Anti-RANTES Antibodies

Abstract

The invention relates to fully human monoclonal antibodies, and fragments thereof, that bind to the chemokine Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES, CCL5), thereby modulating the interaction between RANTES and one of more of its receptors, such as, e.g., CCR1, CCR3, CCR4 and CCR5, and/or modulating the biological activities of RANTES. The invention also relates to the use of these or any anti-RANTES antibodies in the prevention or treatment of immune-related disorders and in the amelioration of one or more symptoms associated with an immune-related disorder.

Description

RELATED APPLICATION

[0001] This application is a continuation of U.S. patent application Ser. No. 13/207,774, filed Aug. 11, 2011 and issued as U.S. Pat. No. 8,673,299, which is a continuation of U.S. patent application Ser. No. 12/221,485, filed on Aug. 4, 2008 and issued as U.S. Pat. No. 8,012,474, which claims the benefit of U.S. Provisional Application No. 60/963,271, filed Aug. 2, 2007, the contents of each of which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] This invention relates generally to fully human monoclonal antibodies that bind to RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted) as well as to methods for use thereof.

INCORPORATION BY REFERENCE

[0003] The contents of the text file named "414C02USSeqList.txt," which was created on Mar. 18, 2014 and is 116 KB in size, are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

[0004] RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted, CCL5) is a chemokine that is a chemoattractant for eosinophils, monocytes, and lymphocytes.

[0005] Elevated levels of RANTES expression has been implicated in a variety of diseases and disorders. Accordingly, there exists a need for therapies that target RANTES activity.

SUMMARY OF THE INVENTION

[0006] The present invention provides monoclonal antibodies, such as fully human monoclonal antibodies, that specifically bind Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES, also referred to herein as CCL5). Exemplary monoclonal antibodies include the antibodies referred to herein as 1D9, 1E4, C8, 3E7, 4D8, 5E1, 6A8, 7B5, CG11, BG11, A9, E6, H6, G2, E10, C10, 2D1, A5, H11, D1 and/or E7. Alternatively, the monoclonal antibody is an antibody that binds to the same epitope as 1D9, 1E4, C8, 3E7, 4D8, 5E1, 6A8, 7B5, CG11, BG11, A9, E6, H6, G2, E10, C10, 2D1, A5, H11, D1 and/or E7. The antibodies are respectively referred to herein as huRANTES antibodies. huRANTES antibodies include fully human monoclonal antibodies, as well as humanized monoclonal antibodies and chimeric antibodies.

[0007] huRANTES antibodies of the invention also include antibodies that include a heavy chain variable amino acid sequence that is at least 90%, 92%, 95%, 97%, 98%, 99% or more identical the amino acid sequence of SEQ ID NO: 2, 18, 22, 38, 48, 52, 56, 60, 68, 84, 100, 116, 132, 148, 164, 180, 200, 216, 232, or 248 and/or a light chain variable amino acid that is at least 90%, 92%, 95%, 97%, 98%, 99% or more identical the amino acid sequence of SEQ ID NO: 4, 24, 40, 62, 70, 86, 102, 118, 134, 150, 166, 182, 196, 202, 218, 234, or 250.

[0008] Preferably, the three heavy chain complementarity determining regions (CDRs) include an amino acid sequence at least 90%, 92%, 95%, 97%, 98%, 99% or more identical to each of: (i) a VH CDR1 sequence selected from SEQ ID NO: 8, 28, 44, 74, 90, 106, 122, 138, 154, and 222; (ii) a VH CDR2 sequence selected from SEQ ID NO: 9, 29, 45, 75, 91, 107, 123, 139, 155, 207, 223, 239, and 255; (iii) a VH CDR3 sequence selected from SEQ ID NOs: 10, 20, 30, 46, 50, 54, 58, 64, 76, 92, 108, 124, 140, 156, 169, 188, 208, 224, and 240; and a light chain with three CDR that include an amino acid sequence at least 90%, 92%, 95%, 97%, 98%, 99% or more identical to each of (iv) a VL CDR1 sequence selected from SEQ ID NO: 14, 34, 77, 96, 112, 128, 144, 160, 176, 190, 192, 212, 228, and 244; (v) a VL CDR2 sequence selected from SEQ ID NO: 15, 35, 81, 97, 113, 129, 145, 161, 177, 191, 193, 213, 229, and 245; and (vi) a VL CDR3 sequence selected from SEQ ID NO: 16, 36, 66, 82, 98, 114, 130, 146, 162, 178, 194, 214, 230, 235 and 246.

[0009] Preferably, the huRANTES antibodies are formatted in an IgG isotype. More preferably, the huRANTES antibodies are formatted in an IgG1 isotype.

[0010] Exemplary IgG1-formatted antibody are the IgG1-formatted 1D9, 1E4 and C8 antibodies comprising the heavy chain sequence and light chain sequence shown below, and the CDR sequences are shown in boxes:

TABLE-US-00001 >1D9 Heavy chain amino acid sequence (SEQ ID NO: 167) ##STR00001## >1D9 Light chain amino acid sequence (SEQ ID NO: 168) ##STR00002## >1E4 Heavy chain amino acid sequence (SEQ ID NO: 238) ##STR00003## >1E4 Light chain amino acid sequence (SEQ ID NO: 254) ##STR00004## >C8 Heavy chain amino acid sequence (SEQ ID NO: 186) ##STR00005## >C8 Light chain amino acid sequence (SEQ ID NO: 187) ##STR00006##

[0011] The closest germline for the huRANTES antibodies described herein are shown below in Table 1:

TABLE-US-00002 TABLE 1 Closest germlines for the huRANTES antibodies. Antibodies marked in italic were derived by an affinity maturation process from antibody 2D1 (Lower part of the table). Clone ID VH dp number VL dp number CG11 Vh1_DP-3_(1-f) Vlambda1_DPL8_(1e) BG11 Vh1_DP-5_(1-24) Vlambda3_DPL16_(31 A9 Vh3_DP-47_(3-23) Vlambda6_6a E6 Vh1_DP-5_(1-24) Vlambda6_6a H6 Vh1_DP-5_(1-24) Vlambda1_DPL8_(1e) G2 Vh1_DP-5_(1-24) Vlambda2_DPL11_(2a2) E10 Vh3_DP-46_(3-30.3) Vlambda3_3h C10 Vh3_DP-47_(3-23) Vlambda3_3h 2D1 Vh1_DP-5_(1-24) Vlambda3_3h A5 Vh1_DP-5_(1-24) Vlambda3_3h H11 VM_DP-10_(1-69) Vlambda1_DPL8_(1e) D1 Vh1_DP-3_(1-f) Vlambda1_DPL8_(1e) E7 VM_DP-10_(1-69) Vlambda1_DPL9_(1f) C8 Vh3_DP-46_(3-30.3) Vlambda3_3h 1D9 Vh1_DP-5_(1-24) Vlambda3_3h 1E4 Vh1_DP-5_(1-24) Vlambda3_3h 3E7 Vh1_DP-5_(1-24) Vlambda3_3h 4D8 Vh1_DP-5_(1-24) Vlambda3_3h 5E1 Vh1_DP-5_(1-24) Vlambda3_3h 6A8 Vh1_DP-5_(1-24) Vlambda3_3h 7B5 Vh1_DP-5_(1-24) Vlambda3_3h

[0012] The invention also provides antibodies that bind human RANTES when human RANTES is bound to glycosaminoglycan (GAG), i.e., bind human RANTES in the context of GAG. In a preferred embodiment, these antibodies include (a) a V_H CDR1 region comprising the amino acid sequence of SEQ ID NO: 8, 28, 44, 90, 106, 122 or 154; (b) a V_H CDR2 region comprising the amino acid sequence of SEQ ID NO: 9, 29, 45, 91, 107, 123, 155, or 207; (c) a V_H CDR3 region comprising the amino acid sequence of SEQ ID NO: 10, 20, 30, 64, 92, 124, 156, 188, or 208, (d) a V_L CDR1 region comprising the amino acid sequence of SEQ ID NO: 14, 34, 96, 128, 160, 176, 192, or 212; (e) a V_L CDR2 region comprising the amino acid sequence of SEQ ID NO: 15, 35, 97, 129, 161, 177, 193, or 213; and (f) a V_L CDR3 region comprising the amino acid sequence of SEQ ID NO: 16, 36, 98, 130, 162, 178, 194, or 214.

[0013] In some embodiments, the antibody is a monoclonal antibody or an antigen-binding fragment thereof. In some embodiments, the antibody is a fully human monoclonal antibody or an antigen-binding fragment thereof. In some embodiments, the antibody is an IgG isotype, such as, for example, an IgG1 isotype.

[0014] The invention also provides antagonist molecules of human RANTES, and in particular, antagonists of human RANTES proteins, polypeptides and/or peptides that include at least amino acid residues 16-18 of the mature amino acid sequence of human RANTES, e.g., SEQ ID NO: 170 shown in FIG. 6. The anti-human RANTES antagonists bind to, or otherwise interact with, a human RANTES protein, polypeptide, and/or peptide to modulate, e.g., reduce, inhibit or otherwise interfere, partially or completely with a biological function of a human RANTES protein, such as for example, the binding of RANTES to a receptor such as CCR1, CCR3, CCR4 and/or CCR5, or the binding of RANTES to glycosaminoglycans (GAG).

[0015] In a preferred embodiment, the ability of the anti-human RANTES antagonists to bind to, or otherwise interact with, human RANTES protein to modulate one or more biological functions of human RANTES is dependent upon the presence of amino acid residues 16-18 of the mature human RANTES sequence such as SEQ ID NO: 170. In this embodiment, the antagonist molecules do not bind a human RANTES polypeptide that lacks amino acid residues 16-18 of SEQ ID NO: 170.

[0016] The anti-RANTES antagonist molecules provided herein completely or partially reduce or otherwise modulate RANTES expression or activity upon binding to, or otherwise interacting with, human RANTES. The reduction or modulation of a biological function of RANTES is complete or partial upon interaction between the antagonist and the human RANTES protein, polypeptide and/or peptide. The anti-huRANTES antagonists are considered to completely inhibit RANTES expression or activity when the level of RANTES expression or activity in the presence of the anti-huRANTES antagonist is decreased by at least 95%, e.g., by 96%, 97%, 98%, 99% or 100% as compared to the level of RANTES expression or activity in the absence of interaction, e.g., binding with an anti-huRANTES antagonist described herein. The anti-huRANTES antagonists are considered to partially inhibit RANTES expression or activity when the level of RANTES expression or activity in the presence of the anti-huRANTES antagonist is decreased by less than 95%, e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90% as compared to the level of RANTES expression or activity in the absence of interaction, e.g., binding with an anti-huRANTES antagonist described herein.

[0017] In some embodiments, the anti-RANTES antagonist molecule is selected from a small molecule inhibitor; a polypeptide, a peptide, a RANTES-derived mutant polypeptide, a RANTES-derived polypeptide variant, a RANTES receptor-derived mutant polypeptide, e.g., a mutated CCR1, CCR3, CCR4 or CCR5 protein, polypeptide or peptide, a RANTES receptor-derived polypeptide variant, e.g., a CCR1, CCR3, CCR4 or CCR5 variant peptide, polypeptide or protein, and a nucleic acid-based antagonist.

[0018] In some embodiments, the anti-RANTES antagonist molecule is an isolated monoclonal anti-human RANTES antibody or antigen-binding fragment thereof. Preferably, the antibody (or antigen-binding fragment thereof) binds to amino acid residues 16-18 of the mature amino acid sequence of human RANTES, e.g., SEQ ID NO: 170 shown in FIG. 6. In some embodiments, the anti-RANTES antibody is a fully human monoclonal anti-human RANTES antibody or antigen-binding fragment thereof. In some embodiments, the antibody is an IgG isotype, such as an IgG1 isotype.

[0019] In some embodiments, the anti-RANTES antagonist molecule is a mutated RANTES polypeptide or RANTES-derived variant polypeptide or a mutated RANTES receptor, for example, selected from CCR1, CCR3, CCR4, and CCR5, or a variant of a RANTES receptor polypeptide, such as CCR1, CCR3, CCR4, or CCR5, that modulates an activity of RANTES selected from the ability of RANTES to bind to a receptor selected from CCR1, CCR3, CCR4, and CCR5, the ability of RANTES to bind a glycosaminoglycan and the ability of RANTES to form oligomers.

[0020] In some embodiments, the anti-RANTES antagonist molecule is a nucleic acid-based antagonist such as, for example, an aptamer or other oligonucleotide capable of interacting with targets, such as proteins, polypeptides, small molecules, carbohydrates, peptides or any other biological molecules, through interactions other than Watson-Crick base pairing.

[0021] The invention also provides methods of treating, preventing, alleviating a symptom of, or otherwise mitigating ischemia, a clinical indication associated with ischemia and/or reperfusion injury in a subject. The invention is based on the discovery that modulation, particularly, inhibition or other reduction of RANTES expression or activity inhibits ischemia and/or reperfusion injury in an animal model for ischemia and reperfusion. Accordingly, the invention provides methods of preventing or inhibiting ischemia, a clinical indication associated with ischemia, reperfusion injury, in a subject, in a bodily tissue and/or in a tissue or organ to be transplanted. In the methods provided herein, the subject to be treated is administered an antagonist of RANTES. Likewise, in the treatment of organs to be transplanted, the organ, or a portion thereof, is contacted with an antagonist of RANTES. The methods provided herein are useful in vivo and ex vivo.

[0022] Suitable antagonists of RANTES include any antibody or fragment thereof that inhibits, neutralizes or otherwise interferes with the expression and/or activity of RANTES, such as, e.g., the huRANTES antibodies provided herein; small molecule inhibitors; proteins, polypeptides, peptides; protein-, polypeptide- and/or peptide-based antagonists such as RANTES mutants and/or other RANTES variants and or RANTES receptor-based mutants and/or variants, such as, for example, mutated or variant versions of CCR1, CCR3, CCR4 or CCR5 polypeptides; nucleic acid based antagonists such as siRNA and/or anti-sense RNA, and/or aptamers; and/or fragments thereof that inhibit, neutralize or otherwise interfere with the expression and/or activity of RANTES.

[0023] Examples of polypeptide-based antagonists of RANTES include modified variants of RANTES that inhibit, neutralize or otherwise interfere with the expression and/or activity of RANTES. Variants of RANTES that are known to antagonize RANTES, for example, by decreasing the ability of RANTES to bind to glycosaminoglycans (GAG), include the RANTES mutants and variants described in PCT Publication Nos. WO 2004/062688; WO 2003/0844562; WO 2003/051921; WO 2002/028419; WO 2000/016796 and WO 1996/017935, each of which is hereby incorporated by reference in its entirety.

[0024] Examples of nucleic acid-based antagonists of RANTES include short interfering RNA (siRNA) mediated gene silencing where expression products of a RANTES gene are targeted by specific double stranded RANTES derived siRNA nucleotide sequences that are complementary to a segment of the RANTES gene transcript, e.g., at least 19-25 nucleotides long, including the 5' untranslated (UT) region, the ORF, or the 3' UT region. See, e.g., PCT applications WO00/44895, WO99/32619, WO01/75164, WO01/92513, WO 01/29058, WO01/89304, WO02/16620, and WO02/29858, each incorporated by reference herein in their entirety. Nucleic-acid based antagonists of RANTES also include antisense nucleic acids. An antisense nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a RANTES protein or fragment thereof. For example, antisense RANTES antagonists comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire RANTES coding strand, or to only a portion thereof.

[0025] Preferably, the RANTES antagonist inhibits, partially or completely, a function of RANTES selected from the ability of RANTES to bind to a corresponding receptor (e.g., CCR1, CCR3, CCR4, and/or CCR5), the ability of RANTES to bind glycosaminoglycans and/or the ability of RANTES to form oligomers. Suitable RANTES antagonists are identified, for example, using the assays and models provided in the Examples below.

[0026] The anti-huRANTES antagonists are considered to completely inhibit RANTES expression or activity when the level of RANTES expression or activity in the presence of the anti-huRANTES antagonist is decreased by at least 95%, e.g., by 96%, 97%, 98%, 99% or 100% as compared to the level of RANTES expression or activity in the absence of interaction, e.g., binding with an anti-huRANTES antagonist described herein. The anti-huRANTES antagonists are considered to partially inhibit RANTES expression or activity when the level of RANTES expression or activity in the presence of the anti-huRANTES antagonist is decreased by less than 95%, e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90% as compared to the level of RANTES expression or activity in the absence of interaction, e.g., binding with an anti-huRANTES antagonist described herein.

[0027] In one aspect, the invention provides methods of treating, preventing or alleviating a symptom of ischemia or a clinical indication associated with ischemia by administering a RANTES antagonist, such as a huRANTES antibody, to a subject in need thereof or by contacting an organ in need thereof with a RANTES antagonist, such as a huRANTES antibody. The ischemia to be treated includes cardiac ischemia, cerebral ischemia, renal ischemia, and related ischemic diseases or events. Clinical indications associated with ischemia and reperfusion include, for example, coronary artery disease, cerebral vascular disease, cardiac ischemia, myocardial ischemia, renal ischemia and peripheral vascular disease. Ischemia is a feature of heart diseases including atherosclerosis, myocardial infarction, transient ischemic attacks, cerebrovascular accidents, ruptured arteriovenous malformations, and peripheral artery occlusive disease. The heart, the kidneys, and the brain are among the organs that are the most sensitive to inadequate blood supply. Ischemia in brain tissue is due, for example, to stroke or head injury. Use of a RANTES antagonist, such as a huRANTES antibody, is also envisioned as part of a protocol for optimizing tissue health during extra-corporeal perfusion of organs and/or tissue prior to transplantation, including, for example, heart, lung, and kidney. The organs to be treated using the methods provided herein are contacted in vivo or ex vivo.

[0028] The antibodies and compositions provided herein are useful in treating, preventing or otherwise delaying the progression of tissue injury or other damage caused by ischemia or a clinical indication associated with ischemia. For example, a huRANTES antibody or other RANTES antagonist of the invention is administered to a subject in need thereof before an ischemic event, during an ischemic event, after an ischemic event or any combination thereof.

[0029] The antibodies, RANTES antagonists and compositions provided herein are also useful in methods of treating, preventing or alleviating a symptom of a reperfusion injury or other tissue damage that occurs in a subject when blood supply returns to a tissue site after a period of ischemia. For example, a RANTES antagonist, such as a huRANTES antibody of the invention, is administered to a subject in need thereof, e.g., during an ischemic event, after an ischemic event or both during and after an ischemic event. In some cases, restoration of blood flow after a period of ischemia can be more damaging than the ischemia. Reintroduction of oxygen causes a greater production of damaging free radicals, resulting in reperfusion injury. With reperfusion injury, tissue damage and/or necrosis can be greatly accelerated. Reperfusion injuries to be treated or prevented include injuries caused by an inflammatory response in the damaged tissue or tissues.

[0030] The subject or organ to be transplanted is suffering from or is predisposed to developing ischemia, an ischemic-related disorder, and/or reperfusion related tissue damage. Preferably, the subject is a mammal, and more preferably, the subject is a human.

[0031] In another aspect, the invention provides methods of treating, preventing or alleviating a symptom of an immune-related disorder by administering a huRANTES antibody to a subject. For example, the huRANTES antibodies are used to treat, prevent or alleviate a symptom associated with an autoimmune disease or inflammatory disorder. Optionally, the subject is further administered with a second agent such as, but not limited to, an anti-cytokine reagent, anti-chemokine reagent, an anti-cytokine reagent or an anti-chemokine receptor that recognizes the ligand or receptor for proteins such as interleukin 1 (IL-1), IL-2, IL-4, IL-6, IL-12, IL-13, IL-15, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-27, IL-31, MIP1 alpha, MIP1 beta, IP-10, MCP1, ITAC, MIG, SDF and fractalkine.

[0032] The subject is suffering from or is predisposed to developing an immune related disorder, such as, for example, an autoimmune disease or an inflammatory disorder. Preferably, the subject is a mammal, and more preferably, the subject is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 is a series of graphs depicting the activity of anti-huRANTES antibodies in chemotaxis assays using L1.2 cells transfected with hCCR5 and 1 nM or 0.2 nM of recombinant human RANTES (FIGS. 1A and B respectively) as well as native human RANTES (FIG. 1C).

[0034] FIG. 2 is a graph depicting the capacity of anti-huRANTES antibodies to bind to huRANTES in the context of glycosaminoglycans in and ELISA assay.

[0035] FIG. 3 is a series of graphs depicting the activity of anti-huRANTES antibody 1E4 in calcium flux assays using: L1.2 cells expressing hCCR1 and 25 nM recombinant human RANTES (FIG. 3A); L1.2 cells expressing hCCR3 and 25 nM recombinant human RANTES (FIG. 3B); L1.2 cells expressing hCCR5 and 4 nM recombinant human RANTES (FIG. 3C).

[0036] FIG. 4 is a series of graphs depicting the activity of anti-huRANTES antibody 1E4 in chemotaxis assays using: L1.2 cells expressing hCCR1 and 2 nM of recombinant human RANTES (FIG. 4A); L1.2 cells expressing hCCR3 and 10 nM of recombinant human RANTES (FIG. 4B); L1.2 cells expressing hCCR5 and 1 nM of recombinant human RANTES (FIG. 4C); L1.2 cells expressing hCCR5 and about 1 nM of native human RANTES (FIG. 4D).

[0037] FIG. 5 is a graph depicting the cross-reactivity profile of antibody 1E4 against a panel of human, cynomolgus, mouse and rat chemokines in an ELISA.

[0038] FIG. 6 is a sequence alignment of mature RANTES protein from human (SEQ ID NO: 170), cynomolgus monkey (SEQ ID NO: 171), mouse (SEQ ID NO: 172) and rat (SEQ ID NO: 206). The arrows indicate positions that are conserved in human and cynomolgus RANTES but not in the mouse or rat sequences and that were targeted by site-directed mutagenesis.

[0039] FIG. 7 is a graph depicting the binding of antibody 1E4 (open bars) or of a polyclonal antibody raised against mouse RANTES (hatched bars) to human RANTES, mouse RANTES and variants of mouse RANTES in which the indicated mouse amino acids have been replaced by the amino acids found in the human sequence at the same position.

[0040] FIG. 8 is an illustration depicting the protocol of a murine ischemia reperfusion model provided herein.

[0041] FIG. 9 is a series of graphs depicting that anti-RANTES treatment decreased infarct size in a murine model of ischemia reperfusion. The data represents 20 mice per group.

[0042] FIG. 10 is a series of graphs depicting that anti-RANTES treatment decreased infarct size in a murine model of ischemia reperfusion in a dose-dependent manner. Data represents 3 mice per group.

[0043] FIG. 11 is an illustration depicting the protocol of a murine ischemia model provided herein.

[0044] FIG. 12 is a series of graphs depicting that anti-RANTES treatment decreased infarct size in a murine model of ischemia. The data represents 10 mice per group.

[0045] FIG. 13 is a series of graphs depicting that anti-RANTES treatment decreased infarct size in a murine model of ischemia in a dose-dependent manner. Data represents 3 mice per group.

DETAILED DESCRIPTION

[0046] The present invention provides fully human monoclonal antibodies specific for the chemokine Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES, CCL5). The terms "RANTES" and "CCL5" are used interchangeably herein. The antibodies are collectively referred to herein as huRANTES antibodies. The huRANTES antibodies specifically bind RANTES. As used herein, the terms "specific for", "specific binding", "directed against" (and all grammatical variations thereof) are used interchangeably in the context of antibodies that recognize and bind to a RANTES epitope when the equilibrium binding constant (K_d) is ≦1 μM, e.g., ≦100 nM, preferably ≦10 nM, and more preferably ≦1 nM. For example, the huRANTES antibodies provided herein exhibit a K_d in the range approximately between ≦10 nM to about 100 μM.

[0047] The huRANTES antibodies are, for example, RANTES antagonists or inhibitors that modulate at least one biological activity of RANTES. Biological activities of RANTES include, for example, binding a RANTES receptor such as, for example, CCR1, CCR3, CCR4, and/or CCR5; chemoattraction of eosinophils, monocytes, and lymphocytes; binding of RANTES to glycosaminoglycans as well as RANTES oligomerization. For example, the huRANTES antibodies completely or partially inhibit RANTES activity by partially or completely blocking the binding of RANTES to a RANTES receptor (e.g., CCR1, CCR3, CCR4, and/or CCR5). The RANTES antibodies are considered to completely inhibit RANTES activity when the level of RANTES activity in the presence of the huRANTES antibody is decreased by at least 95%, e.g., by 96%, 97%, 98%, 99% or 100% as compared to the level of RANTES activity in the absence of binding with a huRANTES antibody described herein. The RANTES antibodies are considered to partially inhibit RANTES activity when the level of RANTES activity in the presence of the huRANTES antibody is decreased by less than 95%, e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90% as compared to the level of RANTES activity in the absence of binding with a huRANTES antibody described herein.

[0048] The huRANTES antibodies of the invention are produced by immunizing an animal with RANTES, such as, for example, murine or human RANTES or an immunogenic fragment, derivative or variant thereof. Alternatively, the animal is immunized with cells transfected with a vector containing a nucleic acid molecule encoding RANTES, such that RANTES is expressed and associated with the surface of the transfected cells. Alternatively, the antibodies are obtained by screening a library that contains antibody or antigen binding domain sequences for binding to RANTES. This library is prepared, e.g., in bacteriophage as protein or peptide fusions to a bacteriophage coat protein that is expressed on the surface of assembled phage particles and the encoding DNA sequences contained within the phage particles (i.e., "phage displayed library").

[0049] huRANTES antibodies of the invention include, for example, the heavy chain complementarity determining regions (CDRs) shown below in Table 2, the light chain CDRs shown in Table 3, and combinations thereof.

TABLE-US-00003 TABLE 2 VH CDR sequences from antibody clones that bind and neutralize RANTES. Clone ID Heavy CDR1 Heavy CDR2 Heavy CDR3 CG11 DYYIH LIDPKDGEIQYAEKFQA EVLSGIRVFPFDP (SEQ NO: 74) (SEQ NO: 75) (SEQ NO: 76) BG11 ELSMH GFDPEDGETIYAQKFQG YSGSSGWWAFDI (SEQ NO: 90) (SEQ NO: 91) (SEQ NO: 92) A9 SYAMS AISGSGGSTYYADSVKG DLGYCTNGVCWGIDY (SEQ NO: 106) (SEQ NO: 107) (SEQ NO: 108) E6 EIAIH SFEPEDAEAIYAQRFQG DPYYASSGSNYMEV (SEQ NO: 122) (SEQ NO: 123) (SEQ NO: 124) H6 KQSMH SSNPEDDETLYAKKFQG DSQGFYYYYGMDV (SEQ NO: 138) (SEQ NO: 139) (SEQ NO: 140) G2 ELSIH GFDPEDGETIYAQNFQG DLTGSRDS (SEQ NO: 154) (SEQ NO: 155) (SEQ NO: 156) E10 SYAMH VISYDGSNKYYADSVKG ETFPHYYYYYMDV (SEQ NO: 28) (SEQ NO: 29) (SEQ NO: 30) C10 SYAMS AISGSGGSTYYADSVKG VRGSSQYDFWSGS (SEQ NO: 106) (SEQ NO: 107) EFDY (SEQ NO: 188) 2D1 DFAMH GYVPEDGDTIYAQKFQG DPLYSGSLSY (SEQ NO: 44) (SEQ NO: 45) (SEQ NO: 64) A5 ELSIH YIDPEDGEPIYAQKFQG VTGSTSDAFDL (SEQ NO: 154) (SEQ NO: 207) (SEQ NO: 208) H11 NYALS GFIPLVDTTNYAQRFQG EQVAVGPGPTSDRG (SEQ NO: 222) (SEQ NO: 223) PDGLDV (SEQ NO: 224) D1 DYYIH LVDSEEDGETLFAETFRG EYGEYGFFQS (SEQ NO: 74) (SEQ NO: 239) (SEQ NO: 240) E7 NYALS AVIPLVETTSYAQRFQG EQVAVGPGPTSNRG (SEQ NO: 222) (SEQ NO: 255) PDGLDV (SEQ NO: 169) C8 SYAMH VISYDGSNKYYADSVKG ETFPHYYYYYMDV (SEQ NO: 28) (SEQ NO: 29) (SEQ NO: 30) 1D9 EFAMH GFVPEDGETIYAQKFQG DPLYPPGLSP (SEQ NO: 8) (SEQ NO: 9) (SEQ NO: 10) 1E4 EFAMH GFVPEDGETIYAQKFQG DPLYEGSFSV (SEQ NO: 8) (SEQ NO: 9) (SEQ NO: 20) 3E7 DFAMH GYVPEDGDTIYAQKFQG DPLYPPGLSP (SEQ NO: 44) (SEQ NO: 45) (SEQ NO: 46) 4D8 DFAMH GYVPEDGDTIYAQKFQG DPLYTPGLYV (SEQ NO: 44) (SEQ NO: 45) (SEQ NO: 50) 5E1 DFAMH GYVPEDGDTIYAQKFQG DYLYIPSLSY (SEQ NO: 44) (SEQ NO: 45) (SEQ NO: 54) 6A8 DFAMH GYVPEDGDTIYAQKFQG DPLYPPGLQP (SEQ NO: 44) (SEQ NO: 45) (SEQ NO: 58) 7B5 DFAMH GYVPEDGDTIYAQKFQG DPLYSGSLSY (SEQ NO: 44) (SEQ NO: 45) (SEQ NO: 64) Antibodies marked in italic were derived by an affinity maturation process from antibody 2D1 (Lower part of the table).

TABLE-US-00004 TABLE 3 VL CDR sequences from antibody clones that bind and neutralize RANTES. Clone ID Light CDR1 Light CDR2 Light CDR3 CG11 TGSSSNIGAGYDVY DTNNRPP QSYDIALSNSNVV (SEQ NO: 77) (SEQ NO: 81) (SEQ NO: 82) BG11 QGDSLRSYYAS GKNNRPS QTWGTGIWV (SEQ NO: 96) (SEQ NO: 97) (SEQ NO: 98) A9 TRSSGSIADNYVQ DDDQRLS QSYDDSNDV (SEQ NO: 112) (SEQ NO: 113) (SEQ NO: 114) E6 TGSGGSISSNYVQ EDDQRPS HSYDGNNRWV (SEQ NO: 128) (SEQ NO: 129) (SEQ NO: 130) H6 TGSSSNIGADYDVH DNINRPS QSYDSSLSGVL (SEQ NO: 144) (SEQ NO: 145) (SEQ NO: 146) G2 TGSRSDIGYYNYVS DVTERPS SSFSSGDTFVV (SEQ NO: 160) (SEQ NO: 161) (SEQ NO: 162) E10 GGGNFDDEGVH DDTGRPS QAWDSSNDHPV (SEQ NO: 176) (SEQ NO: 177) (SEQ NO: 178) C10 GGDNIGGQNVH YDTDRPS QVWDVDSDHPWV (SEQ NO: 192) (SEQ NO: 193) (SEQ NO: 194) 2D1 GGNNIESKSVH DDSDRPS QVWDSNTDHWV (SEQ NO: 14) (SEQ NO: 15) (SEQ NO: 16) A5 GGANLWGLGVH DNSDRAS QVWDSSSDHWV (SEQ NO: 212) (SEQ NO: 213) (SEQ NO: 214) H11 TGSNSNLGADYDVH DNNIRPS QSYDTGLTSSDVI (SEQ NO: 228) (SEQ NO: 229) (SEQ NO: 230) D1 TGSSSNIGADYDVN GDINRPS QSFDNSLSGSVI (SEQ NO: 244) (SEQ NO: 245) (SEQ NO: 246) E7 TGSSSNIGDGYDVH GNSNRPS GTWDDILNGWV (SEQ NO: 190) (SEQ NO: 191) (SEQ NO: 235) C8 EGDDTDIGTVN EDGYRPS QFWDVDSDHPV (SEQ NO: 34) (SEQ NO: 35) (SEQ NO: 36) 1D9 GGNNIESKSVH DDSDRPS QVWDSNTDHWV (SEQ NO: 14) (SEQ NO: 15) (SEQ NO: 16) 1E4 GGNNIESKSVH DDSDRPS QVWDSNTDHWV (SEQ NO: 14) (SEQ NO: 15) (SEQ NO: 16) 3E7 GGNNIESKSVH DDSDRPS QVWDSNTDHWV (SEQ NO: 14) (SEQ NO: 15) (SEQ NO: 16) 4D8 GGNNIESKSVH DDSDRPS QVWDSNTDHWV (SEQ NO: 14) (SEQ NO: 15) (SEQ NO: 16) 5E1 GGNNIESKSVH DDSDRPS QVWDSNTDHWV (SEQ NO: 14) (SEQ NO: 15) (SEQ NO: 16) 6A8 GGNNIESKSVH DDSDRPS QVWDSNTDHWV (SEQ NO: 14) (SEQ NO: 15) (SEQ NO: 16) 7B5 GGNNIESKSVH DDSDRPS QVWDSGPVWWI (SEQ NO: 14) (SEQ NO: 15) (SEQ NO: 66) Antibodies marked in italic were derived by an affinity maturation process from antibody 2D1 (Lower part of the table).

[0050] An exemplary huRANTES monoclonal antibody is the 1D9 antibody described herein. As shown below, the 1D9 antibody includes a heavy chain variable region (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1, and a light chain variable region (SEQ ID NO:4) encoded by the nucleic acid sequence shown in SEQ ID NO:3. The CDR sequences are shown in boxes.

TABLE-US-00005 >1D9 Heavy chain variable domain nucleic acid sequence (SEQ ID NO: 1): ##STR00007## >1D9 Heavy chain variable domain amino acid sequence (SEQ ID NO: 2) ##STR00008## >D9 Light chain variable domain nucleic acid sequence (SEQ ID NO: 3): ##STR00009## >1D9 Light chain variable domain amino acid sequence (SEQ ID NO: 4) ##STR00010##

[0051] The amino acids encompassing the complementarity determining regions (CDR) are as defined by Chothia et al. and E. A. Kabat et al. (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)). The heavy chain CDRs of the 1D9 antibody have the following sequences: EFAMH (SEQ ID NO:8), encoded by the nucleic acid sequence GAGTTCGCCATGCAC (SEQ ID NO: 5); GFVPEDGETIYAQKFQG (SEQ ID NO:9), encoded by the nucleic acid sequence GGTTTTGTTCCTGAAGATGGTGAGACAATCTACGCGCAGAAGTTCCAGGGC (SEQ ID NO: 6); and DPLYTPGLEP (SEQ ID NO:10), encoded by the nucleic acid sequence GATCCCCTGTATACTCCGGGTCTTGAGCCT (SEQ ID NO: 7). The light chain CDRs of the 1D9 antibody have the following sequences: GGNNIESKSVH (SEQ ID NO:14), encoded by the nucleic acid sequence GGGGGAAACAACATTGAAAGTAAAAGTGTGCAC (SEQ ID NO:11); DDSDRPS (SEQ ID NO:15), encoded by the nucleic acid sequence GATGATAGCGACCGGCCCTCA (SEQ ID NO: 12); and QVWDSNTDHWV (SEQ ID NO:16), encoded by the nucleic acid sequence CAGGTGTGGGATAGTAATACTGATCATTGGGTG (SEQ ID NO: 13).

[0052] An exemplary huRANTES monoclonal antibody is the 1E4 antibody described herein. As shown below, the 1E4 antibody includes a heavy chain variable region (SEQ ID NO:18) encoded by the nucleic acid sequence shown in SEQ ID NO:17, and a light chain variable region (SEQ ID NO: 4) encoded by the nucleic acid sequence shown in SEQ ID NO:3. The CDR sequences are shown in boxes.

TABLE-US-00006 >1E4 Heavy Chain variable domain nucleic acid sequence (SEQ ID NO: 17): ##STR00011## >1E4 Heavy chain variable domain amino acid sequence (SEQ ID NO: 18) ##STR00012## >1E4 Light chain nucleic acid sequence (SEQ ID NO: 3): ##STR00013## >1E4 Light chain variable domain amino acid sequence (SEQ ID NO: 4) ##STR00014##

[0053] The amino acids encompassing the complementarity determining regions (CDR) are as defined by Chothia et al. and E. A. Kabat et al. (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)). The heavy chain CDRs of the 1E4 antibody have the following sequences: EFAMH (SEQ ID NO:8), encoded by the nucleic acid sequence GAGTTCGCCATGCAC (SEQ ID NO: 5); GFVPEDGETIYAQKFQG (SEQ ID NO:9), encoded by the nucleic acid sequence GGTTTTGTTCCTGAAGATGGTGAGACAATCTACGCGCAGAAGTTCCAGGGC (SEQ ID NO: 6); and DPLYEGSFSV (SEQ ID NO:20), encoded by the nucleic acid sequence GATCCCCTGTATGAGGGTCCGTTTTCTGTT (SEQ ID NO: 19). The light chain CDRs of the 1E4 antibody have the following sequences: GGNNIESKSVH (SEQ ID NO:14), encoded by the nucleic acid sequence GGGGGAAACAACATTGAAAGTAAAAGTGTGCAC (SEQ ID NO:11); DDSDRPS (SEQ ID NO:15), encoded by the nucleic acid sequence GATGATAGCGACCGGCCCTCA (SEQ ID NO: 12); and QVWDSNTDHWV (SEQ ID NO:16), encoded by the nucleic acid sequence CAGGTGTGGGATAGTAATACTGATCATTGGGTG (SEQ ID NO: 13).

[0054] An exemplary huRANTES monoclonal antibody is the C8 antibody described herein. As shown below, the C8 antibody includes a heavy chain variable region (SEQ ID NO:22) encoded by the nucleic acid sequence shown in SEQ ID NO: 21, and a light chain variable region (SEQ ID NO:24) encoded by the nucleic acid sequence shown in SEQ ID NO: 23. The CDR sequences are shown in boxes.

TABLE-US-00007 >C8 Heavy chain variable domain nucleic acid sequence (SEQ ID NO: 21) ##STR00015## >C8 Heavy chain variable domain amino acid sequence (SEQ ID NO: 22) ##STR00016## >C8 Light chain variable domain nucleic acid sequence (SEQ ID NO: 23): ##STR00017## >C8 Light chain variable domain amino acid sequence (SEQ ID NO: 24) ##STR00018##

[0055] The amino acids encompassing the complementarity determining regions (CDR) are as defined by Chothia et al. and E. A. Kabat et al. (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)). The heavy chain CDRs of the C8 antibody have the following sequences: SYAMH (SEQ ID NO:28), encoded by the nucleic acid sequence AGCTATGCTATGCAC (SEQ ID NO: 25); VISYDGSNKYYADSVKG (SEQ ID NO:29), encoded by the nucleic acid sequence GTTATATCATATGATGGAAGTAATAAATACTACGCAGACTCCGTGAAGGGC (SEQ ID NO: 26); and ETFPHYYYYYMDV (SEQ ID NO:30), encoded by the nucleic acid sequence GAAACTTTCCCCCACTACTACTACTACTACATGGACGTC (SEQ ID NO: 27). The light chain CDRs of the C8 antibody have the following sequences: EGDDTDIGTVN (SEQ ID NO:34), encoded by the nucleic acid sequence GAGGGAGACGACACTGACATTGGTACTGTCAAC (SEQ ID NO:31); EDGYRPS (SEQ ID NO:35), encoded by the nucleic acid sequence GAGGATGGCTACCGGCCCTCA (SEQ ID NO: 32); and QFWDVDSDHPV (SEQ ID NO:36), encoded by the nucleic acid sequence CAGTTCTGGGATGTTGACAGTGATCATCCGGTT (SEQ ID NO: 33).

[0056] An exemplary huRANTES monoclonal antibody is the 3E7 antibody described herein. As shown below, the 3E7 antibody includes a heavy chain variable region (SEQ ID NO:38) encoded by the nucleic acid sequence shown in SEQ ID NO: 37, and a light chain variable region (SEQ ID NO:40) encoded by the nucleic acid sequence shown in SEQ ID NO: 39. The CDR sequences are shown in boxes.

TABLE-US-00008 >3E7 Heavy chain variable domain nucleic acid sequence (SEQ ID NO: 37) ##STR00019## >3E7 Heavy chain variable domain amino acid sequence (SEQ ID NO: 38) ##STR00020## >3E7 Light chain variable domain nucleic acid sequence (SEQ ID NO: 39): ##STR00021## >3E7 Light chain variable domain amino acid sequence (SEQ ID NO: 40) ##STR00022##

[0057] The amino acids encompassing the complementarity determining regions (CDR) are as defined by Chothia et al. and E. A. Kabat et al. (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)). The heavy chain CDRs of the 3E7 antibody have the following sequences: DFAMH (SEQ ID NO:44), encoded by the nucleic acid sequence GACTTCGCCATGCAC (SEQ ID NO: 41); GYVPEDGDTIYAQKFQG (SEQ ID NO:45), encoded by the nucleic acid sequence GGTTATGTTCCTGAAGATGGTGACACAATCTACGCGCAGAAGTTCCAGGGC (SEQ ID NO: 42); and DPLYPPGLSP (SEQ ID NO:46), encoded by the nucleic acid sequence GATCCCCTGTATCCGCCTGGGCTGTCTCCT (SEQ ID NO: 43). The light chain CDRs of the 3E7 antibody have the following sequences: GGNNIESKSVH (SEQ ID NO:14), encoded by the nucleic acid sequence GGGGGAAACAACATTGAAAGTAAAAGTGTGCAC (SEQ ID NO:11); DDSDRPS (SEQ ID NO:15), encoded by the nucleic acid sequence GATGATAGCGACCGGCCCTCA (SEQ ID NO: 12); and QVWDSNTDHWV (SEQ ID NO:16), encoded by the nucleic acid sequence CAGGTGTGGGATAGTAATACTGATCATTGGGTG (SEQ ID NO: 13).

[0058] An exemplary huRANTES monoclonal antibody is the 4D8 antibody described herein. As shown below, the 4D8 antibody includes a heavy chain variable region (SEQ ID NO:48) encoded by the nucleic acid sequence shown in SEQ ID NO: 47, and a light chain variable region (SEQ ID NO:40) encoded by the nucleic acid sequence shown in SEQ ID NO: 39. The CDR sequences are shown in boxes.

TABLE-US-00009 >4D8 Heavy chain variable domain nucleic acid sequence (SEQ ID NO: 47) ##STR00023## >4D8 Heavy chain variable domain amino acid sequence (SEQ ID NO: 48) ##STR00024## >4D8 Light chain variable domain nucleic acid sequence (SEQ ID NO: 39): ##STR00025## >4D8 Light chain variable domain amino acid sequence (SEQ ID NO: 40) ##STR00026##

[0059] The amino acids encompassing the complementarity determining regions (CDR) are as defined by Chothia et al. and E. A. Kabat et al. (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)). The heavy chain CDRs of the 4D8 antibody have the following sequences: DFAMH (SEQ ID NO:44), encoded by the nucleic acid sequence GACTTCGCCATGCAC (SEQ ID NO: 41); GYVPEDGDTIYAQKFQG (SEQ ID NO:45), encoded by the nucleic acid sequence GGTTATGTTCCTGAAGATGGTGACACAATCTACGCGCAGAAGTTCCAGGGC (SEQ ID NO: 42); and DPLYTPGLYV (SEQ ID NO:50), encoded by the nucleic acid sequence GATCCCCTGTATACGCCTGGTCTGTATGTG (SEQ ID NO: 49). The light chain CDRs of the 4D8 antibody have the following sequences: GGNNIESKSVH (SEQ ID NO:14), encoded by the nucleic acid sequence GGGGGAAACAACATTGAAAGTAAAAGTGTGCAC (SEQ ID NO:11); DDSDRPS (SEQ ID NO:15), encoded by the nucleic acid sequence GATGATAGCGACCGGCCCTCA (SEQ ID NO: 12); and QVWDSNTDHWV (SEQ ID NO:16), encoded by the nucleic acid sequence CAGGTGTGGGATAGTAATACTGATCATTGGGTG (SEQ ID NO: 13).

[0060] An exemplary huRANTES monoclonal antibody is the 5E1 antibody described herein. As shown below, the 5E1 antibody includes a heavy chain variable region (SEQ ID NO:52) encoded by the nucleic acid sequence shown in SEQ ID NO: 51, and a light chain variable region (SEQ ID NO:40) encoded by the nucleic acid sequence shown in SEQ ID NO: 39. The CDR sequences are shown in boxes.

TABLE-US-00010 >5E1 Heavy chain variable domain nucleic acid sequence (SEQ ID NO: 51) ##STR00027## >5E1 Heavy chain variable domain amino acid sequence (SEQ ID NO: 52) ##STR00028## >5E1 Light chain variable domain nucleic acid sequence (SEQ ID NO: 39): ##STR00029## >5E1 Light chain variable domain amino acid sequence (SEQ ID NO: 40) ##STR00030##

[0061] The amino acids encompassing the complementarity determining regions (CDR) are as defined by Chothia et al. and E. A. Kabat et al. (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)). The heavy chain CDRs of the 5E1 antibody have the following sequences: DFAMH (SEQ ID NO:44), encoded by the nucleic acid sequence GACTTCGCCATGCAC (SEQ ID NO: 41); GYVPEDGDTIYAQKFQG (SEQ ID NO:45), encoded by the nucleic acid sequence GGTTATGTTCCTGAAGATGGTGACACAATCTACGCGCAGAAGTTCCAGGGC (SEQ ID NO: 42); and DYLYIPSLSY (SEQ ID NO:54), encoded by the nucleic acid sequence GATTATTTGTATATTCCTAGCTTATCCTAC (SEQ ID NO: 53). The light chain CDRs of the 5E1 antibody have the following sequences: GGNNIESKSVH (SEQ ID NO:14), encoded by the nucleic acid sequence GGGGGAAACAACATTGAAAGTAAAAGTGTGCAC (SEQ ID NO:11); DDSDRPS (SEQ ID NO:15), encoded by the nucleic acid sequence GATGATAGCGACCGGCCCTCA (SEQ ID NO: 12); and QVWDSNTDHWV (SEQ ID NO:16), encoded by the nucleic acid sequence CAGGTGTGGGATAGTAATACTGATCATTGGGTG (SEQ ID NO: 13).

[0062] An exemplary huRANTES monoclonal antibody is the 6A8 antibody described herein. As shown below, the 6A8 antibody includes a heavy chain variable region (SEQ ID NO:56) encoded by the nucleic acid sequence shown in SEQ ID NO: 55, and a light chain variable region (SEQ ID NO:40) encoded by the nucleic acid sequence shown in SEQ ID NO: 39. The CDR sequences are shown in boxes.

TABLE-US-00011 >6A8 Heavy chain variable domain nucleic acid sequence (SEQ ID NO: 55) ##STR00031## >6A8 Heavy chain variable domain amino acid sequence (SEQ ID NO: 56) ##STR00032## >6A8 Light chain variable domain nucleic acid sequence (SEQ ID NO: 39): ##STR00033## >6A8 Light chain variable domain amino acid sequence (SEQ ID NO: 40) ##STR00034##

[0063] The amino acids encompassing the complementarity determining regions (CDR) are as defined by Chothia et al. and E. A. Kabat et al. (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)). The heavy chain CDRs of the 6A8 antibody have the following sequences: DFAMH (SEQ ID NO:44), encoded by the nucleic acid sequence GACTTCGCCATGCAC (SEQ ID NO: 41); GYVPEDGDTIYAQKFQG (SEQ ID NO:45), encoded by the nucleic acid sequence GGTTATGTTCCTGAAGATGGTGACACAATCTACGCGCAGAAGTTCCAGGGC (SEQ ID NO: 42); and DPLYPPGLQP (SEQ ID NO:58), encoded by the nucleic acid sequence GATCCCCTGTATCCTCCGGGGCTGCAGCCT (SEQ ID NO: 57). The light chain CDRs of the 6A8 antibody have the following sequences: GGNNIESKSVH (SEQ ID NO:14), encoded by the nucleic acid sequence GGGGGAAACAACATTGAAAGTAAAAGTGTGCAC (SEQ ID NO:11); DDSDRPS (SEQ ID NO:15), encoded by the nucleic acid sequence GATGATAGCGACCGGCCCTCA (SEQ ID NO: 12); and QVWDSNTDHWV (SEQ ID NO:16), encoded by the nucleic acid sequence CAGGTGTGGGATAGTAATACTGATCATTGGGTG (SEQ ID NO: 13).

[0064] An exemplary huRANTES monoclonal antibody is the 7B5 antibody described herein. As shown below, the 7B5 antibody includes a heavy chain variable region (SEQ ID NO:60) encoded by the nucleic acid sequence shown in SEQ ID NO: 59, and a light chain variable region (SEQ ID NO:62) encoded by the nucleic acid sequence shown in SEQ ID NO: 61. The CDR sequences are shown in boxes.

TABLE-US-00012 >7B5 Heavy chain variable domain nucleic acid sequence (SEQ ID NO: 59) ##STR00035## >7B5 Heavy chain variable domain amino acid sequence (SEQ ID NO: 60) ##STR00036## >7B5 Light chain variable domain nucleic acid sequence (SEQ ID NO: 61): ##STR00037## >7B5 Light chain variable domain amino acid sequence (SEQ ID NO: 62) ##STR00038##

[0065] The amino acids encompassing the complementarity determining regions (CDR) are as defined by Chothia et al. and E. A. Kabat et al. (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)). The heavy chain CDRs of the 7B5 antibody have the following sequences: DFAMH (SEQ ID NO:44), encoded by the nucleic acid sequence GACTTCGCCATGCAC (SEQ ID NO: 41); GYVPEDGDTIYAQKFQG (SEQ ID NO:45), encoded by the nucleic acid sequence GGTTATGTTCCTGAAGATGGTGACACAATCTACGCGCAGAAGTTCCAGGGC (SEQ ID NO: 42); and DPLYSGSLSY (SEQ ID NO:64), encoded by the nucleic acid sequence GATCCCCTGTATAGTGGGAGCTTATCCTAC (SEQ ID NO: 63). The light chain CDRs of the 7B5 antibody have the following sequences: GGNNIESKSVH (SEQ ID NO:14), encoded by the nucleic acid sequence GGGGGAAACAACATTGAAAGTAAAAGTGTGCAC (SEQ ID NO:11); DDSDRPS (SEQ ID NO:15), encoded by the nucleic acid sequence GATGATAGCGACCGGCCCTCA (SEQ ID NO: 12); and QVWDSGPVWWI (SEQ ID NO:66), encoded by the nucleic acid sequence TCAGGTGTGGGATAGTGGTCCTGTGTGGTGGATT (SEQ ID NO: 65).

[0066] An exemplary huRANTES monoclonal antibody is the CG11 antibody described herein. As shown below, the CG11 antibody includes a heavy chain variable region (SEQ ID NO:68) encoded by the nucleic acid sequence shown in SEQ ID NO: 67, and a light chain variable region (SEQ ID NO:70) encoded by the nucleic acid sequence shown in SEQ ID NO: 69. The CDR sequences are shown in boxes.

TABLE-US-00013 >CG11 Heavy chain variable domain nucleic acid sequence (SEQ ID NO: 67) ##STR00039## >CG11 Heavy chain variable domain amino acid sequence (SEQ ID NO: 68) ##STR00040## >CG11 Light chain variable domain nucleic acid sequence (SEQ ID NO: 69): ##STR00041## >CG11 Light chain variable domain amino acid sequence (SEQ ID NO: 70) ##STR00042##

[0067] The amino acids encompassing the complementarity determining regions (CDR) are as defined by Chothia et al. and E. A. Kabat et al. (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)). The heavy chain CDRs of the CG11_antibody have the following sequences: DYYIH (SEQ ID NO:74), encoded by the nucleic acid sequence GACTACTACATACAC (SEQ ID NO: 71); LIDPKDGEIQYAEKFQA (SEQ ID NO:75), encoded by the nucleic acid sequence GGTTATGTTCCTGAAGATGGTGACACAATCTACGCGCAGAAGTTCCAGGGC (SEQ ID NO: 72); and EVLSGIRVFPFDP (SEQ ID NO:76), encoded by the nucleic acid sequence GAGGTTTTAAGCGGTATTAGGGTTTTCCCATTCGACCCC (SEQ ID NO: 73). The light chain CDRs of the CG11_antibody have the following sequences: TGSSSNIGAGYDVY (SEQ ID NO:77), encoded by the nucleic acid sequence ACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTATAT (SEQ ID NO:80); DTNNRPP (SEQ ID NO:81), encoded by the nucleic acid sequence GATACCAACAATCGACCCCCA (SEQ ID NO: 78); and QSYDIALSNSNVV (SEQ ID NO:82), encoded by the nucleic acid sequence CAGTCTTATGACATCGCCCTGAGTAACTCGAATGTGGTT (SEQ ID NO: 79).

[0068] An exemplary huRANTES monoclonal antibody is the BG11 antibody described herein. As shown below, the BG11 antibody includes a heavy chain variable region (SEQ ID NO:84) encoded by the nucleic acid sequence shown in SEQ ID NO: 83, and a light chain variable region (SEQ ID NO:86) encoded by the nucleic acid sequence shown in SEQ ID NO: 85. The CDR sequences are shown in boxes.

TABLE-US-00014 >BG11 Heavy chain variable domain nucleic acid sequence (SEQ ID NO: 83) ##STR00043## >BG11 Heavy chain variable domain amino acid sequence (SEQ ID NO: 84) ##STR00044## >BG11 Light chain variable domain nucleic acid sequence (SEQ ID NO: 85): ##STR00045## >BG11 Light chain variable domain amino acid sequence (SEQ ID NO: 86) ##STR00046##

[0069] The amino acids encompassing the complementarity determining regions (CDR) are as defined by Chothia et al. and E. A. Kabat et al. (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)). The heavy chain CDRs of the BG11_antibody have the following sequences: ELSMH (SEQ ID NO:90), encoded by the nucleic acid sequence GAATTATCCATGCAC (SEQ ID NO: 87); GFDPEDGETIYAQKFQG (SEQ ID NO:91), encoded by the nucleic acid sequence GGTTTTGATCCTGAAGATGGTGAAACAATCTACGCACAGAAGTTCCAGGGC (SEQ ID NO: 88); and YSGSSGWWAFDI (SEQ ID NO:92), encoded by the nucleic acid sequence TATTCTGGTAGTAGTGGTTGGTGGGCTTTTGATATC (SEQ ID NO: 89). The light chain CDRs of the BG11_antibody have the following sequences: QGDSLRSYYAS (SEQ ID NO:96), encoded by the nucleic acid sequence CAAGGAGACAGCCTCAGAAGCTATTATGCAAGC (SEQ ID NO:93); GKNNRPS (SEQ ID NO:97), encoded by the nucleic acid sequence GGTAAAAACAACCGGCCCTCA (SEQ ID NO: 94); and QTWGTGIWV (SEQ ID NO:98), encoded by the nucleic acid sequence CAGACCTGGGGCACTGGCATTTGGGTG (SEQ ID NO: 95).

[0070] An exemplary huRANTES monoclonal antibody is the A9 antibody described herein. As shown below, the A9 antibody includes a heavy chain variable region (SEQ ID NO:100) encoded by the nucleic acid sequence shown in SEQ ID NO: 99, and a light chain variable region (SEQ ID NO:102) encoded by the nucleic acid sequence shown in SEQ ID NO:101. The CDR sequences are shown in boxes.

TABLE-US-00015 >A9 Heavy chain variable domain nucleic acid sequence (SEQ ID NO: 99) ##STR00047## >A9 Heavy chain variable domain amino acid sequence (SEQ ID NO: 100) ##STR00048## >A9 Light chain variable domain nucleic acid sequence (SEQ ID NO: 101): ##STR00049## >A9 Light chain variable domain amino acid sequence (SEQ ID NO: 102) ##STR00050##

[0071] The amino acids encompassing the complementarity determining regions (CDR) are as defined by Chothia et al. and E. A. Kabat et al. (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)). The heavy chain CDRs of the A9 antibody have the following sequences: SYAMS (SEQ ID NO:106), encoded by the nucleic acid sequence AGCTATGCCATGAGC (SEQ ID NO: 103); AISGSGGSTYYADSVKG (SEQ ID NO:107), encoded by the nucleic acid sequence GCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGC (SEQ ID NO: 104); and DLGYCTNGVCWGIDY (SEQ ID NO:108), encoded by the nucleic acid sequence GATTTAGGATATTGTACTAATGGTGTATGCTGGGGTATTGACTAC (SEQ ID NO: 105). The light chain CDRs of the A9 antibody have the following sequences: TRSSGSIADNYVQ (SEQ ID NO:112), encoded by the nucleic acid sequence ACCCGCAGCAGTGGCAGCATTGCCGACAACTATGTGCAG (SEQ ID NO:109); DDDQRLS (SEQ ID NO:113), encoded by the nucleic acid sequence GACGATGACCAAAGACTCTCT (SEQ ID NO: 110); and QSYDDSNDV (SEQ ID NO:114), encoded by the nucleic acid sequence CAGTCTTATGATGACTCCAATGATGTG (SEQ ID NO: 111).

[0072] An exemplary huRANTES monoclonal antibody is the E6 antibody described herein. As shown below, the E6 antibody includes a heavy chain variable region (SEQ ID NO:116) encoded by the nucleic acid sequence shown in SEQ ID NO: 115, and a light chain variable region (SEQ ID NO:118) encoded by the nucleic acid sequence shown in SEQ ID NO: 117. The CDR sequences are shown in boxes.

TABLE-US-00016 >E6 Heavy chain variable domain nucleic acid sequence (SEQ ID NO: 115): ##STR00051## >E6 Heavy chain variable domain amino acid sequence (SEQ ID NO: 116): ##STR00052## >E6 Light chain variable domain nucleic acid sequence (SEQ ID NO: 117): ##STR00053## >E6 Light chain variable domain amino acid sequence (SEQ ID NO: 118) ##STR00054##

[0073] The amino acids encompassing the complementarity determining regions (CDR) are as defined by Chothia et al. and E. A. Kabat et al. (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)). The heavy chain CDRs of the E6_antibody have the following sequences: EIAIH (SEQ ID NO:122), encoded by the nucleic acid sequence GAAATAGCCATACAC (SEQ ID NO: 119); SFEPEDAEAIYAQRFQG (SEQ ID NO:123), encoded by the nucleic acid sequence AGTTTTGAGCCTGAAGATGCTGAAGCAATCTACGCACAGAGGTTCCAGGGC (SEQ ID NO: 120); and DPYYASSGSNYMEV (SEQ ID NO:124), encoded by the nucleic acid sequence GATCCCTACTATGCTAGCAGTGGTTCTAACTACATGGAGGTC (SEQ ID NO: 121). The light chain CDRs of the E6 antibody have the following sequences: TGSGGSISSNYVQ (SEQ ID NO:128), encoded by the nucleic acid sequence ACCGGCAGCGGCGGCAGCATTTCCAGCAACTATGTCCAG (SEQ ID NO:125); EDDQRPS (SEQ ID NO:129), encoded by the nucleic acid sequence GAGGATGACCAAAGACCCTCT (SEQ ID NO: 126); and HSYDGNNRWV (SEQ ID NO:130), encoded by the nucleic acid sequence CACTCTTATGATGGCAACAATCGGTGGGTC (SEQ ID NO: 127).

[0074] An exemplary huRANTES monoclonal antibody is the H6 antibody described herein. As shown below, the H6 antibody includes a heavy chain variable region (SEQ ID NO:132) encoded by the nucleic acid sequence shown in SEQ ID NO: 131, and a light chain variable region (SEQ ID NO:133) encoded by the nucleic acid sequence shown in SEQ ID NO: 132. The CDR sequences are shown in boxes.

TABLE-US-00017 >H6 Heavy chain variable domain nucleic acid sequence (SEQ ID NO: 131): ##STR00055## >H6 Heavy chain variable domain amino acid sequence (SEQ ID NO: 132): ##STR00056## >H6 Light chain variable domain nucleic acid sequence (SEQ ID NO: 133): ##STR00057## >H6 Light chain variable domain amino acid sequence (SEQ ID NO: 134) ##STR00058##

[0075] The amino acids encompassing the complementarity determining regions (CDR) are as defined by Chothia et al. and E. A. Kabat et al. (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)). The heavy chain CDRs of the H6_antibody have the following sequences: KQSMH (SEQ ID NO:138), encoded by the nucleic acid sequence AAACAATCCATGCAC (SEQ ID NO: 135); SSNPEDDETLYAKKFQG (SEQ ID NO:139), encoded by the nucleic acid sequence AGTTCTAATCCTGAAGATGATGAAACACTCTACGCAAAGAAGTTCCAGGGC (SEQ ID NO: 136); and DSQGFYYYYGMDV (SEQ ID NO:140), encoded by the nucleic acid sequence GACTCCCAGGGTTTTTACTATTACTACGGTATGGACGTC (SEQ ID NO: 137). The light chain CDRs of the H6 antibody have the following sequences: TGSSSNIGADYDVH (SEQ ID NO:144), encoded by the nucleic acid sequence ACTGGGAGCAGCTCCAACATCGGGGCAGATTATGATGTACAC (SEQ ID NO:141); DNINRPS (SEQ ID NO:145), encoded by the nucleic acid sequence GATAACATCAATCGGCCCTCA (SEQ ID NO: 142); and QSYDSSLSGVL (SEQ ID NO:146), encoded by the nucleic acid sequence CAGTCCTATGACAGCAGCCTGAGTGGTGTGCTA (SEQ ID NO: 143).

[0076] An exemplary huRANTES monoclonal antibody is the G2 antibody described herein. As shown below, the G2 antibody includes a heavy chain variable region (SEQ ID NO:148) encoded by the nucleic acid sequence shown in SEQ ID NO: 147, and a light chain variable region (SEQ ID NO:150) encoded by the nucleic acid sequence shown in SEQ ID NO: 149. The CDR sequences are shown in boxes.

TABLE-US-00018 >G2 Heavy chain variable domain nucleic acid sequence (SEQ ID NO: 147): ##STR00059## >G2 Heavy chain variable domain amino acid sequence (SEQ ID NO: 148): ##STR00060## >G2 Light chain variable domain nucleic acid sequence (SEQ ID NO: 149): ##STR00061## >G2 Light chain variable domain amino acid sequence (SEQ ID NO: 150) ##STR00062##

[0077] The amino acids encompassing the complementarity determining regions (CDR) are as defined by Chothia et al. and E. A. Kabat et al. (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)). The heavy chain CDRs of the G2_antibody have the following sequences: ELSIH (SEQ ID NO:154), encoded by the nucleic acid sequence GAATTATCCATTCAC (SEQ ID NO: 151); GFDPEDGETIYAQNFQG (SEQ ID NO:155), encoded by the nucleic acid sequence GGTTTTGATCCTGAAGATGGTGAAACAATCTACGCACAGAATTTCCAGGGC (SEQ ID NO: 152); and DLTGSRDS (SEQ ID NO:156), encoded by the nucleic acid sequence GATCTAACTGGAAGTAGGGACTCC (SEQ ID NO: 153). The light chain CDRs of the G2 antibody have the following sequences: TGSRSDIGYYNYVS (SEQ ID NO:160), encoded by the nucleic acid sequence ACTGGAAGCAGGAGTGACATTGGTTACTATAACTATGTCTCC (SEQ ID NO:157); DVTERPS (SEQ ID NO:161), encoded by the nucleic acid sequence GATGTCACTGAGCGACCCTCA (SEQ ID NO: 158); and SSFSSGDTFVV (SEQ ID NO:162), encoded by the nucleic acid sequence AGCTCATTTTCAAGTGGCGACACCTTCGTGGTT (SEQ ID NO: 159).

[0078] An exemplary huRANTES monoclonal antibody is the E10 antibody described herein. As shown below, the E10 antibody includes a heavy chain variable region (SEQ ID NO:164) encoded by the nucleic acid sequence shown in SEQ ID NO: 163, and a light chain variable region (SEQ ID NO:166) encoded by the nucleic acid sequence shown in SEQ ID NO: 165. The CDR sequences are shown in boxes.

TABLE-US-00019 >E10 Heavy chain variable domain nucleic acid sequence (SEQ ID NO: 163): ##STR00063## >E10 Heavy chain variable domain amino acid sequence (SEQ ID NO: 164): ##STR00064## >E10 Light chain variable domain nucleic acid sequence (SEQ ID NO: 165): ##STR00065## >E10 Light chain variable domain amino acid sequence (SEQ ID NO: 166) ##STR00066##

[0079] The amino acids encompassing the complementarity determining regions (CDR) are as defined by Chothia et al. and E. A. Kabat et al. (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)). The heavy chain CDRs of the E10 antibody have the following sequences: SYAMH (SEQ ID NO:28), encoded by the nucleic acid sequence AGCTATGCTATGCAC (SEQ ID NO: 25); VISYDGSNKYYADSVKG (SEQ ID NO:29), encoded by the nucleic acid sequence GTTATATCATATGATGGAAGTAATAAATACTACGCAGACTCCGTGAAGGGC (SEQ ID NO: 26); and ETFPHYYYYYMDV (SEQ ID NO:30), encoded by the nucleic acid sequence GAAACTTTCCCCCACTACTACTACTACTACATGGACGTC (SEQ ID NO: 27). The light chain CDRs of the E10 antibody have the following sequences: GGGNFDDEGVH (SEQ ID NO:176), encoded by the nucleic acid sequence GGGGGAGGCAACTTTGACGATGAAGGTGTTCAC (SEQ ID NO:173); DDTGRPS (SEQ ID NO:177), encoded by the nucleic acid sequence GATGATACCGGCCGGCCCTCA (SEQ ID NO: 174); and QAWDSSNDHPV (SEQ ID NO:178), encoded by the nucleic acid sequence CAGGCGTGGGATAGTAGTAATGATCATCCCGTG (SEQ ID NO: 175).

[0080] An exemplary huRANTES monoclonal antibody is the C10 antibody described herein. As shown below, the C10 antibody includes a heavy chain variable region (SEQ ID NO:180) encoded by the nucleic acid sequence shown in SEQ ID NO: 179, and a light chain variable region (SEQ ID NO:182) encoded by the nucleic acid sequence shown in SEQ ID NO: 181. The CDR sequences are shown in boxes.

TABLE-US-00020 >C10 Heavy chain variable domain nucleic acid sequence (SEQ ID NO: 179): ##STR00067## >C10 Heavy chain variable domain amino acid sequence (SEQ ID NO: 180): ##STR00068## >C10 Light chain variable domain nucleic acid sequence (SEQ ID NO: 181): ##STR00069## >C10 Light chain variable domain amino acid sequence (SEQ ID NO: 182) ##STR00070##

[0081] The amino acids encompassing the complementarity determining regions (CDR) are as defined by Chothia et al. and E. A. Kabat et al. (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)). The heavy chain CDRs of the C10 antibody have the following sequences: SYAMS (SEQ ID NO:106), encoded by the nucleic acid sequence AGCTATGCCATGAGC (SEQ ID NO: 103); AISGSGGSTYYADSVKG (SEQ ID NO:107), encoded by the nucleic acid sequence GCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGC (SEQ ID NO: 104); and VRGSSQYDFWSGSEFDY (SEQ ID NO:188), encoded by the nucleic acid sequence GTAAGGGGGAGTTCCCAGTACGATTTTTGGAGTGGGTCCGAGTTTGACTAC (SEQ ID NO: 185). The light chain CDRs of the C10 antibody have the following sequences: GGDNIGGQNVH (SEQ ID NO:192), encoded by the nucleic acid sequence GGGGGAGACAACATTGGAGGTCAAAATGTTCAC (SEQ ID NO:189); YDTDRPS (SEQ ID NO:193), encoded by the nucleic acid sequence TATGATACCGACCGGCCCTCA (SEQ ID NO: 190); and QVWDVDSDHPWV (SEQ ID NO:194), encoded by the nucleic acid sequence CAGGTGTGGGATGTTGATAGTGATCATCCTTGGGTG (SEQ ID NO: 191).

[0082] An exemplary huRANTES monoclonal antibody is the 2D1 antibody described herein. As shown below, the 2D1 antibody includes a heavy chain variable region (SEQ ID NO:60) encoded by the nucleic acid sequence shown in SEQ ID NO: 59, and a light chain variable region (SEQ ID NO:196) encoded by the nucleic acid sequence shown in SEQ ID NO: 195. The CDR sequences are shown in boxes.

TABLE-US-00021 >2D1 Heavy chain variable domain nucleic acid sequence (SEQ ID NO: 59) ##STR00071## >2D1 Heavy chain variable domain amino acid sequence (SEQ ID NO: 60) ##STR00072## >2D1 Light chain variable domain nucleic acid sequence (SEQ ID NO: 195): ##STR00073## >2D1 Light chain variable domain amino acid sequence (SEQ ID NO: 196) ##STR00074##

[0083] The amino acids encompassing the complementarity determining regions (CDR) are as defined by Chothia et al. and E. A. Kabat et al. (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)). The heavy chain CDRs of the 2D1 antibody have the following sequences: DFAMH (SEQ ID NO:44), encoded by the nucleic acid sequence GACTTCGCCATGCAC (SEQ ID NO: 41); GYVPEDGDTIYAQKFQG (SEQ ID NO:45), encoded by the nucleic acid sequence GGTTATGTTCCTGAAGATGGTGACACAATCTACGCGCAGAAGTTCCAGGGC (SEQ ID NO: 42); and DPLYSGSLSY (SEQ ID NO:64), encoded by the nucleic acid sequence GATCCCCTGTATAGTGGGAGCTTATCCTAC (SEQ ID NO: 53). The light chain CDRs of the 2D1 antibody have the following sequences: GGNNIESKSVH (SEQ ID NO:14), encoded by the nucleic acid sequence GGGGGAAACAACATTGAAAGTAAAAGTGTGCAC (SEQ ID NO:11); DDSDRPS (SEQ ID NO:15), encoded by the nucleic acid sequence GATGATAGCGACCGGCCCTCA (SEQ ID NO: 12); and QVWDSNTDHWV (SEQ ID NO:16), encoded by the nucleic acid sequence CAGGTGTGGGATAGTAATACTGATCATTGGGTG (SEQ ID NO: 197).

[0084] An exemplary huRANTES monoclonal antibody is the A5 antibody described herein. As shown below, the A5 antibody includes a heavy chain variable region (SEQ ID NO:200) encoded by the nucleic acid sequence shown in SEQ ID NO: 199, and a light chain variable region (SEQ ID NO:202) encoded by the nucleic acid sequence shown in SEQ ID NO: 201. The CDR sequences are shown in boxes.

TABLE-US-00022 >A5 Heavy chain variable domain nucleic acid sequence (SEQ ID NO: 199) ##STR00075## >A5 Heavy chain variable domain amino acid sequence (SEQ ID NO: 200) ##STR00076## >A5 Light chain variable domain nucleic acid sequence (SEQ ID NO: 201): ##STR00077## >A5 Light chain variable domain amino acid sequence (SEQ ID NO: 202) ##STR00078##

[0085] The amino acids encompassing the complementarity determining regions (CDR) are as defined by Chothia et al. and E. A. Kabat et al. (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)). The heavy chain CDRs of the A5 antibody have the following sequences: ELSIH (SEQ ID NO:154), encoded by the nucleic acid sequence GAATTATCCATACAC (SEQ ID NO: 203); YIDPEDGEPIYAQKFQG (SEQ ID NO:207), encoded by the nucleic acid sequence TATATTGATCCTGAAGATGGTGAACCAATTTACGCACAGAAGTTCCAGGGC (SEQ ID NO: 204); and VTGSTSDAFDL (SEQ ID NO:208), encoded by the nucleic acid sequence GTCACTGGAAGTACTTCGGATGCCTTTGATCTC (SEQ ID NO: 205). The light chain CDRs of the A5 antibody have the following sequences: GGANLWGLGVH (SEQ ID NO:212), encoded by the nucleic acid sequence GGGGGAGCCAATCTTTGGGGTCTAGGTGTCCAT (SEQ ID NO:209); DNSDRAS (SEQ ID NO:213), encoded by the nucleic acid sequence GATAATAGCGACCGGGCCTCA (SEQ ID NO: 210); and QVWDSSSDHWV (SEQ ID NO:214), encoded by the nucleic acid sequence CAGGTGTGGGATAGTAGTAGTGATCACTGGGTG (SEQ ID NO: 211).

[0086] An exemplary huRANTES monoclonal antibody is the H11 antibody described herein. As shown below, the H11 antibody includes a heavy chain variable region (SEQ ID NO:216) encoded by the nucleic acid sequence shown in SEQ ID NO: 215, and a light chain variable region (SEQ ID NO:218) encoded by the nucleic acid sequence shown in SEQ ID NO: 217. The CDR sequences are shown in boxes.

TABLE-US-00023 >H11 Heavy chain variable domain nucleic acid sequence (SEQ ID NO: 215) ##STR00079## >H11 Heavy chain variable domain amino acid sequence (SEQ ID NO: 216) ##STR00080## >H11 Light chain variable domain nucleic acid sequence (SEQ ID NO: 217): ##STR00081## >H11 Light chain variable domain amino acid sequence (SEQ ID NO: 218) ##STR00082##

[0087] The amino acids encompassing the complementarity determining regions (CDR) are as defined by Chothia et al. and E. A. Kabat et al. (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)). The heavy chain CDRs of the H11 antibody have the following sequences: NYALS (SEQ ID NO:222), encoded by the nucleic acid sequence AACTATGCTCTCAGC (SEQ ID NO: 219); GFIPLVDTTNYAQRFQG (SEQ ID NO:223), encoded by the nucleic acid sequence GGGTTCATCCCTCTCGTCGATACTACGAACTACGCACAGAGGTTTCAGGGC (SEQ ID NO: 220); and EQVAVGPGPTSDRGPDGLDV (SEQ ID NO:224), encoded by the nucleic acid sequence GAGCAGGTGGCGGTGGGACCTGGACCCACCTCAGACCGGGGGCCCGATGGTCTTG ATGTC (SEQ ID NO: 221). The light chain CDRs of the H11 antibody have the following sequences: TGSNSNLGADYDVH (SEQ ID NO:228), encoded by the nucleic acid sequence ACTGGGAGCAACTCCAACCTCGGGGCGGATTATGATGTACAC (SEQ ID NO:225); DNNIRPS (SEQ ID NO:229), encoded by the nucleic acid sequence GATAACAACATTCGTCCCTCA (SEQ ID NO: 226); and QSYDTGLTSSDVI (SEQ ID NO:230), encoded by the nucleic acid sequence CAGTCGTATGACACCGGCCTGACTTCTTCGGATGTGATA (SEQ ID NO: 227).

[0088] An exemplary huRANTES monoclonal antibody is the D1 antibody described herein. As shown below, the D1 antibody includes a heavy chain variable region (SEQ ID NO:232) encoded by the nucleic acid sequence shown in SEQ ID NO: 231, and a light chain variable region (SEQ ID NO:234) encoded by the nucleic acid sequence shown in SEQ ID NO: 233. The CDR sequences are shown in boxes.

TABLE-US-00024 >D1 Heavy chain variable domain nucleic acid sequence (SEQ ID NO: 231) ##STR00083## >D1 Heavy chain variable domain amino acid sequence (SEQ ID NO: 232) ##STR00084## >D1 Light chain variable domain nucleic acid sequence (SEQ ID NO: 233): ##STR00085## >D1 Light chain variable domain amino acid sequence (SEQ ID NO: 234) ##STR00086##

[0089] The amino acids encompassing the complementarity determining regions (CDR) are as defined by Chothia et al. and E. A. Kabat et al. (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)). The heavy chain CDRs of the D1 antibody have the following sequences: DYYIH (SEQ ID NO:74), encoded by the nucleic acid sequence GACTACTACATACAC (SEQ ID NO: 71); LVDSEEDGETLFAETFRG (SEQ ID NO:239), encoded by the nucleic acid sequence CTTGTTGATTCTGAAGAAGATGGTGAAACATTATTCGCAGAGACTTTCAGGGGC (SEQ ID NO: 236); and EYGEYGFFQS (SEQ ID NO:240), encoded by the nucleic acid sequence GAATATGGTGAATATGGGTTCTTCCAATCG (SEQ ID NO: 237). The light chain CDRs of the D1 antibody have the following sequences: TGSSSNIGADYDVN (SEQ ID NO:244), encoded by the nucleic acid sequence ACTGGGAGCAGCTCCAACATCGGGGCAGATTATGATGTAAAC (SEQ ID NO:241); GDINRPS (SEQ ID NO:245), encoded by the nucleic acid sequence GGTGACATCAATCGGCCCTCA (SEQ ID NO: 242); and QSFDNSLSGSVI (SEQ ID NO:246), encoded by the nucleic acid sequence CAGTCGTTTGACAACAGCCTGAGTGGGTCTGTGATT (SEQ ID NO: 243).

[0090] An exemplary huRANTES monoclonal antibody is the E7 antibody described herein. As shown below, the E7 antibody includes a heavy chain variable region (SEQ ID NO:248) encoded by the nucleic acid sequence shown in SEQ ID NO: 247, and a light chain variable region (SEQ ID NO:250) encoded by the nucleic acid sequence shown in SEQ ID NO: 249. The CDR sequences are shown in boxes.

TABLE-US-00025 >E7 Heavy chain variable domain nucleic acid sequence (SEQ ID NO: 247) ##STR00087## >E7 Heavy chain variable domain amino acid sequence (SEQ ID NO: 248) ##STR00088## >E7 Light chain variable domain nucleic acid sequence (SEQ ID NO: 249): ##STR00089## >E7 Light chain variable domain amino acid sequence (SEQ ID NO: 250) ##STR00090##

[0091] The amino acids encompassing the complementarity determining regions (CDR) are as defined by Chothia et al. and E. A. Kabat et al. (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)). The heavy chain CDRs of the E7 antibody have the following sequences: NYALS (SEQ ID NO:222), encoded by the nucleic acid sequence AACTACGCTCTGAGC (SEQ ID NO: 251); AVIPLVETTSYAQRFQG (SEQ ID NO:255), encoded by the nucleic acid sequence GCGGTCATCCCTCTCGTCGAGACTACGAGCTACGCACAGAGGTTCCAGGGC (SEQ ID NO: 252); and EQVAVGPGPTSNRGPDGLDV (SEQ ID NO:169), encoded by the nucleic acid sequence GAGCAGGTGGCGGTGGGACCTGGACCCACTTCAAATCGGGGGCCCGATGGCCTAG ATGTC (SEQ ID NO: 253). The light chain CDRs of the E7 antibody have the following sequences: TGSSSNIGDGYDVH (SEQ ID NO:190), encoded by the nucleic acid sequence ACTGGGAGCAGCTCCAACATCGGGGACGGTTATGATGTACAC (SEQ ID NO:183); GNSNRPS (SEQ ID NO:191), encoded by the nucleic acid sequence GGTAACAGTAATCGGCCCTCA (SEQ ID NO: 184); and GTWDDILNGWV (SEQ ID NO:235), encoded by the nucleic acid sequence GGAACATGGGATGACATCCTGAATGGTTGGGTG (SEQ ID NO: 189).

[0092] huRANTES antibodies of the invention also include antibodies that include a heavy chain variable amino acid sequence that is at least 90%, 92%, 95%, 97%, 98%, 99% or more identical the amino acid sequence of SEQ ID NO: 2, 18, 22, 38, 48, 52, 56, 60, 68, 84, 100, 116, 132, 148, 164, 180, 200, 216, 232, or 248 and/or a light chain variable amino acid that is at least 90%, 92%, 95%, 97%, 98%, 99% or more identical the amino acid sequence of SEQ ID NO: 4, 24, 40, 62, 70, 86, 102, 118, 134, 150, 166, 182, 196, 202, 218, 234, or 250.

[0093] Alternatively, the monoclonal antibody is an antibody that binds to the same epitope as 1D9, 1E4, C8, 3E7, 4D8, 5E1, 6A8, 7B5, CG11, BG11, A9, E6, H6, G2, E10, C10, 2D1, A5, H11, D1 and/or E7.

[0094] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. Standard techniques are used for recombinant DNA and oligonucleotide synthesis, as well as tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclatures utilized in connection with, and the laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, delivery and treatment of patients.

[0095] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0096] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F_ab, F_ab' and F.sub.(ab')2 fragments, and antibodies in an F_ab expression library. By "specifically bind" or "immunoreacts with" is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react (i.e., bind) with other polypeptides or binds at much lower affinity (K_d>10^-6) with other polypeptides.

[0097] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ea., 2nd ed. Raven Press, N.Y. (1989)). The variable regions of each light/heavy chain pair form the antibody binding site.

[0098] The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.

[0099] In general, antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG₁, IgG₂, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.

[0100] The term "antigen-binding site," or "binding portion" refers to the part of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as "hypervariable regions," are interposed between more conserved flanking stretches known as "framework regions," or "FRs". Thus, the term "FR" refers to amino acid sequences which are naturally found between, and adjacent to, hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions," or "CDRs." The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989).

[0101] As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or fragment thereof, or a T-cell receptor. The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is ≦1 μM; e.g., ≦100 nM, preferably ≦10 nM and more preferably ≦1 nM.

[0102] As used herein, the terms "immunological binding," and "immunological binding properties" refer to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (K_d) of the interaction, wherein a smaller K_d represents a greater affinity. Immunological binding properties of selected polypeptides are quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the "on rate constant" (K_on) and the "off rate constant" (K_off) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). The ratio of K_off/K_on enables the cancellation of all parameters not related to affinity, and is equal to the dissociation constant K_d. (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). An antibody of the present invention is said to specifically bind to a RANTES epitope when the equilibrium binding constant (K_d) is ≦1 μM, e.g., ≦100 nM, preferably ≦10 nM, and more preferably ≦1 nM, as measured by assays such as radioligand binding assays or surface plasmon resonance (SPR) or similar assays known to those skilled in the art. For example, the huRANTES antibodies provided herein exhibit a K_d in the range approximately between ≦10 nM to about 100 pM.

[0103] Those skilled in the art will recognize that it is possible to determine, without undue experimentation, if a human monoclonal antibody has the same specificity as a human monoclonal antibody of the invention (e.g., monoclonal antibody D9, E4 or C8) by ascertaining whether the former prevents the latter from binding to a RANTES antigen polypeptide. If the human monoclonal antibody being tested competes with a human monoclonal antibody of the invention, as shown by a decrease in binding by the human monoclonal antibody of the invention, then the two monoclonal antibodies bind to the same, or a closely related, epitope. Another way to determine whether a human monoclonal antibody has the specificity of a human monoclonal antibody of the invention is to pre-incubate the human monoclonal antibody of the invention with the RANTES antigen polypeptide with which it is normally reactive, and then add the human monoclonal antibody being tested to determine if the human monoclonal antibody being tested is inhibited in its ability to bind the RANTES antigen polypeptide. If the human monoclonal antibody being tested is inhibited then, in all likelihood, it has the same, or functionally equivalent, epitopic specificity as the monoclonal antibody of the invention.

[0104] Various procedures known within the art are used for the production of the monoclonal antibodies directed against a protein such as a RANTES protein, or against derivatives, fragments, analogs homologs or orthologs thereof. (See, e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated herein by reference). Fully human antibodies are antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or "fully human antibodies" herein. Human monoclonal antibodies are prepared, for example, using the procedures described in the Examples provided below. Human monoclonal antibodies can be also prepared by using the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

[0105] Antibodies are purified by well-known techniques, such as affinity chromatography using protein A or protein G. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immuno affinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

[0106] In some instances, it may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating immune-related diseases. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989)). In a preferred embodiment, the huRANTES antibodies of the invention are not modified with respect to effector function.

[0107] The invention also includes F_v, F_ab, F_ab' and F.sub.(ab')2 huRANTES antibody fragments, single chain huRANTES antibodies, bispecific huRANTES antibodies and heteroconjugate huRANTES antibodies.

[0108] Bispecific antibodies are antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for RANTES. The second binding target is any other antigen, and in some embodiments, the second binding target is an extracellular target such as a cell-surface protein or receptor or receptor subunit.

[0109] Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0110] Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

[0111] Other approaches for generating bispecific antibodies are described, e.g., in WO 96/27011, which is hereby incorporated by reference in its entirety. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')₂ bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. See e.g., Brennan et al., Science 229:81 (1985), which is hereby incorporated by reference in its entirety.

[0112] Additionally, Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. See e.g., Shalaby et al., J. Exp. Med. 175:217-225 (1992), which is hereby incorporated by reference in its entirety.

[0113] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. See e.g., Kostelny et al., J. Immunol. 148(5):1547-1553 (1992), which is hereby incorporated by reference in its entirety. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), which is hereby incorporated by reference in its entirety, has provided an alternative mechanism for making bispecific antibody fragments. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994), which is hereby incorporated by reference in its entirety.

[0114] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. See, Tutt et al., J. Immunol. 147:60 (1991).

[0115] Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, IFNγ, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).

[0116] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.

[0117] The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

[0118] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

[0119] Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. (See WO94/11026).

[0120] Those of ordinary skill in the art will recognize that a large variety of possible moieties can be coupled to the resultant antibodies or to other molecules of the invention. (See, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

[0121] Coupling is accomplished by any chemical reaction that will bind the two molecules so long as the antibody and the other moiety retain their respective activities. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation. The preferred binding is, however, covalent binding. Covalent binding is achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the present invention, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987). Preferred linkers are described in the literature. (See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Pat. No. 5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an antibody by way of an oligopeptide linker Particularly preferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Cat #21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6 [3-(2-pyridyldithio)-propianamide]hexanoate (Pierce Chem. Co. Cat. #2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC.

[0122] The term "isolated polynucleotide" as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the "isolated polynucleotide" (1) is not associated with all or a portion of a polynucleotide in which the "isolated polynucleotide" is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.

[0123] The term "isolated protein" referred to herein means a protein of cDNA, recombinant RNA, or synthetic origin or some combination thereof, which by virtue of its origin, or source of derivation, the "isolated protein" (1) is not associated with proteins found in nature, (2) is free of other proteins from the same source, e.g., free of murine proteins, (3) is expressed by a cell from a different species, or (4) does not occur in nature.

[0124] The term "polypeptide" is used herein as a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence. Hence, native protein fragments, and analogs are species of the polypeptide genus. Preferred polypeptides in accordance with the invention comprise the human heavy chain immunoglobulin molecules presented herein and the human light chain immunoglobulin molecules presented herein, as well as antibody molecules formed by combinations comprising the heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as kappa light chain immunoglobulin molecules, and vice versa, as well as fragments and analogs thereof.

[0125] The term "naturally-occurring" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory or otherwise is naturally-occurring.

[0126] The term "operably linked" as used herein refers to positions of components so described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0127] The term "control sequence" as used herein refers to polynucleotide sequences which are necessary to effect the expression and processing of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. The term "polynucleotide" as referred to herein means a polymeric boron of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.

[0128] The term oligonucleotide referred to herein includes naturally occurring, and modified nucleotides linked together by naturally occurring, and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a polynucleotide subset generally comprising a length of 200 bases or fewer. Preferably oligonucleotides are 10 to 60 bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usually single stranded, e.g., for probes, although oligonucleotides may be double stranded, e.g., for use in the construction of a gene mutant. Oligonucleotides of the invention are either sense or antisense oligonucleotides.

[0129] The term "naturally occurring nucleotides" referred to herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" referred to herein includes nucleotides with modified or substituted sugar groups and the like. The term "oligonucleotide linkages" referred to herein includes Oligonucleotides linkages such as phosphorothioate, phosphorodithioate, phosphoroselerloate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoronmidate, and the like. See e.g., LaPlanche et al. Nucl. Acids Res. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984), Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anti Cancer Drug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90:543 (1990). An oligonucleotide can include a label for detection, if desired.

[0130] The term "selectively hybridize" referred to herein means to detectably and specifically bind. Polynucleotides, oligonucleotides and fragments thereof in accordance with the invention selectively hybridize to nucleic acid strands under hybridization and wash conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids. High stringency conditions can be used to achieve selective hybridization conditions as known in the art and discussed herein. Generally, the nucleic acid sequence homology between the polynucleotides, oligonucleotides, and fragments of the invention and a nucleic acid sequence of interest will be at least 80%, and more typically with preferably increasing homologies of at least 85%, 90%, 95%, 99%, and 100%. Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching gap lengths of 5 or less are preferred with 2 or less being more preferred. Alternatively and preferably, two protein sequences (or polypeptide sequences derived from them of at least 30 amino acids in length) are homologous, as this term is used herein, if they have an alignment score of at more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater. See Dayhoff, M. O., in Atlas of Protein Sequence and Structure, pp. 101-110 (Volume 5, National Biomedical Research Foundation (1972)) and Supplement 2 to this volume, pp. 1-10. The two sequences or parts thereof are more preferably homologous if their amino acids are greater than or equal to 50% identical when optimally aligned using the ALIGN program. The term "corresponds to" is used herein to mean that a polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence. In contradistinction, the term "complementary to" is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For illustration, the nucleotide sequence "TATAC" corresponds to a reference sequence "TATAC" and is complementary to a reference sequence "GTATA".

[0131] The following terms are used to describe the sequence relationships between two or more polynucleotide or amino acid sequences: "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity". A "reference sequence" is a defined sequence used as a basis for a sequence comparison a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 18 nucleotides or 6 amino acids in length, frequently at least 24 nucleotides or 8 amino acids in length, and often at least 48 nucleotides or 16 amino acids in length. Since two polynucleotides or amino acid sequences may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide or amino acid sequence) that is similar between the two molecules, and (2) may further comprise a sequence that is divergent between the two polynucleotides or amino acid sequences, sequence comparisons between two (or more) molecules are typically performed by comparing sequences of the two molecules over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window", as used herein, refers to a conceptual segment of at least 18 contiguous nucleotide positions or 6 amino acids wherein a polynucleotide sequence or amino acid sequence may be compared to a reference sequence of at least 18 contiguous nucleotides or 6 amino acid sequences and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions, deletions, substitutions, and the like (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison, Wis.), Geneworks, or MacVector software packages), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.

[0132] The term "sequence identity" means that two polynucleotide or amino acid sequences are identical (i.e., on a nucleotide-by-nucleotide or residue-by-residue basis) over the comparison window. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U or I) or residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms "substantial identity" as used herein denotes a characteristic of a polynucleotide or amino acid sequence, wherein the polynucleotide or amino acid comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 18 nucleotide (6 amino acid) positions, frequently over a window of at least 24-48 nucleotide (8-16 amino acid) positions, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the comparison window. The reference sequence may be a subset of a larger sequence.

[0133] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology--A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland Mass. (1991)). Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4 hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the lefthand direction is the amino terminal direction and the righthand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0134] Similarly, unless specified otherwise, the lefthand end of single-stranded polynucleotide sequences is the 5' end the lefthand direction of double-stranded polynucleotide sequences is referred to as the 5' direction. The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction sequence regions on the DNA strand having the same sequence as the RNA and which are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences", sequence regions on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences".

[0135] As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity, and most preferably at least 99 percent sequence identity.

[0136] Preferably, residue positions which are not identical differ by conservative amino acid substitutions.

[0137] Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine valine, glutamic-aspartic, and asparagine-glutamine.

[0138] As discussed herein, minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%. In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. The hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. The hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine. Other families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are the amide containing family; (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those of ordinary skill in the art. Preferred amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known. Bowie et al. Science 253:164 (1991). Thus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the invention.

[0139] Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (4) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various muteins of a sequence other than the naturally-occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally-occurring sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et at. Nature 354:105 (1991).

[0140] The term "polypeptide fragment" as used herein refers to a polypeptide that has an amino terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally-occurring sequence deduced, for example, from a full length cDNA sequence. Fragments typically are at least 5, 6, 8 or 10 amino acids long, preferably at least 14 amino acids long' more preferably at least 20 amino acids long, usually at least 50 amino acids long, and even more preferably at least 70 amino acids long. The term "analog" as used herein refers to polypeptides which are comprised of a segment of at least 25 amino acids that has substantial identity to a portion of a deduced amino acid sequence and which has at least one of the following properties: (1) specific binding to RANTES, under suitable binding conditions or (2) ability to block appropriate RANTES binding. Typically, polypeptide analogs comprise a conservative amino acid substitution (or addition or deletion) with respect to the naturally-occurring sequence. Analogs typically are at least 20 amino acids long, preferably at least 50 amino acids long or longer, and can often be as long as a full-length naturally-occurring polypeptide.

[0141] Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics" or "peptidomimetics". Fauchere, J. Adv. Drug Res. 15:29 (1986), Veber and Freidinger TIBS p. 392 (1985); and Evans et al. J. Med. Chem. 30:1229 (1987). Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biochemical property or pharmacological activity), such as human antibody, but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of: --CH₂NH--, --CH₂S--, --CH₂--CH₂--, --CH═CH-(cis and trans), --COCH₂--, CH(OH)CH₂--, and --CH₂SO--, by methods well known in the art. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) may be used to generate more stable peptides. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992)); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.

[0142] The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.

[0143] As used herein, the terms "label" or "labeled" refers to incorporation of a detectable marker, e.g., by incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods). In certain situations, the label or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³5S, ⁹⁰Y, ⁹⁹Te, ¹¹¹In, ¹²5I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. The term "pharmaceutical agent or drug" as used herein refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[0144] Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

[0145] As used herein, "substantially pure" means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present.

[0146] Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95%, and 99%. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.

[0147] The term patient includes human and veterinary subjects. The term subject includes humans and other mammals.

[0148] Human Antibodies and Humanization of Antibodies

[0149] A huRANTES antibody is generated, for example, using the procedures described in the Examples provided below.

[0150] In other, alternative methods, a huRANTES antibody is developed, for example, using phage-display methods using antibodies containing only human sequences. Such approaches are well-known in the art, e.g., in WO92/01047 and U.S. Pat. No. 6,521,404, which are hereby incorporated by reference. In this approach, a combinatorial library of phage carrying random pairs of light and heavy chains are screened using natural or recombinant source of RANTES or fragments thereof. In another approach, a huRANTES antibody can be produced by a process wherein at least one step of the process includes immunizing a transgenic, non-human animal with human RANTES protein. In this approach, some of the endogenous heavy and/or kappa light chain loci of this xenogenic non-human animal have been disabled and are incapable of the rearrangement required to generate genes encoding immunoglobulins in response to an antigen. In addition, at least one human heavy chain locus and at least one human light chain locus have been stably transfected into the animal. Thus, in response to an administered antigen, the human loci rearrange to provide genes encoding human variable regions immunospecific for the antigen. Upon immunization, therefore, the xenomouse produces B-cells that secrete fully human immunoglobulins.

[0151] A variety of techniques are well-known in the art for producing xenogenic non-human animals. For example, see U.S. Pat. No. 6,075,181 and No. 6,150,584, which is hereby incorporated by reference in its entirety. This general strategy was demonstrated in connection with generation of the first XenoMouse® strains as published in 1994. See Green et al. Nature Genetics 7:13-21 (1994), which is hereby incorporated by reference in its entirety. See also, U.S. Pat. Nos. 6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598 and Japanese Patent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2 and European Patent No., EP 0 463 151 B1 and International Patent Applications No. WO 94/02602, WO 96/34096, WO 98/24893, WO 00/76310 and related family members.

[0152] In an alternative approach, others have utilized a "minilocus" approach in which an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus. Thus, one or more VH genes, one or more D_H genes, one or more J_H genes, a mu constant region, and a second constant region (preferably a gamma constant region) are formed into a construct for insertion into an animal. See e.g., U.S. Pat. Nos. 5,545,806; 5,545,807; 5,591,669; 5,612,205; 5,625,825; 5,625,126; 5,633,425; 5,643,763; 5,661,016; 5,721,367; 5,770,429; 5,789,215; 5,789,650; 5,814,318; 5,877; 397; 5,874,299; 6,023,010; and 6,255,458; and European Patent No. 0 546 073 B1; and International Patent Application Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884 and related family members.

[0153] Generation of human antibodies from mice in which, through microcell fusion, large pieces of chromosomes, or entire chromosomes, have been introduced, has also been demonstrated. See European Patent Application Nos. 773 288 and 843 961.

[0154] Human anti-mouse antibody (HAMA) responses have led the industry to prepare chimeric or otherwise humanized antibodies. While chimeric antibodies have a human constant region and a immune variable region, it is expected that certain human anti-chimeric antibody (HACA) responses will be observed, particularly in chronic or multi-dose utilizations of the antibody. Thus, it would be desirable to provide fully human antibodies against RANTES in order to vitiate or otherwise mitigate concerns and/or effects of HAMA or HACA response.

[0155] The production of antibodies with reduced immunogenicity is also accomplished via humanization, chimerization and display techniques using appropriate libraries. It will be appreciated that murine antibodies or antibodies from other species can be humanized or primatized using techniques well known in the art. See e.g., Winter and Harris Immunol Today 14:43 46 (1993) and Wright et al. Crit, Reviews in Immunol. 12125-168 (1992). The antibody of interest may be engineered by recombinant DNA techniques to substitute the CH1, CH2, CH3, hinge domains, and/or the framework domain with the corresponding human sequence (See WO 92102190 and U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and 5,777,085). Also, the use of Ig cDNA for construction of chimeric immunoglobulin genes is known in the art (Liu et al. P.N.A.S. 84:3439 (1987) and J. Immunol. 139:3521 (1987)). mRNA is isolated from a hybridoma or other cell producing the antibody and used to produce cDNA. The cDNA of interest may be amplified by the polymerase chain reaction using specific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library is made and screened to isolate the sequence of interest. The DNA sequence encoding the variable region of the antibody is then fused to human constant region sequences. The sequences of human constant regions genes may be found in Kabat et al. (1991) Sequences of Proteins of immunological Interest, N.I.H. publication no. 91-3242. Human C region genes are readily available from known clones. The choice of isotype will be guided by the desired effecter functions, such as complement fixation, or activity in antibody-dependent cellular cytotoxicity. Preferred isotypes are IgG1, IgG3 and IgG4. Either of the human light chain constant regions, kappa or lambda, may be used. The chimeric, humanized antibody is then expressed by conventional methods.

[0156] Antibody fragments, such as Fv, F(ab')₂ and Fab may be prepared by cleavage of the intact protein, e.g., by protease or chemical cleavage. Alternatively, a truncated gene is designed. For example, a chimeric gene encoding a portion of the F(ab')₂ fragment would include DNA sequences encoding the CH1 domain and hinge region of the H chain, followed by a translational stop codon to yield the truncated molecule.

[0157] Consensus sequences of H and L J regions may be used to design oligonucleotides for use as primers to introduce useful restriction sites into the J region for subsequent linkage of V region segments to human C region segments. C region cDNA can be modified by site directed mutagenesis to place a restriction site at the analogous position in the human sequence.

[0158] Expression vectors include plasmids, retroviruses, YACs, EBV derived episomes, and the like. A convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed. In such vectors, splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C region, and also at the splice regions that occur within the human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions. The resulting chimeric antibody may be joined to any strong promoter, including retroviral LTRs, e.g., SV-40 early promoter, (Okayama et al. Mol. Cell. Bio. 3:280 (1983)), Rous sarcoma virus LTR (Gorman et al. P.N.A.S. 79:6777 (1982)), and moloney murine leukemia virus LTR (Grosschedl et al. Cell 41:885 (1985)). Also, as will be appreciated, native Ig promoters and the like may be used.

[0159] Further, human antibodies or antibodies from other species can be generated through display type technologies, including, without limitation, phage display, retroviral display, ribosomal display, and other techniques, using techniques well known in the art and the resulting molecules can be subjected to additional maturation, such as affinity maturation, as such techniques are well known in the art. Wright et al. Crit, Reviews in Immunol. 12125-168 (1992), Hanes and Pluckthun PNAS USA 94:4937-4942 (1997) (ribosomal display), Parmley and Smith Gene 73:305-318 (1988) (phage display), Scott, TIBS, vol. 17:241-245 (1992), Cwirla et al. PNAS USA 87:6378-6382 (1990), Russel et al. Nucl. Acids Research 21:1081-1085 (1993), Hoganboom et al. Immunol. Reviews 130:43-68 (1992), Chiswell and McCafferty TIBTECH; 10:80-8A (1992), and U.S. Pat. No. 5,733,743. If display technologies are utilized to produce antibodies that are not human, such antibodies can be humanized as described above.

[0160] Using these techniques, antibodies can be generated to RANTES expressing cells, RANTES itself, forms of RANTES, epitopes or peptides thereof, and expression libraries thereto (See e.g., U.S. Pat. No. 5,703,057) which can thereafter be screened as described above for the activities described above.

[0161] Design and Generation of Other Therapeutics

[0162] In accordance with the present invention and based on the activity of the antibodies that are produced and characterized herein with respect to RANTES, the design of other therapeutic modalities beyond antibody moieties is facilitated. Such modalities include, without limitation, advanced antibody therapeutics, such as bispecific antibodies, immunotoxins, and radiolabeled therapeutics, generation of peptide therapeutics, gene therapies, particularly intrabodies, antisense therapeutics, and small molecules.

[0163] For example, in connection with bispecific antibodies, bispecific antibodies can be generated that comprise (i) two antibodies one with a specificity to RANTES and another to a second molecule that are conjugated together, (ii) a single antibody that has one chain specific to RANTES and a second chain specific to a second molecule, or (iii) a single chain antibody that has specificity to RANTES and a second molecule. Such bispecific antibodies are generated using techniques that are well known for example, in connection with (i) and (ii) See e.g., Fanger et al. Immunol Methods 4:72-81 (1994) and Wright et al. Crit, Reviews in Immunol. 12125-168 (1992), and in connection with (iii) See e.g., Traunecker et al. Int. J. Cancer (Suppl.) 7:51-52 (1992).

[0164] In connection with immunotoxins, antibodies can be modified to act as immunotoxins utilizing techniques that are well known in the art. See e.g., Vitetta Immunol Today 14:252 (1993). See also U.S. Pat. No. 5,194,594. In connection with the preparation of radiolabeled antibodies, such modified antibodies can also be readily prepared utilizing techniques that are well known in the art. See e.g., Junghans et al. in Cancer Chemotherapy and Biotherapy 655-686 (2d edition, Chafner and Longo, eds., Lippincott Raven (1996)). See also U.S. Pat. Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (RE 35,500), 5,648,471, and 5,697,902. Each of immunotoxins and radiolabeled molecules would be likely to kill cells expressing RANTES.

[0165] In connection with the generation of therapeutic peptides, through the utilization of structural information related to RANTES and antibodies thereto, such as the antibodies of the invention or screening of peptide libraries, therapeutic peptides can be generated that are directed against RANTES. Design and screening of peptide therapeutics is discussed in connection with Houghten et al. Biotechniques 13:412-421 (1992), Houghten PNAS USA 82:5131-5135 (1985), Pinalla et al. Biotechniques 13:901-905 (1992), Blake and Litzi-Davis BioConjugate Chem. 3:510-513 (1992). Immunotoxins and radiolabeled molecules can also be prepared, and in a similar manner, in connection with peptidic moieties as discussed above in connection with antibodies. Assuming that the RANTES molecule (or a form, such as a splice variant or alternate form) is functionally active in a disease process, it will also be possible to design gene and antisense therapeutics thereto through conventional techniques. Such modalities can be utilized for modulating the function of RANTES. In connection therewith the antibodies of the present invention facilitate design and use of functional assays related thereto. A design and strategy for antisense therapeutics is discussed in detail in International Patent Application No. WO 94/29444. Design and strategies for gene therapy are well known. However, in particular, the use of gene therapeutic techniques involving intrabodies could prove to be particularly advantageous. See e.g., Chen et al. Human Gene Therapy 5:595-601 (1994) and Marasco Gene Therapy 4:11-15 (1997). General design of and considerations related to gene therapeutics is also discussed in International Patent Application No. WO 97/38137.

[0166] Knowledge gleaned from the structure of the RANTES molecule and its interactions with other molecules in accordance with the present invention, such as the antibodies of the invention, and others can be utilized to rationally design additional therapeutic modalities. In this regard, rational drug design techniques such as X-ray crystallography, computer-aided (or assisted) molecular modeling (CAMM), quantitative or qualitative structure-activity relationship (QSAR), and similar technologies can be utilized to focus drug discovery efforts. Rational design allows prediction of protein or synthetic structures which can interact with the molecule or specific forms thereof which can be used to modify or modulate the activity of RANTES. Such structures can be synthesized chemically or expressed in biological systems. This approach has been reviewed in Capsey et al. Genetically Engineered Human Therapeutic Drugs (Stockton Press, NY (1988)). Further, combinatorial libraries can be designed and synthesized and used in screening programs, such as high throughput screening efforts.

[0167] Therapeutic Administration and Formulations

[0168] It will be appreciated that administration of therapeutic entities in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, Pa. (1975)), particularly Chapter 87 by Blaug, Seymour, therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as Lipofectin®), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance." Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. "Lyophilization and development of solid protein pharmaceuticals." Int. J. Pharm. 203(1-2):1-60 (2000), Charman W N "Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts." J Pharm Sci. 89(8):967-78 (2000), Powell et al. "Compendium of excipients for parenteral formulations" PDA J Pharm Sci Technol. 52:238-311 (1998) and the citations therein for additional information related to formulations, excipients and carriers well known to pharmaceutical chemists.

[0169] The RANTES antagonists, huRANTES antibodies and therapeutic formulations of the invention, which include a RANTES antagonist, such as a huRANTES antibody of the invention, are used to treat or alleviate ischemia, a clinical indication associated with ischemia, reperfusion injury, a symptom associated with an immune-related disorder, such as, for example, an autoimmune disease or an inflammatory disorder.

[0170] Autoimmune diseases include, for example, Acquired Immunodeficiency Syndrome (AIDS, which is a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, celiac sprue-dermatitis hepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigold, cold agglutinin disease, crest syndrome, Crohn's disease, Degos' disease, dermatomyositis-juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernacious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomena, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive systemic sclerosis (PSS), also known as systemic sclerosis (SS)), Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vitiligo and Wegener's granulomatosis.

[0171] Inflammatory disorders include, for example, chronic and acute inflammatory disorders. Examples of inflammatory disorders include Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft vs. host disease, hemolytic anemias, osteoarthritis, sepsis, stroke, transplantation of tissue and organs, vasculitis, diabetic retinopathy and ventilator induced lung injury.

[0172] The huRANTES antibodies modulate an immune response in a subject, e.g., in a human subject and transplanted organ. In one embodiment, the RANTES antagonist, huRANTES antibody, fragment thereof or therapeutic formulation thereof is used to treat ischemia, a clinical indication associated with ischemia, reperfusion injury, and/or another immune-related disorder in conjunction with a surgical treatment or other interventional therapy used in the art to treat a given disorder. For example, interventional therapies used in the treatment of ischemia, a clinical indication associated with ischemia, and/or reperfusion injury include surgical intervention or angioplasty. The RANTES antagonist, huRANTES antibody, fragment thereof or therapeutic formulation thereof is administered simultaneously (i.e., during) the interventional therapy, or the RANTES antagonist, huRANTES antibody, fragment thereof or therapeutic formulation thereof is administered at a different time than the interventional therapy. For example, the RANTES antagonist, huRANTES antibody, fragment thereof or therapeutic formulation thereof is administered in some embodiments after an interventional therapy.

[0173] In one embodiment, the RANTES antagonist, huRANTES antibody, fragment thereof or therapeutic formulation thereof used to treat ischemia, a clinical indication associated with ischemia, reperfusion injury, and/or another immune-related disorder are administered in combination with any of a variety of anti-cytokine agents or anti-chemokine agents. Suitable anti-cytokine or anti-chemokine reagents recognize, for example, cytokines such as interleukin 1 (IL-1), IL-2, IL-4, IL-6, IL-12, IL-13, IL-15, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-27 and IL-31, and/or chemokines such as MIP1 alpha, MIP1 beta, RANTES, MCP1, RANTES, ITAC, MIG, SDF and fractalkine.

[0174] In one embodiment, the RANTES antagonist, huRANTES antibody, fragment thereof or therapeutic formulation thereof used to treat ischemia, a clinical indication associated with ischemia, reperfusion injury, and/or another immune-related disorder are administered in conjunction with one or more additional agents, or a combination of additional agents. For example, the RANTES antagonist (e.g., huRANTES antibody) and additional agent are formulated into a single therapeutic composition, and the RANTES antagonist and additional agent are administered simultaneously. Alternatively, the RANTES antagonist and additional agent are separate from each other, e.g., each is formulated into a separate therapeutic composition, and the RANTES antagonist and the additional agent are administered simultaneously, or the RANTES antagonist and the additional agent are administered at different times during a treatment regimen. For example, the RANTES antagonist (e.g., huRANTES antibody) is administered prior to the administration of the additional agent, the RANTES antagonist is administered subsequent to the administration of the additional agent, or the RANTES antagonist and the additional agent are administered in an alternating fashion. As described herein, the RANTES antagonist and additional agent are administered in single doses or in multiple doses.

[0175] For example, in the treatment of coronary artery disease, the RANTES antagonist, huRANTES antibody, fragment thereof or therapeutic formulation thereof, is administered in conjunction with one or more additional agents such as cholesterol-lowering medicines, such as statins; anticoagulants, such as heparin and/or oral anticoagulants such as warfarin and dicumarol; aspirin, and other antiplatelet medicines; ACE (angiotensin-converting enzyme) inhibitors, such as sulfhydryl-containing ACE inhibitors (e.g., Captopril), dicarboxylate-containing ACE inhibitors (e.g., Enalapril, Ramipril, Quinapril, Perindopril, Lisinopril, Benazepril); phosphonate-containing ACE inhibitors (e.g., Fosinopril); beta blockers; calcium channel blockers; nitroglycerin; long-acting nitrates; glycoprotein IIb-IIIa inhibitors; and thrombolytic agents. The RANTES antagonist and the additional agent are administered simultaneously, or RANTES antagonist and the additional agent are administered at different times during a treatment regimen.

[0176] For example, in the treatment of cerebral vascular disease, the RANTES antagonist, huRANTES antibody, fragment thereof or therapeutic formulation thereof, is administered in conjunction with one or more additional agents such as cholesterol-lowering medicines, such as statins, aspirin, and other antiplatelet medicines. The RANTES antagonist and the additional agent are administered simultaneously, or RANTES antagonist and the additional agent are administered at different times during a treatment regimen.

[0177] For example, in the treatment of cardiac ischemia, the RANTES antagonist, huRANTES antibody, fragment thereof or therapeutic formulation thereof, is administered in conjunction with one or more additional agents such as aspirin, and other antiplatelet medicines; ACE (angiotensin-converting enzyme) inhibitors, such as sulfhydryl-containing ACE inhibitors (e.g., Captopril), dicarboxylate-containing ACE inhibitors (e.g., Enalapril, Ramipril, Quinapril, Perindopril, Lisinopril, Benazepril); phosphonate-containing ACE inhibitors (e.g., Fosinopril); beta blockers; calcium channel blockers; nitroglycerin; and long-acting nitrates. The RANTES antagonist and the additional agent are administered simultaneously, or RANTES antagonist and the additional agent are administered at different times during a treatment regimen.

[0178] For example, in the treatment of myocardial ischemia, the RANTES antagonist, huRANTES antibody, fragment thereof or therapeutic formulation thereof is administered in conjunction with one or more additional agents such as beta blockers; calcium channel blockers; nitroglycerin; and long-acting nitrates. The RANTES antagonist and the additional agent are administered simultaneously, or RANTES antagonist and the additional agent are administered at different times during a treatment regimen.

[0179] For example, in the treatment of renal ischemia, the RANTES antagonist, huRANTES antibody, fragment thereof or therapeutic formulation thereof is administered in conjunction with one or more additional agents such as cholesterol-lowering medicines, such as aspirin, and other antiplatelet medicines. The RANTES antagonist and the additional agent are administered simultaneously, or RANTES antagonist and the additional agent are administered at different times during a treatment regimen.

[0180] For example, in the treatment of peripheral vascular disease, the RANTES antagonist, huRANTES antibody, fragment thereof or therapeutic formulation thereof is administered in conjunction with one or more additional agents such as anticoagulants, such as heparin and/or oral anticoagulants such as warfarin and dicumarol; aspirin, and other antiplatelet medicines; pentoxifylline; cilostazol; and thrombolytic agents. The RANTES antagonist and the additional agent are administered simultaneously, or RANTES antagonist and the additional agent are administered at different times during a treatment regimen.

[0181] For example, in the treatment of multiple sclerosis, the huRANTES antibody, or therapeutic formulation thereof, is administered in conjunction with one or more additional agents such as interferon beta 1a, interferon beta 1b, glatiramer acetate, natalizumab, copaxone, and combinations thereof. The huRANTES antibody and the additional agent are administered simultaneously, or the huRANTES antibody and the additional agent are administered at different times during a treatment regimen.

[0182] In the treatment of Crohn's disease, the huRANTES antibody, or therapeutic formulation thereof, is administered in conjunction with one or more additional agents such as an antibiotic, an aminosalicylate, Infliximab, Adalimumab, and combinations thereof. Suitable antibiotics include, e.g., metronidazole and/or ciprofloxacin. Suitable aminosalicylates include, for example, mesalamine and/or sulfasalazine. The huRANTES antibody and the additional agent are administered simultaneously, or the huRANTES antibody and the additional agent are administered at different times during a treatment regimen.

[0183] In the treatment of ulcerative colitis, the huRANTES antibody, or therapeutic formulation thereof, is administered in conjunction with one or more additional agents such as 6-mercaptopurine, azathioprine, Infliximab and combinations thereof. The huRANTES antibody and the additional agent are administered simultaneously, or the huRANTES antibody and the additional agent are administered at different times during a treatment regimen.

[0184] In the treatment of psoriasis, the huRANTES antibody, or therapeutic formulation thereof, is administered in conjunction with one or more additional agents such as alefacept, efalizumab, Adalimumab, Infliximab, cyclosporine, Methotrexate, and combinations thereof. The huRANTES antibody and the additional agent are administered simultaneously, or the huRANTES antibody and the additional agent are administered at different times during a treatment regimen.

[0185] In the treatment of atherosclerosis, the huRANTES antibody, or therapeutic formulation thereof, is administered in conjunction with one or more additional agents such as warfarin, a cholesterol lowering drug, and combinations thereof. Suitable cholesterol lowering drugs include, for example, statins and flbrates. The huRANTES antibody and the additional agent are administered simultaneously, or the huRANTES antibody and the additional agent are administered at different times during a treatment regimen.

[0186] The huRANTES antibodies and therapeutic formulations thereof are used in methods of treating or alleviating a symptom associated with an immune-related disorder. For example, the compositions of the invention are used to treat or alleviate a symptom of any of the autoimmune diseases and inflammatory disorders described herein. Symptoms associated with immune-related disorders include, for example, inflammation, fever, loss of appetite, weight loss, abdominal symptoms such as, for example, abdominal pain, diarrhea or constipation, joint pain or aches (arthralgia), fatigue, rash, anemia, extreme sensitivity to cold (Raynaud's phenomenon), muscle weakness, muscle fatigue, changes in skin or tissue tone, shortness of breath or other abnormal breathing patterns, chest pain or constriction of the chest muscles, abnormal heart rate (e.g., elevated or lowered), light sensitivity, blurry or otherwise abnormal vision, and reduced organ function.

[0187] The RANTES antagonists, such as a huRANTES antibody, and therapeutic formulations thereof are administered to a subject suffering from ischemia, a clinical indication associated with ischemia, reperfusion injury, and/or an immune-related disorder, such as an autoimmune disease or an inflammatory disorder. A subject or organ suffering from ischemia, a clinical indication associated with ischemia, reperfusion injury, an autoimmune disease or an inflammatory disorder is identified by methods known in the art. For example, subjects are identified using any of a variety of clinical and/or laboratory tests such as, physical examination, radiologic examination and blood, urine and stool analysis to evaluate immune status. For example, patients suffering from lupus are identified, e.g., by using the anti-nuclear antibody test (ANA) to determine if auto-antibodies to cell nuclei are present in the blood. Patients suffering from Crohn's are identified, e.g., using an upper gastrointestinal (GI) series and/or a colonoscopy to evaluate the small and large intestines, respectively. Patients suffering from psoriasis are identified, e.g., using microscopic examination of tissue taken from the affected skin patch, while patients suffering from rheumatoid arthritis are identified using, e.g., blood tests and/or x-ray or other imaging evaluation. Patients suffering from atherosclerosis are identified, e.g., using blood tests, electrocardiograms (ECG), stress testing, coronary angiography, ultrasound, and computed tomography (CT).

[0188] Administration of a RANTES antagonist, such as a huRANTES antibody, to a patient suffering from ischemia, a clinical indication associated with ischemia, reperfusion injury, or an immune-related disorder such as an autoimmune disease or an inflammatory disorder is considered successful if any of a variety of laboratory or clinical results is achieved. For example, administration of a huRANTES antibody to a patient suffering from ischemia, a clinical indication associated with ischemia, reperfusion injury, an immune-related disorder such as an autoimmune disease or an inflammatory disorder is considered successful one or more of the symptoms associated with the disorder is alleviated, reduced, inhibited or does not progress to a further, i.e., worse, state. Administration of a huRANTES antibody to a patient suffering from ischemia, a clinical indication associated with ischemia, reperfusion injury, an immune-related disorder such as an autoimmune disease or an inflammatory disorder is considered successful if the disorder, e.g., an autoimmune disorder, enters remission or does not progress to a further, i.e., worse, state.

[0189] Diagnostic and Prophylactic Formulations

[0190] The fully human anti-RANTES MAbs of the invention are used in diagnostic and prophylactic formulations. In one embodiment, a RANTES antagonist, such as a huRANTES MAb of the invention, is administered to patients that are at risk of developing ischemia, a clinical indication associated with ischemia, reperfusion injury, and/or one of the aforementioned autoimmune diseases. A patient's or organ's predisposition to ischemia, a clinical indication associated with ischemia, reperfusion injury, and/or one or more of the aforementioned autoimmune diseases can be determined using genotypic, serological or biochemical markers.

[0191] In another embodiment of the invention, a RANTES antagonist, such as a huRANTES antibody is administered to human individuals diagnosed with a clinical indication associated with ischemia, reperfusion injury, one or more of the aforementioned autoimmune diseases. Upon diagnosis, a RANTES antagonist, such as a huRANTES antibody is administered to mitigate or reverse the effects of the clinical indication associated with ischemia, reperfusion injury, or autoimmunity.

[0192] Antibodies of the invention are also useful in the detection of RANTES in patient samples and accordingly are useful as diagnostics. For example, the huRANTES antibodies of the invention are used in in vitro assays, e.g., ELISA, to detect RANTES levels in a patient sample.

[0193] In one embodiment, a huRANTES antibody of the invention is immobilized on a solid support (e.g., the well(s) of a microtiter plate). The immobilized antibody serves as a capture antibody for any RANTES that may be present in a test sample. Prior to contacting the immobilized antibody with a patient sample, the solid support is rinsed and treated with a blocking agent such as milk protein or albumin to prevent nonspecific adsorption of the analyte.

[0194] Subsequently the wells are treated with a test sample suspected of containing the antigen, or with a solution containing a standard amount of the antigen. Such a sample is, e.g., a serum sample from a subject suspected of having levels of circulating antigen considered to be diagnostic of a pathology. After rinsing away the test sample or standard, the solid support is treated with a second antibody that is detectably labeled. The labeled second antibody serves as a detecting antibody. The level of detectable label is measured, and the concentration of RANTES antigen in the test sample is determined by comparison with a standard curve developed from the standard samples.

[0195] It will be appreciated that based on the results obtained using the huRANTES antibodies of the invention in an in vitro diagnostic assay, it is possible to stage a disease (e.g., a clinical indication associated with ischemia, an autoimmune or inflammatory disorder) in a subject based on expression levels of the RANTES antigen. For a given disease, samples of blood are taken from subjects diagnosed as being at various stages in the progression of the disease, and/or at various points in the therapeutic treatment of the disease. Using a population of samples that provides statistically significant results for each stage of progression or therapy, a range of concentrations of the antigen that may be considered characteristic of each stage is designated.

[0196] All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an admission that any is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. The invention having now been described by way of written description, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description and examples below are for purposes of illustration and not limitation of the claims that follow.

EXAMPLES

[0197] The following examples, including the experiments conducted and results achieved are provided for illustrative purposes only and are not to be construed as limiting upon the present invention.

Example 1

Cloning, Expression and Purification of Human RANTES

Cloning

[0198] The gene encoding the mature protein human RANTES (GenBank Accession No. M21121) or other chemokines were cloned in an expression plasmid pET43 (Novagen Madison, Wis.) by PCR amplification. The sequence for the Factor X protease cleavage site was introduced at the C-terminus of NusA. The sequence for the AviTag (Avidity, Denver Colo.) biotinylation site was introduced at the C-terminus of the chemokine coding sequence. The pET-derived plasmids were used for the co-transformation of bacterial strain Origami B with pACYC184-BirA plasmid that encodes the biotin ligase gene. For expression in mammalian cells, the gene encoding relevant chemokines were cloned from cDNA in the pEAK8 expression vector (Edge Biosystems, Gaithersburg, Md.). An AviTag biotinylation site was introduced at the C-terminus of the protein followed by an internal ribosome entry site (IRES) allowing for the expression of the BirA gene encoding a biotin ligase. This construct allows for the secreted expression of chemokines biotinylated in vivo at a single site.

Expression of NusA-huRANTES Fusion Protein in E. coli

[0199] An overnight culture of bacteria harboring the expression construct was diluted 1:30 into Terrific broth (InvitroGen) containing 50 μg/mL Ampicillin, 10 μg/mL Kanamicin, 5 μg/mL Tetracycline, 20 μg/mL Chloramphenicol and 50 μM Biotin. The culture was incubated at 37° C. with shaking until OD 600=0.7 was reached. IPTG was then added to a final concentration of 1 mM, incubated for 15 min. at 37° C. and overnight at 25° C.

Expression of huRANTES in Mammalian Cells

[0200] PEAK cells were cultures in DMEM (Sigma) supplemented with 10% FCS, 2 mM L-Glutamine (Sigma), 25 μg/ml gentamycin (Sigma) and incubated at 37° C., 5% CO₂. PEAK cells were transfected with the modified pEAK8 vectors using Mirus transfection reagent. Puromycin (Sigma) was added at 1 μg/ml after cell adherence in order to select and maintain transfected cell populations. Biotin (Sigma) was added to production batches at 50 μM. Biotinylated chemokines from the transfected PEAK cell supernatants were shown to be active in chemotaxis assays.

Purification and Cleavage of Fusion Proteins

[0201] Bacterial pellets were resuspended in Bugbuster (Novagen) containing Benzonase Nuclease and protease inhibitor Complete EDTA-free (Roche) and incubated for 1 hour at 4° C. The soluble and insoluble fractions were separated by centrifugation at 10,000 g for 15 min at 4° C. Soluble and insoluble protein fractions were analyzed by SDS-PAGE (Novex gels, InvitroGen). The soluble fraction was diluted 1/2 with Buffer A (Tris-HCl 100 mM pH 8.0, NaCl 600 mM, CaCl₂ 10 mM, Imidazole 40 mM), mixed with 50% (v/v) Ni-NTA agarose (Qiagen) previously equilibrated in Buffer B (Tris-HCl 50 mM pH 8.0, NaCl 300 mM, CaCl₂ 5 mM, Imidazole 20 mM). The mixture was incubated for 30 min at RT with gentle shaking. The beads obtained after centrifugation were loaded in Poly-Prep chromatography columns (Biorad), washed three times with 5 volumes of Buffer B and eluted with Buffer C (Tris-HCl 50 mM pH 8.0, NaCl 200 mM, CaCl₂ 5 mM, Imidazole 400 mM). Elution fractions containing the protein were pooled and desalted using PD-10 columns (Amersham). NusA-chemokine fusion proteins were cleaved by Factor X (Novagen, Madison, Wis.) by incubating 1 mg protein with 25 U Factor X at 30° C. for up to 24 h in cleavage buffer (Tris-HCl 50 mM pH 8.0, NaCl 200 mM, CaCl₂ 5 mM). For some of the fusions proteins, the parameters for optimal cleavage were slightly different but were easily determined by varying incubation time (4-24 h) and/or temperature (25-37° C.). The cleaved protein was analyzed by SDS-PAGE and the activity tested by chemotaxis.

Example 2

Screening of Human scFv Libraries

[0202] General procedures for construction and handling of human scFv libraries are described in Vaughan et al., (Nat. Biotech. 1996, 14:309-314), hereby incorporated by reference in its entirety. Libraries of human scFv were screened against huRANTES according to the following procedure.

Liquid Phase Selections.

[0203] Aliquots of scFv phage libraries (10¹² Pfu) obtained from Cambridge Antibody Technology (Cambridge, UK) were blocked with PBS containing 3% (w/v) skimmed milk for one hour at room temperature on a rotary mixer. Blocked phage was then deselected on streptavidin magnetic beads (Dynal M-280) for one hour at room temperature on a rotary mixer. Deselected phage was then incubated with in vivo biotinylated huRANTES (100 nM) for two hours at room temperature on a rotary mixer. This selection step was performed either on NusA-huRANTES biotinylated fusion protein or on biotinylated-huRANTES released from the fusion by proteolytic cleavage. Beads were captured using a magnetic stand followed by four washes with PBS/0.1% Tween 20 and 3 washes with PBS. Beads were then directly added to 10 ml of exponentially growing TG1 cells and incubated for one hour at 37° C. with slow shaking (100 rpm). An aliquot of the infected TG1 was serial diluted to titer the selection output. The remaining infected TG1 were spun at 3000 rpm for 15 minutes and re-suspended in 0.5 ml 2×TY-AG (2×TY media containing 100 μg/ml ampicilin and 2% glucose) and spread on 2×TYAG agar Bioassay plates. After overnight incubation at 30° C. 10 ml of 2×TYAG was added to the plates and the cells were scraped form the surface and transferred to a 50 ml polypropylene tube. 2×TYAG containing 50% glycerol was added to the cell suspension to obtain a final concentration of 17% glycerol. Aliquots of the selection round were kept at -80° C.

Phage Rescue.

[0204] 100 μl of cell suspension obtained from previous selection rounds were added to 20 ml of 2×TYAG and grown at 37° C. with agitation (240 rpm) until an OD₆₀₀ of 0.3 to 0.5 was reached. The culture was then super-infected with 3.3×10¹⁰ MK13K07 helper phage and incubated for one hour at 37° C. (150 rpm). The medium was then changed by centrifugating the cells at 2000 rpm for 10 minutes, removing the medium and resuspending the pellet in 20 ml of 2×TY-AK (100 μg/ml ampicilin; 50 μg/ml kanamycin). The culture was then grown overnight at 30° C. (240 rpm).

Monoclonal Phage Rescue for ELISA.

[0205] Single clones were picked into a microtiter plate containing 150 μl of 2×TYAG media (2% glucose) per well and grown at 37° C. (100-120 rpm) for 5-6 h. M13K07 helper phage was added to each well to obtain a multiplicity of infection (MOI) of 10 (i.e., 10 phage for each cell in the culture) and incubated at 37° C. (100 rpm) for 1 h. Following growth, plates were centrifuged at 3,200 rpm for 10 min. Supernatant was carefully removed, cells re-suspended in 150 μl 2×TYAK medium and grown overnight at 30° C. (120 rpm). For the ELISA, the phage are blocked by adding 150 μl of 2× concentration PBS containing 5% skimmed milk powder followed by one hour incubation at room temperature. The plates were then centrifuged 10 minutes at 3000 rpm and the phage containing supernatant used for the ELISA.

Phage ELISA.

[0206] ELISA plates (Maxisorb, NUNC) were coated overnight with 2 μg/ml NusA-Rantes fusion protein in PBS. Control plates were coated with 2 μg/ml NusA. Plates were then blocked with 3% skimmed milk/PBS at room temperature for 1 h. Plates were washed 3 times with PBS 0.05% Tween 20 before transferring the pre-blocked phage supernatants and incubation for one hour at room temperature. Plates were then washed 3 times with PBS 0.05% Tween 20. 50 μl of 3% skimmed milk/PBS containing (HRP)-conjugated anti-M13 antibody (Amersham, diluted 1:10,000) to each well. Following incubation at room temperature for 1 hr, the plates were washed 5 times with PBS 0.05% Tween 20. The ELISA was then revealed by adding 50 μl of TMB (Sigma) and 50 μl of 2NH₂SO₄ to stop the reaction. Absorption intensity was read at 450 nm.

Phage Clone Sequencing

[0207] Single clones were placed in a microtiter plate containing 150 μl of 2×TYAG media (2% glucose) per well and grown at 30° C. (120 rpm) overnight. The next day 5 μl of culture was transferred into another plate containing 45 μl of dH₂O and mixed. The plates was then frozen at -20° C. After thawing, 1 μl of this suspension was used for PCR amplification using standard PCR protocols with primer specific for pCANTAB6: mycseq, 5'-CTCTTCTGAGATGAGTTTTTG-3' (SEQ ID NO: 197) and gene3leader, 5'-TTATTATTCGCAATTCCTTTAGTTGTTCCT-3' (SEQ ID NO: 198).

[0208] The PCR reactions were purified in 96 well format using the Montage PCRμ96 system (Millipore). 5 μl of the eluted DNA was sequencing using the mycseq and gene3leader primers.

ScFv Periplasmic Preparation for Functional Tests.

[0209] Individual clones were inoculated into a deep well microtiter plate containing 0.9 ml of 2×TYAG media (0.1% glucose) per well and grown at 37° C. for 5-6 h (250 rpm). 100 μl per well of 0.2 mM IPTG in 2×TY medium were then added to give a final concentration of 0.02 mM IPTG. Plates were then incubated overnight at 30° C. with shaking at 250 rpm. The deep-well plates were centrifuged at 2,500 rpm for 10 min and the supernatant carefully removed. The pellets were re-suspended in 150 μl TES buffer (50 mM Tris/HCl (pH 8), 1 mM EDTA (pH 8), 20% sucrose, complemented with Complete protease inhibitor, Roche). A hypotonic shock was produced by adding 150 μl of diluted TES buffer (1:5 TES:water dilution) and incubation on ice for 30 min. Plates were then centrifuged at 4000 rpm for 10 minutes to remove cells and debris. The supernatants were carefully transferred into another microtiter plate and kept on ice for immediate testing in functional assays or ELISAs.

Large Scale scFv Purification

[0210] A starter culture of 1 ml of 2×TYAG was inoculated with a single colony from a freshly streaked 2×TYAG agar plate and incubated with shaking (240 rpm) at 37° C. for 5 hours. 0.9 ml of this culture was used to inoculate a 400 ml culture of the same media and was grown overnight at 30° C. with vigorous shaking (300 rpm).

[0211] The next day the culture was induced by adding 400 μl of 1M IPTG and incubation was continued for an additional 3 hours. The cells were collected by centrifugation at 5,000 rpm for 10 minutes at 4° C. Pelleted cells were resuspended in 10 ml of ice-cold TES buffer complemented with protease inhibitors as described above. Osmotic shock was achieved by adding 15 ml of 1:5 diluted TES buffer and incubation for 1 hour on ice. Cells were centrifuged at 10,000 rpm for 20 minutes at 4° C. to pellet cell debris. The supernatant was carefully transferred to a fresh tube. Imidazole was added to the supernatant to a final concentration of 10 mM. 1 ml of Ni-NTA resin (Qiagen), equilibrated in PBS was added to each tube and incubated on a rotary mixer at 4° C. (20 rpm) for 1 hour.

[0212] The tubes were centrifuged at 2,000 rpm for 5 minutes and the supernatant carefully removed. The pelleted resin was resuspended in 10 ml of cold (4° C.) Wash buffer 1 (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH to 8.0). The suspension was added to a polyprep column (Biorad). 8 ml of cold Wash Buffer 2 (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, pH to 8.0) were used to wash the column by gravity flow. The scFv were eluted from the column with 2 ml of Elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH to 8.0). Fractions were analyzed by absorption at 280 nm and protein containing fractions were pooled before buffer exchange on a PD10 desalting column (Amersham) equilibrated with PBS. The scFv in PBS were analyzed by SDS-PAGE and quantified by absorption at 280 nm. The purified scFv were aliquoted and stored at -20° C. and at 4° C.

Example 3

Inhibition of huRANTES Induced Calcium Flux Using scFv Extracts

[0213] Periplasmic extracts of various huRANTES scFv were produced as described above. L1.2 cells expressing hCCR5 were cultured in RPMI medium supplemented with 10% FCS. Extracts containing the scFv were incubated with 2-10 nM of huRANTES (Peprotech, Rocky Hill N.J.) for 30 minutes at room temperature. Cells were washed in PBS and loaded with 2 μM Fura 2/AM. 100 μl of loaded cells were added to each well of a 96-well black, transparent flat-bottom plate and calcium flux kinetics were recorded by measuring the fluorescence at 514 nm upon excitation at 340 or 380 nm on a Flex station II instrument (Molecular Devices) upon addition of the chemokine scFv mix. The inhibitory activity of each scFv extract was assessed by comparison to an extract containing an irrelevant scFv.

Example 4

scFv Inhibition of huRANTES-Induced Cell Chemotaxis

[0214] Wild type L1.2 cells and L1.2 cells expressing hCCR5 were cultured in RPMI medium supplemented with 10% FCS. The day before the experiment cells were incubated with 0.6 mg/ml of butyric acid. Different concentrations of purified scFv were incubated with 0.2-10 nM huRANTES and placed in the bottom chamber of chemotaxis 96-well plate (Neuroprobe). The filter plate was placed on top of the chemotaxis plate and each well was overlaid with 20 μl of a 10⁶ cells/ml suspension. The plate was incubated for 2 hours at 37° C. Cells that migrated through the filter were stained with DRAQ5 (Alexis Corporation) and counted on an FMAT 8200 reader (Applied Biosystems, Foster City Calif.). The IC₅₀ (where 50% of the huRANTES induced cell migration is inhibited, i.e., 50% inhibitory concentration), for each candidate antibody was determined (Table 4).

TABLE-US-00026 TABLE 4 Potency of antibodies tested in scFv format in chemotaxis functional assays. Chemotaxis was performed using 1 nM, of huRANTES, Clone ID Chemotaxis IC₅₀ (nM) CG11 3.6 BG11 7 A9 72 E6 72 H6 25 G2 62 E10 9.4 C10 41 2D1 1.3 A5 21 H11 4.3 D1 22 E7 3.8 C8 90 1D9 0.82 1E4 1.25 3E7 0.47 4D8 0.08 5E1 0.2 6A8 0.94 7B5 1.6

Example 5

Reformatting scFv into IgG Format

[0215] The V_H and V_L sequence of selected scFv were amplified with specific oligonucleotides introducing a leader sequence and a HindIII restriction site at the 5' end. An ApaI or an AvrII site was introduced at the 3' end of the heavy and light chain sequence, respectively. The amplified V_H sequences were digested HindIII/ApaI and cloned into the pCon_gamma1 expression vector (LONZA, Basel, Switzerland). The amplified V_L sequences were digested HindIII/AvrII and cloned into the pCon_lambda2 expression vector (LONZA). The constructions were verified by sequencing before transfection into mammalian cells.

[0216] The V_H and V_L cDNA sequences in their appropriate expression vectors were transfected into mammalian cells using the Fugene 6 Transfection Reagent (Roche, Basel, Switzerland). Briefly, Peak cells were cultured in 6-well plates at a concentration of 6×10⁵ cells per well in 2 ml culture media containing fetal bovine serum. The expression vectors, encoding the candidate V_H and V_L sequences, were co-transfected into the cells using the Fugene 6 Transfection Reagent according to manufacturer's instructions. One day following transfection, the culture media was aspirated, and 3 ml of fresh serum-free media was added to cells and cultured for three days at 37° C. Following three days culture period, the supernatant was harvested for IgG purified on protein G-Sepharose 4B fast flow columns (Sigma, St. Louis, Mo.) according to manufacturer's instructions. Briefly, supernatants from transfected cells were incubated overnight at 4° C. with ImmunoPure (G) IgG binding buffer (Pierce, Rockford Ill.). Samples were then passed over Protein G-Sepharose 4B fast flow columns and the IgG consequently purified using elution buffer. The eluted IgG fraction was then dialyzed against PBS and the IgG content quantified by absorption at 280 nm. Purity and IgG integrity were verified by SDS-PAGE.

Example 6

Production of Native Human huRANTES

[0217] THP1 cells were cultured in 10 ml media at a concentration at 1×10⁶/ml with 10 μg/ml LPS. Following overnight incubation at 37° C., cells were centrifuged, supernatant was collected and the concentration of native huRANTES was estimated in a chemotaxis assay as described in Example 4. Not only native huRANTES but also other ligands of CCR5 are produced by THP1 cells when stimulated with LPS as described above. Therefore, when using these supernatants in chemotaxis assays to determine the neutralization potential of anti-huRANTES antibodies, the other ligands of CCR5 were neutralized with a mixture of antibodies against hMIP-1α, hMIP-1β, hMCP-2, hMIP-1δ each at a concentration of 5 μg/ml (R&D Systems).

Example 7

Inhibition of huRANTES-Induced Calcium Flux or Cell Chemotaxis Using Reformatted scFv into IgG1 Format

[0218] scFv were reformatted into an IgG format as described above in Example 5. The neutralizing potential of the IgG on huRANTES-induced calcium flux or cell chemotaxis was evaluated using the cell-based assays described in Example 3 and 4. As shown as examples in FIG. 1 IgGs C8, 1D9 and 1E4 inhibit the activity of both recombinant and native huRANTES in a dose-dependent manner. The IC₅₀ values in these assays for all antibodies are summarized in Tables 5 and 6.

TABLE-US-00027 TABLE 5 Potency of antibodies tested in IgG1 format in chemotaxis and calcium flux functional assays. Chemotaxis was performed using either 1 nM or 0.2 nM of recombinant huRANTES while calcium flux was induced with 10 nM of recombinant huRANTES. Chemotaxis Chemotaxis Calcium Flux Clone IC₅₀ (nM) IC₅₀ (nM) IC₅₀ (nM) ID 1 nM huRANTES 0.2 nM huRANTES rhuRANTES CG11 4.8 ND 9.5 BG11 29 ND 7.7 A9 1 ND 3.3 E6 14 ND 12.7 H6 8.7 ND 9 G2 18.4 ND ND E10 16 ND ND C10 17 ND ND 2D1 1.7 1.3 ND A5 13.2 ND ND H11 1.3 ND ND D1 7 ND ND E7 2.2 ND ND C8 2.1 0.49 ND 1D9 0.35 0.038 ND 1E4 0.46 0.034 ND 3E7 0.68 0.25 ND 4D8 1.16 0.22 ND 5E1 0.82 0.25 ND 6A8 0.74 0.31 ND 7B5 1.1 0.31 ND

TABLE-US-00028 TABLE 6 Potency of antibodies tested in IgG1 format in chemotaxis functional assay performed using native human RANTES produced by THP1 cells at a concentration of <1 nM. Chemotaxis IC₅₀ (nM) >1 nM native Clone ID huRANTES C8 1.6 1D9 0.033 1E4 0.028

Example 8

Antibody Binding to huRANTES Immobilized on Glycosaminoglycan

[0219] As with many chemokines, huRANTES is able to oligomerize and bind to glycosaminoglycans (GAG) expressed at surface of cells such as endothelial cells. In order to make sure that the antibodies were able to bind to huRANTES in this context, they were tested in the following assay. Streptavidin coated 96 well plates (Roche, Basel, Switzerland) were coated with biotin labeled heparin as a prototypic GAG (Sigma, St. Louis, Mo.). After washing the excess heparin huRANTES was added the wells for immobilization GAG. After incubation with the antibodies to be tested, the wells were washed and binding was revealed with an anti-human IgG Fcg specific antibody coupled to HRP (Jackson, West Grove, Pa.). As shown in FIG. 2 some antibodies were able to bind huRANTES when bound to GAG whereas others were unable to do so probably because their epitope on huRANTES was no longer accessible within the oligomeric structure. The capacity of the antibodies to bind huRANTES in the context of GAG is summarized in Table 7.

TABLE-US-00029 TABLE 7 Ability of antibodies to bind to huRANTES immobilized on GAG. Binding to huRANTES Clone ID immobilized on GAGs CG11 No BG11 Yes A9 No E6 Yes H6 No G2 Yes E10 Yes C10 Yes 2D1 Yes A5 Yes H11 No D1 No E7 No C8 Yes 1E4 Yes 1D9 Yes

Example 9

Affinity Maturation of Antibody 2D1

[0220] A selected lead candidate (2D1) was subjected to affinity maturation in order to increase its affinity for huRANTES and its potency in huRANTES neutralization assays. Stretches of 5 residues in the CDR3 of the heavy or light chain were randomized in order to generate 6 libraries (Library size ranging from 5×10⁷ to 10⁹). Three high stringency selection rounds were performed as described in Example 2. Screening for improved variant was performed using scFv periplasmic extracts in an epitope competition assay. Briefly, the parent antibody (2D1) was coated on plates and diluted periplasmic scFv extracts were added to each well. Biotinylated huRANTES was then added and incubated for 2 hours at room temperature. After washing, huRANTES remaining bound to the coated parent antibody was revealed using streptavidin coupled HRP (Jackson, West Grove Pa.). As a reference to identify improved variants 2D1 scFv was used to compete coated 2D1 in an IgG format.

Example 10

Generation of a Stable CHOK1SV Cell Line Expressing 1E4

[0221] The CHOK1SV cell line, property of Lonza Biologics, plc, was used to generate pools through semi-stable transfection for the production of the antibody 1E4. Briefly, exponentially growing cells in the medium CD-CHO (Invitrogen) supplemented with 6 mM of L-glutamine, were electroporated under the following conditions: in a 0.4 cm cuvette, 1.0×10⁷ viable cells in 700 μL of fresh CD-CHO were gently mixed with 40 μg of DNA in 100 μL of Tris EDTA buffer solution, pH 7,4, immediately followed by delivering of a single pulse of 300 volts, 900 μF. The contents of 2 cuvettes were immediately transferred in 200 mL of fresh pre-warmed CD-CHO medium. This cell suspension was subsequently distributed in 4 tissue culture-treated T75 flasks and placed in a humidified incubator set at 10% CO₂ in air and a temperature of 37° C. to generate semi-stable pools. Around twenty-four hours after transfection, selective pressure (by MSX supplementation at 50 μM) was applied. Individual stably transfected clones were then selected using ClonePix technology (Genetix) and screened for 1E4 productivity.

Example 11

Large Scale Purification of 1E4 from CHO Supernatant

[0222] The process involves MabSelect SuRe affinity chromatography (GE Healthcare), retrovirus inactivation by low pH treatment, pH adjustment for SP Sepharose cation exchange chromatography, concentration/diafiltration before Capto Q (GE Healthcare) anion exchange chromatography and concentration/diafiltration into final formulation buffer.

[0223] Briefly, supernatant produced by 25 L Wave Bag fermentation of clone was clarified and captured on MabSelect SuRe Protein A Affinity column with an overall recovery of 95% at 80% of the maximum loading capacity (32 mg of Antibody per mL of matrix). The eluate was proven to be stable at elution pH up to 48 h. The stability of the 1E4 antibody was also evaluated at the low pH (3.7) used for viral inactivation and the Antibody was stable up to 48 h.

[0224] The pool of Protein A eluates was then loaded onto an SP Sepharose cation exchange column after pH adjustment (pH 5). This step was optimised for efficient residual aggregate removal, the optimal elution buffer was found to be 107 mM Sodium Chloride (in 25 mM Sodium Acetate pH 5). A concentration/diafiltration step was then used to buffer exchange the 1E4 antibody into the appropriate buffer for Capto Q Chromatography (25 mM Sodium Acetate, 40 mM Sodium Chloride pH 5). A concentration of about 50 mg/mL was reached without any problems of degradation or aggregation. The Capto Q Chromatography in nonbinding mode was optimised for efficient contaminant removal (Host Cell Proteins, Protein A and DNA). Antibody 1E4 was finally concentrated and diafiltered into the 25 mM Histidine, 125 mM NaCl, pH 6 formulation buffer to a final concentration of about 10 mg/mL.

[0225] Antibody 1E4 did not show a tendency to aggregate throughout the purification process, and presented good stability across the purification process. The final product reached all prerequisite specifications in terms of aggregates levels and residual contamination.

Example 12

Functional Characterization of Antibody 1E4 Purified Form CHO Supernatant

[0226] RANTES is a ligand for the receptors CCR1, CCR3 and CCR5. The capacity of 1E4 purified from CHO supernatants to block the interaction with each one of these receptors was assessed in chemotaxis and calcium flux assays.

Calcium Flux

[0227] L1.2 cells expressing either hCCR1, hCCR3 or hCCR5 were cultured in RPMI medium supplemented with 10% FCS. For optimal results, cells expressing hCCR1 were starved overnight in medium containing 1% of FCS. The day before the experiment all cells were incubated with 0.3 mg/ml of butyric acid. Different concentrations of 1E4 were incubated with 4 to 25 nM of huRANTES (Peprotech, Rocky Hill N.J.) for 30 minutes at room temperature. Cells were washed in PBS and loaded with 2 μM Fura 2/AM. 100 μl of loaded cells were added to each well of a 96-well black, transparent flat-bottom plate and calcium flux kinetics were recorded by measuring the fluorescence at 514 nm upon excitation at 340 or 380 nm on a Flex station II instrument (Molecular Devices) after addition of the chemokine-antibody mix. As shown in FIG. 3, 1E4 was able to inhibit calcium flux in cells that express either hCCR1, hCCR3 or hCCR5 in a dose dependent manner. The IC₅₀ (where 50% of the huRANTES induced calcium flux is inhibited, i.e., 50% inhibitory concentration) was determined (Table 8).

TABLE-US-00030 TABLE 8 Potency of antibody 1E4 purified from CHO supernatant in calcium flux functional assay using cells expressing the one three cognate receptors of RANTES. Cells and concentration of huRANTES used for calcium flux induction IC₅₀ (nM) L1.2-hCCR1; 25 nM huRANTES 4.9 L1.2-hCCR3; 25 nM huRANTES 4.46 L1.2-hCCR5; 4 nM huRANTES 0.54

Chemotaxis

[0228] Wild type L1.2 cells and L1.2 cells expressing either hCCR1, hCCR3 or hCCR5 were cultured in RPMI medium supplemented with 10% FCS. For optimal results, cells expressing hCCR1 were starved overnight in medium containing 1% of FCS. The day before the experiment all cells were incubated with 0.3 mg/ml of butyric acid. For optimal results, cells expressing hCCR1 were starved overnight in medium containing 1% of FCS. Different concentrations of 1E4 were incubated with 1-10 nM of recombinant huRANTES or 1 nM of native huRANTES (generated as described in example 6) and placed in the bottom chamber of chemotaxis 96-well plate (Neuroprobe). The filter plate was placed on top of the chemotaxis plate and each well was overlaid with 20 μl of a 10⁶ cells/ml suspension. The plate was incubated for 2 hours at 37° C. Cells that migrated through the filter were stained with DRAQ5 (Alexis Corporation) and counted on an FMAT 8200 reader (Applied Biosystems, Foster City Calif.). As shown in FIG. 4, 1E4 was able to inhibit calcium flux in cells that express either hCCR1, hCCR3 or hCCR5 in a dose dependent manner. The IC₅₀ (where 50% of the huRANTES induced cell migration is inhibited, i.e., 50% inhibitory concentration) was determined (Table 9).

TABLE-US-00031 TABLE 9 Potency of antibody 1E4 purified from CHO supernatant in chemotaxis functional assay using cells expressing the one three cognate receptors of RANTES. Cells and concentration of huRANTES used for chemotaxis assays IC₅₀ (nM) L1.2-hCCR1; 2 nM huRANTES 0.46 L1.2-hCCR3; l0 nM huRANTES 3.33 L1.2-hCCR5; 1 nM huRANTES 0.2 L1.2-hCCR5; 1 nM native huRANTES 0.09

Example 13

Cross-Reactivity of 1E4 Antibody

[0229] 1E4 was tested for its ability to bind to a panel of chemokines from different species in an ELISA. The panel included the following chemokines: human RANTES, cynomolgus monkey RANTES, rat RANTES, mouse RANTES, human ITAC, human IP-10, cynomolgus monkey IP-10, human MIG, cynomolgus monkey MIG, human MIP1α, human MIP1β, human MCP-1, human MCP-2. Briefly, chemokines cloned from cDNA isolated from human, mouse, rat, and cynomolgus monkey were expressed as fusion proteins and purified as described in Example 1. The chemokines were coated at 5 μg/ml in an maxisopb plate (Nunc, Denmark) and incubated with a concentration range of 1E4. The level of binding was revealed using an anti-human Fc-γ specific antibody coupled to horse radish peroxidase (Jackson) and a fluorescent substrate. As shown in FIG. 5, the antibody 1E4 only binds to human and cynomolgus RANTES and not with RANTES from other species nor with any of the other human chemokines tested. Proper coating of all the chemokines was controlled using monoclonal antibodies directed against each chemokine and all the chemokines tested could be detected in this format.

Example 14

Epitope Mapping of 1E4 Antibody

[0230] In an ELISA, the antibody 1E4 binds with equivalent apparent affinity to both human and cynomolgus RANTES (FIG. 5). In order to identify residues potentially required on huRANTES for binding to 1E4, the RANTES protein sequences from several species were aligned as shown in FIG. 6. In the alignment, residues that are conserved between the human and cynomolgus sequences and that are different in mouse and rat RANTES to which 1E4 is unable to bind were analyzed to identify the following amino acids: A16, R17, P18, G32, P37, R59 and S64. Three mutants of mouse RANTES were generated by site directed mutagenesis in order to introduce the human residues at those positions: [S16A/L17R/A18P]; [S32G/L37P] and [Q59R/Y64S]. These mutant forms of mouse RANTES were expressed and biotinylated in vivo as described in Example 1. These variant of mouse RANTES were captured in streptavidin coated plates (Streptawell, Roche). The coating of the biotinylated chemokine was confirmed using a anti-mouse RANTES polyclonal antibody (R&D Systems). It was then tested whether the introduction of these residues could restore 1E4 binding to mouse RANTES. Briefly, mouse RANTES, human RANTES as well as three mutant forms of mouse RANTES and control supernatants were captured in Streptawell plates (Roche) for 30 minutes at room temperature. After washing, antibody 1E4 was added at a concentration of 1 μg/ml in 1% BSA-PBS and incubated for 1 hour at room temperature. The plate was washed and incubated with a goat anti-human IgG Fcγ-specific antibody coupled to horse radish peroxidase (Jackson). After washing the signal was revealed with TMB (Roche) and stopped with H₂SO₄. The plates were read at 450 nm. As shown in FIG. 7, the [S16A/L17R/A18P] mutant restores binding of 1E4 to mouse RANTES indicating that A16, R17 and P18 are critical for the 1E4 epitope integrity on human RANTES.

Example 15

Affinity and Binding Kinetics of 1E4

[0231] The affinity and binding kinetics of 1E4 on human RANTES and cynomolgus RANTES were characterized on a Biacore 2000 instrument (Biacore AB, Uppsala, Sweden). 433 RU (response unit) of a donkey anti-human IgG polyclonal antibody were immobilized by EDC/NHS chemistry on a C1 Biacore chip. This surface was used to capture antibody 1E4. The surface was regenerated after each cycle by injection of 10 mM glycine pH=2 at 30 μL/min, for 60 s followed by 1 min. of stabilization time in HBS-EP buffer.

[0232] Binding was measured by passing either huRANTES (Peprotech, Rocky Hill N.J.) or a NusA-fusion proteins of human RANTES (NusA-huRANTES) and Cynomolgus RANTES (NusA-cynoRANTES) at various concentrations. All proteins were diluted in the running buffer HBS-EP buffer (Biacore AB, Uppsala, Sweden). Injection was performed at 75 μl/min for 3 min. followed by 15 min. of dissociation time and the temperature was set at 25° C. The data was fitted according to 1:1 Langmuir model and the K_on, K_off and K_D values determined. Very similar values were obtained using huRANTES or the NusA-huRANTES fusion, but better response signals were obtained with the fusion protein due to its larger size that induces a better response on the Biacore. The affinity of antibody 1E4 for huRANTES and cynoRANTES are 0.45 nM and 2.24 nM, respectively. The Affinities and kinetic constants of both antibodies are summarized in Table 10.

TABLE-US-00032 TABLE 10 Kinetic and affinity constants of antibody 1E4 for human and cynomolgus RANTES measured by Biacore. huRANTES NusA-huRANTES NusA-cynoRANTES Ka (1/Ms) 5.36 × 10⁶ 1.87 × 10⁶ 5.46 × 10⁶ Kd (1/s) 2.44 × 10^-31 8.35 × 10^-4 1.22 × 10^-3 KD (M) 4.55 × 10^-10 4.47 × 10^-10 2.24 × 10^-9

Example 16

Animal Model of Ischemia

Materials and Methods

[0233] Animals:

[0234] Eight to 12 week old C57BL/6 mice are used for the experiments. All animal studies were approved by the local ethical Committee.

[0235] Antibodies and In Vivo Treatment:

[0236] C57BL/6 mice were injected either in the peritoneal cavity (i.p.) or intravenously (i.v.). For the ischemia followed by reperfusion model, monoclonal antibodies (mAb) were injected 5 minutes before the end of the occlusion period. For the permanent ligation model, mAbs were injected 5 minutes after the chronic ligature was put in place. The mAbs included: (1) the rat anti-mouse RANTES (mRANTES), mAb478 and (2) the rat anti-mouse isotype mAb control, mAb64. Hybridomas that produced mAb478 or mAb64 were obtained from R&D or the American Tissue Culture Collection, respectively, and all mAb were produced, purified and stored in-house.

[0237] For the i.p. treatment, 1 mg/mouse of the IgG control or anti-mRANTES mAbs was administered. For the i.v. treatment, either 0.1, 0.3, 0.5 or 1 mg/mouse of the anti-mRANTES mAb or 1 mg/mouse of the IgG control (i.e. highest dose of anti-mRANTES) was administered.

In Vivo Ischemia-Reperfusion or Ligation Permanent Model

[0238] Surgery:

[0239] Mice were initially anesthetized with 4% isofluorane then intubated. Mechanical ventilation was performed with a tidal volume of 300 μL at 120 breaths using a rodent respirator (model 683; Harvard Apparatus). Anesthesia was maintained with 2% isofluorane delivered 100% through the ventilator. A thoracotomy was performed in the left fourth intercostal space, and the pericardial sac was then removed. An 8-0 Prolene suture was passed under the left coronary artery at the inferior edge of the left atrium and tied with a slipknot to produce occlusion.

[0240] For the reperfusion model, a small piece of polyethylene tubing was used to secure the ligature without damaging the artery and after 30 minutes of ischemia, the left anterior descending (LAD) coronary artery occlusion was released and reperfusion permitted to occur.

[0241] For the permanent legation model, the LAD coronary artery was irreversibly occluded by using a double knot 8-0 Prolene suture. The chest was then closed and air was evacuated from the chest cavity. The endotracheal tube was then removed and normal respiration restored.

[0242] After 24 hours of reperfusion or after 24 hours of permanent occlusion, animals were euthanized to determine infarct size.

[0243] Evaluation of Risk Zone and Infarct Size:

[0244] At the end of the reperfusion period, mice were re-anesthetized with 0.3 mL ketamine-xylazine and the LAD coronary artery was re-ligated. 3% Evans Blue dye (Sigma) was injected i.v. (retro-orbital administration) to delineate the in vivo risk zone (R). The heart was rapidly excised and rinsed in saline. After removal of the right ventricle and connective tissues, the heart was frozen and then sectioned into 3-mm transverse sections from apex to base (5 slices/heart). Following thawing, the sections were incubated at 37° C. with 1% triphenyltetrazolium chloride in phosphate buffer (pH7.4) for 15 min, fixed in 10% formaldehyde solution and, after 24 hours, photographed with a digital camera to distinguish areas of stained viable versus unstained necrotic tissue. Left ventricular infarct zone (I) was determined using a computerized planimetric technique (MetaMorph6 software, Zeiss) and expressed as a percentage of either the area at risk (AAR) or ventricular area (V).

Example 17

Effect of Inhibiting RANTES in Ischemia Reperfusion Models

Model 1: Ischemia Reperfusion

[0245] A diagram illustrating the protocol of the murine ischemia reperfusion model is shown in FIG. 8. In this protocol, B6 mice are divided into three groups and administered a vehicle control (PBS), an isotype control (mAb 64 described in Example 10) or a rat anti-mRANTES monoclonal antibody according to the following schedule:

[0246] Group 1: PBS administered i.p. or i.v. 5 minutes prior to reperfusion;

[0247] Group 2: rat IgG2a (mAb 64; isotype control) administered i.p. (1 mg/mouse) or i.v. (1.0 mg/mouse) 5 minutes prior to reperfusion;

[0248] Group 3: rat anti-mouse RANTES (mAb 478) administered i.p. (1 mg/mouse) or i.v. (0.1, 0.3, 0.5, 1.0 mg/mouse) 5 minutes prior to reperfusion;

[0249] All animals were killed 24 hours post-reperfusion. Each group of mice was evaluated by assessing the following parameters:

[0250] weight of mice;

[0251] AAR/V=area at risk divided by the total area of heart (ischemic zone);

[0252] I/AAR=infarcted area divided by the area at risk; and

[0253] I/V=infarcted area divided by the total area of the ventricles.

[0254] Both I/AAR and I/V provide data on extent of infarcted tissue.

[0255] As shown in FIG. 9, treatment with the anti-RANTES monoclonal antibody decreased infarct size in the murine model of ischemia reperfusion provided herein. Injecting mAb 478 (1 mg/mouse i.p.) five minutes prior to reperfusion significantly decreased the infarct size as compared to isotype control or PBS treated mice. Data represents 20 mice per group.

[0256] FIG. 10 demonstrates that treatment with the anti-RANTES monoclonal antibody decreased infarct size in the murine model of ischemia reperfusion in a dose-dependent manner. Injecting mAb 478 i.v. (at doses of 0.1, 0.3, 0.5, 1.0 mg/mouse) five minutes prior to reperfusion significantly decreased the infarct size at higher doses as compared to isotype control (1 mg/mouse). Data represents 3 mice per group.

Model 2: Permanent Occlusion

[0257] A diagram illustrating the protocol of the murine permanent occlusion model is shown in FIG. 11. In this protocol, B6 mice are divided into three groups and administered a vehicle control (PBS), an isotype control (mAb 64 described in Example 10) or a rat anti-mRANTES monoclonal antibody according to the following schedule:

[0258] Group 1: PBS administered i.p. or i.v.;

[0259] Group 2: rat IgG2a (mAb 64; isotype control) administered i.p. (1 mg/mouse) or i.v. (1.0 mg/mouse);

[0260] Group 3: rat anti-mouse RANTES (mAb 478) administered i.p. (1 mg/mouse) or i.v. (0.1, 0.3, 0.5, 1.0 mg/mouse).

[0261] Each group of mice was evaluated by assessing the following parameters:

[0262] weight of mice;

[0263] AAR/V=area at risk divided by the total area of heart (ischemic zone);

[0264] I/AAR=infarcted area divided by the area at risk; and

[0265] I/V=infarcted area divided by the total area of the ventricles.

[0266] All animals were killed at 24 hrs post occlusion.

[0267] As shown in FIG. 12, treatment with the anti-RANTES monoclonal antibody decreased infarct size in the murine model of ischemia provided herein. Injecting mAb 478 (1 mg/mouse i.p.) significantly decreased the infarct size as compared to isotype control or PBS treated mice. Data represents 10 mice per group.

[0268] FIG. 13 demonstrates that treatment with the anti-RANTES monoclonal antibody decreased infarct size in the murine model of ischemia in a dose-dependent manner. Injecting mAb 478 i.v. (at doses of 0.1, 0.3, 0.5, 1.0 mg/mouse) significantly decreased the infarct size at higher doses as compared to isotype control (1 mg/mouse). Data represents 3 mice per group.

Other Embodiments

[0269] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Sequence CWU 1

1

2551357DNAArtificial Sequencechemically synthesized 1caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60tcctgcaagg tttccggata caccctcact gagttcgcca tgcactgggt gcgacaggct 120cctggaaaag ggcttgagtg gatgggaggt tttgttcctg aagatggtga gacaatctac 180gcgcagaagt tccagggcag agtcaccatg accgaggaca catctacaga cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aacagatccc 300ctgtatactc cgggtcttga gccttggggg caggggacca cggtcaccgt ctcgagt 3572119PRTArtificial Sequencechemically synthesized 2Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu Phe 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Gly Phe Val Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asp Pro Leu Tyr Thr Pro Gly Leu Glu Pro Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 3324DNAArtificial Sequencechemically synthesized 3tcctatgtgc tgactcagcc accctcggtg tcagtggccc caggacagac ggccaggatt 60acctgtgggg gaaacaacat tgaaagtaaa agtgtgcact ggtaccagca gaagccaggc 120caggcccctg tgctggtggt ctatgatgat agcgaccggc cctcagggat ccctgagcga 180ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240gatgaggccg actattactg tcaggtgtgg gatagtaata ctgatcattg ggtgttcggc 300ggagggacca agctcaccgt ccta 3244108PRTArtificial Sequencechemically synthesized 4Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Glu Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Thr Asp His 85 90 95 Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 515DNAArtificial Sequencechemically synthesized 5gagttcgcca tgcac 15651DNAArtificial Sequencechemically synthesized 6ggttttgttc ctgaagatgg tgagacaatc tacgcgcaga agttccaggg c 51730DNAArtificial Sequencechemically synthesized 7gatcccctgt atactccggg tcttgagcct 3085PRTArtificial Sequencechemically synthesized 8Glu Phe Ala Met His 1 5 917PRTArtificial Sequencechemically synthesized 9Gly Phe Val Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 1010PRTArtificial Sequencechemically synthesized 10Asp Pro Leu Tyr Thr Pro Gly Leu Glu Pro 1 5 10 1133DNAArtificial Sequencechemically synthesized 11gggggaaaca acattgaaag taaaagtgtg cac 331221DNAArtificial Sequencechemically synthesized 12gatgatagcg accggccctc a 211333DNAArtificial Sequencechemically synthesized 13caggtgtggg atagtaatac tgatcattgg gtg 331411PRTArtificial Sequencechemically synthesized 14Gly Gly Asn Asn Ile Glu Ser Lys Ser Val His 1 5 10 157PRTArtificial Sequencechemically synthesized 15Asp Asp Ser Asp Arg Pro Ser 1 5 1611PRTArtificial Sequencechemically synthesized 16Gln Val Trp Asp Ser Asn Thr Asp His Trp Val 1 5 10 17357DNAArtificial Sequencechemically synthesized 17caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60tcctgcaagg tttccggata caccctcact gagttcgcca tgcactgggt gcgacaggct 120cctggaaaag ggcttgagtg gatgggaggt tttgttcctg aagatggtga gacaatctac 180gcgcagaagt tccagggcag agtcaccatg accgaggaca catctacaga cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aacagatccc 300ctgtatgagg gttcgttttc tgtttggggg caggggacca cggtcaccgt ctcgagt 35718119PRTArtificial Sequencechemically synthesized 18Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu Phe 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Gly Phe Val Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asp Pro Leu Tyr Glu Gly Ser Phe Ser Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 1930DNAArtificial Sequencechemically synthesized 19gatcccctgt atgagggtcc gttttctgtt 302010PRTArtificial Sequencechemically synthesized 20Asp Pro Leu Tyr Glu Gly Ser Phe Ser Val 1 5 10 21366DNAArtificial Sequencechemically synthesized 21caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt agctatgcta tgcactgggt ccgccaggct 120ccaggcaagg ggctagagtg ggtggcagtt atatcatatg atggaagtaa taaatactac 180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gagagaaact 300ttcccccact actactacta ctacatggac gtctggggcc ggggcaccct ggtcaccgtc 360tcgagt 36622122PRTArtificial Sequencechemically synthesized 22Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Thr Phe Pro His Tyr Tyr Tyr Tyr Tyr Met Asp Val Trp 100 105 110 Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 23324DNAArtificial Sequencechemically synthesized 23tcctatgtgc tgactcagcc cccctcggtg tcagtggccc cagggcagac ggcccgcatt 60acctgtgagg gagacgacac tgacattggt actgtcaact ggtaccagca gaaaccaggc 120caggcccctg tgttggtcat tagtgaggat ggctaccggc cctcagggat ccctgaacga 180ttctctggct ccaactctgg gaacacggcc acccttacca tctccagggt cgaggccggg 240gatgaggccg actattactg tcagttctgg gatgttgaca gtgatcatcc ggttttcggc 300ggagggaccc agctcaccgt ccta 32424108PRTArtificial Sequencechemically synthesized 24Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Glu Gly Asp Asp Thr Asp Ile Gly Thr Val 20 25 30 Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Ser 35 40 45 Glu Asp Gly Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Phe Trp Asp Val Asp Ser Asp His 85 90 95 Pro Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu 100 105 2515DNAArtificial Sequencechemically synthesized 25agctatgcta tgcac 152651DNAArtificial Sequencechemically synthesized 26gttatatcat atgatggaag taataaatac tacgcagact ccgtgaaggg c 512739DNAArtificial Sequencechemically synthesized 27gaaactttcc cccactacta ctactactac atggacgtc 39285PRTArtificial Sequencechemically synthesized 28Ser Tyr Ala Met His 1 5 2917PRTArtificial Sequencechemically synthesized 29Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 3013PRTArtificial Sequencechemically synthesized 30Glu Thr Phe Pro His Tyr Tyr Tyr Tyr Tyr Met Asp Val 1 5 10 3133DNAArtificial Sequencechemically synthesized 31gagggagacg acactgacat tggtactgtc aac 333221DNAArtificial Sequencechemically synthesized 32gaggatggct accggccctc a 213333DNAArtificial Sequencechemically synthesized 33cagttctggg atgttgacag tgatcatccg gtt 333411PRTArtificial Sequencechemically synthesized 34Glu Gly Asp Asp Thr Asp Ile Gly Thr Val Asn 1 5 10 357PRTArtificial Sequencechemically synthesized 35Glu Asp Gly Tyr Arg Pro Ser 1 5 3611PRTArtificial Sequencechemically synthesized 36Gln Phe Trp Asp Val Asp Ser Asp His Pro Val 1 5 10 37357DNAArtificial Sequencechemically synthesized 37caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60tcctgcaagg tttccggata caccctcaat gacttcgcca tgcactgggt gcgacaggct 120cctggaaaag ggcttgagtg gatgggaggt tatgttcctg aagatggtga cacaatctac 180gcgcagaagt tccagggcag agtcaccatg accgaggaca catctacaga cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aacagatccc 300ctgtatccgc ctgggctgtc tccttggggg caggggacca cggtcaccgt ctcgagt 35738119PRTArtificial Sequencechemically synthesized 38Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Asn Asp Phe 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Gly Tyr Val Pro Glu Asp Gly Asp Thr Ile Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asp Pro Leu Tyr Pro Pro Gly Leu Ser Pro Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 39324DNAArtificial Sequencechemically synthesized 39tcctatgtgc tgactcagcc accctcggtg tcagtggccc caggacagac ggccaggatt 60acctgtgggg gaaacaacat tgaaagtaaa agtgtgcact ggtaccagca gaagccaggc 120caggcccctg tgctggtggt ctatgatgat agcgaccggc cctcagggat ccctgagcga 180ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240gatgaggccg actattactg tcaggtgtgg gatagtaata ctgatcattg ggtgttcggc 300ggagggacca aggtcaccgt ccta 32440108PRTArtificial Sequencechemically synthesized 40Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Glu Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Thr Asp His 85 90 95 Trp Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105 4115DNAArtificial Sequencechemically synthesized 41gacttcgcca tgcac 154251DNAArtificial Sequencechemically synthesized 42ggttatgttc ctgaagatgg tgacacaatc tacgcgcaga agttccaggg c 514330DNAArtificial Sequencechemically synthesized 43gatcccctgt atccgcctgg gctgtctcct 30445PRTArtificial Sequencechemically synthesized 44Asp Phe Ala Met His 1 5 4517PRTArtificial Sequencechemically synthesized 45Gly Tyr Val Pro Glu Asp Gly Asp Thr Ile Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 4610PRTArtificial Sequencechemically synthesized 46Asp Pro Leu Tyr Pro Pro Gly Leu Ser Pro 1 5 10 47357DNAArtificial Sequencechemically synthesized 47caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60tcctgcaagg tttccggata caccctcaat gacttcgcca tgcactgggt gcgacaggct 120cctggaaaag ggcttgagtg gatgggaggt tatgttcctg aagatggtga cacaatctac 180gcgcagaagt tccagggcag agtcaccatg accgaggaca catctacaga cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aacagatccc 300ctgtatacgc ctggtctgta tgtgtggggg caggggacca cggtcaccgt ctcgagt 35748119PRTArtificial Sequencechemically synthesized 48Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Asn Asp Phe 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Gly Tyr Val Pro Glu Asp Gly Asp Thr Ile Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asp Pro Leu Tyr Thr Pro Gly Leu Tyr Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 4930DNAArtificial Sequencechemically synthesized 49gatcccctgt atacgcctgg tctgtatgtg 305010PRTArtificial Sequencechemically synthesized 50Asp Pro Leu Tyr Thr Pro Gly Leu Tyr Val 1 5 10 51357DNAArtificial Sequencechemically synthesized 51caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60tcctgcaagg tttccggata caccctcaat gacttcgcca tgcactgggt gcgacaggct 120cctggaaaag ggcttgagtg gatgggaggt tatgttcctg aagatggtga cacaatctac 180gcgcagaagt tccagggcag agtcaccatg accgaggaca catctacaga cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aacagattat 300ttgtatattc ctagcttatc ctactggggg caggggacca cggtcaccgt ctcgagt 35752119PRTArtificial Sequencechemically synthesized 52Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Asn Asp Phe 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Gly Tyr Val Pro Glu Asp Gly Asp Thr Ile Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asp Tyr Leu Tyr Ile Pro Ser Leu Ser Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 5330DNAArtificial Sequencechemically synthesized 53gattatttgt atattcctag cttatcctac 305410PRTArtificial Sequencechemically synthesized 54Asp Tyr Leu Tyr Ile Pro Ser Leu Ser Tyr 1 5 10 55357DNAArtificial Sequencechemically synthesized 55caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60tcctgcaagg tttccggata

caccctcaat gacttcgcca tgcactgggt gcgacaggct 120cctggaaaag ggcttgagtg gatgggaggt tatgttcctg aagatggtga cacaatctac 180gcgcagaagt tccagggcag agtcaccatg accgaggaca catctacaga cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aacagatccc 300ctgtatcctc cggggctgca gccttggggg caggggacca cggtcaccgt ctcgagt 35756119PRTArtificial Sequencechemically synthesized 56Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Asn Asp Phe 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Gly Tyr Val Pro Glu Asp Gly Asp Thr Ile Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asp Pro Leu Tyr Pro Pro Gly Leu Gln Pro Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 5730DNAArtificial Sequencechemically synthesized 57gatcccctgt atcctccggg gctgcagcct 305810PRTArtificial Sequencechemically synthesized 58Asp Pro Leu Tyr Pro Pro Gly Leu Gln Pro 1 5 10 59357DNAArtificial Sequencechemically synthesized 59caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60tcctgcaagg tttccggata caccctcaat gacttcgcca tgcactgggt gcgacaggct 120cctggaaaag ggcttgagtg gatgggaggt tatgttcctg aagatggtga cacaatctac 180gcgcagaagt tccagggcag agtcaccatg accgaggaca catctacaga cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aacagatccc 300ctgtatagtg ggagcttatc ctactggggg caggggacca cggtcaccgt ctcgagt 35760119PRTArtificial Sequencechemically synthesized 60Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Asn Asp Phe 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Gly Tyr Val Pro Glu Asp Gly Asp Thr Ile Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asp Pro Leu Tyr Ser Gly Ser Leu Ser Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 61324DNAArtificial Sequencechemically synthesized 61tcctatgtgc tgactcagcc accctcggtg tcagtggccc caggacagac ggccaggatt 60acctgtgggg gaaacaacat tgaaagtaaa agtgtgcact ggtaccagca gaagccaggc 120caggcccctg tgctggccgt ctatgatgat agcgaccggc cctcagggat ccctgagcga 180ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240gatgaggccg actattactg tcaggtgtgg gatagtggtc ctgtgtggtg gattttcggc 300ggagggacca aggtcaccgt ccta 32462108PRTArtificial Sequencechemically synthesized 62Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Glu Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Gly Pro Val Trp 85 90 95 Trp Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 6330DNAArtificial Sequencechemically synthesized 63gatcccctgt atagtgggag cttatcctac 306410PRTArtificial Sequencechemically synthesized 64Asp Pro Leu Tyr Ser Gly Ser Leu Ser Tyr 1 5 10 6534DNAArtificial Sequencechemically synthesized 65tcaggtgtgg gatagtggtc ctgtgtggtg gatt 346611PRTArtificial Sequencechemically synthesized 66Gln Val Trp Asp Ser Gly Pro Val Trp Trp Ile 1 5 10 67366DNAArtificial Sequencechemically synthesized 67caggtgcagc tggtgcagtc tgggactgag gtgaagaagc ctggggctac agtgaatgtt 60tcctgcaaga tttccggaca cctcttcacc gactactaca tacactgggt gcaacaggcc 120cctggaaaag ggcttgagtg ggtgggactt attgatccta aagatggtga aatccaatac 180gcagagaaat tccaggccag agtcaccatt acagcggaca cgtccacaga cacagtttac 240atggaattga acagcctgag atctgaagac acggccgtgt attactgtgc aacagaggtt 300ttaagcggta ttagggtttt cccattcgac ccctggggcc agggcaccct ggtcaccgtc 360tcgagt 36668122PRTArtificial Sequencechemically synthesized 68Gln Val Gln Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Asn Val Ser Cys Lys Ile Ser Gly His Leu Phe Thr Asp Tyr 20 25 30 Tyr Ile His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Leu Ile Asp Pro Lys Asp Gly Glu Ile Gln Tyr Ala Glu Lys Phe 50 55 60 Gln Ala Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Val Tyr 65 70 75 80 Met Glu Leu Asn Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Glu Val Leu Ser Gly Ile Arg Val Phe Pro Phe Asp Pro Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 69339DNAArtificial Sequencechemically synthesized 69cagtctgtgc tgactcagcc accctcagtg tctggggccc cagggcagag ggtcaccatc 60tcttgcactg ggagcagctc caacatcggg gcaggttatg atgtatattg gtaccaacag 120tttccaggga aagcccccaa actcctcatc tatgatacca acaatcgacc cccaggggtc 180cctgatcgat tctctggctc caagtctggc acctcagcct ccctggccat cagtgggctc 240cagactgaag atgaggctga ttattactgc cagtcttatg acatcgccct gagtaactcg 300aatgtggttt tcggcggagg gaccaagctg accgtccta 33970113PRTArtificial Sequencechemically synthesized 70Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val Tyr Trp Tyr Gln Gln Phe Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Thr Asn Asn Arg Pro Pro Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Gln Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ile Ala 85 90 95 Leu Ser Asn Ser Asn Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val 100 105 110 Leu 7115DNAArtificial Sequencechemically synthesized 71gactactaca tacac 157251DNAArtificial Sequencechemically synthesized 72ggttatgttc ctgaagatgg tgacacaatc tacgcgcaga agttccaggg c 517339DNAArtificial Sequencechemically synthesized 73gaggttttaa gcggtattag ggttttccca ttcgacccc 39745PRTArtificial Sequencechemically synthesized 74Asp Tyr Tyr Ile His 1 5 7517PRTArtificial Sequencechemically synthesized 75Leu Ile Asp Pro Lys Asp Gly Glu Ile Gln Tyr Ala Glu Lys Phe Gln 1 5 10 15 Ala 7613PRTArtificial Sequencechemically synthesized 76Glu Val Leu Ser Gly Ile Arg Val Phe Pro Phe Asp Pro 1 5 10 7714PRTArtificial Sequencechemically synthesized 77Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val Tyr 1 5 10 7821DNAArtificial Sequencechemically synthesized 78gataccaaca atcgaccccc a 217939DNAArtificial Sequencechemically synthesized 79cagtcttatg acatcgccct gagtaactcg aatgtggtt 398042DNAArtificial Sequencechemically synthesized 80actgggagca gctccaacat cggggcaggt tatgatgtat at 42817PRTArtificial Sequencechemically synthesized 81Asp Thr Asn Asn Arg Pro Pro 1 5 8213PRTArtificial Sequencechemically synthesized 82Gln Ser Tyr Asp Ile Ala Leu Ser Asn Ser Asn Val Val 1 5 10 83363DNAArtificial Sequencechemically synthesized 83caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg tttccggata caccctcact gaattatcca tgcactgggt gcgacaggct 120cctggaaaag ggcttgagtg gatgggaggt tttgatcctg aagatggtga aacaatctac 180gcacagaagt tccagggcag agtcaccatg accgaggaca catctacaga cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aacttattct 300ggtagtagtg gttggtgggc ttttgatatc tggggccaag ggacaatggt caccgtctcg 360agt 36384121PRTArtificial Sequencechemically synthesized 84Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu Leu 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Tyr Ser Gly Ser Ser Gly Trp Trp Ala Phe Asp Ile Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 85318DNAArtificial Sequencechemically synthesized 85tcttctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60acatgccaag gagacagcct cagaagctat tatgcaagct ggtaccagca gaagccagga 120caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240gatgaggctg actattactg tcagacctgg ggcactggca tttgggtgtt cggcggaggg 300accaagctga ccgtccta 31886106PRTArtificial Sequencechemically synthesized 86Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Trp Gly Thr Gly Ile Trp Val 85 90 95 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 8715DNAArtificial Sequencechemically synthesized 87gaattatcca tgcac 158851DNAArtificial Sequencechemically synthesized 88ggttttgatc ctgaagatgg tgaaacaatc tacgcacaga agttccaggg c 518936DNAArtificial Sequencechemically synthesized 89tattctggta gtagtggttg gtgggctttt gatatc 36905PRTArtificial Sequencechemically synthesized 90Glu Leu Ser Met His 1 5 9117PRTArtificial Sequencechemically synthesized 91Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 9212PRTArtificial Sequencechemically synthesized 92Tyr Ser Gly Ser Ser Gly Trp Trp Ala Phe Asp Ile 1 5 10 9333DNAArtificial Sequencechemically synthesized 93caaggagaca gcctcagaag ctattatgca agc 339421DNAArtificial Sequencechemically synthesized 94ggtaaaaaca accggccctc a 219527DNAArtificial Sequencechemically synthesized 95cagacctggg gcactggcat ttgggtg 279611PRTArtificial Sequencechemically synthesized 96Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala Ser 1 5 10 977PRTArtificial Sequencechemically synthesized 97Gly Lys Asn Asn Arg Pro Ser 1 5 989PRTArtificial Sequencechemically synthesized 98Gln Thr Trp Gly Thr Gly Ile Trp Val 1 5 99372DNAArtificial Sequencechemically synthesized 99gaggtgcagc tggtggagtc cgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagagattta 300ggatattgta ctaatggtgt atgctggggt attgactact ggggccaggg gacaatggtc 360accgtctcga gt 372100124PRTArtificial Sequencechemically synthesized 100Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Gly Tyr Cys Thr Asn Gly Val Cys Trp Gly Ile Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 101330DNAArtificial Sequencechemically synthesized 101aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60tcctgcaccc gcagcagtgg cagcattgcc gacaactatg tgcagtggta ccagcagcgc 120ccgggcagtg cccccaccac tatcatctat gacgatgacc aaagactctc tggggtccct 180gatcgattct ctggctccat tgacacttcc tccaactctg cctccctctc catctctgga 240ctgaggactg aggacgaggc tgattactac tgtcagtctt atgatgactc caatgatgtg 300ttcggcggag ggaccaagct gaccgtccta 330102110PRTArtificial Sequencechemically synthesized 102Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Ala Asp Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Ile 35 40 45 Ile Tyr Asp Asp Asp Gln Arg Leu Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Thr Ser Ser Asn Ser Ala Ser Leu Ser Ile Ser Gly 65 70 75 80 Leu Arg Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Asp 85 90 95 Ser Asn Asp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 10315DNAArtificial Sequencechemically synthesized 103agctatgcca tgagc 1510451DNAArtificial Sequencechemically synthesized 104gctattagtg gtagtggtgg tagcacatac tacgcagact ccgtgaaggg c 5110545DNAArtificial Sequencechemically synthesized 105gatttaggat attgtactaa tggtgtatgc tggggtattg actac 451065PRTArtificial Sequencechemically synthesized 106Ser Tyr Ala Met Ser 1 5 10717PRTArtificial Sequencechemically synthesized 107Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 10815PRTArtificial Sequencechemically synthesized 108Asp Leu Gly Tyr Cys Thr Asn Gly Val Cys Trp Gly Ile Asp Tyr 1 5 10 15 10939DNAArtificial Sequencechemically

synthesized 109acccgcagca gtggcagcat tgccgacaac tatgtgcag 3911021DNAArtificial Sequencechemically synthesized 110gacgatgacc aaagactctc t 2111127DNAArtificial Sequencechemically synthesized 111cagtcttatg atgactccaa tgatgtg 2711213PRTArtificial Sequencechemically synthesized 112Thr Arg Ser Ser Gly Ser Ile Ala Asp Asn Tyr Val Gln 1 5 10 1137PRTArtificial Sequencechemically synthesized 113Asp Asp Asp Gln Arg Leu Ser 1 5 1149PRTArtificial Sequencechemically synthesized 114Gln Ser Tyr Asp Asp Ser Asn Asp Val 1 5 115369DNAArtificial Sequencechemically synthesized 115caggtgcagc tggtgcagtc tggggctgag gtggagaagc ctggggcctc agtgaaggtc 60tcctgcaggg tttcgggata ccccctcact gaaatagcca tacactgggt gcgacaggct 120cctggaaaag ggcttgagtg gatgggaagt tttgagcctg aagatgctga agcaatctac 180gcacagaggt tccagggcag agtcacaatg accgaggaaa catctgcaaa cactgcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt atttctgtgc aacagatccc 300tactatgcta gcagtggttc taactacatg gaggtctggg gccgaggaac cctggtcacc 360gtctcgagt 369116123PRTArtificial Sequencechemically synthesized 116Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Glu Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Arg Val Ser Gly Tyr Pro Leu Thr Glu Ile 20 25 30 Ala Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ser Phe Glu Pro Glu Asp Ala Glu Ala Ile Tyr Ala Gln Arg Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Glu Glu Thr Ser Ala Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Thr Asp Pro Tyr Tyr Ala Ser Ser Gly Ser Asn Tyr Met Glu Val 100 105 110 Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 117333DNAArtificial Sequencechemically synthesized 117aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatt 60tcctgcaccg gcagcggcgg cagcatttcc agcaactatg tccagtggta ccgacagcgc 120ccgggcagcg cccccagcac tgtgatctat gaggatgacc aaagaccctc tggggtccct 180gatcggatct ctggctccat cgacagttcc tccaactctg cctccctcac catctctgga 240ctgacaactg aggacgaggc tgactactat tgtcactctt atgatggcaa caatcggtgg 300gtcttcggcg gagggaccaa gctgaccgtc cta 333118111PRTArtificial Sequencechemically synthesized 118Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Gly Ser Gly Gly Ser Ile Ser Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Arg Gln Arg Pro Gly Ser Ala Pro Ser Thr Val 35 40 45 Ile Tyr Glu Asp Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Ile Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Thr Thr Glu Asp Glu Ala Asp Tyr Tyr Cys His Ser Tyr Asp Gly 85 90 95 Asn Asn Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 11915DNAArtificial Sequencechemically synthesized 119gaaatagcca tacac 1512051DNAArtificial Sequencechemically synthesized 120agttttgagc ctgaagatgc tgaagcaatc tacgcacaga ggttccaggg c 5112142DNAArtificial Sequencechemically synthesized 121gatccctact atgctagcag tggttctaac tacatggagg tc 421225PRTArtificial Sequencechemically synthesized 122Glu Ile Ala Ile His 1 5 12317PRTArtificial Sequencechemically synthesized 123Ser Phe Glu Pro Glu Asp Ala Glu Ala Ile Tyr Ala Gln Arg Phe Gln 1 5 10 15 Gly 12414PRTArtificial Sequencechemically synthesized 124Asp Pro Tyr Tyr Ala Ser Ser Gly Ser Asn Tyr Met Glu Val 1 5 10 12539DNAArtificial Sequencechemically synthesized 125accggcagcg gcggcagcat ttccagcaac tatgtccag 3912621DNAArtificial Sequencechemically synthesized 126gaggatgacc aaagaccctc t 2112730DNAArtificial Sequencechemically synthesized 127cactcttatg atggcaacaa tcggtgggtc 3012813PRTArtificial Sequencechemically synthesized 128Thr Gly Ser Gly Gly Ser Ile Ser Ser Asn Tyr Val Gln 1 5 10 1297PRTArtificial Sequencechemically synthesized 129Glu Asp Asp Gln Arg Pro Ser 1 5 13010PRTArtificial Sequencechemically synthesized 130His Ser Tyr Asp Gly Asn Asn Arg Trp Val 1 5 10 131366DNAArtificial Sequencechemically synthesized 131caggtgcagc tggtgcagtc tggggctgag gtgaagaggc ctggggcctc agtgaaggtc 60tcctgcaaag tttccggaaa caccctcagt aaacaatcca tgcactgggt gcgacaggct 120cctggaaaag ggtttgagtg gatgggaagt tctaatcctg aagatgatga aacactctac 180gcaaagaagt tccagggcag agtcaccatg accgaggaca catccacaga cacagcctat 240ttggagttga gcagtctgag gtctgaggac acggccgtgt attattgtgc aacagactcc 300cagggttttt actattacta cggtatggac gtctggggcc agggcaccct ggtcaccgtc 360tcgagt 366132122PRTArtificial Sequencechemically synthesized 132Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Asn Thr Leu Ser Lys Gln 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Phe Glu Trp Met 35 40 45 Gly Ser Ser Asn Pro Glu Asp Asp Glu Thr Leu Tyr Ala Lys Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asp Ser Gln Gly Phe Tyr Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 133333DNAArtificial Sequencechemically synthesized 133cagtctgtgc tgactcagcc gccctcagtg tctggggccc cagggcagag ggtcaccatc 60tcctgcactg ggagcagctc caacatcggg gcagattatg atgtacactg gtaccagcaa 120cttccaggaa cagtccccaa actcctcatc tatgataaca tcaatcggcc ctcaggggtc 180cctgaccgat tctctggctc caagtctggc acctcagcct ccctggccat cactgggctc 240caggctgagg atgaggctga ttattactgc cagtcctatg acagcagcct gagtggtgtg 300ctattcggcg gagggaccaa ggtcaccgtc cta 333134111PRTArtificial Sequencechemically synthesized 134Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Asp 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Val Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn Ile Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser 85 90 95 Leu Ser Gly Val Leu Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105 110 13515DNAArtificial Sequencechemically synthesized 135aaacaatcca tgcac 1513651DNAArtificial Sequencechemically synthesized 136agttctaatc ctgaagatga tgaaacactc tacgcaaaga agttccaggg c 5113739DNAArtificial Sequencechemically synthesized 137gactcccagg gtttttacta ttactacggt atggacgtc 391385PRTArtificial Sequencechemically synthesized 138Lys Gln Ser Met His 1 5 13917PRTArtificial Sequencechemically synthesized 139Ser Ser Asn Pro Glu Asp Asp Glu Thr Leu Tyr Ala Lys Lys Phe Gln 1 5 10 15 Gly 14013PRTArtificial Sequencechemically synthesized 140Asp Ser Gln Gly Phe Tyr Tyr Tyr Tyr Gly Met Asp Val 1 5 10 14142DNAArtificial Sequencechemically synthesized 141actgggagca gctccaacat cggggcagat tatgatgtac ac 4214221DNAArtificial Sequencechemically synthesized 142gataacatca atcggccctc a 2114333DNAArtificial Sequencechemically synthesized 143cagtcctatg acagcagcct gagtggtgtg cta 3314414PRTArtificial Sequencechemically synthesized 144Thr Gly Ser Ser Ser Asn Ile Gly Ala Asp Tyr Asp Val His 1 5 10 1457PRTArtificial Sequencechemically synthesized 145Asp Asn Ile Asn Arg Pro Ser 1 5 14611PRTArtificial Sequencechemically synthesized 146Gln Ser Tyr Asp Ser Ser Leu Ser Gly Val Leu 1 5 10 147351DNAArtificial Sequencechemically synthesized 147caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaggg cttcgggata cgccctcact gaattatcca ttcactgggt gcgacaggct 120cctggaaaag ggcttgagtg gatgggaggt tttgatcctg aagatggtga aacaatctac 180gcacagaatt tccagggcag agtcatcatg accgaggaca catctacaga cacagcctac 240atggagctga gcagcctgaa atctgaggac acggccgtgt attattgtgc gacagatcta 300actggaagta gggactcctg gggccaaggc accctggtca ccgtctcgag t 351148117PRTArtificial Sequencechemically synthesized 148Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Arg Ala Ser Gly Tyr Ala Leu Thr Glu Leu 20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Asn Phe 50 55 60 Gln Gly Arg Val Ile Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asp Leu Thr Gly Ser Arg Asp Ser Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 149333DNAArtificial Sequencechemically synthesized 149cagtctgtgc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60tcctgcactg gaagcaggag tgacattggt tactataact atgtctcctg gtaccaacaa 120cacccaggga aagtccccaa actcataatt tatgatgtca ctgagcgacc ctcaggggtt 180tctgatcgct tctctggctc caagtctgcc aacacggcct ccctgaccat ctctgggctc 240caggctgagg acgaggctga ttattactgc agctcatttt caagtggcga caccttcgtg 300gttttcggcg gagggaccaa gctgaccgtc cta 333150111PRTArtificial Sequencechemically synthesized 150Gln Ser Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Ser Arg Ser Asp Ile Gly Tyr Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu 35 40 45 Ile Ile Tyr Asp Val Thr Glu Arg Pro Ser Gly Val Ser Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Ala Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Phe Ser Ser Gly 85 90 95 Asp Thr Phe Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 15115DNAArtificial Sequencechemically synthesized 151gaattatcca ttcac 1515251DNAArtificial Sequencechemically synthesized 152ggttttgatc ctgaagatgg tgaaacaatc tacgcacaga atttccaggg c 5115324DNAArtificial Sequencechemically synthesized 153gatctaactg gaagtaggga ctcc 241545PRTArtificial Sequencechemically synthesized 154Glu Leu Ser Ile His 1 5 15517PRTArtificial Sequencechemically synthesized 155Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Asn Phe Gln 1 5 10 15 Gly 1568PRTArtificial Sequencechemically synthesized 156Asp Leu Thr Gly Ser Arg Asp Ser 1 5 15742DNAArtificial Sequencechemically synthesized 157actggaagca ggagtgacat tggttactat aactatgtct cc 4215821DNAArtificial Sequencechemically synthesized 158gatgtcactg agcgaccctc a 2115933DNAArtificial Sequencechemically synthesized 159agctcatttt caagtggcga caccttcgtg gtt 3316014PRTArtificial Sequencechemically synthesized 160Thr Gly Ser Arg Ser Asp Ile Gly Tyr Tyr Asn Tyr Val Ser 1 5 10 1617PRTArtificial Sequencechemically synthesized 161Asp Val Thr Glu Arg Pro Ser 1 5 16211PRTArtificial Sequencechemically synthesized 162Ser Ser Phe Ser Ser Gly Asp Thr Phe Val Val 1 5 10 163366DNAArtificial Sequencechemically synthesized 163caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt agctatgcta tgcactgggt ccgccaggct 120ccaggcaagg ggctagagtg ggtggcagtt atatcatatg atggaagtaa taaatactac 180gcagactccg tgaagggccg attctccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gagagaaact 300ttcccccact actactacta ctacatggac gtctggggca aggggacaat ggtcaccgtc 360tcgagt 366164122PRTArtificial Sequencechemically synthesized 164Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Thr Phe Pro His Tyr Tyr Tyr Tyr Tyr Met Asp Val Trp 100 105 110 Gly Lys Gly Thr Met Val Thr Val Ser Ser 115 120 165324DNAArtificial Sequencechemically synthesized 165tcctatgtgc tgactcagcc accctcggtg tccgtggccc cagggcagac ggccagaatt 60tcctgtgggg gaggcaactt tgacgatgaa ggtgttcact ggtaccagca gaccccaggc 120caggcccctg tactggtcgt ctatgatgat accggccggc cctcagggat ccctgagcga 180ttctctggct ccagttctgg gaatacggcc accctgacca tcagccgggt cgaagccggg 240gatgaggccg actattactg tcaggcgtgg gatagtagta atgatcatcc cgtgttcggc 300ggagggaccc agctcaccgt ccta 324166108PRTArtificial Sequencechemically synthesized 166Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Ser Cys Gly Gly Gly Asn Phe Asp Asp Glu Gly Val 20 25 30 His Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Thr Gly Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Asn Asp His 85 90 95 Pro Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu 100 105 167214PRTArtificial Sequencechemically synthesized 167Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Glu Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35

40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Thr Asp His 85 90 95 Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210 168449PRTArtificial Sequencechemically synthesized 168Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu Phe 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Gly Phe Val Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asp Pro Leu Tyr Thr Pro Gly Leu Glu Pro Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys 16920PRTArtificial Sequencechemically synthesized 169Glu Gln Val Ala Val Gly Pro Gly Pro Thr Ser Asn Arg Gly Pro Asp 1 5 10 15 Gly Leu Asp Val 20 17068PRTArtificial Sequencechemically synthesized 170Ser Pro Tyr Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Ile Ala 1 5 10 15 Arg Pro Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly 20 25 30 Lys Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Lys Asn Arg Gln 35 40 45 Val Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser 50 55 60 Leu Glu Met Ser 65 17168PRTArtificial Sequencechemically synthesized 171Ser Pro His Ala Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Ile Ala 1 5 10 15 Arg Pro Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly 20 25 30 Lys Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Lys Asn Arg Gln 35 40 45 Val Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser 50 55 60 Leu Glu Met Ser 65 17268PRTArtificial Sequencechemically synthesized 172Ser Pro Tyr Gly Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Leu Ser 1 5 10 15 Leu Ala Leu Pro Arg Ala His Val Lys Glu Tyr Phe Tyr Thr Ser Ser 20 25 30 Lys Cys Ser Asn Leu Ala Val Val Phe Val Thr Arg Arg Asn Arg Gln 35 40 45 Val Cys Ala Asn Pro Glu Lys Lys Trp Val Gln Glu Tyr Ile Asn Tyr 50 55 60 Leu Glu Met Ser 65 17333DNAArtificial Sequencechemically synthesized 173gggggaggca actttgacga tgaaggtgtt cac 3317421DNAArtificial Sequencechemically synthesized 174gatgataccg gccggccctc a 2117533DNAArtificial Sequencechemically synthesized 175caggcgtggg atagtagtaa tgatcatccc gtg 3317611PRTArtificial Sequencechemically synthesized 176Gly Gly Gly Asn Phe Asp Asp Glu Gly Val His 1 5 10 1777PRTArtificial Sequencechemically synthesized 177Asp Asp Thr Gly Arg Pro Ser 1 5 17811PRTArtificial Sequencechemically synthesized 178Gln Ala Trp Asp Ser Ser Asn Asp His Pro Val 1 5 10 179378DNAArtificial Sequencechemically synthesized 179gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaaaaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc aagagtaagg 300gggagttccc agtacgattt ttggagtggg tccgagtttg actactgggg ccaggggaca 360atggtcaccg tctcgagt 378180126PRTArtificial Sequencechemically synthesized 180Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Arg Gly Ser Ser Gln Tyr Asp Phe Trp Ser Gly Ser Glu 100 105 110 Phe Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 181327DNAArtificial Sequencechemically synthesized 181tcctatgtgc tgactcagcc accctcagtg tcagtggccc caggaaagac ggccagcatt 60tcctgtgggg gagacaacat tggaggtcaa aatgttcact ggtatcagca gaagccaggc 120caggcccctg tgctcgtcat ctattatgat accgaccggc cctcagggat ccctgagcga 180ttctctggct ccaactctgg gaacacggcc accctgtcca tcagcagggt cgaagccgcg 240gatgaggccg actattactg tcaggtgtgg gatgttgata gtgatcatcc ttgggtgttc 300ggcggaggga ccaagctgac cgtccta 327182109PRTArtificial Sequencechemically synthesized 182Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys 1 5 10 15 Thr Ala Ser Ile Ser Cys Gly Gly Asp Asn Ile Gly Gly Gln Asn Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Tyr Asp Thr Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Ser Ile Ser Arg Val Glu Ala Ala 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Val Asp Ser Asp His 85 90 95 Pro Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 18342DNAArtificial Sequencechemically synthesized 183actgggagca gctccaacat cggggacggt tatgatgtac ac 4218421DNAArtificial Sequencechemically synthesized 184ggtaacagta atcggccctc a 2118551DNAArtificial Sequencechemically synthesized 185gtaaggggga gttcccagta cgatttttgg agtgggtccg agtttgacta c 51186452PRTArtificial Sequencechemically synthesized 186Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Thr Phe Pro His Tyr Tyr Tyr Tyr Tyr Met Asp Val Trp 100 105 110 Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys 450 187214PRTArtificial Sequencechemically synthesized 187Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Glu Gly Asp Asp Thr Asp Ile Gly Thr Val 20 25 30 Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Ser 35 40 45 Glu Asp Gly Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Phe Trp Asp Val Asp Ser Asp His 85 90 95 Pro Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210 18817PRTArtificial Sequencechemically synthesized 188Val Arg Gly Ser Ser Gln Tyr Asp Phe Trp Ser Gly Ser Glu Phe Asp 1 5 10 15 Tyr 18933DNAArtificial Sequencechemically synthesized 189ggaacatggg atgacatcct gaatggttgg gtg 3319014PRTArtificial Sequencechemically synthesized 190Thr Gly Ser Ser Ser Asn Ile Gly Asp Gly Tyr Asp Val His 1 5 10 1917PRTArtificial Sequencechemically synthesized 191Gly Asn Ser Asn Arg Pro Ser 1 5 19211PRTArtificial Sequencechemically synthesized 192Gly Gly Asp Asn Ile Gly Gly Gln Asn Val His 1 5 10 1937PRTArtificial Sequencechemically synthesized 193Tyr Asp Thr Asp Arg Pro Ser 1 5 19412PRTArtificial Sequencechemically synthesized 194Gln Val Trp Asp Val Asp Ser Asp His Pro Trp Val 1 5 10 195324DNAArtificial Sequencechemically synthesized 195tcctatgtgc tgactcagcc accctcggtg tcagtggccc caggacagac ggccaggatt 60acctgtgggg gaaacaacat tgaaagtaaa agtgtgcact ggtaccagca gaagccaggc 120caggcccctg tgctggtggt ctatgatgat agcgaccggc cctcagggat ccctgagcga 180ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240gatgaggccg actattactg tcaggtgtgg gatagtaata ctgatcattg ggtgttcggc 300ggagggacca aggtcaccgt ccta 324196108PRTArtificial Sequencechemically synthesized 196Ser Tyr Val Leu

Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Glu Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Thr Asp His 85 90 95 Trp Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105 19721DNAArtificial Sequencechemically synthesized 197ctcttctgag atgagttttt g 2119830DNAArtificial Sequencechemically synthesized 198ttattattcg caattccttt agttgttcct 30199360DNAArtificial Sequencechemically synthesized 199caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg tttccggata cgccctcagt gaattatcca tacactgggt gcgacaggct 120cctggcaaag gccttgagtg gatgtcgtat attgatcctg aagatggtga accaatttac 180gcacagaagt tccagggcag agccaccatg accgaggact catctacaga cacagcctac 240atggagatgg gcagcctgac atctgacgac acggccgttt attactgtgc aggtgtcact 300ggaagtactt cggatgcctt tgatctctgg ggccggggaa ccctggtcac cgtctcgagt 360200120PRTArtificial Sequencechemically synthesized 200Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Ala Leu Ser Glu Leu 20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Ser Tyr Ile Asp Pro Glu Asp Gly Glu Pro Ile Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Ala Thr Met Thr Glu Asp Ser Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Met Gly Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Val Thr Gly Ser Thr Ser Asp Ala Phe Asp Leu Trp Gly Arg 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 201324DNAArtificial Sequencechemically synthesized 201tcctatgtgc tgactcagga cccctcggtg tcagtggccc caggacagac ggccaggatc 60acctgtgggg gagccaatct ttggggtcta ggtgtccatt ggtatcaaca aaagtcaggc 120caggcccctg tgttggtcgt ctctgataat agcgaccggg cctcagggat ccctgagcga 180ttctctggct ccaattctgg gaccacggcc accctgaccc tcagcagggt cgaagtcggc 240gatgaggccg actattactg tcaggtgtgg gatagtagta gtgatcactg ggtgttcggc 300ggcaggacca agctgaccgt ccta 324202108PRTArtificial Sequencechemically synthesized 202Ser Tyr Val Leu Thr Gln Asp Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Ala Asn Leu Trp Gly Leu Gly Val 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Gln Ala Pro Val Leu Val Val Ser 35 40 45 Asp Asn Ser Asp Arg Ala Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Thr Thr Ala Thr Leu Thr Leu Ser Arg Val Glu Val Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Trp Val Phe Gly Gly Arg Thr Lys Leu Thr Val Leu 100 105 20315DNAArtificial Sequencechemically synthesized 203gaattatcca tacac 1520451DNAArtificial Sequencechemically synthesized 204tatattgatc ctgaagatgg tgaaccaatt tacgcacaga agttccaggg c 5120533DNAArtificial Sequencechemically synthesized 205gtcactggaa gtacttcgga tgcctttgat ctc 3320668PRTArtificial Sequencechemically synthesized 206Ser Pro Tyr Gly Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Leu Ser 1 5 10 15 Leu Ala Leu Pro Arg Ala His Val Lys Glu Tyr Phe Tyr Thr Ser Ser 20 25 30 Lys Cys Ser Asn Leu Ala Val Val Phe Val Thr Arg Arg Asn Arg Gln 35 40 45 Val Cys Ala Asn Pro Glu Lys Lys Trp Val Gln Glu Tyr Ile Asn Tyr 50 55 60 Leu Glu Met Ser 65 20717PRTArtificial Sequencechemically synthesized 207Tyr Ile Asp Pro Glu Asp Gly Glu Pro Ile Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 20811PRTArtificial Sequencechemically synthesized 208Val Thr Gly Ser Thr Ser Asp Ala Phe Asp Leu 1 5 10 20933DNAArtificial Sequencechemically synthesized 209gggggagcca atctttgggg tctaggtgtc cat 3321021DNAArtificial Sequencechemically synthesized 210gataatagcg accgggcctc a 2121133DNAArtificial Sequencechemically synthesized 211caggtgtggg atagtagtag tgatcactgg gtg 3321211PRTArtificial Sequencechemically synthesized 212Gly Gly Ala Asn Leu Trp Gly Leu Gly Val His 1 5 10 2137PRTArtificial Sequencechemically synthesized 213Asp Asn Ser Asp Arg Ala Ser 1 5 21411PRTArtificial Sequencechemically synthesized 214Gln Val Trp Asp Ser Ser Ser Asp His Trp Val 1 5 10 215387DNAArtificial Sequencechemically synthesized 215caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcgtc ggtgaaggtc 60tcctgcaagg cctctggagg catctccgac aactatgctc tcagctgggt gcgacaggcc 120cctggccaag gacttgagtg gatgggaggg ttcatccctc tcgtcgatac tacgaactac 180gcacagaggt ttcagggcag actcacgatt accgcggacg actccatgag tacagtctac 240atggaactaa gaagcctgcg atctgacgac acggccatgt attattgtgc gagagagcag 300gtggcggtgg gacctggacc cacctcagac cgggggcccg atggtcttga tgtctggggc 360caagggacaa tggtcaccgt ctcgagt 387216129PRTArtificial Sequencechemically synthesized 216Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Ile Ser Asp Asn Tyr 20 25 30 Ala Leu Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Phe Ile Pro Leu Val Asp Thr Thr Asn Tyr Ala Gln Arg Phe 50 55 60 Gln Gly Arg Leu Thr Ile Thr Ala Asp Asp Ser Met Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Glu Gln Val Ala Val Gly Pro Gly Pro Thr Ser Asp Arg Gly 100 105 110 Pro Asp Gly Leu Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser 115 120 125 Ser 217339DNAArtificial Sequencechemically synthesized 217cagtctgtgc tgactcagcc gtcctcagtg tctggggccc cagggcacag ggtcaccatt 60tcctgcactg ggagcaactc caacctcggg gcggattatg atgtacactg gtatcagcag 120cttccagggt cagcccccaa actcctcatc tatgataaca acattcgtcc ctcaggggtc 180cctgcccgat tctctggctc caagtctggc acctcagcct ccctggccat cactgggctc 240caggctgaag atgaggctga ttattactgc cagtcgtatg acaccggcct gacttcttcg 300gatgtgatat tcggcggagg gaccaagctg accgtccta 339218113PRTArtificial Sequencechemically synthesized 218Gln Ser Val Leu Thr Gln Pro Ser Ser Val Ser Gly Ala Pro Gly His 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Asn Ser Asn Leu Gly Ala Asp 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Ser Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn Asn Ile Arg Pro Ser Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Thr Gly 85 90 95 Leu Thr Ser Ser Asp Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val 100 105 110 Leu 21915DNAArtificial Sequencechemically synthesized 219aactatgctc tcagc 1522051DNAArtificial Sequencechemically synthesized 220gggttcatcc ctctcgtcga tactacgaac tacgcacaga ggtttcaggg c 5122160DNAArtificial Sequencechemically synthesized 221gagcaggtgg cggtgggacc tggacccacc tcagaccggg ggcccgatgg tcttgatgtc 602225PRTArtificial Sequencechemically synthesized 222Asn Tyr Ala Leu Ser 1 5 22317PRTArtificial Sequencechemically synthesized 223Gly Phe Ile Pro Leu Val Asp Thr Thr Asn Tyr Ala Gln Arg Phe Gln 1 5 10 15 Gly 22420PRTArtificial Sequencechemically synthesized 224Glu Gln Val Ala Val Gly Pro Gly Pro Thr Ser Asp Arg Gly Pro Asp 1 5 10 15 Gly Leu Asp Val 20 22542DNAArtificial Sequencechemically synthesized 225actgggagca actccaacct cggggcggat tatgatgtac ac 4222621DNAArtificial Sequencechemically synthesized 226gataacaaca ttcgtccctc a 2122739DNAArtificial Sequencechemically synthesized 227cagtcgtatg acaccggcct gacttcttcg gatgtgata 3922814PRTArtificial Sequencechemically synthesized 228Thr Gly Ser Asn Ser Asn Leu Gly Ala Asp Tyr Asp Val His 1 5 10 2297PRTArtificial Sequencechemically synthesized 229Asp Asn Asn Ile Arg Pro Ser 1 5 23013PRTArtificial Sequencechemically synthesized 230Gln Ser Tyr Asp Thr Gly Leu Thr Ser Ser Asp Val Ile 1 5 10 231360DNAArtificial Sequencechemically synthesized 231gaggtgcagc tggtgcagtc tgggcctgag gtgaagaagc ctggggccac agtgaaaatt 60tcctgcaacg tctctgcaga aaccttcacc gactactaca tacactgggt caaacaggcc 120cctggaagag ggctggagtg gatggggctt gttgattctg aagaagatgg tgaaacatta 180ttcgcagaga ctttcagggg cagagtcgcc ctaaccgcgg acaggtccac aaacaccgcc 240tacatggagt tgcgcagcct gagacatgac gacacggccg tctattattg tgcagcagaa 300tatggtgaat atgggttctt ccaatcgtgg ggccagggaa ccctggtcac cgtctcgagt 360232120PRTArtificial Sequencechemically synthesized 232Glu Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Asn Val Ser Ala Glu Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile His Trp Val Lys Gln Ala Pro Gly Arg Gly Leu Glu Trp Met 35 40 45 Gly Leu Val Asp Ser Glu Glu Asp Gly Glu Thr Leu Phe Ala Glu Thr 50 55 60 Phe Arg Gly Arg Val Ala Leu Thr Ala Asp Arg Ser Thr Asn Thr Ala 65 70 75 80 Tyr Met Glu Leu Arg Ser Leu Arg His Asp Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Ala Glu Tyr Gly Glu Tyr Gly Phe Phe Gln Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 233336DNAArtificial Sequencechemically synthesized 233cagtctgtgc tgactcagcc accctcagtg tctggggccc cagggcagag ggtcaccatc 60tcctgcactg ggagcagctc caacatcggg gcagattatg atgtaaactg gtaccagcag 120cttccaggaa cttcccccaa actcctcatc tatggtgaca tcaatcggcc ctcaggggtc 180cctgaccgat tctctgcctc caagtctggc acctcagcct ccctggccat cactgggctc 240caggctgagg atgaggctga ttattactgc cagtcgtttg acaacagcct gagtgggtct 300gtgattttcg gcggagggac caagctgacc gtccta 336234112PRTArtificial Sequencechemically synthesized 234Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Asp 20 25 30 Tyr Asp Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ser Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asp Ile Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Phe Asp Asn Ser 85 90 95 Leu Ser Gly Ser Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 23511PRTArtificial Sequencechemically synthesized 235Gly Thr Trp Asp Asp Ile Leu Asn Gly Trp Val 1 5 10 23654DNAArtificial Sequencechemically synthesized 236cttgttgatt ctgaagaaga tggtgaaaca ttattcgcag agactttcag gggc 5423730DNAArtificial Sequencechemically synthesized 237gaatatggtg aatatgggtt cttccaatcg 30238449PRTArtificial Sequencechemically synthesized 238Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu Phe 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Gly Phe Val Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asp Pro Leu Tyr Glu Gly Ser Phe Ser Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys 23918PRTArtificial Sequencechemically synthesized 239Leu Val Asp Ser Glu Glu Asp Gly Glu Thr Leu Phe Ala Glu Thr Phe 1 5 10 15 Arg Gly 24010PRTArtificial Sequencechemically synthesized

240Glu Tyr Gly Glu Tyr Gly Phe Phe Gln Ser 1 5 10 24142DNAArtificial Sequencechemically synthesized 241actgggagca gctccaacat cggggcagat tatgatgtaa ac 4224221DNAArtificial Sequencechemically synthesized 242ggtgacatca atcggccctc a 2124336DNAArtificial Sequencechemically synthesized 243cagtcgtttg acaacagcct gagtgggtct gtgatt 3624414PRTArtificial Sequencechemically synthesized 244Thr Gly Ser Ser Ser Asn Ile Gly Ala Asp Tyr Asp Val Asn 1 5 10 2457PRTArtificial Sequencechemically synthesized 245Gly Asp Ile Asn Arg Pro Ser 1 5 24612PRTArtificial Sequencechemically synthesized 246Gln Ser Phe Asp Asn Ser Leu Ser Gly Ser Val Ile 1 5 10 247387DNAArtificial Sequencechemically synthesized 247caggtgcagc tggtgcagtc tggggctgag gtgaagaagc cggggtcgtc ggtgaaggtc 60tcctgcaaga tttctggagg catctccgac aactacgctc tgagctgggt gcgacaggcc 120cctgggcaag gacttgagtg gatgggagcg gtcatccctc tcgtcgagac tacgagctac 180gcacagaggt tccagggcag actcacaatt accgcggacg actccttgaa tacactgtac 240atggaattgg gaagcctgcg atctgacgac acggccatgt attactgtgc gagagagcag 300gtggcggtgg gacctggacc cacttcaaat cgggggcccg atggcctaga tgtctggggc 360agagggacaa tggtcaccgt ctcgagt 387248129PRTArtificial Sequencechemically synthesized 248Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ile Ser Gly Gly Ile Ser Asp Asn Tyr 20 25 30 Ala Leu Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Val Ile Pro Leu Val Glu Thr Thr Ser Tyr Ala Gln Arg Phe 50 55 60 Gln Gly Arg Leu Thr Ile Thr Ala Asp Asp Ser Leu Asn Thr Leu Tyr 65 70 75 80 Met Glu Leu Gly Ser Leu Arg Ser Asp Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Glu Gln Val Ala Val Gly Pro Gly Pro Thr Ser Asn Arg Gly 100 105 110 Pro Asp Gly Leu Asp Val Trp Gly Arg Gly Thr Met Val Thr Val Ser 115 120 125 Ser 249333DNAArtificial Sequencechemically synthesized 249cagtctgtgc tgactcagcc accctcagtg tctggggccc cagggcagag ggtcaccatc 60tcctgcactg ggagcagctc caacatcggg gacggttatg atgtacactg gtatcagcag 120cttccaggaa cagcccccaa actcctcatc tatggtaaca gtaatcggcc ctcaggggtc 180cctgaccgat tctctggctc cacctctggc acctccgcct ccctggccat ccgtgggctc 240cagtctgagg atgaggctga ttactactgt ggaacatggg atgacatcct gaatggttgg 300gtgttcggcg gagggaccaa gctgaccgtc cta 333250111PRTArtificial Sequencechemically synthesized 250Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Asp Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Thr Ser Gly Thr Ser Ala Ser Leu Ala Ile Arg Gly Leu 65 70 75 80 Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Asp Ile 85 90 95 Leu Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 25115DNAArtificial Sequencechemically synthesized 251aactacgctc tgagc 1525251DNAArtificial Sequencechemically synthesized 252gcggtcatcc ctctcgtcga gactacgagc tacgcacaga ggttccaggg c 5125360DNAArtificial Sequencechemically synthesized 253gagcaggtgg cggtgggacc tggacccact tcaaatcggg ggcccgatgg cctagatgtc 60254214PRTArtificial Sequencechemically synthesized 254Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Glu Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Thr Asp His 85 90 95 Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210 25517PRTArtificial Sequencechemically synthesized 255Ala Val Ile Pro Leu Val Glu Thr Thr Ser Tyr Ala Gln Arg Phe Gln 1 5 10 15 Gly

Claims

1. An isolated antagonist molecule of human RANTES, wherein said antagonist molecule binds a human RANTES polypeptide that comprises at least residues acid residues 16-18 of SEQ ID NO: 170 and reduces a biological activity of RANTES activity upon interaction between said antagonist and said human RANTES polypeptide.

2. The antagonist molecule of claim 1, wherein said antagonist molecule does not bind a human RANTES polypeptide that lacks amino acid residues 16-18 of SEQ ID NO: 170.

3. The antagonist molecule of claim 1, wherein said antagonist molecule is selected from a small molecule inhibitor; a polypeptide, a peptide, and a nucleic acid-based antagonist.

4. The antagonist molecule of claim 1, wherein said antagonists is an isolated monoclonal anti-human RANTES antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof binds to an epitope on a mature human RANTES polypeptide, said epitope comprising amino acid residues 16-18 of SEQ ID NO: 170.

5. The antagonist molecule of claim 4, wherein said monoclonal antibody is a fully human monoclonal anti-human RANTES antibody or antigen-binding fragment thereof.

6. The antagonist molecule of claim 5, wherein said antibody is an IgG1 isotype.

7. An isolated fully human monoclonal anti-human RANTES antibody or fragment thereof, wherein said antibody comprises: (a) a V_H CDR1 region comprising the amino acid sequence of SEQ ID NO: 8, 28, 44, 74, 90, 106, 122, 138, 154, or 222; (b) a V_H CDR2 region comprising the amino acid sequence of SEQ ID NO: 9, 29, 45, 75, 91, 107, 123, 139, 155, 207, 223, 239, or 255; (c) a V_H CDR3 region comprising the amino acid sequence of SEQ ID NO: 10, 20, 30, 46, 50, 54, 58, 64, 76, 92, 108, 124, 140, 156, 169, 188, 208, 224, or 240, (d) a V_L CDR1 region comprising the amino acid sequence of SEQ ID NO: 14, 34, 77, 96, 112, 128, 144, 160, 176, 190, 192, 212, 228, or 244; (e) a V_L CDR2 region comprising the amino acid sequence of SEQ ID NO: 15, 35, 81, 97, 113, 129, 145, 161, 177, 191, 193, 213, 229, or 245; and (f) a V_L CDR3 region comprising the amino acid sequence of SEQ ID NO: 16, 36, 66, 82, 98, 114, 130, 146, 162, 178, 194, 214, 230, 235 or 246, wherein said antibody binds RANTES.

8. The antibody of claim 7, wherein said antibody comprises a combination of a V_H CDR1 region, a V_H CDR2 region, a V_H CDR3 region, a V_L CDR1 region, a V_L CDR2 region, and a V_L CDR1 region selected from the group consisting of: (a) a V_H CDR1 region comprising the amino acid sequence of SEQ ID NO: 8; a V_H CDR2 region comprising the amino acid sequence of SEQ ID NO:9, a V_H CDR3 region comprising the amino acid sequence of SEQ ID NO:10; a V_L CDR1 region comprising the amino acid sequence of SEQ ID NO: 14; a V_L CDR2 region comprising the amino acid sequence of SEQ ID NO: 15; and a V_L CDR3 region comprising an amino acid sequence of SEQ ID NO:16; (b) a V_H CDR1 region comprising the amino acid sequence of SEQ ID NO: 8; a V_H CDR2 region comprising the amino acid sequence of SEQ ID NO:9, a V_H CDR3 region comprising the amino acid sequence of SEQ ID NO:20; a V_L CDR1 region comprising the amino acid sequence of SEQ ID NO: 14; a V_L CDR2 region comprising the amino acid sequence of SEQ ID NO: 15; and a V_L CDR3 region comprising an amino acid sequence of SEQ ID NO:16; and (c) a V_H CDR1 region comprising the amino acid sequence of SEQ ID NO: 28; a V_H CDR2 region comprising the amino acid sequence of SEQ ID NO:29, a V_H CDR3 region comprising the amino acid sequence of SEQ ID NO:30; a V_L CDR1 region comprising the amino acid sequence of SEQ ID NO: 34; a V_L CDR2 region comprising the amino acid sequence of SEQ ID NO:35; and a V_L CDR3 region comprising an amino acid sequence of SEQ ID NO:36.

9. The antibody of claim 7, wherein said antibody is an IgG isotype.

10. The antibody of claim 7, wherein said antibody is an IgG1 isotype.

11. The antibody of claim 7, wherein said antibody comprises a heavy chain variable sequence comprising an amino acid sequence selected from SEQ ID NO: 2, 18, 22, 38, 48, 52, 56, 60, 68, 84, 100, 116, 132, 148, 164, 180, 200, 216, 232, or 248 and a light chain variable sequence comprising the amino acid sequence of SEQ ID NO: 4, 24, 40, 62, 70, 86, 102, 118, 134, 150, 166, 182, 196, 202, 218, 234, or 250.

12. The antibody of claim 11, wherein said antibody is an IgG isotype.

13. The antibody of claim 11, wherein said antibody comprises a combination of a heavy chain variable sequence and a light chain variable sequence selected from the group consisting of: (a) a heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO:2 and a light chain variable sequence comprising the amino acid sequence of SEQ ID NO:4; (b) a heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO:18 and a light chain variable sequence comprising the amino acid sequence of SEQ ID NO:4; and (c) a heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO:22 and a light chain variable sequence comprising the amino acid sequence of SEQ ID NO:24.

14. The antibody of claim 13, wherein said antibody comprises a combination of a heavy chain sequence and a light chain variable sequence selected from the group consisting of: (a) a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 167 and a light chain sequence comprising the amino acid sequence of SEQ ID NO: 168; (b) a heavy chain sequence comprising the amino acid sequence of SEQ ID NO:238 and a light chain sequence comprising the amino acid sequence of SEQ ID NO:254; and (c) a heavy chain sequence comprising the amino acid sequence of SEQ ID NO:186 and a light chain sequence comprising the amino acid sequence of SEQ ID NO:187.

15. The antibody of claim 13, wherein said antibody is an IgG isotype.

16. The antibody of claim 15, wherein said antibody is an IgG1 isotype.

17. A pharmaceutical composition comprising the antibody of claim 1 and a carrier.

18. An isolated antibody that binds human RANTES when human RANTES is bound to a glycosaminoglycan (GAG), wherein said antibody comprises: (a) a V_H CDR1 region comprising the amino acid sequence of SEQ ID NO: 8, 28, 44, 90, 106, 122 or 154; (b) a V_H CDR2 region comprising the amino acid sequence of SEQ ID NO: 9, 29, 45, 91, 107, 123, 155, or 207; (c) a V_H CDR3 region comprising the amino acid sequence of SEQ ID NO: 10, 20, 30, 64, 92, 124, 156, 188, or 208, (d) a V_L CDR1 region comprising the amino acid sequence of SEQ ID NO: 14, 34, 96, 128, 160, 176, 192, or 212; (e) a V_L CDR2 region comprising the amino acid sequence of SEQ ID NO: 15, 35, 97, 129, 161, 177, 193, or 213; and (f) a V_L CDR3 region comprising the amino acid sequence of SEQ ID NO: 16, 36, 98, 130, 162, 178, 194, or 214. wherein said antibody binds RANTES in the context of GAG.

19. The antibody of claim 18, wherein said antibody is a monoclonal antibody or an antigen-binding fragment thereof.

20. The antibody of claim 18, wherein said antibody is a fully human monoclonal antibody or an antigen-binding fragment thereof.

21. The antibody of claim 18, wherein said antibody is an IgG isotype.

22. The antibody of claim 18, wherein said antibody is an IgG1 isotype.

23. A method of alleviating a symptom of a clinical indication associated with ischemia or reperfusion injury in a subject, the method comprising administering the antagonist of claim 1 to a subject in need thereof in an amount sufficient to alleviate the symptom of the clinical indication associated with ischemia or reperfusion injury in the subject.

24. The method of claim 23, wherein said subject is a human.

25. The method of claim 23, wherein said antagonist is a monoclonal antibody or an antigen-binding fragment thereof.

26. The method of claim 23, wherein said antagonist is the antibody of claim 7.

27. The method of claim 31, wherein said antagonist is a mutated RANTES polypeptide that modulates an activity of RANTES selected from the ability of RANTES to bind to a receptor selected from CCR1, CCR3, CCR4, and CCR5, the ability of RANTES to bind a glycosaminoglycan and the ability of RANTES to form oligomers.

28. A method of alleviating a symptom of an autoimmune disease or inflammatory disorder, the method comprising administering the antibody of claim 7 to a subject in need thereof in an amount sufficient to alleviate the symptom of the autoimmune disease or inflammatory disorder in the subject.

29. The method of claim 28, wherein said subject is a human.

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