ANTI-EPHA2 ANTIBODY

Abstract

The present invention provides an antibody which recognizes an epitope recognized by an antibody produced by a hybridoma SH348-1 (FERM BP-10836) or a hybridoma SH357-1 (FERM BP-10837), an antibody produced by the hybridoma SH348-1 or the hybridoma SH357-1, an antibody obtained by humanizing the antibody produced by the hybridoma SH348-1 or the hybridoma SH357-1, a pharmaceutical agent comprising the antibody as an active ingredient, etc.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application is a division of U.S. patent application Ser. No. 13/899,996, filed May 22, 2013, which is a division of U.S. patent application Ser. No. 12/713,041, filed Feb. 25, 2010, now U.S. Pat. No. 8,449,882, which is a continuation-in-part of International Application No. PCT/JP2008/065486, filed Aug. 29, 2008, which claims priority from Japanese Patent Application No. 2007-224007, filed Aug. 30, 2007. Each application is incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] The present invention relates to an antibody having an inhibitory activity against cell malignant transformation and/or tumor cell growth. More specifically, the present invention relates to an antibody against EPHA2 and a pharmaceutical composition comprising the antibody.

STATEMENT REGARDING SEQUENCE LISTING

[0003] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is 54617_SEQ_Final_--2015-10-02.txt. The text file is 233 KB; was created on Oct. 2, 2015; and is being submitted via EFS-Web with the filing of the specification.

BACKGROUND

[0004] EPHA2 is a receptor tyrosine kinase that has a molecular weight of 130 kDa and has a single transmembrane domain (Molecular and Cellular Biology, 1990, vol. 10, p. 6316-6324). EPHA2 has a ligand-binding domain and two fibronectin type 3 domains present in the N-terminal extracellular region and a tyrosine kinase domain and a sterile-α-motif (SAM) domain present in the C-terminal intracellular region.

[0005] GPI-anchored plasma membrane proteins ephrin-A1 to ephrin-A5 are known as EPHA2 ligands (Annual Review of Neuroscience, 1998, vol. 21, p. 309-345). The ligand binding to EPHA2 activates the tyrosine kinase domain and phosphorylates tyrosine residues present in the EPHA2 intracellular region, resulting in signal transduction within the cell. It has also been reported that EPHA2 bound with the ligand is internalized into the cell through endocytosis and is eventually degraded by a proteasome (Molecular Cancer Research, 2002, vol. 1, p. 79-87).

[0006] High expression of EPHA2 has been reported clinically in many cancers, particularly, breast cancer, esophagus cancer, prostate cancer, gastric cancer, non-small cell lung cancer, colon cancer, and glioblastoma multiforme (Cancer Research, 2001, vol. 61, p. 2301-2306; International Journal of Cancer, 2003, vol. 103, p. 657-663; The Prostate, 1999, vol. 41, p. 275-280; American Journal of Pathology, 2003, vol. 163, p. 2271-2276; Cancer Science, 2005, vol. 96, p. 42-47; Clinical Cancer Research, 2003, vol. 9, p. 613-618; Oncology Reports, 2004, vol. 11, p. 605-611; and Molecular Cancer Research, 2005, vol. 3, p. 541-551). It has also been reported that: for esophagus cancer, EPHA2 expression-positive patients tend to have a high frequency of regional lymph node metastasis, a large number of lymph node metastases, and a poor degree of tumor differentiation and a low five-year survival rate (International Journal of Cancer, 2003, vol. 103, p. 657-663); for non-small cell lung cancer, patients highly expressing EPHA2 tend to have a low disease-free survival rate and to have recurrence, particularly of brain metastasis (Clinical Cancer Research, 2003, vol. 9, p. 613-618); and for colon cancer, EPHA2 expression-positive patients tend to have liver metastasis, lymphatic vessel invasion, and lymph node metastasis, and many patients with high clinical stage are EPHA2 expression-positive patients (Oncology Reports, 2004, vol. 11, p. 605-611).

[0007] Moreover, it has been reported that by the introduction of EPHA2 genes into cells, non-cancer cells acquire cancer phenotypes such as anchorage-independent growth ability, tubular morphology-forming ability on the extracellular matrix, and in vivo tumor growth ability (Cancer Research, 2001, vol. 61, p. 2301-2306), and cancer cells have enhanced invasiveness through the extracellular matrix (Biochemical and Biophysical Research Communications, 2004, vol. 320, p. 1096-1102; and Oncogene, 2004, vol. 23, p. 1448-1456). In addition, it has been reported that: the invasiveness or anchorage-independent growth of cancer cells and in vivo tumor growth are inhibited by knockdown of EPHA2 expression using siRNA (Oncogene, 2004, vol. 23, p. 1448-1456; and Cancer Research, 2002, vol. 62, p. 2840-2847); and the invasiveness, anchorage-independent growth, and tubular morphology-forming ability of cancer cells are inhibited by activating EPHA2 using fusion proteins of its ligand ephrin-A1 and a human IgG Fc region and inducing EPHA2 degradation through endocytosis (Cancer Research, 2001, vol. 61, p. 2301-2306; Molecular Cancer Research, 2005, vol. 3, p. 541-551; and Biochemical and Biophysical Research Communications, 2004, vol. 320, p. 1096-1102).

[0008] On the other hand, EPHA2 has been reported to be expressed not only in cancer cells but also within tumors or in their surrounding blood vessels (Oncogene, 2000, vol. 19, p. 6043-6052). It has been reported that in mice, EPHA2 signals are involved in angiogenesis induced by ephrin-A1, and particularly, EPHA2 expressed in vascular endothelial cells is required for the tube formation or survival of the vascular endothelial cells (Journal of Cell Science, 2004, vol. 117, p. 2037-2049). It has also been reported that fusion proteins of an EPHA2 extracellular region and a human IgG Fc region inhibit angiogenesis in vivo and exhibit an antitumor effect (Oncogene, 2002, vol. 21, p. 7011-7026).

[0009] Monoclonal antibodies are useful not only as diagnostic drugs but also as therapeutic drugs. Monoclonal antibodies are actively used particularly in the field of cancer therapy, and monoclonal antibodies against receptor tyrosine kinases such as HER2 and EGFR or against CD20 extracellular regions are used in cancer therapy and exhibit excellent effects (The New England Journal of Medicine, 2007, vol. 357, p. 39-51; Oncogene, 2007, vol. 26, p. 3661-3678; and Oncogene, 2007, vol. 26, p. 3603-3613). The mechanisms of action of the monoclonal antibodies used in cancer therapy include apoptosis induction and inhibition of growth signals. In addition, their immunoresponse-mediated action such as ADCC or CDC is also considered to play a very important role. In actuality, it has been reported that anti-HER2 antibodies (trastuzumab) or anti-CD20 antibodies (rituximab) exhibit a much weaker antitumor effect in xenografts of nude mice deficient in FcγRs necessary for ADCC induction than in nude mice that are not deficient in FcγRs, when these antibodies are administered thereto (Nature Medicine, 2000, vol. 6, p. 443-446). It has also been reported that anti-CD20 antibodies (rituximab) exhibit a weaker antitumor effect in mice depleted of complement by the administration of cobra venom than in mice that are not depleted of complement, when the antibody is administered thereto (Blood, 2004, vol. 103, p. 2738-2743).

[0010] For EPHA2, it has been reported that agonistic anti-EPHA2 monoclonal antibodies having an activity of inducing the phosphorylation of EPHA2 tyrosine residues and an activity of inducing EPHA2 degradation, as for the ligands, inhibit the anchorage-independent growth of a breast cancer cell line and the tubular morphology formation thereof on the extracellular matrix (Cancer Research, 2002, vol. 62, p. 2840-2847). It has also been reported that agonistic anti-EPHA2 monoclonal antibodies which recognize an epitope on EPHA2 displayed on cancer cells rather than non-cancer cells and have an activity of inducing the phosphorylation of EPHA2 tyrosine residues and an activity of inducing EPHA2 degradation exhibit an antitumor effect in vivo (Cancer Research, 2003, vol. 63, p. 7907-7912; and the pamphlet of WO 03/094859). On the other hand, Kiewlich et al. have reported that their anti-EPHA2 monoclonal antibodies had an activity of inducing the phosphorylation of EPHA2 tyrosine residues and an activity of inducing EPHA2 degradation but did not exhibit an antitumor effect in vivo (Neoplasia, 2006, vol. 8, p. 18-30).

[0011] Moreover, the pamphlet of WO 2006/084226 discloses anti-EPHA2 monoclonal antibodies LUCA19, SG5, LUCA40, and SPL1 obtained by immunizing mice with cancer cells and discloses that, among these antibodies: LUCA19 and SG5 do not influence the phosphorylation of EPHA2 tyrosine residues; LUCA40 inhibits cancer cell growth in vitro; and LUCA19, SG5, and LUCA40 are internalized into cancer cells in the presence of anti-mouse antibody labeled with toxin (saporin). The document has also reported that LUCA40 and SPL1 exhibit an antitumor effect in vivo. However, the presence or absence of the agonistic activities of these antibodies remains to be clarified.

[0012] Despite these studies, an epitope for an anti-EPHA2 antibody that exhibits an antitumor effect in vivo is still unknown. No previous document has reported that a particular amino acid sequence in an EPHA2 extracellular region is useful as an epitope for a monoclonal antibody intended for cancer therapy.

[0013] Even antibodies against the same antigen differ in their properties depending on differences in epitopes or their sequences. Furthermore, due to this difference in their properties, the antibodies, when administered to humans, would clinically respond in different a manner with respect to drug effectiveness, the frequency of therapeutic response, side effects, the frequency of occurrence of drug resistance, etc.

[0014] Thus, a drug having clinically the best properties may also differ depending on the patient. In many cases, such properties are unknown until the drug is actually administered. Thus, it has been strongly required to develop a drug having a novel mechanism of action. It has also been strongly required to develop an antibody against EPHA2 having properties different from those of conventional antibodies.

DESCRIPTION OF THE DRAWINGS

[0015] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIGS. 1A and 1B are diagrams showing Western blotting results showing the presence or absence of the activity of inducing the phosphorylation of EPHA2 tyrosine residues by an anti-EPHA2 antibody. FIG. 1A) is a diagram showing the results obtained in the absence of a cross-linking antibody, wherein the upper bar shows the results for a 4G10 antibody, and the lower bar shows the results for an anti-EPHA2 antibody (D7). FIG. 1B) is a diagram showing the results obtained in the presence of a cross-linking antibody, wherein the upper bar shows the results of a 4G10 antibody, and the lower bar shows the results of an anti-EPHA2 antibody (D7);

[0017] FIGS. 2A and 2B are diagrams showing Western blotting results showing the presence or absence of the activity of inducing a decrease in EPHA2 protein level by an anti-EPHA2 antibody. FIG. 2A) is a diagram showing the results obtained in the absence of a cross-linking antibody, wherein the upper bar shows the results for an anti-EPHA2 antibody (D7), and the lower bar shows the results for an anti-β-actin antibody. FIG. 2B) is a diagram showing the results obtained in the presence of a cross-linking antibody, wherein the upper bar shows the results for an anti-EPHA2 antibody (D7), and the lower bar shows the results for an anti-β-actin antibody;

[0018] FIGS. 3A-3C are diagrams showing the presence or absence of the ADCC activity of an anti-EPHA2 antibody against various cell lines. In the diagram, "**" represents P<0.01, and "***" represents P<0.001. FIG. 3A) is a diagram showing the ADCC activity against MDA-MB-231 cells. FIG. 3B) is a diagram showing the ADCC activity against A549 cells. FIG. 3C) is a diagram showing the ADCC activity against PC-3 cells;

[0019] FIGS. 4A-4F are diagrams showing the presence or absence of the CDC activity of an anti-EPHA2 antibody against various cells. In the diagram, "***" represents P<0.001. FIG. 4A) is a diagram showing the CDC activity of SH348-1 against MDA-MB-231 cells. FIG. 4B) is a diagram showing the CDC activity of SH348-1 against A549 cells. FIG. 4C) is a diagram showing the CDC activity of SH348-1 against PC-3 cells. FIG. 4D) is a diagram showing the CDC activity of SH357-1 against MDA-MB-231 cells. FIG. 4E) is a diagram showing the CDC activity of SH357-1 against A549 cells. FIG. 4F) is a diagram showing the CDC activity of SH357-1 against PC-3 cells;

[0020] FIG. 5A) is a diagram showing EPHA2 domain structure prediction and the positions, in EPHA2, of EPHA2-ECD, FNIII-NC, FNIII-N, and FNIII-C which are peptides for epitope determination. In the diagram, Ligand-BD represents a ligand-binding domain, FN3 represents a fibronectin type 3 domain, TM represents a transmembrane region, Trk kinase represents a tyrosine kinase domain, and SAM represents a SAM domain;

[0021] FIG. 5B) is a diagram showing the presence or absence of the binding activity of an anti-EPHA2 antibody for EPHA2-ECD, FNIII-NC, FNIII-N, and FNIII-C;

[0022] FIG. 6A) is a diagram showing the antitumor activity of SH348-1 against MDA-MB-231 cell-transplanted mice;

[0023] FIG. 6B) is a diagram showing the antitumor activity of SH357-1 against MDA-MB-231 cell-transplanted mice. In the diagram, the error bar represents a standard error (n=9);

[0024] FIG. 7A) is a diagram showing the binding activity of SH348-1 to an EPHA2 extracellular region polypeptide;

[0025] FIG. 7B) is a diagram showing the binding activity of SH357-1 to an EPHA2 extracellular region polypeptide;

[0026] FIG. 7C) is a diagram showing the binding activity of Ab96-1 to an EPHA2 extracellular region polypeptide;

[0027] FIG. 8 is a diagram showing the ligand binding inhibitory activities of SH348-1, SH357-1, and Ab96-1;

[0028] FIG. 9 is a diagram showing that SH348-1 and SH357-1 have an activity of inhibiting the ephrin-A1-dependent phosphorylation of EPHA2 tyrosine residues;

[0029] FIG. 10 is a diagram showing the outline of deletion mutants of EPHA2 for epitope identification;

[0030] FIG. 11A) is a diagram showing the reactivity of SH348-1 to deletion mutants of EPHA2;

[0031] FIG. 11B) is a diagram showing the detection of the deletion mutants of EPHA2 on a PVDF membrane in FIG. 11A);

[0032] FIG. 11C) is a diagram showing the reactivity of SH357-1 to deletion mutants of EPHA2;

[0033] FIG. 11D) is a diagram showing the detection of the deletion mutants of EPHA2 on a PVDF membrane in FIG. 11C);

[0034] FIG. 12A) is a diagram showing the binding activity of hSH348-1-T1 to an EPHA2 extracellular region polypeptide;

[0035] FIG. 12B) is a diagram showing the binding activity of hSH348-1-T3 to an EPHA2 extracellular region polypeptide;

[0036] FIG. 12C) is a diagram showing the binding activity of hSH357-1-T1 to an EPHA2 extracellular region polypeptide;

[0037] FIG. 12D) is a diagram showing the binding activity of hSH357-1-T3 to an EPHA2 extracellular region polypeptide;

[0038] FIG. 13A) is a diagram showing the competitive inhibitory activities of hSH348-1-T1 and hSH348-1-T3 against the antigen binding of SH348-1;

[0039] FIG. 13B) is a diagram showing the competitive inhibitory activities of hSH357-1-T1 and hSH357-1-T3 against the antigen binding of SH357-1;

[0040] FIG. 14A) is a diagram showing the activity of inhibiting the ephrin-A1-dependent phosphorylation of EPHA2 tyrosine residues by hSH348-1-T1 or hSH348-1-T3; and

[0041] FIG. 14B) is a diagram showing the activity of inhibiting the ephrin-A1-dependent phosphorylation of EPHA2 tyrosine residues by hSH357-1-T1 or hSH357-1-T3.

DETAILED DESCRIPTION

Problems to be Solved by the Invention

[0042] An object of the present invention is to provide an antibody against EPHA2.

[0043] A further object of the present invention is to provide a pharmaceutical composition and the like comprising an anti-EPHA2 antibody having a therapeutic effect on cancer.

[0044] A further object of the present invention is to provide a method for producing the antibody.

[0045] A further object of the present invention is to provide a method for inhibiting tumor growth using the antibody, etc.

Means for Solving the Problems

[0046] The present inventors have conducted diligent studies to attain the objects and consequently successfully obtained a novel anti-EPHA2 monoclonal antibody which has no activity of inducing the phosphorylation of EPHA2 tyrosine residues and has ADCC and CDC activities against EPHA2-expressing cancer cells. Furthermore, the present inventors have examined an epitope for this antibody and consequently found for the first time that an antibody that binds to a region comprising, of two fibronectin type 3 domains present in EPHA2, the c-terminal domain, has an excellent antitumor activity in vivo. Based on these findings, the present invention has been completed.

[0047] Specifically, the present invention comprises:

[0048] (1) An antibody which recognizes an epitope recognized by an antibody produced by the hybridoma SH348-1 (FERM BP-10836);

[0049] (2) The antibody according to (1), wherein the antibody has the following properties a) to d):

[0050] a) having no ability to phosphorylate EPHA2 tyrosine residues;

[0051] b) having an ADCC activity against EPHA2-expressing cells;

[0052] c) having a CDC activity against EPHA2-expressing cells; and

[0053] d) having an antitumor activity in vivo;

[0054] (3) The antibody according to (1), wherein the antibody has the following properties a) to e):

[0055] a) having no ability to phosphorylate EPHA2 tyrosine residues;

[0056] b) exhibiting an effect of decreasing an EPHA2 protein level;

[0057] c) having an ADCC activity against EPHA2-expressing cells;

[0058] d) having a CDC activity against EPHA2-expressing cells; and

[0059] e) having an antitumor activity in vivo;

[0060] (4) An antibody which specifically binds to a polypeptide consisting of an amino acid sequence represented by amino acid Nos. 426 to 534 of SEQ ID NO: 8 in the sequence listing;

[0061] (5) An antibody which specifically binds to a polypeptide consisting of an amino acid sequence represented by amino acid Nos. 426 to 534 of SEQ ID NO: 8 in the sequence listing and has the following properties a) to d):

[0062] a) having no ability to phosphorylate EPHA2 tyrosine residues;

[0063] b) having an ADCC activity against EPHA2-expressing cells;

[0064] c) having a CDC activity against EPHA2-expressing cells; and

[0065] d) having an antitumor activity in vivo;

[0066] (6) An antibody which specifically binds to a peptide consisting of an amino acid sequence represented by amino acid Nos. 426 to 534 of SEQ ID NO: 8 in the sequence listing and has the following properties a) to e):

[0067] a) having no ability to phosphorylate EPHA2 tyrosine residues;

[0068] b) exhibiting an effect of decreasing an EPHA2 protein level;

[0069] c) having an ADCC activity against EPHA2-expressing cells;

[0070] d) having a CDC activity against EPHA2-expressing cells; and

[0071] e) having an antitumor activity in vivo;

[0072] (7) The antibody according to any one of (1) to (6), wherein the antibody specifically binds to a peptide consisting of an amino acid sequence represented by amino acid Nos. 439 to 534 of SEQ ID NO: 8 in the sequence listing;

[0073] (8) The antibody according to any one of (1) to (7), wherein the antibody inhibits the phosphorylation of EPHA2 tyrosine residues induced by an EPHA2 ligand;

[0074] (9) The antibody according to any one of (1) to (7), wherein the antibody does not inhibit EPHA2 ligand binding to EPHA2 but inhibits the phosphorylation of EPHA2 tyrosine residues induced by the ligand;

[0075] (10) An antibody which specifically binds to a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8 in the sequence listing, wherein the antibody has the amino acid sequences represented by SEQ ID NOs: 59, 61, and 63 in the sequence listing, or amino acid sequences having deletion, substitution, or addition of one or more amino acids in the amino acid sequences, as the complementarity determining regions in the heavy chain variable region and has the amino acid sequences represented by SEQ ID NOs: 65, 67, and 69 in the sequence listing, or amino acid sequences having deletion, substitution, or addition of one or more amino acids in the amino acid sequences, as complementarity determining regions in the light chain variable region;

[0076] (11) An antibody which specifically binds to a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8 in the sequence listing, characterized by the following 1) and 2):

[0077] 1) having a heavy chain peptide comprising an amino acid sequence represented by the general formula (I):

-FRH₁-CDRH₁-FRH₂-CDRH₂-FRH₃-CDRH₃-FRH₄- (I)

[0078] wherein FRH₁ represents an arbitrary amino acid sequence consisting of 18 to 30 amino acids; CDRH₁ represents the amino acid sequence represented by SEQ ID NO: 59 in the sequence listing or an amino acid sequence having deletion, substitution, or addition of one or more amino acids in the amino acid sequence; FRH₂ represents an arbitrary amino acid sequence consisting of 14 amino acids; CDRH₂ represents the amino acid sequence represented by SEQ ID NO: 61 in the sequence listing or an amino acid sequence having deletion, substitution, or addition of one or more amino acids in the amino acid sequence; FRH₃ represents an arbitrary amino acid sequence consisting of 32 amino acids; CDRH₃ represents the amino acid sequence represented by SEQ ID NO: 63 in the sequence listing or an amino acid sequence having deletion, substitution, or addition of one or more amino acids in the amino acid sequence; and FRH₄ represents an arbitrary amino acid sequence consisting of 11 amino acids, wherein these amino acids are linked to each other through peptide bonds; and

[0079] 2) having a light chain polypeptide comprising an amino acid sequence represented by the general formula (II):

-FRL₁-CDRL₁-FRL₂-CDRL₂-FRL₃-CDRL₃-FRL₄- (II)

[0080] wherein FRL₁ represents an arbitrary amino acid sequence consisting of 23 amino acids; CDRL₁ represents the amino acid sequence represented by SEQ ID NO: 65 in the sequence listing or an amino acid sequence having deletion, substitution, or addition of one or more amino acids in the amino acid sequence; FRL₂ represents an arbitrary amino acid sequence consisting of 15 amino acids; CDRL₂ represents the amino acid sequence represented by SEQ ID NO: 67 in the sequence listing or an amino acid sequence having deletion, substitution, or addition of one or more amino acids in the amino acid sequence; FRL₃ represents an arbitrary amino acid sequence consisting of 32 amino acids; CDRL₃ represents the amino acid sequence represented by SEQ ID NO: 69 in the sequence listing or an amino acid sequence having deletion, substitution, or addition of one or more amino acids in the amino acid sequence; and FRL₄ represents an arbitrary amino acid sequence consisting of 10 amino acids, wherein these amino acids are linked to each other through peptide bonds;

[0081] (12) An antibody which recognizes an epitope recognized by an antibody produced by the hybridoma SH357-1 (FERM BP-10837);

[0082] (13) The antibody according to (12), wherein the antibody has the following properties a) to d):

[0083] a) having no ability to phosphorylate EPHA2 tyrosine residues;

[0084] b) having an ADCC activity against EPHA2-expressing cells;

[0085] c) having a CDC activity against EPHA2-expressing cells; and

[0086] d) having an antitumor activity in vivo;

[0087] (14) An antibody which specifically binds to a peptide consisting of an amino acid sequence represented by amino acid Nos. 426 to 534 of SEQ ID NO: 8 in the sequence listing and has the following properties a) to e):

[0088] a) having no ability to phosphorylate EPHA2 tyrosine residues;

[0089] b) exhibiting no effect of decreasing an EPHA2 protein level;

[0090] c) having an ADCC activity against EPHA2-expressing cells;

[0091] d) having a CDC activity against EPHA2-expressing cells; and

[0092] e) having an antitumor activity in vivo;

[0093] (15) The antibody according to any one of (12) to (14), wherein the antibody specifically binds to a peptide consisting of an amino acid sequence represented by amino acid Nos. 439 to 534 of SEQ ID NO: 8 in the sequence listing;

[0094] (16) The antibody according to any one of (12) to (15), wherein the antibody inhibits the phosphorylation of EPHA2 tyrosine residues induced by an EPHA2 ligand;

[0095] (17) The antibody according to any one of (12) to (15), wherein the antibody does not inhibit EPHA2 ligand binding to EPHA2 but inhibits the phosphorylation of EPHA2 tyrosine residues induced by the ligand;

[0096] (18) An antibody which specifically binds to a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8 in the sequence listing, wherein the antibody has the amino acid sequences represented by SEQ ID NOs: 71, 73, and 75 in the sequence listing, or amino acid sequences having deletion, substitution, or addition of one or more amino acids in the amino acid sequences, as complementarity determining regions in the heavy chain variable region and has the amino acid sequences represented by SEQ ID NOs: 77, 79, and 81 in the sequence listing, or amino acid sequences having deletion, substitution, or addition of one or more amino acids in the amino acid sequences, as complementarity determining regions in the light chain variable region;

[0097] (19) An antibody which specifically binds to a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8 in the sequence listing, characterized by the following 1) and 2):

[0098] 1) having a heavy chain peptide comprising an amino acid sequence represented by the general formula (I):

-FRH₁-CDRH₁-FRH₂-CDRH₂-FRH₃-CDRH₃-FRH₄- (I)

[0099] wherein FRH₁ represents an arbitrary amino acid sequence consisting of 18 to 30 amino acids; CDRH₁ represents the amino acid sequence represented by SEQ ID NO: 71 in the sequence listing or an amino acid sequence having deletion, substitution, or addition of one or more amino acids in the amino acid sequence; FRH₂ represents an arbitrary amino acid sequence consisting of 14 amino acids; CDRH₂ represents the amino acid sequence represented by SEQ ID NO: 73 in the sequence listing or an amino acid sequence having deletion, substitution, or addition of one or more amino acids in the amino acid sequence; FRH₃ represents an arbitrary amino acid sequence consisting of 32 amino acids; CDRH₃ represents the amino acid sequence represented by SEQ ID NO: 75 in the sequence listing or an amino acid sequence having deletion, substitution, or addition of one or more amino acids in the amino acid sequence; and FRH₄ represents an arbitrary amino acid sequence consisting of 11 amino acids, wherein these amino acids are linked to each other through peptide bonds; and

[0100] 2) having a light chain polypeptide comprising an amino acid sequence represented by the general formula (II):

-FRL₁-CDRL₁-FRL₂-CDRL₂-FRL₃-CDRL₃-FRL₄- (II)

[0101] wherein FRL₁ represents an arbitrary amino acid sequence consisting of 23 amino acids; CDRL₁ represents the amino acid sequence represented by SEQ ID NO: 77 in the sequence listing or an amino acid sequence having deletion, substitution, or addition of one or more amino acids in the amino acid sequence; FRL₂ represents an arbitrary amino acid sequence consisting of 15 amino acids; CDRL₂ represents the amino acid sequence represented by SEQ ID NO: 79 in the sequence listing or an amino acid sequence having deletion, substitution, or addition of one or more amino acids in the amino acid sequence; FRL₃ represents an arbitrary amino acid sequence consisting of 32 amino acids; CDRL₃ represents the amino acid sequence represented by SEQ ID NO: 81 in the sequence listing or an amino acid sequence having deletion, substitution, or addition of one or more amino acids in the amino acid sequence; and FRL₄ represents an arbitrary amino acid sequence consisting of 10 amino acids, wherein these amino acids are linked to each other through peptide bonds;

[0102] (20) The antibody according to any one of (1) to (19), characterized in that the antibody is a humanized antibody;

[0103] (21) The antibody according to any one of (1) to (19), characterized in that the antibody is a human antibody;

[0104] (22) The antibody according to any one of (1) to (19), characterized in that the antibody is an IgG antibody;

[0105] (23) The antibody according to any one of (1) to (9) and (12) to (17), characterized in that the antibody is any selected from Fab, F(ab')2, Fv, scFv, a diabody, a linear antibody, and a multispecific antibody;

[0106] (24) An antibody produced by the hybridoma SH348-1 (FERM BP-10836);

[0107] (25) An antibody produced by the hybridoma SH357-1 (FERM BP-10837);

[0108] (26) An antibody consisting of the following 1) and 2):

[0109] 1) a heavy chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 1 to 119 of SEQ ID NO: 35 in the sequence listing or a heavy chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 1 to 119 of SEQ ID NO: 39 in the sequence listing; and

[0110] 2) a light chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 1 to 112 of SEQ ID NO: 37 in the sequence listing or a light chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 1 to 112 of SEQ ID NO: 41 in the sequence listing;

[0111] (27) An antibody consisting of the following 1) or 2):

[0112] 1) a heavy chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 1 to 119 of SEQ ID NO: 35 in the sequence listing and a light chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 1 to 112 of SEQ ID NO: 37 in the sequence listing; and

[0113] 2) a heavy chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 1 to 119 of SEQ ID NO: 39 in the sequence listing and a light chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 1 to 112 of SEQ ID NO: 41 in the sequence listing;

[0114] (28) An antibody consisting of the following 1) and 2):

[0115] 1) a heavy chain polypeptide comprising the amino acid sequence represented by SEQ ID NO: 35 in the sequence listing or a heavy chain polypeptide comprising the amino acid sequence represented by SEQ ID NO: 39 in the sequence listing; and

[0116] 2) a light chain polypeptide comprising the amino acid sequence represented by SEQ ID NO: 37 in the sequence listing or a light chain polypeptide comprising the amino acid sequence represented by SEQ ID NO: 41 in the sequence listing;

[0117] (29) An antibody consisting of the following 1) or 2):

[0118] 1) a heavy chain polypeptide comprising the amino acid sequence represented by SEQ ID NO: 35 in the sequence listing and a light chain polypeptide comprising the amino acid sequence represented by SEQ ID NO: 37 in the sequence listing; and

[0119] 2) a heavy chain polypeptide comprising the amino acid sequence represented by SEQ ID NO: 39 in the sequence listing and a light chain polypeptide comprising the amino acid sequence represented by SEQ ID NO: 41 in the sequence listing;

[0120] (30) An antibody obtained by humanizing an antibody according to any one of (24) to (29);

[0121] (31) An antibody consisting of the following 1) and 2):

[0122] 1) a heavy chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 20 to 468 of SEQ ID NO: 107 in the sequence listing or a heavy chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 20 to 468 of SEQ ID NO: 115 in the sequence listing; and

[0123] 2) a light chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 21 to 239 of SEQ ID NO: 91 in the sequence listing or a light chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 21 to 239 of SEQ ID NO: 99 in the sequence listing;

[0124] (32) An antibody consisting of the following 1) or 2):

[0125] 1) a heavy chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 20 to 468 of SEQ ID NO: 107 in the sequence listing and a light chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 21 to 239 of SEQ ID NO: 91 in the sequence listing; and

[0126] 2) a heavy chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 20 to 468 of SEQ ID NO: 115 in the sequence listing and a light chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 21 to 239 of SEQ ID NO: 99 in the sequence listing;

[0127] (33) An antibody consisting of the following 1) and 2):

[0128] 1) a heavy chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 20 to 468 of SEQ ID NO: 139 in the sequence listing or a heavy chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 20 to 468 of SEQ ID NO: 147 in the sequence listing; and

[0129] 2) a light chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 21 to 239 of SEQ ID NO: 123 in the sequence listing or a light chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 21 to 239 of SEQ ID NO: 131 in the sequence listing;

[0130] (34) An antibody consisting of the following 1) or 2):

[0131] 1) a heavy chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 20 to 468 of SEQ ID NO: 139 in the sequence listing and a light chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 21 to 239 of SEQ ID NO: 123 in the sequence listing; and

[0132] 2) a heavy chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 20 to 468 of SEQ ID NO: 147 in the sequence listing and a light chain polypeptide comprising an amino acid sequence represented by amino acid Nos. 21 to 239 of SEQ ID NO: 131 in the sequence listing;

[0133] (35) A Fab, F(ab')2, Fv, scFv, a diabody, a linear antibody, or a multispecific antibody derived from antibodies according to any of (24) to (34);

[0134] (36) Any one polypeptide selected from the group consisting of the following 1) to 20):

[0135] 1) a polypeptide comprising an amino acid sequence represented by amino acid Nos. 1 to 119 of SEQ ID NO: 35 in the sequence listing;

[0136] 2) a polypeptide comprising an amino acid sequence represented by amino acid Nos. 1 to 119 of SEQ ID NO: 39 in the sequence listing;

[0137] 3) a polypeptide comprising an amino acid sequence represented by amino acid Nos. 1 to 112 of SEQ ID NO: 37 in the sequence listing;

[0138] 4) a polypeptide comprising an amino acid sequence represented by amino acid Nos. 1 to 112 of SEQ ID NO: 41 in the sequence listing;

[0139] 5) a polypeptide comprising an amino acid sequence represented by amino acid Nos. 20 to 468 of SEQ ID NO: 107 in the sequence listing;

[0140] 6) a polypeptide comprising an amino acid sequence represented by amino acid Nos. 20 to 468 of SEQ ID NO: 115 in the sequence listing;

[0141] 7) a polypeptide comprising an amino acid sequence represented by amino acid Nos. 20 to 138 of SEQ ID NO: 107 in the sequence listing;

[0142] 8) a polypeptide comprising an amino acid sequence represented by amino acid Nos. 20 to 138 of SEQ ID NO: 115 in the sequence listing;

[0143] 9) a polypeptide comprising an amino acid sequence represented by amino acid Nos. 21 to 239 of SEQ ID NO: 91 in the sequence listing;

[0144] 10) a polypeptide comprising an amino acid sequence represented by amino acid Nos. 21 to 239 of SEQ ID NO: 99 in the sequence listing;

[0145] 11) a polypeptide comprising an amino acid sequence represented by amino acid Nos. 21 to 134 of SEQ ID NO: 91 in the sequence listing;

[0146] 12) a polypeptide comprising an amino acid sequence represented by amino acid Nos. 21 to 134 of SEQ ID NO: 99 in the sequence listing;

[0147] 13) a polypeptide comprising an amino acid sequence represented by amino acid Nos. 20 to 468 of SEQ ID NO: 139 in the sequence listing;

[0148] 14) a polypeptide comprising an amino acid sequence represented by amino acid Nos. 20 to 468 of SEQ ID NO: 147 in the sequence listing;

[0149] 15) a polypeptide comprising an amino acid sequence represented by amino acid Nos. 20 to 138 of SEQ ID NO: 139 in the sequence listing;

[0150] 16) a polypeptide comprising an amino acid sequence represented by amino acid Nos. 20 to 138 of SEQ ID NO: 147 in the sequence listing;

[0151] 17) a polypeptide comprising an amino acid sequence represented by amino acid Nos. 21 to 239 of SEQ ID NO: 123 in the sequence listing;

[0152] 18) a polypeptide comprising an amino acid sequence represented by amino acid Nos. 21 to 239 of SEQ ID NO: 131 in the sequence listing;

[0153] 19) a polypeptide comprising an amino acid sequence represented by amino acid Nos. 21 to 134 of SEQ ID NO: 123 in the sequence listing; and

[0154] 20) a polypeptide comprising an amino acid sequence represented by amino acid Nos. 21 to 134 of SEQ ID NO: 131 in the sequence listing;

[0155] (37) The mouse hybridoma SH348-1 (FERM BP-10836);

[0156] (38) The mouse hybridoma SH357-1 (FERM BP-10837);

[0157] (39) A pharmaceutical composition characterized by comprising at least one antibody selected from the antibodies according to (1) to (35);

[0158] (40) A pharmaceutical composition for cancer treatment characterized by comprising at least one antibody selected from the antibodies according to (1) to (35);

[0159] (41) A method for inhibiting tumor growth in a mammal, comprising administering any antibody selected from the group consisting of antibodies according to (1) to (35), (39), and (40);

[0160] (42) The method for inhibiting tumor growth according to (41), characterized in that the tumor is a tumor expressing EPHA2;

[0161] (43) A polynucleotide encoding an antibody or a polypeptide according to any one of (1) to (36);

[0162] (44) A host cell transformed with a polynucleotide according to (43); and

[0163] (45) A method for producing an antibody using a host cell according to (44).

Advantages of the Invention

[0164] According to the present invention, a novel anti-EPHA2 monoclonal antibody has been successfully obtained, which has no activity of inducing the phosphorylation of EPHA2 tyrosine residues and has ADCC and CDC activities against EPHA2-expressing cancer cells. Furthermore, it has been found that the antibody has excellent antitumor activity in vivo.

[0165] Furthermore, a pharmaceutical composition for cancer treatment comprising the antibody has been provided.

BEST MODE FOR CARRYING OUT THE INVENTION

1. Definitions

[0166] In the present specification, the terms "cancer" and "tumor" are used in the same sense.

[0167] In the present specification, the term "gene" is meant to encompass not only DNA but also mRNA thereof, cDNA, and cRNA thereof. Thus, the term "EPHA2 gene" in the present invention encompasses EPHA2 DNA, mRNA, cDNA, and cRNA.

[0168] In the present specification, the term "polynucleotide" is used in the same sense as a nucleic acid and also encompasses DNA, RNA, probes, oligonucleotides, and primers.

[0169] In the present specification, the terms "polypeptide" and "protein" are used without being differentiated therebetween.

[0170] In the present specification, the term "cell" also encompasses cells in individual animal and cultured cells.

[0171] In the present specification, the term "cell malignant transformation" means that cells exhibit abnormal growth, for example, lose sensitivity to contact inhibition or exhibit anchorage-independent growth. Cells exhibiting such abnormal growth are referred to as "cancer cells".

[0172] In the present specification, a protein having equivalent functions to the cell malignant transformation activity and/or cell growth activity or the like of EPHA2 is also referred to as EPHA2.

[0173] In the present specification, the term "phosphorylation of tyrosine residues" means that tyrosine residues contained in the amino acid sequence of a peptide are phosphorylated. Whether or not tyrosine residues are phosphorylated can be examined, for example, based on the affinity of the peptide for an anti-phosphotyrosine antibody (e.g., Anti-Phosphotyrosine, recombinant 4G10 HRP-conjugate (manufactured by Millipore (Upstate), #16-184)). The tyrosine residues can be determined to be phosphorylated, when the peptide binds to the antibody.

[0174] In the present specification, the term "ability to phosphorylate EPHA2 tyrosine residues" refers to an ability to phosphorylate tyrosine residues in the amino acid sequence of EPHA2. Whether or not an antibody has the ability to phosphorylate EPHA2 tyrosine residues can be determined, for example, by incubating the antibody and EPHA2 and then examining the presence or absence of the affinity of the EPHA2 for an anti-phosphotyrosine antibody.

[0175] In the present specification, the phrase "decreasing an EPHA2 protein expression level" means that an EPHA2 protein level is decreased. Whether or not an antibody has an effect of decreasing an EPHA2 protein level can be examined, for example, by incubating the antibody and EPHA2 and then quantifying the EPHA2 level.

[0176] In the present specification, the term "EPHA2 ligand" refers to a substance capable of serving as an EPHA2 ligand. Specific examples thereof can include GPI-anchored plasma membrane proteins ephrin-A1 to ephrin-A5 (Annual Review of Neuroscience, 1998, vol. 21, p. 309-345).

[0177] In the present specification, the term "cytotoxicity" refers to any pathologic change in cells and refers not only to direct injury but also to any structural or functional damage to cells such as DNA cleavage, dimerization of bases, chromosomal breakage, damage of mitotic apparatus, and decrease in various enzyme activities.

[0178] In the present specification, the term "cytotoxic activity" refers to an activity that causes the cytotoxicity.

[0179] In the present specification, ADCC is synonymous with antibody-dependent cellular cytotoxicity and refers to a reaction through which Fcγ receptor-bearing cells adhere via the Fcγ receptors to the Fc portions of antibodies bound with surface antigens in target cells and kill the target cells. An ADCC activity is also referred to as an antibody-dependent cytotoxic activity and refers to an activity that causes the reaction. The ADCC activity can be measured by methods usually performed by those skilled in the art and can be measured, for example, according to a method described in paragraph 3)-2 of Example 3 in the present specification.

[0180] In the present specification, the term "CDC" is synonymous with complement-dependent cytotoxicity. A CDC activity refers to an activity that causes complement-dependent cytotoxicity. The CDC activity can be measured by methods usually performed by those skilled in the art and can be measured, for example, according to a method described in paragraph 3)-3 of Example 3 in the present specification.

[0181] In the present specification, the phrase "having an antitumor activity in vivo" means having the activity of inhibiting or reducing tumor growth in tumor-bearing animal individuals. Whether or not an anti-EPHA2 antibody "has an antitumor activity in vivo" can be examined by methods usually performed by those skilled in the art and can also be examined, for example, according to the following method: an appropriate dose of the anti-EPHA2 antibody is intraperitoneally administered as a test substance to tumor cell (e.g., MDA-MB-231 cell)-subcutaneously transplanted nude mice (e.g., BALB/cAJcl-nu/nu; obtained from CLEA Japan, Inc.), and the time-dependent change in tumor volume is compared between the nude mice and anti-EPHA2 antibody-unadministered controls. The anti-EPHA2 antibody as a test substance can be determined to "have an antitumor activity in vivo", when the tumor volume is significantly smaller in the mice than in the controls.

[0182] Each of the heavy and light chains of an antibody molecule is known to have three complementarity determining regions (CDRs). In the present specification, the complementarity determining regions of an antibody are represented by CDRH₁, CDRH₂, and CDRH₃ for the complementarity determining regions of the heavy chain and by CDRL₁, CDRL₂, and CDRL₃ for the complementarity determining regions of the light chain.

[0183] In the present specification, the term "epitope" means an EPHA2 partial peptide having antigenicity and/or immunogenicity in vivo in animals, preferably, mammals, more preferably mice or humans. The epitope as an EPHA2 partial peptide having antigenicity can be determined by methods well known by those skilled in the art, such as by an immunoassay, and can be determined, for example, according to the following method in which various partial structures of EPHA2 are prepared. For the preparation of the partial structures, oligopeptide synthesis techniques known in the art can be used. For example, a series of sequentially shorter polypeptides of appropriate lengths from the C- or N-terminus of EPHA2 is prepared using a gene recombination technique well known by those skilled in the art. Then, the reactivity of the antibody to these polypeptides is studied. A recognition site is roughly determined, and shorter peptides are then synthesized. The reactivity to these peptides can be studied to thereby determine the epitope.

1. Regarding EPHA2

[0184] (1) EPHA2 Gene

[0185] The nucleotide sequence of the EPHA2 gene and the amino acid sequence thereof are recorded as EPH receptor A2 in GenBank (Accession NOs: NM_--004431 and NP_--004422, respectively). Moreover, the nucleotide sequence of an open reading frame (ORF) in the EPHA2 gene is described in SEQ ID NO: 1 in the sequence listing. The amino acid sequence thereof is described in SEQ ID NO: 2 in the sequence listing.

[0186] In this context, EPHA2 also encompasses proteins which consist of an amino acid sequence derived from the EPHA2 amino acid sequence by the substitution, deletion, or addition of one or more amino acids and have an equivalent biological activity to that of this enzyme.

[0187] (2) Cancer Site-Specific Expression of EPHA2 Gene

[0188] The EPHA2 gene has been reported to be highly expressed in many cancers, particularly, breast cancer, esophagus cancer, prostate cancer, gastric cancer, non-small cell lung cancer, colon cancer, and glioblastoma multiform.

[0189] Specifically, the expression level of EPHA2 in each cell and/or each tissue can be measured to thereby determine the state of malignant transformation and/or cancer cell growth that can be attributed to EPHA2 over-expression in test subjects.

[0190] Moreover, a substance that inhibits the expression level and/or activity of EPHA2 has an activity of inhibiting cell malignant transformation and/or cancer cell growth attributed to EPHA2.

[0191] Thus, test substances are contacted with EPHA2-expressing cells, and a substance that inhibits the expression level and/or activity of EPHA2 can be selected to thereby screen for an antitumor substance.

[0192] In this context, siRNA against EPHA2 inhibits EPHA2 expression and can be used as an antitumor agent. The siRNA against EPHA2 can be produced by: designing, based on the nucleotide sequence of EPHA2 mRNA, RNA consisting of a partial sequence of EPHA2 mRNA (sense RNA) and RNA consisting of a nucleotide sequence complementary to the nucleotide sequence of the RNA (antisense RNA); synthesizing the RNAs by a chemical synthesis method known per se in the art; and hybridizing both the obtained RNAs. It is preferred that a sequence of one or more nucleotides called an overhang sequence should be bound to the 3'-end of each of the sense and antisense RNAs constituting the siRNA. The overhang sequence is not particularly limited as long as it protects the RNA from nuclease. Any sequence of preferably 1 to 10, more preferably 1 to 4, even more preferably 2 nucleotides can be used.

2. Antibody against EPHA2

[0193] (1) Preparation of Antigen

[0194] Examples of antigens for obtaining the antibody of the present invention against EPHA2 can include a full-length polypeptide of EPHA2 and partial polypeptides thereof and can more specifically include a full-length polypeptide of EPHA2 and preferably an EPHA2 extracellular region polypeptide (consisting of an amino acid sequence represented by amino acid Nos. 1 to 534 of SEQ ID NO: 8 in the sequence listing), more preferably a partial polypeptide of the EPHA2 extracellular region polypeptide comprising an amino acid sequence represented by amino acid Nos. 426 to 534 of SEQ ID NO: 8 in the sequence listing, even more preferably a partial polypeptide of the EPHA2 extracellular region polypeptide comprising an amino acid sequence represented by amino acid Nos. 439 to 534 of SEQ ID NO: 8 in the sequence listing, and derivatives obtained by adding an arbitrary amino acid sequence or a carrier to these sequences. Further examples thereof can include polypeptides consisting of consecutive partial amino acid sequences of at least 6 amino acids and derivatives obtained by adding an arbitrary amino acid sequence or a carrier to these sequences.

[0195] In this context, the EPHA2 full-length polypeptide or the partial polypeptides thereof used as an antigen can be obtained by causing the EPHA2 gene or genes of the partial polypeptides to be expressed in host cells by genetic engineering.

[0196] EPHA2 can be directly purified, for use, from human tumor tissues or tumor cells. Moreover, the EPHA2 full-length polypeptide or the partial polypeptides thereof can be synthesized in vitro or obtained by causing host cells by genetic engineering to produce the polypeptide.

[0197] In the genetic engineering, specifically, genes encoding EPHA2 or partial polypeptides thereof are incorporated into vectors capable of expressing the EPHA2 or partial polypeptides thereof, and the EPHA2 or partial polypeptides thereof can then be synthesized in a solution containing enzymes, substrates, and energy substances necessary for transcription and translation. Alternatively, host cells of other prokaryotes or eukaryotes can be transformed therewith and caused to express the EPHA2 or partial polypeptides thereof to obtain the desired protein.

[0198] cDNA of the partial polypeptide of EPHA2 can be obtained, for example, by a so-called polymerase chain reaction (hereinafter, referred to as "PCR") method in which PCR (see Saiki, R. K., et al. Science (1988) 239, p. 487-489) is performed using EPHA2-expressing cDNA libraries as templates and primers specifically amplifying EPHA2 cDNA or DNA encoding the partial polypeptide.

[0199] Examples of in vitro polypeptide synthesis include, but are not limited to, the Rapid Translation System (RTS) manufactured by Roche Diagnostics Corp.

[0200] Examples of the host prokaryotic cells include Escherichia coli and Bacillus subtilis. To transform these host cells with the gene of interest, the host cells are transformed with plasmid vectors comprising a replicon, i.e., a replication origin, and a regulatory sequence derived from a species compatible with the hosts. Moreover, it is preferred that the vectors should have a sequence that can impart phenotypic character (phenotype) selectivity to the transformed cells.

[0201] The host eukaryotic cells encompass cells of vertebrates, insects, yeast, and the like. For example, monkey COS cells (Gluzman, Y. Cell (1981) 23, p. 175-182, ATCC CRL-1650), mouse fibroblasts NIH3T3 (ATCC No. CRL-1658), and dihydrofolate reductase-deficient strains (Urlaub, G. and Chasin, L. A., Proc. Natl. Acad. Sci. USA (1980) 77, p. 4126-4220) of Chinese hamster ovarian cells (CHO cells, ATCC CCL-61) are often used as the vertebrate cells, though the vertebrate cells are not limited thereto.

[0202] The transformants thus obtained can be cultured according to standard methods and are able within the culture to intracellularly or extracellularly produce the polypeptide of interest.

[0203] A medium used in the culture can be selected appropriately according to the adopted host cells from among various media routinely used. For Escherichia coli, for example, an LB medium optionally supplemented with an antibiotic (e.g., ampicillin) or IPTG can be used.

[0204] The recombinant protein intracellularly or extracellularly produced by the transformants in the culture can be separated and purified by various separation procedures known in the art using the physical properties, chemical properties, or the like of the protein.

[0205] The procedures can be exemplified specifically by treatment with usual protein precipitants, ultrafiltration, various liquid chromatography techniques such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, affinity chromatography, and high-performance liquid chromatography (HPLC), dialysis, and combinations thereof.

[0206] Moreover, the recombinant protein to be expressed can be linked to 6 histidine residues to thereby efficiently purify the resulting protein on a nickel affinity column.

[0207] By combining these methods, the polypeptide of interest can be produced easily in large amounts with high yields and high purity.

[0208] The antibody of the present invention can be obtained by immunizing animals with the antigen according to a standard method and collecting antibodies produced in vivo, followed by purification.

[0209] Moreover, antibody-producing cells that produce the antibody against EPHA2 are fused with myeloma cells according to a method known in the art (e.g., Kohler and Milstein, Nature (1975) 256, p. 495-497; and Kennet, R. ed., Monoclonal Antibodies, p. 365-367, Plenum Press, N.Y. (1980)) to thereby establish hybridomas, from which monoclonal antibodies can also be obtained.

[0210] (2) Production of Anti-EPHA2 Monoclonal Antibody

[0211] Examples of antibodies which specifically bind to EPHA2 can include monoclonal antibodies which specifically bind to EPHA2. A method for obtaining the antibodies is as described below.

[0212] For monoclonal antibody production, the following process is generally required:

[0213] (a) the step of purifying biopolymers used as antigens,

[0214] (b) the step of immunizing animals with the antigens through injection, then collecting blood from the animals, assaying the antibody titer thereof to determine the timing of splenectomy, and then preparing antibody-producing cells,

[0215] (c) the step of preparing myeloma cells (hereinafter, referred to as "myelomas"),

[0216] (d) the step of performing cell fusion between the antibody-producing cells and the myelomas,

[0217] (e) the step of selecting a hybridoma group that produces the antibody of interest,

[0218] (f) the step of dividing into single cell clones (cloning),

[0219] (g) the step of culturing the hybridomas for producing monoclonal antibodies in large amounts or raising hybridoma-transplanted animals, according to circumstances,

[0220] (h) the step of studying the bioactivities and binding specificities of the monoclonal antibodies thus produced or assaying properties as labeling reagents, etc.

[0221] Hereinafter, the method for preparing monoclonal antibodies will be described in detail in line with these steps, though the method for preparing antibodies is not limited thereto. For example, antibody-producing cells other than splenic cells and myelomas can also be used.

[0222] (a) Purification of Antigens

[0223] EPHA2 or partial polypeptides thereof prepared by the method as described above can be used as antigens.

[0224] Moreover, partial peptides of the protein of the present invention, which are chemically synthesized using membrane fractions prepared from EPHA2-expressing recombinant somatic cells or the EPHA2-expressing recombinant somatic cells themselves according to a method well known by those skilled in the art, can also be used as antigens.

[0225] (b) Preparation of Antibody-Producing Cells

[0226] The antigens obtained in step (a) are mixed with adjuvants well known by those skilled in the art, for example, complete or incomplete Freund's adjuvants, or other auxiliaries such as potassium aluminum sulfate, and experimental animals are immunized with these immunogens. Animals used in hybridoma preparation methods known in the art can be used as the experimental animals without problems. Specifically, for example, mice, rats, goats, sheep, cow, and horses can be used. However, mice or rats are preferably used as the animals to be immunized, from the viewpoint of the easy availability of myeloma cells to be fused with the extracted antibody-producing cells, etc.

[0227] Moreover, the lineages of the mice and rats actually used are not particularly limited. For example, mouse lineages such as A, AKR, BALB/c, BDP, BA, CE, C3H, 57BL, C57BR, C57L, DBA, FL, HTH, HT1, LP, NZB, NZW, RF, R III, SJL, SWR, WB, and 129 and rat lineages such as Low, Lewis, Sprague-Dawley, ACI, BN, and Fischer can be used.

[0228] These mice and rats can be obtained from, for example, experimental animal growers/distributors such as CLEA Japan, Inc. and Charles River Laboratories Japan, Inc.

[0229] Of these lineages, the mouse BALB/c lineage and the rat Low lineage are particularly preferable as the animals to be immunized, in consideration of fusion compatibility with myeloma cells as described later.

[0230] Moreover, mice having a reduced biological mechanism for autoantibody removal, i.e., autoimmune disease mice, are also preferably used in consideration of the antigenic homology between humans and mice.

[0231] These mice or rats are preferably 5 to 12 weeks old, more preferably 6 to 8 weeks old, at the time of immunization.

[0232] For the immunization of the animals with EPHA2 or the recombinant antigens thereof, methods known in the art described in detail in, for example, Weir, D. M., Handbook of Experimental Immunology Vol. I. II. III., Blackwell Scientific Publications, Oxford (1987), and Kabat, E. A. and Mayer, M. M., Experimental Immunochemistry, Charles C. Thomas Publisher Springfield, Ill. (1964) can be used.

[0233] Of these immunization methods, a method preferable in the present invention is specifically illustrated as described below.

[0234] Specifically, the membrane protein fractions used as antigens or antigen-expressing cells are first administered intradermally or intraperitoneally to the animals.

[0235] However, the combined use of both of the administration routes is preferable for enhancing immunization efficiency. Immunization efficiency can be enhanced particularly by performing intradermal administration in early immunizations and performing intraperitoneal administration in later immunizations or only in the last immunization.

[0236] The administration schedule of the antigens differs depending on the type of animals to be immunized, the individual differences thereof, etc. The antigens are generally administered at 3 to 6 doses preferably at 2- to 6-week intervals, more preferably at 3 to 4 doses at 2- to 4-week intervals.

[0237] Moreover, the dose of the antigens differs depending on the type of animals, the individual differences thereof, etc., and is generally of the order of 0.05 to 5 mg, preferably 0.1 to 0.5 mg.

[0238] A booster is performed 1 to 6 weeks later, preferably 2 to 4 weeks later, more preferably 2 to 3 weeks later, from such antigen administration.

[0239] In this context, the dose of the antigens in the booster differs depending on the type of animals, the size thereof, etc., and is generally of the order of 0.05 to 5 mg, preferably 0.1 to 0.5 mg, more preferably 0.1 to 0.2 mg, for example, for mice.

[0240] 1 to 10 days later, preferably 2 to 5 days later, more preferably 2 to 3 days later, from the booster, splenic cells or lymphocytes containing antibody-producing cells are aseptically extracted from the animals to be immunized.

[0241] In this procedure, their antibody titers are measured, and animals having a sufficiently increased antibody titer can be used as sources of antibody-producing cells to thereby enhance the efficiency of the subsequent procedures.

[0242] Examples of methods for measuring the antibody titers used here can include, but not limited to, RIA and ELISA.

[0243] The antibody titer measurement according to the present invention can be performed by procedures as described below, for example, according to ELISA.

[0244] First, the purified or partially purified antigens are adsorbed onto the surface of a solid phase such as 96-well plates for ELISA. Furthermore, antigen-unadsorbed solid phase surface is covered with proteins unrelated to the antigens, for example, bovine serum albumin (hereinafter, referred to as "BSA"). The surfaces are washed and then contacted with serially diluted samples (e.g., mouse serum) as primary antibodies such that the antibodies in the samples are bound to the antigens.

[0245] Furthermore, enzyme-labeled antibodies against the mouse antibodies are added thereto as secondary antibodies such that the secondary antibodies are bound to the mouse antibodies. After washing, substrates for the enzyme are added thereto, and, for example, the change in absorbance caused by color development based on substrate degradation is measured to thereby calculate antibody titers.

[0246] The antibody-producing cells can be separated from these spleen cells or lymphocytes according to methods known in the art (e.g., Kohler et al., Nature (1975) 256, p. 495; Kohler et al., Eur. J. Immunol. (1977) 6, p. 511; Milstein et al., Nature (1977), 266, p. 550; and Walsh, Nature, (1977) 266, p. 495).

[0247] For example, for the spleen cells, a general method can be adopted, which involves cutting the cells into strips, filtering them through a stainless mesh, and then separating the antibody-producing cells therefrom by floating in Eagle's minimal essential medium (MEM).

[0248] (C) Preparation of Myeloma Cells (Hereinafter, Referred to as "Myelomas")

[0249] Myeloma cells used in cell fusion are not particularly limited and can be selected appropriately, for use, from cell strains known in the art. However, HGPRT (hypoxanthine-guanine phosphoribosyl transferase)-deficient strains for which selection methods have been established are preferably used in consideration of convenient hybridoma selection from fused cells.

[0250] Specific examples thereof include: mouse-derived X63-Ag8 (X63), NS1-ANS/1 (NS1), P3X63-Ag8.U1 (P3U1), X63-Ag8.653 (X63.653), SP2/0-Ag14 (SP2/0), MPC11-45.6TG1.7 (45.6TG), FO, S149/5XXO, and BU.1; rat-derived 210.RSY3.Ag.1.2.3 (Y3); and human-derived U266AR (SKO-007), GM1500.GTG-A12 (GM1500), UC729-6, LICR-LON-HMy2 (HMy2), and 8226AR/NIP4-1 (NP41).

[0251] These HGPRT-deficient strains can be obtained from, for example, American Type Culture Collection (ATCC).

[0252] These cell strains are subcultured in an appropriate medium, for example, an 8-azaguanine medium [RPMI-1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, and fetal bovine serum (hereinafter, referred to as "FBS") and further supplemented with 8-azaguanine], an Iscove's Modified Dulbecco's Medium (hereinafter, referred to as "IMDM"), or a Dulbecco's Modified Eagle Medium (hereinafter, referred to as "DMEM") and subcultured in a normal medium [e.g., an ASF104 medium (manufactured by Ajinomoto Co., Inc.) containing 10% FBS] 3 to 4 days before cell fusion. On the day of fusion, 2×10⁷ or more cells are secured.

[0253] (d) Cell Fusion

[0254] Fusion between the antibody-producing cells and the myeloma cells can be performed appropriately under conditions that do not excessively reduce the cell survival rates, according to methods known in the art (e.g., Weir, D. M., Handbook of Experimental Immunology Vol. I. II. III., Blackwell Scientific Publications, Oxford (1987); and Kabat, E. A. and Mayer, M. M., Experimental Immunochemistry, Charles C. Thomas Publisher Springfield, Ill. (1964)).

[0255] For example, a chemical method which involves mixing the antibody-producing cells and the myeloma cells in a high-concentration polymer (e.g., polyethylene glycol) solution and a physical method which uses electric stimulations can be used as such methods.

[0256] Of these methods, the chemical method is specifically exemplified as described below.

[0257] Specifically, when polyethylene glycol is used as a polymer in the high-concentration polymer solution, the antibody-producing cells and the myeloma cells are mixed in a solution of polyethylene glycol having a molecular weight of 1500 to 6000, preferably 2000 to 4000, at a temperature of 30 to 40° C., preferably 35 to 38° C., for 1 to 10 minutes, preferably 5 to 8 minutes.

[0258] (e) Selection of Hybridoma Group

[0259] A method for selecting the hybridomas obtained by the cell fusion is not particularly limited, and a HAT (hypoxanthine-aminopterin-thymidine) selection method (Kohler et al., Nature (1975) 256, p. 495; and Milstein et al., Nature (1977) 266, p. 550) is usually used.

[0260] This method is effective for obtaining hybridomas using HGPRT-deficient myeloma cell strains that cannot survive in aminopterin.

[0261] Specifically, unfused cells and the hybridomas can be cultured in a HAT medium to thereby cause only aminopterin-resistant hybridomas to remain and grow.

[0262] (f) Dividing into Single Cell Clones (Cloning)

[0263] For example, methods known in the art, such as methylcellulose, soft agarose, and limiting dilution methods can be used as methods for cloning the hybridomas (see e.g., Barbara, B. M. and Stanley, M. S.: Selected Methods in Cellular Immunology, W.H. Freeman and Company, San Francisco (1980)). Of these methods, the limiting dilution method is particularly preferable.

[0264] In this method, feeders such as rat fetus-derived fibroblast strains or normal mouse splenic, thymus, or ascites cells are inoculated onto a microplate.

[0265] On the other hand, the hybridomas are diluted to 0.2 to 0.5 individuals/0.2 ml in advance in a medium. This solution containing the diluted hybridomas floating therein is added at a concentration of 0.1 ml/well, and the hybridomas can be continuously cultured for approximately 2 weeks while approximately 1/3 of the medium is replaced with a new one at regular intervals (e.g., 3-day intervals), to thereby grow hybridoma clones.

[0266] For wells having an observable antibody titer, for example, cloning by the limiting dilution method is repeated 2 to 4 times, and clones whose antibody titer is stably observed can be selected as anti-EPHA2 monoclonal antibody-producing hybridoma strains.

[0267] Examples of the hybridoma strains thus cloned can include hybridoma SH348-1 and hybridoma SH357-1. Hybridoma SH348-1 and hybridoma SH357-1 have been deposited on Jun. 8, 2007 with International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, (address: Tsukuba Central 6, Higashi 1-1-1, Tsukuba, Ibaraki, Japan). Hybridoma SH348-1 has been designated as SH348-1 with Accession No. FERM BP-10836, and hybridoma SH357-1 has been designated as SH357-1 with Accession No. FERM BP-10837.

[0268] In the present specification, an antibody produced by hybridoma SH348-1 is referred to as "SH348-1", and an antibody produced by hybridoma SH357-1 is referred to as "SH357-1".

[0269] (g) Preparation of Monoclonal Antibodies by Hybridoma Culture

[0270] The hybridomas thus selected can be cultured to thereby efficiently obtain monoclonal antibodies. Prior to the culture, it is preferred that hybridomas producing the monoclonal antibody of interest should be screened.

[0271] For this screening, methods known per se in the art can be adopted.

[0272] The antibody titer measurement according to the present invention can be performed, for example, by ELISA as described in paragraph (b).

[0273] The hybridomas obtained by the method as described above can be cryopreserved in liquid nitrogen or in a freezer at -80° C. or lower.

[0274] Moreover, the completely cloned hybridomas can be passaged several times in a HT medium (HAT medium except for aminopterin) and then cultured in a normal medium changed therefrom.

[0275] Large-scale culture is performed by rotational culture using large culture bottles or spinner culture.

[0276] A supernatant obtained in this large-scale culture can be purified according to methods well known by those skilled in the art, such as gel filtration, to obtain monoclonal antibodies which specifically bind to the protein of the present invention.

[0277] Moreover, the hybridomas can be intraperitoneally injected to mice of the same lineage thereas (e.g., BALB/c) or Nu/Nu mice and grown to obtain ascites containing the monoclonal antibody of the present invention in large amounts.

[0278] For the intraperitoneal administration, mineral oil such as 2,6,10,14-tetramethyl pentadecane (pristane) is administered beforehand (3 to 7 days before the administration) to obtain ascites in larger amounts.

[0279] For example, an immunosuppressive agent is intraperitoneally injected in advance to the mice of the same lineage as the hybridomas to inactivate the T cells. 20 days later, 10⁶ to 10⁷ hybridoma clone cells are allowed to float (0.5 ml) in a serum-free medium, and this solution is intraperitoneally administered to the mice. Ascites are usually collected from the mice when abdominal distention occurs by accumulated ascites. By this method, monoclonal antibodies are obtained with a concentration approximately 100 times higher than that in the culture solution.

[0280] The monoclonal antibodies obtained by the method can be purified by methods described in, for example, Weir, D. M.: Handbook of Experimental Immunology, Vol. I, II, III, Blackwell Scientific Publications, Oxford (1978).

[0281] Specific examples thereof include ammonium sulfate precipitation, gel filtration, ion-exchange chromatography, and affinity chromatography.

[0282] For the purification, commercially available monoclonal antibody purification kits (e.g., MAbTrap GII Kit; manufactured by Pharmacia Inc.) and the like can also be used as convenient methods.

[0283] The monoclonal antibodies thus obtained have high antigen specificity for EPHA2.

[0284] (h) Assay of Monoclonal Antibodies

[0285] The isotype and subclass of the monoclonal antibodies thus obtained can be determined as described below.

[0286] First, examples of identification methods include the Ouchterlony method, ELISA, and RIA.

[0287] The Ouchterlony method is convenient but requires a concentration procedure for a low concentration of monoclonal antibodies.

[0288] On the other hand, when the ELISA or RIA is used, the culture supernatant is directly reacted with an antigen-adsorbed solid phase, and further, antibodies compatible with various immunoglobulin isotypes and subclasses can be used as secondary antibodies to thereby identify the isotype or subclass of the monoclonal antibodies.

[0289] Moreover, commercially available kits for identification (e.g., Mouse Typer Kit; manufactured by Bio-Rad Laboratories, Inc.) and the like can also be used as more convenient methods.

[0290] Furthermore, the proteins can be quantified according to a Folin-Lowry method and a calculation method using absorbance at 280 nm [1.4 (OD280)=1 mg/ml immunoglobulin].

[0291] (3) Other Antibodies

[0292] The antibody of the present invention encompasses the monoclonal antibody against EPHA2 as well as genetic recombinant antibodies artificially modified for the purpose of, for example, reducing xenoantigenicity against humans, for example, chimeric, humanized, and human antibodies. These antibodies can be produced according to known methods.

[0293] Examples of the chimeric antibody include an antibody having variable and constant regions derived from species different from each other and specifically include a chimeric antibody comprising mouse-derived variable regions and human-derived constant regions joined together (see Proc. Natl. Acad. Sci. U.S.A., 81, 6851-6855, (1984)).

[0294] Examples of the humanized antibody can include an antibody comprising a human-derived antibody with complementarity determining regions (CDRs) replaced with those of another species (see Nature (1986) 321, p. 522-525) and an antibody comprising a human antibody with CDR sequences and some framework amino acid residues replaced with those of another species by CDR grafting (see WO 90/07861 and U.S. Pat. No. 6,972,323).

[0295] Further examples of the antibody of the present invention can include an anti-human antibody. The anti-EPHA2 human antibody means a human antibody having only the gene sequence of a human chromosome-derived antibody. The anti-EPHA2 human antibody can be obtained by methods using human antibody-producing mice having a human chromosome fragment containing genes of human antibody H and L chains (see e.g., Tomizuka, K. et al., Nature Genetics (1997) 16, p. 133-143; Kuroiwa, Y. et al., Nucl. Acids Res. (1998) 26, p. 3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects vol. 10, p. 69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999; and Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, p. 722-727).

[0296] For such transgenic animals, specifically, genetic recombinant animals in which loci of endogenous immunoglobulin heavy and light chains in non-human mammals are broken and loci of human immunoglobulin heavy and light chains are introduced instead via yeast artificial chromosome (YAC) vectors or the like can be created by preparing knockout animals and transgenic animals and crossing these animals.

[0297] Moreover, eukaryotic cells are transformed with cDNA encoding each of such human antibody heavy and light chains, preferably vectors containing the cDNA, by gene recombination techniques, and transformed cells producing genetic recombinant human monoclonal antibodies can also be cultured to thereby obtain these antibodies from the culture supernatant.

[0298] In this context, for example, eukaryotic cells, preferably mammalian cells such as CHO cells, lymphocytes, and myelomas can be used as hosts.

[0299] Moreover, methods for obtaining phage-displayed human antibodies selected from human antibody libraries are also known (see e.g., Wormstone, I. M. et al., Investigative Ophthalmology & Visual Science. (2002) 43 (7), p. 2301-2308; Carmen, S. et al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), p. 189-203; and Siriwardena, D. et al., Ophthalmology (2002) 109 (3), p. 427-431).

[0300] For example, a phage display method can be used, which involves causing human antibody variable regions to be expressed as a single-chain antibody (scFv) on phage surface and selecting phages binding to antigens (Nature Biotechnology (2005), 23, (9), p. 1105-1116).

[0301] Likewise, another phage display method can also be used, which involves causing human antibody Fab (antigen-binding fragment) to be expressed on the surface of phage and selecting phages binding to antigens (WO 97/08320 and WO 01/05950).

[0302] Genes of the phages selected based on antigen binding can be analyzed to thereby determine DNA sequences encoding human antibody variable regions binding to the antigens.

[0303] When the DNA sequence of scFv or Fab binding to the antigens is clarified, CDR sequences are extracted therefrom, and expression vectors having the sequences can be prepared and introduced into appropriate hosts, followed by gene expression to obtain human antibodies (WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438, WO 95/15388, Annu Rev. Immunol (1994) 12, p. 433-455, and Nature Biotechnology (2005) 23 (9), p. 1105-1116).

[0304] The antibody genes can be temporarily isolated and then introduced into appropriate hosts to prepare antibodies. In such a case, appropriate hosts and expression vectors can be combined for use.

[0305] When eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used.

[0306] Examples of the animal cells can include (1) mammalian cells, for example, monkey COS cells (Gluzman, Y. Cell (1981) 23, p. 175-182, ATCC CRL-1650), mouse fibroblasts NIH3T3 (ATCC No. CRL-1658), and dihydrofolate reductase-deficient strains (Urlaub, G. and Chasin, L. A. Proc. Natl. Acad. Sci. U.S.A. (1980) 77, p. 4126-4220) of Chinese hamster ovarian cells (CHO cells, ATCC CCL-61).

[0307] Moreover, these hosts can also be modified, for use, to express antibodies having a modified sugar chain structure and an enhanced ADCC activity (antibody-dependent cytotoxic activity) or CDC activity. Examples of such hosts can include CHO cells comprising genes incorporated therein which encode antibody molecules producing antibody compositions in which sugar chains having fucose-unbound N-acetylglucosamine at the reducing ends thereof occupy 20% or more of complex-type N-glycoside linked sugar chains binding to antibody Fc regions (see WO 02/31140).

[0308] When prokaryotic cells are used, examples thereof can include Escherichia coli and Bacillus subtilis.

[0309] The antibody gene of interest is introduced into these cells by transformation, and the transformed cells are cultured in vitro to obtain antibodies.

[0310] The isotype of the antibody of the present invention is not limited, and examples thereof include IgG (IgG1, IgG2, IgG3, or IgG4), IgM, IgA (IgA1 or IgA2), IgD, and IgE and can preferably include IgG and IgM.

[0311] Moreover, the antibody of the present invention may be an antibody fragment having the antigen-binding site of the antibody or a modified form thereof

[0312] Examples of the antibody fragment include Fab, F(ab')2, Fv, single-chain Fv (scFv) comprising heavy and light chain Fvs linked via an appropriate linker, a diabody, a linear antibody, and a multispecific antibody formed by antibody fragments.

[0313] Furthermore the antibody of the present invention may be a multispecific antibody having specificity for at least two different antigens.

[0314] Such a molecule usually binds two antigens (i.e., a bispecific antibody). The "multispecific antibody" according to the present invention encompasses antibodies having specificity for more (e.g., three) antigens.

[0315] The multispecific antibody used as the antibody of the present invention may be a full-length antibody or a fragment of such an antibody (e.g., a F(ab')2 bispecific antibody). The bispecific antibody can be prepared by binding heavy and light chains (HL pairs) of two antibodies or can also be prepared by fusing hybridomas producing monoclonal antibodies different from each other to prepare bispecific antibody-producing fused cells (Millstein et al., Nature (1983) 305, p. 537-539).

[0316] The antibody of the present invention may be a single-chain antibody (also referred to as scFv). The single-chain antibody is obtained by linking antibody heavy and light chain V regions via a polypeptide linker (Pluckthun, The Pharmacology of Monoclonal Antibodies, 113 (Rosenberg and Moore ed., Springer Verlag, New York, p. 269-315 (1994)); and Nature Biotechnology (2005), 23, p. 1126-1136).

[0317] Methods for preparing the single-chain antibody are well known in the art (see e.g., U.S. Pat. Nos. 4,946,778, 5,260,203, 5,091,513, and 5,455,030). In this scFv, the heavy and light chain V regions are linked via a linker that does not form a conjugate, preferably a polypeptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988), 85, p. 5879-5883). The heavy and light chain V regions in the scFv may be derived from the same antibodies or may be derived from different antibodies.

[0318] For example, an arbitrary single-chain peptide of 12 to 19 residues is used as the peptide linker for linking the V regions.

[0319] DNA encoding the scFv is obtained by: amplifying, as templates, the full-length sequences or partial sequences (encoding the desired amino acid sequences) of DNA encoding the heavy chain or heavy chain V region of the antibody and DNA encoding the light chain or light chain V region thereof, by a PCR method using primer pairs designed for both ends thereof; and subsequently further amplifying DNA encoding the peptide linker portion in combination with a primer pair designed to respectively link both ends of the linker sequence to the heavy and light chain sequences.

[0320] Moreover, once the DNA encoding the scFv is prepared, expression vectors containing the DNA and hosts transformed with the expression vectors can be obtained according to standard methods. Moreover, by use of the hosts, the scFv can be obtained according to standard methods.

[0321] For these antibody fragments, their genes are obtained and expressed in the same way as above, and the hosts can be allowed to produce the antibody fragments.

[0322] The antibody of the present invention may be a polyclonal antibody, which is a mixture of a plurality of anti-EPHA2 antibodies differing in amino acid sequences. One example of the polyclonal antibody can include a mixture of a plurality of antibodies differing in CDRs. A mixture of cells producing antibodies different from each other is cultured, and antibodies purified from the culture can be used as such polyclonal antibodies (see WO 2004/061104).

[0323] Antibodies obtained by binding the antibody of the present invention with various molecules such as polyethylene glycol (PEG) can also be used as the modified form of the antibody.

[0324] Furthermore, the antibody of the present invention may be a conjugate of these antibodies formed with other drugs (immunoconjugate). Examples of such an antibody can include conjugates obtained by binding these antibodies to radioactive materials or compounds having a pharmacological effect (Nature Biotechnology (2005) 23, p. 1137-1146).

[0325] The obtained antibodies can be purified until homogeneous. In the antibody separation and purification, any separation/purification method used for usual proteins can be used.

[0326] The antibodies can be separated and purified by appropriately selecting and combining, for example, using chromatography columns, filters, ultrafiltration, salting-out, dialysis, polyacrylamide gel electrophoresis for preparation, and isoelectric focusing (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); and Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), though the separation/purification method is not limited thereto.

[0327] Examples of chromatography include affinity chromatography, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, and adsorption chromatography.

[0328] These chromatography techniques can be performed using liquid-phase chromatography such as HPLC or FPLC.

[0329] Examples of columns used in the affinity chromatography include protein A and protein G columns.

[0330] Examples of columns based on the protein A column include Hyper D, POROS, Sepharose F.F. (Pharmacia Inc.).

[0331] Moreover, the antibodies can also be purified through their affinity for antigens using an antigen-immobilized carrier.

3. Properties of Antibody of the Present Invention

[0332] The anti-EPHA2 antibody of the present invention obtained by the method has the following properties:

[0333] (1) one antibody of the present invention has the following properties a) to e):

[0334] a) having no ability to phosphorylate EPHA2 tyrosine residues;

[0335] b) having an ADCC activity against EPHA2-expressing cells;

[0336] c) having a CDC activity against EPHA2-expressing cells;

[0337] d) having an antitumor activity in vivo; and

[0338] e) specifically binding to a polypeptide consisting of an amino acid sequence represented by amino acid Nos. 426 to 534 of SEQ ID NO: 8 in the sequence listing.

[0339] Examples of the antibody having such properties can include any one antibody selected from the group consisting of the following 1) to 8):

[0340] 1) SH348-1,

[0341] 2) an antibody which recognizes an epitope recognized by an antibody produced by hybridoma SH348-1 (FERM BP-10836),

[0342] 3) an antibody which has the amino acid sequences represented by SEQ ID NOs: 59, 61, and 63 in the sequence listing as complementarity determining regions in the heavy chain variable region and has the amino acid sequences represented by SEQ ID NOs: 65, 67, and 69 in the sequence listing as complementarity determining regions in the light chain variable region,

[0343] 4) an antibody characterized by the following i) and ii):

[0344] i) having a heavy chain peptide comprising an amino acid sequence represented by the general formula (I):

-FRH₁-CDRH₁-FRH₂-CDRH₂-FRH₃-CDRH₃-FRH₄- (I)

[0345] wherein FRH₁ represents an arbitrary amino acid sequence consisting of 18 to 30 amino acids; CDRH₁ represents the amino acid sequence represented by SEQ ID NO: 59 in the sequence listing; FRH₂ represents an arbitrary amino acid sequence consisting of 14 amino acids; CDRH₂ represents the amino acid sequence represented by SEQ ID NO: 61 in the sequence listing; FRH₃ represents an arbitrary amino acid sequence consisting of 32 amino acids; CDRH₃ represents the amino acid sequence represented by SEQ ID NO: 63 in the sequence listing; and FRH₄ represents an arbitrary amino acid sequence consisting of 11 amino acids, wherein these amino acids are linked to each other through peptide bonds; and

[0346] ii) having a light chain polypeptide comprising an amino acid sequence represented by the general formula (II):

-FRL₁-CDRL₁-FRL₂-CDRL₂-FRL₃-CDRL₃-FRL₄- (II)

[0347] wherein FRL₁ represents an arbitrary amino acid sequence consisting of 23 amino acids; CDRL₁ represents the amino acid sequence represented by SEQ ID NO: 65 in the sequence listing; FRL₂ represents an arbitrary amino acid sequence consisting of 15 amino acids; CDRL₂ represents the amino acid sequence represented by SEQ ID NO: 67 in the sequence listing; FRL₃ represents an arbitrary amino acid sequence consisting of 32 amino acids; CDRL₃ represents the amino acid sequence represented by SEQ ID NO: 69 in the sequence listing; and FRL₄ represents an arbitrary amino acid sequence consisting of 10 amino acids, wherein these amino acids are linked to each other through peptide bonds.

[0348] 5) SH357-1,

[0349] 6) an antibody which recognizes an epitope recognized by an antibody produced by hybridoma SH357-1 (FERM BP-10837),

[0350] 7) an antibody which has the amino acid sequences represented by SEQ ID NOs: 71, 73, and 75 in the sequence listing as complementarity determining regions in the heavy chain variable region and has the amino acid sequences represented by SEQ ID NOs: 77, 79, and 81 in the sequence listing as complementarity determining regions in the light chain variable region,

[0351] 8) an antibody according to any one of (5) to (7), characterized by the following i) and ii):

[0352] i) having a heavy chain peptide comprising an amino acid sequence represented by the general formula (I):

-FRH₁-CDRH₁-FRH₂-CDRH₂-FRH₃-CDRH₃-FRH₄- (I)

[0353] wherein FRH₁ represents an arbitrary amino acid sequence consisting of 18 to 30 amino acids sequences; CDRH₁ represents the amino acid sequence represented by SEQ ID NO: 71 in the sequence listing; FRH₂ represents an arbitrary amino acid sequence consisting of 14 amino acids; CDRH₂ represents the amino acid sequence represented by SEQ ID NO: 73 in the sequence listing; FRH₃ represents an arbitrary amino acid sequence consisting of 32 amino acids; CDRH₃ represents the amino acid sequence represented by SEQ ID NO: 75 in the sequence listing; and FRH₄ represents an arbitrary amino acid sequence consisting of 11 amino acids, wherein these amino acids are linked to each other through peptide bonds; and

[0354] ii) having a light chain polypeptide comprising an amino acid sequence represented by the general formula (II):

-FRL₁-CDRL₁-FRL₂-CDRL₂-FRL₃-CDRL₃-FRL₄- (II)

[0355] wherein FRL₁ represents an arbitrary amino acid sequence consisting of 23 amino acids; CDRL₁ represents the amino acid sequence represented by SEQ ID NO: 77 in the sequence listing; FRL₂ represents an arbitrary amino acid sequence consisting of 15 amino acids; CDRL₂ represents the amino acid sequence represented by SEQ ID NO: 79 in the sequence listing; FRL₃ represents an arbitrary amino acid sequence consisting of 32 amino acids; CDRL₃ represents the amino acid sequence represented by SEQ ID NO: 81 in the sequence listing; and FRL₄ represents an arbitrary amino acid sequence consisting of 10 amino acids, wherein these amino acids are linked to each other through peptide bonds.

[0356] (2) another antibody of the present invention has the following properties a) to f):

[0357] a) having no ability to phosphorylate EPHA2 tyrosine residues;

[0358] b) exhibiting an effect of decreasing an EPHA2 protein level;

[0359] c) having an ADCC activity against EPHA2-expressing cells;

[0360] d) having a CDC activity against EPHA2-expressing cells;

[0361] e) having an antitumor activity in vivo; and

[0362] f) specifically binding to a polypeptide consisting of an amino acid sequence represented by amino acid Nos. 426 to 534 of SEQ ID NO: 8 in the sequence listing.

[0363] Examples of the antibody having such properties can include any one antibody selected from the group consisting of the following 1) to 4):

[0364] 1) SH348-1,

[0365] 2) an antibody which recognizes an epitope recognized by an antibody produced by hybridoma SH348-1 (FERM BP-10836),

[0366] 3) an antibody which has the amino acid sequences represented by SEQ ID NOs: 59, 61, and 63 in the sequence listing as complementarity determining regions in the heavy chain variable region and has the amino acid sequences represented by SEQ ID NOs: 65, 67, and 69 in the sequence listing as complementarity determining regions in the light chain variable region,

[0367] 4) an antibody characterized by the following i) and ii):

[0368] i) having a heavy chain peptide comprising an amino acid sequence represented by the general formula (I):

-FRH₁-CDRH₁-FRH₂-CDRH₂-FRH₃-CDRH₃-FRH₄- (I)

[0369] wherein FRH₁ represents an arbitrary amino acid sequence consisting of 18 to 30 amino acids; CDRH₁ represents the amino acid sequence represented by SEQ ID NO: 59 in the sequence listing; FRH₂ represents an arbitrary amino acid sequence consisting of 14 amino acids; CDRH₂ represents the amino acid sequence represented by SEQ ID NO: 61 in the sequence listing; FRH₃ represents an arbitrary amino acid sequence consisting of 32 amino acids; CDRH₃ represents the amino acid sequence represented by SEQ ID NO: 63 in the sequence listing; and FRH₄ represents an arbitrary amino acid sequence consisting of 11 amino acids, wherein these amino acids are linked to each other through peptide bonds; and

[0370] ii) having a light chain polypeptide comprising an amino acid sequence represented by the general formula (II):

-FRL₁-CDRL₁-FRL₂-CDRL₂-FRL₃-CDRL₃-FRL₄- (II)

[0371] wherein FRL₁ represents an arbitrary amino acid sequence consisting of 23 amino acids; CDRL₁ represents the amino acid sequence represented by SEQ ID NO: 65 in the sequence listing; FRL₂ represents an arbitrary amino acid sequence consisting of 15 amino acids; CDRL₂ represents the amino acid sequence represented by SEQ ID NO: 67 in the sequence listing; FRL₃ represents an arbitrary amino acid sequence consisting of 32 amino acids; CDRL₃ represents the amino acid sequence represented by SEQ ID NO: 69 in the sequence listing; and FRL₄ represents an arbitrary amino acid sequence consisting of 10 amino acids, wherein these amino acids are linked to each other through peptide bonds.

[0372] (3) another antibody of the present invention has the following properties a) to e):

[0373] a) having no ability to phosphorylate EPHA2 tyrosine residues;

[0374] b) exhibiting no effect of decreasing an EPHA2 protein level;

[0375] c) having an ADCC activity;

[0376] d) having a CDC activity;

[0377] e) having an antitumor activity in vivo; and

[0378] f) specifically binding to a polypeptide consisting of an amino acid sequence represented by amino acid Nos. 426 to 534 of SEQ ID NO: 8 in the sequence listing.

[0379] Examples of the antibody having such properties can include any one antibody selected from the group consisting of the following 1) to 4):

[0380] 1) SH357-1,

[0381] 2) an antibody which recognizes an epitope recognized by an antibody produced by hybridoma SH357-1 (FERM BP-10836),

[0382] 3) an antibody which has the amino acid sequences represented by SEQ ID NOs: 71, 73, and 75 in the sequence listing as complementarity determining regions in the heavy chain variable region and has the amino acid sequences represented by SEQ ID NOs: 77, 79, and 81 in the sequence listing as complementarity determining regions in the light chain variable region,

[0383] 4) an antibody having the following properties i) and ii):

[0384] i) having a heavy chain peptide comprising an amino acid sequence represented by the general formula (I):

-FRH₁-CDRH₁-FRH₂-CDRH₂-FRH₃-CDRH₃-FRH₄- (I)

[0385] wherein FRH₁ represents an arbitrary amino acid sequence consisting of 18 to 30 amino acids; CDRH₁ represents the amino acid sequence represented by SEQ ID NO: 71 in the sequence listing; FRH₂ represents an arbitrary amino acid sequence consisting of 14 amino acids; CDRH₂ represents the amino acid sequence represented by SEQ ID NO: 73 in the sequence listing; FRH₃ represents an arbitrary amino acid sequence consisting of 32 amino acids; CDRH₃ represents the amino acid sequence represented by SEQ ID NO: 75 in the sequence listing; and FRH₄ represents an arbitrary amino acid sequence consisting of 11 amino acids, wherein these amino acids are linked to each other through peptide bonds; and

[0386] ii) having a light chain polypeptide comprising an amino acid sequence represented by the general formula (II):

-FRL₁-CDRL₁-FRL₂-CDRL₂-FRL₃-CDRL₃-FRL₄- (II)

[0387] wherein FRL₁ represents an arbitrary amino acid sequence consisting of 23 amino acids; CDRL₁ represents the amino acid sequence represented by SEQ ID NO: 77 in the sequence listing; FRL₂ represents an arbitrary amino acid sequence consisting of 15 amino acids; CDRL₂ represents the amino acid sequence represented by SEQ ID NO: 79 in the sequence listing; FRL₃ represents an arbitrary amino acid sequence consisting of 32 amino acids; CDRL₃ represents the amino acid sequence represented by SEQ ID NO: 81 in the sequence listing; and FRL₄ represents an arbitrary amino acid sequence consisting of 10 amino acids, wherein these amino acids are linked to each other through peptide bonds.

4. Pharmaceutical Agent Comprising Anti-EPHA2 Antibody

[0388] The anti-EPHA2 antibody of the present invention is useful as a pharmaceutical agent, particularly, a pharmaceutical composition intended for cancer treatment, or as an antibody for immunological diagnosis of such disease.

[0389] Preferable examples of cancer types can include, but not limited to, breast cancer, esophagus cancer, prostate cancer, gastric cancer, non-small cell lung cancer, colon cancer, and glioblastoma multiforme.

[0390] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the anti-EPHA2 antibody and a pharmaceutically acceptable diluent, carrier, solubilizing agent, emulsifying agent, preservative, and/or adjuvant.

[0391] It is preferred that the substances pharmaceutically used that are acceptable in the pharmaceutical composition of the present invention should be nontoxic, at the dose or administration concentration used, to individuals that receive the pharmaceutical composition.

[0392] The pharmaceutical composition of the present invention can contain a pharmaceutical substance for changing, maintaining, or retaining pH, osmotic pressure, viscosity, transparency, color, isotonicity, sterility, stability, the rate of dissolution, the rate of sustained release, absorptivity, or permeability.

[0393] Examples of the pharmaceutical substance can include, but not limited to, the following: amino acids such as glycine, alanine, glutamine, asparagine, arginine, and lysine; antimicrobial agents; antioxidants such as ascorbic acid, sodium sulfate, and sodium bisulfite; buffers such as phosphate, citrate, and borate buffers, hydrogen carbonate, and Tris-HCl solutions; fillers such as mannitol and glycine; chelating agents such as ethylenediaminetetraacetic acid (EDTA); complexing agents such as caffeine, polyvinyl pyrrolidine, β-cyclodextrin, and hydroxypropyl-β-cyclodextrin; extenders such as glucose, mannose, and dextrin; monosaccharides, disaccharides, glucose, mannose, and other hydrocarbons such as dextrin; coloring agents; flavoring agents; diluents; emulsifying agents; hydrophilic polymers such as polyvinyl pyrrolidine; low-molecular-weight polypeptides; salt-forming counterions; antiseptics such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, and hydrogen peroxide; solvents such as glycerin, propylene glycol, and polyethylene glycol; sugar alcohols such as mannitol and sorbitol; suspending agents; surfactants such as PEG, sorbitan ester, polysorbates such as polysorbate 20 and polysorbate 80, Triton, tromethamine, lecithin, and cholesterol; stability enhancers such as sucrose and sorbitol; elasticity enhancers such as sodium chloride, potassium chloride, mannitol, and sorbitol; delivery vehicles; diluents; excipients; and/or pharmaceutical adjuvants.

[0394] The amounts of these pharmaceutical substances added are preferably 0.01 to 100 times, particularly, 0.1 to 10 times higher than the weight of the anti-EPHA2 antibody.

[0395] In this context, the present invention also encompasses a pharmaceutical composition containing an immunoliposome containing the anti-EPHA2 antibody in a liposome or the anti-EPHA2 antibody bound with a liposome (U.S. Pat. No. 6,214,388).

[0396] The preferable composition of the pharmaceutical composition in a preparation can be determined appropriately by those skilled in the art according to applicable disease, an applicable administration route, etc.

[0397] The excipients or carriers in the pharmaceutical composition may be liquid or solid. The appropriate excipients or carriers may be injectable water, saline, cerebrospinal fluids, or other substances usually used in parenteral administration.

[0398] Neutral saline or serum albumin-containing saline can also be used as a carrier. The pharmaceutical composition can also contain a Tris buffer (pH 7.0 to 8.5) or an acetate buffer (pH 4.0 to 5.5) as well as sorbitol or other compounds. The pharmaceutical composition of the present invention is prepared in a freeze-dried or liquid form as an appropriate drug having the selected composition and necessary purity.

[0399] The pharmaceutical composition comprising the anti-EPHA2 antibody can also be prepared in a freeze-dried form using an appropriate excipient such as sucrose.

[0400] The pharmaceutical composition of the present invention can be prepared for parenteral administration or can also be prepared for gastrointestinal absorption.

[0401] The composition and concentration of the preparation can be determined depending on an administration method. When the anti-EPHA2 antibody contained in the pharmaceutical composition of the present invention has higher affinity for EPHA2, i.e., higher affinity (lower Kd value) for EPHA2 with respect to a dissociation constant (Kd value), the drug containing this antibody can be efficacious at a lower dose in humans. Based on this result, the dose of the pharmaceutical composition of the present invention in human can also be determined.

[0402] The dose of the anti-EPHA2 antibody in humans may be usually be approximately 0.1 to 100 mg/kg once per 1 to 180 days.

[0403] Examples of dosage forms of the pharmaceutical composition of the present invention include injections including drip, suppositories, nasal agents, sublingual agents, and transdermally absorbable agents.

[0404] The administration of the pharmaceutical composition of the present invention can inhibit the growth of EPHA2-expressing tumors.

EXAMPLES

[0405] Hereinafter, the present invention will be described more specifically with reference to the Examples. However, the present invention is not intended to be limited to them.

[0406] In the Examples below, procedures related to genetic engineering were performed according to the methods described in "Molecular Cloning", (Sambrook, J., Fritsch, E. F., and Maniatis, T., published by Cold Spring Harbor Laboratory Press, 1989) or methods described in other experimental manuals used by those skilled in the art or according to instructions included in the commercially available reagents or kits used, unless otherwise specified.

Example 1

Preparation of Plasmid

[0407] 1)-1 Preparation of Vectors Expressing Human EPHA2

[0408] 1)-1-1 Preparation of a Vector Expressing Full-Length Human EPHA2

[0409] cDNA encoding human EPHA2 was amplified by PCR reaction using cDNA synthesized from SK-OV-3 cell-derived total RNA as the template and a primer set:

TABLE-US-00001 Primer 1: (sequence listing sequence ID No. 3) 5'-ggggacaagtttgtacaaaaaagcaggcttcggggatcggaccgaga gcgagaag-3'; and Primer 2: (sequence listing sequence ID No. 4) 5'-ggggaccactttgtacaagaaagctgggtcctagatggggatcccca cagtgttcacctggtcctt-3'.

[0410] The PCR product was incorporated into pDONR221 (manufactured by Invitrogen Corp.) using BP Clonase (manufactured by Invitrogen Corp.) to prepare an entry vector. The stop codon was removed from the EPHA2 gene in the entry vector using GeneTailor Site-Directed Mutagenesis System (manufactured by Invitrogen Corp.) and a primer set:

TABLE-US-00002 Primer 3: (sequence listing sequence ID No. 5) 5'-ctgtggggatccccatcgacccagctttc-3'; and Primer 4: (sequence listing sequence ID No. 6) 5'-gatggggatccccacagtgttcacctggtc-3'.

[0411] Recombination reaction between the obtained entry vector and the pcDNA-DEST40 Gateway Vector (manufactured by Invitrogen Corp.) was performed using LR Clonase (manufactured by Invitrogen Corp.) to prepare pcDNA-DEST40-EPHA2 (the present vector has the nucleotide sequence represented by SEQ ID NO: 7 in the sequence listing, between attB1 and attB2 sequences). Moreover, the sequence of the ORF portion of the EPHA2 gene cloned into the present vector is represented by nucleotide Nos. 33 to 2960 of SEQ ID NO: 7 in the sequence listing. Moreover, the amino acid sequence of EPHA2 is represented by SEQ ID NO: 8 in the sequence listing.

[0412] 1)-1-2 Preparation of EPHA2 Extracellular Region Expression Vector

[0413] cDNA encoding a human EPHA2 extracellular region polypeptide (consisting of an amino acid sequence represented by amino acid Nos. 1 to 534 of SEQ ID NO: 8 in the sequence listing; hereinafter, abbreviated to "EPHA2-ECD") was amplified by PCR reaction using a primer set:

TABLE-US-00003 Primer 5: (sequence listing sequence ID No. 9) 5'-aaaaagcttatggagctccaggcagcccgc-3'; and Primer 6: (sequence listing sequence ID No. 10) 5'-aaagggccctcagttgccagatccctccgg-3'.

[0414] The obtained PCR product was cleaved with HindIII and ApaI and cloned into the HindIII/ApaI site of pcDNA3.1 (hereinafter, the resulting vector is abbreviated to "pcDNA3.1-EPHA2-ECD"; and in the description below and the drawings, the recombinant protein expressed by "pcDNA3.1-EPHA2-ECD" is referred to as "rEPHA2-ECD").

[0415] 1)-1-3 Preparation of Expression Vectors of Truncated EPHA2 Proteins

[0416] To construct vectors expressing a region consisting of an amino acid sequence represented by amino acid Nos. 315 to 540 of SEQ ID NO: 8 in the sequence listing of EPHA2 (hereinafter, referred to as "FnIII-NC"), a region consisting of an amino acid sequence represented by amino acid Nos. 315 to 430 thereof (hereinafter, referred to as "FnIII-N"), or a region consisting of an amino acid sequence represented by amino acid Nos. 426 to 540 thereof (hereinafter, referred to as "FnIII-C"), PCR reactions with pcDNA-DEST40-EPHA2 as the templates were performed using each primer set:

TABLE-US-00004 Primer set for FnIII-NC amplification: Primer 7: (sequence listing sequence ID No. 11) 5'-gcaggcttcatcgaaggtcgtgggcgggcacctcaggacccag-3'; and Primer 8: (sequence listing sequence ID No. 12) 5'-gtacaagaaagctgggtgctagccgccaatcaccgccaag-3'; Primer set for FnIII-N amplification: Primer 7, and Primer 9: (sequence listing sequence ID No. 13) 5'-gtacaagaaagctgggtgctaggcagtacggaagctgcgg-3'; Primer set for FnIII-C amplification: Primer 10: (sequence listing sequence ID No. 14) 5'-gcaggcttcatcgaaggtcgtgggagcttccgtactgccagtg-3'; and Primer 8.

[0417] To add attB1 and attB2 sites to both ends of the obtained PCR products, PCR reaction with each PCR product as a template was performed using a primer set:

TABLE-US-00005 Primer 11: (sequence listing sequence ID No. 15) 5'-ggggacaagtttgtacaaaaaagcaggcttcatcgaaggtcgtggg- 3'; and Primer 12: (sequence listing sequence ID No. 16) 5'-ggggaccactttgtacaagaaagctgggt-3'.

[0418] The PCR products obtained in this procedure were incorporated into pDONR221 using BP Clonase to prepare entry vectors. Recombination reactions between each entry vector and a destination vector prepared by cleaving the NdeI and BamHI sites of pET15b (manufactured by Novagen) with restriction enzymes, blunting the cleaved sites, and then ligating Reading Frame Cassette C.1 of Gateway Vector Conversion System (manufactured by Invitrogen Corp.) into the blunted sites were performed using LR Clonase in order to prepare expression vectors (hereinafter, recombinant proteins expressed by the FnIII-NC-, FnIII-N-, and FnIII-C-incorporated expression vectors are referred to as rFnIII-NC, rFnIII-N, and rFnIII-C, respectively).

[0419] 1)-2 Preparation of Human EPHB2 Extracellular Region Expression Vector

[0420] cDNA encoding human EPHB2 was obtained by PCR reaction using cDNA synthesized from HCC70 cell-derived total RNA as the template and a primer set:

TABLE-US-00006 Primer 13: (sequence listing sequence ID No. 17) 5'-ggggacaagtttgtacaaaaaagcaggcttcgccccgggaagcgcag cc-3'; and Primer 14: (sequence listing sequence ID No. 18) 5'-ggggaccactttgtacaagaaagctgggtcctaaacctccacagact gaatctggttcatctg-3'.

[0421] The nucleotide sequence of human EPHB2 cDNA is represented by SEQ ID NO: 19 in the sequence listing. The amino acid sequence thereof is represented by SEQ ID NO: 20 in the sequence listing. PCR reaction was performed using a primer set for amplifying cDNA encoding a human EPHB2 extracellular region (region consisting of an amino acid sequence represented by amino acid Nos. 1 to 542 of SEQ ID NO: 20 in the sequence listing) (hereinafter, abbreviated to "EPHB2-ECD"):

TABLE-US-00007 Primer 15: (sequence listing sequence ID No. 21) 5'-aaaaagcttatggctctgcggaggctgggg-3'; and Primer 16: (sequence listing sequence ID No. 22) 5'-aaagatatctcatggcaacttctcctggat-3'.

[0422] The obtained PCR product was cleaved with HindIII and EcoRV and cloned into the HindIII/EcoRV site of pcDNA3.1 (hereinafter, the resulting vector is abbreviated to "pcDNA3.1-EPHB2-ECD"; and in the description below and the drawings, the recombinant protein expressed by "pcDNA3.1-EPHB2-ECD" is referred to as rEPHB2-ECD).

[0423] 1)-3 Preparation of Human ERBB2 Expression Vector

[0424] PCR reaction with Human clone collection (manufactured by STRATAGENE, #C33830) as the template was performed using a primer set:

TABLE-US-00008 Primer 17: (sequence listing sequence ID No. 23) 5'-caccatggagctggcggccttg-3'; and Primer 18: (sequence listing sequence ID No. 24) 5'-tcccactggcacgtccagacc-3'.

[0425] The obtained PCR product was incorporated into pENTR/D-TOPO (manufactured by Invitrogen Corp.) using pENTR Directional TOPO Cloning kit (manufactured by Invitrogen Corp.) to prepare an entry vector. To repair mutations caused by amino acid substitution, the entry vector was digested with EcoRI, and a fragment containing the pENTR/D-TOPO-derived sequence among the obtained fragments was ligated with the second largest fragment (approximately 1.6 kbp) among fragments obtained by digested Human clone collection (manufactured by STRATAGENE, #C14640) with EcoRI. Recombination reactions between the obtained entry vector and the pcDNA-DEST40 Gateway vector were performed using LR Clonase in order to prepare pcDNA-DEST40-ERBB2 (the vector has the nucleotide sequence represented by SEQ ID NO: 25 in the sequence listing, between attB1 and attB2 sequences).

Example 2

Preparation of Monoclonal Antibody

[0426] 2)-1 Preparation of Antigen

[0427] To express EPHA2-ECD, FreeStyle 293-F cells (manufactured by Invitrogen Corp.) were transfected with pcDNA3.1-EPHA2-ECD using 293fectin (manufactured by Invitrogen Corp.) and cultured at 37° C. in 8% CO₂ for 5 days. After the culture, the culture supernatant was collected by centrifugation and used as a source for rEPHA2-ECD purification. The obtained culture supernatant was dialyzed against 20 mM Tris-HCl, pH 7.5, using a dialysis tube having a molecular cutoff of 15000, then filtered through a filter (0.45 μm, PES), and then applied to HiPrep 16/10 Q XL (manufactured by GE Healthcare Bio-Sciences Corp.) equilibrated with 20 mM Tris-HCl, pH 7.5. Elution was performed with a linear concentration gradient of NaCl (20 mM Tris-HCl, pH 7.5, 0-1 M NaCl). An aliquot of the eluted fractions was separated by SDS-polyacrylamide gel electrophoresis (hereinafter, abbreviated to "SDS-PAGE"). Then, the gel was stained with Coomassie Brilliant Blue (hereinafter, abbreviated to "CBB-stained") to confirm rEPHA2-ECD-containing fractions. Next, the rEPHA2-ECD-containing fractions were combined and applied to HiLoad 26/60 Superdex 200 pg (manufactured by GE Healthcare Bio-Sciences Corp.) equilibrated with PBS. After elution with PBS, an aliquot of the eluted fractions was separated by SDS-PAGE. Then, the gel was CBB-stained to confirm rEPHA2-ECD-containing fractions. The rEPHA2-ECD-containing fractions were combined and used as an antigen for immunization and an antigen for epitope determination. The protein concentration was measured using BCA Protein Assay Reagent (manufactured by PIERCE).

[0428] 2)-2 Immunization

[0429] 4- to 6-week-old BALB/cAnNCrlCrlj mice (Charles River Laboratories Japan, Inc.) were used. On day 0, a mixture of 50 μg of rEPHA2-ECD and Adjuvant Complete Freund H37 Rv (manufactured by Wako Pure Chemical Industries, Ltd.) (1:1 in terms of volume ratio) was subcutaneously administered to the mouse dorsal region. Likewise, a mixture of 50 μg of rEPHA2-ECD and TiterMax Gold Adjuvant (manufactured by Sigma-Aldrich, Inc.) (1:1 in terms of volume ratio) was subcutaneously administered to the dorsal region of another individual. On days 22 and 36, a mixture of 50 μg of rEPHA2-ECD and Adjuvant Incomplete Freund (manufactured by Wako Pure Chemical Industries, Ltd.) (1:1 in terms of volume ratio) was subcutaneously administered to the dorsal region of each mouse. On day 53, 50 μg of rEPHA2-ECD was intraperitoneally administered to each mouse. On day 56, the mouse spleen was collected and used in hybridoma preparation.

[0430] 2)-3 Hybridoma Preparation

[0431] Cell fusion between the spleen cells and mouse myeloma P3X63Ag8U.1 cells was performed using PEG4000 (manufactured IBL (Immuno-Biological Laboratories, Co., Ltd.)) to prepare hybridomas. A culture supernatant of the obtained hybridomas was used for screening anti-EPHA2 antibody-producing hybridomas.

[0432] 2)-4 Antibody Screening

[0433] 2)-4-1 Preparation of Cells Expressing a Gene Encoding an Antigen

[0434] 293T cells were seeded at 5×10⁴ cells/cm² onto a collagen type I-coated flask (manufactured by IWAKI) and cultured overnight at 37° C. in 5% CO₂ in DMEM containing 10% FBS. On the next day, the 293T cells were transfected with pcDNA-DEST40-EPHA2 or pcDNA-DEST40-ERBB2 as a control using Lipofectamine 2000 (manufactured by Invitrogen Corp.) and further incubated overnight at 37° C. in 5% CO₂. On the next day, the transfected 293T cells were treated with trypsin, then washed with DMEM containing 10% FBS, and then suspended in PBS containing 5% FBS. The obtained cell suspension was used in Cell-ELISA and flow cytometry analysis.

[0435] 2)-4-2 Cell-ELISA

[0436] The cell suspension prepared in the paragraph 2)-4-1 was centrifuged, and the supernatant was removed. Then, the EPHA2-expressing 293T cells and the ERBB2-expressing 293T cells were separately suspended by the addition of the hybridoma culture supernatant and incubated at 4° C. for 1 hour. The cells in the wells were washed twice with PBS containing 5% FBS. Then, the cells were suspended by the addition of Goat anti-Mouse IgG, Peroxidase Conjugated (manufactured by Millipore (Chemicon), #AP181P) diluted 500 times with PBS containing 5% FBS, and incubated at 4° C. for 1 hour. The cells in the wells were washed twice with PBS containing 5% FBS. Then, OPD Color Developing Solution (o-phenylenediamine dihydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.) and H₂O₂ were dissolved at concentrations of 0.4 mg/ml and 0.6% (v/v), respectively, in an OPD solution (0.05 M trisodium citrate, 0.1 M disodium hydrogen phosphate dodecahydrate, pH 4.5)) was added at 100 μl/well. Color reaction was performed with stirring and terminated by the addition of 1 M HCl at 100 μl/well. The cells were precipitated by centrifugation, and the supernatant was then transferred to a new 96-well flat-bottom microplate. The absorbance at 490 nm was measured using a plate reader (ARVO, PerkinElmer). To select hybridomas producing antibodies which specifically bind to EPHA2 expressed on the surface of the cell membrane, hybridomas producing a culture supernatant exhibiting higher absorbance in the EPHA2-expressing 293T cells than in the ERBB2-expressing 293T cells (controls) were selected to be positive for production of anti-EPHA2 antibody.

[0437] 2)-4-3 Flow Cytometric Analysis

[0438] To eliminate false positives in Cell-ELISA, antibodies produced by the hybridoma determined to be positive in Cell-ELISA were further examined for their binding specificities for EPHA2 by flow cytometry. The cell suspension prepared in paragraph 2)-4-1 was centrifuged, and the supernatant was removed. Then, the EPHA2-expressing 293T cells and the ERBB2-expressing 293T cells were separately suspended by the addition of the hybridoma culture supernatant and incubated at 4° C. for 1 hour. The cells in the wells were washed twice with PBS containing 5% FBS. Then, the cells were suspended by the addition of "Fluorescein-conjugated goat IgG fraction to mouse IgG" (Whole Molecule) (manufactured by ICN Pharmaceuticals, Inc., #55493) diluted 1000 times with PBS containing 5% FBS, and incubated at 4° C. for 1 hour. The cells were washed twice with PBS containing 5% FBS and then resuspended in PBS containing 5% FBS and further containing 2 μg/ml 7-aminoactinomycin D (manufactured by Invitrogen Corp. (Molecular Probes)), followed by analysis using a flow cytometer (FC500, Beckman Coulter, Inc.). The data was analyzed using Flowjo (Tree Star, Inc.). 7-aminoactinomycin D-positive dead cells were excluded using a gate. Then, the FITC fluorescence intensity histograms of live cells were plotted. Hybridomas producing samples that provided stronger fluorescence intensity in the fluorescence intensity histogram of the EPHA2-expressing 293T cells than in the histogram of the ERBB2-expressing 293T cells as controls were obtained as anti-EPHA2 antibody-producing hybridomas.

[0439] 2)-5 Separation of Hybridoma into Single Clones

[0440] The anti-EPHA2 antibody-producing hybridomas were diluted with ClonaCell-HY Selection Medium D (manufactured by StemCell Technologies, #03804) and cultured, and the formed colonies were collected as single clones. The collected clones were separately cultured and examined for their binding activities for EPHA2 using the culture supernatants in the same way as in paragraph 2)-4-3 to establish hybridomas producing anti-EPHA2 monoclonal antibodies (SH348-1, SH357-1, Ab57-1, Ab65-1, Ab96-1, Ab100-1, Ab105-1, Ab106-13, Ab136-1, Ab148-1, Ab151-4, Ab230-1, Ab373-1, and Ab382-1).

[0441] 2)-6 Confirmation of Binding Activity of Monoclonal Antibody for Cancer Cell Line

[0442] Whether or not the monoclonal antibodies obtained in paragraph 2)-5 bound to cancer cells highly expressing EPHA2 was studied by the flow cytometric method in the same way as in paragraph 2)-4-3. A human breast cancer cell line (MDA-MB-231), a human lung cancer cell line (A549), and a human prostate cancer cell line (PC-3) were used instead of the transfected 293T cells. As a result, all the established monoclonal antibodies were confirmed to bind to these cancer cell lines.

[0443] 2)-7 Isotype Determination of Monoclonal Antibody

[0444] The isotypes of the monoclonal antibodies were determined using Mouse monoclonal isotyping kit (manufactured by AbD Serotec). As a result, the isotypes were IgG1 (Ab57-1 and Ab230-1), IgG2a (SH348-1, SH357-1, Ab65-1, Ab96-1, Ab100-1, Ab136-1, Ab148-1, and Ab151-4), and IgG2b (Ab105-1, Ab106-13, Ab373-1, and Ab382-1).

[0445] 2)-8 Preparation of Monoclonal Antibody

[0446] The monoclonal antibodies were purified from ascites of hybridoma-transplanted mice or a hybridoma culture supernatant (hereinafter, referred to as a "source for antibody purification").

[0447] The mouse ascites were prepared as follows: first, 7- to 8-week-old BALB/cAJcl-nu/nu mice (CLEA Japan, Inc.) were treated with pristane (manufactured by Sigma-Aldrich, Inc.). Approximately 3 weeks later, the hybridomas washed with saline were intraperitoneally transplanted in an amount of 1×10⁷ cells/mouse. 1 to 2 weeks later, ascites accumulated in the peritoneal cavity was collected, then sterilized through a 0.22-μm filter, and used as a source for antibody purification.

[0448] The hybridoma culture supernatant was prepared using CELLine (manufactured by BD Biosciences). The hybridomas were cultured according to the manufacturer's instructions except that ClonaCell-HY Growth Medium E (manufactured by StemCell Technologies, #03805) was used as a medium. The collected culture supernatant was filtered through a 0.45-μm filter and used as a source for antibody purification.

[0449] The antibodies were purified using an affinity column comprising Recombinant Protein A rPA50 (manufactured by RepliGen Corp.) immobilized on Formyl-Cellulofine (manufactured by Seikagaku Corp.) (hereinafter, abbreviated to "Formyl-Cellulofine Protein A") or HiTrap MabSelect SuRe (manufactured by GE Healthcare Bio-Sciences Corp.). For the Formyl-Cellulofine Protein A, the source for antibody purification was diluted three times with Binding Buffer (3 M NaCl, 1.5 M glycine, pH 8.9) and applied to the column, which was then washed with Binding Buffer, followed by elution with 0.1 M citric acid, pH 4.0. On the other hand, for the HiTrap MabSelect SuRe, the source for antibody purification was added to the column, which was then washed with PBS, followed by elution with 2 M arginine-HCl, pH 4.0. The antibody eluate was neutralized, and the buffer was then replaced with PBS.

[0450] The antibody concentrations were determined by eluting the antibodies bound with POROS G 20 μm Column, PEEK, 4.6 mm×100 mm, 1.7 ml (Applied Biosystems) and measuring the absorbance (O.D. 280 nm) of the eluate. Specifically, the antibody sample diluted with PBS was applied to POROS G 20 μm equilibrated with Equilibrating Buffer (30.6 mM sodium dihydrogen phosphate dodecahydrate, 19.5 mM monopotassium phosphate, 0.15 M NaCl, pH 7.0). The column was washed with Equilibrating Buffer, and the antibody bound to the column was then eluted with an eluent (0.1% (v/v) HCl, 0.15 M NaCl). The peak area of the absorbance (O.D. 280 nm) of the eluate was measured, and the concentration was calculated according to the following equation:

Concentration of antibody sample (mg/ml)=(Peak area of antibody sample)/(Peak area of standard (human IgG1))×Concentration of standard (mg/ml)×Dilution factor.

[0451] Moreover, the concentration of endotoxin contained in the obtained antibodies was measured using Endospecy ES-50M Set (Seikagaku Corp., #020150) and Endotoxin Standard CSE-L Set (Seikagaku Corp., #020055) and was confirmed to be 1 EU/mg or lower. The resulting antibodies were used in the subsequent experiments.

Example 3

Properties of SH348-1 and SH357-1

[0452] 3)-1 Study of Anti-EPHA2 Antibody for its Activity of Inducing Phosphorylation of EPHA2 Tyrosine Residues and its Activity of Inducing Decrease in EPHA2 Protein Level

[0453] 3)-1-1 Preparation of antibody-stimulated cell lysate MDA-MB-231 cells suspended in RPMI1640 containing 10% FBS, 50 units/ml penicillin, and 50 μg/ml streptomycin (hereinafter, abbreviated to "10% FBS-containing RPMI1640 (with antibiotics)") were seeded at 6×10⁵ cells/well onto a 6-well dish and cultured overnight at 37° C. in 5% CO₂. On the next day, the medium was discarded, and RPMI1640 was added thereto. The cells were further cultured overnight at 37° C. in 5% CO₂. On the next day, SH348-1, SH357-1, Mouse IgG₂A Isotype Control (in the description below and the drawings, abbreviated to "mIgG2a"; manufactured by R&D Systems, Inc., #MAB003) as an isotype control antibody, Recombinant Mouse Ephrin-A1/Fc Chimera (in the description below and the drawings, abbreviated to "Ephrin-A1/Fc"; manufactured by R&D Systems, Inc., #602-A1-200) as a soluble EPHA2 ligand, and Recombinant Human IgG₁ Fc (in the description below and the drawings, abbreviated to "hG₁Fc"; manufactured by R&D Systems, Inc., #110-HG-100) as a control protein for the soluble ligand were separately diluted at a concentration shown in FIG. 1 or 2 (SH348-1, SH357-1, and mIgG2a: 10 μg/ml or 50 μg/ml in FIG. 1A and 50 μg/ml in FIG. 2A, Ephrin-A1/Fc and hG₁Fc: 1 μg/ml in FIGS. 1 and 2) with RPMI1640. The resulting solution was added to the MDA-MB-231 cells after discarding of the medium and incubated at 37° C. in 5% CO₂ for the predetermined time in 5% CO₂. Moreover, in the experiments in the presence of a cross-linking antibody, SH348-1, SH357-1, or mIgG2a and Goat anti-mouse IgG, Fcγ fragment specific (min X Hu, Bov, Hrs Sr Prot) (manufactured by Jackson ImmunoResearch Laboratories, Inc., #115-005-071) as a cross-linking antibody were mixed at each concentration of 10 μg/ml or 50 μg/ml (FIG. 1B) and 50 μg/ml (FIG. 2B) in RPMI1640. The resulting solution was added to the MDA-MB-231 cells after discarding of the medium and incubated at 37° C. in 5% CO₂ for the predetermined time. At the predetermined time, the supernatant was removed, and the cells were lysed by the addition of 1× Cell Lysis Buffer (manufactured by Cell Signaling Technology, Inc.) containing 1 mM PMSF (manufactured by Sigma-Aldrich, Inc.) and centrifuged at 15000 rpm for 5 minutes. The supernatants of cell lysates were used as samples in immunoprecipitation and Western blotting. The protein concentrations of the samples were measured using BCA Protein Assay Reagent (manufactured by PIERCE).

[0454] 3)-1-2 Verification of Activity of Inducing Phosphorylation of EPHA2 Tyrosine Residues

[0455] To immunoprecipitate EPHA2, first, 8 μg of anti-Eck/EphA2, clone D7 (in the description below and the drawings, abbreviated to "anti-EPHA2 antibody (D7)"; manufactured by Millipore (Upstate), #05-480) was added to 25 μl of a suspension of Protein G magnetic beads (manufactured by NEW ENGLAND BioLabs, Inc.) per sample, and the mixture was inverted for mixing at 4° C. for 2 hours.

[0456] Then, FBS was added thereto at a final concentration of 10%, and the mixture was further inverted for mixing at 4° C. for 30 minutes. The beads were washed three times with 1× Cell Lysis Buffer containing 1 mM PMSF. Then, 200 μg of the cell lysate supernatants prepared in paragraph 3)-1-1 were added thereto, and the mixtures were inverted for mixing overnight at 4° C. On the next day, the beads were washed three times with 1× Cell Lysis Buffer containing 1 mM PMSF. Then, SDS-Sample Buffer (56.3 mM Tris-HCl, pH 6.8, 1.8% (w/v) SDS, 9% glycerol, 0.72 M 2-mercaptoethanol, 0.045 mg/ml bromophenol blue) was added to the beads, and the mixtures were heated at 98° C. for 5 minutes. The proteins dissociated from the beads were separated by SDS-PAGE.

[0457] To perform Western blotting, the proteins were transferred from the gels to polyvinylidene difluoride membranes (hereinafter, abbreviated to a "PVDF membrane"; 0.45 μm in pore size; manufactured by Millipore). After the transfer, the PVDF membranes were blocked by shaking in Blocking Solution (one pouch of Block Ace powder (manufactured by Dainippon Sumitomo Pharma Co., Ltd. (Snow Brand Milk Products Co., Ltd.)) was dissolved in 100 ml of ultrapure water, to which Tween 20 and sodium azide were then added at final concentrations of 0.1% (v/v) and 0.02% (w/v), respectively). First, to detect the immunoprecipitated EPHA2, the PVDF membranes thus blocked were soaked in an anti-EPHA2 antibody (D7) solution diluted to 0.25 μg/ml with Blocking Solution, and shaken at room temperature for 1 hour. The PVDF membranes were washed for 10 minutes three times with TBST (50 mM Tris-HCl, pH 8.0, 138 mM NaCl, 2.7 mM KCl, 0.1% (v/v) Tween 20). Then, the PVDF membranes were soaked in an Anti-Mouse Ig, HRP-Linked Whole Ab Sheep (manufactured by GE Healthcare Bio-Sciences Corp.) solution diluted 3000 times with TBST, and shaken at room temperature for 30 minutes. The PVDF membranes were further washed for 10 minutes three times with TBST. Then, signals were detected on a film for chemiluminescence using ECL Plus (manufactured by GE Healthcare Bio-Sciences Corp.).

[0458] Next, to remove the antibodies from these PVDF membranes, the PVDF membranes were soaked in Stripping Solution (50 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 100 mM 2-mercaptoethanol) and shaken at 55° C. for 30 minutes. Then, the PVDF membranes were soaked in Quenching Solution (TBST containing 1% (v/v) H₂O₂ and 0.1% (w/v) NaN₃), then shaken at room temperature for 20 minutes, and further washed for 10 minutes three times with TBST. To detect the phosphorylated state of the EPHA2 tyrosine residues, these PVDF membranes were blocked by shaking in sodium azide-free Blocking Solution (one pouch of Block Ace powder was dissolved in 100 ml of ultrapure water, to which Tween 20 was then added at a final concentration of 0.1% (v/v)). Then, the PVDF membranes were soaked in an Anti-Phosphotyrosine, recombinant 4G10 HRP-conjugate (in the figures, abbreviated to "4G10 antibody"; manufactured by Millipore (Upstate), #16-184) solution diluted 10000 times with sodium azide-free Blocking Solution, and shaken at room temperature for 1 hour. The PVDF membranes were washed for 10 minutes three times with TBST and then further washed for 5 minutes three times with H₂O. Signals were detected on a film for chemiluminescence using ECL Plus.

[0459] As a result, by the addition of the soluble ligand Ephrin-A1/Fc, EPHA2 tyrosine residues were phosphorylated in 10 minutes. By contrast, when the antibodies SH348-1 and SH357-1 were added at a concentration of 10 μg/ml or 50 μg/ml, the effect of inducing the phosphorylation of EPHA2 tyrosine residues as seen by the ligand was not observed at all in the predetermined times (10 minutes, 30 minutes, and 60 minutes) (FIG. 1A). Likewise, even in the presence of the cross-linking antibody, the effect of inducing the phosphorylation of EPHA2 tyrosine residues as seen in the ligand was not observed in the presence of antibodies SH348-1 and SH357-1 (FIG. 1B).

[0460] 3)-1-3 Verification of Activity of Inducing Decrease in EPHA2 Protein Level

[0461] 10 μg of the cell lysate supernatants prepared in paragraph 3)-1-1 were separated by SDS-PAGE. Then, the proteins in the gel were transferred to PVDF membranes and subjected to Western blotting using an anti-EPHA2 antibody (D7) and Monoclonal Anti-β-Actin clone AC-15 (in the description below and the figures, abbreviated to "anti-β-actin antibody"; manufactured by Sigma-Aldrich, Inc., #A-5441) as a control for the sample protein level. Specifically, after the transfer, the PVDF membranes were blocked by shaking in Blocking Solution and then cut with a razor into two pieces centered around a molecular weight of 70 kDa. The PVDF membranes containing 70 kDa or larger proteins were soaked in an anti-EPHA2 antibody (D7) solution diluted to 0.25 μg/ml with Blocking Solution, while the PVDF membranes containing 70 kDa or smaller proteins were soaked in an anti-β-actin antibody solution diluted 1000 times with Blocking Solution. Each PVDF membrane was shaken at room temperature for 1 hour. Each PVDF membrane was washed for 10 minutes three times with TBST. Then, each PVDF membrane was soaked in an Anti-Mouse Ig, HRP-Linked Whole Ab Sheep solution diluted 3000 times with TBST, and shaken at room temperature for 30 minutes. Each PVDF membrane was washed for 10 minutes three times with TBST. Then, signals were detected using ECL Plus and NightOWL LB983 (Berthold Technologies GmBH & Co. KG). The signal intensity of the bands was quantified using Gel-Pro Analyzer Version 4.5 for Windows (registered trademark; Media Cybernetics, Inc.).

[0462] As a result, by the addition of the soluble ligand Ephrin-A1/Fc, a significant decrease in EPHA2 protein level was observed (FIGS. 2A and 2B). By the addition of the antibody SH348-1, a decrease in EPHA2 protein level, albeit weaker than the activity of the ligand, was observed both in the presence and absence of the cross-linking antibody (FIGS. 2A and 2B). On the other hand, in the antibody SH357-1, almost no change in EPHA2 protein level was observed regardless of the presence or absence of the cross-linking antibody (FIGS. 2A and 2B).

[0463] To analyze the EPHA2 protein level after 24 hours of the SH348-1 addition, an average of {the signal intensity of the EPHA2 band/the signal intensity of the β-actin band} was calculated from three experimental results corrected with a ligand/antibody-nonsupplemented sample. As a result, the value after 24 hours of the SH348-1 addition was 70% in the absence of the cross-linking antibody and 69% in the presence of the cross-linking antibody, when the value after 24 hours of the mIgG2a addition is defined as 100%.

[0464] 3)-2 ADCC Activity

[0465] 3)-2-1 Preparation of Effector Cells

[0466] The spleen was aseptically collected from CAnN.Cg-Foxn1^nu/CrlCrlj nude mice (Charles River Laboratories Japan, Inc.). The collected spleen was homogenized with two slide glasses and hemolyzed using BD Pharm Lyse (manufactured by BD Biosciences, #555899). The obtained spleen cells were suspended in phenol red-free RPMI1640 (manufactured by Invitrogen Corp.) containing 10% Fetal Bovine Serum, Ultra-low IgG (manufactured by Invitrogen Corp.) (hereinafter, abbreviated to a "medium for ADCC") and passed through a cell strainer (40 μm in pore size; manufactured by BD Biosciences). Then, the number of live cells was counted by a trypan blue exclusion test. The spleen cell suspension was centrifuged, and the medium was then removed. The cells were resuspended at a live cell density of 1.5×10⁷ cells/ml in a medium for ADCC and used as effector cells.

[0467] 3)-2-2 Preparation of Target Cells

[0468] MDA-MB-231, A549, or PC-3 cells were treated with trypsin. The cells were washed with RPMI1640 containing 10% FBS and then resuspended in RPMI1640 containing 10% FBS. Each cell (4×10⁶ cells) was mixed with Chromium-51 (5550 kBq) sterilized through a 0.22-μm filter, and labeled therewith at 37° C. in 5% CO₂ for 1 hour. The labeled cells were washed three times with a medium for ADCC and resuspended at a concentration of 2×10⁵ cells/ml in a medium for ADCC to prepare target cells.

[0469] 3)-2-3 ⁵¹Cr Release Assay

[0470] The target cells (2×10⁵ cells/ml) were dispensed at 50 μl/well to a 96-well U-bottom microplate. 50 μl of SH348-1, SH357-1, or an isotype control antibody (mIgG2a) diluted to 2.5 μg/ml (in terms of a final concentration after addition of the effector cells) with a medium for ADCC was added thereto and incubated at 4° C. for 1 hour. 100 μl of the effector cells (1.5×10⁷ cells/ml) was added thereto and incubated overnight at 37° C. in 5% CO₂. On the next day, the supernatant was collected into LumaPlate (manufactured by PerkinElmer). The dose of released gamma rays was measured using a gamma counter. The cell lysis rate attributed to the ADCC activity was calculated according to the following equation:

Cell lysis rate (%)=(A-B)/(C-B)×100

[0471] A: counts in sample wells

[0472] B: an average (n=3) of counts in spontaneous release (antibody/effector cell-nonsupplemented wells). Instead of the antibody and the effector cells, 50 μl and 100 μl, respectively, of a medium for ADCC were added. The other procedures were performed in the same way as in the sample wells.

[0473] C: an average (n=3) of counts in the maximum release (wells containing the target cells dissolved in a detergent). 50 μl of a medium for ADCC was added instead of the antibody, and 100 μl of a medium for ADCC containing 2% (v/v) Triton-X100 was added instead of the effector cells. The other procedures were performed in the same way as in the sample wells.

[0474] FIG. 3 shows an average of three experiments, wherein the error bar represents a standard deviation, and the P value was calculated by Student's t-test. As a result, the antibody SH348-1 exhibited cell lysis activities of 8.2%, 9.1%, and 4.7% against the MDA-MB-231 cells (FIG. 3A), the A549 cells (FIG. 3B), and the PC-3 cells (FIG. 3C), respectively. The antibody SH357-1 exhibited cell lysis activities of 8.8%, 13.0%, and 9.0% against the MDA-MB-231 cells (FIG. 3A), the A549 cells (FIG. 3B), and the PC-3 cells (FIG. 3C), respectively. These results demonstrated that both the antibodies have an ADCC activity against MDA-MB-231 cells, A549 cells, and PC-3 cells.

[0475] 3)-3 CDC Activity

[0476] MDA-MB-231, A549, or PC-3 cells suspended in 10% FBS-containing RPMI1640 (with antibiotics) were seeded at 5000 cells/well onto a 96-well microplate and cultured overnight at 37° C. in 5% CO₂. On the next day, SH348-1, SH357-1, or an isotype control antibody (mIgG2a) diluted to 25 μg/ml (in terms of a final concentration after addition of complements) with 10% FBS-containing RPMI1640 (with antibiotics) was added thereto and incubated at 4° C. for 1 hour. Rabbit complements (manufactured by CEDARLANE, #CL3051) diluted to 30% with RPMI1640 were added thereto at a final concentration of 5%, then incubated at 37° C. in 5% CO₂ for 1 hour, and further left standing at room temperature for 30 minutes. To measure the cell viability, CellTiter-Glo Luminescent Cell Viability Assay (manufactured by Promega Corp.) was added in an amount equal to that of the culture solution, and the mixture was stirred at room temperature for 10 minutes. Then, the amount of light emitted was measured using a plate reader. The cell viability was calculated according to the following equation:

Cell viability (%)=(a-b)/(c-b)×100

[0477] a: the amount of light emitted from sample wells

[0478] b: an average (n=8) of the amount of light emitted as a background (cell/antibody-nonsupplemented wells). 10% FBS-containing RPMI1640 (with antibiotics) was added, instead of the seeded cells, in an amount equal to that of the cell suspension, and 10% FBS-containing RPMI1640 (with antibiotics) was added, instead of the antibody, in an amount equal to that of the antibody dilution. The other procedures were performed in the same way as in the sample wells.

[0479] c: an average (n=3) of the amount of light emitted from antibody-nonsupplemented wells. 10% FBS-containing RPMI1640 (with antibiotics) was added, instead of the antibody, in an amount equal to that of the antibody dilution. The other procedures were performed in the same way as in the sample wells.

[0480] FIG. 4 shows an average of three experiments, wherein the error bar represents a standard deviation, and the P value was calculated by Student's t-test. As a result, the antibody SH348-1 induced 44%, 31%, and 41% decreases in the cell viability of the MDA-MB-231 cells (FIG. 4A), the A549 cells (FIG. 4B), and the PC-3 cells (FIG. 4C), respectively, in the presence of the complements. The antibody SH357-1 also induced 65%, 60%, and 65% decreases in the cell survival rates of the MDA-MB-231 cells (FIG. 4D), the A549 cells (FIG. 4E), and the PC-3 cells (FIG. 4F), respectively, in the presence of the complements. These results demonstrated that both the antibodies have a CDC activity against MDA-MB-231 cells, A549 cells, and PC-3 cells.

[0481] 3)-4 Epitope Determination

[0482] 3)-4-1 Preparation of Truncated EPHA2 Polypeptides (rFnIII-NC, rFnIII-N, and rFnIII-C)

[0483] Escherichia coli BL21 and Escherichia coli Origami (DE3) (manufactured by Novagen) were separately transformed with the expression plasmid prepared in paragraph 1)-1-3, and cultured in an LB medium supplemented with 50 μg/ml ampicillin (manufactured by Sigma-Aldrich, Inc.). Expression of truncated EPHA2 polypeptides was induced using Autoinduction System (manufactured by Novagen) for BL21 and the addition of 0.5 mM IPTG for Origami (DE3). The bacterial cells were collected by centrifugation at 6000 rpm for 20 minutes, then suspended in a homogenizing buffer (50 mM Tris HCl, pH 7.5, 150 mM NaCl, 0.1% (v/v) Triton-X100, 10% (v/v) glycerol), and then sonicated on ice. The supernatant was collected by centrifugation at 14000 rpm for 15 minutes and applied to 0.5 ml of Ni-NTA (manufactured by Invitrogen Corp.). The Ni-NTA was washed with a washing buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 50 mM imidazole, 10% (v/v) glycerol), followed by elution with an eluting buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 400 mM imidazole, 10% (v/v) glycerol). The eluted samples were further purified by gel filtration column chromatography (Superdex 75 10/300; manufactured by GE Healthcare Bio-Sciences Corp.) using PBS as a solvent. The protein concentrations of the obtained recombinant proteins were measured using Protein Assay (manufactured by Bio-Rad Laboratories, Inc).

[0484] 3)-4-2 Preparation of EPHB2 Extracellular Region Polypeptide (rEPHB2-ECD)

[0485] To express EPHB2-ECD, FreeStyle 293-F cells were transfected with pcDNA3.1-EphA2-ECD using 293fectin and cultured at 37° C. in 8% CO₂ for 72 hours. After the culture, the culture solution was collected by centrifugation and used as a source for rEPHB2-ECD purification. The obtained culture supernatant was dialyzed against 20 mM Tris-HCl, pH 7.5, using a dialysis tube having a molecular cutoff of 15000, then filtered through a filter (0.45 μm, PES), and then applied to HiPrep 16/10 Q XL equilibrated with 20 mM Tris-HCl, pH 7.5. Elution was performed with a linear concentration gradient of NaCl (20 mM Tris-HCl, pH 7.5, 0-1 M NaCl). An aliquot of the eluted fractions was separated by SDS-PAGE. Then, the gel was CBB-stained to confirm that there were rEPHB2-ECD-containing fractions. Next, the rEPHB2-ECD-containing fractions were combined and applied to HiLoad 26/60 Superdex 200 pg equilibrated with PBS. After elution with PBS, an aliquot of the elution fractions was separated by SDS-PAGE. Then, the gel was CBB-stained to confirm that there were rEPHB2-ECD-containing fractions. The rEPHB2-ECD-containing fractions were combined and used as an antigen for epitope determination. The protein concentration was measured using BCA Protein Assay Reagent.

[0486] 3)-4-3 Determination of Binding Sites in Antigens by ELISA

[0487] rEPHA2-ECD, rFnIII-NC, rFnIII-N, rFnIII-C, or a control protein rEPHB2-ECD was diluted to 1 μg/ml with PBS, then dispensed at 100 μl/well onto an immunoplate (manufactured by Nunc, #442404), and incubated overnight at 4° C. to thereby adsorb the protein to the plate. On the next day, the solution in the wells was removed, and a Block Ace solution (one pouch of Block Ace powder was dissolved in 100 ml of ultrapure water) diluted 4 times with PBS was dispensed at 200 μl/well and incubated at room temperature for 1 hour. The solution in the wells was removed, and SH348-1, SH357-1, or an isotype control antibody (mIgG2a) diluted to 5 μg/ml with Diluting Buffer (PBS, 0.05% (v/v) Tween 20) was then added at 50 μl/well. The plate was incubated at room temperature for 1 hour. Then, the solution in the wells was removed, and the wells were washed twice with a diluting buffer. Goat anti-Mouse IgG, Peroxidase Conjugated, diluted 3000 times with Diluting Buffer was added at 50 μl/well and incubated at room temperature for 1 hour. The solution in the wells was removed, and the wells were washed twice with Diluting Buffer. Then, a color reaction was performed with stirring by the addition of OPD Color Developing Solution at 100 μl/well. After color development, the color reaction was terminated by the addition of 1 M HCl at 100 μl/well. The absorbance at 490 nm was measured using a plate reader.

[0488] FIG. 5A shows the EPHA2 domain structure prediction (NCBI CDD version 2.11, CBS TMHMM Server v.2.0) and the positions of EPHA2-ECD, FnIII-NC, FnIII-N, and FnIII-C in EPHA2. Ligand-BD represents a ligand-binding domain, FN3 represents a fibronectin type 3 domain, TM represents a transmembrane region, Trk kinase represents a tyrosine kinase domain, and SAM represents a SAM domain.

[0489] Recombinant proteins of the EPHA2 extracellular region (EPHA2-ECD), the region containing two fibronectin type 3 domains (FnIII-NC), the region containing the N-terminal fibronectin type 3 domain (FnIII-N), and the region containing the C-terminal fibronectin type 3 domain (FnIII-C) were prepared and studied for their binding activities with respect to SH348-1 and SH357-1. As a result, the antibodies SH348-1 and SH357-1 exhibited binding activities with respect to rEPHA2-ECD, rFnIII-NC, and rFnIII-C (FIG. 5B). Thus, the antibodies SH348-1 and SH357-1 were shown to bind to a region from amino acids 426 to 534 containing the C-terminal fibronectin type 3 domain (amino acid sequence represented by amino acid Nos. 426 to 534 of SEQ ID NO: 8 in the sequence listing).

Example 4

In Vivo Antitumor Effect

[0490] MDA-MB-231 cells were dissociated from a culture flask by trypsin treatment and then suspended in 10% FBS-containing RPMI1640 (with antibiotics). After centrifugation, the supernatant was removed. The cells were washed twice with the same medium as above, then suspended in BD Matrigel Basement Membrane Matrix (manufactured by BD Biosciences), and subcutaneously transplanted at a concentration of 5×10⁶ cells/mouse into the dorsal region of 6-week-old BALB/cAJcl-nu/nu ((CLEA Japan, Inc.). When the day of transplantation is defined as day 0, SH348-1 or SH357-1 was intraperitoneally administered at a dose of 500 μg/mouse on days 9, 16, 23, and 30. PBS having the same volume (500 μl) as that of the antibody was intraperitoneally administered as a control. The tumor volume was measured on days 9, 13, 16, 20, 23, 28, 30, 34, and 37 to study the antitumor effect attributed to the antibody administration. As a result, tumor growth was significantly inhibited in the SH348-1- and SH357-1-administered groups compared to in the PBS-administered group (in the tumor volume comparison with the PBS-administered group on day 37, P values for SH348-1 and SH357-1 were both P<0.001; the P values were calculated by Student's t-test). Moreover, the tumor growth inhibitory rate (=100-(average of tumor volumes of the antibody-administered group)/(average of tumor volumes of the PBS-administered group)×100) on day 37 was 89.5% for SH348-1 and 84.1% for SH357-1. Their very strong antitumor effects were observed in vivo (FIGS. 6A and 6B).

[0491] Of 14 anti-EPHA2 monoclonal antibodies studied for their antitumor effects on the MDA-MB-231 cell-transplanted mice, only the antibodies SH348-1 and SH357-1 binding to FnIII-C were shown to be effective (Table 1).

TABLE-US-00009 TABLE 1 Inhibitory effect on Binding activity to antigen tumor rEPHA2- growth (in Antibody ECD rFnIII-NC rFnIII-N rFnIII-C vivo) SH 348-1 + + - + + SH 357-1 + + - + + Ab 57-1 + + + - - Ab 65-1 + - - - - Ab 96-1 + - - - - Ab 100-1 + - - - - Ab 105-1 + - - - - Ab 106-13 + - - - - Ab 136-1 + - - - - Ab 148-1 + - - - - Ab 151-4 + - - - - Ab 230-1 + + + - - Ab 373-1 + - - - - Ab 382-1 + - - - - These results demonstrated that SH348-1 and SH357-1 are antibodies which recognize the previously unreported epitope (amino acid sequence represented by amino acid Nos. 426 to 534 of SEQ ID NO: 8 in the sequence listing) and exhibit an antitumor effect. Moreover, the region to which SH348-1 or SH357-1 binds was shown to serve as a promising target of anti-tumor monoclonal antibodies targeted for EPHA2.

Example 5

Identification of SH348-1 and SH357-1 Antibody Genes

[0492] To determine the heavy and light chain N-terminal amino acid sequences of the mouse anti-human EPHA2 antibodies SH348-1 and SH357-1, an aliquot of a solution containing the SH348-1 or SH357-1 purified in paragraph 2)-8 was separated by SDS-PAGE. The proteins in the gel thus separated were transferred from the gel to a PVDF membrane (0.45 μm in pore size; manufactured by Invitrogen Corp.). The PVDF membrane was washed with a washing buffer (25 mM NaCl, 10 mM sodium borate buffer, pH 8.0), then stained by soaking in a staining solution (50% methanol, 20% acetic acid, 0.05% Coomassie Brilliant Blue) for 5 minutes, and then destained with 90% methanol. Band portions corresponding to the heavy and light chains (heavy chain: the band with smaller mobility, light chain: the band with larger mobility) visualized on the PVDF membrane were excised, and an attempt was made to identify their respective N-terminal amino acid sequences by an automatic Edman method (see Edman, P., et al. (1967) Eur. J. Biochem. 1, 80) using Procise (registered trademark) cLC Protein Sequencer Model 492cLC (Applied Biosystems). For the SH348-1 heavy chain, the amino acid sequence could not be identified by the method. Therefore, the N-terminal pyroglutamic acid was removed using Pfu Pyroglutamate Aminopeptidase (manufactured by TAKARA BIO INC.), and the same procedure as above was then performed to identify an amino acid sequence starting at the second amino acid from the N-terminus.

[0493] As a result, the amino acid sequence (starting at the second amino acid from the N-terminus) of the band corresponding to the SH348-1 heavy chain was

[0494] I-Q-L-V-Q-S-G-P (SEQ ID NO: 26 in the sequence listing).

[0495] The N-terminal amino acid sequence of the band corresponding to the SH348-1 light chain was

[0496] D-V-L-M-T-Q-S-P-L-S-L (SEQ ID NO: 27 in the sequence listing).

[0497] The N-terminal amino acid sequence of the band corresponding to the SH357-1 heavy chain was

[0498] Q-I-Q-L-V-Q-S-G-P (SEQ ID NO: 28 in the sequence listing).

[0499] The N-terminal amino acid sequence of the band corresponding to the SH357-1 light chain was

[0500] D-V-L-M-T-Q-T-P-L-S-L-P-V-S-L-G-D-Q-A (SEQ ID NO: 29 in the sequence listing).

[0501] These amino acid sequences were compared with antibody amino acid sequence database prepared by Kabat et al. (see Kabat, E. A. et al., (1991) in Sequences of Proteins of Immunological Interest Vol. I and II, U.S. Department of Health and Human Services). As a result, the subtype of the SH348-1 heavy chain (γ2a chain) was miscellaneous, and the subtype of the light chain was kappa light II. Moreover, the subtype of the SH357-1 heavy chain (γ2a chain) was determined to be miscellaneous, and the subtype of the light chain was determined to be kappa light II.

[0502] Thus, the following oligonucleotide primers were synthesized, which respectively hybridized to the 5'-terminal region of an antibody gene coding region belonging to these mouse subtypes and the 3'-terminal region thereof containing a stop codon (see Kabat et al., ibid; Matti Kartinen et al. (1988) 25, 859-865; and Heinrich, G. et al. (1984) J. Exp. Med. 159, p. 417-435):

TABLE-US-00010 (DB3F1: sequence listing sequence ID No. 30) 5'-cagatccagttggtgcagtctggacct-3' (MIG2AEVR1: sequence listing sequence ID No. 31) 5'-aagatatctcatttacccggagtccgggagaa-3' (MK19EIF1: sequence listing sequence ID No. 32) 5'-aagaattcatgaagttgcctgttagg-3' (KEVR1: sequence listing sequence ID No. 33) 5'-aagatatcttaacactcattcctgttgaagct-3'

[0503] To clone cDNAs encoding the SH348-1 and SH357-1 heavy and light chains, mRNA was prepared from the SH348-1- or SH357-1-producing hybridomas using Quick Prep mRNA Purification Kit (manufactured by GE Healthcare Bio-Sciences Corp., #27-9254-01). From each mRNA thus obtained, cDNA encoding each antibody heavy or light chain was amplified using TaKaRa One Step RNA PCR Kit (AMV) (manufactured by TAKARA BIO INC., #RR024A) and the primer set for the heavy chain (combination of DB3F1 and MIG2AEVR1) or the primer set for the light chain (combination of MK19EIF1 and KEVR1). These cDNAs amplified by PCR were cloned using Zero Blunt TOPO PCR Cloning Kit (manufactured by Invitrogen Corp.). Each of the cloned heavy and light chain nucleotide sequences was determined using a gene sequence analyzer ("ABI PRISM 3700 DNA Analyzer; Applied Biosystems" or "Applied Biosystems 3730xl Analyzer; Applied Biosystems"). In the sequencing reaction, GeneAmp 9700 (Applied Biosystems) was used.

[0504] The determined nucleotide sequence of the cDNA encoding the SH348-1 heavy chain is represented by SEQ ID NO: 34 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 35. The nucleotide sequence of the cDNA encoding the SH348-1 light chain is represented by SEQ ID NO: 36 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 37 in the sequence listing. The nucleotide sequence of the cDNA encoding the SH357-1 heavy chain is represented by SEQ ID NO: 38 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 39. The nucleotide sequence of the cDNA encoding the SH357-1 light chain is represented by SEQ ID NO: 40 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 41. Sequences represented by nucleotide Nos. 1 to 27 and 1327 to 1350 of SEQ ID NO: 34, a sequence represented by nucleotide Nos. 637 to 660 of SEQ ID NO: 36, sequences represented by nucleotide Nos. 1 to 27 and 1327 to 1350 of SEQ ID NO: 38, and a sequence represented by nucleotide Nos. 637 to 660 of SEQ ID NO: 40 are sequences derived from the primers.

[0505] Moreover, the amino acid sequences of these heavy and light chains were analyzed by comparison with antibody amino acid sequence database prepared by Kabat et al. (see Kabat, E. A., et al. (1991) in "Sequence of Proteins of Immunological Interest Vol. I and II"; U.S. Department of Health and Human Services). As a result, the SH348-1 heavy chain was shown to have an amino acid sequence represented by amino acid Nos. 1 to 119 of SEQ ID NO: 35 in the sequence listing as a variable region and have an amino acid sequence represented by amino acid Nos. 120 to 449 thereof as a constant region. Moreover, the SH348-1 light chain was shown to have an amino acid sequence represented by amino acid Nos. 1 to 112 of SEQ ID NO: 37 in the sequence listing as a variable region and have an amino acid sequence represented by amino acid Nos. 113 to 219 thereof as a constant region.

[0506] The SH357-1 heavy chain was shown to have an amino acid sequence represented by amino acid Nos. 1 to 119 of SEQ ID NO: 39 in the sequence listing as a variable region and have an amino acid sequence represented by amino acid Nos. 120 to 449 thereof as a constant region. Moreover, the SH357-1 light chain was shown to have an amino acid sequence represented by amino acid Nos. 1 to 112 of SEQ ID NO: 41 in the sequence listing as a variable region and have an amino acid sequence represented by amino acid Nos. 113 to 219 thereof as a constant region.

[0507] The nucleotide sequence encoding the SH348-1 heavy chain variable region is represented by SEQ ID NO: 42 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 43. The nucleotide sequence encoding the SH348-1 heavy chain constant region is represented by SEQ ID NO: 44, and the amino acid sequence thereof is represented by SEQ ID NO: 45. The nucleotide sequence encoding the SH348-1 light chain variable region is represented by SEQ ID NO: 46 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 47. The nucleotide sequence encoding the SH348-1 light chain constant region is represented by SEQ ID NO: 48, and the amino acid sequence thereof is represented by SEQ ID NO: 49. Sequences represented by nucleotide Nos. 1 to 27 of SEQ ID NO: 42, a sequence represented by nucleotide Nos. 970 to 993 of SEQ ID NO: 44, and sequences represented by nucleotide Nos. 301 to 324 of SEQ ID NO: 48, are sequences derived from the primers.

[0508] Moreover, the nucleotide sequence encoding the SH357-1 heavy chain variable region is represented by SEQ ID NO: 50 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 51. The nucleotide sequence encoding the SH357-1 heavy chain constant region is represented by SEQ ID NO: 52, and the amino acid sequence thereof is represented by SEQ ID NO: 53. The nucleotide sequence encoding the SH357-1 light chain variable region is represented by SEQ ID NO: 54 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 55. The nucleotide sequence encoding the SH357-1 light chain constant region is represented by SEQ ID NO: 56, and the amino acid sequence thereof is represented by SEQ ID NO: 57. Sequences represented by nucleotide Nos. 1 to 27 of SEQ ID NO: 50, a sequence represented by nucleotide Nos. 970 to 993 of SEQ ID NO: 52, and sequences represented by nucleotide Nos. 301 to 324 of SEQ ID NO: 56 are sequences derived from the primers.

[0509] Furthermore, the positions and sequences of CDRs in each of the amino acid sequences of the heavy and light chain variable regions were analyzed and determined by homology comparison with an antibody amino acid sequence database prepared by Kabat et al. (see Kabat, E. A., et al. (1991) ibid). According to the document, even different antibodies have framework regions which have an amino acid length almost equal to each other and are observed to have amino acid sequence commonality in the variable regions, if they are in the same subtype. On the other hand, CDRs are specific sequences flanked by these framework regions. Thus, the amino acid sequences of the heavy and light chains were analyzed by comparison with those of the same subtype thereas. As a result, CDRs in the SH348-1 heavy chain were determined to have amino acid sequences represented by amino acid Nos. 26 to 35 of SEQ ID NO: 35 in the sequence listing (CDRH₁), amino acid Nos. 50 to 66 thereof (CDRH₂), and amino acid Nos. 99 to 108 thereof (CDRH₃). CDRs in the SH348-1 light chain were determined to have amino acid sequences represented by amino acid Nos. 24 to 39 of SEQ ID NO: 37 in the sequence listing (CDRL₁), amino acid Nos. 55 to 61 thereof (CDRL₂), and amino acid Nos. 94 to 102 thereof (CDRL₃). CDRs in the SH357-1 heavy chain were determined to have amino acid sequences represented by amino acid Nos. 26 to 35 of SEQ ID NO: 39 in the sequence listing (CDRH₁), amino acid Nos. 50 to 66 thereof (CDRH₂), and amino acid Nos. 99 to 108 thereof (CDRH₃). CDRs in the SH357-1 light chain were determined to have amino acid sequences represented by amino acid Nos. 24 to 39 of SEQ ID NO: 41 in the sequence listing (CDRL₁), amino acid Nos. 55 to 61 thereof (CDRL₂), and amino acid Nos. 94 to 102 thereof (CDRL₃).

[0510] The nucleotide sequence encoding the SH348-1 CDRH₁ is represented by SEQ ID NO: 58 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 59. The nucleotide sequence encoding the SH348-1 CDRH₂ is represented by SEQ ID NO: 60, and the amino acid sequence thereof is represented by SEQ ID NO: 61. The nucleotide sequence encoding the SH348-1 CDRH₃ is represented by SEQ ID NO: 62, and the amino acid sequence thereof is represented by SEQ ID NO: 63. The nucleotide sequence encoding the SH348-1 CDRL₁ is represented by SEQ ID NO: 64, and the amino acid sequence thereof is represented by SEQ ID NO: 65. The nucleotide sequence encoding the SH348-1 CDRL₂ is represented by SEQ ID NO: 66, and the amino acid sequence thereof is represented by SEQ ID NO: 67. The nucleotide sequence encoding the SH348-1 CDRL₃ is represented by SEQ ID NO: 68, and the amino acid sequence thereof is represented by SEQ ID NO: 69. Moreover, the nucleotide sequence encoding the SH357-1 CDRH₁ is represented by SEQ ID NO: 70, and the amino acid sequence thereof is represented by SEQ ID NO: 71. The nucleotide sequence encoding the SH357-1 CDRH₂ is represented by SEQ ID NO: 72, and the amino acid sequence thereof is represented by SEQ ID NO: 73. The nucleotide sequence encoding the SH357-1 CDRH₃ is represented by SEQ ID NO: 74, and the amino acid sequence thereof is represented by SEQ ID NO: 75. The nucleotide sequence encoding the SH357-1 CDRL₁ is represented by SEQ ID NO: 76, and the amino acid sequence thereof is represented by SEQ ID NO: 77. The nucleotide sequence encoding the SH357-1 CDRL₂ is represented by SEQ ID NO: 78, and the amino acid sequence thereof is represented by SEQ ID NO: 79. The nucleotide sequence encoding the SH357-1 CDRL₃ is represented by SEQ ID NO: 80, and the amino acid sequence thereof is represented by SEQ ID NO: 81.

Example 6

Binding Activity of Anti-EPHA2 Antibody to EPHA2 Extracellular Region

[0511] A solution of an EPHA2 extracellular region polypeptide (manufactured by R&D Systems, Inc., #3035-A2-100) or bovine serum albumin (in the description below and the figures, abbreviated to "BSA") as a control diluted to 1 μg/ml with PBS was dispensed at 100 μl/well onto an immunoplate (manufactured by Nunc, #442404) and incubated overnight at 4° C. to thereby adsorb the protein to the plate.

[0512] On the next day, the solution in the wells was removed, and a Block Ace solution (one pouch of Block Ace powder (manufactured by Dainippon Sumitomo Pharma Co., Ltd. (Show Brand Milk Products Co., Ltd.)) was dissolved in 100 ml of ultrapure water) diluted 4 times with PBS was dispensed at 200 μl/well and incubated at room temperature for 1 hour. The wells were washed twice with Diluting Buffer (PBS, 0.05% (v/v) Tween 20). Then, SH348-1, SH357-1, and Ab96-1 were separately diluted with PBS to a concentration of 1.25×10^-4 μg/ml, 1.25×10^-3 μg/ml, 1.25×10^-2 μg/ml, 1.25×10^-1 μg/ml, 1.25 μg/ml, 12.5 μg/ml, or 125 μg/ml, and the resulting solution (containing 0.05% (v/v) (final concentration) Tween 20) was added at 100 μl/well.

[0513] The plate was incubated at room temperature for 1 hour. Then, the solution in the wells was removed, and the wells were washed twice with Diluting Buffer. Goat anti-Mouse IgG, Peroxidase Conjugated (manufactured by Millipore (Chemicon), #AP181P) diluted 1000 times with Diluting Buffer was added at 100 μl/well and incubated at room temperature for 1 hour. The solution in the wells was removed, and the wells were washed twice with Diluting Buffer. Then, a color reaction was performed with stirring by the addition of OPD Color Developing Solution at 100 μl/well. After color development, the color reaction was terminated by the addition of 1 M HCl at 100 μl/well. The absorbance at 490 nm was measured using a plate reader (FIG. 7).

[0514] FIG. 7A) shows the results of SH348-1, FIG. 7B) shows the results of SH357-1, and FIG. 7C) shows the results of Ab96-1. In each graph, the absorbance is indicated in mean±standard deviation (n=3). Stronger absorbance represents stronger binding activity. As shown in the graphs, all the antibodies SH348-1, SH357-1, and Ab96-1 exhibited no affinity for BSA, demonstrating that they specifically bind to the EPHA2 extracellular region.

Example 7

Influence of Anti-EPHA2 Antibody on Ligand Binding

[0515] An EPHA2 extracellular region polypeptide (manufactured by R&D Systems, Inc., #3035-A2-100)-immobilized immunoplate was prepared according to the method described in Example 6. The immunoplate wells were washed twice with Diluting Buffer. Then, SH348-1, SH357-1, Ab96-1, or Mouse IgG₂A Isotype Control (in the description below and the figures, abbreviated to "mIgG2a"; manufactured by R&D Systems, Inc., #MAB003) as an isotype control antibody diluted to 10 μg/ml or 50 μg/ml with Diluting Buffer was added at 100 μl/well. The plate was incubated at room temperature for 1 hour. Then, Recombinant Mouse Ephrin-A1/Fc Chimera (in the description below and the figures, abbreviated to "Ephrin-A1/Fc"; manufactured by R&D Systems, Inc., #602-A1-200) as a soluble ligand or Recombinant Human IgG₁ Fc (in the description below and the figures, abbreviated to "hG₁Fc"; manufactured by R&D Systems, Inc., #110-HG-100) as a negative control protein for the soluble ligand was added at a final concentration of 1 μg/ml and incubated at room temperature for 1 hour.

[0516] Next, according to the method described in Example 6, the solution in the wells was removed, and the wells were washed with Diluting Buffer. Peroxidase AffiniPure Goat Anti-Human IgG Fcγ Fragment Specific (Jackson ImmunoResearch Laboratories, Inc., #109-035-098) was added thereto. A color reaction was performed using OPD Color Developing Solution. The absorbance at 490 nm was measured using a plate reader (FIG. 8).

[0517] In FIG. 8, the absorbance is indicated in mean±standard deviation (n=3). The antibody Ab96-1 even at a concentration of 10 μg/ml strongly inhibited the binding of the EPHA2 ligand ephrin-A1 to EPHA2. By contrast, the antibodies SH348-1 and SH357-1, even when added at a concentration of 50 μg/ml (five times the concentration of Ab96-1), did not inhibit the binding of Ephrin-A1/Fc to EPHA2. These results demonstrated that the antibodies SH348-1 and SH357-1 do not inhibit the binding of Ephrin-A1/Fc to EPHA2.

Example 8

Verification of Inhibitory Activity of Anti-EPHA2 Antibody Against Ephrin-A1-Dependent Phosphorylation of EPHA2 Tyrosine Residues

[0518] 8)-1 Preparation of Cell Lysates

[0519] MDA-MB-231 cells suspended in RPMI1640 containing 10% FBS, 50 units/ml penicillin, and 50 μg/ml streptomycin (hereinafter, abbreviated to "10% FBS-containing RPMI1640 (with antibiotics)") were seeded at 2.5×10⁵ cells/well onto a 12-well dish and cultured overnight at 37° C. in 5% CO₂. Next, the medium in the wells was discarded, and RPMI1640 was newly added thereto. The cells were further cultured overnight at 37° C. in 5% CO₂. Next, the medium in the wells was removed, and only RPMI1640 or RPMI1640 containing the antibody (mIgG2a, SH348-1, or SH357-1) at a concentration of 10 μg/ml or 50 μg/ml was added to each well and preincubated at 37° C. in 5% CO₂ for 1 hour.

[0520] To the thus-preincubated wells supplemented with only RPMI1640, a 1/50 volume of Ephrin-A1/Fc or hG₁Fc (final concentration of 1 μg/ml) or RPMI1640 with the same volume thereas (in FIG. 9, represented by (-)) were added. Moreover, to the wells supplemented with mIgG2a, SH348-1, or SH357-1, a 1/50 volume of Ephrin-A1/Fc (final concentration of 1 μg/ml) was added.

[0521] The dishes were further incubated at 37° C. in 5% CO₂ for 15 minutes. After discarding of the supernatants, 1× Cell Lysis Buffer (manufactured by Cell Signaling Technology, Inc.) containing 1 mM PMSF (manufactured by Sigma-Aldrich, Inc.) and Protease Inhibitor Cocktail (manufactured by Sigma-Aldrich, Inc., #P8340) (hereinafter, abbreviated to PPCLB) was added thereto to lyse the cells. The lysates were centrifuged at 15000 rpm for 5 minutes, and the obtained supernatants were used as immunoprecipitation samples. The protein concentrations of the samples were measured using BCA Protein Assay Reagent (manufactured by PIERCE).

[0522] 8)-2 Detection of Phosphorylated State of EPHA2 by Immunoprecipitation

[0523] 25 μl of a suspension of Protein G magnetic beads (manufactured by NEW ENGLAND BioLabs, Inc.) and 4 μg of anti-EphA2 Antibody (manufactured by Santa Cruz Biotechnology, Inc., #sc-924) were added per sample, and the mixture was inverted for mixing at 4° C. for 2 hours. Next, FBS was added thereto at a final concentration of 10%, and the mixture was further inverted for mixing at 4° C. for 30 minutes. Next, the beads were washed with PPCLB. Next, 200 μg of the cell lysate supernatants prepared in paragraph 8)-1 were added thereto, and the mixtures were inverted for mixing overnight at 4° C.

[0524] On the next day, the beads were washed three times with PPCLB. Then, SDS-Sample Buffer (56.3 mM Tris-HCl, pH 6.8, 1.8% (w/v) SDS, 9% glycerol, 0.72 M 2-mercaptoethanol, 0.045 mg/ml bromophenol blue) was added to the beads, and the mixtures were heated at 98° C. for 5 minutes. The proteins dissociated from the beads were separated by SDS-PAGE.

[0525] The proteins were transferred from the gel to a PVDF membrane (0.45 μm in pore size; manufactured by Millipore). The PVDF membrane was blocked by shaking in sodium azide-free Blocking Solution (one pouch of Block Ace powder was dissolved in 100 ml of ultrapure water, to which Tween 20 was then added at a final concentration of 0.1% (v/v)).

[0526] To detect the phosphorylated state of the EPHA2 tyrosine residues, the PVDF membrane was soaked in a solution of Anti-Phosphotyrosine, recombinant 4G10 HRP-conjugate (manufactured by Upstate, #16-184) diluted 10000 times with sodium azide-free Blocking Solution, and reacted at room temperature for 1 hour. The PVDF membrane was washed for 10 minutes three times with TBST (50 mM Tris-HCl, pH 8.0, 138 mM NaCl, 2.7 mM KCl, 0.1% (v/v) Tween 20) and then further washed for 5 minutes three times with H₂O. Signals were detected on a film for chemiluminescence using ECL Plus (manufactured by GE Healthcare Bio-Sciences Corp.).

[0527] Next, to detect the immunoprecipitated EPHA2 present on this PVDF membrane, the PVDF membrane after the detection of the phosphorylated state of the EPHA2 tyrosine residues was soaked in Stripping Solution (50 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 100 mM 2-mercaptoethanol) and shaken at 55° C. for 30 minutes. Then, the PVDF membrane was soaked in Quenching Solution (TBST containing 1% (v/v) H₂O₂ and 0.1% (w/v) NaN₃) and shaken at room temperature for 20 minutes to remove the antibody on the PVDF membrane. The PVDF membrane was washed for 10 minutes three times with TBST and blocked in Blocking Solution (one pouch of Block Ace powder was dissolved in 100 ml of ultrapure water, to which Tween 20 and sodium azide were then added at final concentrations of 0.1% (v/v) and 0.02% (w/v), respectively). Then, the PVDF membrane was soaked in a solution of an anti-EphA2 Antibody (manufactured by Santa Cruz Biotechnology, Inc., #sc-924) diluted 4000 times with Blocking Solution, and reacted at room temperature for 1 hour. The PVDF membrane was washed for 10 minutes three times with TBST. Then, the PVDF membrane was soaked in a solution of Anti-rabbit IgG, HRP-linked Antibody (manufactured by Cell Signaling Technology, Inc., #7074) diluted 10000 times with TBST, and reacted at room temperature for 30 minutes. Then, this PVDF membrane was washed for 10 minutes three times with TBST. Then, signals were detected on a film for chemiluminescence using ECL Plus.

[0528] The soluble ligand Ephrin-A1/Fc-induced phosphorylation of EPHA2 tyrosine residues was not inhibited by the isotype control antibody mIgG2a but was inhibited in a dose-dependent manner by the addition of the antibodies SH348-1 and SH357-1 (FIG. 9).

[0529] These results demonstrated that the antibodies SH348-1 and SH357-1 do not inhibit the binding of the ligand ephrin-A1 to EPHA2 but inhibit the ligand-induced phosphorylation of EPHA2 tyrosine residues.

Example 9

Epitope Identification for Anti-EPHA2 Antibody

[0530] Deletion mutants of EPHA2 consisting of a region shown in FIG. 10 were prepared, and a region binding to SH348-1 or SH357-1 was determined.

[0531] 9)-1 Preparation of Deletion Mutants of EPHA2

[0532] To express deletion mutants of EPHA2 as proteins having GST-Tag and His-Tag added to the N-terminus and S-Tag added to the C-terminus, the following primers were synthesized, and gene fragments amplified by a method shown below were cloned into pET-49b (+) (manufactured by Novagen):

TABLE-US-00011 (primer N1: sequence ID No. 82) 5'-attaggatccgagcttccgtactgccagtgtc-3' (primer N2: sequence ID No. 83) 5'-attaggatccgccccccaaggtgaggct-3' (primer N3: sequence ID No. 84) 5'-attaggatccggtcacttaccgcaagaagggaga-3' (primer N4: sequence ID No. 85) 5'-attaggatccggtccaggtgcaggcactgacg-3' (primer C1: sequence ID No. 86) 5'-aattaagcttgccgccaatcaccgccaagtt-3' (primer C2: sequence ID No. 87) 5'-aattaagcttgttgccagatccctccggggac-3' (primer C3: sequence ID No. 88) 5'-aattaagcttcaggtaggtggtgtctggg-3' (primer C4: sequence ID No. 89) 5'-aattaagcttctcgtacttccacactcggc-3'

[0533] Each region was amplified by PCR reaction using pcDNA-DEST40-EPHA2 as the template and a primer set: the primers N1 and C1 for a region consisting of an amino acid sequence represented by amino acid Nos. 426 to 540 of SEQ ID NO: 8 in the sequence listing (hereinafter, referred to as "m1"); the primers N1 and C2 for a region consisting of an amino acid sequence represented by amino acid Nos. 426 to 534 thereof (hereinafter, referred to as "m2"); the primers N1 and C3 for a region consisting of an amino acid sequence represented by amino acid Nos. 426 to 504 thereof (hereinafter, referred to as "m3"); the primers N1 and C4 for a region consisting of an amino acid sequence represented by amino acid Nos. 426 to 470 thereof (hereinafter, referred to as "m4"); the primers N2 and C2 for a region consisting of an amino acid sequence represented by amino acid Nos. 439 to 534 thereof (hereinafter, referred to as "m5"); the primers N3 and C2 for a region consisting of an amino acid sequence represented by amino acid Nos. 471 to 534 thereof (hereinafter, referred to as "m6"); the primers N4 and C2 for a region consisting of an amino acid sequence represented by amino acid Nos. 505 to 534 thereof (hereinafter, referred to as "m7"); the primers N2 and C3 for a region consisting of an amino acid sequence represented by amino acid Nos. 439 to 504 thereof (hereinafter, referred to as "m8"); and the primers N2 and C4 for a region consisting of an amino acid sequence represented by amino acid Nos. 439 to 470 thereof (hereinafter, referred to as "m9"). The obtained PCR products were cleaved with BamHI and HindIII and subcloned into the BamHI/HindIII site of pET-49b (+).

[0534] 9)-2 Expression of Deletion Mutants of EPHA2

[0535] Escherichia coli BL21 (DE3) was transformed with the plasmid DNA constructed in paragraph 9)-1 or pET-49b (+) as a negative control. The obtained transformants were cultured in an LB medium supplemented with 30 μg/ml kanamycin (manufactured by Invitrogen Corp.). Expression of the deletion mutants of EPHA2 was induced using Autoinduction System (manufactured by Novagen). The bacterial cells were collected by centrifugation and washed with PBS. Then, the bacterial cells were lysed with a 2% SDS solution containing 1 mM PMSF and Protease Inhibitor Cocktail (manufactured by Sigma-Aldrich, Inc., #P8340). The supernatant was collected by centrifugation and used in epitope identification.

[0536] The expression level of proteins (consisting of pET-49b (+)-derived GST-Tag, His-Tag, and S-Tag and linker portions for connecting them; in the description below and the figures, referred to as "Vec") expressed in the bacterial cell lysate of Escherichia coli transformed with pET-49b (+) was estimated by a method described below. Dilution series of the Vec-expressing bacterial cell lysate and dilution series of 6×His Protein Ladder (manufactured by QIAGEN) were separately dissolved in SDS-Sample Buffer and heated at 98° C. for 5 minutes. Then, the proteins were separated by SDS-PAGE, and the proteins in the gel were transferred to a PVDF membrane. The PVDF membrane was blocked in TBST containing 5% BSA. Then, the PVDF membrane was soaked in a solution of Penta-His HRP Conjugate (manufactured by QIAGEN) diluted 1000 times with TBST containing 5% BSA, and reacted at room temperature for 1 hour. This PVDF membrane was washed for 10 minutes three times with TBST. Then, signals were detected using ECL Plus. Signal intensity was compared between the dilution series of the Vec-expressing bacterial cell lysate and the dilution series of 6×His Protein Ladder, and the concentration of the Vec protein contained in the Vec-expressing bacterial cell lysate was estimated from the protein level per band contained in 6×His Protein Ladder. Deletion mutants of EPHA2 with an amount that exhibited reactivity equivalent to that of 20 ng of Vec in Western blotting using S-Tag Monoclonal Antibody (Novagen) were used in the subsequent epitope identification experiment.

[0537] 9)-3) Epitope Identification

[0538] The cell lysates containing the deletion mutants of EPHA2 prepared in paragraph 9)-2 were dissolved in SDS-Sample Buffer and heated at 98° C. for 5 minutes. The resulting samples were separated by SDS-PAGE, and the proteins in the gels were transferred to PVDF membranes. After the transfer, the PVDF membranes were blocked by shaking in Blocking Solution (one pouch of Block Ace powder was dissolved in 100 ml of ultrapure water, to which Tween 20 and sodium azide were then added at final concentrations of 0.1% (v/v) and 0.02% (w/v), respectively). Then, these PVDF membranes were reacted overnight at 4° C. in Blocking Solution containing 2 μg/ml SH348-1 or SH357-1. These PVDF membranes were washed for 10 minutes three times with TBST and further reacted at room temperature for 30 minutes in a solution of Anti-Mouse Ig, HRP-Linked Whole Ab Sheep diluted 5000 times with TBST. Subsequently, the PVDF membranes were washed for 10 minutes three times with TBST. Then, signals were detected on a film for chemiluminescence using ECL Plus.

[0539] Next, the PVDF membranes were soaked in Stripping Solution (50 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 100 mM 2-mercaptoethanol), then shaken at 55° C. for 30 minutes, and then washed for 10 minutes three times with TBST. These PVDF membranes were blocked in Blocking Solution and then washed for 10 minutes three times with TBST. The PVDF membranes were soaked in a solution of S-Tag Monoclonal Antibody diluted 10000 times with TBST, and reacted at room temperature for 30 minutes. The PVDF membranes were washed for 10 minutes three times with TBST. Then, the PVDF membranes were soaked in a solution of Anti-Mouse Ig, HRP-Linked Whole Ab Sheep diluted 5000 times with TBST, and reacted at room temperature for 30 minutes. Next, the PVDF membranes were washed for 10 minutes three times with TBST. Then, signals were detected on a film for chemiluminescence using ECL Plus.

[0540] As a result, both the antibodies SH348-1 (FIG. 11A) and SH357-1 (FIG. 11C) exhibited binding activities only to m1, m2, and m5. Moreover, in this procedure, the deletion mutants of EPHA2 were present in an almost constant amount on each of the PVDF membranes (SH348-1: FIG. 11B, SH357-1: FIG. 11D). These results demonstrated that the antibodies SH348-1 and SH357-1 bind to a region consisting of an amino acid sequence represented by amino acid Nos. 439 to 534 of SEQ ID NO: 8 in the sequence listing in the EPHA2 amino acid sequence.

Example 10

Design of Humanized Antibody

[0541] 10)-1 Design of Humanized Antibody of SH348-1

[0542] 10)-1-1 Molecular Modeling of SH348-1 Variable Regions

[0543] The molecular modeling of SH348-1 variable regions was conducted according to homology modeling (Methods in Enzymology, 203, 121-153, (1991)). The primary sequences (three-dimensional structures derived from X-ray crystal structures are available) of human immunoglobulin variable regions registered in Protein Data Bank (Nuc. Acid Res. 35, D301-D303 (2007)) were compared with the SH348-1 variable regions determined in Example 5. As a result, 2JEL and 1A4J were respectively selected as sequences having the highest sequence homology to the SH348-1 light or heavy chain variable regions. The three-dimensional structures of framework regions were prepared based on a "framework model" obtained by combining the coordinates of 2JEL and 1A4J corresponding to the SH348-1 light and heavy chains. For SH348-1 CDRs, CDRL₁, CDRL₂, CDRL₃, CDRH₁, and CDRH₂ were assigned to clusters 16A, 7A, 9A, 10A, and 10A, respectively, according to the classification of Thornton et al. (J. Mol. Biol., 263, 800-815, (1996)). CDRH₃ was classified in e (9) D according to the H3-rules (FEBS letters 399, 1-8 (1996)). Subsequently, the typical conformation of each CDR was incorporated in the framework model.

[0544] Finally, to obtain possible molecular models of the SH348-1 variable regions in terms of energy, an energy calculation was conducted for excluding disadvantageous interatomic contact. These procedures were performed using a commercially available three-dimensional protein structure prediction program Prime and coordinate search program MacroModel (Schrodinger, LLC).

[0545] 10)-1-2 Design of Amino Acid Sequence of Humanized SH348-1

[0546] Humanized SH348-1 antibodies were constructed according to CDR grafting (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). Acceptor antibodies were selected based on amino acid homology within the framework regions. The sequences of the SH348-1 framework regions were compared with those of all human frameworks registered in the Kabat Database (Nuc. Acid Res. 29, 205-206 (2001)) involving antibody amino acid sequences. A GSD2B5B10'CL antibody was selected as an acceptor due to their having, at 72%, the highest sequence homology between their framework regions. The amino acid residues of the framework regions in GSD2B5B10'CL were aligned with the corresponding amino acid residues in SH348-1 to identify positions where different amino acids therebetween were used. The positions of these residues were analyzed using the three-dimensional model of SH348-1 thus constructed. Then, donor residues to be grafted on the acceptor were selected according to the criteria provided by Queen et al. (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). Humanized SH348-1 sequences were determined by transferring some selected donor residues to the acceptor antibody GSD2B5B10'CL. As a result, the humanized sequences of two types of light chains and two types of heavy chains were obtained as shown below. Hereinafter, variable and constant regions and CDRs were classified based on the antibody amino acid sequence database prepared by Kabat et al.

[0547] 10-1-3) Humanization of SH348-1 Light Chain

[0548] 10-1-3-1) hSH348-T1L-Type Light Chain:

[0549] A humanized SH348-1 light chain was designed by substituting amino acid Nos. 2 (valine), 3 (leucine), 14 (serine), 15 (leucine), 17 (aspartic acid), 18 (glutamine), 50 (lysine), 79 (arginine), 88 (leucine), 105 (glycine), 109 (leucine), and 114 (alanine) of SH348-1 light chain variable region shown in SEQ ID NO: 37 of the sequence listing with isoleucine, valine, threonine, proline, glutamic acid, proline, glutamine, lysine, valine, glutamine, valine, and threonine, respectively, and was designated as a "hSH348-T1L-type light chain".

[0550] The nucleotide sequence encoding the hSH348-1-T1L-type light chain is represented by SEQ ID NO: 90 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 91. The nucleotide sequence represented by nucleotide Nos. 1 to 60 of SEQ ID NO: 90 is a secretion signal sequence. The nucleotide sequence represented by nucleotide Nos. 61 to 402 thereof is a variable region. The nucleotide sequence represented by nucleotide Nos. 403 to 717 thereof is a constant region. The nucleotide sequence represented by nucleotide Nos. 130 to 177 thereof is CDRL₁. The nucleotide sequence represented by nucleotide Nos. 223 to 243 thereof is CDRL₂. The nucleotide sequence represented by nucleotide Nos. 340 to 363 thereof is CDRL₃.

[0551] The amino acid sequence represented by amino acid Nos. 1 to 20 of SEQ ID NO: 91 of the sequence listing is a secretion signal sequence. The amino acid sequence represented by amino acid Nos. 21 to 134 thereof is a variable region. The amino acid sequence represented by amino acid Nos. 135 to 239 thereof is a constant region. The amino acid sequence represented by amino acid Nos. 44 to 59 thereof is CDRL₁. The amino acid sequence represented by amino acid Nos. 75 to 81 thereof is CDRL₂. The amino acid sequence represented by amino acid Nos. 114 to 121 thereof is CDRL₃.

[0552] Moreover, the nucleotide sequence encoding the hSH348-1-T1L-type light chain CDRL₁ is represented by SEQ ID NO: 92 in the sequence listing, the amino acid sequence thereof is represented by SEQ ID NO: 93, the nucleotide sequence of CDRL₂ is represented by SEQ ID NO: 94 in the sequence listing, the amino acid sequence thereof is represented by SEQ ID NO: 95, the nucleotide sequence of CDRL₃ is represented by SEQ ID NO: 96 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 97.

[0553] 10-1-3-2) hSH348-T3L-Type Light Chain:

[0554] A humanized SH348-1 light chain was designed by substituting amino acid Nos. 14 (serine), 15 (leucine), 17 (aspartic acid), 18 (glutamine), 50 (lysine), 79 (arginine), 88 (leucine), 105 (glycine), 109 (leucine), and 114 (alanine) of SH348-1 light chain variable region shown in SEQ ID NO: 37 of the sequence listing with threonine, proline, glutamic acid, proline, glutamine, lysine, valine, glutamine, valine, and threonine, respectively, and was designated as a "hSH348-1-T3L-type light chain".

[0555] The nucleotide sequence encoding the hSH348-1-T3L-type light chain is represented by SEQ ID NO: 98 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 99. The nucleotide sequence represented by nucleotide Nos. 1 to 60 of SEQ ID NO: 98 is a secretion signal sequence. The nucleotide sequence represented by nucleotide Nos. 61 to 402 thereof is a variable region. The nucleotide sequence represented by nucleotide Nos. 403 to 717 thereof is a constant region. The nucleotide sequence represented by nucleotide Nos. 130 to 177 thereof is CDRL₁. The nucleotide sequence represented by nucleotide Nos. 223 to 243 thereof is CDRL₂. The nucleotide sequence represented by nucleotide Nos. 340 to 363 thereof is CDRL₃.

[0556] The amino acid sequence represented by amino acid Nos. 1 to 20 of SEQ ID NO: 99 of the sequence listing is a secretion signal sequence. The amino acid sequence represented by amino acid Nos. 21 to 134 thereof is a variable region. The amino acid sequence represented by amino acid Nos. 135 to 239 thereof is a constant region. The amino acid sequence represented by amino acid Nos. 44 to 59 thereof is CDRL₁. The amino acid sequence represented by amino acid Nos. 75 to 81 thereof is CDRL₂. The amino acid sequence represented by amino acid Nos. 114 to 121 thereof is CDRL₃.

[0557] Moreover, the nucleotide sequence encoding the hSH348-1-T3L-type light chain CDRL₁ is represented by SEQ ID NO: 100 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 101, the nucleotide sequence of CDRL₂ is represented by SEQ ID NO: 102 in the sequence listing, the amino acid sequence thereof is represented by SEQ ID NO: 103, the nucleotide sequence of CDRL₃ is represented by SEQ ID NO: 104 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 105.

[0558] 10-1-4) Humanization of SH348-1 Heavy Chain

[0559] 10-1-4-1) hSH348-1-T1H-Type Heavy Chain:

[0560] A humanized SH348-1 heavy chain was designed by substituting amino acid Nos. 2 (isoleucine), 9 (proline), 11 (leucine), 16 (glutamic acid), 17 (threonine), 20 (isoleucine), 38 (lysine), 43 (lysine), 46 (lysine), 68 (phenylalanine), 69 (alanine), 70 (phenylalanine), 71 (serine), 72 (leucine), 73 (glutamic acid), 76 (alanine), 80 (phenylalanine), 82 (glutamine), 83 (isoleucine), 84 (asparagine), 85 (asparagine), 87 (lysine), 88 (asparagine), 93 (threonine), 95 (phenylalanine), 114 (threonine), and 115 (leucine) of SH348-1 heavy chain variable region shown in SEQ ID NO: 43 of the sequence listing with valine, alanine, valine, serine, serine, valine, arginine, glutamine, glutamic acid, valine, threonine, isoleucine, threonine, alanine, aspartic acid, threonine, tyrosine, glutamic acid, leucine, serine, serine, arginine, serine, valine, tyrosine, leucine and valine, respectively, and was designated as a "hSH348-1-T1H-type heavy chain".

[0561] The nucleotide sequence encoding the hSH348-1-T1H-type heavy chain is represented by SEQ ID NO: 106 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 107. The nucleotide sequence represented by nucleotide Nos. 1 to 57 of SEQ ID NO: 106 is a secretion signal sequence. The nucleotide sequence represented by nucleotide Nos. 58 to 414 thereof is a variable region. The nucleotide sequence represented by nucleotide Nos. 415 to 1404 thereof is a constant region. The nucleotide sequence represented by nucleotide Nos. 148 to 162 thereof is CDRH₁. The nucleotide sequence represented by nucleotide Nos. 205 to 255 thereof is CDRH₂. The nucleotide sequence represented by nucleotide Nos. 352 to 381 thereof is CDRL₃.

[0562] The amino acid sequence represented by amino acid Nos. 1 to 19 of SEQ ID NO: 107 of the sequence listing is a secretion signal sequence. The amino acid sequence represented by amino acid Nos. 20 to 138 thereof is a variable region. The amino acid sequence represented by amino acid Nos. 139 to 468 thereof is a constant region. The amino acid sequence represented by amino acid Nos. 50 to 54 thereof is CDRH₁. The amino acid sequence represented by amino acid Nos. 69 to 85 thereof is CDRH₂. The amino acid sequence represented by amino acid Nos. 118 to 127 thereof is CDRH₃.

[0563] Moreover, the nucleotide sequence encoding the hSH348-1-T1H-type heavy chain CDRH₁ is represented by SEQ ID NO: 108 in the sequence listing, the amino acid sequence thereof is represented by SEQ ID NO: 109, the nucleotide sequence of CDRH₂ is represented by SEQ ID NO: 110 in the sequence listing, the amino acid sequence thereof is represented by SEQ ID NO: 111, the nucleotide sequence of CDRH₃ is represented by SEQ ID NO: 112 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 113.

[0564] 10-1-4-2) hSH348-1-T3H-Type Heavy Chain:

[0565] A humanized SH348-1 heavy chain was designed by substituting amino acid Nos. 9 (proline), 11 (leucine), 16 (glutamic acid), 17 (threonine), 20 (isoleucine), 38 (lysine), 43 (lysine), 73 (glutamic acid), 76 (alanine), 80 (phenylalanine), 82 (glutamine), 83 (isoleucine), 84 (asparagine), 85 (asparagine), 87 (lysine), 88 (asparagine), 93 (threonine), 95 (phenylalanine), 114 (threonine), and 115 (leucine) of SH348-1 heavy chain variable domains shown in SEQ ID NO: 43 of the sequence listing with alanine, valine, serine, serine, valine, arginine, glutamine, aspartic acid, threonine, tyrosine, glutamic acid, leucine, serine, serine, arginine, serine, valine, tyrosine, leucine and valine, respectively, and was designated as a "hSH348-1-T3H-type heavy chain".

[0566] The nucleotide sequence encoding the hSH348-1-T3H-type heavy chain is represented by SEQ ID NO: 114 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 115. The nucleotide sequence represented by nucleotide Nos. 1 to 57 of SEQ ID NO: 114 is a secretion signal sequence. The nucleotide sequence represented by nucleotide Nos. 58 to 414 thereof is a variable region. The nucleotide sequence represented by nucleotide Nos. 415 to 1404 thereof is a constant region. The nucleotide sequence represented by nucleotide Nos. 148 to 162 thereof is CDRH₁. The nucleotide sequence represented by nucleotide Nos. 205 to 255 thereof is CDRH₂. The nucleotide sequence represented by nucleotide Nos. 352 to 381 thereof is CDRL₃.

[0567] The amino acid sequence represented by amino acid Nos. 1 to 19 of SEQ ID NO: 115 of the sequence listing is a secretion signal sequence. The amino acid sequence represented by amino acid Nos. 20 to 138 thereof is a variable region. The amino acid sequence represented by amino acid Nos. 139 to 468 thereof is a constant region. The amino acid sequence represented by amino acid Nos. 50 to 54 thereof is CDRH₁. The amino acid sequence represented by amino acid Nos. 69 to 85 thereof is CDRH₂. The amino acid sequence represented by amino acid Nos. 118 to 127 thereof is CDRH₃.

[0568] Moreover, the nucleotide sequence encoding the hSH348-1-T3H-type heavy chain CDRH₁ is represented by SEQ ID NO: 116 in the sequence listing, the amino acid sequence thereof is represented by SEQ ID NO: 117, the nucleotide sequence of CDRH₂ is represented by SEQ ID NO: 118 in the sequence listing, the amino acid sequence thereof is represented by SEQ ID NO: 119, the nucleotide sequence of CDRH₃ is represented by SEQ ID NO: 120 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 121.

[0569] 10)-2 Design of Humanized Antibody of SH357-1

[0570] 10)-2-1 Molecular Modeling of SH357-1 Variable Region

[0571] 2JEL and 1A4J were selected as sequences having the highest sequence homology to the SH357-1 light and heavy chain variable regions, respectively, in the same way as in paragraph 10)-1-1. CDRL₁, CDRL₂, CDRL₃, CDRH₁, and CDRH₂ were assigned to clusters 16A, 7A, 9A, 10A, and 10A, respectively. CDRH₃ was classified in e(9)D.

[0572] 10)-2-2 Design of Amino Acid Sequence for Humanized SH357-1

[0573] A GSD2B5B10'CL antibody was selected as an acceptor in the same way as in paragraph 10)-1-2, and the sequence of humanized SH357-1 was determined. As a result, the humanized sequences of two types of light chains and two types of heavy chains were obtained as shown below.

[0574] 10-2-3) Humanization of SH357-1 Light Chain

[0575] 10-2-3-1) hSH357-1-T1L-Type Light Chain:

[0576] A humanized SH357-1 light chain designed by substituting amino acid Nos. 2 (valine), 3 (leucine), 7 (threonine), 14 (serine), 15 (leucine), 17 (aspartic acid), 18 (glutamine), 50 (lysine), 88 (leucine), 105 (glycine), 109 (leucine), and 114 (alanine) of SH357-1 light chain shown in SEQ ID NO: 41 of the sequence listing with isoleucine, valine, serine, threonine, proline, glutamic acid, proline, glutamine, valine, glutamine, valine, and threonine, respectively, was designated as a "hSH357-1-T1L-type light chain".

[0577] The nucleotide sequence encoding the hSH357-1-T1L-type light chain is represented by SEQ ID NO: 122 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 123. The nucleotide sequence represented by nucleotide Nos. 1 to 60 of SEQ ID NO: 122 is a secretion signal sequence. The nucleotide sequence represented by nucleotide Nos. 61 to 402 thereof is a variable region. The nucleotide sequence represented by nucleotide Nos. 403 to 717 thereof is a constant region. The nucleotide sequence represented by nucleotide Nos. 130 to 177 thereof is CDRL₁. The nucleotide sequence represented by nucleotide Nos. 223 to 243 thereof is CDRL₂. The nucleotide sequence represented by nucleotide Nos. 340 to 363 thereof is CDRL₃.

[0578] The amino acid sequence represented by amino acid Nos. 1 to 20 of SEQ ID NO: 123 of the sequence listing is a secretion signal sequence. The amino acid sequence represented by amino acid Nos. 21 to 134 thereof is a variable region. The amino acid sequence represented by amino acid Nos. 135 to 239 thereof is a constant region. The amino acid sequence represented by amino acid Nos. 44 to 59 thereof is CDRL₁. The amino acid sequence represented by amino acid Nos. 75 to 81 thereof is CDRL₂. The amino acid sequence represented by amino acid Nos. 114 to 121 thereof is CDRL₃.

[0579] Moreover, the nucleotide sequence encoding the hSH357-1-T1L-type light chain CDRL₁ is represented by SEQ ID NO: 124 in the sequence listing, the amino acid sequence thereof is represented by SEQ ID NO: 125, the nucleotide sequence of CDRL₂ is represented by SEQ ID NO: 126 in the sequence listing, the amino acid sequence thereof is represented by SEQ ID NO: 127, the nucleotide sequence of CDRL₃ is represented by SEQ ID NO: 128 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 129.

[0580] 10-2-3-2) hSH357-1-T3L-Type Light Chain:

[0581] A humanized SH357-1 light chain designed by substituting amino acid Nos. 7 (threonine), 14 (serine), 15 (leucine), 17 (aspartic acid), 18 (glutamine), 50 (lysine), 88 (leucine), 105 (glycine), 109 (leucine), and 114 (alanine) of SH357-1 light chain shown in SEQ ID NO: 41 with serine, threonine, proline, glutamic acid, proline, glutamine, valine, glutamine, valine, and threonine, respectively, was designated as a "hSH357-1-T3L-type light chain".

[0582] The nucleotide sequence encoding the hSH357-1-T3L-type light chain is represented by SEQ ID NO: 130 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 131. A nucleotide sequence represented by nucleotide Nos. 1 to 60 of SEQ ID NO: 130 is a secretion signal sequence. A nucleotide sequence represented by nucleotide Nos. 61 to 402 thereof is a variable region. A nucleotide sequence represented by nucleotide Nos. 403 to 717 thereof is a constant region. A nucleotide sequence represented by nucleotide Nos. 130 to 177 thereof is CDRL₁. A nucleotide sequence represented by nucleotide Nos. 223 to 243 thereof is CDRL₂. A nucleotide sequence represented by nucleotide Nos. 340 to 363 thereof is CDRL₃.

[0583] The amino acid sequence represented by amino acid Nos. 1 to 20 of SEQ ID NO: 131 of the sequence listing is a secretion signal sequence. The amino acid sequence represented by amino acid Nos. 21 to 134 thereof is a variable region. The amino acid sequence represented by amino acid Nos. 135 to 239 thereof is a constant region. The amino acid sequence represented by amino acid Nos. 44 to 59 thereof is CDRL₁. The amino acid sequence represented by amino acid Nos. 75 to 81 thereof is CDRL₂. The amino acid sequence represented by amino acid Nos. 114 to 121 thereof is CDRL₃.

[0584] Moreover, the nucleotide sequence encoding the hSH357-1-T3L-type light chain CDRL₁ is represented by SEQ ID NO: 132 in the sequence listing, the amino acid sequence thereof is represented by SEQ ID NO: 133, the nucleotide sequence of CDRL₂ is represented by SEQ ID NO: 134 in the sequence listing, the amino acid sequence thereof is represented by SEQ ID NO: 135, the nucleotide sequence of CDRL₃ is represented by SEQ ID NO: 136 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 137.

[0585] 10-2-4) Humanization of SH357-1 Heavy Chain

[0586] 10-2-4-1) hSH357-1-T1H-Type Heavy Chain:

[0587] A humanized SH357-1 heavy chain was designed by substituting amino acid Nos. 2 (isoleucine), 9 (proline), 11 (leucine), 16 (glutamic acid), 17 (threonine), 20 (isoleucine), 38 (lysine), 43 (lysine), 46 (lysine), 68 (phenylalanine), 69 (alanine), 70 (phenylalanine), 71 (serine), 72 (leucine), 73 (glutamic acid), 76 (alanine), 82 (glutamine), 83 (isoleucine), 85 (asparagine), 87 (lysine), 88 (asparagine), 93 (serine), 95 (phenylalanine), 114 (threonine), and 115 (leucine) of SH357-1 heavy chain variable region shown in SEQ ID NO: 51 of the sequence listing with valine, alanine, valine, alanine, serine, valine, arginine, glutamine, glutamic acid, valine, threonine, isoleucine, threonine, alanine, aspartic acid, threonine, glutamic acid, leucine, serine, arginine, serine, valine, tyrosine, leucine and valine, respectively, and was designated as a "hSH357-1-T1H-type heavy chain".

[0588] The nucleotide sequence encoding the hSH357-1-T1H-type heavy chain is represented by SEQ ID NO: 138 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 139. The nucleotide sequence represented by nucleotide Nos. 1 to 57 of SEQ ID NO: 138 is a secretion signal sequence. The nucleotide sequence represented by nucleotide Nos. 58 to 414 thereof is a variable region. The nucleotide sequence represented by nucleotide Nos. 415 to 1404 thereof is a constant region. The nucleotide sequence represented by nucleotide Nos. 145 to 162 thereof is CDRH₁. The nucleotide sequence represented by nucleotide Nos. 205 to 255 thereof is CDRH₂. The nucleotide sequence represented by nucleotide Nos. 352 to 381 thereof is CDRL₃.

[0589] The amino acid sequence represented by amino acid Nos. 1 to 19 of SEQ ID NO: 139 of the sequence listing is a secretion signal sequence. The amino acid sequence represented by amino acid Nos. 20 to 138 thereof is a variable region. The amino acid sequence represented by amino acid Nos. 139 to 468 thereof is a constant region. The amino acid sequence represented by amino acid Nos. 49 to 54 thereof is CDRH₁. The amino acid sequence represented by amino acid Nos. 69 to 85 thereof is CDRH₂. The amino acid sequence represented by amino acid Nos. 118 to 127 thereof is CDRH₃.

[0590] Moreover, the nucleotide sequence encoding the hSH357-1-T1H-type heavy chain CDRH₁ is represented by SEQ ID NO: 140 in the sequence listing, the amino acid sequence thereof is represented by SEQ ID NO: 141, the nucleotide sequence of CDRH₂ is represented by SEQ ID NO: 142 in the sequence listing, the amino acid sequence thereof is represented by SEQ ID NO: 143, the nucleotide sequence of CDRH₃ is represented by SEQ ID NO: 144 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 145.

[0591] 10-2-4-2) hSH357-1-T3H-Type Heavy Chain:

[0592] A humanized SH357-1 heavy chain was designed by substituting amino acid Nos. 9 (proline), 11 (leucine), 16 (glutamic acid), 17 (threonine), 20 (isoleucine), 38 (lysine), 43 (lysine), 73 (glutamic acid), 76 (alanine), 82 (glutamine), 83 (isoleucine), 85 (asparagine), 87 (lysine), 88 (asparagine), 93 (serine), 95 (phenylalanine), 114 (threonine), and 115 (leucine) of SH357-1 heavy chain variable region shown in SEQ ID NO: 51 with alanine, valine, alanine, serine, valine, arginine, glutamine, aspartic acid, threonine, glutamic acid, leucine, serine, arginine, serine, valine, tyrosine, leucine and valine, respectively, and was designated as a "hSH357-1-T3H-type heavy chain".

[0593] The nucleotide sequence encoding the hSH357-1-T3H-type heavy chain is represented by SEQ ID NO: 146 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 147. The nucleotide sequence represented by nucleotide Nos. 1 to 57 of SEQ ID NO: 146 is a secretion signal sequence. The nucleotide sequence represented by nucleotide Nos. 58 to 414 thereof is a variable region. The nucleotide sequence represented by nucleotide Nos. 415 to 1404 thereof is a constant region. The nucleotide sequence represented by nucleotide Nos. 145 to 162 thereof is CDRH₁. The nucleotide sequence represented by nucleotide Nos. 205 to 255 thereof is CDRH₂. The nucleotide sequence represented by nucleotide Nos. 352 to 381 thereof is CDRL₃.

[0594] The amino acid sequence represented by amino acid Nos. 1 to 19 of SEQ ID NO: 147 of the sequence listing is a secretion signal sequence. The amino acid sequence represented by amino acid Nos. 20 to 138 thereof is a variable region. The amino acid sequence represented by amino acid Nos. 139 to 468 thereof is a constant region. The amino acid sequence represented by amino acid Nos. 49 to 54 thereof is CDRH₁. The amino acid sequence represented by amino acid Nos. 69 to 85 thereof is CDRH₂. The amino acid sequence represented by amino acid Nos. 118 to 127 thereof is CDRH₃.

[0595] Moreover, the nucleotide sequence encoding the hSH357-1-T3H-type heavy chain CDRH₁ is represented by SEQ ID NO: 148 in the sequence listing, the amino acid sequence thereof is represented by SEQ ID NO: 149, the nucleotide sequence of CDRH₂ is represented by SEQ ID NO: 150 in the sequence listing, the amino acid sequence thereof is represented by SEQ ID NO: 151, the nucleotide sequence of CDRH₃ is represented by SEQ ID NO: 152 in the sequence listing, and the amino acid sequence thereof is represented by SEQ ID NO: 153.

Example 11

Preparation of Humanized Anti-EPHA2 Antibodies

[0596] To measure the activities of the humanized SH348-1 and the humanized SH357-1, plasmids having the heavy and light chains of the humanized anti-EPHA2 antibodies obtained in Example 10 were constructed as shown below.

[0597] 11)-1 Construction of Humanized Anti-EPHA2 Antibody Expression Vectors

[0598] 11)-1-1 Preparation of Versatile Humanized Antibody Light Chain Expression Vector (pEF6/KCL)

[0599] A gene encoding a human antibody light chain signal sequence and a human Ig light chain (κ chain) constant region, described in SEQ ID NO: 154 in the sequence listing, was synthesized (Invitrogen Corp.; artificial gene synthesis service) and cleaved with restriction enzymes NheI and PmeI. The cleaved DNA fragment was inserted into the NheI/PmeI site of a vector pEF6/V5-HisB (Invitrogen Corp.) to construct a versatile humanized antibody light chain expression vector (pEF6/KCL).

[0600] 11)-1-2 Preparation of Versatile Humanized Antibody Heavy Chain Expression Vector (pEF1/FCCU-1)

[0601] A gene encoding a human antibody heavy chain signal sequence and a human IgG1 constant region, described in SEQ ID NO: 155 in the sequence listing, was synthesized (Invitrogen Corp.; artificial gene synthesis service) and cleaved with restriction enzymes NheI and PmeI. The cleaved DNA fragment was inserted into the NheI/PmeI site of a vector pEF1/myc-HisB (Invitrogen Corp.) to construct a versatile humanized antibody H chain expression vector (pEF1/FCCU-1).

[0602] 11)-1-3 hSH348-1-T1L and hSH348-1-T3L-Type Light Chain Expression Vectors

[0603] Each DNA containing a gene encoding a hSH348-1-T1L or hSH348-1-T3L-type light chain variable region represented by SEQ ID NO: 156 and 157 of the sequence listing, fused with a secretion signal was synthesized (Invitrogen Corp., Artificial Gene Synthesis Service) and digested with restriction enzymes NdeI and BsiWI. The resulting DNA fragments were separately inserted into sites of the versatile vector for humanized antibody light chain expression (pEF6/KCL) digested in advance with restriction enzymes NdeI and BsiWI to thereby construct hSH348-1-T1L and hSH348-1-T3L-type light chain expression vectors. The obtained expression vectors were designated as "pEF6/KCL/hSH348-1-T1L" and "pEF6/KCL/hSH348-1-T3L", respectively.

[0604] 11)-1-4 Construction of hSH348-1-T1H and hSH348-1-T3H-Type Heavy Chain Expression Vectors

[0605] Each DNA containing a gene encoding a hSH348-1-T1H or hSH348-1-T3H-type heavy chain variable region represented by SEQ ID NO: 158 and 159, respectively, of the sequence listing was synthesized (Invitrogen Corp., Artificial Gene Synthesis Service) and digested with a restriction enzyme BlpI. The resulting DNA fragments were separately inserted into sites of the versatile vector for humanized antibody H chain expression (pEF1/FCCU-1) digested in advance with a restriction enzyme BlpI to thereby construct hSH348-1-T1H and hSH348-1-T3H-type heavy chain expression vectors. The obtained expression vectors were designated as "pEF1/FCCU/hSH348-1-T1H" and "pEF1/FCCU/hSH348-1-T3H", respectively.

[0606] 11)-1-5 Construction of hSH357-1-T1L and hSH357-1-T3L-Type Light Chain Expression Vectors

[0607] Each DNA containing a gene encoding a hSH357-1-T1L or hSH357-1-T3L-type light chain variable region represented by SEQ ID NO: 160 or 161, respectively, of the sequence listing, fused with a secretion signal was synthesized (Invitrogen Corp., Artificial Gene Synthesis Service) and digested with restriction enzymes NdeI and BsiWI. The resulting DNA fragments were separately inserted into sites of the versatile vector for humanized antibody L chain expression (pEF6/KCL) digested in advance with restriction enzymes NdeI and BsiWI to thereby construct hSH357-1-T1L and hSH357-1-T3L-type light chain expression vectors. The obtained expression vectors were designated as "pEF6/KCL/hSH357-1-T1L" and "pEF6/KCL/hSH357-1-T3L", respectively.

[0608] 11)-1-6 Construction of hSH357-1-T1H and hSH357-1-T3H-Type Heavy Chain Expression Vectors

[0609] Each DNA containing a gene encoding a hSH357-1-T1H or hSH357-1-T3H-type heavy chain variable region represented by SEQ ID NO: 162 or 163, respectively, of the sequence listing was synthesized (Invitrogen Corp., Artificial Gene Synthesis Service) and digested with a restriction enzyme BlpI. The resulting DNA fragments were separately inserted into sites of the versatile vector for humanized antibody H chain expression (pEF1/FCCU-1) digested in advance with a restriction enzyme BlpI to thereby construct hSH357-1-T1H and hSH357-1-T3H-type heavy chain expression vectors. The obtained expression vectors were designated as "pEF1/FCCU/hSH357-1-T1H" and "pEF1/FCCU/hSH357-1-T3H", respectively.

[0610] 11)-2 Production of Humanized Antibody

[0611] 1.2×10⁹ cells of 293 FreeStyle cells at the log growth phase were seeded onto 1.2 L of fresh FreeStyle 293 Expression Medium (Invitrogen Corp.) and shake-cultured (125 rpm) in an incubator at 37° C. in 8% CO₂. 12 mg of Polyethyleneimine (Polyscience #24765) was dissolved in 40 mL of an Opti-Pro SFM medium (manufactured by Invitrogen Corp.) and left at room temperature for 5 minutes. An H chain expression plasmid (0.6 mg) and an L chain expression plasmid (1.8 mg) prepared using a PureLink HiPure Plasmid kit (Invitrogen Corp.) were suspended in 40 mL of an Opti-Pro SFM medium (Invitrogen Corp.). 40 mL of the expression plasmid/OptiPro SFM mixed solution was added to 40 mL of the Polyethyleneimine/OptiPro SFM mixed solution thus left at room temperature for 5 minutes and further left at room temperature for 5 minutes. Next, 80 mL of the Polyethyleneimine/expression plasmid/OptiPro SFM mixed solution was added to the 293 FreeStyle cell suspension, and shake-culture was continued. After 7-day culture at 37° C. in 8% CO2, the culture supernatant was collected.

[0612] 11)-3 Purification of Humanized Antibody

[0613] The culture supernatant obtained in paragraph 11)-2 was filtered through a Disposable Capsule Filter (Advantec MFS Inc., #CCS-045-E1H) and then purified by Protein A affinity column chromatography. The culture supernatant was applied to MabSelect SuRe HiTrap 1 mL (manufactured by GE Healthcare Bio-sciences Corp.) equilibrated with PBS, and washed with PBS. Next, a 2 M arginine solution (pH 4.0) was applied thereto to collect antibody-containing fractions. The pH was adjusted to 7, and the antibody-containing fractions were applied to a HiPrep Desalting Column (26/10, 50 mL) (GE Healthcare Bio-sciences Corp.) equilibrated with PBS in advance. After replacement with PBS, the antibody-containing fractions were passed through a 0.2-μm filter to prepare a purified sample.

[0614] The antibody concentration was determined by eluting the antibodies bound to a POROS G 20 μm Column, PEEK, 4.6 mm×100 mm, 1.7 ml (manufactured by Applied Biosystems) and measuring the absorbance (O.D. 280 nm) of the eluate, followed by peak area comparison with a standard (human IgG1).

[0615] A humanized antibody SH348-1 obtained by the combination between pEF6/KCL/hSH348-1-T1L and pEF1/FCCU/hSH348-1-T1H was designated as "hSH348-1-T1"; and a humanized antibody SH348-1 obtained by the combination between pEF6/KCL/hSH348-1-T3L and pEF1/FCCU/hSH348-1-T3H was designated as "hSH348-1-T3".

[0616] Moreover, a humanized antibody SH357-1 obtained by the combination between pEF6/KCL/hSH357-1-T1L and pEF1/FCCU/hSH357-1-T1H was designated as "hSH357-1-T1"; and a humanized antibody SH357-1 obtained by the combination between pEF6/KCL/hSH357-1-T3L and pEF1/FCCU/hSH357-1-T3H was designated as "hSH357-1-T3".

Example 12

Confirmation of Binding Activity of Humanized Anti-EPHA2 Antibody to Antigen

[0617] The abilities of the antibodies hSH348-1-T1, hSH348-1-T3, hSH357-1-T1, and hSH357-1-T3 to bind to the antigen were confirmed according to the method described in Example 6 except that Peroxidase AffiniPure Goat Anti-Human IgG Fcγ Fragment Specific (manufactured by Jackson ImmunoResearch Laboratories, Inc., #109-035-098) was used as a secondary antibody.

[0618] In the graphs of FIGS. 12A) to 12D), the absorbance is indicated in mean±standard deviation (n=3). As a result, all the humanized anti-EPHA2 antibodies hSH348-1-T1, hSH348-1-T3, hSH357-1-T1, and hSH357-1-T3 were confirmed to have binding activity to the EPHA2 extracellular region.

Example 13

Measurement of Competitive Inhibitory Activity Against Binding of SH348-1 or SH357-1 to EPHA2

[0619] The competitive inhibitory activities of hSH348-1-T1 and hSH348-1-T3 against the binding of SH348-1 to EPHA2 as well as the competitive inhibitory activities of hSH357-1-T1 and hSH357-1-T3 against the binding of SH357-1 to EPHA2 were measured by a method described below.

[0620] The mouse monoclonal antibodies SH348-1 and SH357-1 were separately biotinylated using EZ-Link Sulfo-NHS-LC Biotinylation Kit (manufactured by Thermo Fisher Scientific K.K., #21435) according to the protocol included therein (hereinafter, the biotinylated SH348-1 and SH357-1 were referred to as "bSH348-1" and "bSH357-1", respectively). The concentrations of bSH348-1, bSH357-1, and unlabeled antibodies (SH348-1, SH357-1, hSH348-1-T1, hSH348-1-T3, hSH357-1-T1, hSH357-1-T3, and Ab96-1) used in the competitive inhibition experiment were measured using BCA Protein Assay Reagent (manufactured by PIERCE).

[0621] An EPHA2 extracellular region polypeptide (manufactured by R&D Systems, Inc., #3035-A2-100) was diluted to 0.5 μg/ml with PBS, then dispensed at 100 μl/well onto an immunoplate (manufactured by Nunc, #442404), and incubated overnight at 4° C. to thereby adsorb the protein onto the plate. On the next day, the wells were washed once with Diluting Buffer (PBS, 0.05% (v/v) Tween 20). Then, a Block Ace solution (one pouch of Block Ace powder was dissolved in 100 ml of ultrapure water) diluted 4 times with PBS was dispensed at 200 μl/well and incubated at room temperature for 4 hours. The solution in the wells was removed. Then, mixed solutions of the biotinylated antibodies (5 μg/ml) and various concentrations (0 μg/ml, 1 μg/ml, 5 μg/ml, 25 μg/ml, 50 μg/ml, and 125 μg/ml) of the unlabeled antibodies (solvent: PBS containing 0.05% (v/v) (final concentration) Tween 20) were separately dispensed at 100 μl/well and incubated at room temperature for 1 hour. The wells were washed twice with Diluting Buffer (PBS, 0.05% (v/v) Tween 20). Then, Streptavidin-horseradish Peroxidase Conjugate (manufactured by GE Healthcare Bio-Sciences Corp., #RPN1231V) diluted 500 times with Diluting Buffer was added at 100 μl/well and incubated at room temperature for 1 hour. The solution in the wells was removed, and the wells were washed twice with Diluting Buffer. Then, a color reaction was performed with stirring by the addition of OPD Color Developing Solution at 100 μl/well. After color development, the color reaction was terminated by the addition of 1 M HCl at 100 μl/well. The absorbance at 490 nm was measured using a plate reader.

[0622] As a result, the absorbance of the wells supplemented with only bSH348-1 or bSH357-1 was 0.780±0.016 and 0.978±0.007 (mean±standard deviation (n=3)), respectively.

[0623] In the graphs of FIGS. 13A) and 13B), the absorbance is indicated in mean±standard deviation (n=3). The binding of SH348-1 or SH357-1 to EPHA2 was not inhibited by Ab96-1 differing in epitope therefrom. On the other hand, the binding of SH348-1 to EPHA2 was shown to be inhibited by the antibody SH348-1 itself or its humanized antibodies hSH348-1-T1 and hSH348-1-T3 (FIG. 13A). Likewise the binding of SH357-1 to EPHA2 was shown to be inhibited by the antibody SH357-1 itself or its humanized antibodies hSH357-1-T1 and hSH357-1-T3 (FIG. 13B).

Example 14

Inhibitory Effect of Humanized Anti-EPHA2 Antibody on Ephrin-A1-Dependent Phosphorylation of EPHA2 Tyrosine Residues

[0624] The ability of the humanized anti-EPHA2 antibody to inhibit ephrin-A1-dependent phosphorylation of EPHA2 tyrosine residues was examined according to the method described in Example 8. As a result, all the antibodies hSH348-1-T1, hSH348-1-T3, hSH357-1-T1, and hSH357-1-T3 were shown to maintain an activity of inhibiting Ephrin-A1/Fc-induced phosphorylation of EPHA2 tyrosine residues (FIG. 14).

INDUSTRIAL APPLICABILITY

[0625] An anti-EPHA2 antibody of the present invention has an antitumor activity. A pharmaceutical composition comprising the anti-EPHA2 antibody can be used as an anticancer agent.

[0626] While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Sequence CWU 1

1

16312931DNAHomo sapiensCDS(1)..(2931) 1atg gag ctc cag gca gcc cgc gcc tgc ttc gcc ctg ctg tgg ggc tgt 48Met Glu Leu Gln Ala Ala Arg Ala Cys Phe Ala Leu Leu Trp Gly Cys 1 5 10 15 gcg ctg gcc gcg gcc gcg gcg gcg cag ggc aag gaa gtg gta ctg ctg 96Ala Leu Ala Ala Ala Ala Ala Ala Gln Gly Lys Glu Val Val Leu Leu 20 25 30 gac ttt gct gca gct gga ggg gag ctc ggc tgg ctc aca cac ccg tat 144Asp Phe Ala Ala Ala Gly Gly Glu Leu Gly Trp Leu Thr His Pro Tyr 35 40 45 ggc aaa ggg tgg gac ctg atg cag aac atc atg aat gac atg ccg atc 192Gly Lys Gly Trp Asp Leu Met Gln Asn Ile Met Asn Asp Met Pro Ile 50 55 60 tac atg tac tcc gtg tgc aac gtg atg tct ggc gac cag gac aac tgg 240Tyr Met Tyr Ser Val Cys Asn Val Met Ser Gly Asp Gln Asp Asn Trp 65 70 75 80 ctc cgc acc aac tgg gtg tac cga gga gag gct gag cgt atc ttc att 288Leu Arg Thr Asn Trp Val Tyr Arg Gly Glu Ala Glu Arg Ile Phe Ile 85 90 95 gag ctc aag ttt act gta cgt gac tgc aac agc ttc cct ggt ggc gcc 336Glu Leu Lys Phe Thr Val Arg Asp Cys Asn Ser Phe Pro Gly Gly Ala 100 105 110 agc tcc tgc aag gag act ttc aac ctc tac tat gcc gag tcg gac ctg 384Ser Ser Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Ala Glu Ser Asp Leu 115 120 125 gac tac ggc acc aac ttc cag aag cgc ctg ttc acc aag att gac acc 432Asp Tyr Gly Thr Asn Phe Gln Lys Arg Leu Phe Thr Lys Ile Asp Thr 130 135 140 att gcg ccc gat gag atc acc gtc agc agc gac ttc gag gca cgc cac 480Ile Ala Pro Asp Glu Ile Thr Val Ser Ser Asp Phe Glu Ala Arg His 145 150 155 160 gtg aag ctg aac gtg gag gag cgc tcc gtg ggg ccg ctc acc cgc aaa 528Val Lys Leu Asn Val Glu Glu Arg Ser Val Gly Pro Leu Thr Arg Lys 165 170 175 ggc ttc tac ctg gcc ttc cag gat atc ggt gcc tgt gtg gcg ctg ctc 576Gly Phe Tyr Leu Ala Phe Gln Asp Ile Gly Ala Cys Val Ala Leu Leu 180 185 190 tcc gtc cgt gtc tac tac aag aag tgc ccc gag ctg ctg cag ggc ctg 624Ser Val Arg Val Tyr Tyr Lys Lys Cys Pro Glu Leu Leu Gln Gly Leu 195 200 205 gcc cac ttc cct gag acc atc gcc ggc tct gat gca cct tcc ctg gcc 672Ala His Phe Pro Glu Thr Ile Ala Gly Ser Asp Ala Pro Ser Leu Ala 210 215 220 act gtg gcc ggc acc tgt gtg gac cat gcc gtg gtg cca ccg ggg ggt 720Thr Val Ala Gly Thr Cys Val Asp His Ala Val Val Pro Pro Gly Gly 225 230 235 240 gaa gag ccc cgt atg cac tgt gca gtg gat ggc gag tgg ctg gtg ccc 768Glu Glu Pro Arg Met His Cys Ala Val Asp Gly Glu Trp Leu Val Pro 245 250 255 att ggg cag tgc ctg tgc cag gca ggc tac gag aag gtg gag gat gcc 816Ile Gly Gln Cys Leu Cys Gln Ala Gly Tyr Glu Lys Val Glu Asp Ala 260 265 270 tgc cag gcc tgc tcg cct gga ttt ttt aag ttt gag gca tct gag agc 864Cys Gln Ala Cys Ser Pro Gly Phe Phe Lys Phe Glu Ala Ser Glu Ser 275 280 285 ccc tgc ttg gag tgc cct gag cac acg ctg cca tcc cct gag ggt gcc 912Pro Cys Leu Glu Cys Pro Glu His Thr Leu Pro Ser Pro Glu Gly Ala 290 295 300 acc tcc tgc gag tgt gag gaa ggc ttc ttc cgg gca cct cag gac cca 960Thr Ser Cys Glu Cys Glu Glu Gly Phe Phe Arg Ala Pro Gln Asp Pro 305 310 315 320 gcg tcg atg cct tgc aca cga ccc ccc tcc gcc cca cac tac ctc aca 1008Ala Ser Met Pro Cys Thr Arg Pro Pro Ser Ala Pro His Tyr Leu Thr 325 330 335 gcc gtg ggc atg ggt gcc aag gtg gag ctg cgc tgg acg ccc cct cag 1056Ala Val Gly Met Gly Ala Lys Val Glu Leu Arg Trp Thr Pro Pro Gln 340 345 350 gac agc ggg ggc cgc gag gac att gtc tac agc gtc acc tgc gaa cag 1104Asp Ser Gly Gly Arg Glu Asp Ile Val Tyr Ser Val Thr Cys Glu Gln 355 360 365 tgc tgg ccc gag tct ggg gaa tgc ggg ccg tgt gag gcc agt gtg cgc 1152Cys Trp Pro Glu Ser Gly Glu Cys Gly Pro Cys Glu Ala Ser Val Arg 370 375 380 tac tcg gag cct cct cac gga ctg acc cgc acc agt gtg aca gtg agc 1200Tyr Ser Glu Pro Pro His Gly Leu Thr Arg Thr Ser Val Thr Val Ser 385 390 395 400 gac ctg gag ccc cac atg aac tac acc ttc acc gtg gag gcc cgc aat 1248Asp Leu Glu Pro His Met Asn Tyr Thr Phe Thr Val Glu Ala Arg Asn 405 410 415 ggc gtc tca ggc ctg gta acc agc cgc agc ttc cgt act gcc agt gtc 1296Gly Val Ser Gly Leu Val Thr Ser Arg Ser Phe Arg Thr Ala Ser Val 420 425 430 agc atc aac cag aca gag ccc ccc aag gtg agg ctg gag ggc cgc agc 1344Ser Ile Asn Gln Thr Glu Pro Pro Lys Val Arg Leu Glu Gly Arg Ser 435 440 445 acc acc tcg ctt agc gtc tcc tgg agc atc ccc ccg ccg cag cag agc 1392Thr Thr Ser Leu Ser Val Ser Trp Ser Ile Pro Pro Pro Gln Gln Ser 450 455 460 cga gtg tgg aag tac gag gtc act tac cgc aag aag gga gac tcc aac 1440Arg Val Trp Lys Tyr Glu Val Thr Tyr Arg Lys Lys Gly Asp Ser Asn 465 470 475 480 agc tac aat gtg cgc cgc acc gag ggt ttc tcc gtg acc ctg gac gac 1488Ser Tyr Asn Val Arg Arg Thr Glu Gly Phe Ser Val Thr Leu Asp Asp 485 490 495 ctg gcc cca gac acc acc tac ctg gtc cag gtg cag gca ctg acg cag 1536Leu Ala Pro Asp Thr Thr Tyr Leu Val Gln Val Gln Ala Leu Thr Gln 500 505 510 gag ggc cag ggg gcc ggc agc aag gtg cac gaa ttc cag acg ctg tcc 1584Glu Gly Gln Gly Ala Gly Ser Lys Val His Glu Phe Gln Thr Leu Ser 515 520 525 ccg gag gga tct ggc aac ttg gcg gtg att ggc ggc gtg gct gtc ggt 1632Pro Glu Gly Ser Gly Asn Leu Ala Val Ile Gly Gly Val Ala Val Gly 530 535 540 gtg gtc ctg ctt ctg gtg ctg gca gga gtt ggc ttc ttt atc cac cgc 1680Val Val Leu Leu Leu Val Leu Ala Gly Val Gly Phe Phe Ile His Arg 545 550 555 560 agg agg aag aac cag cgt gcc cgc cag tcc ccg gag gac gtt tac ttc 1728Arg Arg Lys Asn Gln Arg Ala Arg Gln Ser Pro Glu Asp Val Tyr Phe 565 570 575 tcc aag tca gaa caa ctg aag ccc ctg aag aca tac gtg gac ccc cac 1776Ser Lys Ser Glu Gln Leu Lys Pro Leu Lys Thr Tyr Val Asp Pro His 580 585 590 aca tat gag gac ccc aac cag gct gtg ttg aag ttc act acc gag atc 1824Thr Tyr Glu Asp Pro Asn Gln Ala Val Leu Lys Phe Thr Thr Glu Ile 595 600 605 cat cca tcc tgt gtc act cgg cag aag gtg atc gga gca gga gag ttt 1872His Pro Ser Cys Val Thr Arg Gln Lys Val Ile Gly Ala Gly Glu Phe 610 615 620 ggg gag gtg tac aag ggc atg ctg aag aca tcc tcg ggg aag aag gag 1920Gly Glu Val Tyr Lys Gly Met Leu Lys Thr Ser Ser Gly Lys Lys Glu 625 630 635 640 gtg ccg gtg gcc atc aag acg ctg aaa gcc ggc tac aca gag aag cag 1968Val Pro Val Ala Ile Lys Thr Leu Lys Ala Gly Tyr Thr Glu Lys Gln 645 650 655 cga gtg gac ttc ctc ggc gag gcc ggc atc atg ggc cag ttc agc cac 2016Arg Val Asp Phe Leu Gly Glu Ala Gly Ile Met Gly Gln Phe Ser His 660 665 670 cac aac atc atc cgc cta gag ggc gtc atc tcc aaa tac aag ccc atg 2064His Asn Ile Ile Arg Leu Glu Gly Val Ile Ser Lys Tyr Lys Pro Met 675 680 685 atg atc atc act gag tac atg gag aat ggg gcc ctg gac aag ttc ctt 2112Met Ile Ile Thr Glu Tyr Met Glu Asn Gly Ala Leu Asp Lys Phe Leu 690 695 700 cgg gag aag gat ggc gag ttc agc gtg ctg cag ctg gtg ggc atg ctg 2160Arg Glu Lys Asp Gly Glu Phe Ser Val Leu Gln Leu Val Gly Met Leu 705 710 715 720 cgg ggc atc gca gct ggc atg aag tac ctg gcc aac atg aac tat gtg 2208Arg Gly Ile Ala Ala Gly Met Lys Tyr Leu Ala Asn Met Asn Tyr Val 725 730 735 cac cgt gac ctg gct gcc cgc aac atc ctc gtc aac agc aac ctg gtc 2256His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Asn Ser Asn Leu Val 740 745 750 tgc aag gtg tct gac ttt ggc ctg tcc cgc gtg ctg gag gac gac ccc 2304Cys Lys Val Ser Asp Phe Gly Leu Ser Arg Val Leu Glu Asp Asp Pro 755 760 765 gag gcc acc tac acc acc agt ggc ggc aag atc ccc atc cgc tgg acc 2352Glu Ala Thr Tyr Thr Thr Ser Gly Gly Lys Ile Pro Ile Arg Trp Thr 770 775 780 gcc ccg gag gcc att tcc tac cgg aag ttc acc tct gcc agc gac gtg 2400Ala Pro Glu Ala Ile Ser Tyr Arg Lys Phe Thr Ser Ala Ser Asp Val 785 790 795 800 tgg agc ttt ggc att gtc atg tgg gag gtg atg acc tat ggc gag cgg 2448Trp Ser Phe Gly Ile Val Met Trp Glu Val Met Thr Tyr Gly Glu Arg 805 810 815 ccc tac tgg gag ttg tcc aac cac gag gtg atg aaa gcc atc aat gat 2496Pro Tyr Trp Glu Leu Ser Asn His Glu Val Met Lys Ala Ile Asn Asp 820 825 830 ggc ttc cgg ctc ccc aca ccc atg gac tgc ccc tcc gcc atc tac cag 2544Gly Phe Arg Leu Pro Thr Pro Met Asp Cys Pro Ser Ala Ile Tyr Gln 835 840 845 ctc atg atg cag tgc tgg cag cag gag cgt gcc cgc cgc ccc aag ttc 2592Leu Met Met Gln Cys Trp Gln Gln Glu Arg Ala Arg Arg Pro Lys Phe 850 855 860 gct gac atc gtc agc atc ctg gac aag ctc att cgt gcc cct gac tcc 2640Ala Asp Ile Val Ser Ile Leu Asp Lys Leu Ile Arg Ala Pro Asp Ser 865 870 875 880 ctc aag acc ctg gct gac ttt gac ccc cgc gtg tct atc cgg ctc ccc 2688Leu Lys Thr Leu Ala Asp Phe Asp Pro Arg Val Ser Ile Arg Leu Pro 885 890 895 agc acg agc ggc tcg gag ggg gtg ccc ttc cgc acg gtg tcc gag tgg 2736Ser Thr Ser Gly Ser Glu Gly Val Pro Phe Arg Thr Val Ser Glu Trp 900 905 910 ctg gag tcc atc aag atg cag cag tat acg gag cac ttc atg gcg gcc 2784Leu Glu Ser Ile Lys Met Gln Gln Tyr Thr Glu His Phe Met Ala Ala 915 920 925 ggc tac act gcc atc gag aag gtg gtg cag atg acc aac gac gac atc 2832Gly Tyr Thr Ala Ile Glu Lys Val Val Gln Met Thr Asn Asp Asp Ile 930 935 940 aag agg att ggg gtg cgg ctg ccc ggc cac cag aag cgc atc gcc tac 2880Lys Arg Ile Gly Val Arg Leu Pro Gly His Gln Lys Arg Ile Ala Tyr 945 950 955 960 agc ctg ctg gga ctc aag gac cag gtg aac act gtg ggg atc ccc atc 2928Ser Leu Leu Gly Leu Lys Asp Gln Val Asn Thr Val Gly Ile Pro Ile 965 970 975 tga 29312976PRTHomo sapiens 2Met Glu Leu Gln Ala Ala Arg Ala Cys Phe Ala Leu Leu Trp Gly Cys 1 5 10 15 Ala Leu Ala Ala Ala Ala Ala Ala Gln Gly Lys Glu Val Val Leu Leu 20 25 30 Asp Phe Ala Ala Ala Gly Gly Glu Leu Gly Trp Leu Thr His Pro Tyr 35 40 45 Gly Lys Gly Trp Asp Leu Met Gln Asn Ile Met Asn Asp Met Pro Ile 50 55 60 Tyr Met Tyr Ser Val Cys Asn Val Met Ser Gly Asp Gln Asp Asn Trp 65 70 75 80 Leu Arg Thr Asn Trp Val Tyr Arg Gly Glu Ala Glu Arg Ile Phe Ile 85 90 95 Glu Leu Lys Phe Thr Val Arg Asp Cys Asn Ser Phe Pro Gly Gly Ala 100 105 110 Ser Ser Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Ala Glu Ser Asp Leu 115 120 125 Asp Tyr Gly Thr Asn Phe Gln Lys Arg Leu Phe Thr Lys Ile Asp Thr 130 135 140 Ile Ala Pro Asp Glu Ile Thr Val Ser Ser Asp Phe Glu Ala Arg His 145 150 155 160 Val Lys Leu Asn Val Glu Glu Arg Ser Val Gly Pro Leu Thr Arg Lys 165 170 175 Gly Phe Tyr Leu Ala Phe Gln Asp Ile Gly Ala Cys Val Ala Leu Leu 180 185 190 Ser Val Arg Val Tyr Tyr Lys Lys Cys Pro Glu Leu Leu Gln Gly Leu 195 200 205 Ala His Phe Pro Glu Thr Ile Ala Gly Ser Asp Ala Pro Ser Leu Ala 210 215 220 Thr Val Ala Gly Thr Cys Val Asp His Ala Val Val Pro Pro Gly Gly 225 230 235 240 Glu Glu Pro Arg Met His Cys Ala Val Asp Gly Glu Trp Leu Val Pro 245 250 255 Ile Gly Gln Cys Leu Cys Gln Ala Gly Tyr Glu Lys Val Glu Asp Ala 260 265 270 Cys Gln Ala Cys Ser Pro Gly Phe Phe Lys Phe Glu Ala Ser Glu Ser 275 280 285 Pro Cys Leu Glu Cys Pro Glu His Thr Leu Pro Ser Pro Glu Gly Ala 290 295 300 Thr Ser Cys Glu Cys Glu Glu Gly Phe Phe Arg Ala Pro Gln Asp Pro 305 310 315 320 Ala Ser Met Pro Cys Thr Arg Pro Pro Ser Ala Pro His Tyr Leu Thr 325 330 335 Ala Val Gly Met Gly Ala Lys Val Glu Leu Arg Trp Thr Pro Pro Gln 340 345 350 Asp Ser Gly Gly Arg Glu Asp Ile Val Tyr Ser Val Thr Cys Glu Gln 355 360 365 Cys Trp Pro Glu Ser Gly Glu Cys Gly Pro Cys Glu Ala Ser Val Arg 370 375 380 Tyr Ser Glu Pro Pro His Gly Leu Thr Arg Thr Ser Val Thr Val Ser 385 390 395 400 Asp Leu Glu Pro His Met Asn Tyr Thr Phe Thr Val Glu Ala Arg Asn 405 410 415 Gly Val Ser Gly Leu Val Thr Ser Arg Ser Phe Arg Thr Ala Ser Val 420 425 430 Ser Ile Asn Gln Thr Glu Pro Pro Lys Val Arg Leu Glu Gly Arg Ser 435 440 445 Thr Thr Ser Leu Ser Val Ser Trp Ser Ile Pro Pro Pro Gln Gln Ser 450 455 460 Arg Val Trp Lys Tyr Glu Val Thr Tyr Arg Lys Lys Gly Asp Ser Asn 465 470 475 480 Ser Tyr Asn Val Arg Arg Thr Glu Gly Phe Ser Val Thr Leu Asp Asp 485 490 495 Leu Ala Pro Asp Thr Thr Tyr Leu Val Gln Val Gln Ala Leu Thr Gln 500 505 510 Glu Gly Gln Gly Ala Gly Ser Lys Val His Glu Phe Gln Thr Leu Ser 515 520 525 Pro Glu Gly Ser Gly Asn Leu Ala Val Ile Gly Gly Val Ala Val Gly 530 535 540 Val Val Leu Leu Leu Val Leu Ala Gly Val Gly Phe Phe Ile His Arg 545 550 555 560 Arg Arg Lys Asn Gln Arg Ala Arg Gln Ser Pro Glu Asp Val Tyr Phe 565 570 575 Ser Lys Ser Glu Gln Leu Lys Pro Leu Lys Thr Tyr Val Asp Pro His 580 585 590 Thr Tyr Glu Asp Pro Asn Gln Ala Val Leu Lys Phe Thr Thr Glu Ile 595 600 605 His Pro Ser Cys Val Thr Arg Gln Lys Val Ile Gly Ala Gly Glu Phe 610 615 620 Gly Glu Val Tyr Lys Gly Met Leu Lys Thr Ser Ser Gly Lys Lys Glu 625 630 635 640 Val Pro Val Ala Ile Lys Thr Leu Lys Ala Gly Tyr Thr Glu Lys Gln 645 650 655 Arg Val Asp

Phe Leu Gly Glu Ala Gly Ile Met Gly Gln Phe Ser His 660 665 670 His Asn Ile Ile Arg Leu Glu Gly Val Ile Ser Lys Tyr Lys Pro Met 675 680 685 Met Ile Ile Thr Glu Tyr Met Glu Asn Gly Ala Leu Asp Lys Phe Leu 690 695 700 Arg Glu Lys Asp Gly Glu Phe Ser Val Leu Gln Leu Val Gly Met Leu 705 710 715 720 Arg Gly Ile Ala Ala Gly Met Lys Tyr Leu Ala Asn Met Asn Tyr Val 725 730 735 His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Asn Ser Asn Leu Val 740 745 750 Cys Lys Val Ser Asp Phe Gly Leu Ser Arg Val Leu Glu Asp Asp Pro 755 760 765 Glu Ala Thr Tyr Thr Thr Ser Gly Gly Lys Ile Pro Ile Arg Trp Thr 770 775 780 Ala Pro Glu Ala Ile Ser Tyr Arg Lys Phe Thr Ser Ala Ser Asp Val 785 790 795 800 Trp Ser Phe Gly Ile Val Met Trp Glu Val Met Thr Tyr Gly Glu Arg 805 810 815 Pro Tyr Trp Glu Leu Ser Asn His Glu Val Met Lys Ala Ile Asn Asp 820 825 830 Gly Phe Arg Leu Pro Thr Pro Met Asp Cys Pro Ser Ala Ile Tyr Gln 835 840 845 Leu Met Met Gln Cys Trp Gln Gln Glu Arg Ala Arg Arg Pro Lys Phe 850 855 860 Ala Asp Ile Val Ser Ile Leu Asp Lys Leu Ile Arg Ala Pro Asp Ser 865 870 875 880 Leu Lys Thr Leu Ala Asp Phe Asp Pro Arg Val Ser Ile Arg Leu Pro 885 890 895 Ser Thr Ser Gly Ser Glu Gly Val Pro Phe Arg Thr Val Ser Glu Trp 900 905 910 Leu Glu Ser Ile Lys Met Gln Gln Tyr Thr Glu His Phe Met Ala Ala 915 920 925 Gly Tyr Thr Ala Ile Glu Lys Val Val Gln Met Thr Asn Asp Asp Ile 930 935 940 Lys Arg Ile Gly Val Arg Leu Pro Gly His Gln Lys Arg Ile Ala Tyr 945 950 955 960 Ser Leu Leu Gly Leu Lys Asp Gln Val Asn Thr Val Gly Ile Pro Ile 965 970 975 355DNAArtificial SequencePCR primer 1 3ggggacaagt ttgtacaaaa aagcaggctt cggggatcgg accgagagcg agaag 55466DNAArtificial SequencePCR primer 2 4ggggaccact ttgtacaaga aagctgggtc ctagatgggg atccccacag tgttcacctg 60gtcctt 66529DNAArtificial SequencePrimer 3 5ctgtggggat ccccatcgac ccagctttc 29630DNAArtificial SequencePrimer 4 6gatggggatc cccacagtgt tcacctggtc 3072961DNAArtificial SequenceEPHA2 mutated sequence 7tcggggatcg gaccgagagc gagaagcgcg gc atg gag ctc cag gca gcc cgc 53 Met Glu Leu Gln Ala Ala Arg 1 5 gcc tgc ttc gcc ctg ctg tgg ggc tgt gcg ctg gcc gcg gcc gcg gcg 101Ala Cys Phe Ala Leu Leu Trp Gly Cys Ala Leu Ala Ala Ala Ala Ala 10 15 20 gcg cag ggc aag gaa gtg gta ctg ctg gac ttt gct gca gct gga ggg 149Ala Gln Gly Lys Glu Val Val Leu Leu Asp Phe Ala Ala Ala Gly Gly 25 30 35 gag ctc ggc tgg ctc aca cac ccg tat ggc aaa ggg tgg gac ctg atg 197Glu Leu Gly Trp Leu Thr His Pro Tyr Gly Lys Gly Trp Asp Leu Met 40 45 50 55 cag aac atc atg aat gac atg ccg atc tac atg tac tcc gtg tgc aac 245Gln Asn Ile Met Asn Asp Met Pro Ile Tyr Met Tyr Ser Val Cys Asn 60 65 70 gtg atg tct ggc gac cag gac aac tgg ctc cgc acc aac tgg gtg tac 293Val Met Ser Gly Asp Gln Asp Asn Trp Leu Arg Thr Asn Trp Val Tyr 75 80 85 cga gga gag gct gag cgt atc ttc att gag ctc aag ttt act gta cgt 341Arg Gly Glu Ala Glu Arg Ile Phe Ile Glu Leu Lys Phe Thr Val Arg 90 95 100 gac tgc aac agc ttc cct ggt ggc gcc agc tcc tgc aag gag act ttc 389Asp Cys Asn Ser Phe Pro Gly Gly Ala Ser Ser Cys Lys Glu Thr Phe 105 110 115 aac ctc tac tat gcc gag tcg gac ctg gac tac ggc acc aac ttc cag 437Asn Leu Tyr Tyr Ala Glu Ser Asp Leu Asp Tyr Gly Thr Asn Phe Gln 120 125 130 135 aag cgc ctg ttc acc aag att gac acc att gcg ccc gat gag atc acc 485Lys Arg Leu Phe Thr Lys Ile Asp Thr Ile Ala Pro Asp Glu Ile Thr 140 145 150 gtc agc agc gac ttc gag gca cgc cac gtg aag ctg aac gtg gag gag 533Val Ser Ser Asp Phe Glu Ala Arg His Val Lys Leu Asn Val Glu Glu 155 160 165 cgc tcc gtg ggg ccg ctc acc cgc aaa ggc ttc tac ctg gcc ttc cag 581Arg Ser Val Gly Pro Leu Thr Arg Lys Gly Phe Tyr Leu Ala Phe Gln 170 175 180 gat atc ggt gcc tgt gtg gca cta ctc tcc gtc cgt gtc tac tac aag 629Asp Ile Gly Ala Cys Val Ala Leu Leu Ser Val Arg Val Tyr Tyr Lys 185 190 195 aag tgc ccc gag ctg ctg cag ggc ctg gcc cac ttc cct gag acc atc 677Lys Cys Pro Glu Leu Leu Gln Gly Leu Ala His Phe Pro Glu Thr Ile 200 205 210 215 gcc ggc tct gat gca cct tcc ctg gcc act gtg gcc ggc acc tgt gtg 725Ala Gly Ser Asp Ala Pro Ser Leu Ala Thr Val Ala Gly Thr Cys Val 220 225 230 gac cat gcc gtg gtg cca ccg ggg ggt gaa gag ccc cgt atg cac tgt 773Asp His Ala Val Val Pro Pro Gly Gly Glu Glu Pro Arg Met His Cys 235 240 245 gca gtg gat ggc gag tgg ctg gtg ccc att ggg cag tgc ctg tgc cag 821Ala Val Asp Gly Glu Trp Leu Val Pro Ile Gly Gln Cys Leu Cys Gln 250 255 260 gca ggc tac gag aag gtg gag gat gcc tgc cag gcc tgc tcg cct gga 869Ala Gly Tyr Glu Lys Val Glu Asp Ala Cys Gln Ala Cys Ser Pro Gly 265 270 275 ttt ttt aag ttt gag gca tct gag agc ccc tgc ttg gag tgc cct gag 917Phe Phe Lys Phe Glu Ala Ser Glu Ser Pro Cys Leu Glu Cys Pro Glu 280 285 290 295 cac acg ctg cca tcc cct gag ggt gcc acc tcc tgc gag tgt gag gaa 965His Thr Leu Pro Ser Pro Glu Gly Ala Thr Ser Cys Glu Cys Glu Glu 300 305 310 ggc ttc ttc cgg gca cct cag gac cca gcg tcg atg cct tgc aca cga 1013Gly Phe Phe Arg Ala Pro Gln Asp Pro Ala Ser Met Pro Cys Thr Arg 315 320 325 ccc cct tcc gcc cca cac tac ctc aca gcc gtg ggc atg ggt gcc aag 1061Pro Pro Ser Ala Pro His Tyr Leu Thr Ala Val Gly Met Gly Ala Lys 330 335 340 gtg gag ctg cgc tgg acg ccc cct cag gac agc ggg ggc cgc gag gac 1109Val Glu Leu Arg Trp Thr Pro Pro Gln Asp Ser Gly Gly Arg Glu Asp 345 350 355 att gtc tac agc gtc acc tgc gaa cag tgc tgg ccc gag tct ggg gaa 1157Ile Val Tyr Ser Val Thr Cys Glu Gln Cys Trp Pro Glu Ser Gly Glu 360 365 370 375 tgc ggg ccg tgt gag gcc agt gtg cgc tac tcg gag cct cct cac gga 1205Cys Gly Pro Cys Glu Ala Ser Val Arg Tyr Ser Glu Pro Pro His Gly 380 385 390 ctg acc cgc acc agt gtg aca gtg agc gac ctg gag ccc cac atg aac 1253Leu Thr Arg Thr Ser Val Thr Val Ser Asp Leu Glu Pro His Met Asn 395 400 405 tac acc ttc acc gtg gag gcc cgc aat ggc gtc tca ggc ctg gta acc 1301Tyr Thr Phe Thr Val Glu Ala Arg Asn Gly Val Ser Gly Leu Val Thr 410 415 420 agc cgc agc ttc cgt act gcc agt gtc agc atc aac cag aca gag ccc 1349Ser Arg Ser Phe Arg Thr Ala Ser Val Ser Ile Asn Gln Thr Glu Pro 425 430 435 ccc aag gtg agg ctg gag ggc cgc agc acc acc tcg ctt agc gtc tcc 1397Pro Lys Val Arg Leu Glu Gly Arg Ser Thr Thr Ser Leu Ser Val Ser 440 445 450 455 tgg agc atc ccc ccg ccg cag cag agc cga gtg tgg aag tac gag gtc 1445Trp Ser Ile Pro Pro Pro Gln Gln Ser Arg Val Trp Lys Tyr Glu Val 460 465 470 act tac cgc aag aag gga gac tcc aac agc tac aat gtg cgc cgc acc 1493Thr Tyr Arg Lys Lys Gly Asp Ser Asn Ser Tyr Asn Val Arg Arg Thr 475 480 485 gag ggt ttc tcc gtg acc ctg gac gac ctg gcc cca gac acc acc tac 1541Glu Gly Phe Ser Val Thr Leu Asp Asp Leu Ala Pro Asp Thr Thr Tyr 490 495 500 ctg gtc cag gtg cag gca ctg acg cag gag ggc cag ggg gcc ggc agc 1589Leu Val Gln Val Gln Ala Leu Thr Gln Glu Gly Gln Gly Ala Gly Ser 505 510 515 aag gtg cac gaa ttc cag acg ctg tcc ccg gag gga tct ggc aac ttg 1637Lys Val His Glu Phe Gln Thr Leu Ser Pro Glu Gly Ser Gly Asn Leu 520 525 530 535 gcg gtg att ggc ggc gtg gct gtc ggt gtg gtc ctg ctt ctg gtg ctg 1685Ala Val Ile Gly Gly Val Ala Val Gly Val Val Leu Leu Leu Val Leu 540 545 550 gca gga gtt ggc ttc ttt atc cac cgc agg agg aag aac cag cgt gcc 1733Ala Gly Val Gly Phe Phe Ile His Arg Arg Arg Lys Asn Gln Arg Ala 555 560 565 cgc cag tcc ccg gag gac gtt tac ttc tcc aag tca gaa caa ctg aag 1781Arg Gln Ser Pro Glu Asp Val Tyr Phe Ser Lys Ser Glu Gln Leu Lys 570 575 580 ccc ctg aag aca tac gtg gac ccc cac aca tat gag gac ccc aac cag 1829Pro Leu Lys Thr Tyr Val Asp Pro His Thr Tyr Glu Asp Pro Asn Gln 585 590 595 gct gtg ttg aag ttc act acc gag atc cat cca tcc tgt gtc act cgg 1877Ala Val Leu Lys Phe Thr Thr Glu Ile His Pro Ser Cys Val Thr Arg 600 605 610 615 cag aag gtg atc gga gca gga gag ttt ggg gag gtg tac aag ggc atg 1925Gln Lys Val Ile Gly Ala Gly Glu Phe Gly Glu Val Tyr Lys Gly Met 620 625 630 ctg aag aca tcc tcg ggg aag aag gag gtg ccg gtg gcc atc aag acg 1973Leu Lys Thr Ser Ser Gly Lys Lys Glu Val Pro Val Ala Ile Lys Thr 635 640 645 ctg aaa gcc ggc tac aca gag aag cag cga gtg gac ttc ctc ggc gag 2021Leu Lys Ala Gly Tyr Thr Glu Lys Gln Arg Val Asp Phe Leu Gly Glu 650 655 660 gcc ggc atc atg ggc cag ttc agc cac cac aac atc atc cgc cta gag 2069Ala Gly Ile Met Gly Gln Phe Ser His His Asn Ile Ile Arg Leu Glu 665 670 675 ggc gtc atc tcc aaa tac aag ccc atg atg atc atc act gag tac atg 2117Gly Val Ile Ser Lys Tyr Lys Pro Met Met Ile Ile Thr Glu Tyr Met 680 685 690 695 gag aat ggg gcc ctg gac aag ttc ctt cgg gag aag gat ggc gag ttc 2165Glu Asn Gly Ala Leu Asp Lys Phe Leu Arg Glu Lys Asp Gly Glu Phe 700 705 710 agc gtg ctg cag ctg gtg ggc atg ctg cgg ggc atc gca gct ggc atg 2213Ser Val Leu Gln Leu Val Gly Met Leu Arg Gly Ile Ala Ala Gly Met 715 720 725 aag tac ctg gcc aac atg aac tat gtg cac cgt gac ctg gct gcc cgc 2261Lys Tyr Leu Ala Asn Met Asn Tyr Val His Arg Asp Leu Ala Ala Arg 730 735 740 aac atc ctc gtc aac agc aac ctg gtc tgc aag gtg tct gac ttt ggc 2309Asn Ile Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly 745 750 755 ctg tcc cgc gtg ctg gag gac gac ccc gag gcc acc tac acc acc agt 2357Leu Ser Arg Val Leu Glu Asp Asp Pro Glu Ala Thr Tyr Thr Thr Ser 760 765 770 775 ggc ggc aag atc ccc atc cgc tgg acc gcc ccg gag gcc att tcc tac 2405Gly Gly Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile Ser Tyr 780 785 790 cgg aag ttc acc tct gcc agc gac gtg tgg agc ttt ggc att gtc atg 2453Arg Lys Phe Thr Ser Ala Ser Asp Val Trp Ser Phe Gly Ile Val Met 795 800 805 tgg gag gtg atg acc tat ggc gag cgg ccc tac tgg gag ttg tcc aac 2501Trp Glu Val Met Thr Tyr Gly Glu Arg Pro Tyr Trp Glu Leu Ser Asn 810 815 820 cac gag gtg atg aaa gcc atc aat gat ggc ttc cgg ctc ccc aca ccc 2549His Glu Val Met Lys Ala Ile Asn Asp Gly Phe Arg Leu Pro Thr Pro 825 830 835 atg gac tgc ccc tcc gcc atc tac cag ctc atg atg cag tgc tgg cag 2597Met Asp Cys Pro Ser Ala Ile Tyr Gln Leu Met Met Gln Cys Trp Gln 840 845 850 855 cag gag cgt gcc cgc cgc ccc aag ttc gct gac atc gtc agc atc ctg 2645Gln Glu Arg Ala Arg Arg Pro Lys Phe Ala Asp Ile Val Ser Ile Leu 860 865 870 gac aag ctc att cgt gcc cct gac tcc ctc aag acc ctg gct gac ttt 2693Asp Lys Leu Ile Arg Ala Pro Asp Ser Leu Lys Thr Leu Ala Asp Phe 875 880 885 gac ccc cgc gtg tct atc cgg ctc ccc agc acg agc ggc tcg gag ggg 2741Asp Pro Arg Val Ser Ile Arg Leu Pro Ser Thr Ser Gly Ser Glu Gly 890 895 900 gtg ccc ttc cgc acg gtg tcc gag tgg ctg gag tcc atc aag atg cag 2789Val Pro Phe Arg Thr Val Ser Glu Trp Leu Glu Ser Ile Lys Met Gln 905 910 915 cag tat acg gag cac ttc atg gcg gcc ggc tac act gcc atc gag aag 2837Gln Tyr Thr Glu His Phe Met Ala Ala Gly Tyr Thr Ala Ile Glu Lys 920 925 930 935 gtg gtg cag atg acc aac gac gac atc aag agg att ggg gtg cgg ctg 2885Val Val Gln Met Thr Asn Asp Asp Ile Lys Arg Ile Gly Val Arg Leu 940 945 950 ccc ggc cac cag aag cgc atc gcc tac agc ctg ctg gga ctc aag gac 2933Pro Gly His Gln Lys Arg Ile Ala Tyr Ser Leu Leu Gly Leu Lys Asp 955 960 965 cag gtg aac act gtg ggg atc ccc atc g 2961Gln Val Asn Thr Val Gly Ile Pro Ile 970 975 8976PRTArtificial SequenceSynthetic Construct 8Met Glu Leu Gln Ala Ala Arg Ala Cys Phe Ala Leu Leu Trp Gly Cys 1 5 10 15 Ala Leu Ala Ala Ala Ala Ala Ala Gln Gly Lys Glu Val Val Leu Leu 20 25 30 Asp Phe Ala Ala Ala Gly Gly Glu Leu Gly Trp Leu Thr His Pro Tyr 35 40 45 Gly Lys Gly Trp Asp Leu Met Gln Asn Ile Met Asn Asp Met Pro Ile 50 55 60 Tyr Met Tyr Ser Val Cys Asn Val Met Ser Gly Asp Gln Asp Asn Trp 65 70 75 80 Leu Arg Thr Asn Trp Val Tyr Arg Gly Glu Ala Glu Arg Ile Phe Ile 85 90 95 Glu Leu Lys Phe Thr Val Arg Asp Cys Asn Ser Phe Pro Gly Gly Ala 100 105 110 Ser Ser Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Ala Glu Ser Asp Leu 115 120 125 Asp Tyr Gly Thr Asn Phe Gln Lys Arg Leu Phe Thr Lys Ile Asp Thr 130 135 140 Ile Ala Pro Asp Glu Ile Thr Val Ser Ser Asp Phe Glu Ala Arg His 145 150 155 160 Val Lys Leu Asn Val Glu Glu Arg Ser Val Gly Pro Leu Thr Arg Lys 165 170 175 Gly Phe Tyr Leu Ala Phe Gln Asp Ile Gly Ala Cys Val Ala Leu Leu 180 185 190 Ser Val Arg Val Tyr Tyr Lys Lys Cys Pro Glu Leu Leu Gln Gly Leu 195 200 205 Ala His Phe Pro Glu Thr Ile Ala Gly Ser Asp Ala Pro Ser Leu Ala 210 215 220 Thr Val Ala Gly Thr Cys Val Asp His Ala Val Val Pro Pro Gly Gly 225 230 235 240 Glu Glu Pro Arg Met His Cys Ala Val Asp Gly Glu Trp Leu Val Pro 245 250

255 Ile Gly Gln Cys Leu Cys Gln Ala Gly Tyr Glu Lys Val Glu Asp Ala 260 265 270 Cys Gln Ala Cys Ser Pro Gly Phe Phe Lys Phe Glu Ala Ser Glu Ser 275 280 285 Pro Cys Leu Glu Cys Pro Glu His Thr Leu Pro Ser Pro Glu Gly Ala 290 295 300 Thr Ser Cys Glu Cys Glu Glu Gly Phe Phe Arg Ala Pro Gln Asp Pro 305 310 315 320 Ala Ser Met Pro Cys Thr Arg Pro Pro Ser Ala Pro His Tyr Leu Thr 325 330 335 Ala Val Gly Met Gly Ala Lys Val Glu Leu Arg Trp Thr Pro Pro Gln 340 345 350 Asp Ser Gly Gly Arg Glu Asp Ile Val Tyr Ser Val Thr Cys Glu Gln 355 360 365 Cys Trp Pro Glu Ser Gly Glu Cys Gly Pro Cys Glu Ala Ser Val Arg 370 375 380 Tyr Ser Glu Pro Pro His Gly Leu Thr Arg Thr Ser Val Thr Val Ser 385 390 395 400 Asp Leu Glu Pro His Met Asn Tyr Thr Phe Thr Val Glu Ala Arg Asn 405 410 415 Gly Val Ser Gly Leu Val Thr Ser Arg Ser Phe Arg Thr Ala Ser Val 420 425 430 Ser Ile Asn Gln Thr Glu Pro Pro Lys Val Arg Leu Glu Gly Arg Ser 435 440 445 Thr Thr Ser Leu Ser Val Ser Trp Ser Ile Pro Pro Pro Gln Gln Ser 450 455 460 Arg Val Trp Lys Tyr Glu Val Thr Tyr Arg Lys Lys Gly Asp Ser Asn 465 470 475 480 Ser Tyr Asn Val Arg Arg Thr Glu Gly Phe Ser Val Thr Leu Asp Asp 485 490 495 Leu Ala Pro Asp Thr Thr Tyr Leu Val Gln Val Gln Ala Leu Thr Gln 500 505 510 Glu Gly Gln Gly Ala Gly Ser Lys Val His Glu Phe Gln Thr Leu Ser 515 520 525 Pro Glu Gly Ser Gly Asn Leu Ala Val Ile Gly Gly Val Ala Val Gly 530 535 540 Val Val Leu Leu Leu Val Leu Ala Gly Val Gly Phe Phe Ile His Arg 545 550 555 560 Arg Arg Lys Asn Gln Arg Ala Arg Gln Ser Pro Glu Asp Val Tyr Phe 565 570 575 Ser Lys Ser Glu Gln Leu Lys Pro Leu Lys Thr Tyr Val Asp Pro His 580 585 590 Thr Tyr Glu Asp Pro Asn Gln Ala Val Leu Lys Phe Thr Thr Glu Ile 595 600 605 His Pro Ser Cys Val Thr Arg Gln Lys Val Ile Gly Ala Gly Glu Phe 610 615 620 Gly Glu Val Tyr Lys Gly Met Leu Lys Thr Ser Ser Gly Lys Lys Glu 625 630 635 640 Val Pro Val Ala Ile Lys Thr Leu Lys Ala Gly Tyr Thr Glu Lys Gln 645 650 655 Arg Val Asp Phe Leu Gly Glu Ala Gly Ile Met Gly Gln Phe Ser His 660 665 670 His Asn Ile Ile Arg Leu Glu Gly Val Ile Ser Lys Tyr Lys Pro Met 675 680 685 Met Ile Ile Thr Glu Tyr Met Glu Asn Gly Ala Leu Asp Lys Phe Leu 690 695 700 Arg Glu Lys Asp Gly Glu Phe Ser Val Leu Gln Leu Val Gly Met Leu 705 710 715 720 Arg Gly Ile Ala Ala Gly Met Lys Tyr Leu Ala Asn Met Asn Tyr Val 725 730 735 His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Asn Ser Asn Leu Val 740 745 750 Cys Lys Val Ser Asp Phe Gly Leu Ser Arg Val Leu Glu Asp Asp Pro 755 760 765 Glu Ala Thr Tyr Thr Thr Ser Gly Gly Lys Ile Pro Ile Arg Trp Thr 770 775 780 Ala Pro Glu Ala Ile Ser Tyr Arg Lys Phe Thr Ser Ala Ser Asp Val 785 790 795 800 Trp Ser Phe Gly Ile Val Met Trp Glu Val Met Thr Tyr Gly Glu Arg 805 810 815 Pro Tyr Trp Glu Leu Ser Asn His Glu Val Met Lys Ala Ile Asn Asp 820 825 830 Gly Phe Arg Leu Pro Thr Pro Met Asp Cys Pro Ser Ala Ile Tyr Gln 835 840 845 Leu Met Met Gln Cys Trp Gln Gln Glu Arg Ala Arg Arg Pro Lys Phe 850 855 860 Ala Asp Ile Val Ser Ile Leu Asp Lys Leu Ile Arg Ala Pro Asp Ser 865 870 875 880 Leu Lys Thr Leu Ala Asp Phe Asp Pro Arg Val Ser Ile Arg Leu Pro 885 890 895 Ser Thr Ser Gly Ser Glu Gly Val Pro Phe Arg Thr Val Ser Glu Trp 900 905 910 Leu Glu Ser Ile Lys Met Gln Gln Tyr Thr Glu His Phe Met Ala Ala 915 920 925 Gly Tyr Thr Ala Ile Glu Lys Val Val Gln Met Thr Asn Asp Asp Ile 930 935 940 Lys Arg Ile Gly Val Arg Leu Pro Gly His Gln Lys Arg Ile Ala Tyr 945 950 955 960 Ser Leu Leu Gly Leu Lys Asp Gln Val Asn Thr Val Gly Ile Pro Ile 965 970 975 930DNAArtificial SequencePCR primer 5 9aaaaagctta tggagctcca ggcagcccgc 301030DNAArtificial SequencePCR primer 6 10aaagggccct cagttgccag atccctccgg 301143DNAArtificial SequencePCR primer 7 11gcaggcttca tcgaaggtcg tgggcgggca cctcaggacc cag 431240DNAArtificial SequencePCR primer 8 12gtacaagaaa gctgggtgct agccgccaat caccgccaag 401340DNAArtificial SequencePCR primer 9 13gtacaagaaa gctgggtgct aggcagtacg gaagctgcgg 401443DNAArtificial SequencePCR primer 10 14gcaggcttca tcgaaggtcg tgggagcttc cgtactgcca gtg 431546DNAArtificial SequencePCR primer 11 15ggggacaagt ttgtacaaaa aagcaggctt catcgaaggt cgtggg 461629DNAArtificial SequencePCR primer 12 16ggggaccact ttgtacaaga aagctgggt 291749DNAArtificial SequencePCR primer 13 17ggggacaagt ttgtacaaaa aagcaggctt cgccccggga agcgcagcc 491863DNAArtificial SequencePCR primer 14 18ggggaccact ttgtacaaga aagctgggtc ctaaacctcc acagactgaa tctggttcat 60ctg 63192961DNAHomo sapiensCDS(1)..(2961) 19atg gct ctg cgg agg ctg ggg gcc gcg ctg ctg ctg ctg ccg ctg ctc 48Met Ala Leu Arg Arg Leu Gly Ala Ala Leu Leu Leu Leu Pro Leu Leu 1 5 10 15 gcc gcc gtg gaa gaa acg cta atg gac tcc act aca gcg act gct gag 96Ala Ala Val Glu Glu Thr Leu Met Asp Ser Thr Thr Ala Thr Ala Glu 20 25 30 ctg ggc tgg atg gtg cat cct cca tca ggg tgg gaa gag gtg agt ggc 144Leu Gly Trp Met Val His Pro Pro Ser Gly Trp Glu Glu Val Ser Gly 35 40 45 tac gat gag aac atg aac acg atc cgc acg tac cag gtg tgc aac gtg 192Tyr Asp Glu Asn Met Asn Thr Ile Arg Thr Tyr Gln Val Cys Asn Val 50 55 60 ttt gag tca agc cag aac aac tgg cta cgg acc aag ttt atc cgg cgc 240Phe Glu Ser Ser Gln Asn Asn Trp Leu Arg Thr Lys Phe Ile Arg Arg 65 70 75 80 cgt ggc gcc cac cgc atc cac gtg gag atg aag ttt tcg gtg cgt gac 288Arg Gly Ala His Arg Ile His Val Glu Met Lys Phe Ser Val Arg Asp 85 90 95 tgc agc agc atc ccc agc gtg cct ggc tcc tgc aag gag acc ttc aac 336Cys Ser Ser Ile Pro Ser Val Pro Gly Ser Cys Lys Glu Thr Phe Asn 100 105 110 ctc tat tac tat gag gct gac ttt gac tcg gcc acc aag acc ttc ccc 384Leu Tyr Tyr Tyr Glu Ala Asp Phe Asp Ser Ala Thr Lys Thr Phe Pro 115 120 125 aac tgg atg gag aat cca tgg gtg aag gtg gat acc att gca gcc gac 432Asn Trp Met Glu Asn Pro Trp Val Lys Val Asp Thr Ile Ala Ala Asp 130 135 140 gag agc ttc tcc cag gtg gac ctg ggt ggc cgc gtc atg aaa atc aac 480Glu Ser Phe Ser Gln Val Asp Leu Gly Gly Arg Val Met Lys Ile Asn 145 150 155 160 acc gag gtg cgg agc ttc gga cct gtg tcc cgc agc ggc ttc tac ctg 528Thr Glu Val Arg Ser Phe Gly Pro Val Ser Arg Ser Gly Phe Tyr Leu 165 170 175 gcc ttc cag gac tat ggc ggc tgc atg tcc ctc atc gcc gtg cgt gtc 576Ala Phe Gln Asp Tyr Gly Gly Cys Met Ser Leu Ile Ala Val Arg Val 180 185 190 ttc tac cgc aag tgc ccc cgc atc atc cag aat ggc gcc atc ttc cag 624Phe Tyr Arg Lys Cys Pro Arg Ile Ile Gln Asn Gly Ala Ile Phe Gln 195 200 205 gaa acc ctg tcg ggg gct gag agc aca tcg ctg gtg gct gcc cgg ggc 672Glu Thr Leu Ser Gly Ala Glu Ser Thr Ser Leu Val Ala Ala Arg Gly 210 215 220 agc tgc atc gcc aat gcg gaa gag gtg gat gta ccc atc aag ctc tac 720Ser Cys Ile Ala Asn Ala Glu Glu Val Asp Val Pro Ile Lys Leu Tyr 225 230 235 240 tgt aac ggg gac ggc gag tgg ctg gtg ccc atc ggg cgc tgc atg tgc 768Cys Asn Gly Asp Gly Glu Trp Leu Val Pro Ile Gly Arg Cys Met Cys 245 250 255 aaa gca ggc ttc gag gcc gtt gag aat ggc acc gtc tgc cga ggt tgt 816Lys Ala Gly Phe Glu Ala Val Glu Asn Gly Thr Val Cys Arg Gly Cys 260 265 270 cca tct ggg act ttc aag gcc aac caa ggg gat gag gcc tgt acc cac 864Pro Ser Gly Thr Phe Lys Ala Asn Gln Gly Asp Glu Ala Cys Thr His 275 280 285 tgt ccc atc aac agc cgg acc act tct gaa ggg gcc acc aac tgt gtc 912Cys Pro Ile Asn Ser Arg Thr Thr Ser Glu Gly Ala Thr Asn Cys Val 290 295 300 tgc cgc aat ggc tac tac aga gca gac ctg gac ccc ctg gac atg ccc 960Cys Arg Asn Gly Tyr Tyr Arg Ala Asp Leu Asp Pro Leu Asp Met Pro 305 310 315 320 tgc aca acc atc ccc tcc gcg ccc cag gct gtg att tcc agt gtc aat 1008Cys Thr Thr Ile Pro Ser Ala Pro Gln Ala Val Ile Ser Ser Val Asn 325 330 335 gag acc tcc ctc atg ctg gag tgg acc cct ccc cgc gac tcc gga ggc 1056Glu Thr Ser Leu Met Leu Glu Trp Thr Pro Pro Arg Asp Ser Gly Gly 340 345 350 cga gag gac ctc gtc tac aac atc atc tgc aag agc tgt ggc tcg ggc 1104Arg Glu Asp Leu Val Tyr Asn Ile Ile Cys Lys Ser Cys Gly Ser Gly 355 360 365 cgg ggt gcc tgc acc cgc tgc ggg gac aat gta cag tac gca cca cgc 1152Arg Gly Ala Cys Thr Arg Cys Gly Asp Asn Val Gln Tyr Ala Pro Arg 370 375 380 cag cta ggc ctg acc gag cca cgc att tac atc agt gac ctg ctg gcc 1200Gln Leu Gly Leu Thr Glu Pro Arg Ile Tyr Ile Ser Asp Leu Leu Ala 385 390 395 400 cac acc cag tac acc ttc gag atc cag gct gtg aac ggc gtt act gac 1248His Thr Gln Tyr Thr Phe Glu Ile Gln Ala Val Asn Gly Val Thr Asp 405 410 415 cag agc ccc ttc tcg cct cag ttc gcc tct gtg aac atc acc acc aac 1296Gln Ser Pro Phe Ser Pro Gln Phe Ala Ser Val Asn Ile Thr Thr Asn 420 425 430 cag gca gct cca tcg gca gtg tcc atc atg cat cag gtg agc cgc acc 1344Gln Ala Ala Pro Ser Ala Val Ser Ile Met His Gln Val Ser Arg Thr 435 440 445 gtg gac agc att acc ctg tcg tgg tcc cag ccg gac cag ccc aat ggc 1392Val Asp Ser Ile Thr Leu Ser Trp Ser Gln Pro Asp Gln Pro Asn Gly 450 455 460 gtg atc ctg gac tat gag ctg cag tac tat gag aag gag ctc agt gag 1440Val Ile Leu Asp Tyr Glu Leu Gln Tyr Tyr Glu Lys Glu Leu Ser Glu 465 470 475 480 tac aac gcc aca gcc ata aaa agc ccc acc aac acg gtc acc gtg cag 1488Tyr Asn Ala Thr Ala Ile Lys Ser Pro Thr Asn Thr Val Thr Val Gln 485 490 495 ggc ctc aaa gcc ggc gcc atc tat gtc ttc cag gtg cgg gca cgc acc 1536Gly Leu Lys Ala Gly Ala Ile Tyr Val Phe Gln Val Arg Ala Arg Thr 500 505 510 gtg gca ggc tac ggg cgc tac agc ggc aag atg tac ttc cag acc atg 1584Val Ala Gly Tyr Gly Arg Tyr Ser Gly Lys Met Tyr Phe Gln Thr Met 515 520 525 aca gaa gcc gag tac cag aca agc atc cag gag aag ttg cca ctc atc 1632Thr Glu Ala Glu Tyr Gln Thr Ser Ile Gln Glu Lys Leu Pro Leu Ile 530 535 540 atc ggc tcc tcg gcc gct ggc ctg gtc ttc ctc att gct gtg gtt gtc 1680Ile Gly Ser Ser Ala Ala Gly Leu Val Phe Leu Ile Ala Val Val Val 545 550 555 560 atc gcc atc gtg tgt aac aga cgg ggg ttt gag cgt gct gac tcg gag 1728Ile Ala Ile Val Cys Asn Arg Arg Gly Phe Glu Arg Ala Asp Ser Glu 565 570 575 tac acg gac aag ctg caa cac tac acc agt ggc cac atg acc cca ggc 1776Tyr Thr Asp Lys Leu Gln His Tyr Thr Ser Gly His Met Thr Pro Gly 580 585 590 atg aag atc tac atc gat cct ttc acc tac gag gac ccc aac gag gca 1824Met Lys Ile Tyr Ile Asp Pro Phe Thr Tyr Glu Asp Pro Asn Glu Ala 595 600 605 gtg cgg gag ttt gcc aag gaa att gac atc tcc tgt gtc aaa att gag 1872Val Arg Glu Phe Ala Lys Glu Ile Asp Ile Ser Cys Val Lys Ile Glu 610 615 620 cag gtg atc gga gca ggg gag ttt ggc gag gtc tgc agt ggc cac ctg 1920Gln Val Ile Gly Ala Gly Glu Phe Gly Glu Val Cys Ser Gly His Leu 625 630 635 640 aag ctg cca ggc aag aga gag atc ttt gtg gcc atc aag acg ctc aag 1968Lys Leu Pro Gly Lys Arg Glu Ile Phe Val Ala Ile Lys Thr Leu Lys 645 650 655 tcg ggc tac acg gag aag cag cgc cgg gac ttc ctg agc gaa gcc tcc 2016Ser Gly Tyr Thr Glu Lys Gln Arg Arg Asp Phe Leu Ser Glu Ala Ser 660 665 670 atc atg ggc cag ttc gac cat ccc aac gtc atc cac ctg gag ggt gtc 2064Ile Met Gly Gln Phe Asp His Pro Asn Val Ile His Leu Glu Gly Val 675 680 685 gtg acc aag agc aca cct gtg atg atc atc acc gag ttc atg gag aat 2112Val Thr Lys Ser Thr Pro Val Met Ile Ile Thr Glu Phe Met Glu Asn 690 695 700 ggc tcc ctg gac tcc ttt ctc cgg caa aac gat ggg cag ttc aca gtc 2160Gly Ser Leu Asp Ser Phe Leu Arg Gln Asn Asp Gly Gln Phe Thr Val 705 710 715 720 atc cag ctg gtg ggc atg ctt cgg ggc atc gca gct ggc atg aag tac 2208Ile Gln Leu Val Gly Met Leu Arg Gly Ile Ala Ala Gly Met Lys Tyr 725 730 735 ctg gca gac atg aac tat gtt cac cgt gac ctg gct gcc cgc aac atc 2256Leu Ala Asp Met Asn Tyr Val His Arg Asp Leu Ala Ala Arg Asn Ile 740 745 750 ctc gtc aac agc aac ctg gtc tgc aag gtg tcg gac ttt ggg ctc tca 2304Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly Leu Ser 755 760 765 cgc ttt cta gag gac gat acc tca gac ccc acc tac acc agt gcc ctg 2352Arg Phe Leu Glu Asp Asp Thr Ser Asp Pro Thr Tyr Thr Ser Ala Leu 770 775 780 ggc gga aag atc ccc atc cgc tgg aca gcc ccg gaa gcc atc cag tac 2400Gly Gly Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile Gln Tyr 785 790 795 800 cgg aag ttc acc tcg gcc agt gat gtg tgg agc tac ggc att gtc atg 2448Arg Lys Phe Thr Ser Ala Ser Asp Val Trp Ser Tyr Gly Ile Val Met 805 810 815 tgg gag gtg atg tcc tat ggg gag cgg ccc tac tgg gac atg acc aac 2496Trp Glu Val Met Ser Tyr Gly Glu Arg Pro Tyr Trp Asp Met Thr Asn 820 825 830 cag gat gta atc aat gcc att gag cag gac tat cgg ctg cca ccg ccc 2544Gln Asp Val Ile Asn Ala Ile Glu Gln Asp Tyr Arg Leu Pro Pro Pro 835 840 845

atg gac tgc ccg agc gcc ctg cac caa ctc atg ctg gac tgt tgg cag 2592Met Asp Cys Pro Ser Ala Leu His Gln Leu Met Leu Asp Cys Trp Gln 850 855 860 aag gac cgc aac cac cgg ccc aag ttc ggc caa att gtc aac acg cta 2640Lys Asp Arg Asn His Arg Pro Lys Phe Gly Gln Ile Val Asn Thr Leu 865 870 875 880 gac aag atg atc cgc aat ccc aac agc ctc aaa gcc atg gcg ccc ctc 2688Asp Lys Met Ile Arg Asn Pro Asn Ser Leu Lys Ala Met Ala Pro Leu 885 890 895 tcc tct ggc atc aac ctg ccg ctg ctg gac cgc acg atc ccc gac tac 2736Ser Ser Gly Ile Asn Leu Pro Leu Leu Asp Arg Thr Ile Pro Asp Tyr 900 905 910 acc agc ttt aac acg gtg gac gag tgg ctg gag gcc atc aag atg ggg 2784Thr Ser Phe Asn Thr Val Asp Glu Trp Leu Glu Ala Ile Lys Met Gly 915 920 925 cag tac aag gag agc ttc gcc aat gcc ggc ttc acc tcc ttt gac gtc 2832Gln Tyr Lys Glu Ser Phe Ala Asn Ala Gly Phe Thr Ser Phe Asp Val 930 935 940 gtg tct cag atg atg atg gag gac att ctc cgg gtt ggg gtc act ttg 2880Val Ser Gln Met Met Met Glu Asp Ile Leu Arg Val Gly Val Thr Leu 945 950 955 960 gct ggc cac cag aaa aaa atc ctg aac agt atc cag gtg atg cgg gcg 2928Ala Gly His Gln Lys Lys Ile Leu Asn Ser Ile Gln Val Met Arg Ala 965 970 975 cag atg aac cag att cag tct gtg gag gtt tag 2961Gln Met Asn Gln Ile Gln Ser Val Glu Val 980 985 20986PRTHomo sapiens 20Met Ala Leu Arg Arg Leu Gly Ala Ala Leu Leu Leu Leu Pro Leu Leu 1 5 10 15 Ala Ala Val Glu Glu Thr Leu Met Asp Ser Thr Thr Ala Thr Ala Glu 20 25 30 Leu Gly Trp Met Val His Pro Pro Ser Gly Trp Glu Glu Val Ser Gly 35 40 45 Tyr Asp Glu Asn Met Asn Thr Ile Arg Thr Tyr Gln Val Cys Asn Val 50 55 60 Phe Glu Ser Ser Gln Asn Asn Trp Leu Arg Thr Lys Phe Ile Arg Arg 65 70 75 80 Arg Gly Ala His Arg Ile His Val Glu Met Lys Phe Ser Val Arg Asp 85 90 95 Cys Ser Ser Ile Pro Ser Val Pro Gly Ser Cys Lys Glu Thr Phe Asn 100 105 110 Leu Tyr Tyr Tyr Glu Ala Asp Phe Asp Ser Ala Thr Lys Thr Phe Pro 115 120 125 Asn Trp Met Glu Asn Pro Trp Val Lys Val Asp Thr Ile Ala Ala Asp 130 135 140 Glu Ser Phe Ser Gln Val Asp Leu Gly Gly Arg Val Met Lys Ile Asn 145 150 155 160 Thr Glu Val Arg Ser Phe Gly Pro Val Ser Arg Ser Gly Phe Tyr Leu 165 170 175 Ala Phe Gln Asp Tyr Gly Gly Cys Met Ser Leu Ile Ala Val Arg Val 180 185 190 Phe Tyr Arg Lys Cys Pro Arg Ile Ile Gln Asn Gly Ala Ile Phe Gln 195 200 205 Glu Thr Leu Ser Gly Ala Glu Ser Thr Ser Leu Val Ala Ala Arg Gly 210 215 220 Ser Cys Ile Ala Asn Ala Glu Glu Val Asp Val Pro Ile Lys Leu Tyr 225 230 235 240 Cys Asn Gly Asp Gly Glu Trp Leu Val Pro Ile Gly Arg Cys Met Cys 245 250 255 Lys Ala Gly Phe Glu Ala Val Glu Asn Gly Thr Val Cys Arg Gly Cys 260 265 270 Pro Ser Gly Thr Phe Lys Ala Asn Gln Gly Asp Glu Ala Cys Thr His 275 280 285 Cys Pro Ile Asn Ser Arg Thr Thr Ser Glu Gly Ala Thr Asn Cys Val 290 295 300 Cys Arg Asn Gly Tyr Tyr Arg Ala Asp Leu Asp Pro Leu Asp Met Pro 305 310 315 320 Cys Thr Thr Ile Pro Ser Ala Pro Gln Ala Val Ile Ser Ser Val Asn 325 330 335 Glu Thr Ser Leu Met Leu Glu Trp Thr Pro Pro Arg Asp Ser Gly Gly 340 345 350 Arg Glu Asp Leu Val Tyr Asn Ile Ile Cys Lys Ser Cys Gly Ser Gly 355 360 365 Arg Gly Ala Cys Thr Arg Cys Gly Asp Asn Val Gln Tyr Ala Pro Arg 370 375 380 Gln Leu Gly Leu Thr Glu Pro Arg Ile Tyr Ile Ser Asp Leu Leu Ala 385 390 395 400 His Thr Gln Tyr Thr Phe Glu Ile Gln Ala Val Asn Gly Val Thr Asp 405 410 415 Gln Ser Pro Phe Ser Pro Gln Phe Ala Ser Val Asn Ile Thr Thr Asn 420 425 430 Gln Ala Ala Pro Ser Ala Val Ser Ile Met His Gln Val Ser Arg Thr 435 440 445 Val Asp Ser Ile Thr Leu Ser Trp Ser Gln Pro Asp Gln Pro Asn Gly 450 455 460 Val Ile Leu Asp Tyr Glu Leu Gln Tyr Tyr Glu Lys Glu Leu Ser Glu 465 470 475 480 Tyr Asn Ala Thr Ala Ile Lys Ser Pro Thr Asn Thr Val Thr Val Gln 485 490 495 Gly Leu Lys Ala Gly Ala Ile Tyr Val Phe Gln Val Arg Ala Arg Thr 500 505 510 Val Ala Gly Tyr Gly Arg Tyr Ser Gly Lys Met Tyr Phe Gln Thr Met 515 520 525 Thr Glu Ala Glu Tyr Gln Thr Ser Ile Gln Glu Lys Leu Pro Leu Ile 530 535 540 Ile Gly Ser Ser Ala Ala Gly Leu Val Phe Leu Ile Ala Val Val Val 545 550 555 560 Ile Ala Ile Val Cys Asn Arg Arg Gly Phe Glu Arg Ala Asp Ser Glu 565 570 575 Tyr Thr Asp Lys Leu Gln His Tyr Thr Ser Gly His Met Thr Pro Gly 580 585 590 Met Lys Ile Tyr Ile Asp Pro Phe Thr Tyr Glu Asp Pro Asn Glu Ala 595 600 605 Val Arg Glu Phe Ala Lys Glu Ile Asp Ile Ser Cys Val Lys Ile Glu 610 615 620 Gln Val Ile Gly Ala Gly Glu Phe Gly Glu Val Cys Ser Gly His Leu 625 630 635 640 Lys Leu Pro Gly Lys Arg Glu Ile Phe Val Ala Ile Lys Thr Leu Lys 645 650 655 Ser Gly Tyr Thr Glu Lys Gln Arg Arg Asp Phe Leu Ser Glu Ala Ser 660 665 670 Ile Met Gly Gln Phe Asp His Pro Asn Val Ile His Leu Glu Gly Val 675 680 685 Val Thr Lys Ser Thr Pro Val Met Ile Ile Thr Glu Phe Met Glu Asn 690 695 700 Gly Ser Leu Asp Ser Phe Leu Arg Gln Asn Asp Gly Gln Phe Thr Val 705 710 715 720 Ile Gln Leu Val Gly Met Leu Arg Gly Ile Ala Ala Gly Met Lys Tyr 725 730 735 Leu Ala Asp Met Asn Tyr Val His Arg Asp Leu Ala Ala Arg Asn Ile 740 745 750 Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly Leu Ser 755 760 765 Arg Phe Leu Glu Asp Asp Thr Ser Asp Pro Thr Tyr Thr Ser Ala Leu 770 775 780 Gly Gly Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile Gln Tyr 785 790 795 800 Arg Lys Phe Thr Ser Ala Ser Asp Val Trp Ser Tyr Gly Ile Val Met 805 810 815 Trp Glu Val Met Ser Tyr Gly Glu Arg Pro Tyr Trp Asp Met Thr Asn 820 825 830 Gln Asp Val Ile Asn Ala Ile Glu Gln Asp Tyr Arg Leu Pro Pro Pro 835 840 845 Met Asp Cys Pro Ser Ala Leu His Gln Leu Met Leu Asp Cys Trp Gln 850 855 860 Lys Asp Arg Asn His Arg Pro Lys Phe Gly Gln Ile Val Asn Thr Leu 865 870 875 880 Asp Lys Met Ile Arg Asn Pro Asn Ser Leu Lys Ala Met Ala Pro Leu 885 890 895 Ser Ser Gly Ile Asn Leu Pro Leu Leu Asp Arg Thr Ile Pro Asp Tyr 900 905 910 Thr Ser Phe Asn Thr Val Asp Glu Trp Leu Glu Ala Ile Lys Met Gly 915 920 925 Gln Tyr Lys Glu Ser Phe Ala Asn Ala Gly Phe Thr Ser Phe Asp Val 930 935 940 Val Ser Gln Met Met Met Glu Asp Ile Leu Arg Val Gly Val Thr Leu 945 950 955 960 Ala Gly His Gln Lys Lys Ile Leu Asn Ser Ile Gln Val Met Arg Ala 965 970 975 Gln Met Asn Gln Ile Gln Ser Val Glu Val 980 985 2130DNAArtificial SequencePCR primer 15 21aaaaagctta tggctctgcg gaggctgggg 302230DNAArtificial SequencePCR primer 16 22aaagatatct catggcaact tctcctggat 302322DNAArtificial SequencePCR primer 17 23caccatggag ctggcggcct tg 222421DNAArtificial SequencePCR primer 18 24tcccactggc acgtccagac c 21253804DNAHomo sapiens 25ccgcggccgc ccccttcacc atggagctgg cggccttgtg ccgctggggg ctcctcctcg 60ccctcttgcc ccccggagcc gcgagcaccc aagtgtgcac cggcacagac atgaagctgc 120ggctccctgc cagtcccgag acccacctgg acatgctccg ccacctctac cagggctgcc 180aggtggtgca gggaaacctg gaactcacct acctgcccac caatgccagc ctgtccttcc 240tgcaggatat ccaggaggtg cagggctacg tgctcatcgc tcacaaccaa gtgaggcagg 300tcccactgca gaggctgcgg attgtgcgag gcacccagct ctttgaggac aactatgccc 360tggccgtgct agacaatgga gacccgctga acaataccac ccctgtcaca ggggcctccc 420caggaggcct gcgggagctg cagcttcgaa gcctcacaga gatcttgaaa ggaggggtct 480tgatccagcg gaacccccag ctctgctacc aggacacgat tttgtggaag gacatcttcc 540acaagaacaa ccagctggct ctcacactga tagacaccaa ccgctctcgg gcctgccacc 600cctgttctcc gatgtgtaag ggctcccgct gctggggaga gagttctgag gattgtcaga 660gcctgacgcg cactgtctgt gccggtggct gtgcccgctg caaggggcca ctgcccactg 720actgctgcca tgagcagtgt gctgccggct gcacgggccc caagcactct gactgcctgg 780cctgcctcca cttcaaccac agtggcatct gtgagctgca ctgcccagcc ctggtcacct 840acaacacaga cacgtttgag tccatgccca atcccgaggg ccggtataca ttcggcgcca 900gctgtgtgac tgcctgtccc tacaactacc tttctacgga cgtgggatcc tgcaccctcg 960tctgccccct gcacaaccaa gaggtgacag cagaggatgg aacacagcgg tgtgagaagt 1020gcagcaagcc ctgtgcccga gtgtgctatg gtctgggcat ggagcacttg cgagaggtga 1080gggcagttac cagtgccaat atccaggagt ttgctggctg caagaagatc tttgggagcc 1140tggcatttct gccggagagc tttgatgggg acccagcctc caacactgcc ccgctccagc 1200cagagcagct ccaagtgttt gagactctgg aagagatcac aggttaccta tacatctcag 1260catggccgga cagcctgcct gacctcagcg tcttccagaa cctgcaagta atccggggac 1320gaattctgca caatggcgcc tactcgctga ccctgcaagg gctgggcatc agctggctgg 1380ggctgcgctc actgagggaa ctgggcagtg gactggccct catccaccat aacacccacc 1440tctgcttcgt gcacacggtg ccctgggacc agctctttcg gaacccgcac caagctctgc 1500tccacactgc caaccggcca gaggacgagt gtgtgggcga gggcctggcc tgccaccagc 1560tgtgcgcccg agggcactgc tggggtccag ggcccaccca gtgtgtcaac tgcagccagt 1620tccttcgggg ccaggagtgc gtggaggaat gccgagtact gcaggggctc cccagggagt 1680atgtgaatgc caggcactgt ttgccgtgcc accctgagtg tcagccccag aatggctcag 1740tgacctgttt tggaccggag gctgaccagt gtgtggcctg tgcccactat aaggaccctc 1800ccttctgcgt ggcccgctgc cccagcggtg tgaaacctga cctctcctac atgcccatct 1860ggaagtttcc agatgaggag ggcgcatgcc agccttgccc catcaactgc acccactcct 1920gtgtggacct ggatgacaag ggctgccccg ccgagcagag agccagccct ctgacgtcca 1980tcatctctgc ggtggttggc attctgctgg tcgtggtctt gggggtggtc tttgggatcc 2040tcatcaagcg acggcagcag aagatccgga agtacacgat gcggagactg ctgcaggaaa 2100cggagctggt ggagccgctg acacctagcg gagcgatgcc caaccaggcg cagatgcgga 2160tcctgaaaga gacggagctg aggaaggtga aggtgcttgg atctggcgct tttggcacag 2220tctacaaggg catctggatc cctgatgggg agaatgtgaa aattccagtg gccatcaaag 2280tgttgaggga aaacacatcc cccaaagcca acaaagaaat cttagacgaa gcatacgtga 2340tggctggtgt gggctcccca tatgtctccc gccttctggg catctgcctg acatccacgg 2400tgcagctggt gacacagctt atgccctatg gctgcctctt agaccatgtc cgggaaaacc 2460gcggacgcct gggctcccag gacctgctga actggtgtat gcagattgcc aaggggatga 2520gctacctgga ggatgtgcgg ctcgtacaca gggacttggc cgctcggaac gtgctggtca 2580agagtcccaa ccatgtcaaa attacagact tcgggctggc tcggctgctg gacattgacg 2640agacagagta ccatgcagat gggggcaagg tgcccatcaa gtggatggcg ctggagtcca 2700ttctccgccg gcggttcacc caccagagtg atgtgtggag ttatggtgtg actgtgtggg 2760agctgatgac ttttggggcc aaaccttacg atgggatccc agcccgggag atccctgacc 2820tgctggaaaa gggggagcgg ctgccccagc cccccatctg caccattgat gtctacatga 2880tcatggtcaa atgttggatg attgactctg aatgtcggcc aagattccgg gagttggtgt 2940ctgaattctc ccgcatggcc agggaccccc agcgctttgt ggtcatccag aatgaggact 3000tgggcccagc cagtcccttg gacagcacct tctaccgctc actgctggag gacgatgaca 3060tgggggacct ggtggatgct gaggagtatc tggtacccca gcagggcttc ttctgtccag 3120accctgcccc gggcgctggg ggcatggtcc accacaggca ccgcagctca tctaccagga 3180gtggcggtgg ggacctgaca ctagggctgg agccctctga agaggaggcc cccaggtctc 3240cactggcacc ctccgaaggg gctggctccg atgtatttga tggtgacctg ggaatggggg 3300cagccaaggg gctgcaaagc ctccccacac atgaccccag ccctctacag cggtacagtg 3360aggaccccac agtacccctg ccctctgaga ctgatggcta cgttgccccc ctgacctgca 3420gcccccagcc tgaatatgtg aaccagccag atgttcggcc ccagccccct tcgccccgag 3480agggccctct gcctgctgcc cgacctgctg gtgccactct ggaaagggcc aagactctct 3540ccccagggaa gaatggggtc gtcaaagacg tttttgcctt tgggggtgcc gtggagaacc 3600ccgagtactt gacaccccag ggaggagctg cccctcagcc ccaccctcct cctgccttca 3660gcccagcctt cgacaacctc tattactggg accaggaccc accagagcgg ggggctccac 3720ccagcacctt caaagggaca cctacggcag agaacccaga gtacctgggt ctggacgtgc 3780cagtgggaaa gggtgggcgc gccg 3804268PRTMus musculus 26Ile Gln Leu Val Gln Ser Gly Pro 1 5 2711PRTMus musculus 27Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu 1 5 10 289PRTMus musculus 28Gln Ile Gln Leu Val Gln Ser Gly Pro 1 5 2919PRTMus musculus 29Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala 3027DNAArtificial SequencePCR primer DB3F1 30cagatccagt tggtgcagtc tggacct 273132DNAArtificial SequencePCR primer MIG2AEVR1 31aagatatctc atttacccgg agtccgggag aa 323226DNAArtificial SequencePCR primer MK19EIF1 32aagaattcat gaagttgcct gttagg 263332DNAArtificial SequencePCR primer KEVR1 33aagatatctt aacactcatt cctgttgaag ct 32341350DNAMus musculusCDS(1)..(1350) 34cag atc cag ttg gtg cag tct gga cct gag ctg aag aag cct gga gag 48Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 aca gtc aag atc tcc tgc aag gct tct ggg tat acc ttc aca cac tat 96Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr 20 25 30 tca atg cac tgg gtg aag cag gct cca gga aag ggt tta aag tgg atg 144Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 ggc tgg ata aac acc tac act gga gag cca aca tat gct gat gac ttc 192Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 aag gga cgg ttt gcc ttc tct ttg gaa acc tct gcc agc act gcc ttt 240Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Phe 65 70 75 80 ttg cag atc aac aac ctc aaa aat gag gac acg gct aca tat ttc tgt 288Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 gca acc tac tat agg tac gaa aga gac ttt gac tac tgg ggc caa ggc 336Ala Thr Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 acc act ctc aca gtc tcc tca gcc aaa aca aca gcc cca tcg gtc tat 384Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr 115 120 125 cca ctg gcc cct gtg tgt gga gat aca act ggc tcc tcg gtg act cta 432Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu 130 135 140 gga tgc ctg gtc aag ggt tat ttc cct gag cca gtg acc ttg acc tgg 480Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp 145 150 155 160 aac tct gga tcc ctg tcc agt ggt gtg cac acc ttc cca gct gtc ctg 528Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 cag tct gac ctc tac acc ctc

agc agc tca gtg act gta acc tcg agc 576Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser 180 185 190 acc tgg ccc agc cag tcc atc acc tgc aat gtg gcc cac ccg gca agc 624Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 agc acc aag gta gac aag aaa att gag ccc aga ggg ccc aca atc aag 672Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys 210 215 220 ccc tgt cct cca tgc aaa tgc cca gca cct aac ctc ttg ggt gga cca 720Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro 225 230 235 240 tcc gtc ttc atc ttc cct cca aag atc aag gat gta ctc atg atc tcc 768Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser 245 250 255 ctg agc ccc ata gtc aca tgt gtg gtg gtg gat gtg agc gag gat gac 816Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp 260 265 270 cca gat gtc cag atc agc tgg ttt gtg aac aac gtg gaa gta cac aca 864Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr 275 280 285 gct cag aca caa acc cat aga gag gat tac aac agt act ctc cgg gtg 912Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val 290 295 300 gtc agt gcc ctc ccc atc cag cac cag gac tgg atg agt ggc aag gag 960Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu 305 310 315 320 ttc aaa tgc aag gtc aac aac aaa gac ctc cca gcg ccc atc gag aga 1008Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg 325 330 335 acc atc tca aaa ccc aaa ggg tca gta aga gct cca cag gta tat gtc 1056Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val 340 345 350 ttg cct cca cca gaa gaa gag atg act aag aaa cag gtc act ctg acc 1104Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr 355 360 365 tgc atg gtc aca gac ttc atg cct gaa gac att tac gtg gag tgg acc 1152Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr 370 375 380 aac aac ggg aaa aca gag cta aac tac aag aac act gaa cca gtc ctg 1200Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu 385 390 395 400 gac tct gat ggt tct tac ttc atg tac agc aag ctg aga gtg gaa aag 1248Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys 405 410 415 aag aac tgg gtg gaa aga aat agc tac tcc tgt tca gtg gtc cac gag 1296Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu 420 425 430 ggt ctg cac aat cac cac acg act aag agc ttc tcc cgg act ccg ggt 1344Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly 435 440 445 aaa tga 1350Lys 35449PRTMus musculus 35Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr 20 25 30 Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Phe 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Thr Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser 180 185 190 Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys 210 215 220 Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser 245 250 255 Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp 260 265 270 Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr 275 280 285 Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val 290 295 300 Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu 305 310 315 320 Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg 325 330 335 Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val 340 345 350 Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr 355 360 365 Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr 370 375 380 Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys 405 410 415 Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu 420 425 430 Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly 435 440 445 Lys 36660DNAMus musculusCDS(1)..(660) 36gat gtt ttg atg acc caa agt cca ctc tcc ctg cct gtc agt ctt gga 48Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 gat caa gcc tcc atc tct tgc aga tct agt cag agc att gta cat agt 96Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 aat gga aac acc tat tta gaa tgg tac ctg cag aaa cca ggc cag tct 144Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 cca aag ctc ctg atc tac aaa gtt tcc aac cga ttt tct ggg gtc cca 192Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 gac agg ttc agt ggc agt gga tca ggg aca gat ttc aca ctc agg atc 240Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 agc aga gtg gag gct gag gat ctg gga gtt tat tac tgc ttt caa ggt 288Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 tca cat gtt ccg tac acg ttc gga ggg ggg acc aag ctg gaa ata aaa 336Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 cgg gct gat gct gca cca act gta tcc atc ttc cca cca tcc agt gag 384Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 115 120 125 cag tta aca tct gga ggt gcc tca gtc gtg tgc ttc ttg aac aac ttc 432Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 130 135 140 tac ccc aaa gac atc aat gtc aag tgg aag att gat ggc agt gaa cga 480Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 145 150 155 160 caa aat ggc gtc ctg aac agt tgg act gat cag gac agc aaa gac agc 528Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 165 170 175 acc tac agc atg agc agc acc ctc acg ttg acc aag gac gag tat gaa 576Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 180 185 190 cga cat aac agc tat acc tgt gag gcc act cac aag aca tca act tca 624Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 195 200 205 ccc att gtc aag agc ttc aac agg aat gag tgt taa 660Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 210 215 37219PRTMus musculus 37Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 115 120 125 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 130 135 140 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 145 150 155 160 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 180 185 190 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 195 200 205 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 210 215 381350DNAMus musculusCDS(1)..(1350) 38cag atc cag ttg gtg cag tct gga cct gag ctg aag aag cct gga gag 48Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 aca gtc aag atc tcc tgc aag gct tct ggg tat acc ttc ata gac tat 96Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Asp Tyr 20 25 30 tca atg cac tgg gtg aag cag gct cca gga aag ggt tta aag tgg atg 144Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 ggc tgg ata aac acc tac act gga gag cca aca tat tct gat gac ttc 192Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ser Asp Asp Phe 50 55 60 aag gga cgg ttt gcc ttc tct ttg gaa acc tct gcc agc act gcc tat 240Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 ttg cag atc agc aac ctc aaa aat gag gac acg gct tca tat ttc tgt 288Leu Gln Ile Ser Asn Leu Lys Asn Glu Asp Thr Ala Ser Tyr Phe Cys 85 90 95 gca acc tac tat agg tac gaa aga gac ttt gac tac tgg ggc caa ggc 336Ala Thr Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 acc act ctc aca gtc tcc tca gcc aaa aca aca gcc cca tcg gtc tat 384Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr 115 120 125 cca ctg gcc cct gtg tgt gga gat aca act ggc tcc tcg gtg act cta 432Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu 130 135 140 gga tgc ctg gtc aag ggt tat ttc cct gag cca gtg acc ttg acc tgg 480Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp 145 150 155 160 aac tct gga tcc ctg tcc agt ggt gtg cac acc ttc cca gct gtc ctg 528Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 cag tct gac ctc tac acc ctc agc agc tca gtg act gta acc tcg agc 576Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser 180 185 190 acc tgg ccc agc cag tcc atc acc tgc aat gtg gcc cac ccg gca agc 624Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 agc acc aag gtg gac aag aaa att gag ccc aga ggg ccc aca atc aag 672Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys 210 215 220 ccc tgt cct cca tgc aaa tgc cca gca cct aac ctc ttg ggt gga cca 720Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro 225 230 235 240 tcc gtc ttc atc ttc cct cca aag atc aag gat gta ctc atg atc tcc 768Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser 245 250 255 ctg agc ccc ata gtc aca tgt gtg gtg gtg gac gtg agc gag gat gac 816Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp 260 265 270 cca gat gtc cag atc agc tgg ttt gtg aac aac gtg gaa gta cac aca 864Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr 275 280 285 gct cag aca caa acc cat aga gag gat tac aac agt act ctc cgg gtg 912Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val 290 295 300 gtc agt gcc ctc ccc atc cag cac cag gac tgg atg agt ggc aag gag 960Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu 305 310 315 320 ttc aaa tgc aag gtc aac aac aaa gac ctc cca gcg ccc atc gag aga 1008Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg 325 330 335 acc atc tca aaa ccc aaa ggg tca gta aga gct cca cag gta tat gtc 1056Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val 340 345 350 ttg cct cca cca gaa gaa gag atg act aag aaa cag gtc act ctg acc 1104Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr 355 360 365 tgc atg gtc aca gac ttc atg cct gaa gac att tac gtg gag tgg acc 1152Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr 370 375 380 aac aac ggg aaa aca gag cta aac tac aag aac act gaa cca gtc ctg 1200Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu 385 390 395 400 gac tct gat ggt tct tac ttc atg tac agc aag ctg aga gtg gaa aag 1248Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys 405 410 415 aag aac tgg gtg gaa aga aat agc tac tcc tgt tca gtg gtc cac gag 1296Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu 420 425 430 ggt ctg cac aat cac cac acg act aag agc ttc tcc cgg act ccg ggt 1344Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly 435 440 445 aaa tga 1350Lys 39449PRTMus musculus 39Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly

Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Asp Tyr 20 25 30 Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ser Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Asn Leu Lys Asn Glu Asp Thr Ala Ser Tyr Phe Cys 85 90 95 Ala Thr Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser 180 185 190 Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys 210 215 220 Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser 245 250 255 Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp 260 265 270 Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr 275 280 285 Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val 290 295 300 Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu 305 310 315 320 Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg 325 330 335 Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val 340 345 350 Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr 355 360 365 Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr 370 375 380 Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys 405 410 415 Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu 420 425 430 Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly 435 440 445 Lys 40660DNAMus musculusCDS(1)..(660) 40gat gtt ttg atg acc caa act cca ctc tcc ctg cct gtc agt ctt gga 48Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 gat caa gcc tcc atc tct tgc aga tct agt cag agc att gta cat agt 96Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 agt gga atc acc tat tta gaa tgg tac ctg cag aaa cca ggc cag tct 144Ser Gly Ile Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 cca aag ctc ctg atc tac aaa gtt tcc aac cga ttt tct ggg gtc cca 192Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 gac agg ttc agt ggc agt gga tca ggg aca gat ttc aca ctc aag atc 240Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 agc aga gtg gag gct gag gat ctg gga gtt tat tac tgc ttt caa ggt 288Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 tca cat gtt ccg tac acg ttc gga ggg ggg acc aag ctg gaa ata aaa 336Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 cgg gct gat gct gca cca act gta tcc atc ttc cca cca tcc agt gag 384Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 115 120 125 cag tta aca tct gga ggt gcc tca gtc gtg tgc ttc ttg aac aac ttc 432Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 130 135 140 tac ccc aaa gac atc aat gtc aag tgg aag att gat ggc agt gaa cga 480Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 145 150 155 160 caa aat ggc gtc ctg aac agt tgg act gat cag gac agc aaa gac agc 528Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 165 170 175 acc tac agc atg agc agc acc ctc acg ttg acc aag gac gag tat gaa 576Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 180 185 190 cga cat aac agc tat acc tgt gag gcc act cac aag aca tca act tca 624Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 195 200 205 ccc att gtc aag agc ttc aac agg aat gag tgt taa 660Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 210 215 41219PRTMus musculus 41Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Ser Gly Ile Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 115 120 125 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 130 135 140 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 145 150 155 160 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 180 185 190 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 195 200 205 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 210 215 42357DNAMus musculusCDS(1)..(357) 42cag atc cag ttg gtg cag tct gga cct gag ctg aag aag cct gga gag 48Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 aca gtc aag atc tcc tgc aag gct tct ggg tat acc ttc aca cac tat 96Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr 20 25 30 tca atg cac tgg gtg aag cag gct cca gga aag ggt tta aag tgg atg 144Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 ggc tgg ata aac acc tac act gga gag cca aca tat gct gat gac ttc 192Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 aag gga cgg ttt gcc ttc tct ttg gaa acc tct gcc agc act gcc ttt 240Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Phe 65 70 75 80 ttg cag atc aac aac ctc aaa aat gag gac acg gct aca tat ttc tgt 288Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 gca acc tac tat agg tac gaa aga gac ttt gac tac tgg ggc caa ggc 336Ala Thr Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 acc act ctc aca gtc tcc tca 357Thr Thr Leu Thr Val Ser Ser 115 43119PRTMus musculus 43Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr 20 25 30 Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Phe 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Thr Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 44993DNAMus musculusCDS(1)..(993) 44gcc aaa aca aca gcc cca tcg gtc tat cca ctg gcc cct gtg tgt gga 48Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly 1 5 10 15 gat aca act ggc tcc tcg gtg act cta gga tgc ctg gtc aag ggt tat 96Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 20 25 30 ttc cct gag cca gtg acc ttg acc tgg aac tct gga tcc ctg tcc agt 144Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser 35 40 45 ggt gtg cac acc ttc cca gct gtc ctg cag tct gac ctc tac acc ctc 192Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu 50 55 60 agc agc tca gtg act gta acc tcg agc acc tgg ccc agc cag tcc atc 240Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile 65 70 75 80 acc tgc aat gtg gcc cac ccg gca agc agc acc aag gta gac aag aaa 288Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys 85 90 95 att gag ccc aga ggg ccc aca atc aag ccc tgt cct cca tgc aaa tgc 336Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys 100 105 110 cca gca cct aac ctc ttg ggt gga cca tcc gtc ttc atc ttc cct cca 384Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro 115 120 125 aag atc aag gat gta ctc atg atc tcc ctg agc ccc ata gtc aca tgt 432Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys 130 135 140 gtg gtg gtg gat gtg agc gag gat gac cca gat gtc cag atc agc tgg 480Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp 145 150 155 160 ttt gtg aac aac gtg gaa gta cac aca gct cag aca caa acc cat aga 528Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg 165 170 175 gag gat tac aac agt act ctc cgg gtg gtc agt gcc ctc ccc atc cag 576Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln 180 185 190 cac cag gac tgg atg agt ggc aag gag ttc aaa tgc aag gtc aac aac 624His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn 195 200 205 aaa gac ctc cca gcg ccc atc gag aga acc atc tca aaa ccc aaa ggg 672Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly 210 215 220 tca gta aga gct cca cag gta tat gtc ttg cct cca cca gaa gaa gag 720Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu 225 230 235 240 atg act aag aaa cag gtc act ctg acc tgc atg gtc aca gac ttc atg 768Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met 245 250 255 cct gaa gac att tac gtg gag tgg acc aac aac ggg aaa aca gag cta 816Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu 260 265 270 aac tac aag aac act gaa cca gtc ctg gac tct gat ggt tct tac ttc 864Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe 275 280 285 atg tac agc aag ctg aga gtg gaa aag aag aac tgg gtg gaa aga aat 912Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn 290 295 300 agc tac tcc tgt tca gtg gtc cac gag ggt ctg cac aat cac cac acg 960Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr 305 310 315 320 act aag agc ttc tcc cgg act ccg ggt aaa tga 993Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys 325 330 45330PRTMus musculus 45Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly 1 5 10 15 Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu 50 55 60 Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile 65 70 75 80 Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys 85 90 95 Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys 100 105 110 Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro 115 120 125 Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys 130 135 140 Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp 145 150 155 160 Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg 165 170 175 Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln 180 185 190 His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn 195 200 205 Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly 210 215 220 Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu 225 230 235 240 Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met 245 250 255 Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu 260 265 270 Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe 275 280 285 Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn 290 295 300 Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr 305 310 315 320 Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys 325 330 46336DNAMus musculusCDS(1)..(336) 46gat gtt ttg atg acc caa agt cca ctc tcc ctg cct gtc agt ctt gga 48Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val

Ser Leu Gly 1 5 10 15 gat caa gcc tcc atc tct tgc aga tct agt cag agc att gta cat agt 96Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 aat gga aac acc tat tta gaa tgg tac ctg cag aaa cca ggc cag tct 144Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 cca aag ctc ctg atc tac aaa gtt tcc aac cga ttt tct ggg gtc cca 192Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 gac agg ttc agt ggc agt gga tca ggg aca gat ttc aca ctc agg atc 240Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 agc aga gtg gag gct gag gat ctg gga gtt tat tac tgc ttt caa ggt 288Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 tca cat gtt ccg tac acg ttc gga ggg ggg acc aag ctg gaa ata aaa 336Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 47112PRTMus musculus 47Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 48324DNAMus musculusCDS(1)..(324) 48cgg gct gat gct gca cca act gta tcc atc ttc cca cca tcc agt gag 48Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 1 5 10 15 cag tta aca tct gga ggt gcc tca gtc gtg tgc ttc ttg aac aac ttc 96Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 20 25 30 tac ccc aaa gac atc aat gtc aag tgg aag att gat ggc agt gaa cga 144Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 35 40 45 caa aat ggc gtc ctg aac agt tgg act gat cag gac agc aaa gac agc 192Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 50 55 60 acc tac agc atg agc agc acc ctc acg ttg acc aag gac gag tat gaa 240Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 65 70 75 80 cga cat aac agc tat acc tgt gag gcc act cac aag aca tca act tca 288Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 85 90 95 ccc att gtc aag agc ttc aac agg aat gag tgt taa 324Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 100 105 49107PRTMus musculus 49Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 1 5 10 15 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 20 25 30 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 35 40 45 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 65 70 75 80 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 85 90 95 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 100 105 50357DNAMus musculusCDS(1)..(357) 50cag atc cag ttg gtg cag tct gga cct gag ctg aag aag cct gga gag 48Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 aca gtc aag atc tcc tgc aag gct tct ggg tat acc ttc ata gac tat 96Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Asp Tyr 20 25 30 tca atg cac tgg gtg aag cag gct cca gga aag ggt tta aag tgg atg 144Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 ggc tgg ata aac acc tac act gga gag cca aca tat tct gat gac ttc 192Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ser Asp Asp Phe 50 55 60 aag gga cgg ttt gcc ttc tct ttg gaa acc tct gcc agc act gcc tat 240Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 ttg cag atc agc aac ctc aaa aat gag gac acg gct tca tat ttc tgt 288Leu Gln Ile Ser Asn Leu Lys Asn Glu Asp Thr Ala Ser Tyr Phe Cys 85 90 95 gca acc tac tat agg tac gaa aga gac ttt gac tac tgg ggc caa ggc 336Ala Thr Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 acc act ctc aca gtc tcc tca 357Thr Thr Leu Thr Val Ser Ser 115 51119PRTMus musculus 51Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Asp Tyr 20 25 30 Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ser Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Asn Leu Lys Asn Glu Asp Thr Ala Ser Tyr Phe Cys 85 90 95 Ala Thr Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 52993DNAMus musculusCDS(1)..(993) 52gcc aaa aca aca gcc cca tcg gtc tat cca ctg gcc cct gtg tgt gga 48Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly 1 5 10 15 gat aca act ggc tcc tcg gtg act cta gga tgc ctg gtc aag ggt tat 96Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 20 25 30 ttc cct gag cca gtg acc ttg acc tgg aac tct gga tcc ctg tcc agt 144Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser 35 40 45 ggt gtg cac acc ttc cca gct gtc ctg cag tct gac ctc tac acc ctc 192Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu 50 55 60 agc agc tca gtg act gta acc tcg agc acc tgg ccc agc cag tcc atc 240Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile 65 70 75 80 acc tgc aat gtg gcc cac ccg gca agc agc acc aag gtg gac aag aaa 288Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys 85 90 95 att gag ccc aga ggg ccc aca atc aag ccc tgt cct cca tgc aaa tgc 336Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys 100 105 110 cca gca cct aac ctc ttg ggt gga cca tcc gtc ttc atc ttc cct cca 384Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro 115 120 125 aag atc aag gat gta ctc atg atc tcc ctg agc ccc ata gtc aca tgt 432Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys 130 135 140 gtg gtg gtg gac gtg agc gag gat gac cca gat gtc cag atc agc tgg 480Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp 145 150 155 160 ttt gtg aac aac gtg gaa gta cac aca gct cag aca caa acc cat aga 528Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg 165 170 175 gag gat tac aac agt act ctc cgg gtg gtc agt gcc ctc ccc atc cag 576Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln 180 185 190 cac cag gac tgg atg agt ggc aag gag ttc aaa tgc aag gtc aac aac 624His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn 195 200 205 aaa gac ctc cca gcg ccc atc gag aga acc atc tca aaa ccc aaa ggg 672Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly 210 215 220 tca gta aga gct cca cag gta tat gtc ttg cct cca cca gaa gaa gag 720Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu 225 230 235 240 atg act aag aaa cag gtc act ctg acc tgc atg gtc aca gac ttc atg 768Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met 245 250 255 cct gaa gac att tac gtg gag tgg acc aac aac ggg aaa aca gag cta 816Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu 260 265 270 aac tac aag aac act gaa cca gtc ctg gac tct gat ggt tct tac ttc 864Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe 275 280 285 atg tac agc aag ctg aga gtg gaa aag aag aac tgg gtg gaa aga aat 912Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn 290 295 300 agc tac tcc tgt tca gtg gtc cac gag ggt ctg cac aat cac cac acg 960Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr 305 310 315 320 act aag agc ttc tcc cgg act ccg ggt aaa tga 993Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys 325 330 53330PRTMus musculus 53Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly 1 5 10 15 Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu 50 55 60 Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile 65 70 75 80 Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys 85 90 95 Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys 100 105 110 Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro 115 120 125 Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys 130 135 140 Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp 145 150 155 160 Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg 165 170 175 Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln 180 185 190 His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn 195 200 205 Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly 210 215 220 Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu 225 230 235 240 Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met 245 250 255 Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu 260 265 270 Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe 275 280 285 Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn 290 295 300 Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr 305 310 315 320 Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys 325 330 54336DNAMus musculusCDS(1)..(336) 54gat gtt ttg atg acc caa act cca ctc tcc ctg cct gtc agt ctt gga 48Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 gat caa gcc tcc atc tct tgc aga tct agt cag agc att gta cat agt 96Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 agt gga atc acc tat tta gaa tgg tac ctg cag aaa cca ggc cag tct 144Ser Gly Ile Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 cca aag ctc ctg atc tac aaa gtt tcc aac cga ttt tct ggg gtc cca 192Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 gac agg ttc agt ggc agt gga tca ggg aca gat ttc aca ctc aag atc 240Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 agc aga gtg gag gct gag gat ctg gga gtt tat tac tgc ttt caa ggt 288Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 tca cat gtt ccg tac acg ttc gga ggg ggg acc aag ctg gaa ata aaa 336Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 55112PRTMus musculus 55Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Ser Gly Ile Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 56324DNAMus musculusCDS(1)..(324) 56cgg gct gat gct gca cca act gta tcc atc ttc cca cca tcc agt gag 48Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 1 5 10 15 cag tta aca tct gga ggt gcc tca gtc gtg tgc ttc ttg aac aac ttc 96Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 20 25 30 tac ccc aaa gac atc aat gtc aag tgg aag att gat ggc agt gaa cga 144Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 35 40 45 caa aat ggc gtc ctg aac agt tgg act gat cag gac agc aaa gac agc 192Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 50 55 60 acc tac agc atg agc agc acc ctc acg ttg acc aag gac gag tat gaa 240Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 65 70 75 80 cga cat aac agc tat acc tgt gag gcc act cac aag aca tca act tca 288Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 85 90 95 ccc att gtc aag agc ttc aac agg aat

gag tgt taa 324Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 100 105 57107PRTMus musculus 57Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 1 5 10 15 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 20 25 30 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 35 40 45 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 65 70 75 80 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 85 90 95 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 100 105 5830DNAMus musculusCDS(1)..(30) 58ggg tat acc ttc aca cac tat tca atg cac 30Gly Tyr Thr Phe Thr His Tyr Ser Met His 1 5 10 5910PRTMus musculus 59Gly Tyr Thr Phe Thr His Tyr Ser Met His 1 5 10 6051DNAMus musculusCDS(1)..(51) 60tgg ata aac acc tac act gga gag cca aca tat gct gat gac ttc aag 48Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys 1 5 10 15 gga 51Gly 6117PRTMus musculus 61Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys 1 5 10 15 Gly 6230DNAMus musculusCDS(1)..(30) 62tac tat agg tac gaa aga gac ttt gac tac 30Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr 1 5 10 6310PRTMus musculus 63Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr 1 5 10 6448DNAMus musculusCDS(1)..(48) 64aga tct agt cag agc att gta cat agt aat gga aac acc tat tta gaa 48Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 6516PRTMus musculus 65Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 6621DNAMus musculusCDS(1)..(21) 66aaa gtt tcc aac cga ttt tct 21Lys Val Ser Asn Arg Phe Ser 1 5 677PRTMus musculus 67Lys Val Ser Asn Arg Phe Ser 1 5 6827DNAMus musculusCDS(1)..(27) 68ttt caa ggt tca cat gtt ccg tac acg 27Phe Gln Gly Ser His Val Pro Tyr Thr 1 5 699PRTMus musculus 69Phe Gln Gly Ser His Val Pro Tyr Thr 1 5 7030DNAMus musculusCDS(1)..(30) 70ggg tat acc ttc ata gac tat tca atg cac 30Gly Tyr Thr Phe Ile Asp Tyr Ser Met His 1 5 10 7110PRTMus musculus 71Gly Tyr Thr Phe Ile Asp Tyr Ser Met His 1 5 10 7251DNAMus musculusCDS(1)..(51) 72tgg ata aac acc tac act gga gag cca aca tat tct gat gac ttc aag 48Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ser Asp Asp Phe Lys 1 5 10 15 gga 51Gly 7317PRTMus musculus 73Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ser Asp Asp Phe Lys 1 5 10 15 Gly 7430DNAMus musculusCDS(1)..(30) 74tac tat agg tac gaa aga gac ttt gac tac 30Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr 1 5 10 7510PRTMus musculus 75Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr 1 5 10 7648DNAMus musculusCDS(1)..(48) 76aga tct agt cag agc att gta cat agt agt gga atc acc tat tta gaa 48Arg Ser Ser Gln Ser Ile Val His Ser Ser Gly Ile Thr Tyr Leu Glu 1 5 10 15 7716PRTMus musculus 77Arg Ser Ser Gln Ser Ile Val His Ser Ser Gly Ile Thr Tyr Leu Glu 1 5 10 15 7821DNAMus musculusCDS(1)..(21) 78aaa gtt tcc aac cga ttt tct 21Lys Val Ser Asn Arg Phe Ser 1 5 797PRTMus musculus 79Lys Val Ser Asn Arg Phe Ser 1 5 8027DNAMus musculusCDS(1)..(27) 80ttt caa ggt tca cat gtt ccg tac acg 27Phe Gln Gly Ser His Val Pro Tyr Thr 1 5 819PRTMus musculus 81Phe Gln Gly Ser His Val Pro Tyr Thr 1 5 8232DNAArtificial Sequenceprimer N1 82attaggatcc gagcttccgt actgccagtg tc 328328DNAArtificialprimer N2 83attaggatcc gccccccaag gtgaggct 288434DNAArtificialprimer N3 84attaggatcc ggtcacttac cgcaagaagg gaga 348532DNAArtificialprimer N4 85attaggatcc ggtccaggtg caggcactga cg 328631DNAArtificialprimer C1 86aattaagctt gccgccaatc accgccaagt t 318732DNAArtificialprimer C2 87aattaagctt gttgccagat ccctccgggg ac 328829DNAArtificialprimer C3 88aattaagctt caggtaggtg gtgtctggg 298930DNAArtificialprimer C4 89aattaagctt ctcgtacttc cacactcggc 3090720DNAArtificialhSH-348-T1L 90atg gtg ctg cag acc cag gtg ttc atc tcc ctg ctg ctg tgg atc tcc 48Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 ggc gca tat ggc gac ata gtg atg acc cag agc ccc ctg agc ctg ccc 96Gly Ala Tyr Gly Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro 20 25 30 gtg acc ccc ggc gag ccc gcc tcc atc agc tgc cgg agc agc cag agc 144Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 atc gtg cac agc aac ggc aac acc tac ctg gag tgg tac ctg cag aag 192Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys 50 55 60 ccc ggc cag agc ccc cag ctg ctg atc tac aag gtg agc aac cgg ttc 240Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe 65 70 75 80 agc ggc gtg ccc gac cgg ttc agc ggc agc ggc agc ggc acc gac ttc 288Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 acc ctg aag atc agc cgg gtg gag gcc gag gac gtg ggc gtg tac tac 336Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 tgc ttc cag ggc tcc cac gtg ccc tac acc ttc ggc cag ggc acc aag 384Cys Phe Gln Gly Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys 115 120 125 gtg gag atc aag cgt acg gtg gcc gcc ccc tcc gtg ttc atc ttc ccc 432Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 ccc tcc gac gag cag ctg aag tcc ggc acc gcc tcc gtg gtg tgc ctg 480Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 ctg aat aac ttc tac ccc aga gag gcc aag gtg cag tgg aag gtg gac 528Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 aac gcc ctg cag tcc ggg aac tcc cag gag agc gtg acc gag cag gac 576Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 agc aag gac agc acc tac agc ctg agc agc acc ctg acc ctg agc aaa 624Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 gcc gac tac gag aag cac aag gtg tac gcc tgc gag gtg acc cac cag 672Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220 ggc ctg agc tcc ccc gtc acc aag agc ttc aac agg ggg gag tgt tag 720Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 91239PRTArtificialSynthetic Construct 91Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro 20 25 30 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys 50 55 60 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 Cys Phe Gln Gly Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys 115 120 125 Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 9248DNAArtificialhSH348-1-T1L CDRL1 92cgg agc agc cag agc atc gtg cac agc aac ggc aac acc tac ctg gag 48Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 9316PRTArtificialSynthetic Construct 93Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 9421DNAArtificialhSH348-1-T1L CDRL2 94aag gtg agc aac cgg ttc agc 21Lys Val Ser Asn Arg Phe Ser 1 5 957PRTArtificialSynthetic Construct 95Lys Val Ser Asn Arg Phe Ser 1 5 9624DNAArtificialhSH348-1-T1L CDRL3 96ttc cag ggc tcc cac gtg ccc tac 24Phe Gln Gly Ser His Val Pro Tyr 1 5 978PRTArtificialSynthetic Construct 97Phe Gln Gly Ser His Val Pro Tyr 1 5 98720DNAArtificialhSH348-1-T3L 98atg gtg ctg cag acc cag gtg ttc atc tcc ctg ctg ctg tgg atc tcc 48Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 ggc gca tat ggc gac gtg ctg atg acc cag agc ccc ctg agc ctg ccc 96Gly Ala Tyr Gly Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro 20 25 30 gtg acc ccc ggc gag ccc gcc tcc atc agc tgc cgg agc agc cag agc 144Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 atc gtg cac agc aac ggc aac acc tac ctg gag tgg tac ctg cag aag 192Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys 50 55 60 ccc ggc cag agc ccc cag ctg ctg atc tac aag gtg agc aac cgg ttc 240Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe 65 70 75 80 agc ggc gtg ccc gac cgg ttc agc ggc agc ggc agc ggc acc gac ttc 288Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 acc ctg aag atc agc cgg gtg gag gcc gag gac gtg ggc gtg tac tac 336Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 tgc ttc cag ggc tcc cac gtg ccc tac acc ttc ggc cag ggc acc aag 384Cys Phe Gln Gly Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys 115 120 125 gtg gag atc aag cgt acg gtg gcc gcc ccc tcc gtg ttc atc ttc ccc 432Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 ccc tcc gac gag cag ctg aag tcc ggc acc gcc tcc gtg gtg tgc ctg 480Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 ctg aat aac ttc tac ccc aga gag gcc aag gtg cag tgg aag gtg gac 528Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 aac gcc ctg cag tcc ggg aac tcc cag gag agc gtg acc gag cag gac 576Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 agc aag gac agc acc tac agc ctg agc agc acc ctg acc ctg agc aaa 624Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 gcc gac tac gag aag cac aag gtg tac gcc tgc gag gtg acc cac cag 672Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220 ggc ctg agc tcc ccc gtc acc aag agc ttc aac agg ggg gag tgt tag 720Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 99239PRTArtificialSynthetic Construct 99Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro 20 25 30 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys 50 55 60 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 Cys Phe Gln Gly Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys 115 120 125 Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 10048DNAArtificialhSH348-1-T3L CDRL1 100cgg agc agc cag agc atc gtg cac agc aac ggc aac acc tac ctg gag 48Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 10116PRTArtificialSynthetic Construct

101Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 10221DNAArtificialhSH348-1-T3L CDRL2 102aag gtg agc aac cgg ttc agc 21Lys Val Ser Asn Arg Phe Ser 1 5 1037PRTArtificialSynthetic Construct 103Lys Val Ser Asn Arg Phe Ser 1 5 10424DNAArtificialhSH348-1-T3L CDRL3 104ttc cag ggc tcc cac gtg ccc tac 24Phe Gln Gly Ser His Val Pro Tyr 1 5 1058PRTArtificialSynthetic Construct 105Phe Gln Gly Ser His Val Pro Tyr 1 5 1061407DNAArtificialhSH348-1-T1H 106atg aaa cac ctg tgg ttc ttc ctc ctg ctg gtg gca gct ccc aga tgg 48Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 gtg ctg agc cag gtg cag ctg gtg cag agc ggc gcc gag gtg aag aag 96Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 ccc ggc agc agc gtg aag gtg agc tgc aag gcc agc ggc tac acc ttc 144Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 acc cac tac agc atg cac tgg gtg cgg cag gcc ccc ggc cag ggc ctg 192Thr His Tyr Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 gag tgg atg ggc tgg atc aac acc tac acc ggc gag ccc acc tac gcc 240Glu Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala 65 70 75 80 gac gac ttc aag ggc cgg gtg acc atc acc gcc gac acc tcc acc tcc 288Asp Asp Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser 85 90 95 acc gcc tac ctg gaa ctg agc agc ctg cgg agc gag gac acc gcc gtg 336Thr Ala Tyr Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 tac tac tgc gcc acc tac tac cgg tac gag cgg gac ttc gac tac tgg 384Tyr Tyr Cys Ala Thr Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr Trp 115 120 125 ggc cag ggc acc ctg gtg acc gtg agc tca gcc tcc acc aag ggc cca 432Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140 agc gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggc ggc aca 480Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 145 150 155 160 gcc gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccc gtg acc 528Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175 gtg agc tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccc 576Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190 gct gtc ctg cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc 624Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205 gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat 672Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220 cac aag ccc agc aac acc aag gtg gac aag aga gtt gag ccc aaa tct 720His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 225 230 235 240 tgt gac aaa act cac aca tgc cca ccc tgc cca gca cct gaa ctc ctg 768Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255 ggg gga ccc tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc 816Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc 864Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag 912His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 gtg cat aat gcc aag aca aag ccc cgg gag gag cag tac aac agc acg 960Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310 315 320 tac cgg gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat 1008Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc 1056Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350 atc gag aaa acc atc tcc aaa gcc aaa ggc cag ccc cgg gaa cca cag 1104Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 gtg tac acc ctg ccc cca tcc cgg gag gag atg acc aag aac cag gtc 1152Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 370 375 380 agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg 1200Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 gag tgg gag agc aat ggc cag ccc gag aac aac tac aag acc acc cct 1248Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc 1296Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 gtg gac aag agc agg tgg cag cag ggc aac gtc ttc tca tgc tcc gtg 1344Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 atg cat gag gct ctg cac aac cac tac acc cag aag agc ctc tcc ctg 1392Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 tct ccc ggc aaa tga 1407Ser Pro Gly Lys 465 107468PRTArtificialSynthetic Construct 107Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr His Tyr Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala 65 70 75 80 Asp Asp Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser 85 90 95 Thr Ala Tyr Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Thr Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 145 150 155 160 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 225 230 235 240 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310 315 320 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 370 375 380 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Pro Gly Lys 465 10815DNAArtificialhSH348-1-T1H CDRH1 108cac tac agc atg cac 15His Tyr Ser Met His 1 5 1095PRTArtificialSynthetic Construct 109His Tyr Ser Met His 1 5 11051DNAArtificialhSH348-1-T1H_CDRH2 110tgg atc aac acc tac acc ggc gag ccc acc tac gcc gac gac ttc aag 48Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys 1 5 10 15 ggc 51Gly 11117PRTArtificialSynthetic Construct 111Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys 1 5 10 15 Gly 11230DNAArtificialhSH348-1-T1H CDRH3 112tac tac cgg tac gag cgg gac ttc gac tac 30Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr 1 5 10 11310PRTArtificialSynthetic Construct 113Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr 1 5 10 1141407DNAArtificialhSH348-1-T3H 114atg aaa cac ctg tgg ttc ttc ctc ctg ctg gtg gca gct ccc aga tgg 48Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 gtg ctg agc cag atc cag ctg gtg cag agc ggc gcc gag gtg aag aag 96Val Leu Ser Gln Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 ccc ggc agc agc gtg aag gtg agc tgc aag gcc agc ggc tac acc ttc 144Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 acc cac tac agc atg cac tgg gtg cgg cag gcc ccc ggc cag ggc ctg 192Thr His Tyr Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 aag tgg atg ggc tgg atc aac acc tac acc ggc gag ccc acc tac gcc 240Lys Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala 65 70 75 80 gac gac ttc aag ggc cgg ttc gcc ttc agc ctg gac acc tcc acc tcc 288Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Thr Ser 85 90 95 acc gcc tac ctg gaa ctg agc agc ctg cgg agc gag gac acc gcc gtg 336Thr Ala Tyr Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 tac tac tgc gcc acc tac tac cgg tac gag cgg gac ttc gac tac tgg 384Tyr Tyr Cys Ala Thr Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr Trp 115 120 125 ggc cag ggc acc ctg gtg acc gtg agc tca gcc tcc acc aag ggc cca 432Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140 agc gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggc ggc aca 480Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 145 150 155 160 gcc gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccc gtg acc 528Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175 gtg agc tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccc 576Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190 gct gtc ctg cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc 624Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205 gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat 672Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220 cac aag ccc agc aac acc aag gtg gac aag aga gtt gag ccc aaa tct 720His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 225 230 235 240 tgt gac aaa act cac aca tgc cca ccc tgc cca gca cct gaa ctc ctg 768Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255 ggg gga ccc tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc 816Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc 864Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag 912His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 gtg cat aat gcc aag aca aag ccc cgg gag gag cag tac aac agc acg 960Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310 315 320 tac cgg gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat 1008Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc 1056Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350 atc gag aaa acc atc tcc aaa gcc aaa ggc cag ccc cgg gaa cca cag 1104Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 gtg tac acc ctg ccc cca tcc cgg gag gag atg acc aag aac cag gtc 1152Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 370 375 380 agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg 1200Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 gag tgg gag agc aat ggc cag ccc gag aac aac tac aag acc acc cct 1248Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc 1296Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 gtg gac aag agc agg tgg cag cag ggc aac gtc ttc tca tgc tcc gtg 1344Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445

atg cat gag gct ctg cac aac cac tac acc cag aag agc ctc tcc ctg 1392Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 tct ccc ggc aaa tga 1407Ser Pro Gly Lys 465 115468PRTArtificialSynthetic Construct 115Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr His Tyr Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Lys Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala 65 70 75 80 Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Thr Ser 85 90 95 Thr Ala Tyr Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Thr Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 145 150 155 160 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 225 230 235 240 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310 315 320 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 370 375 380 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Pro Gly Lys 465 11615DNAArtificialhSH348-1-T3H CDRH1 116cac tac agc atg cac 15His Tyr Ser Met His 1 5 1175PRTArtificialSynthetic Construct 117His Tyr Ser Met His 1 5 11851DNAArtificialhSH348-1-T3H CDRH2 118tgg atc aac acc tac acc ggc gag ccc acc tac gcc gac gac ttc aag 48Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys 1 5 10 15 ggc 51Gly 11917PRTArtificialSynthetic Construct 119Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys 1 5 10 15 Gly 12030DNAArtificialhSH348-1-T3H CDRH3 120tac tac cgg tac gag cgg gac ttc gac tac 30Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr 1 5 10 12110PRTArtificialSynthetic Construct 121Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr 1 5 10 122720DNAArtificialhSH357-1-T1L 122atg gtg ctg cag acc cag gtg ttc atc tcc ctg ctg ctg tgg atc tcc 48Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 ggc gca tat ggc gac ata gtg atg acc cag agc ccc ctg agc ctg ccc 96Gly Ala Tyr Gly Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro 20 25 30 gtg acc ccc ggc gag ccc gcc agc atc agc tgc cgg agc agc cag agc 144Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 atc gtg cac agc agc ggc atc acc tac ctg gag tgg tac ctg cag aag 192Ile Val His Ser Ser Gly Ile Thr Tyr Leu Glu Trp Tyr Leu Gln Lys 50 55 60 ccc ggc cag agc ccc cag ctg ctg atc tac aag gtg agc aac cgg ttc 240Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe 65 70 75 80 agc ggc gtg ccc gac cgg ttc agc ggc agc ggc agc ggc acc gac ttc 288Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 acc ctg aag atc agc cgg gtg gag gcc gag gac gtg ggc gtg tac tac 336Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 tgc ttc cag ggc agc cac gtg ccc tac acc ttc ggc cag ggc acc aag 384Cys Phe Gln Gly Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys 115 120 125 gtg gag atc aag cgt acg gtg gcc gcc ccc tcc gtg ttc atc ttc ccc 432Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 ccc tcc gac gag cag ctg aag tcc ggc acc gcc tcc gtg gtg tgc ctg 480Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 ctg aat aac ttc tac ccc aga gag gcc aag gtg cag tgg aag gtg gac 528Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 aac gcc ctg cag tcc ggg aac tcc cag gag agc gtg acc gag cag gac 576Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 agc aag gac agc acc tac agc ctg agc agc acc ctg acc ctg agc aaa 624Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 gcc gac tac gag aag cac aag gtg tac gcc tgc gag gtg acc cac cag 672Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220 ggc ctg agc tcc ccc gtc acc aag agc ttc aac agg ggg gag tgt tag 720Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 123239PRTArtificialSynthetic Construct 123Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro 20 25 30 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 Ile Val His Ser Ser Gly Ile Thr Tyr Leu Glu Trp Tyr Leu Gln Lys 50 55 60 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 Cys Phe Gln Gly Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys 115 120 125 Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 12448DNAArtificialhSH357-1-T1L CDRL1 124cgg agc agc cag agc atc gtg cac agc agc ggc atc acc tac ctg gag 48Arg Ser Ser Gln Ser Ile Val His Ser Ser Gly Ile Thr Tyr Leu Glu 1 5 10 15 12516PRTArtificialSynthetic Construct 125Arg Ser Ser Gln Ser Ile Val His Ser Ser Gly Ile Thr Tyr Leu Glu 1 5 10 15 12621DNAArtificialhSH357-1-T1L CDRL2 126aag gtg agc aac cgg ttc agc 21Lys Val Ser Asn Arg Phe Ser 1 5 1277PRTArtificialSynthetic Construct 127Lys Val Ser Asn Arg Phe Ser 1 5 12824DNAArtificialhSH357-1-T1L_CDRL3 128ttc cag ggc agc cac gtg ccc tac 24Phe Gln Gly Ser His Val Pro Tyr 1 5 1298PRTArtificialSynthetic Construct 129Phe Gln Gly Ser His Val Pro Tyr 1 5 130720DNAArtificialhSH357-1-T3L 130atg gtg ctg cag acc cag gtg ttc atc tcc ctg ctg ctg tgg atc tcc 48Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 ggc gca tat ggc gac gtg ctg atg acc cag agc ccc ctg agc ctg ccc 96Gly Ala Tyr Gly Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro 20 25 30 gtg acc ccc ggc gag ccc gcc agc atc agc tgc cgg agc agc cag agc 144Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 atc gtg cac agc agc ggc atc acc tac ctg gag tgg tac ctg cag aag 192Ile Val His Ser Ser Gly Ile Thr Tyr Leu Glu Trp Tyr Leu Gln Lys 50 55 60 ccc ggc cag agc ccc cag ctg ctg atc tac aag gtg agc aac cgg ttc 240Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe 65 70 75 80 agc ggc gtg ccc gac cgg ttc agc ggc agc ggc agc ggc acc gac ttc 288Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 acc ctg aag atc agc cgg gtg gag gcc gag gac gtg ggc gtg tac tac 336Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 tgc ttc cag ggc agc cac gtg ccc tac acc ttc ggc cag ggc acc aag 384Cys Phe Gln Gly Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys 115 120 125 gtg gag atc aag cgt acg gtg gcc gcc ccc tcc gtg ttc atc ttc ccc 432Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 ccc tcc gac gag cag ctg aag tcc ggc acc gcc tcc gtg gtg tgc ctg 480Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 ctg aat aac ttc tac ccc aga gag gcc aag gtg cag tgg aag gtg gac 528Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 aac gcc ctg cag tcc ggg aac tcc cag gag agc gtg acc gag cag gac 576Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 agc aag gac agc acc tac agc ctg agc agc acc ctg acc ctg agc aaa 624Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 gcc gac tac gag aag cac aag gtg tac gcc tgc gag gtg acc cac cag 672Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220 ggc ctg agc tcc ccc gtc acc aag agc ttc aac agg ggg gag tgt tag 720Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 131239PRTArtificialSynthetic Construct 131Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro 20 25 30 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 Ile Val His Ser Ser Gly Ile Thr Tyr Leu Glu Trp Tyr Leu Gln Lys 50 55 60 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 Cys Phe Gln Gly Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys 115 120 125 Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 13248DNAArtificialhSH357-1-T3L CDRL1 132cgg agc agc cag agc atc gtg cac agc agc ggc atc acc tac ctg gag 48Arg Ser Ser Gln Ser Ile Val His Ser Ser Gly Ile Thr Tyr Leu Glu 1 5 10 15 13316PRTArtificialSynthetic Construct 133Arg Ser Ser Gln Ser Ile Val His Ser Ser Gly Ile Thr Tyr Leu Glu 1 5 10 15 13421DNAArtificialhSH357-1-T3L CDRL2 134aag gtg agc aac cgg ttc agc 21Lys Val Ser Asn Arg Phe Ser 1 5 1357PRTArtificialSynthetic Construct 135Lys Val Ser Asn Arg Phe Ser 1 5 13624DNAArtificialhSH357-1-T3L CDRL3 136ttc cag ggc agc cac gtg ccc tac 24Phe Gln Gly Ser His Val Pro Tyr 1 5 1378PRTArtificialSynthetic Construct 137Phe Gln Gly Ser His Val Pro Tyr 1 5 1381407DNAArtificialhSH357-1-T1H 138atg aaa cac ctg tgg ttc ttc ctc ctg ctg gtg gca gct ccc aga tgg 48Met Lys His Leu Trp Phe Phe Leu Leu Leu

Val Ala Ala Pro Arg Trp 1 5 10 15 gtg ctg agc cag gtg cag ctg gtg cag tcc ggc gcc gag gtg aag aag 96Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 ccc ggc gcc tcc gtg aag gtg tcc tgt aag gcc tcc ggc tac acc ttc 144Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 atc gac tac tcc atg cac tgg gtg agg cag gcc ccc ggc cag ggc ctg 192Ile Asp Tyr Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 gag tgg atg ggc tgg atc aac acc tac acc ggc gag ccc acc tac tcc 240Glu Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ser 65 70 75 80 gac gac ttc aag ggc agg gtg acc atc acc gcc gac acc tcc acc agc 288Asp Asp Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser 85 90 95 acc gcc tac ctg gag ctg tcc tcc ctg agg tcc gag gac acc gcc gtg 336Thr Ala Tyr Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 tac tac tgt gcc acc tac tac agg tac gag agg gac ttc gac tac tgg 384Tyr Tyr Cys Ala Thr Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr Trp 115 120 125 ggc cag ggc acc ctg gtg acg gtg agc tca gcc tcc acc aag ggc cca 432Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140 agc gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggc ggc aca 480Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 145 150 155 160 gcc gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccc gtg acc 528Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175 gtg agc tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccc 576Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190 gct gtc ctg cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc 624Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205 gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat 672Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220 cac aag ccc agc aac acc aag gtg gac aag aga gtt gag ccc aaa tct 720His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 225 230 235 240 tgt gac aaa act cac aca tgc cca ccc tgc cca gca cct gaa ctc ctg 768Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255 ggg gga ccc tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc 816Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc 864Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag 912His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 gtg cat aat gcc aag aca aag ccc cgg gag gag cag tac aac agc acg 960Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310 315 320 tac cgg gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat 1008Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc 1056Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350 atc gag aaa acc atc tcc aaa gcc aaa ggc cag ccc cgg gaa cca cag 1104Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 gtg tac acc ctg ccc cca tcc cgg gag gag atg acc aag aac cag gtc 1152Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 370 375 380 agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg 1200Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 gag tgg gag agc aat ggc cag ccc gag aac aac tac aag acc acc cct 1248Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc 1296Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 gtg gac aag agc agg tgg cag cag ggc aac gtc ttc tca tgc tcc gtg 1344Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 atg cat gag gct ctg cac aac cac tac acc cag aag agc ctc tcc ctg 1392Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 tct ccc ggc aaa tga 1407Ser Pro Gly Lys 465 139468PRTArtificialSynthetic Construct 139Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Ile Asp Tyr Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ser 65 70 75 80 Asp Asp Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser 85 90 95 Thr Ala Tyr Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Thr Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 145 150 155 160 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 225 230 235 240 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310 315 320 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 370 375 380 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Pro Gly Lys 465 14018DNAArtificialhSH357-1-T1H CDRH1 140atc gac tac tcc atg cac 18Ile Asp Tyr Ser Met His 1 5 1416PRTArtificialSynthetic Construct 141Ile Asp Tyr Ser Met His 1 5 14251DNAArtificialhSH357-1-T1H CDRH2 142tgg atc aac acc tac acc ggc gag ccc acc tac tcc gac gac ttc aag 48Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ser Asp Asp Phe Lys 1 5 10 15 ggc 51Gly 14317PRTArtificialSynthetic Construct 143Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ser Asp Asp Phe Lys 1 5 10 15 Gly 14430DNAArtificialhSH357-1-T1H CDRH3 144tac tac agg tac gag agg gac ttc gac tac 30Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr 1 5 10 14510PRTArtificialSynthetic Construct 145Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr 1 5 10 1461407DNAArtificialhSH357-1-T3H 146atg aaa cac ctg tgg ttc ttc ctc ctg ctg gtg gca gct ccc aga tgg 48Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 gtg ctg agc cag atc cag ctg gtg cag tcc ggc gcc gag gtg aag aag 96Val Leu Ser Gln Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 ccc ggc gcc tcc gtg aag gtg tcc tgt aag gcc tcc ggc tac acc ttc 144Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 atc gac tac tcc atg cac tgg gtg agg cag gcc ccc ggc cag ggc ctg 192Ile Asp Tyr Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 aag tgg atg ggc tgg atc aac acc tac acc ggc gag ccc acc tac tcc 240Lys Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ser 65 70 75 80 gac gac ttc aag ggc agg ttc gcc ttc tcc ctg gac acc tcc acc agc 288Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Thr Ser 85 90 95 acc gcc tac ctg gag ctg tcc tcc ctg agg tcc gag gac acc gcc gtg 336Thr Ala Tyr Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 tac tac tgt gcc acc tac tac agg tac gag agg gac ttc gac tac tgg 384Tyr Tyr Cys Ala Thr Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr Trp 115 120 125 ggc cag ggc acc ctg gtg acg gtg agc tca gcc tcc acc aag ggc cca 432Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140 agc gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggc ggc aca 480Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 145 150 155 160 gcc gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccc gtg acc 528Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175 gtg agc tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccc 576Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190 gct gtc ctg cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc 624Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205 gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat 672Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220 cac aag ccc agc aac acc aag gtg gac aag aga gtt gag ccc aaa tct 720His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 225 230 235 240 tgt gac aaa act cac aca tgc cca ccc tgc cca gca cct gaa ctc ctg 768Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255 ggg gga ccc tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc 816Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc 864Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag 912His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 gtg cat aat gcc aag aca aag ccc cgg gag gag cag tac aac agc acg 960Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310 315 320 tac cgg gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat 1008Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc 1056Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350 atc gag aaa acc atc tcc aaa gcc aaa ggc cag ccc cgg gaa cca cag 1104Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 gtg tac acc ctg ccc cca tcc cgg gag gag atg acc aag aac cag gtc 1152Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 370 375 380 agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg 1200Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 gag tgg gag agc aat ggc cag ccc gag aac aac tac aag acc acc cct 1248Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc 1296Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 gtg gac aag agc agg tgg cag cag ggc aac gtc ttc tca tgc tcc gtg 1344Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 atg cat gag gct ctg cac aac cac tac acc cag aag agc ctc tcc ctg 1392Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 tct ccc ggc aaa tga 1407Ser Pro Gly Lys 465 147468PRTArtificialSynthetic Construct 147Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Ile Asp Tyr Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Lys Trp Met Gly Trp Ile Asn Thr

Tyr Thr Gly Glu Pro Thr Tyr Ser 65 70 75 80 Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Thr Ser 85 90 95 Thr Ala Tyr Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Thr Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 145 150 155 160 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 225 230 235 240 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310 315 320 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 370 375 380 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Pro Gly Lys 465 14818DNAArtificialhSH357-1-T3H CDRH1 148atc gac tac tcc atg cac 18Ile Asp Tyr Ser Met His 1 5 1496PRTArtificialSynthetic Construct 149Ile Asp Tyr Ser Met His 1 5 15051DNAArtificialhSH357-1-T3H CDRH2 150tgg atc aac acc tac acc ggc gag ccc acc tac tcc gac gac ttc aag 48Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ser Asp Asp Phe Lys 1 5 10 15 ggc 51Gly 15117PRTArtificialSynthetic Construct 151Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ser Asp Asp Phe Lys 1 5 10 15 Gly 15230DNAArtificialhSH357-1-T3H CDRH3 152tac tac agg tac gag agg gac ttc gac tac 30Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr 1 5 10 15310PRTArtificialSynthetic Construct 153Tyr Tyr Arg Tyr Glu Arg Asp Phe Asp Tyr 1 5 10 154520DNAArtificialIgGL constant+signal 154ctaggtaagc ttggtaccac ccaagctggc taggtaagct tgctagcgcc accatggtgc 60tgcagaccca ggtgttcatc tccctgctgc tgtggatctc cggcgcatat ggcgatatcg 120tgatgattaa acgtacggtg gccgccccct ccgtgttcat cttccccccc tccgacgagc 180agctgaagtc cggcaccgcc tccgtggtgt gcctgctgaa taacttctac cccagagagg 240ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg gaactcccag gagagcgtga 300ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc ctgagcaaag 360ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc ctgagctccc 420ccgtcaccaa gagcttcaac aggggggagt gttaggggcc cgtttaaacg ggtggcatcc 480ctgtgacccc tccccagtgc ctctcctggc cctggaagtt 5201551140DNAArtificialIgGH constant+signal 155ttggtaccac ccaagctggc taggtaagct tgctagcgcc accatgaaac acctgtggtt 60cttcctcctg ctggtggcag ctcccagatg ggtgctgagc caggtgcaat tgtgcaggcg 120gttagctcag cctccaccaa gggcccaagc gtcttccccc tggcaccctc ctccaagagc 180acctctggcg gcacagccgc cctgggctgc ctggtcaagg actacttccc cgaacccgtg 240accgtgagct ggaactcagg cgccctgacc agcggcgtgc acaccttccc cgctgtcctg 300cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc 360acccagacct acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaga 420gttgagccca aatcttgtga caaaactcac acatgcccac cctgcccagc acctgaactc 480ctggggggac cctcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 540cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 600ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc ccgggaggag 660cagtacaaca gcacgtaccg ggtggtcagc gtcctcaccg tcctgcacca ggactggctg 720aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 780accatctcca aagccaaagg ccagccccgg gaaccacagg tgtacaccct gcccccatcc 840cgggaggaga tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 900agcgacatcg ccgtggagtg ggagagcaat ggccagcccg agaacaacta caagaccacc 960cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1020agcaggtggc agcagggcaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1080cactacaccc agaagagcct ctccctgtct cccggcaaat gagatatcgg gcccgtttaa 1140156442DNAArtificialhSH348-1-T1L+signal 156aggtaagctt gctagcgcca ccatggtgct gcagacccag gtgttcatct ccctgctgct 60gtggatctcc ggcgcatatg gcgacatagt gatgacccag agccccctga gcctgcccgt 120gacccccggc gagcccgcct ccatcagctg ccggagcagc cagagcatcg tgcacagcaa 180cggcaacacc tacctggagt ggtacctgca gaagcccggc cagagccccc agctgctgat 240ctacaaggtg agcaaccggt tcagcggcgt gcccgaccgg ttcagcggca gcggcagcgg 300caccgacttc accctgaaga tcagccgggt ggaggccgag gacgtgggcg tgtactactg 360cttccagggc tcccacgtgc cctacacctt cggccagggc accaaggtgg agatcaagcg 420tacggtggcc gccccctccg tg 442157442DNAArtificialhSH348-1-T3L+signal 157aggtaagctt gctagcgcca ccatggtgct gcagacccag gtgttcatct ccctgctgct 60gtggatctcc ggcgcatatg gcgacgtgct gatgacccag agccccctga gcctgcccgt 120gacccccggc gagcccgcct ccatcagctg ccggagcagc cagagcatcg tgcacagcaa 180cggcaacacc tacctggagt ggtacctgca gaagcccggc cagagccccc agctgctgat 240ctacaaggtg agcaaccggt tcagcggcgt gcccgaccgg ttcagcggca gcggcagcgg 300caccgacttc accctgaaga tcagccgggt ggaggccgag gacgtgggcg tgtactactg 360cttccagggc tcccacgtgc cctacacctt cggccagggc accaaggtgg agatcaagcg 420tacggtggcc gccccctccg tg 442158460DNAArtificialhSH348-1-T1H+signal 158gtaagcttgc tagcgcacca tgaagcacct gtggttcttc ctgctgctgg tggccgcccc 60cagatgggtg ctgagccagg tgcagctggt gcagagcggc gccgaggtga agaagcccgg 120cagcagcgtg aaggtgagct gcaaggccag cggctacacc ttcacccact acagcatgca 180ctgggtgcgg caggcccccg gccagggcct ggagtggatg ggctggatca acacctacac 240cggcgagccc acctacgccg acgacttcaa gggccgggtg accatcaccg ccgacacctc 300cacctccacc gcctacctgg aactgagcag cctgcggagc gaggacaccg ccgtgtacta 360ctgcgccacc tactaccggt acgagcggga cttcgactac tggggccagc gcaccctggt 420gaccgtgagc tcagcctcct ccaccaaggg cccctccgtg 460159460DNAArtificialhSH348-1-T3H+signal 159gtaagcttgc tagcgcacca tgaagcacct gtggttcttc ctgctgctgg tggccgcccc 60cagatgggtg ctgagccaga tccagctggt gcagagcggc gccgaggtga agaagcccgg 120cagcagcgtg aaggtgagct gcaaggccag cggctacacc ttcacccact acagcatgca 180ctgggtgcgg caggcccccg gccagggcct gaagtggatg ggctggatca acacctacac 240cggcgagccc acctacgccg acgacttcaa gggccggttc gccttcagcc tggacacctc 300cacctccacc gcctacctgg aactgagcag cctgcggagc gaggacaccg ccgtgtacta 360ctgcgccacc tactaccggt acgagcggga cttcgactac tggggccagc gcaccctggt 420gaccgtgagc tcagcctcct ccaccaaggg cccctccgtg 460160442DNAArtificialhSH357-1-T1L+signal 160aggtaagctt gctagcgcca ccatggtgct gcagacccag gtgttcatct ccctgctgct 60gtggatctcc ggcgcatatg gcgacatagt gatgacccag agccccctga gcctgcccgt 120gacccccggc gagcccgcca gcatcagctg ccggagcagc cagagcatcg tgcacagcag 180cggcatcacc tacctggagt ggtacctgca gaagcccggc cagagccccc agctgctgat 240ctacaaggtg agcaaccggt tcagcggcgt gcccgaccgg ttcagcggca gcggcagcgg 300caccgacttc accctgaaga tcagccgggt ggaggccgag gacgtgggcg tgtactactg 360cttccagggc agccacgtgc cctacacctt cggccagggc accaaggtgg agatcaagcg 420tacggtggcc gccccctccg tg 442161442DNAArtificialhSH357-1-T3L+signal 161aggtaagctt gctagcgcca ccatggtgct gcagacccag gtgttcatct ccctgctgct 60gtggatctcc ggcgcatatg gcgacgtgct gatgacccag agccccctga gcctgcccgt 120gacccccggc gagcccgcca gcatcagctg ccggagcagc cagagcatcg tgcacagcag 180cggcatcacc tacctggagt ggtacctgca gaagcccggc cagagccccc agctgctgat 240ctacaaggtg agcaaccggt tcagcggcgt gcccgaccgg ttcagcggca gcggcagcgg 300caccgacttc accctgaaga tcagccgggt ggaggccgag gacgtgggcg tgtactactg 360cttccagggc agccacgtgc cctacacctt cggccagggc accaaggtgg agatcaagcg 420tacggtggcc gccccctccg tg 442162460DNAArtificialhSH357-1-T1H+signal 162gtaagcttgc tagcgcacca tgaagcacct gtggttcttc ctgctgctgg tggccgcccc 60cagatgggtg ctgagccagg tgcagctggt gcagtccggc gccgaggtga agaagcccgg 120cgcctccgtg aaggtgtcct gtaaggcctc cggctacacc ttcatcgact actccatgca 180ctgggtgagg caggcccccg gccagggcct ggagtggatg ggctggatca acacctacac 240cggcgagccc acctactccg acgacttcaa gggcagggtg accatcaccg ccgacacctc 300caccagcacc gcctacctgg agctgtcctc cctgaggtcc gaggacaccg ccgtgtacta 360ctgtgccacc tactacaggt acgagaggga cttcgactac tggggccagg gcaccctggt 420gacggtgagc tcagcctcct ccaccaaggg cccctccgtg 460163460DNAArtificialhSH357-1-T3H+signal 163gtaagcttgc tagcgcacca tgaagcacct gtggttcttc ctgctgctgg tggccgcccc 60cagatgggtg ctgagccaga tccagctggt gcagtccggc gccgaggtga agaagcccgg 120cgcctccgtg aaggtgtcct gtaaggcctc cggctacacc ttcatcgact actccatgca 180ctgggtgagg caggcccccg gccagggcct gaagtggatg ggctggatca acacctacac 240cggcgagccc acctactccg acgacttcaa gggcaggttc gccttctccc tggacacctc 300caccagcacc gcctacctgg agctgtcctc cctgaggtcc gaggacaccg ccgtgtacta 360ctgtgccacc tactacaggt acgagaggga cttcgactac tggggccagg gcaccctggt 420gacggtgagc tcagcctcct ccaccaaggg cccctccgtg 460

Claims

1. An isolated antibody which binds specifically to EPHA2 and recognizes an epitope recognized by an antibody produced by the hybridoma SH357-1 (FERM BP-10837), wherein the antibody has the following properties a) to d): (a) having no ability to phosphorylate EPHA2 tyrosine residues; (b) having an ADCC activity against EPHA2-expressing cells; (c) having a CDC activity against EPHA2-expressing cells; and (d) having an antitumor activity in vivo.

2. The isolated antibody according to claim 1 which specifically binds to a peptide consisting of an amino acid sequence represented by amino acid Nos. 426 to 534 of SEQ ID NO:8 in the sequence listing and has the following properties a) to e): (a) having no ability to phosphorylate EPHA2 tyrosine residues; (b) exhibiting no effect of decreasing an EPHA2 protein level; (c) having an ADCC activity against EPHA2-expressing cells; (d) having a CDC activity against EPHA2-expressing cells; and (e) having an antitumor activity in vivo.

3. The isolated antibody according to claim 2, wherein the antibody specifically binds to a peptide consisting of an amino acid sequence represented by amino acid Nos. 439 to 534 of SEQ ID NO:8 in the sequence listing.

4. The isolated antibody according to claim 2, wherein the antibody inhibits the phosphorylation of EPHA2 tyrosine residues induced by an EPHA2 ligand.

5. The isolated antibody according to claim 1, wherein the antibody does not inhibit EPHA2 ligand binding to EPHA2 but inhibits the phosphorylation of EPHA2 tyrosine residues induced by the ligand.

6. The isolated antibody according to claim 1 which specifically binds to a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:8 in the sequence listing, wherein the antibody is characterized by the following 1) and 2): 1) having a heavy chain peptide comprising an amino acid sequence represented by the general formula (I): -FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4- (I) wherein FRH1 represents an arbitrary amino acid sequence consisting of 18 to 30 amino acids; CDRH1 represents the amino acid sequence represented by SEQ ID NO:71 in the sequence listing or an amino acid sequence having deletion, substitution, or addition of one or more amino acids in the amino acid sequence; FRH2 represents an arbitrary amino acid sequence consisting of 14 amino acids; CDRH2 represents the amino acid sequence represented by SEQ ID NO:73 in the sequence listing or an amino acid sequence having deletion, substitution, or addition of one or more amino acids in the amino acid sequence; FRH3 represents an arbitrary amino acid sequence consisting of 32 amino acids; CDRH3 represents the amino acid sequence represented by SEQ ID NO:75 in the sequence listing or an amino acid sequence having deletion, substitution, or addition of one or more amino acids in the amino acid sequence; and FRH4 represents an arbitrary amino acid sequence consisting of 11 amino acids, wherein these amino acids are linked to each other through peptide bonds; and 2) having a light chain polypeptide comprising an amino acid sequence represented by the general formula (II): -FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4- (II) wherein FRL1 represents an arbitrary amino acid sequence consisting of 23 amino acids; CDRL1 represents the amino acid sequence represented by SEQ ID NO:77 in the sequence listing or an amino acid sequence having deletion, substitution, or addition of one or more amino acids in the amino acid sequence; FRL2 represents an arbitrary amino acid sequence consisting of 15 amino acids; CDRL2 represents the amino acid sequence represented by SEQ ID NO:79 in the sequence listing or an amino acid sequence having deletion, substitution, or addition of one or more amino acids in the amino acid sequence; FRL3 represents an arbitrary amino acid sequence consisting of 32 amino acids; CDRL3 represents the amino acid sequence represented by SEQ ID NO:81 in the sequence listing or an amino acid sequence having deletion, substitution, or addition of one or more amino acids in the amino acid sequence; and FRL4 represents an arbitrary amino acid sequence consisting of 10 amino acids, wherein these amino acids are linked to each other through peptide bonds.

7. The isolated antibody according to any one of claims 1 to 6, wherein the antibody is a humanized antibody, a human antibody, or an IgG antibody.

8. The isolated antibody according to any one of claims 1 to 6, wherein the antibody is a Fab, F(ab')2, Fv, scFv, a diabody, a linear antibody, or a multispecific antibody.

9. A polypeptide comprising: 1) an amino acid sequence represented by amino acid nos. 1 to 119 of SEQ ID NO:39 in the sequence listing; 2) an amino acid sequence represented by amino acid nos. 1 to 112 of SEQ ID NO:41 in the sequence listing; 3) an amino acid sequence represented by amino acid nos. 20 to 468 of SEQ ID NO:139 in the sequence listing; 4) an amino acid sequence represented by amino acid nos. 20 to 468 of SEQ ID NO:147 in the sequence listing; 5) an amino acid sequence represented by amino acid nos. 20 to 138 of SEQ ID NO:139 in the sequence listing; 6) an amino acid sequence represented by amino acid nos. 20 to 138 of SEQ ID NO:147 in the sequence listing; 7) an amino acid sequence represented by amino acid nos. 21 to 239 of SEQ ID NO:123 in the sequence listing; 8) an amino acid sequence represented by amino acid nos. 21 to 239 of SEQ ID NO:131 in the sequence listing; 9) an amino acid sequence represented by amino acid nos. 21 to 134 of SEQ ID NO:123 of the sequence listing; or 10) an amino acid sequence represented by amino acid nos. 21 to 134 of SEQ ID NO:131 in the sequence listing.

10. A pharmaceutical composition comprising at least one antibody selected from the antibodies according to any of claims 1 to 6 and a pharmaceutically acceptable diluent, carrier, solubilizing agent, emulsifying agent, preservative, and/or adjuvant.

11. A pharmaceutical composition comprising at least one antibody selected from the antibodies according to one of claims 1 to 6 formulated for cancer treatment.

12. A method for inhibiting tumor growth in a mammal, comprising administering any antibody selected from the group consisting of antibodies according to any one of claims 1 to 6.

13. The method according to claim 12, wherein the tumor is a tumor expressing EPHA2.

14. A polynucleotide encoding an antibody or a polypeptide according to any one of claims 1 to 6.

15. A host cell transformed with a polynucleotide according to claim 14.

16. A method for producing an antibody using a host cell according to claim 15.

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