thr Ala Gln Ile Leu Gln Pro Asp Ser Gly Thr Thr Leu Ser Ser1340 1345 1350Pro Pro Val135521569PRTHomo sapiens 21Met Lys Val Leu Leu Arg Leu Ile Cys Phe Ile Ala Leu Leu Ile Ser1 5 10 15Ser Leu Glu Ala Asp Lys Cys Lys Glu Arg Glu Glu Lys Ile Ile Leu20 25 30Val Ser Ser Ala Asn Glu Ile Asp Val Arg Pro Cys Pro Leu Asn Pro35 40 45Asn Glu His Lys Gly Thr Ile Thr Trp Tyr Lys Asp Asp Ser Lys Thr50 55 60Pro Val Ser Thr Glu Gln Ala Ser Arg Ile His Gln His Lys Glu Lys65 70 75 80Leu Trp Phe Val Pro Ala Lys Val Glu Asp Ser Gly His Tyr Tyr Cys85 90 95Val Val Arg Asn Ser Ser Tyr Cys Leu Arg Ile Lys Ile Ser Ala Lys100 105 110Phe Val Glu Asn Glu Pro Asn Leu Cys Tyr Asn Ala Gln Ala Ile Phe115 120 125Lys Gln Lys Leu Pro Val Ala Gly Asp Gly Gly Leu Val Cys Pro Tyr130 135 140Met Glu Phe Phe Lys Asn Glu Asn Asn Glu Leu Pro Lys Leu Gln Trp145 150 155 160Tyr Lys Asp Cys Lys Pro Leu Leu Leu Asp Asn Ile His Phe Ser Gly165 170 175Val Lys Asp Arg Leu Ile Val Met Asn Val Ala Glu Lys His Arg Gly180 185 190Asn Tyr Thr Cys His Ala Ser Tyr Thr Tyr Leu Gly Lys Gln Tyr Pro195 200 205Ile Thr Arg Val Ile Glu Phe Ile Thr Leu Glu Glu Asn Lys Pro Thr210 215 220Arg Pro Val Ile Val Ser Pro Ala Asn Glu Thr Met Glu Val Asp Leu225 230 235 240Gly Ser Gln Ile Gln Leu Ile Cys Asn Val Thr Gly Gln Leu Ser Asp245 250 255Ile Ala Tyr Trp Lys Trp Asn Gly Ser Val Ile Asp Glu Asp Asp Pro260 265 270Val Leu Gly Glu Asp Tyr Tyr Ser Val Glu Asn Pro Ala Asn Lys Arg275 280 285Arg Ser Thr Leu Ile Thr Val Leu Asn Ile Ser Glu Ile Glu Ser Arg290 295 300Phe Tyr Lys His Pro Phe Thr Cys Phe Ala Lys Asn Thr His Gly Ile305 310 315 320Asp Ala Ala Tyr Ile Gln Leu Ile Tyr Pro Val Thr Asn Phe Gln Lys325 330 335His Met Ile Gly Ile Cys Val Thr Leu Thr Val Ile Ile Val Cys Ser340 345 350Val Phe Ile Tyr Lys Ile Phe Lys Ile Asp Ile Val Leu Trp Tyr Arg355 360 365Asp Ser Cys Tyr Asp Phe Leu Pro Ile Lys Ala Ser Asp Gly Lys Thr370 375 380Tyr Asp Ala Tyr Ile Leu Tyr Pro Lys Thr Val Gly Glu Gly Ser Thr385 390 395 400Ser Asp Cys Asp Ile Phe Val Phe Lys Val Leu Pro Glu Val Leu Glu405 410 415Lys Gln Cys Gly Tyr Lys Leu Phe Ile Tyr Gly Arg Asp Asp Tyr Val420 425 430Gly Glu Asp Ile Val Glu Val Ile Asn Glu Asn Val Lys Lys Ser Arg435 440 445Arg Leu Ile Ile Ile Leu Val Arg Glu Thr Ser Gly Phe Ser Trp Leu450 455 460Gly Gly Ser Ser Glu Glu Gln Ile Ala Met Tyr Asn Ala Leu Val Gln465 470 475 480Asp Gly Ile Lys Val Val Leu Leu Glu Leu Glu Lys Ile Gln Asp Tyr485 490 495Glu Lys Met Pro Glu Ser Ile Lys Phe Ile Lys Gln Lys His Gly Ala500 505 510Ile Arg Trp Ser Gly Asp Phe Thr Gln Gly Pro Gln Ser Ala Lys Thr515 520 525Arg Phe Trp Lys Asn Val Arg Tyr His Met Pro Val Gln Arg Arg Ser530 535 540Pro Ser Ser Lys His Gln Leu Leu Ser Pro Ala Thr Lys Glu Lys Leu545 550 555 560Gln Arg Glu Ala His Val Pro Leu Gly56522570PRTHomo sapiens 22Met Thr Leu Leu Trp Cys Val Val Ser Leu Tyr Phe Tyr Gly Ile Leu1 5 10 15Gln Ser Asp Ala Ser Glu Arg Cys Asp Asp Trp Gly Leu Asp Thr Met20 25 30Arg Gln Ile Gln Val Phe Glu Asp Glu Pro Ala Arg Ile Lys Cys Pro35 40 45Leu Phe Glu His Phe Leu Lys Phe Asn Tyr Ser Thr Ala His Ser Ala50 55 60Gly Leu Thr Leu Ile Trp Tyr Trp Thr Arg Gln Asp Arg Asp Leu Glu65 70 75 80Glu Pro Ile Asn Phe Arg Leu Pro Glu Asn Arg Ile Ser Lys Glu Lys85 90 95Asp Val Leu Trp Phe Arg Pro Thr Leu Leu Asn Asp Thr Gly Asn Tyr100 105 110Thr Cys Met Leu Arg Asn Thr Thr Tyr Cys Ser Lys Val Ala Phe Pro115 120 125Leu Glu Val Val Gln Lys Asp Ser Cys Phe Asn Ser Pro Met Lys Leu130 135 140Pro Val His Lys Leu Tyr Ile Glu Tyr Gly Ile Gln Arg Ile Thr Cys145 150 155 160Pro Asn Val Asp Gly Tyr Phe Pro Ser Ser Val Lys Pro Thr Ile Thr165 170 175Trp Tyr Met Gly Cys Tyr Lys Ile Gln Asn Phe Asn Asn Val Ile Pro180 185 190Glu Gly Met Asn Leu Ser Phe Leu Ile Ala Leu Ile Ser Asn Asn Gly195 200 205Asn Tyr Thr Cys Val Val Thr Tyr Pro Glu Asn Gly Arg Thr Phe His210 215 220Leu Thr Arg Thr Leu Thr Val Lys Val Val Gly Ser Pro Lys Asn Ala225 230 235 240Val Pro Pro Val Ile His Ser Pro Asn Asp His Val Val Tyr Glu Lys245 250 255Glu Pro Gly Glu Glu Leu Leu Ile Pro Cys Thr Val Tyr Phe Ser Phe260 265 270Leu Met Asp Ser Arg Asn Glu Val Trp Trp Thr Ile Asp Gly Lys Lys275 280 285Pro Asp Asp Ile Thr Ile Asp Val Thr Ile Asn Glu Ser Ile Ser His290 295 300Ser Arg Thr Glu Asp Glu Thr Arg Thr Gln Ile Leu Ser Ile Lys Lys305 310 315 320Val Thr Ser Glu Asp Leu Lys Arg Ser Tyr Val Cys His Ala Arg Ser325 330 335Ala Lys Gly Glu Val Ala Lys Ala Ala Lys Val Lys Gln Lys Val Pro340 345 350Ala Pro Arg Tyr Thr Val Glu Leu Ala Cys Gly Phe Gly Ala Thr Val355 360 365Leu Leu Val Val Ile Leu Ile Val Val Tyr His Val Tyr Trp Leu Glu370 375 380Met Val Leu Phe Tyr Arg Ala His Phe Gly Thr Asp Glu Thr Ile Leu385 390 395 400Asp Gly Lys Glu Tyr Asp Ile Tyr Val Ser Tyr Ala Arg Asn

Ala Glu405 410 415Glu Glu Glu Phe Val Leu Leu Thr Leu Arg Gly Val Leu Glu Asn Glu420 425 430Phe Gly Tyr Lys Leu Cys Ile Phe Asp Arg Asp Ser Leu Pro Gly Gly435 440 445Ile Val Thr Asp Glu Thr Leu Ser Phe Ile Gln Lys Ser Arg Arg Leu450 455 460Leu Val Val Leu Ser Pro Asn Tyr Val Leu Gln Gly Thr Gln Ala Leu465 470 475 480Leu Glu Leu Lys Ala Gly Leu Glu Asn Met Ala Ser Arg Gly Asn Ile485 490 495Asn Val Ile Leu Val Gln Tyr Lys Ala Val Lys Glu Thr Lys Val Lys500 505 510Glu Leu Lys Arg Ala Lys Thr Val Leu Thr Val Ile Lys Trp Lys Gly515 520 525Glu Lys Ser Lys Tyr Pro Gln Gly Arg Phe Trp Lys Gln Leu Gln Val530 535 540Ala Met Pro Val Lys Lys Ser Pro Arg Arg Ser Ser Ser Asp Glu Gln545 550 555 560Gly Leu Ser Tyr Ser Ser Leu Lys Asn Val565 570231367PRTHomo sapiens 23Met Lys Ser Gly Ser Gly Gly Gly Ser Pro Thr Ser Leu Trp Gly Leu1 5 10 15Leu Phe Leu Ser Ala Ala Leu Ser Leu Trp Pro Thr Ser Gly Glu Ile20 25 30Cys Gly Pro Gly Ile Asp Ile Arg Asn Asp Tyr Gln Gln Leu Lys Arg35 40 45Leu Glu Asn Cys Thr Val Ile Glu Gly Tyr Leu His Ile Leu Leu Ile50 55 60Ser Lys Ala Glu Asp Tyr Arg Ser Tyr Arg Phe Pro Lys Leu Thr Val65 70 75 80Ile Thr Glu Tyr Leu Leu Leu Phe Arg Val Ala Gly Leu Glu Ser Leu85 90 95Gly Asp Leu Phe Pro Asn Leu Thr Val Ile Arg Gly Trp Lys Leu Phe100 105 110Tyr Asn Tyr Ala Leu Val Ile Phe Glu Met Thr Asn Leu Lys Asp Ile115 120 125Gly Leu Tyr Asn Leu Arg Asn Ile Thr Arg Gly Ala Ile Arg Ile Glu130 135 140Lys Asn Ala Asp Leu Cys Tyr Leu Ser Thr Val Asp Trp Ser Leu Ile145 150 155 160Leu Asp Ala Val Ser Asn Asn Tyr Ile Val Gly Asn Lys Pro Pro Lys165 170 175Glu Cys Gly Asp Leu Cys Pro Gly Thr Met Glu Glu Lys Pro Met Cys180 185 190Glu Lys Thr Thr Ile Asn Asn Glu Tyr Asn Tyr Arg Cys Trp Thr Thr195 200 205Asn Arg Cys Gln Lys Met Cys Pro Ser Thr Cys Gly Lys Arg Ala Cys210 215 220Thr Glu Asn Asn Glu Cys Cys His Pro Glu Cys Leu Gly Ser Cys Ser225 230 235 240Ala Pro Asp Asn Asp Thr Ala Cys Val Ala Cys Arg His Tyr Tyr Tyr245 250 255Ala Gly Val Cys Val Pro Ala Cys Pro Pro Asn Thr Tyr Arg Phe Glu260 265 270Gly Trp Arg Cys Val Asp Arg Asp Phe Cys Ala Asn Ile Leu Ser Ala275 280 285Glu Ser Ser Asp Ser Glu Gly Phe Val Ile His Asp Gly Glu Cys Met290 295 300Gln Glu Cys Pro Ser Gly Phe Ile Arg Asn Gly Ser Gln Ser Met Tyr305 310 315 320Cys Ile Pro Cys Glu Gly Pro Cys Pro Lys Val Cys Glu Glu Glu Lys325 330 335Lys Thr Lys Thr Ile Asp Ser Val Thr Ser Ala Gln Met Leu Gln Gly340 345 350Cys Thr Ile Phe Lys Gly Asn Leu Leu Ile Asn Ile Arg Arg Gly Asn355 360 365Asn Ile Ala Ser Glu Leu Glu Asn Phe Met Gly Leu Ile Glu Val Val370 375 380Thr Gly Tyr Val Lys Ile Arg His Ser His Ala Leu Val Ser Leu Ser385 390 395 400Phe Leu Lys Asn Leu Arg Leu Ile Leu Gly Glu Glu Gln Leu Glu Gly405 410 415Asn Tyr Ser Phe Tyr Val Leu Asp Asn Gln Asn Leu Gln Gln Leu Trp420 425 430Asp Trp Asp His Arg Asn Leu Thr Ile Lys Ala Gly Lys Met Tyr Phe435 440 445Ala Phe Asn Pro Lys Leu Cys Val Ser Glu Ile Tyr Arg Met Glu Glu450 455 460Val Thr Gly Thr Lys Gly Arg Gln Ser Lys Gly Asp Ile Asn Thr Arg465 470 475 480Asn Asn Gly Glu Arg Ala Ser Cys Glu Ser Asp Val Leu His Phe Thr485 490 495Ser Thr Thr Thr Ser Lys Asn Arg Ile Ile Ile Thr Trp His Arg Tyr500 505 510Arg Pro Pro Asp Tyr Arg Asp Leu Ile Ser Phe Thr Val Tyr Tyr Lys515 520 525Glu Ala Pro Phe Lys Asn Val Thr Glu Tyr Asp Gly Gln Asp Ala Cys530 535 540Gly Ser Asn Ser Trp Asn Met Val Asp Val Asp Leu Pro Pro Asn Lys545 550 555 560Asp Val Glu Pro Gly Ile Leu Leu His Gly Leu Lys Pro Trp Thr Gln565 570 575Tyr Ala Val Tyr Val Lys Ala Val Thr Leu Thr Met Val Glu Asn Asp580 585 590His Ile Arg Gly Ala Lys Ser Glu Ile Leu Tyr Ile Arg Thr Asn Ala595 600 605Ser Val Pro Ser Ile Pro Leu Asp Val Leu Ser Ala Ser Asn Ser Ser610 615 620Ser Gln Leu Ile Val Lys Trp Asn Pro Pro Ser Leu Pro Asn Gly Asn625 630 635 640Leu Ser Tyr Tyr Ile Val Arg Trp Gln Arg Gln Pro Gln Asp Gly Tyr645 650 655Leu Tyr Arg His Asn Tyr Cys Ser Lys Asp Lys Ile Pro Ile Arg Lys660 665 670Tyr Ala Asp Gly Thr Ile Asp Ile Glu Glu Val Thr Glu Asn Pro Lys675 680 685Thr Glu Val Cys Gly Gly Glu Lys Gly Pro Cys Cys Ala Cys Pro Lys690 695 700Thr Glu Ala Glu Lys Gln Ala Glu Lys Glu Glu Ala Glu Tyr Arg Lys705 710 715 720Val Phe Glu Asn Phe Leu His Asn Ser Ile Phe Val Pro Arg Pro Glu725 730 735Arg Lys Arg Arg Asp Val Met Gln Val Ala Asn Thr Thr Met Ser Ser740 745 750Arg Ser Arg Asn Thr Thr Ala Ala Asp Thr Tyr Asn Ile Thr Asp Pro755 760 765Glu Glu Leu Glu Thr Glu Tyr Pro Phe Phe Glu Ser Arg Val Asp Asn770 775 780Lys Glu Arg Thr Val Ile Ser Asn Leu Arg Pro Phe Thr Leu Tyr Arg785 790 795 800Ile Asp Ile His Ser Cys Asn His Glu Ala Glu Lys Leu Gly Cys Ser805 810 815Ala Ser Asn Phe Val Phe Ala Arg Thr Met Pro Ala Glu Gly Ala Asp820 825 830Asp Ile Pro Gly Pro Val Thr Trp Glu Pro Arg Pro Glu Asn Ser Ile835 840 845Phe Leu Lys Trp Pro Glu Pro Glu Asn Pro Asn Gly Leu Ile Leu Met850 855 860Tyr Glu Ile Lys Tyr Gly Ser Gln Val Glu Asp Gln Arg Glu Cys Val865 870 875 880Ser Arg Gln Glu Tyr Arg Lys Tyr Gly Gly Ala Lys Leu Asn Arg Leu885 890 895Asn Pro Gly Asn Tyr Thr Ala Arg Ile Gln Ala Thr Ser Leu Ser Gly900 905 910Asn Gly Ser Trp Thr Asp Pro Val Phe Phe Tyr Val Gln Ala Lys Thr915 920 925Gly Tyr Glu Asn Phe Ile His Leu Ile Ile Ala Leu Pro Val Ala Val930 935 940Leu Leu Ile Val Gly Gly Leu Val Ile Met Leu Tyr Val Phe His Arg945 950 955 960Lys Arg Asn Asn Ser Arg Leu Gly Asn Gly Val Leu Tyr Ala Ser Val965 970 975Asn Pro Glu Tyr Phe Ser Ala Ala Asp Val Tyr Val Pro Asp Glu Trp980 985 990Glu Val Ala Arg Glu Lys Ile Thr Met Ser Arg Glu Leu Gly Gln Gly995 1000 1005Ser Phe Gly Met Val Tyr Glu Gly Val Ala Lys Gly Val Val Lys1010 1015 1020Asp Glu Pro Glu Thr Arg Val Ala Ile Lys Thr Val Asn Glu Ala1025 1030 1035Ala Ser Met Arg Glu Arg Ile Glu Phe Leu Asn Glu Ala Ser Val1040 1045 1050Met Lys Glu Phe Asn Cys His His Val Val Arg Leu Leu Gly Val1055 1060 1065Val Ser Gln Gly Gln Pro Thr Leu Val Ile Met Glu Leu Met Thr1070 1075 1080Arg Gly Asp Leu Lys Ser Tyr Leu Arg Ser Leu Arg Pro Glu Met1085 1090 1095Glu Asn Asn Pro Val Leu Ala Pro Pro Ser Leu Ser Lys Met Ile1100 1105 1110Gln Met Ala Gly Glu Ile Ala Asp Gly Met Ala Tyr Leu Asn Ala1115 1120 1125Asn Lys Phe Val His Arg Asp Leu Ala Ala Arg Asn Cys Met Val1130 1135 1140Ala Glu Asp Phe Thr Val Lys Ile Gly Asp Phe Gly Met Thr Arg1145 1150 1155Asp Ile Tyr Glu Thr Asp Tyr Tyr Arg Lys Gly Gly Lys Gly Leu1160 1165 1170Leu Pro Val Arg Trp Met Ser Pro Glu Ser Leu Lys Asp Gly Val1175 1180 1185Phe Thr Thr Tyr Ser Asp Val Trp Ser Phe Gly Val Val Leu Trp1190 1195 1200Glu Ile Ala Thr Leu Ala Glu Gln Pro Tyr Gln Gly Leu Ser Asn1205 1210 1215Glu Gln Val Leu Arg Phe Val Met Glu Gly Gly Leu Leu Asp Lys1220 1225 1230Pro Asp Asn Cys Pro Asp Met Leu Phe Glu Leu Met Arg Met Cys1235 1240 1245Trp Gln Tyr Asn Pro Lys Met Arg Pro Ser Phe Leu Glu Ile Ile1250 1255 1260Ser Ser Ile Lys Glu Glu Met Glu Pro Gly Phe Arg Glu Val Ser1265 1270 1275Phe Tyr Tyr Ser Glu Glu Asn Lys Leu Pro Glu Pro Glu Glu Leu1280 1285 1290Asp Leu Glu Pro Glu Asn Met Glu Ser Val Pro Leu Asp Pro Ser1295 1300 1305Ala Ser Ser Ser Ser Leu Pro Leu Pro Asp Arg His Ser Gly His1310 1315 1320Lys Ala Glu Asn Gly Pro Gly Pro Gly Val Leu Val Leu Arg Ala1325 1330 1335Ser Phe Asp Glu Arg Gln Pro Tyr Ala His Met Asn Gly Gly Arg1340 1345 1350Lys Asn Glu Arg Ala Leu Pro Leu Pro Gln Ser Ser Thr Cys1355 1360 136524825PRTHomo sapiens 24Met Gly Trp Leu Cys Ser Gly Leu Leu Phe Pro Val Ser Cys Leu Val1 5 10 15Leu Leu Gln Val Ala Ser Ser Gly Asn Met Lys Val Leu Gln Glu Pro20 25 30Thr Cys Val Ser Asp Tyr Met Ser Ile Ser Thr Cys Glu Trp Lys Met35 40 45Asn Gly Pro Thr Asn Cys Ser Thr Glu Leu Arg Leu Leu Tyr Gln Leu50 55 60Val Phe Leu Leu Ser Glu Ala His Thr Cys Ile Pro Glu Asn Asn Gly65 70 75 80Gly Ala Gly Cys Val Cys His Leu Leu Met Asp Asp Val Val Ser Ala85 90 95Asp Asn Tyr Thr Leu Asp Leu Trp Ala Gly Gln Gln Leu Leu Trp Lys100 105 110Gly Ser Phe Lys Pro Ser Glu His Val Lys Pro Arg Ala Pro Gly Asn115 120 125Leu Thr Val His Thr Asn Val Ser Asp Thr Leu Leu Leu Thr Trp Ser130 135 140Asn Pro Tyr Pro Pro Asp Asn Tyr Leu Tyr Asn His Leu Thr Tyr Ala145 150 155 160Val Asn Ile Trp Ser Glu Asn Asp Pro Ala Asp Phe Arg Ile Tyr Asn165 170 175Val Thr Tyr Leu Glu Pro Ser Leu Arg Ile Ala Ala Ser Thr Leu Lys180 185 190Ser Gly Ile Ser Tyr Arg Ala Arg Val Arg Ala Trp Ala Gln Cys Tyr195 200 205Asn Thr Thr Trp Ser Glu Trp Ser Pro Ser Thr Lys Trp His Asn Ser210 215 220Tyr Arg Glu Pro Phe Glu Gln His Leu Leu Leu Gly Val Ser Val Ser225 230 235 240Cys Ile Val Ile Leu Ala Val Cys Leu Leu Cys Tyr Val Ser Ile Thr245 250 255Lys Ile Lys Lys Glu Trp Trp Asp Gln Ile Pro Asn Pro Ala Arg Ser260 265 270Arg Leu Val Ala Ile Ile Ile Gln Asp Ala Gln Gly Ser Gln Trp Glu275 280 285Lys Arg Ser Arg Gly Gln Glu Pro Ala Lys Cys Pro His Trp Lys Asn290 295 300Cys Leu Thr Lys Leu Leu Pro Cys Phe Leu Glu His Asn Met Lys Arg305 310 315 320Asp Glu Asp Pro His Lys Ala Ala Lys Glu Met Pro Phe Gln Gly Ser325 330 335Gly Lys Ser Ala Trp Cys Pro Val Glu Ile Ser Lys Thr Val Leu Trp340 345 350Pro Glu Ser Ile Ser Val Val Arg Cys Val Glu Leu Phe Glu Ala Pro355 360 365Val Glu Cys Glu Glu Glu Glu Glu Val Glu Glu Glu Lys Gly Ser Phe370 375 380Cys Ala Ser Pro Glu Ser Ser Arg Asp Asp Phe Gln Glu Gly Arg Glu385 390 395 400Gly Ile Val Ala Arg Leu Thr Glu Ser Leu Phe Leu Asp Leu Leu Gly405 410 415Glu Glu Asn Gly Gly Phe Cys Gln Gln Asp Met Gly Glu Ser Cys Leu420 425 430Leu Pro Pro Ser Gly Ser Thr Ser Ala His Met Pro Trp Asp Glu Phe435 440 445Pro Ser Ala Gly Pro Lys Glu Ala Pro Pro Trp Gly Lys Glu Gln Pro450 455 460Leu His Leu Glu Pro Ser Pro Pro Ala Ser Pro Thr Gln Ser Pro Asp465 470 475 480Asn Leu Thr Cys Thr Glu Thr Pro Leu Val Ile Ala Gly Asn Pro Ala485 490 495Tyr Arg Ser Phe Ser Asn Ser Leu Ser Gln Ser Pro Cys Pro Arg Glu500 505 510Leu Gly Pro Asp Pro Leu Leu Ala Arg His Leu Glu Glu Val Glu Pro515 520 525Glu Met Pro Cys Val Pro Gln Leu Ser Glu Pro Thr Thr Val Pro Gln530 535 540Pro Glu Pro Glu Thr Trp Glu Gln Ile Leu Arg Arg Asn Val Leu Gln545 550 555 560His Gly Ala Ala Ala Ala Pro Val Ser Ala Pro Thr Ser Gly Tyr Gln565 570 575Glu Phe Val His Ala Val Glu Gln Gly Gly Thr Gln Ala Ser Ala Val580 585 590Val Gly Leu Gly Pro Pro Gly Glu Ala Gly Tyr Lys Ala Phe Ser Ser595 600 605Leu Leu Ala Ser Ser Ala Val Ser Pro Glu Lys Cys Gly Phe Gly Ala610 615 620Ser Ser Gly Glu Glu Gly Tyr Lys Pro Phe Gln Asp Leu Ile Pro Gly625 630 635 640Cys Pro Gly Asp Pro Ala Pro Val Pro Val Pro Leu Phe Thr Phe Gly645 650 655Leu Asp Arg Glu Pro Pro Arg Ser Pro Gln Ser Ser His Leu Pro Ser660 665 670Ser Ser Pro Glu His Leu Gly Leu Glu Pro Gly Glu Lys Val Glu Asp675 680 685Met Pro Lys Pro Pro Leu Pro Gln Glu Gln Ala Thr Asp Pro Leu Val690 695 700Asp Ser Leu Gly Ser Gly Ile Val Tyr Ser Ala Leu Thr Cys His Leu705 710 715 720Cys Gly His Leu Lys Gln Cys His Gly Gln Glu Asp Gly Gly Gln Thr725 730 735Pro Val Met Ala Ser Pro Cys Cys Gly Cys Cys Cys Gly Asp Arg Ser740 745 750Ser Pro Pro Thr Thr Pro Leu Arg Ala Pro Asp Pro Ser Pro Gly Gly755 760 765Val Pro Leu Glu Ala Ser Leu Cys Pro Ala Ser Leu Ala Pro Ser Gly770 775 780Ile Ser Glu Lys Ser Lys Ser Ser Ser Ser Phe His Pro Ala Pro Gly785 790 795 800Asn Ala Gln Ser Ser Ser Gln Thr Pro Lys Ile Val Asn Phe Val Ser805 810 815Val Gly Pro Thr Tyr Met Arg Val Ser820 82525569PRTHomo sapiens 25Met Lys Val Leu Leu Arg Leu Ile Cys Phe Ile Ala Leu Leu Ile Ser1 5 10 15Ser Leu Glu Ala Asp Lys Cys Lys Glu Arg Glu Glu Lys Ile Ile Leu20 25 30Val Ser Ser Ala Asn Glu Ile Asp Val Arg Pro Cys Pro Leu Asn Pro35 40 45Asn Glu His Lys Gly Thr Ile Thr Trp Tyr Lys Asp Asp Ser Lys Thr50 55 60Pro Val Ser Thr Glu Gln Ala Ser Arg Ile His Gln His Lys Glu Lys65 70 75 80Leu Trp Phe Val Pro Ala Lys Val Glu Asp Ser Gly His Tyr Tyr Cys85 90 95Val Val Arg Asn Ser Ser Tyr Cys Leu Arg Ile Lys Ile Ser Ala Lys100 105 110Phe Val Glu Asn Glu Pro Asn Leu Cys Tyr Asn Ala Gln Ala Ile Phe115 120 125Lys Gln Lys Leu Pro Val Ala Gly Asp Gly Gly Leu Val Cys Pro Tyr130 135 140Met Glu Phe Phe Lys Asn Glu Asn Asn Glu Leu Pro Lys Leu Gln Trp145 150 155 160Tyr Lys Asp Cys Lys Pro Leu Leu Leu Asp Asn Ile His Phe Ser Gly165 170 175Val Lys Asp Arg Leu Ile Val Met Asn Val Ala Glu Lys His Arg Gly180 185 190Asn Tyr Thr Cys His Ala Ser Tyr Thr Tyr Leu Gly Lys Gln Tyr Pro195 200 205Ile Thr Arg Val Ile Glu Phe Ile Thr Leu Glu Glu Asn Lys Pro Thr210 215 220Arg Pro Val Ile Val Ser Pro Ala Asn Glu Thr Met Glu Val Asp Leu225 230 235 240Gly Ser Gln Ile Gln Leu Ile Cys Asn Val Thr Gly Gln Leu Ser Asp245 250 255Ile Ala Tyr Trp Lys Trp Asn Gly Ser Val Ile Asp Glu Asp Asp Pro260 265 270Val Leu Gly Glu Asp Tyr Tyr

Ser Val Glu Asn Pro Ala Asn Lys Arg275 280 285Arg Ser Thr Leu Ile Thr Val Leu Asn Ile Ser Glu Ile Glu Ser Arg290 295 300Phe Tyr Lys His Pro Phe Thr Cys Phe Ala Lys Asn Thr His Gly Ile305 310 315 320Asp Ala Ala Tyr Ile Gln Leu Ile Tyr Pro Val Thr Asn Phe Gln Lys325 330 335His Met Ile Gly Ile Cys Val Thr Leu Thr Val Ile Ile Val Cys Ser340 345 350Val Phe Ile Tyr Lys Ile Phe Lys Ile Asp Ile Val Leu Trp Tyr Arg355 360 365Asp Ser Cys Tyr Asp Phe Leu Pro Ile Lys Ala Ser Asp Gly Lys Thr370 375 380Tyr Asp Ala Tyr Ile Leu Tyr Pro Lys Thr Val Gly Glu Gly Ser Thr385 390 395 400Ser Asp Cys Asp Ile Phe Val Phe Lys Val Leu Pro Glu Val Leu Glu405 410 415Lys Gln Cys Gly Tyr Lys Leu Phe Ile Tyr Gly Arg Asp Asp Tyr Val420 425 430Gly Glu Asp Ile Val Glu Val Ile Asn Glu Asn Val Lys Lys Ser Arg435 440 445Arg Leu Ile Ile Ile Leu Val Arg Glu Thr Ser Gly Phe Ser Trp Leu450 455 460Gly Gly Ser Ser Glu Glu Gln Ile Ala Met Tyr Asn Ala Leu Val Gln465 470 475 480Asp Gly Ile Lys Val Val Leu Leu Glu Leu Glu Lys Ile Gln Asp Tyr485 490 495Glu Lys Met Pro Glu Ser Ile Lys Phe Ile Lys Gln Lys His Gly Ala500 505 510Ile Arg Trp Ser Gly Asp Phe Thr Gln Gly Pro Gln Ser Ala Lys Thr515 520 525Arg Phe Trp Lys Asn Val Arg Tyr His Met Pro Val Gln Arg Arg Ser530 535 540Pro Ser Ser Lys His Gln Leu Leu Ser Pro Ala Thr Lys Glu Lys Leu545 550 555 560Gln Arg Glu Ala His Val Pro Leu Gly56526576PRTMus sp. 26Met Glu Asn Met Lys Val Leu Leu Gly Leu Ile Cys Leu Met Val Pro1 5 10 15Leu Leu Ser Leu Glu Ile Asp Val Cys Thr Glu Tyr Pro Asn Gln Ile20 25 30Val Leu Phe Leu Ser Val Asn Glu Ile Asp Ile Arg Lys Cys Pro Leu35 40 45Thr Pro Asn Lys Met His Gly Asp Thr Ile Ile Trp Tyr Lys Asn Asp50 55 60Ser Lys Thr Pro Ile Ser Ala Asp Arg Asp Ser Arg Ile His Gln Gln65 70 75 80Asn Glu His Leu Trp Phe Val Pro Ala Lys Val Glu Asp Ser Gly Tyr85 90 95Tyr Tyr Cys Ile Val Arg Asn Ser Thr Tyr Cys Leu Lys Thr Lys Val100 105 110Thr Val Thr Val Leu Glu Asn Asp Pro Gly Leu Cys Tyr Ser Thr Gln115 120 125Ala Thr Phe Pro Gln Arg Leu His Ile Ala Gly Asp Gly Ser Leu Val130 135 140Cys Pro Tyr Val Ser Tyr Phe Lys Asp Glu Asn Asn Glu Leu Pro Glu145 150 155 160Val Gln Trp Tyr Lys Asn Cys Lys Pro Leu Leu Leu Asp Asn Val Ser165 170 175Phe Phe Gly Val Lys Asp Lys Leu Leu Val Arg Asn Val Ala Glu Glu180 185 190His Arg Gly Asp Tyr Ile Cys Arg Met Ser Tyr Thr Phe Arg Gly Lys195 200 205Gln Tyr Pro Val Thr Arg Val Ile Gln Phe Ile Thr Ile Asp Glu Asn210 215 220Lys Arg Asp Arg Pro Val Ile Leu Ser Pro Arg Asn Glu Thr Ile Glu225 230 235 240Ala Asp Pro Gly Ser Met Ile Gln Leu Ile Cys Asn Val Thr Gly Gln245 250 255Phe Ser Asp Leu Val Tyr Trp Lys Trp Asn Gly Ser Glu Ile Glu Trp260 265 270Asn Asp Pro Phe Leu Ala Glu Asp Tyr Gln Phe Val Glu His Pro Ser275 280 285Thr Lys Arg Lys Tyr Thr Leu Ile Thr Thr Leu Asn Ile Ser Glu Val290 295 300Lys Ser Gln Phe Tyr Arg Tyr Pro Phe Ile Cys Val Val Lys Asn Thr305 310 315 320Asn Ile Phe Glu Ser Ala His Val Gln Leu Ile Tyr Pro Val Pro Asp325 330 335Phe Lys Asn Tyr Leu Ile Gly Gly Phe Ile Ile Leu Thr Ala Thr Ile340 345 350Val Cys Cys Val Cys Ile Tyr Lys Val Phe Lys Val Asp Ile Val Leu355 360 365Trp Tyr Arg Asp Ser Cys Ser Gly Phe Leu Pro Ser Lys Ala Ser Asp370 375 380Gly Lys Thr Tyr Asp Ala Tyr Ile Leu Tyr Pro Lys Thr Leu Gly Glu385 390 395 400Gly Ser Phe Ser Asp Leu Asp Thr Phe Val Phe Lys Leu Leu Pro Glu405 410 415Val Leu Glu Gly Gln Phe Gly Tyr Lys Leu Phe Ile Tyr Gly Arg Asp420 425 430Asp Tyr Val Gly Glu Asp Thr Ile Glu Val Thr Asn Glu Asn Val Lys435 440 445Lys Ser Arg Arg Leu Ile Ile Ile Leu Val Arg Asp Met Gly Gly Phe450 455 460Ser Trp Leu Gly Gln Ser Ser Glu Glu Gln Ile Ala Ile Tyr Asn Ala465 470 475 480Leu Ile Gln Glu Gly Ile Lys Ile Val Leu Leu Glu Leu Glu Lys Ile485 490 495Gln Asp Tyr Glu Lys Met Pro Asp Ser Ile Gln Phe Ile Lys Gln Lys500 505 510His Gly Val Ile Cys Trp Ser Gly Asp Phe Gln Glu Arg Pro Gln Ser515 520 525Ala Lys Thr Arg Phe Trp Lys Asn Leu Arg Tyr Gln Met Pro Ala Gln530 535 540Arg Arg Ser Pro Leu Ser Lys His Arg Leu Leu Thr Leu Asp Pro Val545 550 555 560Arg Asp Thr Lys Glu Lys Leu Pro Ala Ala Thr His Leu Pro Leu Gly565 570 57527576PRTRattus sp. 27Met Glu Asn Met Lys Val Leu Leu Gly Phe Ile Cys Leu Ile Val Pro1 5 10 15Leu Leu Ser Leu Glu Thr Asp Lys Cys Thr Glu Tyr Pro Asn Glu Val20 25 30Ile Ser Phe Ser Ser Val Asn Glu Ile Asp Ile Arg Ser Cys Pro Leu35 40 45Thr Pro Asn Glu Met His Gly Gly Thr Ile Ile Trp Tyr Lys Asn Asp50 55 60Ser Lys Thr Pro Ile Ser Ala Asp Lys Asp Ser Arg Ile His Gln Gln65 70 75 80Asn Glu His Leu Trp Phe Val Pro Ala Lys Met Glu Asp Ser Gly Tyr85 90 95Tyr Tyr Cys Ile Met Arg Asn Ser Thr Tyr Cys Leu Lys Thr Lys Ile100 105 110Thr Met Ser Val Leu Glu Asn Asp Pro Gly Leu Cys Tyr Asn Thr Gln115 120 125Ala Ser Phe Ile Gln Arg Leu His Val Ala Gly Asp Gly Ser Leu Val130 135 140Cys Pro Tyr Leu Asp Phe Phe Lys Asp Glu Asn Asn Glu Leu Pro Lys145 150 155 160Val Gln Trp Tyr Lys Asn Cys Lys Pro Leu Pro Leu Asp Asp Gly Asn165 170 175Phe Phe Gly Phe Lys Asn Lys Leu Met Val Met Asn Val Ala Glu Glu180 185 190His Arg Gly Asn Tyr Thr Cys Arg Thr Ser Tyr Thr Tyr Gln Gly Lys195 200 205Gln Tyr Pro Val Thr Arg Val Ile Thr Phe Ile Thr Ile Asp Asp Ser210 215 220Lys Arg Asp Arg Pro Val Ile Met Ser Pro Arg Asn Glu Thr Met Glu225 230 235 240Ala Asp Pro Gly Ser Thr Ile Gln Leu Ile Cys Asn Val Thr Gly Gln245 250 255Phe Thr Asp Leu Val Tyr Trp Lys Trp Asn Gly Ser Glu Ile Glu Trp260 265 270Asp Asp Pro Ile Leu Ala Glu Asp Tyr Gln Phe Leu Glu His Pro Ser275 280 285Ala Lys Arg Lys Tyr Thr Leu Ile Thr Thr Leu Asn Val Ser Glu Val290 295 300Lys Ser Gln Phe Tyr Arg Tyr Pro Phe Ile Cys Phe Val Lys Asn Thr305 310 315 320His Ile Leu Glu Thr Ala His Val Arg Leu Val Tyr Pro Val Pro Asp325 330 335Phe Lys Asn Tyr Leu Ile Gly Gly Phe Ala Ile Phe Thr Ala Thr Ala340 345 350Val Phe Cys Ala Cys Ile Tyr Lys Val Phe Lys Val Asp Ile Val Leu355 360 365Trp Tyr Arg Asp Ser Cys Ser Asp Phe Leu Pro Arg Lys Ala Ser Asp370 375 380Gly Arg Thr Tyr Asp Ala Tyr Val Leu Tyr Pro Lys Thr Tyr Gly Glu385 390 395 400Gly Ser Phe Ala Tyr Leu Asp Thr Phe Val Phe Lys Leu Leu Pro Glu405 410 415Val Leu Glu Gly Gln Phe Gly Tyr Lys Leu Phe Ile Cys Gly Arg Asp420 425 430Asp Tyr Val Gly Glu Asp Thr Ile Glu Val Thr Asn Glu Asn Val Lys435 440 445Arg Ser Arg Arg Leu Ile Ile Ile Leu Val Arg Asp Met Gly Ser Phe450 455 460Ser Cys Leu Gly Gln Ser Ser Glu Glu Gln Ile Ala Ile Tyr Asp Ala465 470 475 480Leu Ile Arg Glu Gly Ile Lys Ile Ile Leu Leu Glu Leu Glu Lys Ile485 490 495Gln Asp Tyr Glu Lys Met Pro Glu Ser Ile Gln Phe Ile Lys Gln Lys500 505 510His Gly Ala Ile Cys Trp Ser Gly Asp Phe Lys Glu Arg Pro Gln Ser515 520 525Ala Lys Thr Arg Phe Trp Lys Asn Leu Arg Tyr Gln Met Pro Ala Gln530 535 540Arg Arg Ser Pro Leu Ser Lys His His Leu Leu Thr Leu Asp Pro Val545 550 555 560Leu Asp Thr Lys Glu Lys Leu Gln Ala Glu Thr His Leu Pro Leu Gly565 570 57528555PRTEquus sp. 28Met His Lys Met Thr Ser Thr Phe Leu Leu Ile Gly His Leu Ile Leu1 5 10 15Leu Ile Pro Leu Phe Ser Ala Glu Glu Cys Val Ile Cys Asn Tyr Phe20 25 30Val Leu Val Gly Glu Pro Thr Ala Ile Ser Cys Pro Val Ile Thr Leu35 40 45Pro Met Leu His Ser Asp Tyr Asn Leu Thr Trp Tyr Arg Asn Gly Ser50 55 60Asn Met Pro Ile Thr Thr Glu Arg Arg Ala Arg Ile His Gln Arg Lys65 70 75 80Gly Leu Leu Trp Phe Ile Pro Ala Ala Leu Glu Asp Ser Gly Leu Tyr85 90 95Glu Cys Glu Val Arg Ser Leu Asn Arg Ser Lys Gln Lys Ile Ile Asn100 105 110Leu Lys Val Phe Lys Asn Asp Asn Gly Leu Cys Phe Asn Gly Glu Met115 120 125Lys Tyr Asp Gln Ile Val Lys Ser Ala Asn Ala Gly Lys Ile Ile Cys130 135 140Pro Asp Leu Glu Asn Phe Lys Asp Glu Asp Asn Ile Asn Pro Glu Ile145 150 155 160His Trp Tyr Lys Glu Cys Lys Ser Gly Phe Leu Glu Asp Lys Arg Leu165 170 175Val Leu Ala Glu Gly Glu Asn Ala Ile Leu Ile Leu Asn Val Thr Ile180 185 190Gln Asp Lys Gly Asn Tyr Thr Cys Arg Met Val Tyr Thr Tyr Met Gly195 200 205Lys Gln Tyr Asn Val Ser Arg Thr Met Asn Leu Glu Val Lys Glu Ser210 215 220Pro Leu Lys Met Arg Pro Glu Phe Ile Tyr Pro Asn Asn Asn Thr Ile225 230 235 240Glu Val Glu Leu Gly Ser His Val Val Met Glu Cys Asn Val Ser Ser245 250 255Gly Val Tyr Gly Leu Leu Pro Tyr Trp Gln Val Asn Asp Glu Asp Val260 265 270Asp Ser Phe Asp Ser Thr Tyr Arg Glu Gln Phe Tyr Glu Glu Gly Met275 280 285Pro His Gly Ile Ala Val Ser Gly Thr Lys Phe Asn Ile Ser Glu Val290 295 300Lys Leu Lys Asp Tyr Ala Tyr Lys Phe Phe Cys His Phe Ile Tyr Asp305 310 315 320Ser Gln Glu Phe Thr Ser Tyr Ile Lys Leu Glu His Pro Val Gln Asn325 330 335Ile Arg Gly Tyr Leu Ile Gly Gly Gly Ile Ser Leu Ile Phe Leu Leu340 345 350Phe Leu Ile Leu Ile Val Tyr Lys Ile Phe Lys Ile Asp Ile Val Leu355 360 365Trp Tyr Arg Ser Ser Cys His Pro Leu Leu Gly Lys Lys Val Ser Asp370 375 380Gly Lys Ile Tyr Asp Ala Tyr Val Leu Tyr Pro Lys Asn Arg Glu Ser385 390 395 400Cys Leu Tyr Ser Ser Asp Ile Phe Ala Leu Lys Ile Leu Pro Glu Val405 410 415Leu Glu Arg Gln Cys Gly Tyr Asn Leu Phe Ile Phe Gly Arg Asn Asp420 425 430Leu Ala Gly Glu Ala Val Ile Asp Val Thr Asp Glu Lys Ile His Gln435 440 445Ser Arg Arg Val Ile Ile Ile Leu Val Pro Glu Pro Ser Cys Tyr Gly450 455 460Ile Leu Glu Asp Ala Ser Glu Lys His Leu Ala Val Tyr Asn Ala Leu465 470 475 480Ile Gln Asp Gly Ile Lys Ile Ile Leu Ile Glu Leu Glu Lys Ile Glu485 490 495Asp Tyr Ala Asn Met Pro Glu Ser Ile Lys Tyr Val Lys Gln Lys Tyr500 505 510Gly Ala Ile Arg Trp Thr Gly Asp Phe Ser Glu Arg Ser His Ser Ala515 520 525Ser Thr Arg Phe Trp Lys Lys Val Arg Tyr His Met Pro Ser Arg Lys530 535 540His Gly Ser Ser Ser Gly Phe His Leu Ser Ser545 550 55529825PRTHomo sapiens 29Met Gly Trp Leu Cys Ser Gly Leu Leu Phe Pro Val Ser Cys Leu Val1 5 10 15Leu Leu Gln Val Ala Ser Ser Gly Asn Met Lys Val Leu Gln Glu Pro20 25 30Thr Cys Val Ser Asp Tyr Met Ser Ile Ser Thr Cys Glu Trp Lys Met35 40 45Asn Gly Pro Thr Asn Cys Ser Thr Glu Leu Arg Leu Leu Tyr Gln Leu50 55 60Val Phe Leu Leu Ser Glu Ala His Thr Cys Ile Pro Glu Asn Asn Gly65 70 75 80Gly Ala Gly Cys Val Cys His Leu Leu Met Asp Asp Val Val Ser Ala85 90 95Asp Asn Tyr Thr Leu Asp Leu Trp Ala Gly Gln Gln Leu Leu Trp Lys100 105 110Gly Ser Phe Lys Pro Ser Glu His Val Lys Pro Arg Ala Pro Gly Asn115 120 125Leu Thr Val His Thr Asn Val Ser Asp Thr Leu Leu Leu Thr Trp Ser130 135 140Asn Pro Tyr Pro Pro Asp Asn Tyr Leu Tyr Asn His Leu Thr Tyr Ala145 150 155 160Val Asn Ile Trp Ser Glu Asn Asp Pro Ala Asp Phe Arg Ile Tyr Asn165 170 175Val Thr Tyr Leu Glu Pro Ser Leu Arg Ile Ala Ala Ser Thr Leu Lys180 185 190Ser Gly Ile Ser Tyr Arg Ala Arg Val Arg Ala Trp Ala Gln Cys Tyr195 200 205Asn Thr Thr Trp Ser Glu Trp Ser Pro Ser Thr Lys Trp His Asn Ser210 215 220Tyr Arg Glu Pro Phe Glu Gln His Leu Leu Leu Gly Val Ser Val Ser225 230 235 240Cys Ile Val Ile Leu Ala Val Cys Leu Leu Cys Tyr Val Ser Ile Thr245 250 255Lys Ile Lys Lys Glu Trp Trp Asp Gln Ile Pro Asn Pro Ala Arg Ser260 265 270Arg Leu Val Ala Ile Ile Ile Gln Asp Ala Gln Gly Ser Gln Trp Glu275 280 285Lys Arg Ser Arg Gly Gln Glu Pro Ala Lys Cys Pro His Trp Lys Asn290 295 300Cys Leu Thr Lys Leu Leu Pro Cys Phe Leu Glu His Asn Met Lys Arg305 310 315 320Asp Glu Asp Pro His Lys Ala Ala Lys Glu Met Pro Phe Gln Gly Ser325 330 335Gly Lys Ser Ala Trp Cys Pro Val Glu Ile Ser Lys Thr Val Leu Trp340 345 350Pro Glu Ser Ile Ser Val Val Arg Cys Val Glu Leu Phe Glu Ala Pro355 360 365Val Glu Cys Glu Glu Glu Glu Glu Val Glu Glu Glu Lys Gly Ser Phe370 375 380Cys Ala Ser Pro Glu Ser Ser Arg Asp Asp Phe Gln Glu Gly Arg Glu385 390 395 400Gly Ile Val Ala Arg Leu Thr Glu Ser Leu Phe Leu Asp Leu Leu Gly405 410 415Glu Glu Asn Gly Gly Phe Cys Gln Gln Asp Met Gly Glu Ser Cys Leu420 425 430Leu Pro Pro Ser Gly Ser Thr Ser Ala His Met Pro Trp Asp Glu Phe435 440 445Pro Ser Ala Gly Pro Lys Glu Ala Pro Pro Trp Gly Lys Glu Gln Pro450 455 460Leu His Leu Glu Pro Ser Pro Pro Ala Ser Pro Thr Gln Ser Pro Asp465 470 475 480Asn Leu Thr Cys Thr Glu Thr Pro Leu Val Ile Ala Gly Asn Pro Ala485 490 495Tyr Arg Ser Phe Ser Asn Ser Leu Ser Gln Ser Pro Cys Pro Arg Glu500 505 510Leu Gly Pro Asp Pro Leu Leu Ala Arg His Leu Glu Glu Val Glu Pro515 520 525Glu Met Pro Cys Val Pro Gln Leu Ser Glu Pro Thr Thr Val Pro Gln530 535 540Pro Glu Pro Glu Thr Trp Glu Gln Ile Leu Arg Arg Asn Val Leu Gln545 550 555 560His Gly Ala Ala Ala Ala Pro Val Ser Ala Pro Thr Ser Gly Tyr Gln565 570 575Glu Phe Val His Ala Val Glu Gln Gly Gly Thr Gln Ala Ser Ala Val580 585 590Val Gly Leu Gly Pro Pro Gly Glu Ala Gly Tyr Lys Ala Phe Ser Ser595 600 605Leu Leu Ala Ser Ser Ala Val Ser Pro Glu Lys Cys Gly Phe Gly Ala610 615 620Ser Ser Gly Glu Glu Gly Tyr Lys Pro Phe Gln Asp Leu Ile Pro Gly625 630 635 640Cys Pro Gly Asp Pro Ala Pro Val Pro Val Pro Leu Phe Thr Phe

Gly645 650 655Leu Asp Arg Glu Pro Pro Arg Ser Pro Gln Ser Ser His Leu Pro Ser660 665 670Ser Ser Pro Glu His Leu Gly Leu Glu Pro Gly Glu Lys Val Glu Asp675 680 685Met Pro Lys Pro Pro Leu Pro Gln Glu Gln Ala Thr Asp Pro Leu Val690 695 700Asp Ser Leu Gly Ser Gly Ile Val Tyr Ser Ala Leu Thr Cys His Leu705 710 715 720Cys Gly His Leu Lys Gln Cys His Gly Gln Glu Asp Gly Gly Gln Thr725 730 735Pro Val Met Ala Ser Pro Cys Cys Gly Cys Cys Cys Gly Asp Arg Ser740 745 750Ser Pro Pro Thr Thr Pro Leu Arg Ala Pro Asp Pro Ser Pro Gly Gly755 760 765Val Pro Leu Glu Ala Ser Leu Cys Pro Ala Ser Leu Ala Pro Ser Gly770 775 780Ile Ser Glu Lys Ser Lys Ser Ser Ser Ser Phe His Pro Ala Pro Gly785 790 795 800Asn Ala Gln Ser Ser Ser Gln Thr Pro Lys Ile Val Asn Phe Val Ser805 810 815Val Gly Pro Thr Tyr Met Arg Val Ser820 825309PRTArtificialsynthetic peptide 30Phe Val Phe Ala Arg Thr Met Pro Ala1 53110PRTArtificialsynthetic peptide 31Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr1 5 10329PRTArtificialsynthetic peptide 32Ala Thr Gly Gln Val Cys His Ala Leu1 53311PRTArtificialsynthetic peptide 33Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu1 5 10349PRTArtificialsynthetic peptide 34Trp His Asn Ser Tyr Arg Glu Pro Phe1 53510PRTArtificialsynthetic peptide 35Tyr Arg Glu Pro Phe Glu Gln His Leu Leu1 5 10369PRTArtificialsynthetic peptide 36Ser Asp Thr Leu Leu Leu Thr Trp Ser1 5378PRTArtificialsynthetic peptide 37Ile Tyr Asn Val Thr Tyr Leu Glu1 53811PRTArtificialsynthetic peptide 38Ile Ala Ala Ser Thr Leu Lys Ser Gly Ile Ser1 5 10399PRTArtificialsynthetic peptide 39Lys Pro Ser Glu His Val Lys Pro Arg1 54011PRTArtificialsynthetic peptide 40Phe Thr Cys Glu Glu Asp Phe Tyr Phe Pro Trp1 5 10419PRTArtificialsynthetic peptide 41Ser Val Asp Glu Ile Val Gln Pro Asp1 54212PRTArtificialsynthetic peptide 42Met Asp Pro Ile Asp Thr Thr Ser Val Pro Val Tyr1 5 104312PRTArtificialsynthetic peptide 43Ile Asp Ala Ala Tyr Ile Gln Leu Ile Tyr Pro Val1 5 104412PRTArtificialsynthetic peptide 44Leu Ile Tyr Pro Val Thr Asn Phe Gln Lys His Met1 5 104510PRTArtificialsynthetic peptide 45Leu Glu Glu Asn Lys Pro Thr Arg Pro Val1 5 104610PRTArtificialsynthetic peptide 46Asn Lys Pro Thr Arg Pro Val Ile Val Ser1 5 104710PRTArtificialsynthetic peptide 47Val Ala Glu Lys His Arg Gly Asn Tyr Thr1 5 10489PRTArtificialsynthetic peptide 48Trp Asn Gly Ser Val Ile Asp Glu Asp1 54910PRTArtificialsynthetic peptide 49Val Pro Ala Pro Arg Tyr Thr Val Glu Leu1 5 10509PRTArtificialsynthetic peptide 50Ala Pro Arg Tyr Thr Val Glu Leu Ala1 55111PRTArtificialsynthetic peptide 51Val Gln Lys Asp Ser Cys Phe Asn Ser Pro Met1 5 10529PRTArtificialsynthetic peptide 52Met Lys Leu Pro Val His Lys Leu Tyr1 55311PRTArtificialsynthetic peptide 53Val Gly Ser Pro Lys Asn Ala Val Pro Pro Val1 5 105410PRTArtificialsynthetic peptide 54Val Thr Tyr Pro Glu Asn Gly Arg Thr Phe1 5 105510PRTArtificialsynthetic peptide 55Ile His Ser Pro Asn Asp His Val Val Tyr1 5 10569PRTArtificialsynthetic peptide 56Leu Ile Ser Asn Asn Gly Asn Tyr Thr1 55711PRTArtificialsynthetic peptide 57Val Trp Trp Thr Ile Asp Gly Lys Lys Pro Asp1 5 105811PRTArtificialsynthetic peptide 58Trp Thr Ile Asp Gly Lys Lys Pro Asp Asp Ile1 5 105911PRTArtificialsynthetic peptide 59His Ser Arg Thr Glu Asp Glu Thr Arg Thr Gln1 5 1060810PRTMus sp. 60Met Gly Arg Leu Cys Thr Lys Phe Leu Thr Ser Val Gly Cys Leu Ile1 5 10 15Leu Leu Leu Val Thr Gly Ser Gly Ser Ile Lys Val Leu Gly Glu Pro20 25 30Thr Cys Phe Ser Asp Tyr Ile Arg Thr Ser Thr Cys Glu Trp Phe Leu35 40 45Asp Ser Ala Val Asp Cys Ser Ser Gln Leu Cys Leu His Tyr Arg Leu50 55 60Met Phe Phe Glu Phe Ser Glu Asn Leu Thr Cys Ile Pro Arg Asn Ser65 70 75 80Ala Ser Thr Val Cys Val Cys His Met Glu Met Asn Arg Pro Val Gln85 90 95Ser Asp Arg Tyr Gln Met Glu Leu Trp Ala Glu His Arg Gln Leu Trp100 105 110Gln Gly Ser Phe Ser Pro Ser Gly Asn Val Lys Pro Leu Ala Pro Asp115 120 125Asn Leu Thr Leu His Thr Asn Val Ser Asp Glu Trp Leu Leu Thr Trp130 135 140Asn Asn Leu Tyr Pro Ser Asn Asn Leu Leu Tyr Lys Asp Leu Ile Ser145 150 155 160Met Val Asn Ile Ser Arg Glu Asp Asn Pro Ala Glu Phe Ile Val Tyr165 170 175Asn Val Thr Tyr Lys Glu Pro Arg Leu Ser Phe Pro Ile Asn Ile Leu180 185 190Met Ser Gly Val Tyr Tyr Thr Ala Arg Val Arg Val Arg Ser Gln Ile195 200 205Leu Thr Gly Thr Trp Ser Glu Trp Ser Pro Ser Ile Thr Trp Tyr Asn210 215 220His Phe Gln Leu Pro Leu Ile Gln Arg Leu Pro Leu Gly Val Thr Ile225 230 235 240Ser Cys Leu Cys Ile Pro Leu Phe Cys Leu Phe Cys Tyr Phe Ser Ile245 250 255Thr Lys Ile Lys Lys Ile Trp Trp Asp Gln Ile Pro Thr Pro Ala Arg260 265 270Ser Pro Leu Val Ala Ile Ile Ile Gln Asp Ala Gln Val Pro Leu Trp275 280 285Asp Lys Gln Thr Arg Ser Gln Glu Ser Thr Lys Tyr Pro His Trp Lys290 295 300Thr Cys Leu Asp Lys Leu Leu Pro Cys Leu Leu Lys His Arg Val Lys305 310 315 320Lys Lys Thr Asp Phe Pro Lys Ala Ala Pro Thr Lys Ser Leu Gln Ser325 330 335Pro Gly Lys Ala Gly Trp Cys Pro Met Glu Val Ser Arg Thr Val Leu340 345 350Trp Pro Glu Asn Val Ser Val Ser Val Val Arg Cys Met Glu Leu Phe355 360 365Glu Ala Pro Val Gln Asn Val Glu Glu Glu Glu Asp Glu Ile Val Lys370 375 380Glu Asp Leu Ser Met Ser Pro Glu Asn Ser Gly Gly Cys Gly Phe Gln385 390 395 400Glu Ser Gln Ala Asp Ile Met Ala Arg Leu Thr Glu Asn Leu Phe Ser405 410 415Asp Leu Leu Glu Ala Glu Asn Gly Gly Leu Gly Gln Ser Ala Leu Ala420 425 430Glu Ser Cys Ser Pro Leu Pro Ser Gly Ser Gly Gln Ala Ser Val Ser435 440 445Trp Ala Cys Leu Pro Met Gly Pro Ser Glu Glu Ala Thr Cys Gln Val450 455 460Thr Glu Gln Pro Ser His Pro Gly Pro Leu Ser Gly Ser Pro Ala Gln465 470 475 480Ser Ala Pro Thr Leu Ala Cys Thr Gln Val Pro Leu Val Leu Ala Asp485 490 495Asn Pro Ala Tyr Arg Ser Phe Ser Asp Cys Cys Ser Pro Ala Pro Asn500 505 510Pro Gly Glu Leu Ala Pro Glu Gln Gln Gln Ala Asp His Leu Glu Glu515 520 525Glu Glu Pro Pro Ser Pro Ala Asp Pro His Ser Ser Gly Pro Pro Met530 535 540Gln Pro Val Glu Ser Trp Glu Gln Ile Leu His Met Ser Val Leu Gln545 550 555 560His Gly Ala Ala Ala Gly Ser Thr Pro Ala Pro Ala Gly Gly Tyr Gln565 570 575Glu Phe Val Gln Ala Val Lys Gln Gly Ala Ala Gln Asp Pro Gly Val580 585 590Pro Gly Val Arg Pro Ser Gly Asp Pro Gly Tyr Lys Ala Phe Ser Ser595 600 605Leu Leu Ser Ser Asn Gly Ile Arg Gly Asp Thr Ala Ala Ala Gly Thr610 615 620Asp Asp Gly His Gly Gly Tyr Lys Pro Phe Gln Asn Pro Val Pro Asn625 630 635 640Gln Ser Pro Ser Ser Val Pro Leu Phe Thr Phe Gly Leu Asp Thr Glu645 650 655Leu Ser Pro Ser Pro Leu Asn Ser Asp Pro Pro Lys Ser Pro Pro Glu660 665 670Cys Leu Gly Leu Glu Leu Gly Leu Lys Gly Gly Asp Trp Val Lys Ala675 680 685Pro Pro Pro Ala Asp Gln Val Pro Lys Pro Phe Gly Asp Asp Leu Gly690 695 700Phe Gly Ile Val Tyr Ser Ser Leu Thr Cys His Leu Cys Gly His Leu705 710 715 720Lys Gln His His Ser Gln Glu Glu Gly Gly Gln Ser Pro Ile Val Ala725 730 735Ser Pro Gly Cys Gly Cys Cys Tyr Asp Asp Arg Ser Pro Ser Leu Gly740 745 750Ser Leu Ser Gly Ala Leu Glu Ser Cys Pro Glu Gly Ile Pro Pro Glu755 760 765Ala Asn Leu Met Ser Ala Pro Lys Thr Pro Ser Asn Leu Ser Gly Glu770 775 780Gly Lys Gly Pro Gly His Ser Pro Val Pro Ser Gln Thr Thr Glu Val785 790 795 800Pro Val Gly Ala Leu Gly Ile Ala Val Ser805 81061810PRTEquus sp. 61Met Gly Arg Leu Cys Thr Lys Phe Leu Thr Ser Val Gly Cys Leu Ile1 5 10 15Leu Leu Leu Val Thr Gly Ser Gly Ser Ile Lys Val Leu Gly Glu Pro20 25 30Thr Cys Phe Ser Asp Tyr Ile Arg Thr Ser Thr Cys Glu Trp Phe Leu35 40 45Asp Ser Ala Val Asp Cys Ser Ser Gln Leu Cys Leu His Tyr Arg Leu50 55 60Met Phe Phe Glu Phe Ser Glu Asn Leu Ile Cys Ile Pro Arg Asn Ser65 70 75 80Ala Ser Thr Val Cys Val Cys His Met Glu Met Asn Arg Pro Val Gln85 90 95Ser Asp Arg Tyr Gln Met Glu Leu Trp Ala Glu His Arg Gln Leu Trp100 105 110Gln Gly Ser Phe Ser Pro Ser Gly Asn Val Lys Pro Leu Ala Pro Asp115 120 125Asn Leu Thr Leu His Thr Asn Val Ser Asp Glu Trp Leu Leu Thr Trp130 135 140Asn Asn Leu Tyr Pro Ser Asn Asn Leu Leu Tyr Lys Asp Leu Ile Ser145 150 155 160Met Val Asn Ile Ser Arg Glu Asp Asn Pro Ala Glu Phe Ile Val Tyr165 170 175Asn Val Thr Tyr Lys Glu Pro Arg Leu Ser Phe Pro Ile Asn Ile Leu180 185 190Met Ser Gly Val Tyr Tyr Thr Ala Arg Val Arg Val Arg Ser Gln Ile195 200 205Leu Thr Gly Thr Trp Ser Glu Trp Ser Pro Ser Ile Thr Trp Tyr Asn210 215 220His Phe Gln Leu Pro Leu Ile Gln Arg Leu Pro Leu Gly Val Thr Ile225 230 235 240Ser Cys Leu Cys Ile Pro Leu Phe Cys Leu Phe Cys Tyr Phe Ser Ile245 250 255Thr Lys Ile Lys Lys Ile Trp Trp Asp Gln Ile Pro Thr Pro Ala Arg260 265 270Ser Pro Leu Val Ala Ile Ile Ile Gln Asp Ala Gln Val Pro Leu Trp275 280 285Asp Lys Gln Thr Arg Ser Gln Glu Ser Thr Lys Tyr Pro His Trp Lys290 295 300Thr Cys Leu Asp Lys Leu Leu Pro Cys Leu Leu Lys His Arg Val Lys305 310 315 320Lys Lys Thr Asp Phe Pro Lys Ala Ala Pro Thr Lys Ser Pro Gln Ser325 330 335Pro Gly Lys Ala Gly Trp Cys Pro Met Glu Val Ser Arg Thr Val Leu340 345 350Trp Pro Glu Asn Val Ser Val Ser Val Val Arg Cys Met Glu Leu Phe355 360 365Glu Ala Pro Val Gln Asn Val Glu Glu Glu Glu Asp Glu Ile Val Lys370 375 380Glu Asp Leu Ser Met Ser Pro Glu Asn Ser Gly Gly Cys Gly Phe Gln385 390 395 400Glu Ser Gln Ala Asp Ile Met Ala Arg Leu Thr Glu Asn Leu Phe Ser405 410 415Asp Leu Leu Glu Ala Glu Asn Gly Gly Leu Gly Gln Ser Ala Leu Ala420 425 430Glu Ser Cys Ser Pro Leu Pro Ser Gly Ser Gly Gln Ala Ser Val Ser435 440 445Trp Ala Cys Leu Pro Met Gly Pro Ser Glu Glu Ala Thr Cys Gln Val450 455 460Thr Glu Gln Pro Ser His Pro Gly Pro Leu Ser Gly Ser Pro Ala Gln465 470 475 480Ser Ala Pro Thr Leu Ala Cys Thr Gln Val Pro Leu Val Leu Ala Asp485 490 495Asn Pro Ala Tyr Arg Ser Phe Ser Asp Cys Cys Ser Pro Ala Pro Asn500 505 510Pro Gly Glu Leu Ala Pro Glu Gln Gln Gln Ala Asp His Leu Glu Glu515 520 525Glu Glu Pro Pro Ser Pro Ala Asp Pro His Ser Ser Gly Pro Pro Met530 535 540Gln Pro Val Glu Ser Trp Glu Gln Ile Leu His Met Ser Val Leu Gln545 550 555 560His Gly Ala Ala Ala Gly Ser Thr Pro Ala Pro Ala Gly Gly Tyr Gln565 570 575Glu Phe Val Gln Ala Val Lys Gln Gly Ala Ala Gln Asp Pro Gly Val580 585 590Pro Gly Val Arg Pro Ser Gly Asp Pro Gly Tyr Lys Ala Phe Ser Ser595 600 605Leu Leu Ser Ser Asn Gly Ile Arg Gly Asp Thr Ala Ala Ala Gly Thr610 615 620Asp Asp Gly His Gly Gly Tyr Lys Pro Phe Gln Asn Pro Val Pro Asn625 630 635 640Gln Ser Pro Ser Ser Val Pro Leu Phe Thr Phe Gly Leu Asp Thr Glu645 650 655Leu Ser Pro Ser Pro Leu Asn Ser Asp Pro Pro Lys Ser Pro Pro Glu660 665 670Cys Leu Gly Leu Glu Leu Gly Leu Lys Gly Gly Asp Trp Val Lys Ala675 680 685Pro Pro Pro Ala Asp Gln Val Pro Lys Pro Phe Gly Asp Asp Leu Gly690 695 700Phe Gly Ile Val Tyr Ser Ser Leu Thr Cys His Leu Cys Gly His Leu705 710 715 720Lys Gln His His Ser Gln Glu Glu Gly Gly Gln Ser Pro Ile Val Ala725 730 735Ser Pro Gly Cys Gly Cys Cys Tyr Asp Asp Arg Ser Pro Ser Leu Gly740 745 750Ser Leu Ser Gly Ala Leu Glu Ser Cys Pro Glu Gly Ile Pro Pro Glu755 760 765Ala Asn Leu Met Ser Ala Pro Lys Thr Pro Ser Asn Leu Ser Gly Glu770 775 780Gly Lys Gly Pro Gly His Ser Pro Val Pro Ser Gln Thr Thr Glu Val785 790 795 800Pro Val Gly Ala Leu Gly Ile Ala Val Ser805 810621356PRTHomo sapiens 62Met Gln Ser Lys Val Leu Leu Ala Val Ala Leu Trp Leu Cys Val Glu1 5 10 15Thr Arg Ala Ala Ser Val Gly Leu Pro Ser Val Ser Leu Asp Leu Pro20 25 30Arg Leu Ser Ile Gln Lys Asp Ile Leu Thr Ile Lys Ala Asn Thr Thr35 40 45Leu Gln Ile Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro50 55 60Asn Asn Gln Ser Gly Ser Glu Gln Arg Val Glu Val Thr Glu Cys Ser65 70 75 80Asp Gly Leu Phe Cys Lys Thr Leu Thr Ile Pro Lys Val Ile Gly Asn85 90 95Asp Thr Gly Ala Tyr Lys Cys Phe Tyr Arg Glu Thr Asp Leu Ala Ser100 105 110Val Ile Tyr Val Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser115 120 125Val Ser Asp Gln His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys130 135 140Thr Val Val Ile Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser145 150 155 160Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg165 170 175Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile180 185 190Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser195 200 205Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr210 215 220Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu225 230 235 240Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile245 250 255Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu260 265 270Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe275 280 285Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu290 295 300Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr305 310 315 320Phe Val Arg Val His Glu Lys Pro Phe Val Ala Phe Gly Ser Gly Met325 330 335Glu Ser Leu Val Glu Ala Thr Val Gly Glu Arg Val Arg Ile Pro Ala340 345 350Lys Tyr Leu Gly Tyr Pro Pro Pro Glu

Ile Lys Trp Tyr Lys Asn Gly355 360 365Ile Pro Leu Glu Ser Asn His Thr Ile Lys Ala Gly His Val Leu Thr370 375 380Ile Met Glu Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr Val Ile Leu385 390 395 400Thr Asn Pro Ile Ser Lys Glu Lys Gln Ser His Val Val Ser Leu Val405 410 415Val Tyr Val Pro Pro Gln Ile Gly Glu Lys Ser Leu Ile Ser Pro Val420 425 430Asp Ser Tyr Gln Tyr Gly Thr Thr Gln Thr Leu Thr Cys Thr Val Tyr435 440 445Ala Ile Pro Pro Pro His His Ile His Trp Tyr Trp Gln Leu Glu Glu450 455 460Glu Cys Ala Asn Glu Pro Ser Gln Ala Val Ser Val Thr Asn Pro Tyr465 470 475 480Pro Cys Glu Glu Trp Arg Ser Val Glu Asp Phe Gln Gly Gly Asn Lys485 490 495Ile Glu Val Asn Lys Asn Gln Phe Ala Leu Ile Glu Gly Lys Asn Lys500 505 510Thr Val Ser Thr Leu Val Ile Gln Ala Ala Asn Val Ser Ala Leu Tyr515 520 525Lys Cys Glu Ala Val Asn Lys Val Gly Arg Gly Glu Arg Val Ile Ser530 535 540Phe His Val Thr Arg Gly Pro Glu Ile Thr Leu Gln Pro Asp Met Gln545 550 555 560Pro Thr Glu Gln Glu Ser Val Ser Leu Trp Cys Thr Ala Asp Arg Ser565 570 575Thr Phe Glu Asn Leu Thr Trp Tyr Lys Leu Gly Pro Gln Pro Leu Pro580 585 590Ile His Val Gly Glu Leu Pro Thr Pro Val Cys Lys Asn Leu Asp Thr595 600 605Leu Trp Lys Leu Asn Ala Thr Met Phe Ser Asn Ser Thr Asn Asp Ile610 615 620Leu Ile Met Glu Leu Lys Asn Ala Ser Leu Gln Asp Gln Gly Asp Tyr625 630 635 640Val Cys Leu Ala Gln Asp Arg Lys Thr Lys Lys Arg His Cys Val Val645 650 655Arg Gln Leu Thr Val Leu Glu Arg Val Ala Pro Thr Ile Thr Gly Asn660 665 670Leu Glu Asn Gln Thr Thr Ser Ile Gly Glu Ser Ile Glu Val Ser Cys675 680 685Thr Ala Ser Gly Asn Pro Pro Pro Gln Ile Met Trp Phe Lys Asp Asn690 695 700Glu Thr Leu Val Glu Asp Ser Gly Ile Val Leu Lys Asp Gly Asn Arg705 710 715 720Asn Leu Thr Ile Arg Arg Val Arg Lys Glu Asp Glu Gly Leu Tyr Thr725 730 735Cys Gln Ala Cys Ser Val Leu Gly Cys Ala Lys Val Glu Ala Phe Phe740 745 750Ile Ile Glu Gly Ala Gln Glu Lys Thr Asn Leu Glu Ile Ile Ile Leu755 760 765Val Gly Thr Ala Val Ile Ala Met Phe Phe Trp Leu Leu Leu Val Ile770 775 780Ile Leu Arg Thr Val Lys Arg Ala Asn Gly Gly Glu Leu Lys Thr Gly785 790 795 800Tyr Leu Ser Ile Val Met Asp Pro Asp Glu Leu Pro Leu Asp Glu His805 810 815Cys Glu Arg Leu Pro Tyr Asp Ala Ser Lys Trp Glu Phe Pro Arg Asp820 825 830Arg Leu Lys Leu Gly Lys Pro Leu Gly Arg Gly Ala Phe Gly Gln Val835 840 845Ile Glu Ala Asp Ala Phe Gly Ile Asp Lys Thr Ala Thr Cys Arg Thr850 855 860Val Ala Val Lys Met Leu Lys Glu Gly Ala Thr His Ser Glu His Arg865 870 875 880Ala Leu Met Ser Glu Leu Lys Ile Leu Ile His Ile Gly His His Leu885 890 895Asn Val Val Asn Leu Leu Gly Ala Cys Thr Lys Pro Gly Gly Pro Leu900 905 910Met Val Ile Val Glu Phe Cys Lys Phe Gly Asn Leu Ser Thr Tyr Leu915 920 925Arg Ser Lys Arg Asn Glu Phe Val Pro Tyr Lys Thr Lys Gly Ala Arg930 935 940Phe Arg Gln Gly Lys Asp Tyr Val Gly Ala Ile Pro Val Asp Leu Lys945 950 955 960Arg Arg Leu Asp Ser Ile Thr Ser Ser Gln Ser Ser Ala Ser Ser Gly965 970 975Phe Val Glu Glu Lys Ser Leu Ser Asp Val Glu Glu Glu Glu Ala Pro980 985 990Glu Asp Leu Tyr Lys Asp Phe Leu Thr Leu Glu His Leu Ile Cys Tyr995 1000 1005Ser Phe Gln Val Ala Lys Gly Met Glu Phe Leu Ala Ser Arg Lys1010 1015 1020Cys Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu Ser Glu1025 1030 1035Lys Asn Val Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile1040 1045 1050Tyr Lys Asp Pro Asp Tyr Val Arg Lys Gly Asp Ala Arg Leu Pro1055 1060 1065Leu Lys Trp Met Ala Pro Glu Thr Ile Phe Asp Arg Val Tyr Thr1070 1075 1080Ile Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile1085 1090 1095Phe Ser Leu Gly Ala Ser Pro Tyr Pro Gly Val Lys Ile Asp Glu1100 1105 1110Glu Phe Cys Arg Arg Leu Lys Glu Gly Thr Arg Met Arg Ala Pro1115 1120 1125Asp Tyr Thr Thr Pro Glu Met Tyr Gln Thr Met Leu Asp Cys Trp1130 1135 1140His Gly Glu Pro Ser Gln Arg Pro Thr Phe Ser Glu Leu Val Glu1145 1150 1155His Leu Gly Asn Leu Leu Gln Ala Asn Ala Gln Gln Asp Gly Lys1160 1165 1170Asp Tyr Ile Val Leu Pro Ile Ser Glu Thr Leu Ser Met Glu Glu1175 1180 1185Asp Ser Gly Leu Ser Leu Pro Thr Ser Pro Val Ser Cys Met Glu1190 1195 1200Glu Glu Glu Val Cys Asp Pro Lys Phe His Tyr Asp Asn Thr Ala1205 1210 1215Gly Ile Ser Gln Tyr Leu Gln Asn Ser Lys Arg Lys Ser Arg Pro1220 1225 1230Val Ser Val Lys Thr Phe Glu Asp Ile Pro Leu Glu Glu Pro Glu1235 1240 1245Val Lys Val Ile Pro Asp Asp Asn Gln Thr Asp Ser Gly Met Val1250 1255 1260Leu Ala Ser Glu Glu Leu Lys Thr Leu Glu Asp Arg Thr Lys Leu1265 1270 1275Ser Pro Ser Phe Gly Gly Met Val Pro Ser Lys Ser Arg Glu Ser1280 1285 1290Val Ala Ser Glu Gly Ser Asn Gln Thr Ser Gly Tyr Gln Ser Gly1295 1300 1305Tyr His Ser Asp Asp Thr Asp Thr Thr Val Tyr Ser Ser Glu Glu1310 1315 1320Ala Glu Leu Leu Lys Leu Ile Glu Ile Gly Val Gln Thr Gly Ser1325 1330 1335Thr Ala Gln Ile Leu Gln Pro Asp Ser Gly Thr Thr Leu Ser Ser1340 1345 1350Pro Pro Val1355631367PRTMus sp. 63Met Glu Ser Lys Ala Leu Leu Ala Val Ala Leu Trp Phe Cys Val Glu1 5 10 15Thr Arg Ala Ala Ser Val Gly Leu Pro Gly Asp Phe Leu His Pro Pro20 25 30Lys Leu Ser Thr Gln Lys Asp Ile Leu Thr Ile Leu Ala Asn Thr Thr35 40 45Leu Gln Ile Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro50 55 60Asn Ala Gln Arg Asp Ser Glu Glu Arg Val Leu Val Thr Glu Cys Gly65 70 75 80Gly Gly Asp Ser Ile Phe Cys Lys Thr Leu Thr Ile Pro Arg Val Val85 90 95Gly Asn Asp Thr Gly Ala Tyr Lys Cys Ser Tyr Arg Asp Val Asp Ile100 105 110Ala Ser Thr Val Tyr Val Tyr Val Arg Asp Tyr Arg Ser Pro Phe Ile115 120 125Ala Ser Val Ser Asp Gln His Gly Ile Val Tyr Ile Thr Glu Asn Lys130 135 140Asn Lys Thr Val Val Ile Pro Cys Arg Gly Ser Ile Ser Asn Leu Asn145 150 155 160Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly165 170 175Asn Arg Ile Ser Trp Asp Ser Glu Ile Gly Phe Thr Leu Pro Ser Tyr180 185 190Met Ile Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp195 200 205Glu Thr Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg210 215 220Ile Tyr Asp Val Ile Leu Ser Pro Pro His Glu Ile Glu Leu Ser Ala225 230 235 240Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val245 250 255Gly Leu Asp Phe Thr Trp His Ser Pro Pro Ser Lys Ser His His Lys260 265 270Lys Ile Val Asn Arg Asp Val Lys Pro Phe Pro Gly Thr Val Ala Lys275 280 285Met Phe Leu Ser Thr Leu Thr Ile Glu Ser Val Thr Lys Ser Asp Gln290 295 300Gly Glu Tyr Thr Cys Val Ala Ser Ser Gly Arg Met Ile Lys Arg Asn305 310 315 320Arg Thr Phe Val Arg Val His Thr Lys Pro Phe Ile Ala Phe Gly Ser325 330 335Gly Met Lys Ser Leu Val Glu Ala Thr Val Gly Ser Gln Val Arg Ile340 345 350Pro Val Lys Tyr Leu Ser Tyr Pro Ala Pro Asp Ile Lys Trp Tyr Arg355 360 365Asn Gly Arg Pro Ile Glu Ser Asn Tyr Thr Met Ile Val Gly Asp Glu370 375 380Leu Thr Ile Met Glu Val Thr Glu Arg Asp Ala Gly Asn Tyr Thr Val385 390 395 400Ile Leu Thr Asn Pro Ile Ser Met Glu Lys Gln Ser His Met Val Ser405 410 415Leu Val Val Asn Val Pro Pro Gln Ile Gly Glu Lys Ala Leu Ile Ser420 425 430Pro Met Asp Ser Tyr Gln Tyr Gly Thr Met Gln Thr Leu Thr Cys Thr435 440 445Val Tyr Ala Asn Pro Pro Leu His His Ile Gln Trp Tyr Trp Gln Leu450 455 460Glu Glu Ala Cys Ser Tyr Arg Pro Gly Gln Thr Ser Pro Tyr Ala Cys465 470 475 480Lys Glu Trp Arg His Val Glu Asp Phe Gln Gly Gly Asn Lys Ile Glu485 490 495Val Thr Lys Asn Gln Tyr Ala Leu Ile Glu Gly Lys Asn Lys Thr Val500 505 510Ser Thr Leu Val Ile Gln Ala Ala Asn Val Ser Ala Leu Tyr Lys Cys515 520 525Glu Ala Ile Asn Lys Ala Gly Arg Gly Glu Arg Val Ile Ser Phe His530 535 540Val Ile Arg Gly Pro Glu Ile Thr Val Gln Pro Ala Ala Gln Pro Thr545 550 555 560Glu Gln Glu Ser Val Ser Leu Leu Cys Thr Ala Asp Arg Asn Thr Phe565 570 575Glu Asn Leu Thr Trp Tyr Lys Leu Gly Ser Gln Ala Thr Ser Val His580 585 590Met Gly Glu Ser Leu Thr Pro Val Cys Lys Asn Leu Asp Ala Leu Trp595 600 605Lys Leu Asn Gly Thr Met Phe Ser Asn Ser Thr Asn Asp Ile Leu Ile610 615 620Val Ala Phe Gln Asn Ala Ser Leu Gln Asp Gln Gly Asp Tyr Val Cys625 630 635 640Ser Ala Gln Asp Lys Lys Thr Lys Lys Arg His Cys Leu Val Lys Gln645 650 655Leu Ile Ile Leu Glu Arg Met Ala Pro Met Ile Thr Gly Asn Leu Glu660 665 670Asn Gln Thr Thr Thr Ile Gly Glu Thr Ile Glu Val Thr Cys Pro Ala675 680 685Ser Gly Asn Pro Thr Pro His Ile Thr Trp Phe Lys Asp Asn Glu Thr690 695 700Leu Val Glu Asp Ser Gly Ile Val Leu Arg Asp Gly Asn Arg Asn Leu705 710 715 720Thr Ile Arg Arg Val Arg Lys Glu Asp Gly Gly Leu Tyr Thr Cys Gln725 730 735Ala Cys Asn Val Leu Gly Cys Ala Arg Ala Glu Thr Leu Phe Ile Ile740 745 750Glu Gly Ala Gln Glu Lys Thr Asn Leu Glu Val Ile Ile Leu Val Gly755 760 765Thr Ala Val Ile Ala Met Phe Phe Trp Leu Leu Leu Val Ile Leu Val770 775 780Arg Thr Val Lys Arg Ala Asn Glu Gly Glu Leu Lys Thr Gly Tyr Leu785 790 795 800Ser Ile Val Met Asp Pro Asp Glu Leu Pro Leu Asp Glu Arg Cys Glu805 810 815Arg Leu Pro Tyr Asp Ala Ser Lys Trp Glu Phe Pro Arg Asp Arg Leu820 825 830Lys Leu Gly Lys Pro Leu Gly Arg Gly Ala Phe Gly Gln Val Ile Glu835 840 845Ala Asp Ala Phe Gly Ile Asp Lys Thr Ala Thr Cys Lys Thr Val Ala850 855 860Val Lys Met Leu Lys Glu Gly Ala Thr His Ser Glu His Arg Ala Leu865 870 875 880Met Ser Glu Leu Lys Ile Leu Ile His Ile Gly His His Leu Asn Val885 890 895Val Asn Leu Leu Gly Ala Cys Thr Lys Pro Gly Gly Pro Leu Met Val900 905 910Ile Val Glu Phe Ser Lys Phe Gly Asn Leu Ser Thr Tyr Leu Arg Gly915 920 925Lys Arg Asn Glu Phe Val Pro Tyr Lys Ser Lys Gly Ala Arg Phe Arg930 935 940Gln Gly Lys Asp Tyr Val Gly Glu Leu Ser Val Asp Leu Lys Arg Arg945 950 955 960Leu Asp Ser Ile Thr Ser Ser Gln Ser Ser Ala Ser Ser Gly Phe Val965 970 975Glu Glu Lys Ser Leu Ser Asp Val Glu Glu Glu Glu Ala Ser Glu Glu980 985 990Leu Tyr Lys Asp Phe Leu Thr Leu Glu His Leu Ile Cys Tyr Ser Phe995 1000 1005Gln Val Ala Lys Gly Met Glu Phe Leu Ala Ser Arg Lys Cys Ile1010 1015 1020His Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu Ser Glu Lys Asn1025 1030 1035Val Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys1040 1045 1050Asp Pro Asp Tyr Val Arg Lys Gly Asp Ala Arg Leu Pro Leu Lys1055 1060 1065Trp Met Ala Pro Glu Thr Ile Phe Asp Arg Val Tyr Thr Ile Gln1070 1075 1080Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Phe Ser1085 1090 1095Leu Gly Ala Ser Pro Tyr Pro Gly Val Lys Ile Asp Glu Glu Phe1100 1105 1110Cys Arg Arg Leu Lys Glu Gly Thr Arg Met Arg Ala Pro Asp Tyr1115 1120 1125Thr Thr Pro Glu Met Tyr Gln Thr Met Leu Asp Cys Trp His Glu1130 1135 1140Asp Pro Asn Gln Arg Pro Ser Phe Ser Glu Leu Val Glu His Leu1145 1150 1155Gly Asn Leu Leu Gln Ala Asn Ala Gln Gln Asp Gly Lys Asp Tyr1160 1165 1170Ile Val Leu Pro Met Ser Glu Thr Leu Ser Met Glu Glu Asp Ser1175 1180 1185Gly Leu Ser Leu Pro Thr Ser Pro Val Ser Cys Met Glu Glu Glu1190 1195 1200Glu Val Cys Asp Pro Lys Phe His Tyr Asp Asn Thr Ala Gly Ile1205 1210 1215Ser His Tyr Leu Gln Asn Ser Lys Arg Lys Ser Arg Pro Val Ser1220 1225 1230Val Lys Thr Phe Glu Asp Ile Pro Leu Glu Glu Pro Glu Val Lys1235 1240 1245Val Ile Pro Asp Asp Ser Gln Thr Asp Ser Gly Met Val Leu Ala1250 1255 1260Ser Glu Glu Leu Lys Thr Leu Glu Asp Arg Asn Lys Leu Ser Pro1265 1270 1275Ser Phe Gly Gly Met Met Pro Ser Lys Ser Arg Glu Ser Val Ala1280 1285 1290Ser Glu Gly Ser Asn Gln Thr Ser Gly Tyr Gln Ser Gly Tyr His1295 1300 1305Ser Asp Asp Thr Asp Thr Thr Val Tyr Ser Ser Asp Glu Ala Gly1310 1315 1320Leu Leu Lys Met Val Asp Ala Ala Val His Ala Asp Ser Gly Thr1325 1330 1335Thr Leu Gln Leu Thr Ser Cys Leu Asn Gly Ser Gly Pro Val Pro1340 1345 1350Ala Pro Pro Pro Thr Pro Gly Asn His Glu Arg Gly Ala Ala1355 1360 1365641343PRTRattus sp. 64Met Glu Ser Arg Ala Leu Leu Ala Val Ala Leu Trp Phe Cys Val Glu1 5 10 15Thr Arg Ala Ala Ser Val Gly Leu Pro Gly Asp Ser Leu His Pro Pro20 25 30Lys Leu Ser Thr Gln Lys Asp Ile Leu Thr Ile Leu Ala Asn Thr Thr35 40 45Leu Gln Ile Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro50 55 60Asn Thr Pro Arg Asp Ser Glu Glu Arg Val Leu Val Thr Glu Cys Gly65 70 75 80Asp Ser Ile Phe Cys Lys Thr Leu Thr Val Pro Arg Val Val Gly Asn85 90 95Asp Thr Gly Ala Tyr Lys Cys Phe Tyr Arg Asp Thr Asp Val Ser Ser100 105 110Ile Val Tyr Val Tyr Val Gln Asp His Arg Ser Pro Phe Ile Ala Ser115 120 125Val Ser Asp Glu His Gly Ile Val Tyr Ile Thr Glu Asn Lys Asn Lys130 135 140Thr Val Val Ile Pro Cys Arg Gly Ser Ile Ser Asn Leu Asn Val Ser145 150 155 160Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg165 170 175Ile Ser Trp Asp Ser Glu Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile180 185 190Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Thr195 200 205Tyr Gln Ser Ile Met Tyr Ile Val Leu Val Val Gly Tyr Arg Ile Tyr210 215 220Asp Val Val Leu Ser Pro Pro His Glu Ile Glu Leu Ser Ala Gly Glu225 230 235 240Lys Leu Val Leu Asn Cys Thr Ala Arg

Thr Glu Leu Asn Val Gly Leu245 250 255Asp Phe Ser Trp Gln Phe Pro Ser Ser Lys His Gln His Lys Lys Ile260 265 270Val Asn Arg Asp Val Lys Ser Leu Pro Gly Thr Val Ala Lys Met Phe275 280 285Leu Ser Thr Leu Thr Ile Asp Ser Val Thr Lys Ser Asp Gln Gly Glu290 295 300Tyr Thr Cys Thr Ala Tyr Ser Gly Leu Met Thr Lys Lys Asn Lys Thr305 310 315 320Phe Val Arg Val His Thr Lys Pro Phe Ile Ala Phe Gly Ser Gly Met325 330 335Lys Ser Leu Val Glu Ala Thr Val Gly Ser Gln Val Arg Ile Pro Val340 345 350Lys Tyr Leu Ser Tyr Pro Ala Pro Asp Ile Lys Trp Tyr Arg Asn Gly355 360 365Arg Pro Ile Glu Ser Asn Tyr Thr Met Ile Val Gly Asp Glu Leu Thr370 375 380Ile Met Glu Val Ser Glu Arg Asp Ala Gly Asn Tyr Thr Val Ile Leu385 390 395 400Thr Asn Pro Ile Ser Met Glu Lys Gln Ser His Met Val Ser Leu Val405 410 415Val Asn Val Pro Pro Gln Ile Gly Glu Lys Ala Leu Ile Ser Pro Met420 425 430Asp Ser Tyr Gln Tyr Gly Thr Met Gln Thr Leu Thr Cys Thr Val Tyr435 440 445Ala Asn Pro Pro Leu His His Ile Gln Trp Tyr Trp Gln Leu Glu Glu450 455 460Ala Cys Ser Tyr Arg Pro Ser Gln Thr Asn Pro Tyr Thr Cys Lys Glu465 470 475 480Trp Arg His Val Lys Asp Phe Gln Gly Gly Asn Lys Ile Glu Val Thr485 490 495Lys Asn Gln Tyr Ala Leu Ile Glu Gly Lys Asn Lys Thr Val Ser Thr500 505 510Leu Val Ile Gln Ala Ala Tyr Val Ser Ala Leu Tyr Lys Cys Glu Ala515 520 525Ile Asn Lys Ala Gly Arg Gly Glu Arg Val Ile Ser Phe His Val Ile530 535 540Arg Gly Pro Glu Ile Thr Val Gln Pro Ala Thr Gln Pro Thr Glu Arg545 550 555 560Glu Ser Met Ser Leu Leu Cys Thr Ala Asp Arg Asn Thr Phe Glu Asn565 570 575Leu Thr Trp Tyr Lys Leu Gly Ser Gln Ala Thr Ser Val His Met Gly580 585 590Glu Ser Leu Thr Pro Val Cys Lys Asn Leu Asp Ala Leu Trp Lys Leu595 600 605Asn Gly Thr Val Phe Ser Asn Ser Thr Asn Asp Ile Leu Ile Val Ala610 615 620Phe Gln Asn Ala Ser Leu Gln Asp Gln Gly Asn Tyr Val Cys Ser Ala625 630 635 640Gln Asp Lys Lys Thr Lys Lys Arg His Cys Leu Val Lys Gln Leu Val645 650 655Ile Leu Glu Arg Met Ala Pro Met Ile Thr Gly Asn Leu Glu Asn Gln660 665 670Thr Thr Thr Ile Gly Glu Thr Ile Glu Val Val Cys Pro Thr Ser Gly675 680 685Asn Pro Thr Pro Leu Ile Thr Trp Phe Lys Asp Asn Glu Thr Leu Val690 695 700Glu Asp Ser Gly Ile Val Leu Lys Asp Gly Asn Arg Asn Leu Thr Ile705 710 715 720Arg Arg Val Arg Lys Glu Asp Gly Gly Leu Tyr Thr Cys Gln Ala Cys725 730 735Asn Val Leu Gly Cys Ala Arg Ala Glu Thr Leu Phe Ile Ile Glu Gly740 745 750Val Gln Glu Lys Thr Asn Leu Glu Val Ile Ile Leu Val Gly Thr Ala755 760 765Val Ile Ala Met Phe Phe Trp Leu Leu Leu Val Ile Leu Val Arg Thr770 775 780Val Lys Arg Ala Asn Glu Gly Glu Leu Lys Thr Gly Tyr Leu Ser Ile785 790 795 800Val Met Asp Pro Asp Glu Leu Pro Leu Asp Glu Arg Cys Glu Arg Leu805 810 815Pro Tyr Asp Ala Ser Lys Trp Glu Phe Pro Arg Asp Arg Leu Lys Leu820 825 830Gly Lys Pro Leu Gly Arg Gly Ala Phe Gly Gln Val Ile Glu Ala Asp835 840 845Ala Phe Gly Ile Asp Lys Thr Ala Thr Cys Lys Thr Val Ala Val Lys850 855 860Met Leu Lys Glu Gly Ala Thr His Ser Glu His Arg Ala Leu Met Ser865 870 875 880Glu Leu Lys Ile Leu Ile His Ile Gly His His Leu Asn Val Val Asn885 890 895Leu Leu Gly Ala Cys Thr Lys Pro Gly Gly Pro Leu Met Val Ile Val900 905 910Glu Phe Cys Lys Phe Gly Asn Leu Ser Thr Tyr Leu Arg Gly Lys Arg915 920 925Asn Glu Phe Val Pro Tyr Lys Ser Lys Gly Ala Arg Phe Arg Ser Gly930 935 940Lys Asp Tyr Val Gly Glu Leu Ser Val Asp Leu Lys Arg Arg Leu Asp945 950 955 960Ser Ile Thr Ser Ser Gln Ser Ser Ala Ser Ser Gly Phe Val Glu Glu965 970 975Lys Ser Leu Ser Asp Val Glu Glu Glu Glu Ala Ser Glu Glu Leu Tyr980 985 990Lys Asp Phe Leu Thr Leu Glu His Leu Ile Cys Tyr Ser Phe Gln Val995 1000 1005Ala Lys Gly Met Glu Phe Leu Ala Ser Arg Lys Cys Ile His Arg1010 1015 1020Asp Leu Ala Ala Arg Asn Ile Leu Leu Ser Glu Lys Asn Val Val1025 1030 1035Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asp Pro1040 1045 1050Asp Tyr Val Arg Lys Gly Asp Pro Arg Leu Pro Leu Lys Trp Met1055 1060 1065Ala Pro Glu Thr Ile Phe Asp Arg Ile Tyr Thr Ile Gln Ser Gly1070 1075 1080Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Phe Ser Leu Gly1085 1090 1095Ala Ser Pro Tyr Pro Gly Val Lys Ile Asp Glu Lys Phe Cys Arg1100 1105 1110Arg Leu Lys Glu Gly Thr Arg Met Arg Ala Pro Asp Tyr Thr Thr1115 1120 1125Pro Glu Met Tyr Gln Thr Met Leu Asp Cys Trp His Glu Asp Pro1130 1135 1140Asn Gln Arg Pro Ala Phe Ser Glu Leu Val Glu His Leu Gly Asn1145 1150 1155Leu Leu Gln Ala Asn Ala Gln Gln Asp Gly Lys Asp Tyr Ile Val1160 1165 1170Leu Pro Met Ser Glu Thr Leu Ser Met Glu Glu Asp Ser Gly Leu1175 1180 1185Ser Leu Pro Thr Ser Pro Val Ser Cys Met Glu Glu Glu Glu Val1190 1195 1200Cys Asp Pro Lys Phe His Tyr Asp Asn Thr Ala Gly Ile Ser His1205 1210 1215Tyr Leu Gln Asn Ser Lys Arg Lys Ser Arg Pro Val Ser Val Lys1220 1225 1230Thr Phe Glu Asp Ile Pro Leu Glu Glu Pro Glu Val Lys Val Ile1235 1240 1245Pro Asp Asp Ser Gln Thr Asp Ser Gly Met Val Leu Ala Ser Glu1250 1255 1260Glu Leu Lys Thr Leu Glu Asp Arg Asn Lys Leu Ser Pro Ser Phe1265 1270 1275Gly Gly Met Met Pro Ser Lys Ser Arg Glu Ser Val Ala Ser Glu1280 1285 1290Gly Ser Asn Gln Thr Ser Gly Tyr Gln Ser Gly Tyr His Ser Asp1295 1300 1305Asp Thr Asp Thr Thr Val Tyr Ser Ser Asp Glu Ala Gly Leu Leu1310 1315 1320Lys Leu Val Asp Val Ala Gly His Val Asp Ser Gly Thr Thr Leu1325 1330 1335Arg Ser Ser Pro Val1340651348PRTCallipepla sp. 65Met Glu Leu Gly Pro Leu Arg Val Leu Thr Val Leu Leu Cys Leu Ala1 5 10 15Pro Val Phe Ala Gly Leu Phe Ile Ser Met Asp Gln Pro Thr Leu Ser20 25 30Ile Gln Lys Ser Val Leu Thr Ile Thr Thr Asn Asp Thr Leu Asn Ile35 40 45Thr Cys Ser Gly Gln Arg Ala Val Tyr Trp Ser Trp Pro Asn Asn Gln50 55 60Ser Ser Val Glu Lys Arg Leu Ala Val Thr Gly Cys Ser Glu Gly Pro65 70 75 80Phe Cys Lys Thr Leu Thr Leu Leu Arg Val Ile Gly Asn Asp Thr Gly85 90 95Asp Tyr Arg Cys Leu Tyr Gly Asp Ser Gln Ala Ala Thr Thr Ile Tyr100 105 110Val Tyr Val Gln Asp Tyr Arg Ser Pro Phe Val Thr Ser Val Gly Asp115 120 125Gln Leu Gly Ile Val Tyr Ile Thr Lys Asn Lys Thr Val Val Val Pro130 135 140Cys Leu Gly Thr Val Ser Asn Leu Asn Val Ser Leu His Ala Lys Tyr145 150 155 160Pro Glu Lys Val Phe Val Pro Asp Gly Lys Ser Ile Ser Trp Asp Asn165 170 175Lys Lys Gly Phe Thr Ile Pro Ser His Leu Ile Asn Tyr Ala Gly Met180 185 190Val Phe Cys Glu Ala Lys Ile Asp Asn Glu Ser Tyr Gln Ser Val Ile195 200 205Tyr Ile Val Ala Val Val Gly Tyr Arg Ile Tyr Asp Leu Thr Met Asn210 215 220Pro His Tyr Gln Val Glu Leu Ala Val Gly Glu Lys Leu Val Leu Asn225 230 235 240Cys Thr Val Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Arg Trp Asp245 250 255Tyr Pro Ser Ile Lys Glu Arg Arg Ala Thr Ile Arg Asp Leu Lys Thr260 265 270Thr Ala Gly Glu Ile Lys Thr Phe Val Ser Thr Leu Thr Ile Glu Ser275 280 285Val Asn Leu Ser Asp Lys Gly Arg Tyr Thr Cys Ala Ala Ser Ser Gly290 295 300Arg Met Asn Met Lys Asn Ser Ser Tyr Phe Ile Ile His Glu Ser Pro305 310 315 320Phe Ile His Leu Glu Lys Met Glu Asn Val Val Glu Met Lys Leu Gly325 330 335Asp Thr Val Ser Ile Pro Val Lys Phe Lys Gly Tyr Pro Pro Pro Glu340 345 350Ala Lys Trp Tyr Lys Asn Gly Lys Val Ile Asn Ala Asn His Thr Val355 360 365Lys Leu Gly Tyr Ala Leu Val Ile Thr Glu Ala Thr Glu Lys Asp Ala370 375 380Gly Asn Tyr Thr Val Val Leu Thr Asn Pro Thr Asn Lys Met Gln Lys385 390 395 400Arg His Thr Phe Thr Leu Leu Val Asn Val Pro Pro Gln Ile Gly Glu405 410 415Asn Ala Leu Met Ala Pro Val Asp Ser Tyr Lys Tyr Gly Ser Thr Gln420 425 430Ala Leu Thr Cys Thr Ile Tyr Ala Val Pro Pro Pro Ala Ala Val Leu435 440 445Trp Tyr Trp Gln Leu Glu Glu Glu Cys Thr Phe Ser Pro Gln Lys Val450 455 460Arg Leu Gly Ala Asn Pro Tyr Ala Cys Arg Lys Trp Lys Val Ile Ser465 470 475 480Glu Arg Lys Gly Gly Asn Gln Val Glu Ile Lys Gln Arg Val Val Thr485 490 495Ile Ala Gly Lys Thr Lys Thr Val Ser Thr Leu Val Ile Gln Ala Ala500 505 510Asn Val Ser Ala Leu Tyr Arg Cys Met Ala Thr Asn Arg Ala Gly Ser515 520 525Ser Glu Arg Val Ile Ser Phe His Val Thr Arg Gly Leu Glu Ile Asn530 535 540Leu Gln Pro Arg Ser Gln Leu Thr Glu Lys Asp Asn Thr Ser Leu Gln545 550 555 560Cys Thr Ala Asp Lys Phe Thr Phe Glu Lys Leu Ser Trp Tyr Lys Leu565 570 575Ser Thr His Val Ser Gln Thr Pro Phe Gly Gly Leu Pro Met Pro Val580 585 590Cys Lys Asn Leu Asp Ala Leu Gln Lys Leu Asn Ala Thr Val Ser Asn595 600 605Val Asn Gly Glu Asn Val Thr Leu Glu Leu Ile Leu Arg Asn Ile Ser610 615 620Leu Gln Asp Gly Gly Asp Tyr Val Cys Ile Ala Gln Asp Lys Lys Ala625 630 635 640Lys Thr Gln His Cys Leu Val Lys His Leu Thr Val Gln Glu Pro Leu645 650 655His Pro Arg Leu Val Gly Asn Leu Glu Asn Gln Thr Thr Asn Ile Gly660 665 670Glu Thr Ile Glu Val Leu Cys Thr Val Asn Gly Val Pro Pro Pro Asn675 680 685Ile Thr Trp Phe Lys Asn Ser Glu Thr Leu Phe Glu Asp Ser Gly Ile690 695 700Val Leu Lys Asp Gly Asn Lys Thr Leu Thr Ile Arg Arg Val Arg Lys705 710 715 720Glu Asp Gly Gly Leu Tyr Thr Cys Leu Ala Cys Asn Ile Leu Gly Cys725 730 735Lys Lys Ala Glu Ala Phe Phe Ser Val Gln Gly Ala Glu Glu Lys Thr740 745 750Asn Leu Glu Leu Ile Ile Leu Val Gly Thr Ala Val Ile Ala Met Phe755 760 765Phe Trp Leu Leu Leu Val Ile Ile Leu Arg Thr Val Lys Arg Ala Asn770 775 780Gly Gly Asp Met Lys Thr Gly Tyr Leu Ser Ile Ile Met Asp Pro Asp785 790 795 800Glu Val Pro Ile Asp Glu His Cys Glu Arg Leu Pro Tyr Asp Ala Ser805 810 815Lys Trp Glu Phe Pro Arg Asp Arg Leu Lys Leu Gly Lys Pro Leu Gly820 825 830Arg Gly Ala Phe Gly Gln Val Ile Glu Ala Asp Ala Phe Gly Ile Asp835 840 845Lys Thr Ala Thr Cys Arg Thr Val Ala Val Lys Met Leu Lys Glu Gly850 855 860Ala Thr His Ser Glu His Arg Ala Leu Met Ser Glu Leu Lys Ile Leu865 870 875 880Ile His Ile Gly His His Leu Asn Val Val Asn Leu Leu Gly Ala Cys885 890 895Thr Lys Pro Gly Gly Pro Leu Met Val Ile Val Glu Tyr Cys Lys Phe900 905 910Gly Asn Leu Ser Ala Tyr Leu Arg Ser Lys Arg Ser Glu Phe Ile Pro915 920 925Tyr Lys Met Lys Ser Ala Arg Phe Arg Gln Gly Lys Glu Asn Tyr Thr930 935 940Gly Asp Ile Ser Thr Asp Leu Lys Gln Arg Leu Asp Ser Ile Thr Ser945 950 955 960Ser Gln Ser Ser Thr Ser Ser Gly Phe Val Glu Glu Arg Ser Leu Ser965 970 975Asp Val Glu Glu Glu Asp Ala Gly Ser Glu Asp Leu Cys Lys Asn Pro980 985 990Leu Thr Met Glu Asp Leu Ile Cys Tyr Ser Phe Gln Val Ala Arg Gly995 1000 1005Met Glu Phe Leu Ala Ser Arg Lys Cys Ile His Arg Asp Leu Ala1010 1015 1020Ala Arg Asn Ile Leu Leu Ser Asp Asn Asn Val Val Lys Ile Cys1025 1030 1035Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asp Pro Asp Tyr Val1040 1045 1050Arg Lys Gly Asp Ala Arg Leu Pro Leu Lys Trp Met Ala Pro Glu1055 1060 1065Thr Ile Phe Asp Arg Val Tyr Thr Ile Gln Ser Asp Val Trp Ser1070 1075 1080Phe Gly Val Leu Leu Trp Glu Ile Phe Ser Leu Gly Ala Ser Pro1085 1090 1095Tyr Pro Gly Val Lys Ile Asp Glu Glu Phe Cys Arg Arg Leu Lys1100 1105 1110Glu Gly Thr Arg Met Arg Ala Pro Asp Tyr Thr Thr Pro Glu Met1115 1120 1125Tyr Gln Thr Met Leu Asp Cys Trp His Gly Asp Pro Lys Gln Arg1130 1135 1140Pro Thr Phe Ser Glu Leu Val Glu His Leu Gly Asn Leu Leu Gln1145 1150 1155Ala Asn Val Arg Gln Asp Gly Lys Asp Tyr Val Val Leu Pro Leu1160 1165 1170Ser Val Ser Leu Asn Met Glu Glu Asp Ser Gly Leu Ser Leu Pro1175 1180 1185Thr Ser Pro Ala Ser Cys Lys Glu Glu Glu Glu Val Cys Asp Pro1190 1195 1200Lys Phe His Tyr Asp Asn Thr Ala Gly Ile Ser Gln Tyr Arg Gln1205 1210 1215Gly Ser Lys Arg Lys Ser Arg Pro Val Ser Val Lys Thr Phe Glu1220 1225 1230Asp Ile Pro Leu Val Thr Thr Val Lys Val Val Gln Glu Glu Asn1235 1240 1245Gln Thr Asp Ser Gly Met Val Leu Ala Ser Glu Glu Leu Lys Thr1250 1255 1260Leu Glu Glu Gln Asp Lys Gln Val Lys Ile Pro Phe Ser Thr Leu1265 1270 1275Ala Pro Ser Lys Ser Asn Glu Ser Val Met Ser Glu Ala Ser Asn1280 1285 1290Gln Thr Ser Gly Tyr Gln Ser Gly Tyr His Ser Asp Asp Met Asp1295 1300 1305Asn Met Val Cys Ser Ser Glu Asp Thr Glu Leu Leu Cys Ala Gln1310 1315 1320Glu Ala Ser Pro Thr Leu Pro Arg Cys Ala Trp Pro Gly Ile Tyr1325 1330 1335Ser Pro Ala Pro Val Ala Ser Leu Pro Leu1340 1345

Claims

1. A method for identifying a non-competitive peptide which inhibits the activity of a cytokine receptor, said method comprising selecting a candidate peptide containing from about 7 to about 20 amino acids derived from a flexible region of the receptor, and determining the ability of the candidate peptide to inhibit the oligomerization and/or activity of the receptor by measuring a biological activity of said receptor in the absence or presence of the candidate peptide, wherein a non-competitive peptide is selected when the activity of the receptor is measurably lower in the presence of the candidate peptide as compared to in the absence thereof.

2-37. (canceled)

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