RU2824285C2 - Cytotoxicity-inducing therapeutic agent - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is a bispecific antibody having cellular cytotoxicity, which includes: a first light chain comprising a first VL domain and a first CL domain; a second light chain comprising a second VL domain and a second CL domain. First heavy chain contains the first variable region of the heavy chain and the first constant region of the heavy chain. First constant region of the heavy chain is a non-wild type constant region containing one of the sequences SEQ ID NO: 23, 24, 25 and 26 with one or more amino acid substitutions. Second heavy chain comprises a second variable region of the heavy chain and a second constant region of the heavy chain. Second constant region of the heavy chain is a non-wild type constant region of the heavy chain containing one of the sequences SEQ ID NO: 23, 24, 25 and 26 with one or more amino acid substitutions. Amino acid sequence of the second constant region of the heavy chain has the same isotype as the first constant region of the heavy chain. First light chain and the first heavy chain are bound to form a first antigen-binding domain which binds to CD3. Second light chain and the second heavy chain are bound to form a second antigen-binding domain which does not bind to CD3 and which binds to a cancer antigen which is not CD3. First constant region of the heavy chain binds to a second constant region of the heavy chain, where, when measured by surface plasmon resonance, the ability of the associated first and second constant regions of the heavy chain to bind to this human receptor Fcγ reduced compared to the ability of a human wild-type IgG antibody of the same isotype as the bispecific antibody, to bind to the human Fcγ receptor as a result of at least one of one or more amino acid substitutions within the first and second constant regions of the heavy chain. At least one substitution in the first and second constant regions of the heavy chain is a change in the amino acid sequence compared with the amino acid sequence of the heavy chain constant regions of the wild-type human IgG antibody and wherein at least one of the amino acids which leads to a reduced ability to bind to the human receptor Fcγ, is in a position selected from the following positions according to EU numbering in each of the first and second constant regions of the heavy chain: 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331 and 332. Also disclosed is a pharmaceutical composition containing said antibody and having anti-cancer activity.

EFFECT: antibody according to the invention retains strong anti-cancer activity and has improved safety characteristics inherent in BiTE, has a long half-life in blood and induces cytotoxicity in relation to various cells.

48 cl, 26 dwg, 3 tbl, 11 ex

Description

Field of technology to which the invention relates

The present invention relates to polypeptide complexes that enable cancer treatment due to their ability to bring T cells closer to cancer target cells and to use the cytotoxic activity of T cells in relation to cancer target cells, to methods for producing polypeptide complexes and to therapeutic agents that contain said polypeptide complex as an active substance for inducing cellular cytotoxicity. The present invention also relates to pharmaceutical compositions intended for treating or preventing various types of cancer that contain the above-mentioned therapeutic agent for inducing cellular cytotoxicity as an active substance, and to therapeutic methods consisting in using said pharmaceutical compositions.

Prerequisites for the creation of an invention

To date, many therapeutic antibodies with very high antitumor activity have been developed as pharmaceutical agents for the treatment of cancer (Non-Patent Document 1). These therapeutic antibodies are known to exert their antitumor effects on cancer cells by inhibiting signals that are critical for the growth of cancer cells, inducing cell death signals, antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) (Non-Patent Document 2). ADCC is cytotoxicity caused by effector cells such as NK cells and macrophages against antibody-bound target cancer cells when the Fc region of the antibody binds to the Fc receptor on the effector cells. In this case, the complement complex binds to the complement-binding site in the structure of the antibody. CDC is cytotoxicity that occurs when a component of the complement system in a complex forms a pore in the cell membrane of the antibody-bound cell, enhancing the influx of water or ions into the cell. Although conventional therapeutic antibodies have very high potency, administration of these antibodies has so far resulted in only unsatisfactory therapeutic outcomes. Thus, there is a need to develop therapeutic antibodies with greater cancer cell killing ability.

In addition to the above antibodies that acquire ADCC as their antitumor mechanism by recruiting NK cells or macrophages as effector cells, T cell recruitment-inducing antibodies (TR antibodies) that acquire cytotoxicity as their antitumor mechanism by recruiting T cells as effector cells have been known since the 1980s (Non-Patent Documents 3-5). A TR antibody is a bispecific antibody that comprises an antibody to any of the subunits that form the T cell receptor (TCR) complex on T cells, in particular, an antibody that binds to the epsilon chain of CD3 and an antibody that binds to an antigen on target cancer cells. The T cell approaches the cancer cell when the TR antibody simultaneously binds to both the CD3 epsilon chain and the cancer antigen, and this results in an antitumor effect on the cancer cell due to the cytotoxic activity of the T cell.

An antibody designated as a "trifunctional antibody" is also known as a TR antibody (Non-patent documents 6 and 7). The trifunctional antibody is a complete bispecific IgG antibody in which one arm contains a Fab binding to a cancer antigen, and the other arm contains a Fab binding to the epsilon chain of CD3. The therapeutic effect on malignant ascites has been demonstrated by administering catumaxomab, a trifunctional antibody to EpCAM, into the peritoneal cavities of patients with malignant ascites that have cancer cells positive for EpCAM expression. The use of catumaxomab is approved in the European Union for the above-mentioned treatment.

In addition, it has now been established that a TR antibody designated as a "bispecific T cell-engaging antibody (BiTE)" has a potent antitumor effect (Non-patent Documents 8 and 9). BiTE is a TR antibody with a molecular form in which an scFv of an antibody to a cancer antigen is linked to an scFv of an antibody to the epsilon chain of CD3 via a short polypeptide linker. BiTEs are known to have antitumor activity superior to that of various known TR antibodies (Non-patent Documents 9 and 10). In particular, compared with other TR antibodies, BiTEs have antitumor activity even when administered at a significantly lower concentration and at a lower effector cell/cancer cell ratio (ET ratio). It has also been demonstrated that their action can be successfully exerted without the need for prior activation of effector cells by IL-2, CD28 agonist antibody, etc. Blinatumomab (MT103), which is a BiTE to CD19, has a more potent cytotoxic activity against cancer cells in vitro than Rituxan, which is known to have very good clinical activity. In addition, extremely high activity of blinatumomab has been demonstrated in phase I and II clinical trials currently underway (Non-patent document 11).

Based on the fact that catumaxomab is approved as a therapeutic agent for which clinical efficacy has been demonstrated, and that multiple BiTEs, including blinatumomab, have potent antitumor activity, it has been suggested that TR antibodies that recruit T cells as effector cells may have significantly greater potential as antitumor agents than conventional antibodies whose mechanism of action is related to ADCC.

However, it is known that the trifunctional antibody simultaneously binds to both T cell and cell such as NK cell or macrophage in a cancer antigen-independent manner, and as a result, the receptors expressed on the cells are cross-linked, and the expression of various cytokines is induced in a cancer antigen-independent manner. It is assumed that systemic administration of the trifunctional antibody causes cytokine storm-type side effects due to said induction of cytokine expression. In fact, in a phase I clinical trial, it was found that the maximum tolerated dose of systemic administration of catumaxomab in patients with non-small cell lung cancer was a very low dose of 5 μg/body, and that administration of a higher dose caused serious side effects (Non-patent Document 12). When catumaxomab was administered at this low dose, its blood level could never reach the effective value. This means that the expected antitumor effect cannot be achieved when catumaxomab is administered at the indicated low dose.

However, unlike catumaxomab, BiTE does not contain an Fcγ receptor binding site and therefore does not cross-link with receptors expressed on T cells and cells such as NK cells and macrophages in a cancer antigen-dependent manner. Thus, it has been demonstrated that BiTE does not cause cancer antigen-independent cytokine induction, which was found with catumaxomab. However, since BiTE is a modified low-molecular-weight antibody molecule without the Fc region, the problem is that its half-life in the blood after administration to a patient is significantly shorter than that of IgG antibodies, which are usually used as therapeutic antibodies. In fact, according to published data, the blood half-life of BiTE when administered in vivo is approximately several hours (Non-patent documents 13 and 14). In clinical trials of blinatumomab, it was administered by continuous intravenous infusion using a minipump. This method of administration is not only extremely inconvenient for patients, but also has the potential risk of medical complications related to device malfunction, etc. Therefore, this method of administration cannot be considered desirable.

Documents characterizing the known level of technology

[Non-patent documents]

[Non-patent document 1]: Clin Cancer Res. 16(1), 2010, pp. 11-20.

[Non-patent document 2]: Drug Des Devel Ther 3, 2009, pp. 7-16.

[Non-patent document 3]: Nature 314 (6012), 1985, pp. 628-631.

[Non-patent document 4]: Int J Cancer 41(4), 1988, pp. 609-615.

[Non-patent document 5]: Proc Natl Acad Sci USA 83(5), 1986, pp. 1453-1457.

[Non-patent document 6]: Cancer Treat Rev. 36(6), 2010, pp. 458-467.

[Non-patent document 7]: Expert Opin Biol Ther 10(8), 2010, pp. 1259-1269.

[Non-Patent Document 8]: Proc Natl Acad Sci USA. 92(15), 1995, pp. 7021-7025.

[Non-patent document 9]: Drug Discov Today, 10 (18), 2005, pp. 1237-1244.

[Non-patent document 10]: Trends Biotechnol 22(5), 2004, pp. 238-244.

[Non-Patent Document 11]: Science 321 (5891), 2008, pp. 974-977.

[Non-Patent Document 12]: Cancer Immunol Immunother 56 (10), 2007, cc. 1637-1644.0

[Non-Patent Document 13]: Cancer Immunol Immunother. 55 (5), 2006, cc. 503-514.

[Non-Patent Document 14]: Cancer Immunol Immunother. 58 (1), 2009, cc. 95-109.

Brief summary of the invention

Objectives underlying the present invention

The present invention was created taking into account the above circumstances. The present invention is based on the task of creating polypeptide complexes suitable for cancer treatment, which have the ability to bring T cells closer to a cancer target cell and allow the use of the cytotoxic activity of T cells in relation to cancer target cells, methods for obtaining polypeptide complexes and therapeutic agents that contain the said polypeptide complex as an active substance for inducing cellular cytotoxicity. Another task of the present invention is to develop pharmaceutical compositions intended for the treatment or prevention of various types of cancer, which contain the above-mentioned therapeutic agent for inducing cytotoxicity as an active substance, and therapeutic methods consisting in using the said pharmaceutical compositions. Means for solving the said problems

In the process of creating the invention, new polypeptide complexes were discovered that retain the strong antitumor activity inherent in BiTE and have a long half-life in the blood, as well as very high safety, which leads to the absence of induction of a "cytokine storm" independent of the cancer antigen, etc. In the process of creating the present invention, it was also established that when replacing the antigen-binding domains of the polypeptide complexes, the polypeptide complexes can damage various target cells. Based on the above data, in the process of creating the present invention, it was demonstrated that the polypeptide complexes proposed in the present invention damage cancer cells. In the process of creating the present invention, it was also established that by regulating the association at the CH1/CL interface and introducing " knobs - into - holes" (KiH) modifications into the polypeptide complexes, more effective cellular cytotoxicity is achieved. In addition, the present invention has demonstrated that various types of cancer can be treated or prevented using therapeutic agents for inducing cellular cytotoxicity that contain the polypeptide complex proposed in the present invention as an active ingredient.

More specifically, the present invention provides:

[1] a polypeptide complex that contains:

(1) antigen-binding domain;

(2) a domain containing an Fc region that has reduced Fcγ receptor binding activity; and

(3) T-cell receptor complex binding domain;

[2] the polypeptide complex according to claim [1], wherein the T cell receptor complex binding domain is a T cell receptor binding domain;

[3] the polypeptide complex according to claim [1], wherein the T cell receptor complex binding domain is a CD3 binding domain;

[4] a polypeptide complex according to one of paragraphs [1]-[3], in which the antigen-binding domain is a bivalent antigen-binding domain;

[5] the polypeptide complex according to claim [4], wherein the divalent antigen-binding domain is a domain having the structure F(ab')2;

[6] a polypeptide complex according to claim [5], in which two polypeptides forming the constant region of the heavy chain domain that has the structure F(ab')2 are individually linked to either of the two polypeptides forming the Fc domain;

[7] the polypeptide complex according to claim [6], wherein the CD3-binding domain is linked to either or both of the CH3s forming the Fc domain;

[8] the polypeptide complex according to claim [7], in which the Fv fragment of the heavy chain, forming the CD3-binding domain, is linked to one of the CH3s forming the Fc-domain, and the Fv fragment of the light chain, forming the CD3-binding domain, is linked to the other CH3 forming the Fc-domain;

[9] the polypeptide complex according to claim [8], in which the CH1 domain of the antibody is linked to the Fv fragment of the heavy chain, forming a CD3-binding domain, and the CL domain of the antibody is linked to the Fv fragment of the light chain;

[10] the polypeptide complex according to claim [6], wherein the CD3-binding domain is linked to either or both of the CLs that form F(ab')2;

[11] the polypeptide complex according to claim [6], wherein the CD3-binding domain is linked to either or both of the VHs that form F(ab')2;

[12] the polypeptide complex according to claim [6], wherein the CD3-binding domain is linked to either or both of the VLs that form F(ab')2;

[13] a polypeptide complex according to one of claims [1] to [12], wherein the CD3-binding domain is Fv;

[14] a polypeptide complex according to one of [1]-[7] and [10]-[12], in which the CD3-binding domain is Fab;

[15] a polypeptide complex according to one of [1]-[7] and [10]-[12], in which the CD3-binding domain is an scFv;

[16] a polypeptide complex according to one of claims [1] to [15], wherein the CD3-binding domain is monovalent;

[17] a polypeptide complex according to one of claims [1] to [3], wherein the antigen-binding domain is a monovalent scFv and a monovalent Fab;

[18] the polypeptide complex according to claim [17], wherein the monovalent scFv is linked to one of the polypeptides forming the Fc domain via an scFv that forms the CD3-binding domain; the Fv fragment of the heavy chain of the monovalent Fab is linked to one of the polypeptides forming the Fc domain via the CH1 domain; and the Fv fragment of the light chain of the Fab is linked to the CL domain;

[19] a polypeptide complex according to one of claims [1] to [3], wherein the antigen-binding domain is a divalent scFv;

[20] the polypeptide complex according to claim [19], in which one monovalent scFv is linked to one of the polypeptides forming the Fc domain via the Fv fragment of the heavy chain forming the CD3-binding domain, and another monovalent scFv is linked to another polypeptide forming the Fc domain via the Fv fragment of the light chain forming the CD3-binding domain;

[21] the polypeptide complex according to claim [19], in which one monovalent scFv is linked to one of the polypeptides forming the Fc domain via an scFv forming the CD3 binding domain, and the other monovalent scFv is linked to another polypeptide forming the Fc domain;

[22] a polypeptide complex according to one of claims [1] to [3], wherein the antigen-binding domain and the T-cell receptor complex-binding domain are each a monovalent Fab;

[23] the polypeptide complex according to claim [22], wherein the Fv fragment of the heavy chain of a monovalent Fab forming an antigen-binding domain is linked to one of the polypeptides forming the Fc domain via the CH1 domain and the Fv fragment of the light chain of the Fab is linked to the CL domain; and the Fv fragment of the heavy chain of the Fab forming the T-cell receptor-binding domain is linked to another polypeptide forming the Fc domain via the CH1 domain and the Fv fragment of the light chain of the Fab is linked to the CL domain;

[24] the polypeptide complex according to claim [22], in which the Fv fragment of a monovalent Fab forming an antigen-binding domain is linked to one of the polypeptides forming an Fc domain via a CH1 domain and the Fv fragment of the light chain of the Fab is linked to a CL domain; and the Fv fragment of the light chain of the Fab forming a T-cell receptor-binding domain is linked to another polypeptide forming an Fc domain via a CH1 domain and the Fv fragment of the heavy chain of the Fab is linked to a CL domain;

[25] the polypeptide complex according to claim [22], wherein the Fv fragment of the heavy chain of a monovalent Fab forming an antigen-binding domain is linked to one of the polypeptides forming the Fc domain via a CH1 domain and the Fv fragment of the light chain of the Fab is linked to a CL domain; and the Fv fragment of the heavy chain of the Fab forming a T-cell receptor-binding domain is linked to another polypeptide forming the Fc domain via a CL domain and the Fv fragment of the light chain of the Fab is linked to a CH1 domain;

[26] the polypeptide complex according to claim [22], wherein the Fv fragment of the heavy chain of a monovalent Fab forming a T-cell receptor binding domain is linked to one of the polypeptides forming the Fc domain via the CH1 domain and the Fv fragment of the light chain of the Fab is linked to the CL domain; and the Fv fragment of the light chain of the Fab forming the antigen-binding domain is linked to another polypeptide forming the Fc domain via the CH1 domain and the Fv fragment of the heavy chain of the Fab is linked to the CL domain;

[27] the polypeptide complex according to claim [22], wherein the Fv fragment of the heavy chain of a monovalent Fab forming a T-cell receptor binding domain is linked to one of the polypeptides forming the Fc domain via the CH1 domain; and the Fv fragment of the light chain of the Fab is linked to the CL domain; and the Fv fragment of the heavy chain of the Fab forming the antigen-binding domain is linked to another polypeptide forming the Fc domain via the CL domain, and the Fv fragment of the light chain of the Fab is linked to the CH1 domain;

[28] a polypeptide complex according to claim [22] which contains:

(1) an antigen-binding domain in which the Fv fragment of the heavy chain of the monovalent Fab structure that binds to the antigen is linked through the CH1 domain to one of the polypeptides forming the Fc domain, and the Fv fragment of the light chain of the Fab structure is linked to the CL domain; and

(2) a T cell receptor complex binding domain in which the Fv fragment of the heavy chain of the monovalent Fab structure, which binds to the T cell receptor complex, is linked via CH1 to another polypeptide forming the Fc domain, and the Fv fragment of the light chain of the Fab structure is linked to the CL domain;

wherein the electrical charges of the CH1 and CL domains are controlled in such a way that the Fv fragment of the heavy chain of the antigen-binding domain is connected to the Fv fragment of the light chain of the antigen-binding domain or the Fv fragment of the heavy chain of the T-cell receptor-binding domain is connected to the Fv fragment of the light chain of the T-cell receptor-binding domain;

[29] the polypeptide complex according to claim [28], wherein the amino acid residue in the CH1 domain linked to the Fv fragment of the heavy chain of the T cell receptor complex binding domain has the same type of electrical charge as the amino acid residue in the CL domain linked to the Fv fragment of the antigen binding domain;

[30] the polypeptide complex according to claim [28], wherein the amino acid residue in the CH1 domain linked to the Fv fragment of the heavy chain of the antigen-binding domain has the same type of electrical charge as the amino acid residue in the CL domain linked to the Fv fragment of the light chain of the T-cell receptor complex binding domain;

[31] the polypeptide complex according to [28], in which the amino acid residue in the CH1 domain linked to the Fv fragment of the heavy chain of the T cell receptor complex-binding domain has the same type of electrical charge as the amino acid in the CL domain linked to the Fv fragment of the light chain of the antigen-binding domain, and the amino acid residue in the CH1 domain linked to the Fv fragment of the heavy chain of the antigen-binding domain has the same type of electrical charge as the amino acid in the CL domain linked to the Fv fragment of the light chain of the T cell receptor complex-binding domain;

[32] the polypeptide complex according to claim [29] or claim [31], wherein the amino acid residue in the CH1 domain linked to the Fv fragment of the heavy chain of the T cell receptor binding domain of the complex has an electrical charge opposite to the charge of the amino acid residue in the CL domain linked to the Fv fragment of the light chain of the T cell receptor binding domain;

[33] the polypeptide complex according to claim [30] or claim [31], in which the amino acid residue in the CH1 domain linked to the Fv fragment of the heavy chain of the antigen-binding domain has an electrical charge opposite to the charge of the amino acid residue in the CL domain linked to the Fv fragment of the light chain of the antigen-binding domain;

[34] a polypeptide complex according to one of claims [22] to [33], wherein the T cell receptor complex binding domain is a T cell receptor binding domain;

[35] the polypeptide complex according to claim [34], wherein the T-cell receptor binding domain is a CD3 binding domain;

[36] a polypeptide complex according to claim [32] or claim [33], wherein the amino acid residues in the CH1 and CL domains are one, two or more combinations of amino acid residues selected from the group consisting of combinations of:

(a) the amino acid residue at position 147 (EU numbering) in the CH1 domain and the amino acid residue at position 180 (EU numbering) in the CL domain;

(b) the amino acid residue at position 147 (EU numbering) in the CH1 domain and the amino acid residue at position 131 (EU numbering) in the CL domain;

(c) the amino acid residue at position 147 (EU numbering) in the CH1 domain and the amino acid residue at position 164 (EU numbering) in the CL domain;

(d) the amino acid residue at position 147 (EU numbering) in the CH1 domain and the amino acid residue at position 138 (EU numbering) in the CL domain;

(d) the amino acid residue at position 147 (EU numbering) in the CH1 domain and the amino acid residue at position 123 (EU numbering) in the CL domain; and

(e) the amino acid residue at position 175 (EU numbering) in the CH1 domain and the amino acid residue at position 160 (EU numbering) in the CL domain;

and in which the amino acid residue in the CH1 domain has an electrical charge opposite to the charge of the amino acid residue in the CL domain;

[37] a polypeptide complex according to claim [36], wherein the amino acid residues are selected from a group further comprising a combination of amino acid residues:

(g) the amino acid residue at position 213 (EU numbering) in the CH1 domain and the amino acid residue at position 123 (EU numbering) in the CL domain;

[38] a polypeptide complex according to claim [36] or claim [37], wherein the amino acid residue having the opposite electrical charge is selected from an amino acid residue belonging to any of the groups:

(X) glutamic acid (E) and aspartic acid (D); or

(Y) lysine (K), arginine (R) and histidine (H);

[39] a polypeptide complex according to one of paragraphs [36]-[38], in which the amino acid residues having opposite electrical charges are:

Lys at position 175 (EU numbering) in the CH1 domain and Glu at positions 131, 160 and 180 (EU numbering) in the CL domain;

[40] a polypeptide complex according to one of claims [36]-[38], in which the amino acid residues having opposite electrical charges are:

Glu at positions 147 and 175 (EU numbering) in the CH1 domain and Lys at positions 131, 160 and 180 (EU numbering) in the CL domain;

[41] the polypeptide complex according to claim [40], wherein the amino acid residue at position 213 (EU numbering) in the CH1 domain is Glu and the amino acid residue at position 123 (EU numbering) in the CL domain is Lys;

[42] a polypeptide complex according to one of claims [1] to [41], wherein the Fc domain is characterized by reduced Fcγ receptor binding activity in relation to FcγI, FcγIIA, FcγIIB, FcγIIIA and/or FcγIIIB;

[43] a polypeptide complex according to one of claims [1] to [42], wherein the Fc domain is any of the Fc domains having SEQ ID NOs: 23, 24, 25 and 26, wherein the amino acid(s) forming the Fc domain is(are) altered by mutation;

[44] the polypeptide complex according to claim [43], wherein the Fc domain comprises any of the following amino acids:

an amino acid sequence extending from position 118 to position 260 (EU numbering), which is the sequence SEQ ID NO: 24; or

the amino acid sequence extending from position 261 to position 447 (EU numbering), which is the sequence SEQ ID NO: 26;

[45] the polypeptide complex according to claim [43], wherein the amino acids forming the Fc domain comprise a mutation at any of the following positions: 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331 and 332 (EU numbering);

[46] the polypeptide complex according to claim [45], wherein the Fc domain comprises a mutation in the amino acids of SEQ ID NO: 23 that form the Fc domain;

[47] the polypeptide complex of claim [46], wherein the Fc domain is an Fc domain comprising an amino acid substitution at position 233, 234, 235, 236, 237, 327, 330 or 331 (EU numbering) with an amino acid present at the corresponding position (EU numbering) in the corresponding IgG2 or IgG4;

[48] the polypeptide complex according to claim [46], wherein the Fc domain comprises an amino acid mutation at position 234, 235 or 297 (EU numbering);

[49] the polypeptide complex according to claim [48], wherein the amino acid(s) at position 234, 235 and/or 297 is(are) replaced by alanine;

[50] a polypeptide complex according to one of paragraphs [43]-[49], in which the sequences of the two polypeptides forming the Fc domain differ from each other;

[51] a polypeptide complex according to one of paragraphs [1] to [50], in which the amino acid at position 349 is replaced by cysteine and the amino acid at position 366 (EU numbering) is replaced by tryptophan in the sequence of amino acid residues of one of the two polypeptides forming the Fc domain; and in which the amino acid at position 356 is replaced by cysteine, the amino acid at position 366 is replaced by serine, the amino acid at position 368 is replaced by alanine and the amino acid at position 407 (EU numbering) is replaced by valine in the sequence of amino acid residues of the other polypeptide;

[52] a polypeptide complex according to one of paragraphs [1] to [50], in which the amino acid at position 356 (EU numbering) is replaced by lysine in the sequence of amino acid residues of one of the two polypeptides forming the Fc domain; the amino acid at position 439 (EU numbering) is replaced by glutamic acid in the other polypeptide; and the amino acid at position 435 (EU numbering) is replaced by arginine in the sequence of amino acid residues of any of the two polypeptides;

[53] a polypeptide complex according to claim [51] or claim [52], in which the GK sequence is removed as a result of deletion from the carboxyl termini of the two polypeptides forming the Fc domain;

[54] a polypeptide complex according to one of claims [1] to [53], in which the antigen-binding domains bind to the same epitope;

[55] the polypeptide complex according to [54], wherein said epitope is present in a protein comprising the amino acid sequence of SEQ ID NO: 2;

[56] the polypeptide complex according to [54], wherein said epitope is present in a protein comprising the amino acid sequence of SEQ ID NO: 4;

[57] a polypeptide complex according to one of paragraphs [1] to [53], in which each of the antigen-binding domains binds to a different (different) epitope;

[58] the polypeptide complex according to claim [57], wherein said other epitope is present in a protein comprising the amino acid sequence of SEQ ID NO: 2;

[59] the polypeptide complex according to claim [57], wherein the other epitope is present in a protein comprising the amino acid sequence of SEQ ID NO: 4;

[60] a polynucleotide encoding a polypeptide complex according to one of paragraphs [1]-[59];

[61] a vector containing the polynucleotide of claim [60]; [62] a cell containing the vector of claim [61];

[63] a method for obtaining a polypeptide complex, which consists of culturing a cell according to paragraph [62] and isolating the polypeptide complex from the culture supernatant;

[64] a therapeutic agent for inducing cellular cytotoxicity, which contains as an active substance a polypeptide complex according to one of claims [1]-[59];

[65] a therapeutic agent according to claim [64], wherein the therapeutic agent for inducing cellular cytotoxicity is a therapeutic agent used for cancer;

[66] a therapeutic agent according to claim [65], wherein the cancer is liver cancer or lung cancer;

[67] a method for treating or preventing cancer, which comprises administering the polypeptide complex according to one of paragraphs [1]-[59] to a patient in need thereof; and

[68] a therapeutic or prophylactic method according to claim [67], wherein the cancer is liver cancer or lung cancer.

The present invention also relates to kits intended for use in the method of the present invention, which comprise a polypeptide complex of the present invention or a polypeptide complex obtained by the method of the present invention. The present invention also relates to the use of a polypeptide complex of the present invention or a polypeptide complex obtained by the method of the present invention for the preparation of a therapeutic agent for inducing cellular cytotoxicity. The present invention also relates to polypeptide complexes of the present invention or polypeptide complexes obtained by the methods of the invention for use in the method of the present invention.

Results of the invention

The present invention provides novel polypeptide complexes that retain the strong antitumor activity of BiTE and have a long half-life in blood, as well as very high safety, which results in the absence of induction of a cancer antigen-independent "cytokine storm" or the like. When the antigen-binding domain of the polypeptide complex proposed in the present invention is changed, therapeutic agents that contain the polypeptide complex as an active substance for inducing cellular cytotoxicity can specifically affect and damage various cells, including cancer cells. Thus, various types of cancer can be treated or prevented. This provides the desired treatment, which is highly safe and convenient and reduces the physical burden on patients.

Brief description of the drawings

The drawings show:

Fig. 1 is a graph showing a comparison of the cytotoxic activity of the following antibodies to GPC3 (an antigen belonging to the GPI-anchored receptor family): GPC3 ERY1 (GPC3 BiTE), GPC3 ERY2, and IgG-type antibodies to GPC3. The filled square shaded triangle and an unpainted square the cytotoxic activity of GPC3 ERY1 (GPC3 BiTE), GPC3 ERY2 and IgG-type antibodies to GPC3 are indicated, respectively;

Fig. 2 shows a graph that compares the cytotoxic activity of GPC3 BiTE and GPC3 ERY5. The filled square and an unpainted circle the cytotoxic activity of GPC3 BiTE and GPC3 ERY5 is indicated, respectively;

Fig. 3 is a graph showing a comparison of the cytotoxic activity of GPC3 BiTE and GPC3 ERY6. The filled square and a shaded triangle the cytotoxic activity of GPC3 BiTE and GPC3 ERY6, respectively, is indicated;

Fig. 4 shows a graph that compares the cytotoxic activity of GPC3 BiTE and GPC3 ERY7. The filled square and a painted rhombus the cytotoxic activity of GPC3 BiTE and GPC3 ERY7, respectively, is indicated;

Fig. 5 is a graph showing a comparison of the cytotoxic activity of GPC3 BiTE, GPC3 ERY8-2, GPC3 ERY9-1 and GPC3 ERY10-1. The filled square shaded triangle open circle (○) and open square (□) indicate the cytotoxic activity of GPC3 BiTE, GPC3 ERY8-2, GPC3 ERY9-1 and GPC3 ERY10-1, respectively;

Fig. 6 is a graph demonstrating the in vivo antitumor activity of GPC3 ERY8-2 on the pre-mixed PC-10 model. The open square (□) and filled diamond changes in tumor volume in the GPC3 ERY7-treated group and the control (PBS-treated) group, respectively, are indicated;

Fig. 7 is a graph demonstrating the in vivo antitumor activity of GPC3 ERY10-1 on the pre-mixed PC-10 model. The open square (□) and filled diamond changes in tumor volume in the group treated with GPC3 ERY10-1 and the control (PBS treatment) group, respectively, are indicated;

Fig. 8 is a graph demonstrating the in vivo antitumor activity of GPC3 ERY10-1 in a PC-10 T-cell transfer model. The open square (□) and filled diamond changes in tumor volume in the group treated with GPC3 ERY10-1 and the control (PBS treatment) group, respectively, are indicated;

Fig. 9 is a graph showing the time dependence of plasma concentrations of GPC3 ERY9-1 and GPC3 ERY10-1 determined using GPC3-expressing Ba/F3 cells. The filled diamond and the open square (□) indicates the time dependence of the plasma concentrations of GPC3 ERY9-1 and GPC3 ERY10-1, respectively;

Fig. 10 is a graph showing the time dependence of plasma concentrations of GPC3 ERY9-1 and GPC3 ERY10-1 determined using CD3-expressing Ba/F3 cells. The filled diamond and an unpainted square the time dependence of plasma concentrations of GPC3 ERY9-1 and GPC3 ERY10-1 is shown, respectively;

Fig. 11 is a graph demonstrating the ability of GPC3 BiTE, GPC3 ERY9-1, GPC3 ERY10-1, GPC3 ERY15-1 and catumaxomab to induce cytokines in a cancer antigen-independent manner;

Fig. 12 is a graph showing the in vitro cytotoxicity of GPC3 ERY18 LI, GPC3 ERY18L2, GPC3 ERY18L3, GPC3 ERY18L4 and GPC3 ERY18S1. The filled triangle filled circle filled square unpainted square and an unpainted diamond the cytotoxic activity of GPC3 ERY18 LI, GPC3 ERY18 L2, GPC3 ERY18 L3, GPC3 ERY18 L4 and GPC3 ERY18 S1, respectively, is indicated;

Fig. 13 is a graph showing a comparison of the in vitro cytotoxic activity of GPC3 ERY18 L3 and GPC3 ERY10-1. The filled square and the open square (□) indicates the cytotoxic activity of GPC3 ERY18 L3 and GPC3 ERY10-1, respectively;

Fig. 14 shows a graph comparing the in vitro cytotoxic activity of GPC3 ERY19-3 and GPC3 BiTE. The unshaded square and a filled square the cytotoxic activity of GPC3 ERY19-3 and GPC3 BiTE, respectively, is indicated;

Fig. 15A is a chromatogram showing the results of an analysis using gel filtration of SM in which NTAlL/NTAlR/GC33-k0 is expressed. Fig. 15B is a chromatogram showing the results of an analysis using gel filtration of SM in which NTA2L/NTA2R/GC33-k0 is expressed;

Fig. 16 shows diagrams showing the domains that form the following polypeptide complexes described in the examples provided herein: GPC3 BiTE, GPC3 ERY2, GPC3 ERY5, GPC3 ERY6, GPC3 ERY7, GPC3 ERY8-2, GPC3 ERY9-1, GPC3 ERY 10-1, GPC3 ERY15, GPC3 ERY18, and GPC3 ERY19-3. The domain shaded with cross lines represents the variable region of the H chain of an antibody to a cancer antigen (GPC3, EpCAM, EGFR); the domain with diagonal lines represents the variable region of the L chain of an antibody to a cancer antigen (GPC3, EpCAM, EGFR); the domain with dotted lines represents the variable region of the H chain of an antibody to CD3; The shaded domain represents the variable region of the CD3 antibody L chain; the unshaded domain represents the constant region of the antibody; the cross represents a silent mutation in Fc and the asterisk represents a mutation that enhances Fc heteromeric association;

Fig. 17 - diagrams of GPC3 BiTE (A); GPC3 ERY 10 (B); GPC3 ERY2 (C); GPC3 ERY5 (D); GPC3 ERY6 (E); GPC3 ERY7 (F); GPC3 ERY8-2 (G); GPC3 ERY9-1 (3); GPC3 ERY10-1 (I); GPC3 ERY15 (K); GPC3 ERY18 (L) and GPC3 ERY19-3 (M);

in Fig. 18 the amino acid residues forming the Fc domains of IgG1, IgG2, IgG3 and IgG4 and their EU numbering according to Cabot (in the context of the present description, also designated as the “EU index”);

Fig. 19 is a diagram showing the domains that form the polypeptide complexes described in the examples provided in the present description: GPC3 ERY17-2, GPC3 ERY17-3, EpCAM ERY17-2 and EpCAM ERY17-3. The domain shaded with cross lines represents the variable region of the H chain of the antibody to a cancer antigen (GPC3, EpCAM, EGFR); the domain with diagonal lines represents the variable region of the L chain of the antibody to a cancer antigen (GPC3, EpCAM, EGFR); the domain with dotted lines represents the variable region of the H chain of the antibody to CD3; the filled domain represents the variable region of the L chain of the antibody to CD3; the unfilled domain represents the constant region of the antibody; the cross denotes a silent mutation in Fc and the asterisk denotes a mutation that enhances Fc heteromeric association;

Fig. 20 shows a graph comparing the cytotoxic activity of GPC3 BiTE, GPC3 ERY17-2, GPC3 ERY17-3 and GPC3 ERY10-1. The filled square shaded triangle open circle (○) and open square (□) indicate the cytotoxic activity of GPC3 BiTE, GPC3 ERY17-2, GPC3 ERY17-3 and GPC3 ERY10-1, respectively;

Fig. 21 is a graph demonstrating the in vivo antitumor activity of GPC3 ERY17-2 in a PC-10 T-cell transfer model. The open square (□) and filled diamond changes in tumor volume in the group treated with GPC3 ERY17-2 and the control (PBS treatment) group, respectively, are indicated;

Fig. 22 is a graph showing a comparison of the cytotoxic activity of GPC3 ERY17-2 and GPC3 ERY17-2-M20. The filled triangle and an unpainted circle the cytotoxic activity of GPC3 ERY17-2 and GPC3 ERY17-2-M20, respectively, is indicated;

Fig. 23 shows a graph that compares the cytotoxic activity of EpCAM ERY17-2 and EpCAM ERY17-3. The filled triangle and an unpainted square the cytotoxic activity of EpCAM ERY17-2 and EpCAM ERY17-3, respectively, is indicated;

Fig. 24 shows diagrams showing the domains that form the polypeptide complexes described in the examples provided in the present description: GM1, GM2 and GM0. In "A" a polypeptide complex is shown in which association at the CH1/CL interface is regulated and knob-and-hole (KiH) modifications are introduced. In "B" a polypeptide complex is shown in which association at the CH1/CL interface is not regulated or KiH modifications are not introduced. The domain shaded with cross lines denotes the variable region of the H chain of an antibody to a cancer antigen (GPC3, EpCAM, EGFR); the domain with diagonal lines denotes the variable region of the L chain of an antibody to a cancer antigen (GPC3, EpCAM, EGFR); the domain with dotted lines denotes the variable region of the H chain of an antibody to CD3; The shaded domain represents the variable region of the CD3 antibody L chain; the unshaded domain represents the constant region of the antibody; the cross represents a silent mutation in Fc and the asterisk represents a mutation that enhances Fc heteromeric association;

and the donut-shaped symbol represents a mutation that regulates interactions at the CH1/CL interface;

Fig. 25 is a graph showing a comparison of the cytotoxic activity of GM1, GM2 and GM0. The filled triangle an unpainted square (□) and an unpainted circle the cytotoxic activity of GM1, GM2 and GM3 is indicated, respectively;

Fig. 26 shows a graph demonstrating the cytotoxic activity of EGFR ERY17-2. The filled triangle The cytotoxic activity of EGFR ERY17-2 is indicated.

Embodiments of the invention

The following definitions are provided for the purpose of explaining the present invention. Antibody

As used herein, "antibody" refers to a naturally occurring immunoglobulin or an immunoglobulin produced in whole or in part by synthesis. Antibodies can be isolated from naturally occurring sources, such as naturally occurring plasma and serum, or from culture supernatants of antibody-producing hybridomas. Alternatively, antibodies can be partially or completely synthesized using techniques such as genetic recombination. Preferred antibodies are, for example, antibodies belonging to an immunoglobulin isotype or subclass thereof. Known human immunoglobulins include antibodies of the following nine classes (isotypes): IgG1, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, and IgM. Of these isotypes, antibodies of the invention include IgG1, IgG2, IgG3, and IgG4.

Methods for obtaining an antibody with the desired binding activity are known to those skilled in the art. An example is given below, which describes a method for obtaining an antibody (anti-GPC3 antibody) that binds to GPC3, which belongs to the GPI-anchored receptor family (Int J Cancer. 103(4), 2003, pp.455-465). According to the example described below, antibodies that bind to an antigen other than GPC3 can also be obtained.

Anti-GPC3 antibodies can be produced as polyclonal or monoclonal antibodies using known methods. Anti-GPC3 antibodies are preferably produced as mammalian-derived monoclonal antibodies. Said mammalian-derived antibodies include antibodies produced using hybridomas or host cells transformed with an expression vector that carries an antibody gene created using genetic engineering methods.

Hybridomas producing monoclonal antibodies can be obtained using known methods, such as those described below. Specifically, mammals are immunized using conventional immunization methods using the GPC3 protein as a sensitizing antigen. The resulting immune cells are fused with known parent cells using conventional fusion methods. Hybridomas producing an antibody to GPC3 can then be selected by screening for monoclonal antibody-producing cells using conventional screening methods.

In particular, monoclonal antibodies are produced according to the method described below. First, the GPC3 gene, the nucleotide sequence of which is presented in RefSeq under the registration number NM_001164617.1 (SEQ ID NO: 1), can be expressed to obtain the GPC3 protein, the sequence of which is presented in RefSeq under the registration number NP_001158089.1 (SEQ ID NO: 2), which can be used as a sensitizing antigen for producing an antibody. For this, the gene sequence encoding GPC3 is inserted into a known expression vector and the corresponding host cells are transformed with this vector. The desired human GPC3 protein is purified from the host cells or culture supernatants using known methods. For example, to obtain soluble GPC3 from culture supernatants, the amino acid positions 564-580, which form a hydrophobic region corresponding to the GPI-anchoring sequence used to anchor GPC3 to the cell membrane, are removed from the GPC3 polypeptide sequence of SEQ ID NO: 2 by deletion, and the resulting protein is then expressed in place of the GPC3 protein of SEQ ID NO: 2. Alternatively, purified naturally occurring GPC3 protein can be used as the sensitizing antigen.

The purified GPC3 protein can be used as a sensitizing antigen for immunizing mammals. A partial peptide of GPC3 can also be used as a sensitizing antigen. In this case, the partial peptide can be obtained by chemical synthesis based on the amino acid sequence of human GPC3 or by inserting a part of the GPC3 gene into an expression vector for expression. Alternatively, the partial peptide can be obtained by cleaving the GPC3 protein with a protease. Specific embodiments of the invention do not impose limitations on the length and region of the partial peptide of GPC3. The preferred region can be arbitrarily selected from among the amino acid residues of the amino acid sequence located at positions 564-580 in the amino acid sequence of SEQ ID NO: 2. The number of amino acids forming the peptide that can be used as a sensitizing agent is preferably at least five or more, six or more, or seven or more. More specifically, a peptide consisting of 8 to 50 residues, more preferably 10 to 30 residues, can be used as a sensitizing antigen.

Alternatively, a fusion protein obtained by fusing a desired partial polypeptide or peptide of the GPC3 protein with another polypeptide can be used as a sensitizing antigen. For example, Fc fragments of an antibody and peptide tags are preferably used to produce fusion proteins for use as sensitizing antigens. Vectors for expressing said fusion proteins can be constructed by fusing in frame genes encoding two or more desired polypeptide fragments and inserting the fusion gene into an expression vector as described above. Methods for producing fusion proteins are described in Molecular Cloning, 2nd ed. (Sambrook J. et al., Molecular Cloning, 2nd ed. 1989, 9.47-9.58, Cold Spring Harbor Lab. Press). Methods for producing GPC3 for use as a sensitizing antigen and methods of immunization using GPC3 are specifically described in WO 2003/000883, WO 2004/022754 and WO 2006/006693.

There is no specific limitation regarding the mammals to be immunized with the sensitizing antigen. However, it is preferable to select mammals based on their compatibility with the parent cells used for cell fusion. In general, rodents such as mice, rats, as well as hamsters, rabbits, and monkeys are preferably used.

The above animals are immunized with a sensitizing antigen by known methods. Common immunization methods include, for example, intraperitoneal or subcutaneous injection of a mammal with a sensitizing antigen. In particular, the sensitizing antigen can be appropriately diluted in PBS (phosphate-buffered saline), physiological saline, or the like. If necessary, a common adjuvant such as Freund's complete adjuvant is mixed with the antigen and the mixture is emulsified. Then, the sensitizing antigen is administered to the mammal several times at 4-21 day intervals. Appropriate carriers can be used for immunization with a sensitizing antigen. In particular, when a low molecular weight partial peptide is used as a sensitizing antigen, it is sometimes necessary to combine the peptide, which is the sensitizing antigen, with a carrier protein, such as albumin or keyhole limpet hemocyanin, for immunization.

Alternatively, hybridomas producing the desired antibody can be obtained by DNA immunization, as described below. DNA immunization is an immunization method that provides immunostimulation by expressing a sensitizing antigen in the body of an immunized animal as a result of the introduction of a DNA vector designed to provide expression of a gene encoding an antigenic protein in the animal. Compared with conventional immunization methods in which a protein antigen is administered to animals to be immunized, DNA immunization appears to have the following advantages:

- it is possible to achieve immunostimulation while maintaining the structure of a membrane protein such as GPC3; and

- there is no need to purify the antigen for immunization. To obtain the monoclonal antibody proposed in this

According to the invention, DNA immunization is first administered to an animal to be immunized with DNA expressing the GPC3 protein. The DNA encoding GPC3 can be synthesized using known methods, such as PCR. The resulting DNA is inserted into an appropriate expression vector and then administered to the animal to be immunized. Expression vectors preferably used for this purpose include, for example, commercially available expression vectors such as pcDNA3.1. The vectors can be introduced into the body using conventional methods. For example, DNA immunization is carried out using a gene gun to introduce gold particles coated with the expression vector into the body cells of the animal to be immunized. Antibodies recognizing GPC3 can also be obtained using the methods described in WO 2003/104453.

After the above-described immunization of a mammal, an elevated titer of GPC3-binding antibody is confirmed in the serum of the mammal. Immune cells are then obtained from the mammal and used for cell fusion. In particular, splenocytes are preferably used as immune cells.

A mammalian myeloma cell is used as a cell to be fused with the above-mentioned immunocyte. The myeloma cells preferably contain a suitable selection marker for screening. The selection marker imparts characteristics to the cells that ensure their survival (or death) under specific culture conditions. Hypoxanthine-guanine phosphoribosyltransferase deficiency (abbreviated hereinafter as HGPRT deficiency) and thymidine kinase deficiency (abbreviated hereinafter as TK deficiency) are known as selection markers. Cells deficient in HGPRT or TK have hypoxanthine-aminopterin-thymidine sensitivity (abbreviated hereinafter as HAT sensitivity). HAT-sensitive cells cannot synthesize DNA in HAT selection medium and die as a result. However, when the cells are fused with healthy cells, they can continue to synthesize DNA using the healthy cells' "salvage" pathway and as a result they can grow even in HAT selection medium.

HGPRT-deficient and TK-deficient cells can be selected in a medium containing 6-thioguanine, 8-azaguanine (abbreviated hereinafter as 8AG) or 5'-bromodeoxyuridine, respectively. Healthy cells are killed because they incorporate these pyrimidine analogues into their DNA. However, cells deficient in these enzymes can survive in the selection medium because they cannot incorporate these pyrimidine analogues. In addition, resistance to G418, which is provided by the neomycin resistance gene, which confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogues), is a selection marker. Various types of myeloma cells are known that can be used for cell fusion.

For example, the following cells can be preferably used as myeloma cells:

P3(P3x63Ag8.653) (J. Immunol. 123 (4), 1979, pp. 1548-1550);

P3x63Ag8U.1 (Current Topics in Microbiology and Immunology 81, 1978, pp. 1-7);

NS-1 (C. Eur. J. Immunol. 6 (7), 1976, cc. 511-519);

MPC-11 (Cell 8 (3), 1976, pp. 405-415);

SP2/0 (Nature 276 (5685), 1978, pp. 269-270);

FO (J. Immunol. Methods 35 (1-2), 1980, cc. 1-21);

S194/5.XX0.BU.1 (J. Exp. Med. 148 (1), 1978, cc. 313-323);

R210 (Nature 277 (5692), 1979, pp. 131-133), etc.

Cell fusion of immunocytes and myeloma cells is usually accomplished using known methods, such as that described by Kohler and Milstein et al. (Methods Enzymol. 73, 1981, pp. 3-46).

More specifically, cell fusion can be carried out, for example, in a conventional culture medium in the presence of a cell fusion-enhancing agent. Cell fusion-enhancing agents include, for example, polyethylene glycol (PEG) and Sendai virus (Hemagglutinating mouse virus of Japan) (HVJ). If necessary, an auxiliary substance such as dimethyl sulfoxide is also added to increase the fusion efficiency.

The ratio of immunocytes to myeloma cells can be determined at the discretion of the investigator, preferably, for example, one myeloma cell for every 1-10 immunocytes. Culture media used for cell fusions include, for example, media suitable for growing myeloma cell lines, such as RPMI1640 medium and MEM medium, as well as other conventional culture media used for this type of cell culture. In addition, serum supplements such as fetal calf serum (FCS) can be preferably added to the culture medium.

For cell fusion, the above immune cells and myeloma cells taken in predetermined amounts are mixed well in the above culture medium. Then, a PEG solution (e.g., an average molecular weight of about 1,000 to 6,000) preheated to about 37°C is added thereto at a concentration of generally 30 to 60% (w/v). The mixture is gently mixed until the desired fused cells (hybridomas) are obtained. Then, the above culture medium is gradually added to the cells and centrifuged again to remove the supernatant. In this way, cell fusion agents that are undesirable for the growth of hybridomas can be removed.

The hybridomas thus obtained can be selected by culturing using a conventional selective medium, such as a HAT medium (a culture medium containing hypoxanthine, aminopterin and thymidine). Cells other than the desired hybridomas (unfused cells) can be destroyed by subsequent culturing in the above-mentioned HAT medium for a sufficient period of time. Typically, the period is from several days to several weeks. Hybridomas producing the desired antibody are then screened and individually cloned using conventional serial dilution methods.

The hybridomas thus obtained can be selected using a selection medium based on a selection marker carried by the myeloma cells used for cell fusion. For example, HGPRT- or TK-deficient cells can be selected by culturing using a HAT medium (a culture medium containing hypoxanthine, aminopterin and thymidine). In particular, when HAT-sensitive myeloma cells are used for cell fusion, cells that are characterized by successful fusion with healthy cells can be selectively expanded in the HAT medium. Cells other than the desired hybridomas (non-fused cells) can be destroyed by culturing in the above-mentioned HAT medium for a sufficient period of time. In particular, the desired hybridomas can be selected by culturing, as a rule, for a period of several days to several weeks. Hybridomas producing the desired antibody are then screened and individually cloned using standard serial dilution techniques.

The desired antibodies can preferably be selected and individually cloned using screening methods based on a known antigen/antibody reaction. For example, a GPC3-binding monoclonal antibody can bind to GPC3 expressed on the cell surface. These monoclonal antibodies can be screened using a fluorescence excitation cell separation (FACS) method. FACS is a system that allows for the assessment of antibody binding to the cell surface by analyzing cells in contact with a fluorescent antibody using a laser beam and by assessing the fluorescence emitted by individual cells.

For screening using FACS for hybridomas that produce the monoclonal antibody of the present invention, GPC3-expressing cells are first obtained. Cells that are preferably used for screening are mammalian cells in which GPC3 is forced to be expressed. As a control, the ability of an antibody to bind to GPC3 located on the cell surface can be selectively determined using non-transformed mammalian cells as host cells. In particular, hybridomas that produce a monoclonal antibody to GPC3 can be isolated by selecting hybridomas that produce an antibody that binds to cells that forcefully express GPC3 but not to host cells.

Alternatively, the ability of an antibody to bind to immobilized GPC3-expressing cells can be assessed based on the ELISA principle. For example, GPC3-expressing cells are immobilized on wells of an ELISA plate. Hybridoma culture supernatants are contacted with the immobilized cells in the wells, and antibodies that bind to the immobilized cells are detected. When the monoclonal antibodies are of murine origin, the antibodies bound to the cells can be detected using an antibody to murine immunoglobulin. Hybridomas producing the desired antibody that has antigen-binding activity are selected by the above-described screening and can be cloned by a limiting dilution method or a similar method.

The resulting monoclonal antibody-producing hybridomas can be subcultured in conventional culture medium and stored in liquid nitrogen for extended periods of time.

The above hybridomas are cultured by a conventional method and the desired monoclonal antibodies can be obtained from the culture supernatants. Alternatively, the hybridomas are introduced and grown in suitable mammals and the monoclonal antibodies are obtained from the ascites. The former method is suitable for obtaining antibodies with a high degree of purity.

Preferably, antibodies encoded by antibody genes cloned from antibody-producing cells such as the above-mentioned hybridomas can also be used. The cloned antibody gene is inserted into an appropriate vector and introduced into the host to express the gene-encoded antibody. Methods for isolating antibody genes, inserting the genes into vectors, and transforming host cells have been previously developed (see, for example, Vandamme et al., Eur. J. Biochem. 192(3), 1990, pp. 767-775). Methods for producing recombinant antibodies are also known and are described below.

For example, cDNA encoding the variable region (V region) of an antibody to GPC3 is obtained from hybridoma cells expressing the antibody to GPC3. For this purpose, total RNA is first extracted from the hybridomas. Methods used to extract mRNA from cells include, for example:

- the method of ultracentrifugation in the presence of guanidine (Biochemistry 18(24), 1979, pp. 5294-5299) and

- AGPC method (Anal. Biochem. 162(1), 1987, pp. 156-159).

The extracted mRNAs can be purified using an mRNA Purification Kit (GE Healthcare Bioscience) or a similar kit. Alternatively, commercially available kits for extracting mRNA directly from cells, such as a QuickPrep mRNA Purification Kit (GE Healthcare Bioscience), can also be used. mRNA can be obtained from hybridomas using such kits. Antibody V region-encoding cDNAs can be synthesized from the obtained mRNAs using reverse transcriptase. The cDNA can be synthesized using a Reverse Transcriptase First-strand cDNA Synthesis Kit containing AMV (Seikagaku Co.) or a similar kit. In addition, the SMART RACE cDNA Amplification Kit (Clontech) and the PCR-based 5'-RACE (rapid amplification of cDNA ends) method (Proc. Natl. Acad. Sci. USA 85(23), 1988: 8998-9002; Nucleic Acids Res. 17(8), 1989: 2919-2932) can be used to synthesize and amplify cDNA. In this process of cDNA synthesis, the corresponding restriction enzyme recognition sites described below can be introduced at both ends of the cDNA.

The cDNA fragment of interest is purified from the resulting PCR product and then inserted into a DNA vector by ligation. In this way, a recombinant vector is created and introduced into E. coli or a similar host. After colony selection, the desired recombinant vector can be obtained from colony-forming E. coli. Then, using a known method such as the nucleotide chain termination method, it is determined whether the recombinant vector has the cDNA nucleotide sequence of interest.

The 5'-RACE method, which uses primers to amplify a variable region gene, is generally used to isolate a gene encoding a variable region. First, a cDNA library used for 5'-RACE (5'-RACE cDNA library) is constructed using RNA extracted from hybridoma cells as a template. A commercially available kit such as the SMART RACE cDNA Amplification Kit can be used to synthesize the 5'-RACE cDNA library.

The antibody gene is amplified by PCR using the obtained 5'-RACE cDNA library as a template. Primers for amplification of the mouse antibody gene can be designed based on known antibody gene sequences. The nucleotide sequences of the primers vary depending on the immunoglobulin subclass. Thus, it is preferable to pre-determine the subclass using a commercially available kit such as the Iso Strip mouse monoclonal antibody isotyping kit (Roche Diagnostics).

In particular, for example, to isolate genes encoding mouse IgG, primers are used that provide amplification of genes encoding heavy chains γ1, γ2a, γ2b and γ3 and light chains κ and λ. In general, a primer whose hybridization site ("annealing") with the constant region is located near the variable region is used as a 3'-terminal primer for amplification of the IgG variable region gene. In this case, a primer attached to a set of 5' RACE cDNA library constructs is used as a 5'-terminal primer.

The PCR products amplified in this manner are used to reconstruct immunoglobulins consisting of a combination of heavy and light chains. The desired antibody can be selected using the GPC3-binding activity of the reconstructed immunoglobulin as an indicator. For example, when the objective is to isolate an antibody to GPC3, it is more preferable that the binding of the antibody to GPC3 is specific. GPC3-binding antibodies can be screened, for example, using the following steps:

(1) contacting a GPC3-expressing cell with an antibody containing a V region encoded by cDNA isolated from the hybridoma;

(2) determine the binding of the antibody to a GPC3-expressing cell; and

(3) select an antibody that binds to a GPC3-expressing cell.

Methods for determining the binding of an antibody to GPC3-expressing cells are known. In particular, the binding of an antibody to GPC3-expressing cells can be determined using the above-described techniques, such as FACS. Immobilized samples of GPC3-expressing cells can be used to assess the binding activity of the antibody.

Preferred antibody screening methods that use binding activity as an indicator also include phage vector-based panning methods. Phage vector-based screening methods are advantageous when antibody genes are isolated from heavy chain and light chain subclass libraries from a population of cells expressing polyclonal antibodies. The genes encoding the variable regions of the heavy chain and light chain can be ligated using a suitable linker sequence to form a single-chain Fv (scFv). Phages displaying the scFv on their surface can be produced by inserting the gene encoding the scFv into a phage vector. The phages are contacted with the antigen of interest. DNA encoding the scFv that has the binding activity of interest can then be isolated by collecting phages bound to the antigen. This process can be repeated as needed to enrich for scFv that have the binding activity of interest.

After isolation of the cDNA encoding the V region of the anti-GPC3 antibody of interest, the cDNA is digested with restriction enzymes that recognize restriction sites introduced at both ends of the cDNA. Preferred restriction enzymes recognize and cleave a nucleotide sequence that occurs at low frequency in the nucleotide sequence of the antibody gene. In addition, to insert the single-copy digested fragment in the correct orientation, a restriction site for the enzyme that forms the sticky end is preferably introduced into the vector of interest. The cDNA encoding the V region of the anti-GPC3 antibody is digested according to the above-described method and inserted into a suitable expression vector to generate an antibody expression vector. In the case where the gene encoding the constant region (C region) of the antibody and the gene encoding the above-mentioned V region are fused in frame, a chimeric antibody is obtained. In the context of the present description, "chimeric antibody" means that the constant region and the variable region are different in origin. Thus, in addition to mouse/human heterochimeric antibodies, the chimeric antibodies of the present invention also include human/human allochimeric antibodies. An expression vector of a chimeric antibody can be created by inserting the above-mentioned V region gene into an expression vector that already contains a constant region. In particular, for example,

the sequence recognized by the restriction enzyme that cuts out the above-mentioned V-region gene should preferably be placed in the 5' region of an expression vector carrying DNA that encodes the constant region (C-region) of the desired antibody. The chimeric antibody expression vector is created by fusing in frame two genes that have been digested with the same combination of restriction enzymes.

To obtain a monoclonal antibody to GPC3, the antibody genes are inserted into an expression vector in such a way that the expression of the genes occurs under the control of an expression regulatory region. The expression regulatory region intended for the expression of antibodies includes, for example, enhancers and promoters. In addition, an appropriate signal sequence can be attached to the amino terminus in such a way that the antibody is secreted from the cells to the outside. In the examples described below, a peptide that has the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 72) is used as a signal sequence. Along with it, other suitable signal sequences can be attached. The expressed polypeptide is cleaved at the carboxyl terminus of the above sequence and the resulting polypeptide is secreted from the cells to the outside as a mature polypeptide. Then, suitable host cells are transformed with the expression vector and recombinant cells expressing DNA that codes for the antibody to GPC3 are obtained.

DNAs encoding the antibody heavy chain (H chain) and the antibody light chain (L chain) are separately inserted into different expression vectors for expression of the antibody gene. An antibody molecule having H and L chains can be expressed by cotransfecting the same host cell with vectors into which the H chain and L chain genes, respectively, have been inserted. Alternatively, the host cells can be transformed with a single expression vector into which DNAs encoding the H and L chains have been inserted (see WO 94/11523).

Various combinations of host cells/expression vectors are known for producing antibodies by introducing isolated antibody genes into appropriate hosts. All of these expression systems can be used to isolate the antigen-binding domains and CD3-binding domains of the present invention. Suitable eukaryotic cells that are used as host cells are animal cells, plant cells and fungal cells. In particular, animal cells are, for example, the following cells:

(1) mammalian cells: CHO, COS, myeloma, baby hamster kidney (BHK), HeLa, Vero, etc.;

(2) amphibian cells: oocytes of the clawed frog (Xenopus) or the like, and

(3) insect cells: sf9, sf21, Tn5, etc.

In addition, an antibody gene expression system is known as a plant cell, which uses cells obtained from the genus Nicohana, such as Nicotiana tabacum. Cultured callus cells can be used to transform plant cells.

In addition, the following cells can be used as mushroom cells:

yeasts: of the genus Saccharomyces, such as Saccharomyces cerevisiae, and of the genus Pichia, such as Pichia pastoris, and

filamentous fungi: of the genus Aspergillus, such as Aspergillus niger.

In addition, antibody gene expression systems are known in which prokaryotic cells are used. For example, bacterial cells, i.e., E. coli cells, Bacillus subtilis cells, etc., can be used according to the present invention. Expression vectors that carry antibody genes of interest are introduced into these cells by transfection. The transfected cells are cultured in vitro, and the desired antibody can be obtained from the culture of the transformed cells.

In addition to the above-mentioned host cells, transgenic animals can also be used to produce a recombinant antibody. This means that the antibody can be obtained from an animal into which a gene encoding the antibody of interest has been introduced. For example, the antibody gene can be created as a fusion gene by inserting it in frame into a gene encoding a protein that is specifically produced in milk. For example, goat β-casein can be used as a protein secreted into milk. DNA fragments containing the fusion gene, which includes the antibody gene, are injected into a goat embryo, which is then introduced into a female goat. The desired antibodies can be obtained as a protein fused with a milk protein from the milk of a transgenic goat born from the goat that is the recipient of the embryo (or its offspring). In addition, to increase the volume of milk containing the desired antibody produced by the transgenic goat, hormones can be administered to the transgenic goat if necessary (Ebert K. M. et al., Bio/Technology 12 (7), 1994, pp. 699-702).

When the polypeptide complex provided in the present description is administered to a human, an antigen-binding domain derived from an antibody obtained by genetic recombination, which is artificially modified to reduce heterologous antigenicity with respect to humans and other animals, can also be used as the antigen-binding domain of the complex. Said antibodies obtained by genetic recombination include, for example, humanized antibodies. These modified antibodies can be obtained using known methods.

The variable region of an antibody used to form the antigen-binding domain of the polypeptide complex described herein typically consists of three complementarity determining regions (CDRs) separated by four framework regions (FRs). A CDR is a region that largely determines the binding specificity of an antibody. The amino acid sequences of the CDRs are highly variable. On the other hand, the amino acid sequences that form the FRs often have high identity, even among antibodies with different binding specificities. Thus, the binding specificity of a particular antibody can typically be introduced into another antibody by CDR grafting.

A humanized antibody is also called a reconstituted human antibody. In particular, humanized antibodies obtained by transplanting a CDR of an antibody of an animal other than a human, such as a mouse antibody, into a human antibody, etc., are known. Also known are generally accepted genetic engineering techniques for obtaining humanized antibodies. In particular, for example, PCR with overlapping primers is a known method for transplanting a CDR of a mouse antibody into a human FR. When performing PCR with overlapping primers, a nucleotide sequence that codes for a CDR of a mouse antibody to be transplanted is added to primers intended for synthesizing an FR of a human antibody. Primers are obtained for each of the four FRs. It is generally accepted that when transplanting a mouse CDR into a human FR, it is advisable to select a human FR that has a high level of identity with the mouse FR in order to maintain the CDR function. Thus, it is generally preferred to use a human FR that contains an amino acid sequence that has a high level of identity to the amino acid sequence of the FR that is adjacent to the mouse CDR to be transplanted.

The nucleotide sequences to be ligated are designed to be joined to each other in reading frame. The human FRs are individually synthesized using appropriate primers. This results in products in which the DNA encoding the mouse CDR is fused to the individual FR encoding DNAs. The nucleotide sequences encoding the mouse CDR of each product are designed to overlap each other. A complementary strand synthesis reaction is then performed to anneal the overlapping CDR regions of the products synthesized using the human antibody gene as a template. This reaction inserts the human FRs by ligation via the mouse CDR sequences.

The full-length V region gene, into which three CDRs and four FRs are finally ligated, is amplified using primers hybridizing to the 5' or 3' end, which are added with sequences recognized by suitable restriction enzymes. An expression vector for a humanized antibody can be obtained by inserting the DNA obtained according to the above method and the DNA encoding the C region of the human antibody into the expression vector so as to ligate them in frame. After transfection of the host with the recombinant vector to generate recombinant cells, the recombinant cells are cultured and the DNA encoding the humanized antibody is expressed to produce a humanized antibody in cell culture (see European Patent Publication EP 239400 and International Patent Application Publication WO 1996/002576).

By qualitatively or quantitatively evaluating and measuring the antigen-binding activity of the humanized antibody obtained according to the above method, FRs of the human antibody that enable the CDRs to form a preferable antigen-binding site when ligated via the CDRs can be selected. Amino acid residues in the FRs can be replaced as necessary so that the CDRs of the reshaped human antibody form an acceptable antigen-binding site. For example, amino acid sequence mutations can be introduced into the FRs using a PCR method used for transplanting a mouse CDR into a human FR. More specifically, partial nucleotide sequence mutations can be introduced into primers hybridizing to the FRs. The nucleotide sequence mutations are introduced into the FRs synthesized using these primers. Mutant FR sequences having the desired characteristics can be selected by measuring and evaluating the antigen-binding activity of the mutant antibody with the amino acid substitution using the above-mentioned method (Sato K. et al., Cancer Res. 53, 1993, pp. 85 1-856).

Alternatively, the desired human antibodies can be produced by immunizing transgenic animals having the full repertoire of human antibody genes (see WO 1993/012227; WO 1992/003918; WO 1994/002602; WO 1994/025585; WO 1996/034096; WO 1996/033735) using DNA immunization.

In addition, methods for producing human antibodies by panning using libraries of human antibodies are known. For example, the V region of a human antibody is expressed as a single-chain antibody (scFv) on the surface of a phage using a phage display method. Phages expressing an scFv that binds to an antigen can be selected. The DNA sequence encoding the V region of a human antibody that binds to an antigen can be determined by analyzing the genes of the selected phages. The DNA sequence of the scFv that binds to the antigen is determined. An expression vector is obtained by fusing the V region sequence in frame to the C region sequence of the desired human antibody and then inserting it into a suitable expression vector. The expression vector is introduced into a cell suitable for the above expression. A human antibody can be obtained by expressing a gene encoding a human antibody in cells. Such methods have already been described (see WO 1992/001047; WO 1992/020791; WO 1993/006213; WO 1993/011236; WO 1993/019172; WO 1995/001438; WO 1995/015388).

Antigen binding domain

As used herein, "antigen-binding domain" refers to a portion of an antibody that contains a region that specifically binds to and is complementary to the entire antigen or a portion thereof. When the molecular weight of the antigen is large, the antibody can bind only to a specific portion of the antigen. Said specific portion is called an "epitope". The antigen-binding domain can be formed by one or more variable domains of the antibody. Preferably, the antigen-binding domains comprise both the variable region of the light chain (VL) of the antibody and the variable region of the heavy chain (VH) of the antibody. Said preferred antigen-binding domains include, for example, "single-chain Fv (scFv)", "single-chain antibody", "Fv", "single-chain Fv2 (scFv2)", "Fab" and "F(ab')2".

The antigen-binding domains of the polypeptide complexes of the present invention may bind to the same epitope. The epitope may be present in a protein that comprises the amino acid sequence of SEQ ID NO: 2 or 4. Alternatively, the antigen-binding domains of the polypeptide complexes of the present invention may individually bind to different epitopes. The epitope may be present in a protein that comprises the amino acid sequence of SEQ ID NO: 2 or 4. Specificity

"Specificity" means that a molecule that binds specifically to one or more binding partners does not have any significant ability to bind to molecules other than these partners. In addition, the term "specificity" is also used when an antigen-binding domain is specific for a particular epitope from several epitopes contained in an antigen. When the epitope to which the antigen-binding domain binds is contained in several different antigens, the polypeptide complex containing the antigen-binding domain can bind to different antigens that contain the epitope.

Antigen

The present disclosure is not limited to any particular antigen, and any antigen except CD3 can be used. The antigens mentioned include, for example, receptors, cancer antigens, MHC antigens, and differentiation antigens. Receptors include receptors belonging to the hematopoietic growth factor receptor family, the cytokine receptor family, the tyrosine kinase receptor family, the serine/threonine kinase receptor family, the TNF receptor family, the G protein-coupled receptor family, the GPI-linked receptor family, the tyrosine phosphatase receptor family, the adhesion factor family, and the hormone receptor family. Receptors belonging to these receptor families and their characteristics are described in various documents, for example, in reviews such as New Comprehensive Biochemistry, ed. Cooke V.A., King R.J.B., van der Molen H.J., vol. 18B “Hormones and their Actions Part II”, 1988, cc. 1-46, publishing house) Elsevier Science Publishers BV.; and SAIBO KOGAKU (Cell Technology) Supplementary Volume Handbook Series “Secchaku Inshi Handbook (Handbook for Adhesion factors)” ed. M. Miyasaka, 1994, Shujunsha, Tokyo, Japan; and Patthy, Cell, 61(1), 1990, pp. 13-14; Ullrich et al., Cell 61(2), 1990, pp. 203-212; Massague, Cell 69(6), 1992, pp.1067-1070; Miyajima et al., Annu. Rev. Immunol. 10, 1992, pp. 295-331; Taga et al., FASEB J. 6, 1992, pp. 3387-3396); Fantl et al., Annu. Rev. Biochem. 62, 1993, pp. 453-481; Smith et al., Cell 76(6), 1994, pp. 959-962, Flower D.R. in Biochim. Biophys. Acta: Flower Biochim. Biophys. Acta, 1422(3), 1999, pp. 207-234.

In particular, receptors belonging to the above-mentioned receptor families preferably include, for example, human and mouse erythropoietin (EPO) receptors (Blood 76(1), 1990, pp. 31-35; Cell 57(2), 1989, pp. 277-285), human and mouse granulocyte colony-stimulating factor (G-CSF) receptors (Proc. Natl. Acad. Sci. USA. 87(22), 1990, pp. 8702-8706; mG-CSFR, Cell 61(2), 1990, pp. 341-350), human and mouse thrombopoietin (TPO) receptors (Proc. Natl. Acad. Sci. USA 89(12), 1992, pp. 5640–5644; EMBO J. 12(7), 1993, pp. 2645–53), human and mouse insulin receptors (Nature 313(6005), 1985, pp. 756–761), human and mouse Flt-3 ligand receptors (Proc. Natl. Acad. Sci. USA 91(2), 1994, pp. 459–463), human and mouse platelet-derived growth factor (PDGF) receptors (Proc. Natl. Acad. Sci. USA. 85(10), 1988, pp. 3435–3439), human and mouse interferon (IFN)-α and -β receptors (Cell 60(2), 1990, pp. 225–234; and Cell 77(3), 1994, pp.391-400), human and mouse leptin receptors, human and mouse growth hormone (GH) receptors, human and mouse interleukin (IL)-10 receptors, human and mouse insulin-like growth factor (IGF)-I receptors, human and mouse leukemia inhibitory factor (LIF) receptors, and human and mouse ciliary neurotrophic factor (CNTF) receptors.

Cancer antigens are antigens that are expressed when cells become cancerous and are also referred to as "tumor-specific antigens." In addition, abnormal sugar chains that are expressed on cell surfaces or protein molecules when cells become cancerous are also cancer antigens and are referred to as "cancer-associated carbohydrate antigens." Such cancer antigens include, for example, GPC3 (Int J Cancer. 103(4):455-465 (2003)), Ep C AM (Proc. Natl. Acad. Sci. USA. 86(1):27-31 (1989)), EGFR, CA19-9, CA15-3, and sialyl sulfoxide (SLX). GPC3 belongs to the above-mentioned family of GPI-linked receptors and is expressed in several types of cancer, including liver cancer. EpCAM is expressed in multiple cancers, including lung cancer, and its polynucleotide and polypeptide sequences are submitted to the RefSeq database under the accession numbers NM_002354.2 (SEQ ID NO: 3) and NP_002345.2 (SEQ ID NO: 4), respectively.

Generally, MHC antigens are divided into MHC class I and class II antigens. MHC class I antigens include HLA-A, -B, -C, -E, -F, -G, and -H, and MHC class II antigens include HLA-DR, -DQ, and -DP.

Differentiated antigens include CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15s, CD16, CD18, CD19, CD20, CD21, CD23, CD25, CD28, CD29, CD30, CD32, CD33, CD34, CD35, CD38, CD40, CD41a, CD41b, CD42a, CD42b, CD43, CD44, CD45, CD45RO, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, CD54, CD55, CD56, CD57, CD58, CD61, CD62E, CD62L, CD62P, CD64, CD69, CD71, CD73, CD95, CD102, CD106, CD122, CD126 and CDw130.

Epitope

"Epitope" means an antigenic determinant in an antigen and refers to an antigenic site to which the antigen-binding domain of a polypeptide complex described herein binds. For example, an epitope can be characterized based on its structure. Alternatively, an epitope can be characterized based on the antigen-binding activity of a polypeptide complex that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope can be defined by the amino acid residues that form the epitope. Alternatively, when the epitope is a sugar chain, the epitope can be defined by its specific sugar chain structure.

A linear epitope is an epitope that contains an epitope that recognizes a primary amino acid sequence. Said linear epitope typically contains at least three and most often at least five, such as about 8 to 10 or 6 to 20 amino acid residues in a particular sequence.

In contrast to a linear epitope, a "conformational epitope" is an epitope in which the primary amino acid sequence that constitutes the epitope is not the sole determinant of the epitope recognized (e.g., the primary amino acid sequence of a conformational epitope is not required to be recognized by an epitope-specific antibody). Conformational epitopes may contain a larger number of amino acid residues than linear epitopes. An antibody that recognizes a conformational epitope recognizes the three-dimensional structure of a peptide or protein. For example, when a protein molecule folds and forms a three-dimensional structure, the amino acids and/or polypeptide backbones that form the conformational epitope align and the epitope becomes recognizable to the antibody. Methods for determining epitope conformations include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance, site-specific spin labeling, and electron paramagnetic resonance (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Morris, ed., vol. 66, 1996).

Examples of methods for assessing epitope binding by a test polypeptide complex containing a GPC3 antigen-binding domain are described below. The examples of methods for assessing epitope binding by a test polypeptide complex containing an antigen-binding domain for an antigen other than GPC3 described below may also be used.

For example, to confirm that a test polypeptide complex containing the GPC3 antigen-binding domain recognizes a linear epitope in a GPC3 molecule, the following method can be used. For the above purpose, a linear peptide containing an amino acid sequence that forms the extracellular domain of GPC3 is synthesized. The peptide can be chemically synthesized or produced by genetic engineering using a region in the GPC3 cDNA that encodes the amino acid sequence corresponding to the extracellular domain.

The test polypeptide complex containing the GPC3 antigen binding domain is then assessed for its binding activity to a linear peptide that contains the amino acid sequence that forms the extracellular domain. For example, the immobilized linear peptide can be used as an antigen in an ELISA to assess the binding activity of the polypeptide complex to the peptide. Alternatively, the binding activity to the linear peptide can be assessed by the level of inhibition of the linear peptide from binding the polypeptide complex to GPC3-expressing cells. These assays can demonstrate the binding activity of the polypeptide complex to the linear peptide.

Whether a polypeptide complex comprising a GPC3 antigen binding domain has the ability to recognize a conformational epitope can be determined as follows. For the above purpose, GPC3-expressing cells are obtained. A test polypeptide complex comprising a GPC3 antigen binding domain is considered to recognize a conformational epitope if it, upon contact, is characterized by strong binding to GPC3-expressing cells, but weakly binds to an immobilized linear peptide that contains an amino acid sequence forming an extracellular domain of GPC3. In the context of the present description, "slightly binds" means that the binding activity is 80% or less, typically 50% or less, preferably 30% or less, and most preferably 15% or less, compared to the binding activity to cells expressing human GPC3.

Methods for assessing the binding activity of a test polypeptide complex containing a GPC3 antigen-binding domain to GPC3-expressing cells include, for example, those described in Antibodies: A Laboratory Manual (ed. Harlow, David Lane, Cold Spring Harbor Laboratory, 1988, pp. 359-420). In particular, the assessment can be carried out based on the ELISA principle or the fluorescence excitation cell separation (FACS) method using GPC3-expressing cells as an antigen.

In the ELISA format, the binding activity of a test polypeptide complex containing a GPC3 antigen binding domain to GPC3-expressing cells can be quantified by comparing the signal levels generated during the enzymatic reaction. Specifically, the test polypeptide complex is added to an ELISA plate on which GPC3-expressing cells are immobilized. The test polypeptide complex bound to the cells is then detected using an enzyme-labeled antibody that recognizes the test polypeptide complex. Alternatively, when FACS is used, serial dilutions of the test polypeptide complex are prepared and the titer of the antibody binding to GPC3-expressing cells can be determined by comparing the binding activity to the GPC3-expressing cells.

The binding of the test polypeptide complex to an antigen expressed on the surface of cells suspended in a buffer or similar medium can be detected using a flow cytometer. Known flow cytometers include, for example, the following devices:

FACSCanto™ II,

FACSAria™,

FACSArray™,

FACSVantage™ SE

FACSCalibur™ (all trademarks of BD Biosciences),

EPICS ALTRA HyPerSort,

Cytomics FC 500,

EPICS XL-MCL ADC EPICS XL ADC,

Cell Lab Quanta/Cell Lab Quanta SC (all trademarks of Beckman Coulter).

Preferred methods for assessing the binding activity of a test polypeptide complex containing the antigen-binding domain of GPC3 to an antigen include, for example, the following method. First, GPC3-expressing cells are reacted with a test polypeptide complex, and then the complex is stained with a FITC-labeled secondary antibody that recognizes the polypeptide complex. The test polypeptide complex is appropriately diluted with an appropriate buffer to obtain a complex at a desired concentration. For example, the complex can be used at a concentration of 10 μg/ml to 10 ng/ml. Then, the fluorescence intensity and the number of cells are determined using a FACSCalibur (BD). The fluorescence intensity determined by assays based on the CELL QUEST program (BD), namely, expressed as geometric mean values, reflects the level of binding of the antibody to the cells. This means that the binding activity of the test polypeptide complex, which is characterized by the amount of bound test polypeptide complex, can be estimated by determining the geometric mean value.

Whether a test polypeptide complex containing the GPC3 antigen binding domain shares an epitope with another polypeptide complex can be assessed by competition between the two complexes for the same epitope. Competition between polypeptide complexes can be determined by a cross-blockade assay or a similar assay. For example, a preferred cross-blockade assay is a competitive ELISA assay.

In particular, when performing a cross-blockade assay, the GPC3 protein immobilized in the wells of a microtiter plate is pre-incubated in the presence of a polypeptide complex considered as a competitor candidate or without it, and then the test polypeptide complex is added. The amount of the test polypeptide complex bound to the GPC3 protein in the wells is inversely correlated with the binding activity of the polypeptide complex considered as a competitor candidate that competes for binding to the same epitope. This means that the higher the affinity of the polypeptide complex considered as a competitor candidate for the same epitope, the lower the binding activity of the test polypeptide complex to the wells coated with the GPC3 protein.

The amount of the test polypeptide complex bound via the GPC3 protein to the wells is easily determined by pre-labeling the polypeptide complex. For example, a biotin-labeled polypeptide complex is assayed using an avidin/peroxidase conjugate and an appropriate substrate. In particular, a cross-blockade assay using enzymes such as peroxidase as labels is called a "competitive ELISA assay". The polypeptide complex can also be labeled using other labeling substances that can be detected or assessed. In particular, radioactive labels, fluorescent labels, etc. are known.

When a polypeptide complex considered as a candidate competitor can block the binding of a test polypeptide complex that contains a GPC3 antigen binding domain by at least 20%, preferably by at least 20-50%, and preferably by at least 50% compared to the binding activity determined in a control experiment carried out in the absence of the polypeptide complex considered as a candidate competitor, then the test polypeptide complex is considered to have a significant ability to bind to the same epitope as the polypeptide complex considered as a candidate competitor, or to compete for binding to the same epitope.

If the structure of the epitope associated with a test polypeptide complex that contains a GPC3 antigen-binding domain has already been identified, then to decide whether the test and control polypeptide complexes have the same epitope, the binding activities of the two polypeptide complexes can be compared with a peptide obtained by introducing amino acid mutations into the peptide that forms the epitope.

To evaluate the above binding activities, for example, the binding activities of the test and control polypeptide complexes to the linear peptide into which the mutation has been introduced are compared using the above ELISA format. In addition to the ELISA method, the binding activity with respect to the mutant peptide bound to the column can be determined by passing the test and control polypeptide complexes through the column and then evaluating the amount of the polypeptide complex eluted into the elution solution. Methods are known for adsorbing the mutant peptide on the column, for example in the form of a peptide fused to GST.

Alternatively, when the identified epitope is a conformational epitope, the following method can be used to decide whether the test and control polypeptide complexes share a common epitope. First, GPC3-expressing cells and cells expressing GPC3 with a mutation introduced into the epitope are prepared. The test and control polypeptide complexes are added to a cell suspension obtained by suspending the cells in a suitable buffer, such as PBS. The cell suspensions are then appropriately washed with buffer and an FITC-labeled antibody that recognizes the test and control polypeptide complexes is added. The fluorescence intensity and the number of cells stained with the labeled antibody are determined using a FACSCalibur (BD). The test and control polypeptide complexes are appropriately diluted with a suitable buffer and used at a desired concentration. For example, they can be used at a concentration of 10 μg/ml to 10 ng/ml. The fluorescence intensity is determined by an assay using the CELL QUEST program (BD) to evaluate the results, namely, the geometric mean is determined, which reflects the amount of labeled antibody bound to the cells. This means that the binding activities of the test and control polypeptide complexes, which are characterized by the amount of bound labeled antibody, can be determined by evaluating the geometric mean.

In carrying out the above method, for example, the following method can be used to decide whether a polypeptide complex exhibits "low binding to cells expressing mutant GPC3". First, the test and control polypeptide complexes bound to cells expressing mutant GPC3 are stained with a labeled antibody. Then, the fluorescence intensity in the cells is determined. When a flow pitometer is used to evaluate fluorescence, the fluorescence intensity can be analyzed using the CELL QUEST program. Based on the geometric mean values in the presence and without the polypeptide complex, the value used for comparison (AGeo-Mean), which characterizes the degree of increase in fluorescence intensity due to binding to the polypeptide complex, can be calculated according to the formula below.

ΔGeo-Mean = Geo-Mean (in the presence of the polypeptide complex)/Geo-Mean (in the absence of the polypeptide complex)

The geometric mean value used for comparison (the AGeo-Mean value for the mutant GPC3 molecule), determined by the assay described above, which reflects the amount of the test polypeptide complex bound to cells expressing the mutant GPC3, is compared with the relative ΔGeo-Mean value, which reflects the amount of the test polypeptide complex bound to GPC3-expressing cells. In this case, the concentrations of the test polypeptide complex used to determine the relative ΔGeo-Mean values for GPC3-expressing cells and mutant GPC3-expressing cells are most preferably adjusted so that they are the same or substantially the same. A polypeptide complex that has been confirmed to recognize an epitope in GPC3 is used as a control polypeptide complex.

If the ΔGeo-Mean value of the test polypeptide complex relative to the mutant GPC3-expressing cells, which is used for comparison, is lower than the ΔGeo-Mean value of the test polypeptide complex relative to the GPC3-expressing cells, which is used for comparison, by at least 80%, preferably by 50%, more preferably by 30%, and most preferably by 15%, then the test polypeptide complex is considered to "negligibly bind to the mutant GPC3-expressing cells". The formula for determining the Geo-Mean value is described in the CELL QUEST software manual (BD biosciences). When the relative values are found to be substantially equivalent by comparison, then the test and control polypeptide complexes can be considered to have the same epitope. Variable fragment (Fv)

As used herein, the term "variable fragment (Fv)" refers to the smallest unit of an antibody-derived antigen-binding domain that consists of a pair of antibody light chain variable region (VL) and antibody heavy chain variable region (VH). In 1988, Skerra and Pluckthun discovered that homogeneous and active antibodies could be produced from the periplasmic fraction of E. coli by inserting an antibody gene downstream of a bacterial signal sequence and inducing expression of the gene in E. coli (Science 240(4855), 1988, pp. 1038-1041). In Fv derived from the periplasmic fraction, VH associates with VL so as to bind antigen.

According to the present description, Fv preferably includes, for example, a pair of Fv, which are a polypeptide complex or the like, comprising:

(1) a bivalent antigen-binding domain, which is a bivalent scFv in which one monovalent scFv of the bivalent scFv is linked to one Fc domain-forming polypeptide via a heavy chain Fv fragment forming a CD3-binding domain, and another monovalent scFv is linked to another Fc domain-forming polypeptide via a light chain Fv fragment forming a CD3-binding domain;

(2) a domain containing an Fc domain that does not have the ability to bind an Fcγ receptor and that is derived from the amino acids that form the Fc domain of IgG1, IgG2a, IgG3, or IgG4, and

(3) at least one monovalent CD3-binding domain in which the Fv fragments of the light chain and heavy chain as a result

associations form a CD3-binding domain that can bind to the CD3 antigen.

scFv, single chain antibody and sc(Fv)2

As used herein, the terms "scFv", "single chain antibody" and "sc(Fv)2" all refer to a single polypeptide chain fragment of an antibody that contains variable regions derived from the heavy and light chains but does not contain a constant region. Typically, a single chain antibody also contains a polypeptide linker between the VH and VL domains to form the desired structure, which is likely to mediate antigen binding. Single chain antibody is described in detail by Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, 1994, pp. 269-315" (see also International Patent Publication No. WO 1988/001649; US Patent Nos. 4,946,778 and 5,260,203). In a particular embodiment of the invention, the single chain antibody may be bispecific and/or humanized.

scFv is an antigen-binding domain in which the VH and VL that form the Fv are linked to each other by a peptide linker (Proc. Natl. Acad. Sci. U.S.A. 85(16), 1988, pp. 5879-5883). VH and VL can be held in close proximity by a peptide linker.

sc(Fv)2 is a single chain antibody in which four variable regions of two VL and two VH are linked by linkers such as peptide linkers to form a single chain (J Immunol. Methods 231(1-2), 1999: 177-189). The two VH and two VL can be derived from different monoclonal antibodies. Said sc(Fv)2 preferably include, for example, a bispecific sc(Fv)2 that recognizes two epitopes present in a single antigen, as described in Journal of Immunology 152(11), 1994: 5368-5374. sc(Fv)2 can be produced by methods known to those skilled in the art. For example, sc(Fv)2 can be produced by linking scFv via a linker such as a peptide linker.

According to the present description, the form of the antigen-binding domain forming sc(Fv)2 refers to an antibody in which two VH units and two VL units are arranged in the following order: VH, VL, VH and VL ([VH]-linker-[VL]-linker-[VH]-linker-[VL]), starting from the N-terminus of the single-chain polypeptide. The order of the arrangement of the two VH units and the two VL units is not limited to the above form, and they can be arranged in any

order. An example of the order of arrangement in various forms is given below.

[VL]-linker-[VH]-linker-[VH]-linker-[VL]

[VH]-linker-[VL]-linker-[VL]-linker-[VH]

[VH]-linker-[VHJ-linker-[VL]-linker-[VL]

[VL]-linker-[VL]-linker-[VH]-linker-[VH]

[VL]-linker-[VH]-linker-[VL]-linker-[VH].

The molecular form of sc(Fv)2 is also described in detail in WO 2006/132352. Thus, according to these descriptions, specialists in this field can obtain the desired sc(Fv)2 intended for creating the polypeptide complexes presented in the present description.

In addition, the polypeptide complexes of the present invention can be conjugated to a carrier polymer such as PEG or an organic compound such as an anticancer agent. Alternatively, an additional sugar chain sequence is preferably incorporated into the polypeptide complexes so that the sugar chain provides the desired effect.

Linkers for linking the variable regions of the antibody are arbitrary peptide linkers which can be introduced by genetic engineering, synthetic linkers and linkers described, for example, in Protein Engineering, 9(3), 1996, pp.299-305. However, for the purposes of the present invention, peptide linkers are most preferable. The length of the peptide linkers is not particularly limited, and can be selected by those skilled in the art depending on the task. The preferable length is five or more amino acids (with the upper limit being (but not limited to) usually up to 30 amino acids or less, preferably 20 amino acids or less), and most preferably 15 amino acids. When sc(Fv)2 contains three peptide linkers, their lengths may be the same or different.

For example, the peptide linkers mentioned include:

Sir,

Gly⋅Ser,

Gly⋅Gly⋅Ser,

Ser⋅Gly⋅Gly,

Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 5),

Ser⋅Gly⋅Gly⋅Gly (SEQ ID NO: 6),

Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 7),

Ser⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 8),

Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 9),

Ser⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 10),

Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 11),

Ser⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 12),

(Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 7))n,

(Ser⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 8))n,

where n is an integer of 1 or greater. Those skilled in the art may select the length or sequence of peptide linkers depending on the task at hand.

As a rule, synthetic linkers (chemical cross-linking agents) are used for cross-linking; they are, for example:

N-hydroxysuccinimide (NHS),

disuccinimidyl suberate (DSS),

bis(sulfosuccinimidyl)suberate (BS ³ ),

dithiobis(succinimidyl propionate) (DSP),

dithiobis(sulfosuccinimidyl propionate) (DTSSP),

ethylene glycolbis(succinimidyl succinate) (EGS),

ethylene glycolbis(sulfosuccinimidyl succinate) (sulfo-EGS),

disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST),

bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES) and

and bis[2-(sulfosuccinimideoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES). These cross-linking agents are commercially available.

Typically, three linkers are required to join together the four variable regions of an antibody. The linkers used may be of the same type or different types.

Fab, F(ab')2 and Fab'

"Fab" consists of one light chain and the CH1 domain and variable region of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bridges with the heavy chain of another molecule.

"F(ab')2" or "Fab" is produced by treating immunoglobulin (monoclonal antibody) with protease such as pepsin and papain, and it refers to the antibody fragment produced by cleaving immunoglobulin (monoclonal antibody) near the disulfide bridges present between the hinge regions in each of the two H chains. For example, papain cleaves IgG before the disulfide bridges present between the hinge regions in each of the two H chains to form two homologous antibody fragments in which the L chain containing VL (the variable region of the L chain) and CL (the constant region of the L chain) are linked to an H chain fragment containing VH (the variable domain of the H chain) and CHγ1 (the γ1 region in the constant region of the H chain) via a disulfide bridge at their C-terminal regions. Each of these two homologous antibody fragments is designated Fab'.

"F(ab')2" is composed of two light chains and two heavy chains containing a CH1 domain and a part of CH2 domains in the constant region, as a result of which disulfide bridges are formed between the two heavy chains. F(ab')2 forming the polypeptide complex described herein can be preferably produced as follows. A complete monoclonal antibody or a similar antibody containing a desired antigen-binding domain is partially cleaved with a protease such as pepsin; and Fc fragments are removed by adsorption on a protein A column. There is no limitation on a specific protease as long as it has the ability to selectively cleave the complete antibody to form F(ab')2 under appropriate reaction conditions such as pH for the enzyme. Such proteases include, for example, pepsin and ficin.

Fc domain

The Fc domain that forms the polypeptide complex described herein can be preferably produced as follows. An antibody such as a monoclonal antibody is partially cleaved with a protease such as pepsin. The resulting fragment is then adsorbed onto a protein A or protein G column and eluted with an appropriate elution buffer. There is no limitation on a specific protease as long as it has the ability to selectively cleave antibodies such as monoclonal antibodies under reaction conditions such as a pH value appropriate for the enzyme. These proteases are, for example, pepsin and ficin.

The polypeptide complexes described herein comprise an Fc domain with reduced Fcγ receptor binding activity that includes amino acids that form the Fc domain of IgG1, IgG2, IgG3, or IgG4.

The isotype of an antibody is determined based on the structure of the constant region. The constant regions of the IgG1, IgG2, IgG3, and IgG4 isotypes are referred to as Cγ1, Cγ2, Cγ3, and Cγ4, respectively. The amino acid sequences of the Fc domain of the polypeptides that form human Cγ1, Cγ2, Cγ3, and Cγ4 are exemplified in SEQ ID NOs: 23, 24, 25, and 26, respectively. The relationship between the amino acid residues that form each amino acid sequence and the Kabot EU numbering (referred to herein as the EU index) is shown in Fig. 18.

The term "Fc domain" refers to a region outside F(ab')2 that contains two light chains and two heavy chains containing a portion of the constant region that includes the CH1 domain and a region between the CH1 and CH2 domains that allows disulfide bridges to form between the two heavy chains. The Fc domain that forms the polypeptide complex described herein can be preferably produced as follows. A monoclonal antibody of IgG1, IgG2, IgG3 or IgG4 or a similar antibody is partially cleaved with a protease such as pepsin, followed by elution of the fraction adsorbed on a protein A column. There is no limitation on a specific protease as long as it has the ability to selectively cleave the entire antibody to form F(ab')2 under reaction conditions such as pH appropriate for the enzyme. These proteases include, for example, pepsin and ficin.

Fcγ receptor

The term "Fcγ receptor" refers to a receptor that has the ability to bind to the Fc domain of monoclonal antibodies of the IgG1, IgG2, IgG3, or IgG4 species, and includes all members of the family of proteins encoded primarily by the Fcγ receptor gene. In humans, the family includes FcγRI (CD64), including the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including the isoforms FcγRIIa (including the H131 and R131 allotype), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including the FcγRIIIa isoform (including the V158 and F158 allotypes), and FcγRIIIb (including the FcγRIIIb-NA1 and FcγRIIIb-NA2 allotypes); as well as all unidentified human FcγRs, FcγR isoforms, and allotypes thereof. However, the Fcγ receptor is not limited to these examples. FcγRs include, but are not limited to, FcγRs derived from human, mouse, rat, rabbit, and monkey antibodies. FcγRs can be derived from any organism. Murine FcγR includes, but is not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as all unidentified murine FcγRs, FcγR isoforms, and allotypes thereof. Said preferred Fcγ receptors include, for example, human FcγI (CD64), FcγIIA (CD32), FcγIIB (CD32), FcγIIIA (CD16), and/or FcγIIIB (CD16). The polynucleotide sequence and amino acid sequence of FcγI are set forth in SEQ ID NOs: 13 (NM_000566.3) and 14 (NP_000557.1), respectively; the polynucleotide sequence and amino acid sequence of FcγIIA are presented in SEQ ID NOs: 15 (BC020823.1) and 16 (AAH20823.1), respectively; the polynucleotide sequence and amino acid sequence of FcγIIB are presented in SEQ ID NOs: 17 (BC146678.1) and 18 (AAI46679.1), respectively; the polynucleotide sequence and amino acid sequence of FcγIIIA are presented in SEQ ID NOs: 19 (BC033678.1) and 20 (AAH33678.1), respectively; and the polynucleotide sequence and amino acid sequence of FcγIIIB are presented in SEQ ID NOs: 21 (BC128562.1) and 22 (AAI28563.1), respectively (the RefSeq accession number of each sequence is given in parentheses). Whether the Fcγ receptor has the ability to bind to the Fc domain of an IgG1, IgG2, IgG3 or IgG4 monoclonal antibody can be assessed using the ALPHA Screen® assay (Amplified Luminescent Proximity Homogeneous Assay), the BIACORE method based on surface plasmon resonance (SPR), etc. (Proc. Natl. Acad. Sci. USA 103(11), 2006, pp. 4005-4010), in addition to the FACS and ELISA formats described above.

In this context, the term "Fc ligand" or "effector ligand" refers to a molecule, and preferably a polypeptide, that binds to the Fc domain of an antibody to form an Fc/Fc ligand complex. The molecule may be obtained from any organism. Binding of the Fc ligand to Fc preferably induces one or more effector functions. Such ligands include, but are not limited to, Fc receptors, FcγR, FcaR, FcsR, FcRn, C1q and C3, mannan-binding lectin, mannose receptor, Staphylococcus protein A, Staphylococcus protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRH) (Davis et al., Immunological Reviews 190, 2002, pp. 123-136), which are a family of Fc receptors homologous to FcγR. Fc ligands also include unidentified molecules that bind to Fc.

Fcγ receptor binding activity

Impaired Fc domain binding activity to any of the Fcγ receptors FcγI, FcγIIA, FcγIIB, FcγIIIA and/or FcγIIIB can be assessed using the FACS and ELISA formats described above, as well as the ALPHA Screen assay (homogeneous proximity-enhanced luminescence assay, BIACORE surface plasmon resonance (SPR) method) (Proc. Natl. Acad. Sci. USA 103(11), 2006, pp. 4005-4010).

ALPHA Screen analysis is based on the ALPHA technology, which is based on the principle described below, using two types of beads. The luminescent signal becomes detectable only when the molecules bound to the donor beads interact biologically with the molecules bound to the acceptor beads and when both beads are in close proximity to each other. The photosensitizer in the donor beads, excited by the laser beam, converts oxygen into excited singlet oxygen. When singlet oxygen diffuses from the donor beads and reaches the acceptor beads located in close proximity, a chemiluminescent reaction is induced in the acceptor beads. This reaction ultimately results in the emission of light. If the molecules bound to the donor beads do not interact with the acceptor beads, the singlet oxygen produced by the donor beads does not reach the acceptor beads and the chemiluminescent reaction does not occur.

For example, a biotin-labeled polypeptide complex is immobilized on donor beads and a glutathione-8-transferase (GST)-labeled Fcγ receptor is immobilized on acceptor beads. In the absence of a polypeptide complex containing a competing mutant Fc domain, the Fcγ receptor interacts with the polypeptide complex containing the wild-type Fc domain, resulting in a signal with a wavelength of 520 to 620 nm. The polypeptide complex containing the unlabeled mutant Fc domain competes with the polypeptide complex containing the wild-type Fc domain for interaction with the Fcγ receptor. The relative binding affinity can be estimated by quantifying the decrease in fluorescence due to competition. Methods for biotinylation of polypeptide complexes, such as antibodies, with sulfo-NHS-biotin or similar agents are known. Suitable methods for introducing a GST tag into the Fcγ receptor include methods that involve in-frame fusion of polypeptides encoding Fey and GST, expression of the fused gene using cells into which the vector carrying the gene has been introduced, and subsequent purification using a glutathione-containing column. The induced signal can preferably be analyzed, for example, by fitting a single-site competition model based on nonlinear regression analysis using a program such as GRAPHPAD PRISM (GraphPad; San Diego).

One of the substances to be studied for their interaction is immobilized as a ligand on a thin gold layer of the sensor chip. When light enters the back surface of the sensor chip so that total reflection occurs at the interface between the thin gold layer and the glass, the intensity of the reflected light at a particular site is partially reduced (SPR signal). Another substance to be studied for its interaction is injected as an analyte onto the surface of the sensor chip. When the analyte binds to the ligand, the mass of the immobilized ligand molecule increases. This changes the refractive index of the solvent on the surface of the sensor chip. The change in refractive index causes a positive shift in the SPR signal (and vice versa, dissociation shifts the signal back to its original position). In the Biacore system, the level of the above-described shift (i.e., the change in mass on the surface of the sensor chip) is plotted along the vertical axis, and thus a graph of the change in mass versus time (sensogram) is obtained as quantitative data. The kinetic parameters (the association rate constant (ka) and the dissociation rate constant (kd)) are determined from the sensorgram curves, and the affinity (KD) is determined as the ratio of these two constants. BIACORE methods are preferably used for inhibition assays. Examples of such inhibition assays are described in Proc. Natl. Acad. Sci. USA 103(11), 2006, pp. 4005-4010.

In the context of the present description, "reduced Fcγ receptor binding activity" means, for example, that when using the assay described above, the competitive activity of the test polypeptide complex is 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, or 15% or less, and most preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% less, compared to the competitive activity of a control polypeptide complex.

Polypeptide complexes comprising the Fc domain of a monoclonal antibody in the form of IgG1, IgG2, IgG3 or IgG4 can be used as control polypeptide complexes, respectively. The Fc domain structures are shown in SEQ ID NO: 23 (A added at the N-terminus of the sequence provided in RefSeq under the accession number AAC82527.1), SEQ ID NO: 24 (A added at the N-terminus of the sequence provided in RefSeq under the accession number AAB59393.1), SEQ ID NO: 25 (A added at the N-terminus of the sequence provided in RefSeq under the accession number CAA27268.1) and SEQ ID NO: 26 (A added at the N-terminus of the sequence provided in RefSeq under the accession number AAB59394.1). In addition, when a polypeptide complex containing a mutant Fc domain of an antibody of a specific isotype is used as a test substance, the effect of the mutant mutation on the binding activity to the Fcγ receptor is evaluated using a polypeptide complex containing the Fc domain of the same isotype as a control. As described above, polypeptide complexes containing a mutant Fc domain with a truly reduced binding activity to the Fcγ receptor are accordingly prepared.

Such known mutants include, for example, mutants with deletion of amino acids 231A-238S (EU numbering) (WO 2009/011941), as well as mutants C226S, C229S, P238S, (C220S) (J. Rheumatol 34, 2007, p. 11); C226S and C229S (Hum. Antibod. Hybridomas 1(1), 1990, pp. 47-54); C226S, C229S, E233P, L234V, and L235A (Blood 109, 2007, pp. 1185-1192).

Preferred polypeptide complexes are complexes that contain an Fc domain with an amino acid substitution at position 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331 or 332 (EU numbering) in the amino acids that form the Fc domain of an antibody of a particular isotype. The invention is not limited by the isotype of the antibody from which the Fc domain is obtained, and the corresponding Fc domain derived from a monoclonal antibody in the form of IgG1, IgG2, IgG3 or IgG4 can be used. Preferably, the Fc domain derived from antibodies in the form of IgG1 should be used.

Preferred polypeptide complexes are, for example, complexes that contain an Fc domain that contains one of the following substitutions, the position of which is determined according to EU numbering (each number denotes the position of an amino acid residue according to EU numbering; and the single-letter amino acid symbol that is located before the number denotes the amino acid residue before its substitution, and the single-letter amino acid symbol located after the number denotes the amino acid residue after the substitution), amino acids that form the Fc domain of an antibody in the form of IgG1:

(a) L234F, L235E, P331S;

(b) C226S, C229S, P238S;

(c) C226S, C229S or

(d) C226S, C229S, E233P, L234V, L235A;

as well as complexes that contain an Fc domain with a deletion of amino acids at positions 231-238.

In addition, preferred polypeptide complexes are also complexes that contain an Fc domain that contains one of the following substitutions, the position of which is determined according to EU numbering, of the amino acids that form the Fc domain of an antibody in the form of IgG2:

(e) H268Q, V309L, A330S and P331S;

(e) V234A;

(g) G237A;

(z) V234A and G237A;

(and) A235E and G237A or

(k) V234A, A235E and G237A. Each number represents the position of an amino acid residue according to EU numbering; and the single-letter amino acid symbol preceding the number represents the amino acid residue before its substitution, and the single-letter amino acid symbol following the number represents the amino acid residue after the substitution.

In addition, preferred polypeptide complexes are also complexes that contain an Fc domain that contains one of the following substitutions, the position of which is determined according to EU numbering, of the amino acids that form the Fc domain of an antibody in the form of IgG3:

(l) F241A;

(m) D265A or

(n) V264A. Each number represents the position of an amino acid residue according to EU numbering; and the single-letter amino acid symbol preceding the number represents the amino acid residue before its substitution, and the single-letter amino acid symbol following the number represents the amino acid residue after the substitution.

In addition, preferred polypeptide complexes are also complexes that contain an Fc domain that contains one of the following substitutions, the position of which is determined according to EU numbering, of the amino acids that form the Fc domain of an antibody in the form of IgG4:

(o) L235A, G237A and E318A;

(p) L235E or

(p) F234A and L235A. Each number represents the position of an amino acid residue according to EU numbering; and the single-letter amino acid symbol preceding the number represents the amino acid residue before its substitution, and the single-letter amino acid symbol following the number represents the amino acid residue after the substitution.

Other preferred polypeptide complexes are complexes comprising an Fc domain in which any amino acid at position 233, 234, 235, 236, 237, 327, 330 or 331 (EU numbering) in the amino acids forming the Fc domain of an IgG1 antibody is replaced by an amino acid that is at the corresponding position according to EU numbering in the corresponding IgG2 or IgG4.

Preferred polypeptide complexes are also complexes comprising an Fc domain in which one or more amino acids at positions 234, 235 and 297 (EU numbering) in the amino acids forming the Fc domain of the antibody in the form of IgG1 are(are) replaced by other amino acids. The invention is not limited to the type of amino acid after the replacement; however, the most preferred are polypeptide complexes comprising an Fc domain in which one or more amino acids at positions 234, 235 and 297 are(are) replaced by alanine.

Preferred polypeptide complexes are also complexes comprising an Fc domain in which the amino acid at position 265 (EU numbering) in the amino acids forming the Fc domain of an antibody in the form of IgG1 is replaced by another amino acid. The invention is not limited to the type of amino acid after the replacement; however, the most preferred are polypeptide complexes comprising an Fc domain in which the amino acid at position 265 is replaced by alanine.

Fc domain derived from a bispecific antibody

According to the present description, the corresponding Fc domains derived from the bispecific antibody can also be used as an Fc domain with reduced Fcγ receptor binding activity. A bispecific antibody is an antibody that has two different specificities. An IgG-type bispecific antibody can be secreted from a hybrid hybridoma (quadroma) obtained by fusing two types of hybridomas that produce an antibody in the form of IgG (Milstein C. et al., Nature 305, 1983, pp. 537-540).

Alternatively, the IgG-type bispecific antibody can also be secreted by introducing into cells a total of four genes, i.e., genes of L chains and H chains forming the two types of IgG of interest, and coexpressing them. However, there are theoretically ten combinations of H chains and L chains of IgG that can be obtained by such methods. It is difficult to purify IgG consisting of the desired combination of H chains and L chains from ten types of IgG. In addition, the theoretical amount of IgG secreted with the desired combination is also greatly reduced, so large-scale cultivation is required. This further increases the production cost.

In such a case, an appropriate amino acid substitution can be introduced into the CH3 domain of the H chain forming the Fc domain in order to preferentially secrete IgG with a heterologous combination of H chains. Specifically, this method can be carried out by replacing an amino acid that has a larger side chain (a knob (meaning "bulge")) with an amino acid in the CH3 domain of one of the H chains, and replacing an amino acid that has a smaller side chain (a hole (meaning "hollow")) with an amino acid in the CH3 domain of the other H chain so that the knob fits into the hole. This promotes the formation of the heteromeric H chain and simultaneously inhibits the formation of the homomeric H chain (WO 1996/027011; Ridgway JB et al., Protein Engineering 9, 1996, pp. 617-621; Merchant AM et al., Nature Biotechnology 16, 1998, pp. 677-681).

On the other hand, regarding the L chain, the variable region of the L chain is less polymorphic than the variable region of the H chain, and therefore it can be expected to obtain a common L chain which can have the ability to bind to both H chains. In order to achieve efficient expression of bispecific IgG, it is possible to introduce genes of such a common L chain and two H chains into cells which express IgG (Nature Biotechnology 16, 1998, pp. 677-681). However, this idea is difficult to implement since the probability of obtaining two types of antibodies containing the same L chain by random selection is low. Therefore, a method for selecting a common L chain which is characterized by a strong ability to bind to any different H chains has been proposed (WO 2004/065611).

In addition, there are also known methods for producing a bispecific antibody by applying methods for controlling polypeptide association or association of heteromeric multimers formed from polypeptides to the association between two polypeptides that form an Fc domain. In particular, methods for controlling polypeptide association can be used to produce a bispecific antibody (WO 2006/106905), in which the amino acid residues that form the interface between two polypeptides that form an Fc domain are altered to inhibit association between Fc domains that have the same sequence and to ensure the formation of polypeptide complexes formed by two Fc domains that have different sequences.

The above-mentioned two polypeptides that form an Fc domain derived from a bispecific antibody can be suitably used as a domain comprising an Fc domain according to the present invention. More specifically, the above-mentioned two preferred polypeptides that form an Fc domain include polypeptides in which the amino acids at positions 349 and 366 (EU numbering) in the amino acid sequence of one polypeptide are cysteine and tryptophan, respectively, and the amino acids at positions 356, 366, 368 and 407 (EU numbering) in the amino acid sequence of the other polypeptide are cysteine, serine, alanine and valine, respectively.

In another embodiment of the invention, preferred domains comprising the Fc domain of the present invention comprise two polypeptides that form an Fc domain in which the amino acid at position 409 (EU numbering) in the amino acid sequence of one polypeptide is aspartic acid and the amino acid at position 399 (EU numbering) in the amino acid sequence of the other polypeptide is lysine. According to the above embodiment of the invention, the amino acid at position 409 may be glutamic acid instead of aspartic acid, and the amino acid at position 399 may be arginine instead of lysine. In addition, aspartic acid at position 360 or 392 may also preferably be used in combination with lysine at position 399.

In another embodiment of the invention, preferred domains comprising the Fc domain of the present invention comprise two polypeptides that form an Fc domain, in which the amino acid at position 370 (EU numbering) in the amino acid sequence of one polypeptide is glutamic acid and the amino acid at position 357 (EU numbering) in the amino acid sequence of the other polypeptide is lysine.

In a further embodiment of the invention, preferred domains comprising the Fc domain of the present invention comprise two polypeptides that form an Fc domain in which the amino acid at position 439 (EU numbering) in the amino acid sequence of one polypeptide is glutamic acid and the amino acid at position 356 (EU numbering) in the amino acid sequence of the other polypeptide is lysine.

Furthermore, preferred domains comprising the Fc domain of the present invention include variants with a combination thereof:

two polypeptides form an Fc domain in which the amino acids at positions 409 and 370 (EU numbering) in the amino acid sequence of one polypeptide are aspartic acid and glutamic acid, respectively, and the amino acids at positions 399 and 357 (EU numbering) in the amino acid sequence of the other polypeptide are both lysine (in this embodiment, the glutamic acid at position 370 can be replaced with aspartic acid, and the glutamic acid at position 370 can be replaced with aspartic acid at position 392);

two polypeptides form an Fc domain in which the amino acids at positions 409 and 439 (EU numbering) in the amino acid sequence of one polypeptide are aspartic acid and glutamic acid, respectively, and the amino acids at positions 399 and 356 (EU numbering) in the amino acid sequence of the other polypeptide are both lysine (in this embodiment of the invention, glutamic acid at position 439 can be replaced by aspartic acid at position 360, 392, or 439);

two polypeptides form an Fc domain in which the amino acids at positions 370 and 439 (EU numbering) in the amino acid sequence of one polypeptide are both glutamic acid and the amino acids at positions 357 and 356 (EU numbering) in the amino acid sequence of the other polypeptide are both lysine; and

two polypeptides form an Fc domain in which the amino acids at positions 409, 370 and 439 (EU numbering) in the amino acid sequence of one polypeptide are aspartic acid, glutamic acid and glutamic acid, respectively, and the amino acids at positions 399, 357 and 356 (EU numbering) in the amino acid sequence of the other polypeptide are all lysine (in this embodiment, the amino acid at position 370 cannot be substituted with glutamic acid; alternatively, instead of substituting the amino acid at position 370 with glutamic acid, the glutamic acid at position 439 can be substituted with aspartic acid or the glutamic acid at position 439 can be substituted with aspartic acid at position 392).

In a further embodiment of the invention, preferred domains comprising the Fc domain of the present invention comprise two polypeptides that form an Fc domain, in which the amino acid at position 356 (EU numbering) in the amino acid sequence of one polypeptide is lysine, and the amino acids at positions 435 and 439 (EU numbering) in the amino acid sequence of the other polypeptide are arginine and glutamic acid, respectively.

When the two above-mentioned polypeptides that form an Fc domain derived from a bispecific antibody are used as a domain comprising the Fc domain of the present invention, the antigen-binding domains and/or CD3-binding domains of the present invention can be arranged in a desired combination.

Fc domain with reduced C-terminal heterogeneity

According to the present disclosure, Fc domains characterized by improved C-terminal heterogeneity in addition to the above characteristics can be used as Fc domains with reduced Fcγ receptor binding activity. More specifically, the present invention provides Fc domains in which glycine and lysine at positions 446 and 447 (EU numbering), respectively, in the amino acid sequences of two polypeptides forming an Fc domain that is derived from IgG1, IgG2, IgG3 or IgG4 are removed by deletion.

T cell receptor complex binding domain

In the context of the present description, "T-cell receptor complex binding domain" refers to a portion of an antibody to the T-cell receptor complex that contains a region that specifically binds to and is complementary to all or part of the T-cell receptor complex. Said T-cell receptor complex may be the T-cell receptor itself or an adaptor molecule that, in combination with the T-cell receptor, forms a T-cell receptor complex. A preferred adaptor is CD3.

T cell receptor binding domain

As used herein, a "T cell receptor binding domain" refers to a portion of a T cell receptor antibody that contains a region that specifically binds to and is complementary to all or a portion of the T cell receptor.

The variable region or constant region of the T-cell receptor can be used. However, the preferred epitopes to which the CD3-binding domain binds are those located in the constant region. Constant region sequences include, for example, the T cell receptor α chain (RefSeq accession number CAA26636.1; SEQ ID NO: 67), the T cell receptor β chain (RefSeq accession number C25777; SEQ ID NO: 68), the T cell receptor yl chain (RefSeq accession number A26659; SEQ ID NO: 69), the T cell receptor y2 chain (RefSeq accession number AAB63312.1; SEQ ID NO: 70), and the T cell receptor γ chain (RefSeq accession number AAA61033.1; SEQ ID NO: 71).

CD3 binding domain

As used herein, a "CD3 binding domain" refers to a portion of an anti-CD3 antibody comprising a region that specifically binds to and is complementary to all or a portion of CD3. The CD3 binding domain may be derived from one or more variable domains of the antibody. Preferably, the CD3 binding domain comprises both the light chain variable region (VL) of an anti-CD3 antibody and the heavy chain variable region (VH) of an anti-CD3 antibody. Said preferred CD3 binding domains include, for example, "single-chain Fv (scFv)", "single-chain antibody", "Fv", "single-chain Fv2 (scFv2)", "Fab" and "F(ab')2".

The CD3 binding domain of the present invention can bind to any epitope, as long as the epitope is located within the y chain, s chain or s chain of the sequence that makes up human CD3. Preferred CD3 binding domains according to the present invention include those that comprise the variable region of the light chain (VL) of an anti-CD3 antibody and the variable region of the heavy chain (VH) of an anti-CD3 antibody that bind to an epitope located in the extracellular domain of the s chain of the human CD3 complex. Said preferred CD3 binding domains include those that comprise the variable region of the light chain (VL) of an anti-CD3 antibody and the variable region of the heavy chain (VH) of the OKT3 antibody (Proc. Natl. Acad. Sci. USA 77:4914-4917 (1980)) or various known anti-CD3 antibodies. In addition, said suitable CD3-binding domains include domains derived from an anti-CD3 antibody with the desired characteristics, which are obtained by immunizing the desired animal with the γ-chain, δ-chain or ε-chain forming human CD3, using the methods described above. Suitable anti-CD3 antibodies from which the CD3-binding domain is derived are human antibodies and antibodies humanized according to the method described above. The structures of the γ-chain, δ-chain and ε-chain that form CD3 are represented as the polynucleotide sequences SEQ ID NO: 27 (NM 000073.2), SEQ ID NO: 29 (NM 000732.4) and SEQ ID NO: 31 (NM 000733.3), respectively, and as the polypeptide sequences SEQ ID NO: 28 (NP 000064.1), SEQ ID NO: 30 (NP 000723.1) and SEQ ID NO: 32 (NP 000724.1), respectively (RefSeq accession numbers are given in brackets).

Polypeptide complex

The structure of the polypeptide complex proposed in the present invention is not limited if it contains

(1) antigen-binding domain;

(2) a domain containing an Fc domain with reduced Fcγ receptor binding activity; and

(3) the T-cell receptor complex binding domain mentioned above.

According to the present invention, a preferred domain binding the T cell receptor complex is a T cell receptor binding domain or a CD3 binding domain. Each of the above-described domains can be linked directly via a peptide bond. For example, (1) F(ab')2 is used as an antigen-binding domain and (2) an Fc domain with reduced Fcγ receptor binding activity is used as a domain that contains an Fc domain with reduced Fcγ receptor binding activity. In this case, when the antigen-binding domain (1) is linked via a peptide bond to a domain that contains the Fc domain (2), the linked polypeptide forms an antibody structure. The antibody can be obtained by purifying the culture media of the above-described hybridomas or purifying the culture media of the desired host cells stably bearing the polynucleotide that codes for the antibody-forming polypeptide.

When the CD3-binding domain (3) is coupled to the antibody structure, the CD3-binding domain may be coupled via a peptide bond to the C-terminus of the constant region of the antibody structure. In another embodiment, the CD3-binding domain is coupled via a peptide bond to the N-terminus of the variable region of the heavy chain or the variable region of the light chain of the antibody structure. In another embodiment, the CD3-binding domain may be coupled via a peptide bond to the C-terminus of the constant region of the light chain of the antibody structure. The CD3-binding domain to be coupled may have any desired structure; however, said suitable CD3-binding domain preferably includes Fv and more preferably scFv. There are no restrictions on the valency of the CD3-binding domain that binds to the antibody structure. In order to couple the bivalent CD3 binding domain to the antibody structure, the monovalent CD3 binding domain can be coupled via a peptide bond to the respective C-termini of the two Fc domains forming the constant region of the antibody structure. Alternatively, in order to couple the bivalent CD3 binding domain to the antibody structure, the bivalent scFv (i.e. sc(Fv)2) can be coupled via a peptide bond to the C-terminus of one of the two Fc domains. In this case, it is possible to successfully obtain a polypeptide complex in which the bivalent scFv (i.e. sc(Fv)2) is coupled to the C-terminus of only one of the two Fc domains forming the constant region of the antibody structure, using the above-mentioned Fc domain derived from the bispecific antibody. Alternatively, to connect the monovalent CD3-binding domain to the antibody structure, the monovalent scFv can be connected via a peptide bond to the C-terminus of one of the two Fc domains. In this case, the polypeptide complex proposed in the present invention can be successfully prepared, in which the monovalent scFv is linked to the C-terminus of only one of the two Fc domains forming the constant region of the antibody structure, using the above-mentioned Fc domain derived from the bispecific antibody.

Furthermore, when the CD3-binding domain (3) is coupled via a peptide bond to the C-terminus of the constant region of the antibody structure, suitable polypeptide complexes include complexes in which the Fv of the heavy chain forming the CD3-binding domain is coupled to the C-terminus of one constant region (CH3 domain) forming the Fc domain, and the Fv fragment of the light chain forming the CD3-binding domain is coupled to the C-terminus of another constant region (CH3 domain) forming the Fc domain. In this case, a suitable linker, such as Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 7), is introduced to couple the Fv fragment of the heavy chain or the light chain to the C-terminus of the constant region (CH3 domain). The number of repeats in the linker is not critical; however, they can be selected from 1-10, preferably 2-8, or more preferably 2-6. In particular, it is possible to insert a suitable linker in which the number of repeats of [Gly-Gly-Gly-Gly-Ser] (SEQ ID NO: 7) is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

Alternatively, when a polypeptide complex is obtained in which the Fv fragment of the heavy chain forming the CD3-binding domain is linked to the C-terminus of one constant region (CH3 domain) forming the Fc domain, and the Fv fragment of the light chain of the CD3-binding domain is linked to the C-terminus of another constant region (CH3 domain) forming the Fc domain, then in order to increase the association between the Fv fragment of the heavy chain and the Fv fragment of the light chain, appropriate changes in the amino acid residues can be made that allow the formation of disulfide bridges between the Fv fragment of the heavy chain and the Fv fragment of the light chain.

In another embodiment of the invention, when a polypeptide complex according to the present invention is prepared, in which the Fv fragment of the heavy chain forming the CD3-binding domain is linked to the C-terminus of one constant region (CH3 domain) forming the Fc domain, and the Fv fragment of the light chain forming the CD3-binding domain is linked to the C-terminus of another constant region (CH3 domain) forming the Fc domain, then in order to increase the association between the Fv fragment of the heavy chain and the Fv fragment of the light chain, the CH1 and CL domains can be linked to both the Fv fragment of the heavy chain and the Fv fragment of the light chain.

According to another embodiment of the invention, in order to connect the bivalent CD3 binding domain to the antibody structure, the monovalent CD3 binding domain can be connected via a peptide bond to the corresponding C-termini of the two constant regions of the light chains or the corresponding N-termini of the variable regions of the light chains of the antibody structure. Alternatively, in order to connect the bivalent CD3 binding domain to the antibody structure, the bivalent scFv (i.e. sc(Fv)2) can be connected via a peptide bond to the corresponding C-termini of the two constant regions of the light chains or the corresponding N-termini of the variable regions of the light chains of the antibody structure. In this case, using the above-described Fc domain derived from the bispecific antibody, it is possible to efficiently prepare polypeptide complexes in which the bivalent scFv (i.e. sc(Fv)2) is linked to the C- or N-terminus of one of the two variable regions of the light chains of the antibody structure. Alternatively, in order to connect the monovalent CD3 binding domain to the antibody structure, the monovalent scFv can be linked via a peptide bond to the C- or N-terminus of one of the two variable regions of the light chains. In this case, using the above-described Fc domain derived from the bispecific antibody, it is possible to efficiently prepare polypeptide complexes in which the monovalent scFv is linked to the N- or C-terminus of one variable region of the light chain of the two variable regions of the light chains of the antibody structure.

In another embodiment of the invention, in order to connect the bivalent CD3 binding domain to the antibody structure, the monovalent CD3 binding domain can be connected via a peptide bond to the respective N termini of the two variable regions of the heavy chains of the antibody structure. Alternatively, in order to connect the bivalent CD3 binding domain to the antibody structure, the bivalent scFv (i.e. sc(Fv)2) can be connected via a peptide bond to the N terminus of one of the two variable regions of the heavy chains. In this case, using the above-described Fc domain derived from the bispecific antibody, it is possible to efficiently prepare polypeptide complexes in which the bivalent scFv (i.e. sc(Fv)2) is linked to the N terminus of only one of the two variable regions of the heavy chains of the antibody structure. Alternatively, in order to connect the monovalent CD3-binding domain to the antibody structure, the monovalent scFv can be linked via a peptide bond to the N-terminus of one of the two variable regions of the heavy chains. In this case, using the above-described Fc-domain derived from the bispecific antibody, it is possible to efficiently obtain polypeptide complexes in which the monovalent scFv is linked to the N-terminus of one of the two variable regions of the heavy chains of the antibody structure.

In addition, the above-described polypeptide complex can be obtained by directly connecting each domain via a peptide bond or by peptide binding via a peptide linker. In this case, as a linker to be used, for example, the above-described linker and corresponding linkers with a peptide tag, for example, a His tag, an HA tag or a FLAG tag, can be used. In addition, the ability to mutually bind based on a hydrogen bond, a disulfide bond, a covalent bond or an ionic interaction, or a combination thereof can be preferably used. For example, the affinity between CH1 and CL of an antibody can be used, or the above-described Fc domains derived from a bispecific antibody can be used for heteromeric association of Fc domains. In addition, intradomain disulfide bridges can be preferably used, as described in the examples.

In another structure of the polypeptide complex according to the present invention, for example, a monovalent Fv and a monovalent Fab are preferably used as (1) the antigen-binding domain. In this case, the following structure is used. The Fv fragment of the heavy chain (VH) or the Fv fragment of the light chain (VL) of the monovalent Fv is linked via a peptide bond to the CH1 domain of the heavy chain. The CH1 domain of the heavy chain is linked via a peptide bond to one of (2) two Fc domains with reduced binding activity to the Fcγ receptor, which form the polypeptide complex according to the present invention. The other VL or VH fragment of the monovalent Fv is linked via a peptide bond to the CL domain of the light chain, which is linked via a disulfide bridge to the CH1 domain of the heavy chain. Thus, VH and VL, respectively, connected to the ends of the CH1 domain of the heavy chain and the CL domain of the light chain, form the binding domain of the antibody. sc(Fv)2, which forms both (1) the binding domain of the antibody and (3) the CD3 binding domain, can be connected via a peptide bond to the N-terminus of the other Fc domain of the two domains described above. In this case, a polypeptide complex, which has a structure in which the CH1 domain of the heavy chain is connected via a peptide bond to one of the two Fc domains forming the polypeptide complex, and sc(Fv)2 is connected via a peptide bond to the other Fc domain, can be obtained using the above-described Fc domain derived from a bispecific antibody. The above-described polypeptide complex can be obtained by connecting each domain directly via a peptide bond or by peptide linkage via a peptide linker. In this case, the linker to be used is, for example, the linkers described above as an example and the corresponding linkers with a peptide tag, for example with a His tag, an HA tag or a FLAG tag.

In another preferred structure of the polypeptide complex according to the present invention, for example, a bivalent scFv is also used as (1) the antigen-binding domain. In one embodiment of the structure, a polypeptide complex can also be obtained in which one of the bivalent scFvs is linked via a peptide bond via a VH forming a (3) CD3-binding domain to one of the two (2) Fc-domains with reduced binding activity to the Fcγ-receptor, and the other bivalent scFv is linked via a peptide bond via a VL forming a (3) CD3-binding domain to one of the two (2) Fc-domains with reduced binding activity to the Fcγ-receptor. In this case, the above-described Fc-domain derived from a bispecific antibody can be used. The above-described polypeptide complex can be obtained by linking each domain directly via a peptide bond or by peptide linkage via a peptide linker. In this case, the linker to be used is, for example, the linkers described above as an example and the corresponding linkers with a peptide tag, for example with a His tag, an HA tag or a FLAG tag.

In another embodiment of the structure, for the creation of which a bivalent scFv is used as (1) an antigen-binding domain, it is possible to obtain a polypeptide complex in which one of the bivalent scFvs is linked via a peptide bond of an scFv forming a (3) CD3-binding domain to one of two (2) Fc-domains with reduced binding activity to the Fcγ-receptor, and the other bivalent scFv is linked via a peptide bond to the other (2) Fc-domain with reduced binding activity to the Fcγ-receptor. In this case, using the above-described Fc domain derived from the bispecific antibody, it is possible to obtain a polypeptide complex in which the scFv forming the antigen-binding domain is linked via a peptide bond by means of the scFv forming the CD3-binding domain to one of the two Fc domains forming the polypeptide complex, and the scFv forming the antigen-binding domain is linked via a peptide bond to the other Fc domain. The above-described polypeptide complex can be obtained by linking each domain directly via a peptide bond or by peptide bonding via a peptide linker. In this case, the linker that can be used is, for example, the linkers described above as an example and corresponding linkers with a peptide tag, for example, a His tag, an HA tag or a FLAG tag.

In another preferred structure of the polypeptide complex according to the present invention, for example, both the antigen-binding domain and the T-cell receptor complex-binding domain are each present in a monovalent Fab structure. To create one of the variants of the structure, it is possible to prepare a polypeptide complex in which the Fv fragment of the heavy chain of the monovalent Fab forming the antigen-binding domain is linked via the CH1 domain to one of the polypeptides forming the Fc domain, and the Fv fragment of the light chain of the Fab is linked to the CL domain, while the Fv fragment of the heavy chain of the Fab forming the T-cell receptor binding domain is linked via the CH1 domain to another polypeptide forming the Fc domain, and the Fv fragment of the light chain of the Fab is linked to the CL domain.

To create another variant of the structure, it is possible to obtain a polypeptide complex in which the Fv fragment of the heavy chain of a monovalent Fab, forming an antigen-binding domain, is connected via a CH1 domain to one polypeptide forming an Fc domain, and the Fv fragment of the light chain of Fab is connected to a CL domain, and the Fv fragment of the light chain of Fab, forming a T-cell receptor-binding domain, is connected via a CH1 domain to another polypeptide forming an Fc domain, and the Fv fragment of the heavy chain of Fab is connected to a CL domain. Alternatively, it is also possible to obtain a polypeptide complex in which the Fv fragment of the heavy chain of a monovalent Fab forming a T-cell receptor binding domain is linked via a CH1 domain to one polypeptide forming an Fc domain, and the Fv fragment of the light chain of the Fab is linked to a CL domain, the Fv fragment of the light chain of the Fab forming an antigen-binding domain is linked via a CH1 domain to another polypeptide forming an Fc domain, and the Fv fragment of the heavy chain of the Fab is linked to a CL domain.

To create another variant of the structure, it is possible to obtain a polypeptide complex in which the Fv fragment of the heavy chain of a monovalent Fab, forming an antigen-binding domain, is linked via a CH1 domain to one polypeptide forming an Fc domain, and the Fv fragment of the light chain of Fab is linked to a CL domain, and the Fv fragment of the light chain of Fab, forming a T-cell receptor-binding domain, is linked via a CL domain to another polypeptide forming an Fc domain, and the Fv fragment of the light chain of Fab is linked to a CH1 domain. Alternatively, it is also possible to obtain a polypeptide complex in which the Fv fragment of the heavy chain of a monovalent Fab forming a T-cell receptor binding domain is linked via a CH1 domain to one polypeptide forming an Fc domain, and the Fv fragment of the light chain of the Fab is linked to a CL domain, and the Fv fragment of the heavy chain of the Fab forming an antigen-binding domain is linked via a CL domain to another polypeptide forming an Fc domain, and the Fv fragment of the light chain of the Fab is linked to a CH1 domain.

In another embodiment of the structure of the polypeptide complex of the present invention, in which both the antigen-binding domain and the T-cell receptor complex-binding domain each have a monovalent Fab structure, preferred polypeptides of the present invention include polypeptides that have:

(1) an antigen-binding domain in which the Fv fragment of the heavy chain of the monovalent Fab structure that binds to the antigen is linked via the CH1 domain of one of the above-described polypeptides forming the Fc domain, and the Fv fragment of the Fab structure is linked to the CL domain; and

(2) a T cell receptor complex binding domain in which the heavy chain Fv fragment of the monovalent Fab structure that binds to the T cell receptor complex is linked via a CH1 domain to another polypeptide forming an Fc domain, and the light chain Fv fragment of the Fab structure is linked to a CL domain;

and in which the electrical charges of the CH1 and CL domains are controlled such that there is an association of the Fv fragment of the heavy chain of the antigen-binding domain with the Fv fragment of the light chain of the antigen-binding domain, or an association of the Fv fragment of the heavy chain of the T-cell receptor binding domain with the Fv fragment of the light chain of the T-cell receptor binding domain. In this embodiment of the invention, the structure (structure with controlled association) of the polypeptide complex is not limited to a specific structure, if the electrical charges of the CH1 and CL domains are controlled such that there is an association of the Fv fragment of the heavy chain of the antigen-binding domain with the Fv fragment of the light chain of the antigen-binding domain, or an association of the Fv fragment of the heavy chain of the T-cell receptor binding domain with the Fv fragment of the light chain of the T-cell receptor binding domain.

To create a variant of the structure with controlled association, it is possible to obtain a polypeptide complex in which the amino acid residues of the CH1 domain linked to the Fv fragment of the heavy chain of the domain binding the T-cell receptor complex have the same electrical charges as the amino acid residues of the CL domain linked to the Fv fragment of the light chain of the antigen-binding domain.

To create a variant of the structure with controlled association, it is possible to obtain a polypeptide complex in which the amino acid residues of the CH1 domain linked to the Fv fragment of the heavy chain of the antigen-binding domain have the same electrical charges as the amino acid residues of the CL domain linked to the Fv fragment of the light chain of the domain binding the T-cell receptor complex.

To create a variant of the structure with controlled association, it is possible to obtain a polypeptide complex in which the amino acid residues of the CH1 domain linked to the Fv fragment of the heavy chain of the domain binding the T-cell receptor complex have the same electrical charges as the amino acid residues of the CL domain linked to the Fv fragment of the light chain of the antigen-binding domain, and the amino acid residues of the CH1 domain linked to the Fv fragment of the heavy chain of the antigen-binding domain have the same electrical charges as the amino acid residues of the CL domain linked to the Fv fragment of the light chain of the domain binding the T-cell receptor complex.

To create another variant of the structure with controlled association, it is possible to obtain a polypeptide complex in which the amino acid residues of the CH1 domain linked to the Fv fragment of the heavy chain of the domain binding the T-cell receptor complex have the same electrical charges as the amino acid residues of the CL domain linked to the Fv fragment of the light chain of the antigen-binding domain, and the amino acid residues of the CH1 domain linked to the Fv fragment of the heavy chain of the domain binding the T-cell receptor complex have electrical charges opposite to the charges of the amino acid residues of the CL domain linked to the Fv fragment of the light chain of the domain binding the T-cell receptor.

To create another variant of the structure with controlled association, it is possible to obtain a polypeptide complex in which the amino acid residues of the CH1 domain linked to the Fv fragment of the heavy chain of the domain binding the T-cell receptor complex have the same electrical charges as the amino acid residues of the CL domain linked to the Fv fragment of the light chain of the antigen-binding domain; the amino acid residues of the CH1 domain linked to the Fv fragment of the heavy chain of the antigen-binding domain have the same electrical charges as the residues of the CL domain linked to the Fv fragment of the light chain of the domain binding the T-cell receptor complex; The amino acid residues of the CH1 domain linked to the Fv fragment of the heavy chain of the T-cell receptor binding domain have electrical charges opposite to the charges of the amino acid residues of the CL domain linked to the Fv fragment of the light chain of the T-cell receptor binding domain.

To create another variant of the structure with controlled association, it is possible to obtain a polypeptide complex in which the amino acid residues of the CH1 domain linked to the Fv fragment of the heavy chain of the antigen-binding domain have the same electrical charges as the amino acid residues of the CL domain linked to the Fv fragment of the light chain of the domain binding the T-cell receptor complex, and the amino acid residues of the CH1 domain linked to the Fv fragment of the heavy chain of the antigen-binding domain have electrical charges opposite to the charges of the amino acid residues of the CL domain linked to the Fv fragment of the light chain of the antigen-binding domain.

To create an alternative version of the structure with controlled association, it is possible to obtain a polypeptide complex in which the amino acid residues of the CH1 domain linked to the Fv fragment of the heavy chain of the T cell receptor complex-binding domain have the same electrical charges as the amino acid residues of the CL domain linked to the Fv fragment of the light chain of the antigen-binding complex; the amino acid residues of the CH1 domain linked to the Fv fragment of the heavy chain of the T cell receptor complex-binding domain have the same electrical charges as the amino acid residues of the CL domain linked to the Fv fragment of the light chain of the T cell receptor complex-binding domain; and the amino acid residues of the CRP domain linked to the Fv fragment of the heavy chain of the antigen-binding domain have electrical charges opposite to the charges of the amino acid residues of the CL domain linked to the Fv fragment of the light chain of the antigen-binding domain.

To create another variant of the structure with controlled association, it is possible to obtain a polypeptide complex in which the amino acid residues of the CH1 domain linked to the Fv fragment of the heavy chain of the domain binding the T-cell receptor complex have the same electrical charges as the amino acid residues of the CL domain linked to the Fv fragment of the light chain of the antigen-binding domain; the amino acid residues of the CH1 domain linked to the Fv fragment of the heavy chain of the antigen-binding domain have the same electrical charges as the amino acid residues of the CL domain linked to the Fv fragment of the light chain of the domain binding the T-cell receptor complex; the amino acid residues of the CH1 domain linked to the Fv fragment of the heavy chain of the T cell receptor binding domain complex have electrical charges opposite to the charges of the amino acid residues of the CL domain linked to the Fv fragment of the light chain of the T cell receptor binding domain; and the amino acid residues of the CH1 domain linked to the Fv fragment of the heavy chain of the antigen-binding domain have electrical charges opposite to the charges of the amino acid residues of the CL domain linked to the Fv fragment of the light chain of the antigen-binding domain.

Control of electrical charges of CH1 and CL domains

In order to obtain a bispecific polypeptide complex that recognizes an epitope of the T cell receptor binding domain via the heavy and light chains of the T cell receptor binding domain and an epitope of an antigen via the heavy and light chains of the antigen binding domain, ten types of polypeptide complex molecules can theoretically be obtained if each of the four chains is expressed when creating the polypeptide complex.

However, the desired polypeptide complex molecule can preferably be obtained, for example, by implementing such control of the domains that leads to inhibition of the association between the heavy chain of the T-cell receptor binding domain and the light chain of the antigen binding domain and/or the association between the heavy chain of the antigen binding domain and the light chain of the T-cell receptor binding domain.

Examples include changing the amino acid residues that form the interface between the CH1 of the heavy chain of the T cell receptor binding domain and the CL of the light chain of the antigen binding domain to positively charged amino acid residues, and changing the amino acid residues that form the interface between the CH1 of the heavy chain and the CL of the light chain of the T cell receptor binding domain to negatively charged amino acid residues. As a result of such changes, an undesirable association between the CH1 of the heavy chain of the T cell receptor binding domain and the CL of the light chain of the antigen binding domain is inhibited, since the electrical charges of the amino acid residues that form the interface are positive in both cases, and an undesirable association between the CH1 of the heavy chain of the antigen binding domain and the CL of the light chain of the T cell receptor binding domain is also inhibited, since the electrical charges of the amino acid residues that form the interface are negative in both cases. The desired polypeptide complex according to the present invention can be effectively created as a result of the desired association between CH1 of the heavy chain of the T-cell receptor binding domain and CL of the light chain of the T-cell receptor binding domain, as well as as a result of the desired association between CH1 of the heavy chain of the antigen-binding domain and CL of the light chain of the antigen-binding domain. In addition, the desired association between the heavy and light chains of the T-cell receptor binding domain is preferably enhanced because the amino acid residues that form the interface have opposite electrical charges to each other. The desired association between the heavy and light chains of the antigen-binding domain is also preferably enhanced because the amino acid residues that form the interface have opposite electrical charges to each other. This makes it possible to effectively create the polypeptide complex according to the present invention with the desired association.

In addition, the association control proposed in the present invention can also be used to inhibit the association between CH1 domains (heavy chains of the T cell receptor binding domain and the antigen binding domain) or between CL domains (light chains of the T cell receptor binding domain and the antigen binding domain).

Those skilled in the art can appropriately select a desired polypeptide complex in which the association is controlled according to the present invention by the type of amino acid residues localized in close proximity to the CH1/CL interface during association.

In addition, using public databases or the like, persons skilled in the art can appropriately select suitable CH1 and CL sequences of antibodies in an organism such as a human, a monkey, a mouse, or a rabbit. More specifically, the amino acid sequence information of CH1 and CL can be obtained by the methods described in the examples below.

In particular, as demonstrated in the examples described below, specific examples of combinations of amino acid residues located in close proximity (adjacent or contacting) at the CH1/CL interface during association between CH1 and CL, which are correspondingly linked to the VH and VL forming the T cell receptor binding domain or antigen binding domain, include:

- lysine (K) at position 147 (EU numbering) in CH1 (e.g. at position 147 in the amino acid sequence SEQ ID NO: 1) and threonine (T) at position 180 (EU numbering) in the adjacent (contacting) CL;

- lysine (K) at position 147 (EU numbering) in CH1 and series (S) at position 131 (EU numbering) in the adjacent (contacting) CL;

- lysine (K) at position 147 (EU numbering) in CH1 and threonine (T) at position 164 (EU numbering) in the adjacent (contacting) CL;

- lysine (K) at position 147 (EU numbering) in CH1 and asparagine (N) at position 138 (EU numbering) in CL, which are adjacent (in contact) with each other;

- lysine (K) at position 147 (EU numbering) in CH1 and glutamic acid (E) at position 123 (EU numbering) in the adjacent (contacting) CL;

- glutamine (Q) at position 175 (EU numbering) in CH1 and glutamine (Q) at position 160 (EU numbering) in the adjacent (contacting) CL;

or

- lysine (K) at position 213 (EU numbering) in CH1 and glutamic acid (E) at position 123 (EU numbering) in the adjacent (contacting) CL.

These positions are numbered according to the guidelines of Kabat et al. (Kabat E.A. et al., Sequence of Proteins of Immunological Interest. NIH, 1991).

In the context of the present description, the numbers designated by the EU index correspond to the EU numbering (Sequences of proteins of immunological interest, NIH Publication No. 91-3242). According to the present invention, the terms "amino acid residue at position X (EU numbering)" and "amino acid at position X (EU numbering)" (where X denotes an arbitrary number) are used interchangeably with the terms "amino acid residue corresponding to position X (EU numbering)", "amino acid corresponding to position X (EU numbering)".

As described in the examples below, the desired polypeptide complex can preferably be obtained by substituting amino acid residues and using the methods provided in the present invention.

With respect to the above-mentioned amino acid residues, it is known that they are highly conserved in humans and mice (J. Mol. Recognit. 16, 2003, pp. 113-120). Thus, by changing the amino acid residues corresponding to the above-mentioned amino acid residues, it is also possible to control the association between CH1 and CL in the constant region of the polypeptide complex proposed in the present invention, which differs from the polypeptide complexes described in the examples.

In particular, the present invention provides polypeptide complexes with controlled association between a heavy chain and a light chain, in which one, two or more pairs selected from the group consisting of pairs of amino acid residues specified below in subparagraphs (a) to (e) have the same electrical charges:

(a) the amino acid residue at position 147 (EU numbering) in CH1 and the amino acid residue at position 180 (EU numbering) in CL;

(b) the amino acid residue at position 147 (EU numbering) in CH1 and the amino acid residue at position 131 (EU numbering) in CL;

(c) the amino acid residue at position 147 (EU numbering) in CH1 and the amino acid residue at position 164 (EU numbering) in CL;

(d) the amino acid residue at position 147 (EU numbering) in CH1 and the amino acid residue at position 138 (EU numbering) in CL;

(d) the amino acid residue at position 147 (EU numbering) in CH1 and the amino acid residue at position 123 (EU numbering) in CL and

(e) the amino acid residue at position 175 (EU numbering) in CH1 and the amino acid residue at position 160 (EU numbering) in CL.

In addition, another embodiment of the present invention are antibodies in which the amino acid residues in the pair of amino acid residues specified below in subparagraph (g) have the same electrical charges:

(g) the amino acid residue at position 213 (EU numbering) in CH1 and the amino acid residue at position 123 (EU numbering) in CL.

The amino acid residues in each of the above pairs are localized in close proximity to each other upon association, as described in the examples below. Using homology-based modeling methods or other methods using commercially available programs, those skilled in the art can appropriately identify amino acid positions in the desired CH1 or CL corresponding to the amino acid residues specified in subparagraphs (a) to (g) above, and can appropriately change the amino acid residues at these positions.

Said "electrically charged amino acid residue" in the above-described antibody is preferably selected, for example, from amino acid residues belonging to the following group (X) or (Y):

(X) glutamic acid (E) and aspartic acid (D); and (Y) lysine (K), arginine (R), histidine (H).

With respect to the polypeptide complexes described above, the term "having the same electrical charge" means, for example, that all of the two or more amino acid residues belong to one of the groups (X) and (Y) described above. On the other hand, the term "having an opposite electrical charge" means, for example, that at least one of the two or more amino acid residues is an amino acid residue belonging to one of the groups (X) and (Y) described above, and the other amino acid residues are amino acid residues that belong to another group.

Preferred embodiments of the present invention are also methods for producing the above-described polypeptide complexes and methods proposed in the present invention for controlling association by replacing amino acid residues from the groups indicated above in subparagraphs (a)-(g) with amino acid residues that have the same electrical charge.

In the context of the present invention, the amino acid residues "to be replaced" are not limited to the amino acid residues of the constant region as defined above. Using the homology-based modeling method or other methods based on the use of commercially available programs, those skilled in the art can appropriately identify amino acid residues that form an interface in a mutant polypeptide or heteromeric multimer and appropriately replace amino acid residues at such positions to control association.

In methods for inhibiting an undesirable association between a heavy chain and a light chain by introducing repulsive charges at the interface between the variable regions of the heavy chain and the light chain, the amino acid residues that contact each other at the interface between the variable region of the heavy chain (VH) and the variable region of the light chain (VL) are, for example, glutamine (Q) at position 39 (e.g., at position 39 in the amino acid sequence SEQ ID NO: 6 described in WO 2006/106905) in the variable region of the heavy chain FR2 and glutamine (Q) at position 38 (e.g., at position 44 in the amino acid sequence SEQ ID NO: 8 described in WO 2006/106905) in the adjacent (contacting) variable region of the light chain FR2. Such preferred amino acid residues are also, for example, leucine (L) at position 45 (e.g. at position 45 in the amino acid sequence SEQ ID NO: 6, described in WO 2006/106905) in the variable region of the heavy chain FR2 and proline (P) at position 44 (e.g. at position 44 in the amino acid sequence SEQ ID NO: 8, presented in WO 2006/106905) in the adjacent (contacting) variable region of the light chain FR2. These positions are numbered according to the guidelines of Kabat et al. (Kabat E. A. et al., Sequence of Proteins of Immunological Interest, NIH Press, 1991).

With respect to the above-mentioned amino acid residues, it is known that they are highly conserved in humans and mice (J. Mol. Recognit. 16, 2003, pp. 113-120). Thus, by changing the amino acid residues corresponding to the above-mentioned amino acid residues, it is also possible to control the association between the variable regions VH and VL of antibodies that differ from the polypeptide complexes described in the examples.

More specifically, said antibodies that have variable regions of heavy chains and light chains include antibodies in which the following amino acid residues (1) and (2) or (3) and (4) have the same electrical charges:

(1) the amino acid residue corresponding to position 39 (EU numbering) in the variable region of the heavy chain;

(2) the amino acid residue corresponding to position 38 (EU numbering) in the variable region of the light chain;

(3) the amino acid residue corresponding to position 45 (EU numbering) in the variable region of the heavy chain;

(4) the amino acid residue corresponding to position 44 (EU numbering) in the variable region of the light chain.

The above amino acid residues (1) and (2) or (3) and (4) are localized in close proximity to each other upon association. By means of a homology-based modeling method or other methods using commercially available programs, persons skilled in the art can appropriately identify amino acid positions in a desired variable region of a heavy chain or a light chain corresponding to the amino acid residues indicated above in ((4), and can appropriately change the amino acid residues at the indicated positions.

In the above antibody, the "electrically charged amino acid residue" is preferably selected from, for example, amino acid residues belonging to the following group (X) or (Y):

(X) glutamic acid (E) and aspartic acid (D); and (Y) lysine (K), arginine (R), histidine (H).

In human and mouse antibodies, the above amino acid residues (1)-(4) are typically:

(1) glutamine (Q);

(2) glutamine (Q);

(3) leucine (L) and

(4) proline (P), respectively.

In preferred embodiments of the present invention, the above amino acid residues are changed (e.g., replaced with a charged amino acid). The types of the above amino acid residues (1) to (4) are not limited to those mentioned above. These amino acids may be any other amino acids corresponding to those mentioned above. For example, regarding a human antibody, an amino acid corresponding to the amino acid at position 38 (EU numbering) in the variable region of a light chain may be histidine (H). Based on published documents (e.g., J. Mol. Recognit. 16, 2003, pp. 113-120) or the like, those skilled in the art can determine the type of amino acid residue that corresponds to any position in the light chain, and can therefore change the amino acid residue accordingly (e.g., replaced with a charged amino acid).

In methods for inhibiting the unwanted association between a heavy chain and a light chain by replacing amino acid residues that form a hydrophobic core at the interface between the variable regions of the heavy chain and the light chain with electrically charged polar amino acids, preferred amino acid residues that have the ability to form a hydrophobic core at the interface between the variable region of the heavy chain (VH) and the variable region of the light chain (VL) are leucine (L) at position 45 in the variable region of the heavy chain and proline (P) at position 44, bordering the variable region of the light chain.

In general, the term "hydrophobic core" refers to the region in which the side chains of hydrophobic amino acids are combined within the associated polypeptide. Examples of hydrophobic amino acids include alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. However, amino acid residues other than these hydrophobic amino acids (e.g., tyrosine) may participate in the formation of the hydrophobic core. In combination with the hydrophilic surface formed by the protruding side chains of the hydrophilic amino acids, the hydrophobic core serves as a "driving force" accelerating the association of water-soluble polypeptides. When hydrophobic amino acids of two different domains are present on the surface of the molecule and are accessible to water molecules, the entropy increases, resulting in an increase in free energy. As a result, the association of the two domains with each other must be such as to reduce the free energy, which is required for stabilization. Hydrophobic amino acids at the interface are immersed into the molecule to form a hydrophobic core.

It is believed that when the hydrophobic amino acids that form the hydrophobic core during polypeptide association are changed to polar amino acids that carry an electrical charge, the formation of the hydrophobic core is inhibited. This leads to the inhibition of polypeptide association.

Other known techniques can also be used to create the polypeptide complexes proposed in the present invention. For example, in order to enhance the association of the first VH (VH1) and the first VL (VL1) and/or the second VH (VH2) and the second VL (VL2), in addition to the "changes" proposed in the present invention, amino acids in one of the variable regions of the H chain are replaced with amino acids that have a larger side chain ("protrusion"; "bump"), and amino acids in the variable region of the other H chain are replaced with amino acids with a smaller side chain ("hole"; "void"), as a result of which the "protrusion" is placed in the "hole". This enhances the association of VH1 and VL1 and/or VH2 and VL2, resulting in further inhibition of the association between VH1 and VL2 polypeptides and/or between VH2 and VL1 (WO 1996/027011; Ridgway JB et al., Protein Engineering 9:617-621 (1996); Merchant AM et al., Nature Biotechnology 16:677-681 (1998)).

In creating the polypeptide complex described above, each domain can be directly connected via a peptide bond or the peptides can be linked via a peptide linker. In this case, the linker used can be the linker described above as an example and corresponding linkers with a peptide tag, for example, with a His tag, an HA tag, a myc tag or a FLAG tag. In addition, the ability to mutually bind based on hydrogen bonds, a disulfide bond, a covalent bond or based on ionic interaction, or a combination thereof can be preferably used. For example, the affinity between CH1 and CL of the antibody can be used, or the Fc domains described above, derived from the bispecific antibody, can be used for heteromeric association of Fc domains. In addition, interdomain disulfide bridges can be preferably used, as described in the examples.

The polypeptide complexes provided in the present invention include, for example, the variants shown in Figs. 17, 19 and 24.

The polypeptide complexes provided in the present invention can be prepared by the same methods as those described above for the preparation of recombinant antibodies.

In addition, the present invention relates to polynucleotides encoding the polypeptide complex of the present invention. The polypeptide complex of the present invention can be inserted into any expression vectors. To obtain cells expressing the polypeptide complex, an appropriate host is transformed with an expression vector. The polypeptide complex encoded by the polynucleotide can be obtained by culturing cells expressing the polypeptide complex and collecting the expression product from the culture supernatant. In particular, the present invention relates to vectors carrying a polynucleotide that encodes the polypeptide complex of the present invention, cells that contain the vectors, and methods for obtaining the polypeptide complex of the present invention, which consist in culturing the cells and collecting the polypeptide complex from the culture supernatant. For the above-described purposes, the same techniques can be used as those described above for recombinant antibodies.

Pharmaceutical composition

Another object of the present invention is pharmaceutical compositions that contain as an active substance a polypeptide complex that includes:

(1) antigen-binding domain;

(2) a domain containing an Fc domain with reduced Fcγ receptor binding activity and

(3) CD3-binding domain.

The present invention also relates to therapeutic agents for inducing cell cytotoxicity, which contain as an active substance the above-described complex (therapeutic agents for inducing cell cytotoxicity), cell growth suppressing agents and anticancer agents. The pharmaceutical compositions proposed in the present invention can be used as a means for carrying out therapy or prevention of cancer. According to the present invention, the therapeutic agents for inducing cell cytotoxicity, cell growth suppressing agents and anticancer agents proposed in the present invention are preferably administered to individuals suffering from cancer or having a risk of recurrence of cancer.

According to the present invention, therapeutic agents for inducing cellular cytotoxicity, cell growth suppressing agents and anticancer agents, which contain as an active substance a polypeptide complex that includes:

(1) antigen-binding domain;

(2) a domain containing an Fc domain with reduced Fcγ receptor binding activity and

(3) CD3-binding domain,

can also be considered as a method for preventing or treating cancer, which consists in carrying out a step in which the polypeptide complex is administered to an individual, or the polypeptide complex can be used to prepare therapeutic agents for inducing cellular cytotoxicity, agents that suppress cell growth, or anticancer agents.

In the context of the present description, the term “contains as an active substance a polypeptide complex that includes:

(1) antigen-binding domain;

(2) a domain containing an Fc domain with reduced Fcγ receptor binding activity and

(3) CD3-binding domain",

means that the composition contains a polypeptide complex as the main active ingredient; however, the relative content of the polypeptide complex is not specifically indicated.

The pharmaceutical composition of the present invention, the therapeutic agent for inducing cellular cytotoxicity, the cell growth suppressing agent, or the anticancer agent of the present invention can be prepared, if necessary, using various types of polypeptide complexes. For example, the cytotoxic effect on cells expressing an antigen can be enhanced using a mixture of a plurality of polypeptide complexes of the present invention that bind to the same antigen. Alternatively, the therapeutic effect can be enhanced by preparing a polypeptide complex of the present invention that contains an antigen-binding domain that binds to a cancer antigen in combination with other polypeptide complexes of the present invention that contain an antigen-binding domain for another antigen.

If desired, the polypeptide complexes of the present invention can be encapsulated in microcapsules (microcapsules made of hydroxymethylcellulose, gelatin, poly(methyl methacrylate), etc.) and can be included in components of colloidal systems intended for the administration of medicinal substances (liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) (for example, see Remington's Pharmaceutical Science 16th ed., Oslo, 1980)). In addition, methods for preparing agents in the form of sustained release agents are known and can be used for the polypeptide complexes of the present invention (J. Biomed. Mater. Res. 15, 1981, pp. 267-277; Chemtech. 12, 1982, pp. 98-105; US No. 3,773,719; published European patents (EP) EP 58481 and EP 133988; Biopolymers 22, 1983, pp. 547-556).

The pharmaceutical compositions, cell growth inhibitory agents or anticancer agents of the present invention can be administered to a patient either orally or parenterally. Parenteral administration is preferable. Specifically, these administration methods include injection, nasal administration, transpulmonary administration and transdermal administration. Injections include, for example, intravenous injections, intramuscular injections, intraperitoneal injections and subcutaneous injections. For example, the pharmaceutical compositions, therapeutic agents for inducing cellular cytotoxicity, cell growth inhibitory agents or anticancer agents of the present invention can be administered locally or systemically by injection. In addition, suitable administration methods can be selected depending on the patient's age and symptoms. The administered dose can be selected, for example, from the following range: from 0.0001 to 1000 mg per kg of body weight per administration. Alternatively, the dose can be selected, for example, from the following range: from 0.001 to 100,000 mg/patient. However, the dose of the pharmaceutical composition proposed in the present invention is not limited to the indicated doses.

The pharmaceutical compositions of the present invention can be prepared by conventional methods (see, for example, Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A.), and can also contain pharmaceutically acceptable carriers and additives. Examples include, but are not limited to, surfactants, excipients, colorants, flavors, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow agents, and flavors, and other conventional carriers can be used. Specific examples of carriers are light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, calcium carmellose, sodium carmellose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salt, etc.

The present invention also provides methods for damaging cells expressing a cancer antigen or inhibiting cell growth, comprising contacting cells expressing a cancer antigen with a polypeptide complex of the present invention that binds to a cancer antigen. Monoclonal antibodies that bind to a cancer antigen are described above as a cancer antigen-binding polypeptide complex of the present invention, which is included in the therapeutic agents for inducing cellular cytotoxicity, cell growth inhibitory agents, or anticancer agents of the present invention. The invention is not limited to cells that are bound by the cancer antigen-binding polypeptide complex of the present invention if they express a cancer antigen. In particular, in the present invention, preferred cancer antigen-expressing cells are ovarian cancer cells, prostate cancer cells, breast cancer cells, uterine cancer cells, liver cancer cells, lung cancer cells, pancreatic cancer cells, gastric cancer cells, bladder cancer cells and colon cancer cells. If the cancer antigen is GPC3, the invention is not limited to specific cells as long as they express GPC3. However, preferred cancer cells are hepatocarcinoma cells, lung cancer cells and ovarian cancer cells.

According to the present invention, "bringing into contact" can be carried out, for example, by adding the cancer antigen-binding polypeptide complex of the invention to the culture media of cells expressing the cancer antigen, which are cultured in vitro. In this case, the added polypeptide complex can be used in a form suitable for this purpose, such as a solution or a solid substance obtained by lyophilization, or the like. When the polypeptide complex of the present invention is added as an aqueous solution, the solution may be a pure aqueous solution containing only the polypeptide complex, or a solution containing, for example, the above-mentioned surfactant, excipient, colorant, flavoring agent, preservative, stabilizer, buffer, suspending agent, isotonicity agent, binder, disintegrant, lubricant, flowability agent and corr. The concentration of the added complex is not critical; however, the final concentration in the culture medium is preferably from 1 pg/ml to 1 g/ml, more preferably from 1 ng/ml to 1 mg/ml, and even more preferably from 1 μg/ml to 1 mg/ml.

According to another embodiment of the present invention, "bringing into contact" can also be carried out in vivo by treating non-human animals transplanted with cancer antigen-expressing cells or animals in which cancer antigen expression occurs endogenously. The method of administration can be oral or parenteral. Parenteral administration is preferred. In particular, said methods of administration include injection, nasal administration, pulmonary administration and transdermal administration. Injections include, for example, intravenous injections, intramuscular injections, intraperitoneal injections and subcutaneous injections. For example, the pharmaceutical compositions, therapeutic agents for inducing cellular cytotoxicity, cell growth inhibitory agents or anticancer agents provided in the present invention can be applied locally or systemically by injection. In addition, suitable methods of administration can be selected depending on the age of the animal and the symptoms. When the polypeptide complex according to the present invention is added as an aqueous solution, the solution may be a pure aqueous solution containing only the polypeptide complex, or a solution containing, for example, the above-mentioned surfactant, excipient, colorant, flavoring agent, preservative, stabilizer, buffer, suspending agent, isotonicity agent, binder, disintegrant, lubricant, flowability agent and flavoring agent. The administered dose can be selected, for example, from the following range: from 0.0001 to 1000 mg per kg of body weight per administration. Alternatively, the dose can be selected, for example, from the following range: from 0.001 to 100,000 mg/patient. However, the dose of the polypeptide complex according to the present invention is not limited to these examples.

The methods described below are preferably used as a method for evaluating or determining cellular cytotoxicity caused by contacting the polypeptide complex of the present invention with antigen-expressing cells to which the antigen-binding domain contained in the polypeptide complex of the present invention binds. The methods for evaluating or determining cytotoxic activity in vitro include methods for determining the activity of cytotoxic T cells or the like. Whether or not the polypeptide complex of the present invention has the ability to induce T cell-mediated cellular cytotoxicity can be determined by known methods (see, for example, Current protocols in Immunology, Chapter 7. Immunologic studies in humans, edited by John E, Coligan et al., John Wiley & Sons, Inc., 1993). In the cytotoxicity assay, a polypeptide complex whose antigen-binding domain binds to an antigen different from the antigen recognized by the antigen-binding domain of the polypeptide complex of the present invention and which is not expressed on cells is used as a control polypeptide complex. The control polypeptide complex is assayed in the same manner. Activity is then assessed by determining whether the polypeptide complex of the present invention has a more pronounced cytotoxic activity than the control polypeptide complex.

The cytotoxic activity in vivo is assessed or determined, for example, by the following procedure. Cells expressing an antigen to which the antigen-binding domain included in the polypeptide complex of the present invention binds are transplanted intradermally or subcutaneously into an animal other than a human. Then, starting from the day of transplantation or after it, the test polypeptide complex is administered into a vein or peritoneal cavity every day or at intervals of several days. Over time, the tumor size is determined. The difference in the change in tumor size can be considered as cytotoxic activity. As in the in vitro assay, a control polypeptide complex is administered. The polypeptide complex of the present invention can be considered to have cytotoxic activity when the tumor size is smaller in the group treated with the polypeptide complex of the present invention than in the group treated with the control polypeptide complex.

The MTT method and the measurement of isotope-labeled thymidine uptake into cells are preferably used to evaluate or determine the effect of contact with the polypeptide complex of the present invention on the suppression of growth of cells expressing the antigen to which the antigen-binding domain of the polypeptide complex binds. In this case, methods similar to those described above for evaluating or determining cytotoxic activity in vivo can preferably be used to evaluate or determine the ability to suppress cell growth in vivo.

The present invention also provides kits that can be used in the method of the present invention, which contain the polypeptide complex of the present invention or the polypeptide complex obtained by the method of the present invention. The kits can be packaged together with an additional pharmaceutically acceptable carrier or medium, or with instructions including instructions for use of the kits, etc.

Furthermore, the present invention relates to polypeptide complexes according to the present invention or polypeptide complexes obtained by the method according to the present invention for use in the method according to the present invention.

All documents cited in this description that characterize the prior art are incorporated herein by reference.

Examples

The present invention is described below with reference to specific examples, which do not limit the scope of the invention.

Example 1. Construction and study of GPC3 ERY2

(1) Basic principles

There is a well-known method for prolonging the blood half-life of an administered protein in vivo, which is based on FcRn-mediated recycling using the protein of interest conjugated to the Fc domain of an antibody. However, conjugation of natural-type Fc to BiTE may result in the induction of different cytokines, since the same molecule can bind to a T cell via the anti-CD3 scFv fragment of its BiTE moiety and simultaneously bind to FcgR (Fcγ receptor) on the cell membrane of, for example, an NK cell or a macrophage via its Fc domain, and the resulting cross-linking may activate these cells in a cancer antigen-independent manner. Thus, a molecule designated ERY2 was prepared in which the BiTE antibody was linked via a polypeptide linker to an Fc domain having reduced binding activity to the Fcγ receptor (silent Fc), and the activity of ERY2 was assessed by comparison with the activity of BiTE. An anti-CD3 epsilon chain scFv was linked via a short peptide linker to an anti-glypican 3 (GPC3) scFv, which is a GPI-linked protein known to be highly expressed on liver cancer cells, to produce anti-GPC3 BiTE (GPC3 BiTE) (Fig. 17A). This was then linked to a silent Fc to produce anti-GPC3 ERY2 (GPC3 ERY2) (Fig. 17B). In addition, anti-GPC3 conventional IgG antibodies were generated for comparison. The IgG-type anti-GPC3 antibody was produced as an antibody with a reduced content of fucose in the sugar chain fragment, i.e., an antibody with a low fucose level, which is known to have increased ADCC activity.

(2) Construction of GPC3 BiTE

By PCR amplification using the expression vector for the anti-GPC3 antibody as a template, cDNAs were obtained, each encoding either the variable region of the H chain (anti-GPC3 VH) or the variable region of the L chain (anti-GPC3 VL). PCR was carried out using primers containing the appropriate additional sequences and the above-mentioned cDNAs as templates to construct a cDNA fragment encoding an anti-GPC3 scFv having an amino acid sequence in which the anti-GPC3 VH and anti-GPC3 VL are linked by a linker with three repeats of Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 7).

In addition, a series of oligonucleotides were prepared, each having a nucleotide sequence encoding a partial sequence of the H chain variable region (M12 VH) or the L chain variable region (M12 VL) of an antibody to CD3 (M12) and having complementary sequences at their ends. The oligonucleotides were designed so that they could be linked to each other through the regions of complementary sequences by a polymerase chain reaction to synthesize a polynucleotide corresponding to the H chain variable region (M12 VH) and the L chain variable region (M12 VL). The oligonucleotides were mixed and then linked to each other by PCR to produce two cDNAs encoding the amino acid sequences of the corresponding variable regions. PCR was performed using primers containing the appropriate additional sequences and the above-mentioned cDNAs as templates to construct a cDNA fragment encoding scFv M12, having an amino acid sequence in which M12 VL and M12 VH are linked via a linker having three repeats of Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 7).

Then, by PCR using primers containing the appropriate additional sequences and the above-mentioned cDNA fragments, each encoding either the anti-GPC3 scFv or the M12 scFv, as templates, a cDNA fragment was constructed that encoded an amino acid sequence in which the anti-GPC3 scFv and the M12 scFv were joined via a linker consisting of Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 7) and with a His-tag at the C-terminus (eight histidine residues) (SEQ ID NO: 33 without the 19 amino acids at the amino terminus).

PCR was performed using primers containing the appropriate additional sequences and a cDNA fragment encoding the amino acid sequence of SEQ ID NO: 33 lacking the 19 amino-terminal amino acids as a template to obtain a cDNA fragment in which an EcoRI cleavable sequence, a Kozak sequence, and a nucleotide sequence encoding a secretory signal sequence were added to the 5' end of the above cDNA fragment and a NotI cleavable sequence was added to the 3' end. The resulting cDNA fragment was digested with EcoRI and NotI and inserted into a mammalian cell expression vector to obtain an expression vector for GPC3 BiTE (SEQ ID NO: 33; the mature sequence lacks the 19 amino acids at the amino terminus that serve as a signal sequence).

The vector was introduced into CHO DG44 cells by electroporation. After serial dilution, the cells were cultured in the presence of 1 mg/ml geneticin to isolate drug-resistant cell lines. The culture supernatant of the resulting cell lines was analyzed by Western blotting using an anti-His-tag antibody to select cell lines expressing GPC3 BiTE.

The culture supernatant obtained by large-scale culturing of the cell line was loaded onto a column packed with SP Sepharose FF column (GE Healthcare). After washing the column, the fraction containing GPC3 BiTE was eluted using a NaCl concentration gradient. The fraction was loaded onto a HisTrap HP column (GE Healthcare). After washing the column, the fraction containing GPC3 BiTE was eluted using an imidazole concentration gradient. The fraction was concentrated by ultrafiltration, and then the concentrate was loaded onto a column packed with Superdex 200 (GE Healthcare). Only the monomeric fraction containing GPC3 BiTE was collected to obtain purified GPC3 BiTE.

(3) Construction of GPC3 ERY2

PCR was performed using primers containing the same corresponding additional sequences as in the method described above and using a method well known to those skilled in the art, such as site-directed mutagenesis using the QuikChange Site-Directed Mutagenesis Kit (Stratagene), to generate expression vectors into which a polynucleotide encoding GPC3 ERY2_Hk (SEQ ID NO: 34; mature sequence that lacks the 19 amino acids at the amino terminus that serve as a signal sequence) or GPC3 ERY2 Hh (SEQ ID NO: 35; mature sequence that lacks the 19 amino acids at the amino terminus that serve as a signal sequence) was inserted.

These expression vectors were co-introduced into FreeStyle293-F cells (Invitrogen) for transient expression of GPC3 ERY2. The resulting culture supernatant was loaded onto a column containing Anti FLAG M2 (Sigma). After washing, the column was eluted using FLAG peptide at a concentration of 10 mg/ml (Sigma). The fraction containing GPC3 ERY2 was loaded onto a HisTrap HP column (GE Healthcare). After washing, the column was eluted using an imidazole concentration gradient. The fraction containing GPC3 ERY2 was concentrated by ultrafiltration, and then the concentrate was loaded onto a column packed with Superdex 200 (GE Healthcare). Only the monomeric fraction containing GPC3 ERY2 was collected to obtain purified GPC3 ERY2.

(4) Construction of low-fucose anti-GPC3 antibody

The expression vector for the anti-GPC3 antibody (designated as humanized GC33 antibody in WO 2006/006693) was introduced into GDP-fucose gene knockout CHO DXB11 cells (Cancer Sci. 101(10), 2227-2233, 2010) by electroporation. After serial dilution, the cells were cultured in the presence of 0.5 mg/ml geneticin to select for drug-resistant lines, and cell lines expressing the low-fucose GPC3 antibody were obtained. The culture supernatant obtained from the culture of these cells was subjected to routine purification by affinity chromatography using a Hitrap® protein A column (Pharmacia). The antibody-containing fraction was then purified by gel filtration using Superdex 20026/60 (Pharmacia). The monomeric fraction was collected from the eluate to obtain the low-fucose anti-GPC3 antibody.

(5) Cytotoxicity assay using human peripheral blood mononuclear cells

(5-1) Obtaining a suspension of human peripheral blood mononuclear cells (PBMC)

Peripheral blood samples of 50 ml were obtained from healthy volunteers (adults) using syringes pre-filled with 100 μl (1000 units/ml) of heparin solution (Novo-Heparin 5000 units per injection; Novo Nordisk). Peripheral blood was diluted two-fold with PBS(-), divided into four equal aliquots and added to Leucosep lymphocyte separation tubes (catalog no. 227290; Greiner bio-one system) that had been pre-injected with 15 ml of Ficoll-paque PLUS and centrifuged. After centrifugation of the separation tubes (2150 rpm, 10 min, room temperature), the mononuclear cell fraction layer was collected. The cells in the mononuclear cell fraction were washed once with Dulbecco's modified Eagle's medium (SIGMA) containing 10% FBS (hereinafter referred to as 10%FBS/D-MEM), and then the cell density was adjusted to 4× ¹⁰⁶ cells/mL with 10%FBS/D-MEM. The resulting cell suspension was used as a human PBMC suspension in subsequent experiments.

(5-2) Analysis of cytotoxic activity

Cytotoxic activity was assessed based on cell growth inhibition using xCELLigence Real-Time Cell Analyzer (Roche Diagnostics). The target cells used were SK-pca13a cell line, which was derived from SK-HEP-1 cell line overexpressing human GPC3. SK-pca13a cells were detached from the plates and seeded in E-Plate 96 (Roche Diagnostics) at 1× ¹⁰⁴ cells/well (100 μl/well). Then, viable cell analysis was started using xCELLigence Real-Time Cell Analyzer. The next day, the plate was removed from xCELLigence Real-Time Cell Analyzer and 50 μl of each prepared antibody at different concentrations (0.004, 0.04, 0.4, and 4 nM) were added to the plate. After reacting for 15 min at room temperature, 50 μl of human PBMC suspension (2× ¹⁰⁵ cells/well) prepared according to the method described in section (5-1) were added. The cells were again placed into the xCELLigence Real-Time Cell Analyzer to start the viable cell analysis. The reaction was carried out under 5% nitrogen dioxide gas at 37°C. The cell growth inhibition rate (%) was determined by the following formula using the cell index value determined 72 h after the addition of human PBMC. The cell index value used for the calculation was standardized such that the cell index value determined immediately before the addition of the antibody was taken as 1.

Degree of cell growth inhibition (%) = (A-B) × 100/(A-1)

A represents the average cell index in the well without antibody (target cell and human PBMC only), and B represents the average cell index in each well. The measurement was performed in triplicate.

The cytotoxic activity of GPC3 BiTE, GPC3 ERY2 and anti-GPC3 IgG antibody was evaluated using human blood-derived PBMCs (peripheral blood mononuclear cells) as effector cells. GPC3 BiTE showed very strong activity (Fig. 1). This activity was significantly higher than that of the low-fucose anti-GPC3 antibody. Thus, GPC3 BiTE may serve as an effective therapeutic agent that is more active than the IgG antibody. On the other hand, the activity of GPC3 ERY2 was not as strong as that of GPC3 BiTE, although it was still higher than that of the anti-GPC3 IgG antibody. This suggests that the addition of Fc alone to BiTE does not generate the desired molecule.

Example 2. Construction and evaluation of GPC3 ERY5. GPC3 ERY6 and GPC3 ERY7

Next, in order to improve the specific activity, the cancer agent binding domain (GPC3) was converted into a bivalent one to enhance the cancer cell binding activity. Another anti-GPC3 scFv was added to GPC3 ERY2, creating GPC3 ERY5 (Fig. 17D). In addition, a Fab-type GPC3 binding domain was added instead of the scFv, creating GPC3 ERY7 (Fig. 17E). In addition, GPC3 ERY6 (Fig. 17E) was also constructed, in which the scFv to the CD3 epsilon chain of GPC3 ERY5 was cleaved into two arms.

Specifically, a method known to those skilled in the art, such as PCR using primers containing the same corresponding additional sequences as in the above-described method, was used to obtain a series of expression vectors into which a polynucleotide encoding GPC3 ERY5 Hh, GPC3 ERY6 Hk, GPC3 ERY6 Hh, GPC3 ERY7 Hh or GPC3 ERY7 L was inserted.

For short-term expression of each engineered molecule, the following combinations of expression vectors were introduced into FreeStyle293-F cells:

A. Engineered Molecule: GPC3 ERY5

Polypeptides encoded by polynucleotides inserted into expression vectors: GPC3 ERY5_Hh (SEQ ID NO: 36; mature sequence that lacks the 19 amino acid residues at the amino terminus that serve as a signal sequence) and GPC3 ERY2_Hk.

B. Engineered Molecule: GPC3 ERY6

Polypeptides encoded by polynucleotides inserted into expression vectors: GPC3 ERY6_Hk (SEQ ID NO: 37; mature sequence lacking the 19 amino acids at the amino terminus serving as a signal sequence) and GPC3 ERY6 Hh (SEQ ID NO: 38; mature sequence lacking the 19 amino acids at the amino terminus serving as a signal sequence).

B. Engineered Molecule: GPC3 ERY7

Polypeptides encoded by polynucleotides inserted into expression vectors: GPC3 ERY7 Hh (SEQ ID NO: 39; mature sequence lacking the 19 amino acids at the amino terminus serving as a signal sequence), GPC3 ERY7 L (SEQ ID NO: 40; mature sequence lacking the 19 amino acids at the amino terminus serving as a signal sequence), and GPC3 ERY2_Hk.

The resulting culture supernatant was loaded onto an Anti FLAG M2 column (Sigma). After washing, the column was eluted with FLAG peptide at a concentration of 0.1 mg/ml (Sigma). The fraction containing the created molecule was loaded onto a HisTrap HP column (GE Healthcare). After washing, the column was eluted with an imidazole concentration gradient. The fraction containing the created molecule was concentrated by ultrafiltration. Then, the fraction was loaded onto a Superdex 200 column (GE Healthcare). Only the monomeric fraction was collected from the eluate, obtaining each purified created molecule.

These polypeptide complexes were compared with GPC3 BiTE in terms of cytotoxic activity. As a result, it was demonstrated that the cytotoxic activity of these polypeptide complexes was not as high as that of GPC3 BiTE (Fig. 2-4). This result suggests that adding Fc to the BiTE structure or a structure mimicking the said structure and having a configuration that allows bivalent binding to a cancer antigen does not lead to the creation of the desired molecule.

Example 3. Construction and evaluation of GPC3 ERY8-2. GPC3 ERY9-1 and GPC3 ERY10-1

(1) Construction of GPC3 ERY8-2, GPC3 ERY9-1 and GPC3 ERY10-1

Next, molecules without the BiTE structure but possessing the required activity were created. IgG to a cancer antigen (GPC3) was used as a backbone, and a molecule was created in which scFv to the epsilon chain of CD3 was added to this backbone. The IgG Fc used as a backbone, as in the cases described above, was a silent Fc with reduced binding activity to FcgR (Fcγ receptor). GPC3 ERY8-2 (Fig. 17G), GPC3 ERY10-1 (Fig. 17I), and GPC3 ERY9-1 (Fig. 17B) were constructed, in which scFv to the epsilon chain of CD3 was attached to the N-terminus of the H-chain, the C-terminus of the H-chain, and the C-terminus of the L-chain of an IgG-type antibody to GPC3, respectively.

Specifically, a method known to those skilled in the art, such as PCR using primers containing the same corresponding additional sequences as in the above-described method, was used to obtain a series of expression vectors into which a polynucleotide encoding GPC3 ERY8-2_Hk (SEQ ID NO: 41; a mature sequence that does not contain the 19 amino acids at the amino terminus serving as a signal sequence), GPC3 ERY8-2 Hh (SEQ ID NO: 42; a mature sequence that does not contain the 19 amino acids at the amino terminus serving as a signal sequence), GPC3 ERY9-1H (SEQ ID NO: 43; a mature sequence that does not contain the 19 amino acids at the amino terminus serving as a signal sequence), GPC3 ERY9-1_L-His (SEQ ID NO: 44; a mature sequence that lacks the 19 amino acids at the amino terminus that serve as a signal sequence), GPC3 ERY9-1L-FLAG (SEQ ID NO: 45; mature sequence that lacks the 19 amino acids at the amino terminus that serve as a signal sequence), or GPC3 ERY10-1_Hh (SEQ ID NO: 46; mature sequence that lacks the 19 amino acids at the amino terminus that serve as a signal sequence).

For short-term expression of each engineered molecule, the following combinations of expression vectors were introduced into FreeStyle293-F cells.

G. Engineered Molecule: GPC3 ERY8-2

Polypeptides encoded by polynucleotides inserted into expression vectors: GPC3 ERY8-2 Hk (SEQ ID NO: 41; mature sequence lacking the 19 amino acids at the amino terminus serving as a signal sequence), GPC3 ERY8-2 Hh (SEQ ID NO: 42; mature sequence lacking the 19 amino acids at the amino terminus serving as a signal sequence), and GPC3 ERY7L.

D. Engineered Molecule: GPC3 ERY9-1

Polypeptides encoded by polynucleotides inserted into expression vectors: GPC3 ERY9-1 H (SEQ ID NO: 43; mature sequence lacking the 19 amino acids at the amino terminus serving as a signal sequence), GPC3 ERY9-1_L-His (SEQ ID NO: 44; mature sequence lacking the 19 amino acids at the amino terminus serving as a signal sequence), and GPC3 ERY9-1L-FLAG (SEQ ID NO: 45; mature sequence lacking the 19 amino acids at the amino terminus serving as a signal sequence). E. Engineered Molecule: GPC3 ERY10-1 Polypeptides encoded by polynucleotides inserted into expression vectors: GPC3 ERY10-1_Hh (SEQ ID NO: 46; mature sequence that lacks the 19 amino acid residues at the amino terminus that serve as a signal sequence) and GPC3 ERY8-2_Hk, GPC3 ERY7 L.

The resulting culture supernatant was loaded onto an Anti FLAG M2 column (Sigma). After washing, the column was eluted with FLAG peptide at a concentration of 0.1 mg/ml (Sigma). The fraction containing the created molecule was loaded onto a HisTrap HP column (GE Healthcare). After washing, the column was eluted with an imidazole concentration gradient. The fraction containing the created molecule was concentrated by ultrafiltration. Then, the fraction was loaded onto a Superdex 200 column (GE Healthcare). Only the monomeric fraction was collected from the eluate, obtaining each purified created molecule.

These molecules were evaluated for cytotoxic activity in vitro. The result showed that all the molecules had cytotoxic activity comparable to or higher than that of GPC3 BiTE (Fig. 5). In particular, it was found that GPC3 ERY9-1 and GPC3 ERY10-1 had significantly higher cytotoxic activity than GPC3 BiTE. The present invention demonstrated for the first time that molecules created by adding scFv to the CD3 epsilon chain of IgG to a cancer antigen had cytotoxic activity comparable to or higher than that of BiTE. In particular, it was unexpectedly found in the invention that molecules such as GPC3 ERY9-1 and GPC3 ERY10-1 had significantly higher cytotoxic activity than BiTE, despite the large distance between their cancer antigen-binding domain and the CD3 epsilon chain-binding domain.

(2) In vivo efficacy evaluation of GPC3 ERY8-2 and GPC3 ERY10-1:

The in vivo efficacy of GPC3 ERY8-2 and GPC3 ERY10-1, which demonstrated comparable or superior cytotoxic activity to GPC3 BiTE when assessed using the in vitro assay described in section (1), was determined. GPC3-expressing human lung cancer cell line PC-10 cells were mixed with human PBMCs and then transplanted into NOD severe combined immunodeficiency mice. Mice were treated with either GPC3 ERY8-2 or GPC3 ERY10-1 (referred to as the “pre-mix” model).

In particular, the efficacy of GPC3 ERY8-2 in the “pre-mix” model using PC-10 was assessed as follows. PBMCs were isolated from healthy donors. NK cells were isolated from PBMCs using CD56 MicroBeads for human blood separation (MCAS Miltenyi biotec). Human squamous cell lung carcinoma cell line PC-10 (Immuno-Biological Laboratories Co., Ltd.) (5× ¹⁰⁶ cells), human NK cell-deficient PBMCs (4.5× ¹⁰⁶ cells), and the (artificial) basement membrane Matrigel Matrix (BD) were mixed and then transplanted subcutaneously into the inguinal region of NOD mice with severe combined immunodeficiency (CLEA Japan Inc.; 7-week-old females). The day of transplantation was designated as day 0. One day before transplantation, mice were intraperitoneally injected with anti-asialo-ganglioside M1 (asialo-GM1) antibody (Wako Pure Chemical Industries) at 0.2 mg/mice. Two hours after transplantation, GPC ERY8-2 was intraperitoneally injected at 30 μg/mice. GPC ERY8-2 was injected a total of five times during the period from day 0 to day 4.

In addition, the efficacy of GPC3 ERY10-1 in the “pre-mix” model using PC-10 was evaluated as follows. PBMCs were isolated from the blood of healthy donors. NK cells were isolated from PBMCs using CD56 MicroBeads for human blood separation (MCAS Miltenyi biotec). Human squamous cell lung carcinoma cell line PC-10 (Immuno-Biological Laboratories Co., Ltd.) (5× ¹⁰⁶ cells), human NK cell-deficient PBMCs (4.5× ¹⁰⁶ cells), and Matrigel Matrix basement membrane (BD) were mixed and then transplanted subcutaneously into the inguinal region of NOD severe combined immunodeficiency mice (CLEA Japan Inc.; 7-week-old females). The day of transplantation was designated as day 0. One day before transplantation, mice were intraperitoneally injected with anti-asialo-GM1 antibody (Wako Pure Chemical Industries) at 0.2 mg/mice. Two hours after transplantation, GPC ERY10-1 was intraperitoneally injected at 30 μg/mice. GPC ERY10-1 was injected a total of 13 times during the time periods of days 0–4, days 7–11, and days 14–16.

The result showed that in the GPC3 ERY8-2 and GPC3 ERY10-1 treated groups, tumor growth was significantly suppressed compared with the vehicle (PBS) treated group (Figs. 6 and 7).

In addition, the in vivo efficacy of GPC3 ERY10-1 was also assessed using an alternative model. Specifically, T cells were grown by culturing human PBMC in vitro and then introduced into NOD mice with severe combined immunodeficiency that had developed tumors following PC-10 transplantation. The mice were treated by injecting GPC3 ERY10-1 (designated as the T cell transfer model).

Specifically, the efficacy of GPC3 ERY10-1 was evaluated in the T cell transfer model using the PC-10 cell line as follows. T cells were culture expanded using a human cell activation/expansion kit (MACS Miltenyi biotec) and PBMCs isolated from the blood of healthy volunteers. The human squamous cell lung carcinoma cell line PC-10 (Immuno-Biological Laboratories Co., Ltd.) (1× ¹⁰⁷ cells) was mixed with the basement membrane Matrigel Matrix (BD) and then subcutaneously transplanted into the inguinal region of NOD severe combined immunodeficiency mice (CLEA Japan Inc.; 7-week-old females). The day of transplantation was designated as day 0. Mice were intraperitoneally injected with anti-asialo-GM1 antibody (Wako Pure Chemical Industries) at 0.2 mg/mice 1 day before transplantation and on days 6, 8, 12, 16, and 20. On day 6 after transplantation, mice were grouped according to tumor size and body weight and then transplanted with T cells obtained by culture expansion as described above at 1 × 10 ⁷ cells/mice into the peritoneal cavity. GPC ERY10-1 was intraperitoneally injected at 30 μg/mice 2 h after transplantation. GPC ERY10-1 was injected a total of 5 times on days 7, 8, 12, 16, and 17.

The result indicated that, using this model, the GPC3 ERY10-1-treated group also had a significant antitumor effect compared with the vehicle (PBS)-treated group (Fig. 8).

The data described above demonstrate that a series of molecules in which one scFv of an antibody to the epsilon chain of CD3 is added to an IgG scaffold having a silent Fc have a pronounced antitumor effect in vivo.

(3) Evaluation of retention in plasma

To address the question of whether molecules such as GPC3 ERY8-2, GPC3 ERY9-1, and GPC3 ERY10-1 have a significantly longer plasma half-life compared to GPC3 BiTE, GPC3 ERY9-1 and GPC3 ERY10-1 were administered at 30 μg/molecular dose to NOD severe combined immunodeficiency mice that had not been transplanted with cancer cells, and their plasma concentrations were determined over time.

Specifically, the pharmacokinetic (PK) analysis was performed as follows. GPC3 ERY9-1 and GPC3 ERY10-1 were intraperitoneally administered to NOD mice with severe combined immunodeficiency (CLEA Japan Inc.; 8-week-old females) at a dose of 30 μg/mice. Blood samples were collected from the buccal vein of the mice using a hematocrit capillary (Terumo) at 15 min, 2 h, 1 day, 2 days, and 7 days after administration. Plasma was obtained from the blood.

GPC3 ERY9-1 and GPC3 ERY10-1 were diluted accordingly and added to GPC3-expressing Ba/F3 cells (GPC3/BaF) or human CD3 epsilon chain-expressing Ba/F3 cells (CD3/BaF) to react GPC3 ERY9-1 or GPC3 ERY10-1 with GPC3/BaF and CD3/BaF. After washing the cells, FITC-labeled secondary antibody was added for further interaction. After washing the cells, the fluorescence intensity of the label on the cells was measured using an Epics XL flow cytometer (Beckman Coulter) to obtain a calibration curve for each antibody.

Blood samples from mice treated with GPC3 ERY9-1 or GPC3 ERY10-1 were collected over a period of time. Plasma was obtained from the blood and diluted accordingly. Using a method similar to that used to prepare calibration curves, the plasma samples were reacted with GPC3/BaF or CD3/BaF to determine the amount of GPC3 ERY9-1 and GPC3 ERY10-1 bound to each cell in the plasma. Based on the measured values, the concentration of each antibody in the plasma was calculated, and calibration curves were constructed according to the method described above.

The result indicated that the concentration of both GPC3 ERY9-1 and GPC3 ERY10-1 in blood was maintained at a level greater than 10 nM 2 days after administration (Fig. 9 and 10). These data indicate that molecules such as GPC3 ERY9-1 and GPC3 ERY10-1 have significantly longer plasma half-lives than BiTE.

(4) Effect of Silenced Fc on Cancer Antigen-Independent Cytokine Induction

(4-1) Construction of GPC3 ERY15-1 with FcgR-binding Fc

To address the question of whether molecules such as GPC3 ERY8-2, GPC3 ERY9-1, and GPC3 ERY10-1 could induce cancer antigen-independent cytokine induction, GPC3 ERY15-1 was constructed to have an FcgR-binding Fc (Fig. 17K).

In particular, similar to the method described above, PCR using primers containing the appropriate additional sequences and a method well known to those skilled in the art, such as the QuikChange Site-Directed Mutagenesis Kit (Stratagene), were used to create expression vectors into which a polynucleotide encoding GPC3 ERY15-1_Hh (SEQ ID NO: 47; a mature sequence that does not contain the 19 amino acids at the amino terminus that serve as a signal sequence) or GPC3 ERY15-1_Hk (SEQ ID NO: 48; a mature sequence that does not contain the 19 amino acids at the amino terminus that serve as a signal sequence) was inserted.

Expression vectors for GPC3 ERY15-1_Hh (SEQ ID NO: 47; mature sequence lacking the 19 amino acids at the amino terminus serving as a signal sequence), GPC3 ERY15-1_Hk (SEQ ID NO: 48; mature sequence lacking the 19 amino acids at the amino terminus serving as a signal sequence), and GPC3 ERY7L were co-introduced into FreeStyle293-F cells for transient expression of GPC3 ERY15-1. The resulting culture supernatant was loaded onto an Anti FLAG M2 column (Sigma). After washing, the column was eluted with 0.1 mg/mL FLAG peptide (Sigma). The fraction containing GPC3 ERY15-1 was loaded onto a HisTrap HP column (GE Healthcare). After washing, the column was eluted with an imidazole concentration gradient. The fraction containing the created molecule was concentrated by ultrafiltration. Then, the fraction containing GPC3 ERY15-1 was loaded onto a Superdex 200 column (GE Healthcare). Only the monomeric fraction of GPC3 ERY15-1 was collected from the eluate, obtaining the purified GPC3 ERY15-1 molecule.

(4-2) Analysis of the ability to induce cancer antigen-independent cytokine induction

The ability of GPC3 ERY15-1 to induce cancer antigen-independent cytokine induction was compared with that of GPC3 BiTE, GPC3 ERY9-1, GPC3 ERY10-1, and catumaxomab. PBMCs were isolated from healthy volunteers using the method described above. Fifty microliters of each antibody adjusted to 40 nM were added to 50 microliters of human PBMC suspension (2 x 105 cells/well), followed by the addition of 100 microliters of 10% FBS/D-MEM. The reaction mixture was incubated in an atmosphere containing 5% nitrogen dioxide gas at 37°C. After incubation for 72 h, the culture supernatant was collected and the cytokines secreted into the culture supernatant were quantified using Cytometric Beads Array (CBA) technology using the Human Th1/Th2/Thl7 kit (BD). The analysis was performed in triplicate using the method specified in the attached protocol.

It was found that GPC3 ERY15-1 and catumaxomab, which have an FcgR-binding Fc, had a strong cytokine-inducing ability. In contrast, GPC3 BiTE, which lacks Fc, and GPC3 ERY9-1 and GPC3 ERY10-1, which have a silent Fc, did not show cytokine-inducing ability (Fig. 11). This result suggests that molecules that have a silent Fc, such as GPC3 ERY8-2, GPC3 ERY9-1, and GPC3 ERY10-1, are highly safe molecules that do not have the ability to induce cytokines in a cancer antigen-independent manner.

Example 4. Construction and evaluation of GPC3 ERY18 L1, L2, L3, L4 and S1

Molecules having a CD3-binding domain different in structure from scFv were evaluated. GPC3 ERY18 was constructed (Fig. 17L) in which the VH and VL domains of the CD3 antibody were fused to the C termini of two IgG H chains to the GPC3 cancer antigen. This construction was used to create a series of molecules (GPC3 ERY18 LI, L2, L3, and L4) that had 1–4 linker units (Gly-Gly-Gly-Gly-Ser) at the junction. At the same time, another molecule (GPC3 ERY18 S1) was constructed in which the amino acids at the corresponding positions were replaced by Cys, allowing the introduction of a disulfide bridge.

In particular, a method known in the art, such as PCR using primers containing, as in the method described above, appropriate additional sequences, was carried out to create a series of expression vectors into which a polynucleotide encoding GPC3 ERY18 L1_Hh (SEQ ID NO: 49; a mature sequence that does not contain the 19 amino acids at the amino terminus serving as a signal sequence), GPC3 ERY18 LIHk (SEQ ID NO: 50; a mature sequence that does not contain the 19 amino acids at the amino terminus serving as a signal sequence), GPC3 ERY18 L2_Hh (SEQ ID NO: 51; a mature sequence that does not contain the 19 amino acids at the amino terminus serving as a signal sequence), GPC3 ERY18 L2_Hk (SEQ ID NO: 52; a mature sequence that does not contain the 19 amino acids at the amino terminus that serve as a signal sequence), GPC3 ERY18 L3_Hh (SEQ ID NO: 53; mature sequence that does not contain the 19 amino acids at the amino terminus that serve as a signal sequence), GPC3 ERY18 L3_Hk (SEQ ID NO: 54; a mature sequence that does not contain the 19 amino acids at the amino terminus that serve as a signal sequence), GPC3 ERY18 L4_Hh (SEQ ID NO: 55; a mature sequence that does not contain the 19 amino acids at the amino terminus that serve as a signal sequence), GPC3 ERY18 L4_Hk (SEQ ID NO: 56; a mature sequence that does not contain the 19 amino acids at the amino terminus that serve as a signal sequence), GPC3 ERY18 SIHh (SEQ ID NO: 57; a mature sequence that does not contain the 19 amino acids at the amino terminus that serve as a signal sequence), or GPC3 ERY18 SIHk (SEQ ID NO: 58; a mature sequence that does not contain the 19 amino acids at the amino terminus that serve as a signal sequence).

For short-term expression of the engineered molecules, the following combinations of expression vectors were introduced into FreeStyle293-F cells.

G. Designed molecule: GPC3 ERY18 L1

Expression vectors: GPC3 ERY18 LI Hh (SEQ ID NO: 49; mature sequence lacking the 19 amino acids at the amino terminus that serve as a signal sequence), GPC3 ERY18 LIHk (SEQ ID NO: 50; mature sequence lacking the 19 amino acids at the amino terminus that serve as a signal sequence), and GPC3 ERY7 L.

3. Engineered Molecule: GPC3 ERY18 L2

Expression vectors: GPC3 ERY18 L2_Hh (SEQ ID NO: 51; mature sequence lacking the 19 amino acids at the amino terminus that serve as a signal sequence), GPC3 ERY18 L2 Hk (SEQ ID NO: 52; mature sequence lacking the 19 amino acids at the amino terminus that serve as a signal sequence), and GPC3 ERY7 L.

I. Engineered Molecule: GPC3 ERY18 L3

Expression vectors: GPC3 ERY18 L3_Hh (SEQ ID NO: 53; mature sequence lacking the 19 amino acids at the amino terminus that serve as a signal sequence), GPC3 ERY18 L3_Hk (SEQ ID NO: 54; mature sequence lacking the 19 amino acids at the amino terminus that serve as a signal sequence), and GPC3 ERY7 L.

K. Designed Molecule: GPC3 ERY18 L4

Expression vectors: GPC3 ERY18 L4_Hh (SEQ ID NO: 55; mature sequence that lacks the 19 amino acid residues at the amino terminus that serve as a signal sequence), GPC3 ERY18 L4_Hk (SEQ ID NO: 56; mature sequence that lacks the 19 amino acid residues at the amino terminus that serve as a signal sequence),

19 amino acids serving as a signal sequence) and GPC3 ERY7 L.

L. Designed molecule: GPC3 ERY18 S1

Expression vectors: GPC3 ERY18 SI Hh (SEQ ID NO: 57; mature sequence lacking the 19 amino acids at the amino terminus that serve as a signal sequence), GPC3 ERY18 SIHk (SEQ ID NO: 58; mature sequence lacking the 19 amino acids at the amino terminus that serve as a signal sequence), and GPC3 ERY7 L.

The resulting culture supernatant was loaded onto an Anti FLAG M2 column (Sigma). After washing, the column was eluted with FLAG peptide at a concentration of 0.1 mg/ml (Sigma). The fraction containing the created molecule was loaded onto a HisTrap HP column (GE Healthcare). After washing, the column was eluted with an imidazole concentration gradient. The fraction containing the created molecule was concentrated by ultrafiltration. Then, the fraction was loaded onto a Superdex 200 column (GE Healthcare). Only the monomeric fraction was collected from the eluate, obtaining each purified created molecule.

For each of the molecules GPC3 ERY18 LI, GPC3 ERY18L2, GPC3 ERY18L3, GPC3 ERY18L4 and GPC3 ERY18S1, the in vitro cytotoxic activity was evaluated (Fig. 12 and 13). The result indicates that all molecules except GPC3 ERY18 L1 had an activity comparable to that of GPC3 ERY10-1. This result demonstrates that molecules that have a structure different from that of scFv have comparable cytotoxic activity. The structure in which both the VH and VL domains of the anti-CD3 antibody are linked to the C-termini of two H chains of IgG to cancer antigen (GPC3) is likely to participate in the stabilization of the polypeptide complexes of the present invention.

Example 5. Construction and evaluation of GPC3 ERY19-3

Molecules having a Fab-like CD3-binding domain were then examined. GPC3 ERY19-3 (Fig. 17M) was constructed in which the VH and CH1 domains and the VL and CL domains of the CD3 antibody were all linked to the C termini of two H chains of the IgG anti-cancer antigen (GPC3) antibody. In particular, a method known in the art, such as PCR using primers containing, as in the method described above, the corresponding additional sequences, was carried out, creating expression vectors into which a polynucleotide encoding GPC3 ERY19-3 Hh (SEQ ID NO: 59; a mature sequence that does not contain 19 amino acids at the amino terminus serving as a signal sequence) or GPC3 ERY19-3_Hk (SEQ ID NO: 60; a mature sequence that does not contain 19 amino acids at the amino terminus serving as a signal sequence) was inserted.

Expression vectors for GPC3 ERY19-3 Hh (SEQ ID NO: 59; mature sequence lacking the 19 amino acid signal sequence at the amino terminus), GPC3 ERY19-3 Hk (SEQ ID NO: 60; mature sequence lacking the 19 amino acid signal sequence at the amino terminus), and GPC3 ERY7L were co-introduced into FreeStyle293-F cells for transient expression of GPC3 ERY19-3. The resulting culture supernatant was applied to a HiTrap rProtein A FF column (GE Healthcare). After washing, the column was eluted with acid. The fraction containing GPC3 ERY19-3 was concentrated by ultrafiltration and then loaded onto a Superdex 200 column (GE Healthcare). Only the monomeric fraction of GPC3 ERY19-3 was collected from the eluate to obtain the purified GPC3 ERY19-3 molecule.

The cytotoxic activity of the GPC3 ERY19-3 molecule was evaluated in vitro. The result demonstrated that the molecule had an activity comparable to that of GPC3 BiTE (Fig. 14). The CD3-binding domain with a Fab-like structure apparently participates in the stabilization of the molecules of the polypeptide complexes proposed in the present invention.

Example 6. Preparation of polypeptide complexes using only the purification step on protein A by introducing a mutation into the CH3 domain of GPC3 ERY 10-1

(1) Basic principles

In GPC3 ERY10-1 prepared according to the method described in Example 3, the CH3 domain has a knob-and-hole structure. The desired GPC3 ERY10-1 molecule in which two H chains are heteromerically associated was purified by two types of affinity purification using a His tag and a FLAG tag attached to the C terminus of each H chain. When the GPC3 ERY10-1 molecule is prepared as a pharmaceutical agent, protein A chromatography of the culture supernatant of GPC3 ERY10-1 expressing cells is first performed to purify the polypeptide complex having the Fc domain. This step is followed by an additional chromatographic purification step using His tag-based affinity chromatography and FLAG tag-based affinity chromatography. This increases the cost of the purification process. Thus, in the example below, molecular modifications were investigated that allow the purification of the desired GPC3 ERY10-1 molecule, which has two heteromerically associated H chains, by protein A chromatography alone without the use of a His-tag and FLAG-tag.

In particular, modifications that eliminate protein A binding in one of the two H chains were investigated. As a result of such modifications, in which there was a homomeric association of the non-protein A binding H chains, the molecule could not bind to protein A and therefore passed through the protein A column during chromatography. On the other hand, a molecule in which there was a heteromeric association of the non-protein A binding H chain with the protein A binding H chain and a molecule in which there was a homomeric association of the protein A binding H chains could be separated by protein A chromatography based on differences in affinity for protein A. However, in the Fc domain of the antibody, the protein A binding site overlaps with the FcRn binding site, which is critical for the plasma retention time of the antibody. Thus, it is necessary to selectively reduce only the protein A binding activity while maintaining the FcRn binding activity. A substitution of His at position 435 (EU numbering) to Arg was identified as such a modification. The combination of this mutation with the mutations described in WO 2006/106905 (substitution of Asp at position 356 (EU numbering) in one of the H chains to Lys and Lys at position 439 (EU numbering) in the other H chain to Glu), which enhance the heteromeric association of the two H chains, was assessed to see whether this could facilitate the purification of polypeptide complexes such as GPC3 ERY10-1 using protein A chromatography alone.

(2) Construction of expression vectors carrying antibody genes and expression of corresponding antibodies

To obtain the variable region of the H chain of the antibody, a gene encoding GC33(2)H (variable region of the H chain of the anti-human glypican antibody, SEQ ID NO: 61; a mature sequence that does not contain 19 amino acids at the amino terminus serving as a signal sequence) was constructed by a method known to those skilled in the art. Similarly, to obtain the L chain of the antibody, a gene encoding GC33-k0 (L chain of the anti-human glypican antibody, SEQ ID NO: 62; a mature sequence that does not contain 19 amino acids at the amino terminus serving as a signal sequence) was constructed by a method known to those skilled in the art. Then, to obtain the constant region of the H chain of the antibody, the following genes were constructed by a method known to those skilled in the art.

M. Engineered Molecule: LALA-Gld

LALA-Gld (SEQ ID NO: 63; mature sequence that lacks the 19 amino acids at the amino terminus that serve as a signal sequence), in which Leu at positions 234 and 235 (EU numbering) are replaced by Ala, Asn at position 297 is replaced by Ala, and Gly and Lys at the C-terminus are deleted from the IgG1 sequence.

H. Engineered Molecule: LALA-Gld-CD3

LALA-Gld-CD3 (SEQ ID NO: 64; mature sequence that lacks the 19 amino acids at the amino terminus that serve as a signal sequence), in which a CD3 scFv (the variable region of the H chain of an antibody to human CD3 and the variable region of the L chain of an antibody to human CD3 are connected via a polypeptide linker) is linked to the C-terminus of LALA-Gld (SEQ ID NO: 63; mature sequence that lacks the 19 amino acids at the amino terminus that serve as a signal sequence).

O. Engineered Molecule: LALA-G3S3E-G1d

LALA-G3S3E-Gld (SEQ ID NO: 65; mature sequence lacking the 19 amino acids at the amino terminus that serve as a signal sequence), in which His at position 435 (EU numbering) is replaced by Arg and Lys at position 439 (EU numbering) is replaced by Glu in the sequence LALA-G1d (SEQ ID NO: 63; mature sequence lacking the 19 amino acids at the amino terminus that serve as a signal sequence).

P. Designed molecule: LALA-S3K-G1d-CD3

LALA-S3K-G1d-CD3 (SEQ ID NO: 66; mature sequence lacking the 19 amino acids at the amino terminus that serve as a signal sequence), in which Asp at position 356 (EU numbering) is replaced by Lys in the sequence of LALA-G1d-CD3 (SEQ ID NO: 64; mature sequence lacking the 19 amino acids at the amino terminus that serve as a signal sequence).

The NTA1L and NTA1R genes of the anti-human GPC3 antibody H chains were constructed by attaching LALA-G1d-CD3 or LALA-G1d downstream of GC33(2)H, respectively. And the NTA2L and NTA2R genes of the anti-human GPC3 antibody H chains were constructed by attaching LALA-S3K-Gld-CD3 or LALA-G3S3E-Gld downstream of GC33(2)H, respectively.

Expression vectors for NTA1L, NTA1R, NTA2L, NTA2R (H chains), and GC33-k0 (L chain) were constructed by inserting each gene into an expression vector suitable for animal cells. These vectors were assembled as described below and introduced into FreeStyle293 cells (Invitrogen) by a method known to those skilled in the art to transiently express the polypeptide complexes described below. As indicated below, the polypeptide complexes were designated by the names of the introduced genes, combined in the following order [first H chain/second H chain/E chain].

NTA1L/NTAlR/GC33-k0

NTA2L/NTA2R/GC33-k0

(3) Purification of expressed samples and evaluation of their ability to form heteromeric complexes FreeStyle293-mieTOK cell supernatant (hereinafter referred to as CM) containing the following polypeptide complex was used as a sample.

NTA1L/NTAlR/GC33-k0

NTA2L/NTA2R/GC33-k0

The CM was filtered through a 0.22 μm filter and loaded onto a column packed with rProtein A-Sepharose Fast Flow (GE Healthcare) equilibrated with D-PBS. Wash steps 1 and 2 and elution step 1 were performed using the buffers listed in Table 1. The amount of CM loaded was adjusted so that the amount of antibody loaded was 20 mg/ml resin. The eluted fractions were collected and analyzed by gel filtration to identify the components in the fractions.

The results of gel filtration analysis of each eluted fraction are shown in Fig. 15 and Table 2. The values given represent the area of the eluted peak in percent. When NTA1L/NTA1R/GC33-k0 or NTA2L/NTA2R/GC33-k0 was expressed, homomeric anti-GPC3 antibody (NTA1L/GC33-k0 or NTA2L/GC33-k0) was almost undetectable in SM. Moreover, the content of homomeric anti-GPC3 antibody (NTA2R/GC33-k0) in SM was only about 2% when NTA2L/NTA2R/GC33-k0 was expressed, while its content in SM was about 76% when NTA1L/NTAlR/GC33-k0 was expressed. This result demonstrates that when His at position 435 (EU numbering) is replaced by Arg, and to ensure efficient formation of a heteromeric molecule containing the corresponding H chains, Asp at position 356 (EU numbering) in the polypeptide sequence of one of the H chains is replaced by Lys, and Lys at position 439 (EU numbering) in the polypeptide sequence of the other H chain is replaced by Glu, then heteromeric polypeptide complexes having the same molecular form as GPC3 ERY10-1 can be efficiently purified to a purity of greater than 98% by only one purification step using protein A.

Example 7. Construction and evaluation of GPC3 ERY 17-2 and GPC3 ERY 17-3

(1) Construction of GPC3 ERY 17-2 and GPC3 ERY 17-3

Next, a molecule was constructed by using an IgG to an anticancer antigen (GPC3) as a scaffold and replacing one of the Fabs with a CD3 epsilon-binding domain. As in the above embodiments, the Fc of the IgG used as a scaffold was a silent Fc with reduced FcgR (Fcγ receptor) binding activity. For the CD3 epsilon-binding domains, the VH and VL domains of the CD3 epsilon Fab were altered to produce GPC3 ERY17-2 (Fig. 19A), and the CH1 and CL domains were altered to produce GPC3 ERY17-3 (Fig. 19B).

In particular, using a method well known to those skilled in the art, such as PCR using primers containing the same corresponding additional sequences as in the method described above, a series of expression vectors were constructed into which a polynucleotide encoding ERY17-2 Hh (SEQ ID NO: 73; a mature sequence that does not contain the 19 amino acids at the amino terminus serving as a signal sequence), ERY17-2L (SEQ ID NO: 74; a mature sequence that does not contain the 19 amino acids at the amino terminus serving as a signal sequence), ERY17-3_Hh (SEQ ID NO: 75; a mature sequence that does not contain the 19 amino acids at the amino terminus serving as a signal sequence) or ERY17-3 L (SEQ ID NO: 76; a mature sequence that does not contain the 19 amino acids at the amino terminus serving as a signal sequence) was inserted. amino acids that serve as a signal sequence).

For short-term expression of each engineered molecule, the following combinations of expression vectors were introduced into FreeStyle293-F cells.

R. Engineered Molecule: GPC3 ERY17-2

Polypeptides encoded by polynucleotides inserted into expression vectors: GPC3 ERY8-2 Hk, GPC3 ERY7 L, ERY17-2 Hh (SEQ ID NO: 73; mature sequence lacking the 19 amino acids at the amino terminus that serve as a signal sequence) and ERY17-2_L (SEQ ID NO: 74; mature sequence lacking the 19 amino acids at the amino terminus that serve as a signal sequence).

C. Engineered Molecule: GPC3 ERY17-3

Polypeptides encoded by polynucleotides inserted into expression vectors: GPC3 ERY8-2_Hk, GPC3 ERY7L, ERY17-3_Hh (SEQ ID NO: 75; mature sequence lacking the 19 amino acids at the amino terminus that serve as a signal sequence) and ERY17-3 L (SEQ ID NO: 76; mature sequence lacking the 19 amino acids at the amino terminus that serve as a signal sequence).

(2) Cleaning GPC3 ERY 17-2 and GPC3 ERY 17-3

The resulting culture supernatant was loaded onto an Anti FLAG M2 column (Sigma). After washing, the column was eluted with FLAG peptide at a concentration of 0.1 mg/ml (Sigma). The fraction containing the created molecule was loaded onto a HisTrap HP column (GE Healthcare). After washing, the column was eluted with an imidazole concentration gradient. The fraction containing the created molecule was concentrated by ultrafiltration. Then, the fraction was loaded onto a Superdex 200 column (GE Healthcare). Only the monomeric fraction was collected from the eluate, obtaining each purified created molecule.

(3) Cytotoxic activity of GPC3 ERY 17-2 and GPC3 ERY 17-3

For GPC3 ERY 17-2 and GPC3 ERY 17-3, the cytotoxic activity in vitro was evaluated (Fig. 20). The result demonstrated that both molecules had remarkably higher cytotoxic activity than GPC3 BiTE. Thus, the present invention demonstrated for the first time that molecules in which IgG to an anticancer antigen was used as a scaffold and one of the Fab was replaced with the CD3 epsilon chain binding domain had cytotoxic activity comparable to or higher than that of BiTE.

(4) Evaluation of the efficacy of GPC3 ERY17-2 based on PC-10 T cell transfer model

For GPC3 ERY17-2, which demonstrated cytotoxic activity comparable to or greater than that of GPC3 BiTE in an in vitro assay, the in vivo efficacy was evaluated using a T cell transfer model using PC-10 cells. Specifically, the efficacy of GPC3 ERY17-2 using a T cell transfer model using PC-10 cells was evaluated as follows. T cells were culture expanded using a human cell activation/expansion kit (MACS Miltenyi biotec) and PBMCs isolated from the blood of healthy volunteers. The human squamous cell lung carcinoma cell line PC-10 (Immuno-Biological Laboratories Co., Ltd.) (1× ¹⁰⁷ cells) was mixed with Matrigel Matrix (BD) and then subcutaneously transplanted into the inguinal region of NOD severe combined immunodeficiency mice (CLEA Japan Inc.; 7-week-old females). The day of transplantation was designated as day 0. Anti-asialo-GM1 antibody (Wako Pure Chemical Industries) was intraperitoneally injected to mice at 0.2 mg/mine 1 day before transplantation and on days 13, 17, 21, and 25. On day 13 after transplantation, the mice were grouped according to tumor size and body weight. On day 14 post-transplantation, T cells obtained by culture expansion as described above were transplanted at 3× ¹⁰⁷ cells/individual into the peritoneal cavity. Two hours after transplantation, GPC ERY17-2 was administered intravenously at 30 μg/individual. GPC ERY10-1 was administered a total of 5 times on days 14, 15, 16, 17, and 18.

The result demonstrated that using this model, there was also a significant anti-cancer effect in the GPC3 ERY17-2-treated group compared with the vehicle-treated group (Fig. 21).

The above data demonstrated that molecules in which anti-cancer IgG was used as a scaffold and one of the Fab was replaced with the CD3 epsilon-binding domain had a pronounced anti-cancer effect in vivo.

Example 8. Design and evaluation of GPC3 ERY17-2-M20

(1) Design of GPC3 ERY17-2-M20

Next, a molecule was constructed that retains the required activity even after changes in the CD3 epsilon-binding domain. GPC3 ERY17-2-M20 (Fig. 19A) was created in which the sequence of the CD3 epsilon-binding domain was changed. In particular, using an expression vector for an anti-CD3 antibody (M20) as a template, a series of expression vectors were prepared using a method well known to those skilled in the art, such as PCR using primers containing the same corresponding additional sequences as in the method described above, into which a polynucleotide encoding ERY17-2-M20 Hh (SEQ ID NO: 77; a mature sequence that does not contain the 19 amino acids at the amino terminus serving as a signal sequence) or ERY17-2-M20L (SEQ ID NO: 78; a mature sequence that does not contain the 19 amino acids at the amino terminus serving as a signal sequence) was inserted.

(2) Cleaning GPC3 ERY17-2-M20

Expression vectors for GPC3 ERY8-2 Hk, GPC3 ERY7 L, ERY17-2-M20_Hh (SEQ ID NO: 77), and ERY17-2-M20L (SEQ ID NO: 78) were co-introduced into FreeStyle293-F cells for transient expression of GPC3 ERY17-2-M20. The resulting culture supernatant was filtered through a 0.22 μm filter and then loaded onto an equilibrated column packed with rProtein A-Sepharose Fast Flow (GE Healthcare). Purified GPC3 ERY17-2-M20 was obtained after wash steps 1 and 2 and elution step 1 using the buffers listed in Table 3.

(3) Cytotoxic activity of GPC3 ERY17-2-M20

The cytotoxic activity of GPC3 ERY17-2-M20 was assessed in vitro and it was found that the cytotoxic activity was comparable to that of GPC3 ERY17-2 (Fig. 22). These data demonstrate that even molecules with altered CD3 epsilon binding domain sequence have comparable cytotoxic activity.

Example 9. Design and evaluation of EpCAM ERY17-2 and EpCAM ERY17-3

(1) Construction of EpCAM ERY17-2 and EpCAM ERY17-3 Molecules targeting a different cancer antigen but retaining the desired activity were then constructed. The resulting molecules were EpCAM ERY17-2 (Fig. 19A), in which the anti-GPC3 Fab in GPC3 ERY17-2 was replaced by anti-EpCAM Fab, and EpCAM ERY17-3 (Fig. 19B), in which the anti-GPC3 Fab in GPC3 ERY17-3 was replaced by anti-EpCAM Fab. In particular, using an expression vector for an antibody to EpCAM as a template, a series of expression vectors were constructed using a method well known to those skilled in the art, such as PCR using primers containing the same corresponding additional sequences as in the method described above, into which a polynucleotide encoding EpCAM ERY17 Hk (SEQ ID NO: 79; a mature sequence that does not contain the 19 amino acids at the amino terminus serving as a signal sequence) or EpCAM ERY17 L (SEQ ID NO: 80; a mature sequence that does not contain the 19 amino acids at the amino terminus serving as a signal sequence) was inserted.

For short-term expression of each engineered molecule, the following combinations of expression vectors were introduced into FreeStyle293-F cells.

T. Engineered Molecule: EpCAM ERY17-2 Polypeptides encoded by polynucleotides inserted into expression vectors: EpCAM ERY17 Hk (SEQ ID NO: 79; mature sequence lacking the 19 amino acids at the amino terminus serving as a signal sequence), EpCAM ERY17L (SEQ ID NO: 80; mature sequence lacking the 19 amino acids at the amino terminus serving as a signal sequence), ERY17-2 Hh and ERY17-2 L

U. Engineered molecule: EpCAM ERY17-3

Polypeptides encoded by polynucleotides inserted into expression vectors: EpCAM ERY17 Hk, EpCAM ERY17 L, ERY17-3 Hh and ERY17-3 L.

(2) Purification of EpCAM ERY17-2 and EpCAM ERY17-3

The resulting culture supernatant was loaded onto an Anti FLAG M2 column (Sigma). After washing, the column was eluted with FLAG peptide at a concentration of 0.1 mg/ml (Sigma). The fraction containing the created molecule was loaded onto a HisTrap HP column (GE Healthcare). After washing, the column was eluted with an imidazole concentration gradient. The fraction containing the created molecule was concentrated by ultrafiltration. Then, the fraction was loaded onto a Superdex 200 column (GE Healthcare). Only the monomeric fraction was collected from the eluate, obtaining each purified created molecule.

(3) Cytotoxic activity of EpCAM ERY17-2 and EpCAM ERY17-3

For EpCAM ERY17-2 and EpCAM ERY17-3, the cytotoxic activity was evaluated in vitro, and it was found that both molecules had strong cytotoxic activity (Fig. 23). Thus, the present invention demonstrated that molecules in which IgG to an anticancer antigen was used as a scaffold and one of the Fab was replaced with the CD3 epsilon chain binding domain had cytotoxic activity, even when the type of cancer antigen was changed.

Example 10. Construction and evaluation of bispecific antibodies with modulated association at the CH1/CL interface

(1) Creation of a bispecific antibody

By introducing mutations into each of the CH1 and CL domains of the bispecific antibody and thereby modulating the association at the CH1/CL interface using the electrical charge repulsion at the CH1/CL interface, it is possible to create conditions for specific association to occur between the H chain and the L chain of the anti-GPC3 antibody and between the H chain and the L chain of the anti-CD3 antibody. In order to modulate the association at the CH1/CL interface using the electrical charge repulsion, amino acid residues in the CH1 of the H chains or in the CL of the L chains were replaced with Lys, which is a positively charged amino acid, or with Glu, which is a negatively charged amino acid.

(2) Construction of expression vectors carrying antibody genes and expression of corresponding antibodies

The bispecific antibody (Fig. 24A) was generated by modulating the association at the CH1/CL interface of the anti-CD3 antibody M12 (H chain, SEQ ID NO: 81; L chain, SEQ ID NO: 82) and the anti-GPC3 antibody GC33(2) (H chain, SEQ ID NO: 83; L chain, SEQ ID NO: 84) and further introducing knob-and-hole (KiH) modifications (WO 1996/027011; Ridgway JB et al., Protein Engineering 9:617-621 (1996); Merchant AM et al., Nat. Biotechnol. 16:677-681 (1998)) to avoid association of the H chains with each other. A control bispecific antibody (Fig. 24B) was also constructed in which neither the modulation of the association at the CH1/CL interface nor the knob-and-hole (KiH) modifications were introduced. Specifically, expression vectors were constructed using a method known to those skilled in the art that carried as an insert a polynucleotide encoding M12 TH2h (SEQ ID NO: 85), in which several amino acids in the CH1 H chain of the M12 antibody (SEQ ID NO: 81) were replaced with Lys, or M12 TL17 (SEQ ID NO: 86), in which several amino acids in the CL L chain (SEQ ID NO: 82) were replaced with Glu. Similarly, using a method known to those skilled in the art, expression vectors were constructed carrying as an insert a polynucleotide encoding GC33(2)_TH13k (SEQ ID NO: 87) or GC33(2)_TH15k (SEQ ID NO: 88), in which several amino acids in the CH1 H chain of the GC33(2) antibody (SEQ ID NO: 83) are replaced with Glu, or GC33(2)_TL16 (SEQ ID NO: 89) or GC33(2)_TL19 (SEQ ID NO: 90), in which several amino acids in the CL L chain (SEQ ID NO: 84) are replaced with Lys.

For short-term expression of each engineered molecule, the following combinations of expression vectors were introduced into FreeStyle293-F cells.

F. Designed Molecule: GM1

Expression Vectors: M12_TH2h (SEQ ID NO: 85), M12TL17 (SEQ ID NO: 86), GC33(2) TH13k (SEQ ID NO: 87) and GC33(2) TL16 (SEQ ID NO: 89)

X. Designed by: GM2

Expression vectors: M12_TH2h (SEQ ID NO: 85), M12TL17 (SEQ ID NO: 86), GC33(2)_TH15k (SEQ ID NO: 88) and GC33(2) TL19 (SEQ ID NO: 90) C. Designed molecule : GMO

Expression vectors: M12 H chain (SEQ ID NO: 81), M12 L chain (SEQ ID NO: 82), GC33(2) H chain (SEQ ID NO: 83) and GC33(2) L chain (SEQ ID NO: 84).

Antibodies were purified from the resulting cell supernatant using a method known to those skilled in the art using an rProtein A Sepharose™ Fast Flow column (GE Healthcare).

(3) Cytotoxic activity of GM1, GM2 and GMO

The cytotoxic activity of the polypeptide complexes GM1, GM2 and GMO was evaluated in vitro. The result demonstrates that GM1 and GM2 had comparable cytotoxic activity, and this activity is higher than that of GM0 (Fig. 25). Thus, the present invention demonstrates that the combination of modulation of the association at the CH1/CL interface and KiH modifications allows for the efficient production of bispecific antibodies.

Example 11. Construction and evaluation of EGFR ERY17-2

(1) Construction of EGFR ERY17-2

In addition, a molecule with the desired activity targeting another cancer antigen was created. EGFR ERY 17-2 (Fig. 19A) was constructed by replacing the anti-GPC3 Fab antibody GPC3 ERY17-2 with anti-EGFR Fab. Specifically, using an expression vector for an antibody to EGFR as a template, a series of expression vectors were constructed using a method well known to those skilled in the art, such as PCR using primers containing the same corresponding additional sequences as in the method described above, into which a polynucleotide encoding EGFR ERY 17 Hk (SEQ ID NO: 91; a mature sequence that does not contain the 19 amino acids at the amino terminus serving as a signal sequence) or EGFR ERY17L (SEQ ID NO: 92: a mature sequence that does not contain the 19 amino acids at the amino terminus serving as a signal sequence) was inserted.

For transient expression of each engineered molecule, the following combinations of expression vectors were introduced into FreeStyle293-F cells.

Ch. Engineered Molecule: EGFR ERY17-2

Polypeptides encoded by polynucleotides inserted into expression vectors: EGFR ERY17_Hk (SEQ ID NO: 91: mature sequence that lacks the 19 amino acids at the amino terminus that serve as a signal sequence), EGFR ERY17L (SEQ ID NO: 92: mature sequence that lacks the 19 amino acids at the amino terminus that serve as a signal sequence), ERY17-2_Hh and ERY17-2L.

(2) Purification of EGFR ERY17-2

The resulting culture supernatant was loaded onto an Anti FLAG M2 column (Sigma). After washing, the column was eluted with FLAG peptide at a concentration of 0.1 mg/ml (Sigma). The fraction containing the created molecule was loaded onto a HisTrap HP column (GE Healthcare). After washing, the column was eluted with an imidazole concentration gradient. The fraction containing the created molecule was concentrated by ultrafiltration. Then, the fraction was loaded onto a Superdex 200 column (GE Healthcare). Only the monomeric fraction was collected from the eluate, obtaining each purified created molecule.

(3) Cytotoxic activity of EGFR ERY17-2

For EGFR ERY17-2, the cytotoxic activity was assessed in vitro, and in this case, strong cytotoxic activity was found (Fig. 26). Thus, the present invention demonstrated that molecules in which IgG to an anticancer antigen served as a scaffold and one of the Fab was replaced with the CD3 epsilon chain binding domain had cytotoxic activity, even when the cancer antigen, which was GPC3 or EpCAM, was additionally replaced with another type of antigen.

Industrial applicability

The present invention provides new polypeptide complexes that retain the strong anticancer activity of BiTE and have a long half-life in the blood, and are also characterized by very high safety, which is manifested in the absence of induction of a cytokine storm independent of a cancer antigen, or the like. When replacing the antigen-binding domain of the polypeptide complex proposed in the present invention, therapeutic agents that contain the polypeptide complex for inducing cellular cytotoxicity as an active substance can target and damage various cells, including cancer cells. Thus, various types of cancer can be treated or prevented. This allows for the required treatment to be carried out with high safety and convenience, and to reduce the physical burden on patients.

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SEQUENCE LIST

<110> CHUGAI SEYYAKU KABUSHIKI KAISHA

<120> CYTOTOXICITY-INDUCING THERAPEUTIC AGENT

<130> C1-A1010Y2P

<150> JP 2010-266760

<151> 2010-11-30

<150> JP 2011-121771

<151> 2011-05-31

<150> JP 2011-238818

<151> 2011-10-31

<160> 92

<170> PatentIn version 3.5

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Val Thr Arg Ile Phe Leu Gln Ala Leu Asn Leu Gly Ile Glu Val Ile

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Asp Pro Lys Phe Ile Thr Ser Ile Leu Tyr Glu Asn Asn Val Ile Thr

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Ile Asp Leu Val Gln Asn Ser Ser Gln Lys Thr Gln Asn Asp Val Asp

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Ile Ala Asp Val Ala Tyr Tyr Phe Glu Lys Asp Val Lys Gly Glu Ser

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Leu Phe His Ser Lys Lys Met Asp Leu Thr Val Asn Gly Glu Gln Leu

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Asp Leu Asp Pro Gly Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys Ala

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Pro Glu Phe Ser Met Gln Gly Leu Lys Ala Gly Val Ile Ala Val Ile

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Val Val Val Val Ile Ala Val Val Ala Gly Ile Val Val Leu Val Ile

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Ser Arg Lys Lys Arg Met Ala Lys Tyr Glu Lys Ala Glu Ile Lys Glu

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ccactcctgg aggggaatct ggtcaccctg agctgtgaaa caaagttgct cttgcagagg

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cctggtttgc agctttactt ctccttctac atgggcagca agaccctgcg aggcaggaac

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gaggctgcca cagaggatgg aaatgtcctt aagcgcagcc ctgagttgga gcttcaagtg

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cttggcctcc agttaccaac tcctgtctgg tttcatgtcc ttttctatct ggcagtggga

900

ataatgtttt tagtgaacac tgttctctgg gtgacaatac gtaaagaact gaaaagaaag

960

aaaaagtggg atttagaaat ctctttggat tctggtcatg agaagaaggt aatttccagc

1020

cttcaagaag acagacattt agaagaagag ctgaaatgtc aggaacaaaa agaagaacag

1080

ctgcaggaag gggtgcaccg gaaggagccc caggggggcca cgtag 1125

<210> 14

<211> 374

<212> PRT

<213> Homo sapiens

<400> 14

Met Trp Phe Leu Thr Thr Leu Leu Leu Trp Val Pro Val Asp Gly Gln

1 5 10 15

Val Asp Thr Thr Lys Ala Val Ile Thr Leu Gln Pro Pro Trp Val Ser

20 25 30

Val Phe Gln Glu Glu Thr Val Thr Leu His Cys Glu Val Leu His Leu

35 40 45

Pro Gly Ser Ser Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln

50 55 60

Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser

65 70 75 80

Gly Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile

85 90 95

Gln Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg

100 105 110

Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys

115 120 125

Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys Ala Phe

130 135 140

Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu Lys Thr Asn Ile

145 150 155 160

Ser His Asn Gly Thr Tyr His Cys Ser Gly Met Gly Lys His Arg Tyr

165 170 175

Thr Ser Ala Gly Ile Ser Val Thr Val Lys Glu Leu Phe Pro Ala Pro

180 185 190

Val Leu Asn Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val

195 200 205

Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln

210 215 220

Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn

225 230 235 240

Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser Gly

245 250 255

Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val Leu Lys Arg

260 265 270

Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Leu Gln Leu Pro Thr Pro

275 280 285

Val Trp Phe His Val Leu Phe Tyr Leu Ala Val Gly Ile Met Phe Leu

290 295 300

Val Asn Thr Val Leu Trp Val Thr Ile Arg Lys Glu Leu Lys Arg Lys

305 310 315 320

Lys Lys Trp Asp Leu Glu Ile Ser Leu Asp Ser Gly His Glu Lys Lys

325 330 335

Val Ile Ser Ser Leu Gln Glu Asp Arg His Leu Glu Glu Glu Leu Lys

340 345 350

Cys Gln Glu Gln Lys Glu Glu Gln Leu Gln Glu Gly Val His Arg Lys

355 360 365

Glu Pro Gln Gly Ala Th

370

<210> 15

<211> 951

<212> DNA

<213> Homo sapiens

<400> 15

atgactatgg agacccaaat gtctcagaat gtatgtccca gaaacctgtg gctgcttcaa

60

ccattgacag ttttgctgct gctggcttct gcagacagtc aagctgctcc cccaaaggct

120

gtgctgaaac ttgagccccc gtggatcaac gtgctccagg aggactctgt gactctgaca

180

tgccagggggg ctcgcagccc tgagagcgac tccattcagt ggttccacaa tgggaatctc

240

attcccaccc acacgcagcc cagctacagg ttcaaggcca acaacaatga cagcggggag

300

tacacgtgcc agactggcca gaccagcctc agcgaccctg tgcatctgac tgtgctttcc

360

gaatggctgg tgctccagac ccctcacctg gagttccagg agggagaaac catcatgctg

420

aggtgccaca gctggaagga caagcctctg gtcaaggtca cattcttcca gaatggaaaa

480

tcccagaaat tctcccattt ggatcccacc ttctccatcc cacaagcaaa ccacagtcac

540

agtggtgatt accactgcac aggaaacata ggctacacgc tgttctcatc caagcctgtg

600

accatcactg tccaagtgcc cagcatgggc agctcttcac caatgggggt cattgtggct

660

gtggtcattg cgactgctgt agcagccatt gttgctgctg tagtggcctt gatctactgc

720

aggaaaaagc ggatttcagc caattccact gatcctgtga aggctgccca atttgagcca

780

cctggacgtc aaatgattgc catcagaaag agacaacttg aagaaaccaa caatgactat

840

gaaacagctg acggcggcta catgactctg aaccccaggg cacctactga cgatgataaa

900

aacatctacc tgactcttcc tcccaacgac catgtcaaca gtaataacta a 951

<210> 16

<211> 316

<212> PRT

<213> Homo sapiens

<400> 16

Met Thr Met Glu Thr Gln Met Ser Gln Asn Val Cys Pro Arg Asn Leu

1 5 10 15

Trp Leu Leu Gln Pro Leu Thr Val Leu Leu Leu Leu Ala Ser Ala Asp

20 25 30

Ser Gln Ala Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Pro Trp

35 40 45

Ile Asn Val Leu Gln Glu Asp Ser Val Thr Leu Thr Cys Gln Gly Ala

50 55 60

Arg Ser Pro Glu Ser Asp Ser Ile Gln Trp Phe His Asn Gly Asn Leu

65 70 75 80

Ile Pro Thr His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn

85 90 95

Asp Ser Gly Glu Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp

100 105 110

Pro Val His Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr Pro

115 120 125

His Leu Glu Phe Gln Glu Gly Glu Thr Ile Met Leu Arg Cys His Ser

130 135 140

Trp Lys Asp Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly Lys

145 150 155 160

Ser Gln Lys Phe Ser His Leu Asp Pro Thr Phe Ser Ile Pro Gln Ala

165 170 175

Asn His Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr

180 185 190

Thr Leu Phe Ser Ser Lys Pro Val Thr Ile Thr Val Gln Val Pro Ser

195 200 205

Met Gly Ser Ser Ser Pro Met Gly Val Ile Val Ala Val Val Ile Ala

210 215 220

Thr Ala Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr Cys

225 230 235 240

Arg Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala Ala

245 250 255

Gln Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg Gln

260 265 270

Leu Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr Met

275 280 285

Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr Leu

290 295 300

Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn

305 310 315

<210> 17

<211> 876

<212> DNA

<213> Homo sapiens

<400> 17

atgggaatcc tgtcattctt acctgtcctt gccactgaga gtgactgggc tgactgcaag

60

tccccccagc cttggggtca tatgcttctg tggacagctg tgctattcct ggctcctgtt

120

gctgggacac ctgcagctcc cccaaaggct gtgctgaaac tcgagcccca gtggatcaac

180

gtgctccagg aggactctgt gactctgaca tgccggggga ctcacagccc tgagagcgac

240

tccattcagt ggttccacaa tgggaatctc attcccaccc acacgcagcc cagctacagg

300

ttcaaggcca acaacaatga cagcggggag tacacgtgcc agactggcca gaccagcctc

360

agcgaccctg tgcatctgac tgtgctttct gagtggctgg tgctccagac ccctcacctg

420

gagttccagg agggagaaac catcgtgctg aggtgccaca gctggaagga caagcctctg

480

gtcaaggtca cattcttcca gaatggaaaa tccaagaaat tttcccgttc ggatcccaac

540

ttctccatcc cacaagcaaa ccacagtcac agtggtgatt accactgcac aggaaacata

600

ggctacacgc tgtactcatc caagcctgtg accatcactg tccaagctcc cagctcttca

660

ccgatgggga tcattgtggc tgtggtcact gggattgctg tagcggccat tgttgctgct

720

gtagtggcct tgatctactg caggaaaaag cggatttcag ccaatcccac taatcctgat

780

gaggctgaca aagttggggc tgagaacaca atcacctatt cacttctcat gcacccggat

840

gctctggaag agcctgatga ccagaaccgt atttag 876

<210> 18

<211> 291

<212> PRT

<213> Homo sapiens

<400> 18

Met Gly Ile Leu Ser Phe Leu Pro Val Leu Ala Thr Glu Ser Asp Trp

1 5 10 15

Ala Asp Cys Lys Ser Pro Gln Pro Trp Gly His Met Leu Leu Trp Thr

20 25 30

Ala Val Leu Phe Leu Ala Pro Val Ala Gly Thr Pro Ala Ala Pro Pro

35 40 45

Lys Ala Val Leu Lys Leu Glu Pro Gln Trp Ile Asn Val Leu Gln Glu

50 55 60

Asp Ser Val Thr Leu Thr Cys Arg Gly Thr His Ser Pro Glu Ser Asp

65 70 75 80

Ser Ile Gln Trp Phe His Asn Gly Asn Leu Ile Pro Thr His Thr Gln

85 90 95

Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn Asp Ser Gly Glu Tyr Thr

100 105 110

Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp Pro Val His Leu Thr Val

115 120 125

Leu Ser Glu Trp Leu Val Leu Gln Thr Pro His Leu Glu Phe Gln Glu

130 135 140

Gly Glu Thr Ile Val Leu Arg Cys His Ser Trp Lys Asp Lys Pro Leu

145 150 155 160

Val Lys Val Thr Phe Phe Gln Asn Gly Lys Ser Lys Lys Phe Ser Arg

165 170 175

Ser Asp Pro Asn Phe Ser Ile Pro Gln Ala Asn His Ser His Ser Gly

180 185 190

Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr Thr Leu Tyr Ser Ser Lys

195 200 205

Pro Val Thr Ile Thr Val Gln Ala Pro Ser Ser Ser Pro Met Gly Ile

210 215 220

Ile Val Ala Val Val Thr Gly Ile Ala Val Ala Ala Ile Val Ala Ala

225 230 235 240

Val Val Ala Leu Ile Tyr Cys Arg Lys Lys Arg Ile Ser Ala Asn Pro

245 250 255

Thr Asn Pro Asp Glu Ala Asp Lys Val Gly Ala Glu Asn Thr Ile Thr

260 265 270

Tyr Ser Leu Leu Met His Pro Asp Ala Leu Glu Glu Pro Asp Asp Gln

275 280 285

Asn Arg Ile

290

<210> 19

<211> 765

<212> DNA

<213> Homo sapiens

<400> 19

atgtggcagc tgctcctccc aactgctctg ctacttctag tttcagctgg catgcggact

60

gaagatctcc caaaggctgt ggtgttcctg gagcctcaat ggtacagggt gctcgagaag

120

gacagtgtga ctctgaagtg ccagggagcc tactcccctg aggacaattc cacacagtgg

180

tttcacaatg agagcctcat ctcaagccag gcctcgagct acttcattga cgctgccaca

240

gttgacgaca gtggagagta caggtgccag acaaacctct ccaccctcag tgacccggtg

300

cagctagaag tccatatcgg ctggctgttg ctccaggccc ctcggtgggt gttcaaggag

360

gaagacccta ttcacctgag gtgtcacagc tggaagaaca ctgctctgca taaggtcaca

420

tatttacaga atggcaaagg caggaagtat tttcatcata attctgactt ctacattcca

480

aaagccacac tcaaagacag cggctcctac ttctgcaggg ggcttgttgg gagtaaaaat

540

gtgtcttcag agactgtgaa catcaccatc actcaaggtt tgtcagtgtc aaccatctca

600

tcattctttc cacctgggta ccaagtctct ttctgcttgg tgatggtact cctttttgca

660

gtggacacag gactatattt ctctgtgaag acaaacattc gaagctcaac aagagactgg

720

aaggaccata aatttaaatg gagaaaggac cctcaagaca aatga 765

<210> 20

<211> 254

<212> PRT

<213> Homo sapiens

<400> 20

Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala

1 5 10 15

Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro

20 25 30

Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln

35 40 45

Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu

50 55 60

Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr

65 70 75 80

Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu

85 90 95

Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln

100 105 110

Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys

115 120 125

His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn

130 135 140

Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro

145 150 155 160

Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val

165 170 175

Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln

180 185 190

Gly Leu Ser Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln

195 200 205

Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly

210 215 220

Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp

225 230 235 240

Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys

245 250

<210> 21

<211> 702

<212> DNA

<213> Homo sapiens

<400> 21

atgtggcagc tgctcctccc aactgctctg ctacttctag tttcagctgg catgcggact

60

gaagatctcc caaaggctgt ggtgttcctg gagcctcaat ggtacagcgt gcttgagaag

120

gacagtgtga ctctgaagtg ccagggagcc tactcccctg aggacaattc cacacagtgg

180

tttcacaatg agagcctcat ctcaagccag gcctcgagct acttcattga cgctgccaca

240

gtcaacgaca gtggagagta caggtgccag acaaacctct ccaccctcag tgacccggtg

300

cagctagaag tccatatcgg ctggctgttg ctccaggccc ctcggtgggt gttcaaggag

360

gaagacccta ttcacctgag gtgtcacagc tggaagaaca ctgctctgca taaggtcaca

420

tatttacaga atggcaaaga caggaagtat tttcatcata attctgactt ccacattcca

480

aaagccacac tcaaagatag cggctcctac ttctgcaggg ggcttgttgg gagtaaaaat

540

gtgtcttcag agactgtgaa catcaccatc actcaaggtt tggcagtgtc aaccatctca

600

tcattctctc cacctgggta ccaagtctct ttctgcttgg tgatggtact cctttttgca

660

gtggacacag gactatattt ctctgtgaag acaaacattt ga 702

<210> 22

<211> 233

<212> PRT

<213> Homo sapiens

<400> 22

Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala

1 5 10 15

Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro

20 25 30

Gln Trp Tyr Ser Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln

35 40 45

Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu

50 55 60

Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr

65 70 75 80

Val Asn Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu

85 90 95

Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln

100 105 110

Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys

115 120 125

His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn

130 135 140

Gly Lys Asp Arg Lys Tyr Phe His His Asn Ser Asp Phe His Ile Pro

145 150 155 160

Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val

165 170 175

Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln

180 185 190

Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Ser Pro Pro Gly Tyr Gln

195 200 205

Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly

210 215 220

Leu Tyr Phe Ser Val Lys Thr Asn Ile

225 230

<210> 23

<211> 330

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 23

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 24

<211> 326

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 24

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 25

<211> 377

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 25

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro

100 105 110

Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg

115 120 125

Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys

130 135 140

Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

145 150 155 160

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

165 170 175

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

180 185 190

Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr

195 200 205

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

210 215 220

Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His

225 230 235 240

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

245 250 255

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln

260 265 270

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

275 280 285

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

290 295 300

Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn

305 310 315 320

Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

325 330 335

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile

340 345 350

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln

355 360 365

Lys Ser Leu Ser Leu Ser Pro Gly Lys

370 375

<210> 26

<211> 327

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 26

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 27

<211> 549

<212> DNA

<213> Homo sapiens

<400> 27

atggaacagg ggaagggcct ggctgtcctc atcctggcta tcattcttct tcaaggtact

60

ttggcccagt caatcaaagg aaaccacttg gttaaggtgt atgactatca agaagatggt

120

tcggtacttc tgacttgtga tgcagaagcc aaaaatatca catggtttaa agatgggaag

180

atgatcggct tcctaactga agataaaaaa aaatggaatc tgggaagtaa tgccaaggac

240

cctcgaggga tgtatcagtg taaaggatca cagaacaagt caaaaccact ccaagtgtat

300

tacagaatgt gtcagaactg cattgaacta aatgcagcca ccatatctgg ctttctcttt

360

gctgaaatcg tcagcatttt cgtccttgct gttggggtct acttcattgc tggacaggat

420

ggagttcgcc agtcgagagc ttcagacaag cagactctgt tgcccaatga ccagctctac

480

cagcccctca aggatcgaga agatgaccag tacagccacc ttcaaggaaa ccagttgagg

540

aggaattga 549

<210> 28

<211> 182

<212> PRT

<213> Homo sapiens

<400> 28

Met Glu Gln Gly Lys Gly Leu Ala Val Leu Ile Leu Ala Ile Ile Leu

1 5 10 15

Leu Gln Gly Thr Leu Ala Gln Ser Ile Lys Gly Asn His Leu Val Lys

20 25 30

Val Tyr Asp Tyr Gln Glu Asp Gly Ser Val Leu Leu Thr Cys Asp Ala

35 40 45

Glu Ala Lys Asn Ile Thr Trp Phe Lys Asp Gly Lys Met Ile Gly Phe

50 55 60

Leu Thr Glu Asp Lys Lys Lys Trp Asn Leu Gly Ser Asn Ala Lys Asp

65 70 75 80

Pro Arg Gly Met Tyr Gln Cys Lys Gly Ser Gln Asn Lys Ser Lys Pro

85 90 95

Leu Gln Val Tyr Tyr Arg Met Cys Gln Asn Cys Ile Glu Leu Asn Ala

100 105 110

Ala Thr Ile Ser Gly Phe Leu Phe Ala Glu Ile Val Ser Ile Phe Val

115 120 125

Leu Ala Val Gly Val Tyr Phe Ile Ala Gly Gln Asp Gly Val Arg Gln

130 135 140

Ser Arg Ala Ser Asp Lys Gln Thr Leu Leu Pro Asn Asp Gln Leu Tyr

145 150 155 160

Gln Pro Leu Lys Asp Arg Glu Asp Asp Gln Tyr Ser His Leu Gln Gly

165 170 175

Asn Gln Leu Arg Arg Asn

180

<210> 29

<211> 516

<212> DNA

<213> Homo sapiens

<400> 29

atggaacata gcacgtttct ctctggcctg gtactggcta cccttctctc gcaagtgagc

60

cccttcaaga tacctataga ggaacttgag gacagagtgt ttgtgaattg caataccagc

120

atcacatggg tagagggaac ggtgggaaca ctgctctcag acattacaag actggacctg

180

ggaaaacgca tcctggaccc acgaggaata tataggtgta atgggacaga tatatacaag

240

gacaaagaat ctaccgtgca agttcattat cgaatgtgcc agagctgtgt ggagctggat

300

ccagccaccg tggctggcat cattgtcact gatgtcattg ccactctgct ccttgctttg

360

ggagtcttct gctttgctgg acatgagact ggaaggctgt ctggggctgc cgacacacaa

420

gctctgttga ggaatgacca ggtctatcag cccctccgag atcgagatga tgctcagtac

480

agccaccttg gaggaaactg ggctcggaac aagtga 516

<210> 30

<211> 171

<212> PRT

<213> Homo sapiens

<400> 30

Met Glu His Ser Thr Phe Leu Ser Gly Leu Val Leu Ala Thr Leu Leu

1 5 10 15

Ser Gln Val Ser Pro Phe Lys Ile Pro Ile Glu Glu Leu Glu Asp Arg

20 25 30

Val Phe Val Asn Cys Asn Thr Ser Ile Thr Trp Val Glu Gly Thr Val

35 40 45

Gly Thr Leu Leu Ser Asp Ile Thr Arg Leu Asp Leu Gly Lys Arg Ile

50 55 60

Leu Asp Pro Arg Gly Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys

65 70 75 80

Asp Lys Glu Ser Thr Val Gln Val His Tyr Arg Met Cys Gln Ser Cys

85 90 95

Val Glu Leu Asp Pro Ala Thr Val Ala Gly Ile Ile Val Thr Asp Val

100 105 110

Ile Ala Thr Leu Leu Leu Ala Leu Gly Val Phe Cys Phe Ala Gly His

115 120 125

Glu Thr Gly Arg Leu Ser Gly Ala Ala Asp Thr Gln Ala Leu Leu Arg

130 135 140

Asn Asp Gln Val Tyr Gln Pro Leu Arg Asp Arg Asp Asp Ala Gln Tyr

145 150 155 160

Ser His Leu Gly Gly Asn Trp Ala Arg Asn Lys

165 170

<210> 31

<211> 624

<212> DNA

<213> Homo sapiens

<400> 31

atgcagtcgg gcactcactg gagagttctg ggcctctgcc tcttatcagt tggcgtttgg

60

gggcaagatg gtaatgaaga aatgggtggt attacacaga caccatataa agtctccatc

120

tctggaacca cagtaatatt gacatgccct cagtatcctg gatctgaaat actatggcaa

180

cacaatgata aaaacatagg cggtgatgag gatgataaaa acataggcag tgatgaggat

240

cacctgtcac tgaaggaatt ttcagaattg gagcaaagtg gttattatgt ctgctacccc

300

agaggaagca aaccagaaga tgcgaacttt tatctctacc tgagggcaag agtgtgtgag

360

aactgcatgg agatggatgt gatgtcggtg gccacaattg tcatagtgga catctgcatc

420

actgggggct tgctgctgct ggtttactac tggagcaaga atagaaaggc caaggccaag

480

cctgtgacac gaggagcggg tgctggcggc aggcaaaggg gacaaaacaa ggagaggcca

540

ccacctgttc ccaacccaga ctatgagccc atccggaaag gccagcggga cctgtattct

600

ggcctgaatc agagacgcat ctga 624

<210> 32

<211> 207

<212> PRT

<213> Homo sapiens

<400> 32

Met Gln Ser Gly Thr His Trp Arg Val Leu Gly Leu Cys Leu Leu Ser

1 5 10 15

Val Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr

20 25 30

Gln Thr Pro Tyr Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr

35 40 45

Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys

50 55 60

Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp

65 70 75 80

His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr

85 90 95

Val Cys Tyr Pro Arg Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu

100 105 110

Tyr Leu Arg Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp Val Met

115 120 125

Ser Val Ala Thr Ile Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu

130 135 140

Leu Leu Leu Val Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys

145 150 155 160

Pro Val Thr Arg Gly Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn

165 170 175

Lys Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg

180 185 190

Lys Gly Gln Arg Asp Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile

195 200 205

<210> 33

<211> 523

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 33

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His 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

Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu

145 150 155 160

Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln

165 170 175

Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln

180 185 190

Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg

195 200 205

Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

210 215 220

Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr

225 230 235 240

Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr

245 250 255

Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln

260 265 270

Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr Leu Thr Cys

275 280 285

Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val

290 295 300

Gln Glu Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly Gly Thr Asn

305 310 315 320

Lys Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly

325 330 335

Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala

340 345 350

Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly

355 360 365

Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly

370 375 380

Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Leu Glu Ser Gly Gly Gly

385 390 395 400

Leu Val Gln Pro Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly

405 410 415

Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly

420 425 430

Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr

435 440 445

Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg

450 455 460

Asp Asp Ser Gln Ser Ile Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr

465 470 475 480

Glu Asp Thr Ala Met Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn

485 490 495

Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

500 505 510

Val Ser Ala His His His His His His

515 520

<210> 34

<211> 753

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 34

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His 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

Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu

145 150 155 160

Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln

165 170 175

Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln

180 185 190

Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg

195 200 205

Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

210 215 220

Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr

225 230 235 240

Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr

245 250 255

Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln

260 265 270

Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr Leu Thr Cys

275 280 285

Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val

290 295 300

Gln Glu Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly Gly Thr Asn

305 310 315 320

Lys Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly

325 330 335

Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala

340 345 350

Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly

355 360 365

Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly

370 375 380

Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Leu Glu Ser Gly Gly Gly

385 390 395 400

Leu Val Gln Pro Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly

405 410 415

Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly

420 425 430

Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr

435 440 445

Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg

450 455 460

Asp Asp Ser Gln Ser Ile Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr

465 470 475 480

Glu Asp Thr Ala Met Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn

485 490 495

Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

500 505 510

Val Ser Ala Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro

515 520 525

Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro

530 535 540

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

545 550 555 560

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

565 570 575

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

580 585 590

Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

595 600 605

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

610 615 620

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

625 630 635 640

Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp

645 650 655

Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe

660 665 670

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

675 680 685

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

690 695 700

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

705 710 715 720

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

725 730 735

Thr Gln Lys Ser Leu Ser Leu Ser Pro His His His His His His

740 745 750

His

<210> 35

<211> 257

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 35

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro

20 25 30

Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro

35 40 45

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

50 55 60

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

65 70 75 80

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

85 90 95

Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

100 105 110

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

115 120 125

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

130 135 140

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Cys

145 150 155 160

Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe

165 170 175

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

180 185 190

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

195 200 205

Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

210 215 220

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

225 230 235 240

Thr Gln Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp

245 250 255

Lys

<210> 36

<211> 514

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 36

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His 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

Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu

145 150 155 160

Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln

165 170 175

Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln

180 185 190

Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg

195 200 205

Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

210 215 220

Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr

225 230 235 240

Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr

245 250 255

Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

260 265 270

Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro

275 280 285

Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe

290 295 300

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

305 310 315 320

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

325 330 335

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

340 345 350

Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

355 360 365

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

370 375 380

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

385 390 395 400

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

405 410 415

Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly

420 425 430

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

435 440 445

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

450 455 460

Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

465 470 475 480

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

485 490 495

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp

500 505 510

Asp Lys

<210> 37

<211> 629

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 37

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His 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

Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu

145 150 155 160

Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln

165 170 175

Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln

180 185 190

Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg

195 200 205

Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

210 215 220

Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr

225 230 235 240

Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr

245 250 255

Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Glu Val Lys Leu Leu Glu

260 265 270

Ser Gly Gly Gly Leu Val Gln Pro Lys Gly Ser Leu Lys Leu Ser Cys

275 280 285

Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg

290 295 300

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys

305 310 315 320

Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe

325 330 335

Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile Leu Tyr Leu Gln Met Asn

340 345 350

Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys Val Arg His Gly

355 360 365

Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly

370 375 380

Thr Leu Val Thr Val Ser Ala Glu Pro Lys Ser Ser Asp Lys Thr His

385 390 395 400

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

405 410 415

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

420 425 430

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

435 440 445

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

450 455 460

Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser

465 470 475 480

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

485 490 495

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

500 505 510

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro

515 520 525

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

530 535 540

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

545 550 555 560

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

565 570 575

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

580 585 590

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

595 600 605

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro His His His

610 615 620

His His His His

625

<210> 38

<211> 613

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 38

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His 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

Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu

145 150 155 160

Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln

165 170 175

Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln

180 185 190

Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg

195 200 205

Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

210 215 220

Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr

225 230 235 240

Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr

245 250 255

Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln

260 265 270

Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr Leu Thr Cys

275 280 285

Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val

290 295 300

Gln Glu Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly Gly Thr Asn

305 310 315 320

Lys Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly

325 330 335

Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala

340 345 350

Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly

355 360 365

Gly Thr Lys Leu Thr Val Leu Glu Pro Lys Ser Ser Asp Lys Thr His

370 375 380

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

385 390 395 400

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

405 410 415

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

420 425 430

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

435 440 445

Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser

450 455 460

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

465 470 475 480

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

485 490 495

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

500 505 510

Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala

515 520 525

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

530 535 540

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

545 550 555 560

Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg

565 570 575

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

580 585 590

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys

595 600 605

Asp Asp Asp Asp Lys

610

<210> 39

<211> 470

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 39

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His 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

Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

130 135 140

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

145 150 155 160

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

165 170 175

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

180 185 190

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

195 200 205

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

210 215 220

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

225 230 235 240

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

275 280 285

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

305 310 315 320

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

340 345 350

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

355 360 365

Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys

370 375 380

Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Asp Tyr

450 455 460

Lys Asp Asp Asp Asp Lys

465 470

<210> 40

<211> 238

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 40

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val

20 25 30

Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu

35 40 45

Val His Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Ala Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser

65 70 75 80

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

100 105 110

Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu

115 120 125

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

145 150 155 160

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

165 170 175

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

180 185 190

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

195 200 205

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

210 215 220

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 41

<211> 470

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 41

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His 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

Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

130 135 140

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

145 150 155 160

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

165 170 175

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

180 185 190

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

195 200 205

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

210 215 220

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

225 230 235 240

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

275 280 285

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

305 310 315 320

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

340 345 350

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu

355 360 365

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys

370 375 380

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro His His

450 455 460

His His His His His

465 470

<210> 42

<211> 734

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 42

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser

20 25 30

Pro Gly Glu Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val

35 40 45

Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu

50 55 60

Phe Thr Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile

85 90 95

Thr Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp

100 105 110

Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly Ser

145 150 155 160

Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala

165 170 175

Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

180 185 190

Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser

195 200 205

Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile Leu

210 215 220

Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr

225 230 235 240

Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala

245 250 255

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly

260 265 270

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val

275 280 285

Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser

290 295 300

Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu Met His Trp Ile

305 310 315 320

Arg Gln Pro Pro Gly Gln Gly Leu Glu Trp Ile Gly Ala Ile Asp Pro

325 330 335

Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys Gly Arg Val Thr

340 345 350

Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser

355 360 365

Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser

370 375 380

Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

385 390 395 400

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

405 410 415

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

420 425 430

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

435 440 445

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

450 455 460

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

465 470 475 480

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

485 490 495

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

500 505 510

Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

515 520 525

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

530 535 540

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

545 550 555 560

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

565 570 575

Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

580 585 590

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

595 600 605

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

610 615 620

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr

625 630 635 640

Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser

645 650 655

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

660 665 670

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val

675 680 685

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

690 695 700

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

705 710 715 720

Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp Lys

725 730

<210> 43

<211> 462

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 43

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His 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

Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

130 135 140

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

145 150 155 160

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

165 170 175

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

180 185 190

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

195 200 205

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

210 215 220

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

225 230 235 240

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

275 280 285

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

305 310 315 320

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

340 345 350

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

355 360 365

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

370 375 380

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

450 455 460

<210> 44

<211> 246

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 44

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val

20 25 30

Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu

35 40 45

Val His Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Ala Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser

65 70 75 80

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

100 105 110

Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu

115 120 125

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

145 150 155 160

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

165 170 175

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

180 185 190

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

195 200 205

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

210 215 220

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys His His

225 230 235 240

His His His His His

245

<210> 45

<211> 505

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 45

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val

20 25 30

Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu

35 40 45

Val His Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Ala Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser

65 70 75 80

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

100 105 110

Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu

115 120 125

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

145 150 155 160

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

165 170 175

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

180 185 190

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

195 200 205

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

210 215 220

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly

225 230 235 240

Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln Glu Ser

245 250 255

Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr Leu Thr Cys Arg Ser

260 265 270

Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu

275 280 285

Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly Gly Thr Asn Lys Arg

290 295 300

Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys

305 310 315 320

Ala Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr

325 330 335

Phe Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr

340 345 350

Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

355 360 365

Gly Gly Gly Ser Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val

370 375 380

Gln Pro Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr

385 390 395 400

Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly

405 410 415

Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr

420 425 430

Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp

435 440 445

Ser Gln Ser Ile Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp

450 455 460

Thr Ala Met Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr

465 470 475 480

Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

485 490 495

Ala Asp Tyr Lys Asp Asp Asp Asp Lys

500 505

<210> 46

<211> 729

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 46

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His 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

Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

130 135 140

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

145 150 155 160

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

165 170 175

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

180 185 190

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

195 200 205

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

210 215 220

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

225 230 235 240

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

275 280 285

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

305 310 315 320

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

340 345 350

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

355 360 365

Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys

370 375 380

Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly

450 455 460

Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln Glu Ser

465 470 475 480

Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr Leu Thr Cys Arg Ser

485 490 495

Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu

500 505 510

Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly Gly Thr Asn Lys Arg

515 520 525

Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys

530 535 540

Ala Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr

545 550 555 560

Phe Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr

565 570 575

Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

580 585 590

Gly Gly Gly Ser Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val

595 600 605

Gln Pro Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr

610 615 620

Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly

625 630 635 640

Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr

645 650 655

Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp

660 665 670

Ser Gln Ser Ile Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp

675 680 685

Thr Ala Met Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr

690 695 700

Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

705 710 715 720

Ala Asp Tyr Lys Asp Asp Asp Asp Lys

725

<210> 47

<211> 729

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 47

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His 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

Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

130 135 140

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

145 150 155 160

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

165 170 175

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

180 185 190

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

195 200 205

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

210 215 220

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

225 230 235 240

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

275 280 285

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

305 310 315 320

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

340 345 350

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

355 360 365

Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys

370 375 380

Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly

450 455 460

Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln Glu Ser

465 470 475 480

Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr Leu Thr Cys Arg Ser

485 490 495

Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu

500 505 510

Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly Gly Thr Asn Lys Arg

515 520 525

Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys

530 535 540

Ala Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr

545 550 555 560

Phe Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr

565 570 575

Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

580 585 590

Gly Gly Gly Ser Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val

595 600 605

Gln Pro Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr

610 615 620

Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly

625 630 635 640

Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr

645 650 655

Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp

660 665 670

Ser Gln Ser Ile Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp

675 680 685

Thr Ala Met Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr

690 695 700

Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

705 710 715 720

Ala Asp Tyr Lys Asp Asp Asp Asp Lys

725

<210> 48

<211> 470

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 48

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His 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

Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

130 135 140

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

145 150 155 160

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

165 170 175

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

180 185 190

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

195 200 205

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

210 215 220

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

225 230 235 240

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

275 280 285

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

305 310 315 320

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

340 345 350

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu

355 360 365

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys

370 375 380

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro His His

450 455 460

His His His His His

465 470

<210> 49

<211> 600

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 49

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His 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

Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

130 135 140

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

145 150 155 160

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

165 170 175

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

180 185 190

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

195 200 205

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

210 215 220

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

225 230 235 240

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

275 280 285

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

305 310 315 320

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

340 345 350

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

355 360 365

Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys

370 375 380

Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly

450 455 460

Gly Gly Ser Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

465 470 475 480

Pro Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

485 490 495

Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

500 505 510

Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr

515 520 525

Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser

530 535 540

Gln Ser Ile Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr

545 550 555 560

Ala Met Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val

565 570 575

Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

580 585 590

Asp Tyr Lys Asp Asp Asp Asp Lys

595 600

<210> 50

<211> 584

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 50

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His 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

Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

130 135 140

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

145 150 155 160

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

165 170 175

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

180 185 190

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

195 200 205

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

210 215 220

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

225 230 235 240

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

275 280 285

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

305 310 315 320

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

340 345 350

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu

355 360 365

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys

370 375 380

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly

450 455 460

Gly Gly Ser Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser

465 470 475 480

Pro Gly Glu Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val

485 490 495

Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu

500 505 510

Phe Thr Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro

515 520 525

Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile

530 535 540

Thr Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp

545 550 555 560

Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

565 570 575

His His His His His His His

580

<210> 51

<211> 605

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 51

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His 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

Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

130 135 140

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

145 150 155 160

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

165 170 175

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

180 185 190

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

195 200 205

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

210 215 220

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

225 230 235 240

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

275 280 285

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

305 310 315 320

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

340 345 350

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

355 360 365

Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys

370 375 380

Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly

450 455 460

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Leu Glu Ser Gly

465 470 475 480

Gly Gly Leu Val Gln Pro Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala

485 490 495

Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala

500 505 510

Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn

515 520 525

Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile

530 535 540

Ser Arg Asp Asp Ser Gln Ser Ile Leu Tyr Leu Gln Met Asn Asn Leu

545 550 555 560

Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys Val Arg His Gly Asn Phe

565 570 575

Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu

580 585 590

Val Thr Val Ser Ala Asp Tyr Lys Asp Asp Asp Asp Lys

595 600 605

<210> 52

<211> 589

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 52

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His 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

Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

130 135 140

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

145 150 155 160

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

165 170 175

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

180 185 190

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

195 200 205

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

210 215 220

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

225 230 235 240

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

275 280 285

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

305 310 315 320

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

340 345 350

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu

355 360 365

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys

370 375 380

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly

450 455 460

Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln Glu Ser

465 470 475 480

Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr Leu Thr Cys Arg Ser

485 490 495

Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu

500 505 510

Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly Gly Thr Asn Lys Arg

515 520 525

Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys

530 535 540

Ala Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr

545 550 555 560

Phe Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr

565 570 575

Lys Leu Thr Val Leu His His His His His His His

580 585

<210> 53

<211> 610

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 53

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His 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

Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

130 135 140

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

145 150 155 160

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

165 170 175

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

180 185 190

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

195 200 205

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

210 215 220

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

225 230 235 240

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

275 280 285

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

305 310 315 320

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

340 345 350

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

355 360 365

Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys

370 375 380

Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly

450 455 460

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys

465 470 475 480

Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly Ser Leu Lys

485 490 495

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn

500 505 510

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile

515 520 525

Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys

530 535 540

Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile Leu Tyr Leu

545 550 555 560

Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys Val

565 570 575

Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp

580 585 590

Gly Gln Gly Thr Leu Val Thr Val Ser Ala Asp Tyr Lys Asp Asp Asp

595 600 605

Asp Lys

610

<210> 54

<211> 594

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 54

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His 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

Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

130 135 140

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

145 150 155 160

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

165 170 175

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

180 185 190

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

195 200 205

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

210 215 220

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

225 230 235 240

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

275 280 285

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

305 310 315 320

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

340 345 350

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu

355 360 365

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys

370 375 380

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly

450 455 460

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val

465 470 475 480

Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr

485 490 495

Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala

500 505 510

Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly

515 520 525

Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser

530 535 540

Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu

545 550 555 560

Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val

565 570 575

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu His His His His His

580 585 590

His His

<210> 55

<211> 615

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 55

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His 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

Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

130 135 140

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

145 150 155 160

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

165 170 175

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

180 185 190

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

195 200 205

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

210 215 220

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

225 230 235 240

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

275 280 285

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

305 310 315 320

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

340 345 350

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

355 360 365

Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys

370 375 380

Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly

450 455 460

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

465 470 475 480

Gly Ser Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro

485 490 495

Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn

500 505 510

Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

515 520 525

Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr

530 535 540

Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln

545 550 555 560

Ser Ile Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala

565 570 575

Met Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser

580 585 590

Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Asp

595 600 605

Tyr Lys Asp Asp Asp Asp Lys

610 615

<210> 56

<211> 599

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 56

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His 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

Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

130 135 140

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

145 150 155 160

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

165 170 175

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

180 185 190

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

195 200 205

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

210 215 220

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

225 230 235 240

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

275 280 285

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

305 310 315 320

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

340 345 350

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu

355 360 365

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys

370 375 380

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly

450 455 460

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

465 470 475 480

Gly Ser Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro

485 490 495

Gly Glu Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr

500 505 510

Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe

515 520 525

Thr Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala

530 535 540

Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr

545 550 555 560

Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr

565 570 575

Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu His

580 585 590

His His His His His His

595

<210> 57

<211> 610

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 57

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His 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

Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

130 135 140

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

145 150 155 160

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

165 170 175

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

180 185 190

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

195 200 205

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

210 215 220

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

225 230 235 240

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

275 280 285

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

305 310 315 320

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

340 345 350

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

355 360 365

Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys

370 375 380

Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly

450 455 460

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys

465 470 475 480

Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly Ser Leu Lys

485 490 495

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn

500 505 510

Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val Ala Arg Ile

515 520 525

Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys

530 535 540

Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile Leu Tyr Leu

545 550 555 560

Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys Val

565 570 575

Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp

580 585 590

Gly Gln Gly Thr Leu Val Thr Val Ser Ala Asp Tyr Lys Asp Asp Asp

595 600 605

Asp Lys

610

<210> 58

<211> 594

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 58

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His 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

Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

130 135 140

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

145 150 155 160

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

165 170 175

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

180 185 190

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

195 200 205

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

210 215 220

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

225 230 235 240

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

275 280 285

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

305 310 315 320

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

340 345 350

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu

355 360 365

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys

370 375 380

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly

450 455 460

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val

465 470 475 480

Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr

485 490 495

Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala

500 505 510

Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly

515 520 525

Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser

530 535 540

Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu

545 550 555 560

Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val

565 570 575

Phe Gly Cys Gly Thr Lys Leu Thr Val Leu His His His His His

580 585 590

His His

<210> 59

<211> 704

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 59

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His 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

Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

130 135 140

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

145 150 155 160

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

165 170 175

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

180 185 190

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

195 200 205

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

210 215 220

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

225 230 235 240

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

275 280 285

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

305 310 315 320

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

340 345 350

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

355 360 365

Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys

370 375 380

Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly

450 455 460

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys

465 470 475 480

Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly Ser Leu Lys

485 490 495

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn

500 505 510

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile

515 520 525

Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys

530 535 540

Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile Leu Tyr Leu

545 550 555 560

Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys Val

565 570 575

Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp

580 585 590

Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro

595 600 605

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

610 615 620

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

625 630 635 640

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

645 650 655

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

660 665 670

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

675 680 685

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

690 695 700

<210> 60

<211> 692

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 60

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His 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

Thr Asp Tyr Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

130 135 140

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

145 150 155 160

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

165 170 175

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

180 185 190

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

195 200 205

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

210 215 220

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

225 230 235 240

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

275 280 285

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

305 310 315 320

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

340 345 350

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu

355 360 365

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys

370 375 380

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly

450 455 460

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val

465 470 475 480

Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr

485 490 495

Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala

500 505 510

Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly

515 520 525

Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser

530 535 540

Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu

545 550 555 560

Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val

565 570 575

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg Thr Val Ala Ala Pro

580 585 590

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

595 600 605

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

610 615 620

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

625 630 635 640

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

645 650 655

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

660 665 670

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

675 680 685

Asn Arg Gly Glu

690

<210> 61

<211> 115

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 61

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 62

<211> 219

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 62

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln 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 Val Gly Val Tyr Tyr Cys Ser Gln Asn

85 90 95

Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 63

<211> 328

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 63

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 64

<211> 587

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 64

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Gly Gly Gly

325 330 335

Gly Ser Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro

340 345 350

Gly Glu Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr

355 360 365

Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe

370 375 380

Thr Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala

385 390 395 400

Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr

405 410 415

Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr

420 425 430

Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

450 455 460

Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly Ser Leu

465 470 475 480

Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met

485 490 495

Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg

500 505 510

Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val

515 520 525

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile Leu Tyr

530 535 540

Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys

545 550 555 560

Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr

565 570 575

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

580 585

<210> 65

<211> 328

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 65

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Tyr Thr

305 310 315 320

Gln Glu Ser Leu Ser Leu Ser Pro

325

<210> 66

<211> 587

<212> PRT

<213> Artificial

<220>

<223> Artificially synthesized sequence

<400> 66

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Gly Gly Gly

325 330 335

Gly Ser Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro

340 345 350

Gly Glu Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr

355 360 365

Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe

370 375 380

Thr Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala

385 390 395 400

Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr

405 410 415

Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr

420 425 430

Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

450 455 460

Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly Ser Leu

465 470 475 480

Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met

485 490 495

Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg

500 505 510

Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val

515 520 525

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile Leu Tyr

530 535 540

Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys

545 550 555 560

Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr

565 570 575

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

580 585

<210> 67

<211> 141

<212> PRT

<213> Homo sapiens

<400> 67

Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys

1 5 10 15

Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr

20 25 30

Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr

35 40 45

Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala

50 55 60

Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser

65 70 75 80

Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp

85 90 95

Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe

100 105 110

Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala

115 120 125

Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

130 135 140

<210> 68

<211> 179

<212> PRT

<213> Homo sapiens

<400> 68

Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro

1 5 10 15

Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu

20 25 30

Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

35 40 45

Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys

50 55 60

Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu

65 70 75 80

Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys

85 90 95

Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp

100 105 110

Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg

115 120 125

Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser

130 135 140

Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala

145 150 155 160

Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp

165 170 175

Ser Arg Gly

<210> 69

<211> 173

<212> PRT

<213> Homo sapiens

<400> 69

Asp Lys Gln Leu Asp Ala Asp Val Ser Pro Lys Pro Thr Ile Phe Leu

1 5 10 15

Pro Ser Ile Ala Glu Thr Lys Leu Gln Lys Ala Gly Thr Tyr Leu Cys

20 25 30

Leu Leu Glu Lys Phe Phe Pro Asp Val Ile Lys Ile His Trp Gln Glu

35 40 45

Lys Lys Ser Asn Thr Ile Leu Gly Ser Gln Glu Gly Asn Thr Met Lys

50 55 60

Thr Asn Asp Thr Tyr Met Lys Phe Ser Trp Leu Thr Val Pro Glu Lys

65 70 75 80

Ser Leu Asp Lys Glu His Arg Cys Ile Val Arg His Glu Asn Asn Lys

85 90 95

Asn Gly Val Asp Gln Glu Ile Ile Phe Pro Pro Ile Lys Thr Asp Val

100 105 110

Ile Thr Met Asp Pro Lys Asp Asn Cys Ser Lys Asp Ala Asn Asp Thr

115 120 125

Leu Leu Leu Gln Leu Thr Asn Thr Ser Ala Tyr Tyr Met Tyr Leu Leu

130 135 140

Leu Leu Leu Lys Ser Val Val Tyr Phe Ala Ile Ile Thr Cys Cys Leu

145 150 155 160

Leu Arg Arg Thr Ala Phe Cys Cys Asn Gly Glu Lys Ser

165 170

<210> 70

<211> 204

<212> PRT

<213> Homo sapiens

<400> 70

Lys Gln Leu Asp Ala Asp Val Ser Pro Lys Pro Thr Ile Phe Leu Pro

1 5 10 15

Ser Ile Ala Glu Thr Lys Leu Gln Lys Ala Gly Thr Tyr Leu Cys Leu

20 25 30

Leu Glu Lys Phe Phe Pro Asp Ile Ile Lys Ile His Trp Gln Glu Lys

35 40 45

Lys Ser Asn Thr Ile Leu Gly Ser Gln Glu Gly Asn Thr Met Lys Thr

50 55 60

Asn Asp Thr Tyr Met Lys Phe Ser Trp Leu Thr Val Pro Glu Glu Ser

65 70 75 80

Leu Asp Lys Glu His Arg Cys Ile Val Arg His Glu Asn Asn Lys Asn

85 90 95

Gly Ile Asp Gln Glu Ile Ile Phe Pro Pro Ile Lys Thr Asp Val Thr

100 105 110

Thr Val Asp Pro Lys Asp Ser Tyr Ser Lys Asp Ala Asn Asp Val Thr

115 120 125

Thr Val Asp Pro Lys Tyr Asn Tyr Ser Lys Asp Ala Asn Asp Val Ile

130 135 140

Thr Met Asp Pro Lys Asp Asn Trp Ser Lys Asp Ala Asn Asp Thr Leu

145 150 155 160

Leu Leu Gln Leu Thr Asn Thr Ser Ala Tyr Tyr Met Tyr Leu Leu Leu

165 170 175

Leu Leu Lys Ser Val Val Tyr Phe Ala Ile Ile Thr Cys Cys Leu Leu

180 185 190

Gly Arg Thr Ala Phe Cys Cys Asn Gly Glu Lys Ser

195 200

<210> 71

<211> 177

<212> PRT

<213> Homo sapiens

<400> 71

Pro Ser Tyr Thr Gly Gly Tyr Ala Asp Lys Leu Ile Phe Gly Lys Gly

1 5 10 15

Thr Arg Val Thr Val Glu Pro Arg Ser Gln Pro His Thr Lys Pro Ser

20 25 30

Val Phe Val Met Lys Asn Gly Thr Asn Val Ala Cys Leu Val Lys Glu

35 40 45

Phe Tyr Pro Lys Asp Ile Arg Ile Asn Leu Val Ser Ser Lys Lys Ile

50 55 60

Thr Glu Phe Asp Pro Ala Ile Val Ile Ser Pro Ser Gly Lys Tyr Asn

65 70 75 80

Ala Val Lys Leu Gly Lys Tyr Glu Asp Ser Asn Ser Val Thr Cys Ser

85 90 95

Val Gln His Asp Asn Lys Thr Val His Ser Thr Asp Phe Glu Val Lys

100 105 110

Thr Asp Ser Thr Asp His Val Lys Pro Lys Glu Thr Glu Asn Thr Lys

115 120 125

Gln Pro Ser Lys Ser Cys His Lys Pro Lys Ala Ile Val His Thr Glu

130 135 140

Lys Val Asn Met Met Ser Leu Thr Val Leu Gly Leu Arg Met Leu Phe

145 150 155 160

Ala Lys Thr Val Ala Val Asn Phe Leu Leu Thr Ala Lys Leu Phe Phe

165 170 175

Leu

<210> 72

<211> 19

<212> PRT

<213> Artificial

<220>

<223> signal sequence

<400> 72

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser

<210> 73

<211> 466

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized sequence

<400> 73

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser

20 25 30

Pro Gly Glu Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val

35 40 45

Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu

50 55 60

Phe Thr Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile

85 90 95

Thr Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp

100 105 110

Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

115 120 125

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

130 135 140

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

145 150 155 160

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

165 170 175

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

180 185 190

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

195 200 205

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

210 215 220

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

225 230 235 240

Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe

245 250 255

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

260 265 270

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

275 280 285

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

290 295 300

Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

305 310 315 320

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

325 330 335

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

340 345 350

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

355 360 365

Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly

370 375 380

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

385 390 395 400

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

405 410 415

Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

420 425 430

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

435 440 445

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp

450 455 460

Asp Lys

465

<210> 74

<211> 251

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized sequence

<400> 74

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr

65 70 75 80

Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser

85 90 95

Gln Ser Ile Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr

100 105 110

Ala Met Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val

115 120 125

Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

130 135 140

Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

145 150 155 160

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

165 170 175

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

180 185 190

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

195 200 205

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

210 215 220

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

225 230 235 240

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

245 250

<210> 75

<211> 484

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized sequence

<400> 75

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr

65 70 75 80

Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser

85 90 95

Gln Ser Ile Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr

100 105 110

Ala Met Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val

115 120 125

Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

130 135 140

Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

145 150 155 160

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

165 170 175

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

180 185 190

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

195 200 205

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

210 215 220

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

225 230 235 240

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Asp Lys Thr His Thr

245 250 255

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe

260 265 270

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

275 280 285

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

290 295 300

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

305 310 315 320

Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val

325 330 335

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

340 345 350

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

355 360 365

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

370 375 380

Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val

385 390 395 400

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

405 410 415

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

420 425 430

Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

435 440 445

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

450 455 460

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp

465 470 475 480

Asp Asp Asp Lys

<210> 76

<211> 233

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized sequence

<400> 76

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser

20 25 30

Pro Gly Glu Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val

35 40 45

Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu

50 55 60

Phe Thr Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile

85 90 95

Thr Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp

100 105 110

Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

115 120 125

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

130 135 140

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

145 150 155 160

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

165 170 175

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

180 185 190

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

195 200 205

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

210 215 220

Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230

<210> 77

<211> 466

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized sequence

<400> 77

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser

20 25 30

Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val

35 40 45

Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Gly Leu Ile Gly Gly Thr Lys Phe Leu Ala Pro Gly Thr Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu

85 90 95

Ser Gly Val Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp

100 105 110

Tyr Ser Asn Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

115 120 125

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

130 135 140

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

145 150 155 160

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

165 170 175

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

180 185 190

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

195 200 205

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

210 215 220

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

225 230 235 240

Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe

245 250 255

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

260 265 270

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

275 280 285

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

290 295 300

Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

305 310 315 320

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

325 330 335

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

340 345 350

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

355 360 365

Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly

370 375 380

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

385 390 395 400

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

405 410 415

Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

420 425 430

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

435 440 445

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp

450 455 460

Asp Lys

465

<210> 78

<211> 251

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized sequence

<400> 78

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Asn Lys Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr

65 70 75 80

Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser

85 90 95

Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile

115 120 125

Ser Tyr Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

130 135 140

Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

145 150 155 160

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

165 170 175

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

180 185 190

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

195 200 205

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

210 215 220

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

225 230 235 240

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

245 250

<210> 79

<211> 475

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized sequence

<400> 79

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val

20 25 30

Arg Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala

35 40 45

Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly

50 55 60

Leu Glu Trp Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn Ile His Tyr

65 70 75 80

Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser

85 90 95

Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala

100 105 110

Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro Met Asp Tyr

115 120 125

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly

130 135 140

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

145 150 155 160

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

165 170 175

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

180 185 190

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

195 200 205

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

210 215 220

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

225 230 235 240

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala

245 250 255

Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

260 265 270

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

275 280 285

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

290 295 300

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser

305 310 315 320

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

325 330 335

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

340 345 350

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

355 360 365

Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

370 375 380

Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

385 390 395 400

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

405 410 415

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

420 425 430

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

435 440 445

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

450 455 460

Leu Ser Pro His His His His His His

465 470 475

<210> 80

<211> 239

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized sequence

<400> 80

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val

20 25 30

Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys

115 120 125

Leu 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

<210> 81

<211> 453

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized sequence

<400> 81

Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

325 330 335

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

355 360 365

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Pro

450

<210> 82

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized sequence

<400> 82

Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 83

<211> 443

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized sequence

<400> 83

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440

<210> 84

<211> 219

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized sequence

<400> 84

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala

35 40 45

Pro Arg 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 Val Gly Val Tyr Tyr Cys Ser Gln Asn

85 90 95

Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 85

<211> 453

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized sequence

<400> 85

Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Lys Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

325 330 335

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr Lys

355 360 365

Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser

405 410 415

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Pro

450

<210> 86

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized sequence

<400> 86

Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

115 120 125

Ser Gly Thr Ala Glu Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Glu Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Glu Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 87

<211> 443

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized sequence

<400> 87

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Glu Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440

<210> 88

<211> 443

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized sequence

<400> 88

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Glu Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440

<210> 89

<211> 219

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized sequence

<400> 89

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala

35 40 45

Pro Arg 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 Val Gly Val Tyr Tyr Cys Ser Gln Asn

85 90 95

Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Lys Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Lys Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Lys Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 90

<211> 219

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized sequence

<400> 90

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala

35 40 45

Pro Arg 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 Val Gly Val Tyr Tyr Cys Ser Gln Asn

85 90 95

Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Lys

115 120 125

Gln Leu Lys Ser Gly Thr Ala Lys Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Lys Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Lys Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 91

<211> 474

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized sequence

<400> 91

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln

20 25 30

Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu

35 40 45

Thr Asn Tyr Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu

50 55 60

Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr

65 70 75 80

Pro Phe Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln

85 90 95

Val Phe Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr

100 105 110

Tyr Cys Ala Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp

115 120 125

Gly Gln Gly Thr Leu Val Thr Val Ser Ala 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 Lys Val Glu Pro Lys Ser

225 230 235 240

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

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 Ala 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 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

370 375 380

Ser Leu Trp 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 His His His His His His His

465 470

<210> 92

<211> 233

<212> PRT

<213> Artificial sequence

<220>

<223> Artificially synthesized sequence

<400> 92

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val

20 25 30

Ser Pro Gly Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile

35 40 45

Gly Thr Asn Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg

50 55 60

Leu Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser

85 90 95

Val Glu Ser Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn

100 105 110

Trp Pro Thr Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr

115 120 125

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

130 135 140

Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro

145 150 155 160

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

165 170 175

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

180 185 190

Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

195 200 205

Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

210 215 220

Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<---

Claims (74)

1. A bispecific antibody with cellular cytotoxicity that includes: a first light chain comprising a first VL domain and a first CL domain; a second light chain comprising a second VL domain and a second CL domain; a first heavy chain comprising a first heavy chain variable region and a first heavy chain constant region, wherein the first heavy chain constant region is a non-wild-type constant region comprising one of the sequences of SEQ ID NO: 23, 24, 25 and 26 with one or more amino acid substitutions and optionally a deletion of amino acids at positions 446 and 447 according to EU numbering; and a second heavy chain comprising a second heavy chain variable region and a second heavy chain constant region, wherein the second heavy chain constant region is a non-wild-type heavy chain constant region comprising one of the sequences of SEQ ID NO: 23, 24, 25 and 26 with one or more amino acid substitutions and optionally a deletion of the amino acids at positions 446 and 447 according to EU numbering, wherein the amino acid sequence of the second heavy chain constant region is the same as or different from the amino acid sequence of the first heavy chain constant region and has the same isotype as the first heavy chain constant region; wherein the first light chain and the first heavy chain associate to form a first antigen-binding domain that binds to CD3, wherein the second light chain and the second heavy chain associate to form a second antigen-binding domain that does not bind to CD3 and that binds to a cancer antigen that is not CD3, wherein the first heavy chain constant region associates with the second heavy chain constant region, wherein, when measured by surface plasmon resonance, the ability of the associated first and second heavy chain constant regions to bind to the human Fcγ receptor is reduced compared to the ability of a wild-type human IgG antibody of the same isotype as the bispecific antibody to bind to the human Fcγ receptor, as a result of at least one of the one or more amino acid substitutions within the first and second heavy chain constant regions, wherein the at least one substitution within the first and second heavy chain constant regions is a change in amino acid sequence compared to the amino acid sequence of the heavy chain constant regions of the human IgG antibody wild type and wherein at least one of the amino acids that results in reduced ability to bind to the human Fcγ receptor is at a position selected from the following positions according to EU numbering in each of the first and second constant regions of the heavy chain: 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331 and 332. 2. Bispecific antibody according to claim 1, where the amino acid sequence at positions 118 to 260 (according to EU numbering) of the first heavy chain constant region and the second heavy chain constant region is the sequence of the corresponding part of SEQ ID NO: 24; or the amino acid sequence at positions 261 to 447 (according to EU numbering) of the first heavy chain constant region and the second heavy chain constant region is the sequence of the corresponding part of SEQ ID NO: 26. 3. The bispecific antibody of claim 1, wherein the first heavy chain constant region and the second heavy chain constant region each comprise the sequence of SEQ ID NO: 23 with one or more substitutions and, optionally, a deletion of two residues at the carboxyl terminus of SEQ ID NO: 23. 4. The bispecific antibody of claim 3, wherein the one or more substitutions comprise a substitution in both the first and second constant regions of the heavy chain at a position selected from positions 233, 234, 235, 236, 327, 330 and 331 (according to EU numbering), wherein the substitution at the selected position is an amino acid from the corresponding position in IgG2 or IgG4. 5. The bispecific antibody of claim 3, wherein the one or more substitutions comprise a substitution in both the first and second constant regions of the heavy chain at position 234, 235 or 297 (according to EU numbering). 6. The bispecific antibody of claim 5, wherein one or more substitutions comprise alanine substitution. 7. The bispecific antibody of claim 1, wherein the amino acid sequence of the second constant region of the heavy chain differs from the amino acid sequence of the first constant region of the heavy chain. 8. Bispecific antibody according to claim 7, where: in one of the two constant regions of the heavy chain of the bispecific antibody, the amino acids at positions 349 and 366 (according to EU numbering) are cysteine and tryptophan, respectively; and in the other constant region of the heavy chain of the bispecific antibody, the amino acids at positions 356, 366, 368 and 407 (according to EU numbering) are cysteine, serine, alanine and valine, respectively. 9. Bispecific antibody according to claim 7, where: in one of the two constant regions of the heavy chain of the bispecific antibody, the amino acid at position 356 is lysine, in the other constant region of the heavy chain of the bispecific antibody, the amino acid at position 439 is glutamic acid; and in one of the two constant regions of the heavy chain of the bispecific antibody, but not both, the amino acid at position 435 is arginine (according to EU numbering). 10. The bispecific antibody of claim 8, wherein the amino acids according to EU numbering at positions 446 and 447 are deleted in both constant regions of the heavy chain of the bispecific antibody. 11. The bispecific antibody according to claim 1, wherein the human Fcγ receptor is selected from FcγI, FcγIIA, FcγIIB, FcγIIIA and FcγIIIB. 12. The bispecific antibody of claim 1, wherein the amino acid sequence of the second constant region of the heavy chain is the same as the amino acid sequence of the first constant region of the heavy chain. 13. The bispecific antibody of claim 1, wherein the amino acid sequence of the second constant region of the heavy chain differs from the amino acid sequence of the first constant region of the heavy chain and wherein the percentage of heterodimers formed in a mixture of the first and second heavy chains is higher than the percentage of heterodimers that would be associated if the two constant regions of the heavy chain were identical and corresponded to one of SEQ ID NOs: 23-26. 14. The bispecific antibody of claim 13, wherein the difference between the sequences of the first and second constant regions of the heavy chain comprises a difference in one or more positions of the CH3 domain. 15. The bispecific antibody of claim 14, wherein the difference between the sequences of the first and second constant regions of the heavy chain further comprises a difference in one or more positions of the CH1 domain, wherein one or more positions of the CH1 domain interact with the CL domain. 16. The bispecific antibody of claim 1, wherein the amino acid sequence of the second constant region of the heavy chain differs from the amino acid sequence of the first constant region of the heavy chain and this difference in sequences includes a difference in one or more positions of the CH1 domain of the two constant regions of the heavy chain, wherein one or more positions of the CH1 domain interact with the CL domain. 17. The bispecific antibody of claim 1, wherein the human Fcγ receptor is selected from FcγI, FcIIIγA and FcγIIIB. 18. The bispecific antibody of claim 1, wherein, when measured by surface plasmon resonance, the ability of the bispecific antibody to bind to each of the human Fcγ, FcγI, FcγIIIA and FcγIIIB receptors is reduced compared to the ability of a wild-type human IgG antibody of the same isotype, such as the bispecific antibody, to bind to the same human Fcγ receptors. 19. The bispecific antibody of claim 1, wherein, when measured by surface plasmon resonance, the ability of the bispecific antibody to bind to each of the human Fcγ, FcγI, FcγIIA, FcγIIB, FcγIIIA and FcγIIIB receptors is reduced compared to the ability of a wild-type human IgG antibody of the same isotype, such as the bispecific antibody, to bind to the same human Fcγ receptor. 20. The bispecific antibody of claim 1, wherein the constant region of the first and second heavy chains is a constant region of a human heavy chain. 21. The bispecific antibody of claim 1, wherein the associated first and second heavy chain constant regions have substantially no detectable ability to bind to the human Fcγ receptor as measured by surface plasmon resonance. 22. The bispecific antibody of claim 1, wherein the associated first and second heavy chain constant regions have substantially no detectable ability to bind to human Fcγ, FcγI, FcγIIA, FcγIIB, FcγIIIA and FcγIIIB receptors when measured by surface plasmon resonance. 23. The bispecific antibody of claim 3, wherein the one or more substitutions comprise one of the following sets of substitutions in both the first and second constant regions of the heavy chain (according to EU numbering): (a) L234F, L235E, P331S, (b) C226S, C229S, P238S; (c) C226S, C229S; (d) C226S, C229S, E233P, L234V, L235A. 24. The bispecific antibody of claim 1, wherein each first and second constant region of the heavy chains comprises the sequence of SEQ ID NO: 24 with one or more substitutions and optionally a deletion of two residues at the carboxyl terminus of SEQ ID NO: 24. 25. The bispecific antibody of claim 24, wherein the one or more substitutions comprise one of the following sets of substitutions in both the first and second constant regions of the heavy chain (according to EU numbering): (a) H268Q, V309L, A330S and P331 S; (b) V234A; (c) G237A; (d) V234A and G237A; (e) A235E and G237A; or (f) V234A, A235E and G237A. 26. The bispecific antibody of claim 1, wherein the first heavy chain constant region and the second heavy chain constant region comprise the sequence of SEQ ID NO: 25 with one or more substitutions and optionally a deletion of two residues at the carboxyl terminus of SEQ ID NO: 25. 27. The bispecific antibody of claim 26, wherein the one or more substitutions comprise one of the following substitutions in both the first and second constant regions of the heavy chain (according to EU numbering): (a) F241A; (b) D265A; or (c) V264A. 28. The bispecific antibody of claim 1, wherein the first heavy chain constant region and the second heavy chain constant region comprise the sequence of SEQ ID NO: 26 with one or more substitutions and optionally a deletion of two residues at the carboxyl terminus of SEQ ID NO: 26. 29. The bispecific antibody of claim 28, wherein the one or more substitutions comprise one of the following sets of substitutions in both the first and second constant regions of the heavy chain (according to EU numbering): (a) L235A, G237A and E318A; (b) L235E; or (c) F234A and L235A. 30. The bispecific antibody of claim 1, wherein one or more substitutions comprise a substitution at position 234 (according to EU numbering) in both the first and second constant regions of the heavy chain. 31. The bispecific antibody of claim 1, wherein one or more substitutions comprise a substitution at position 235 (according to EU numbering) in both the first and second constant regions of the heavy chain. 32. The bispecific antibody of claim 1, wherein one or more substitutions comprise a substitution at position 239 (according to EU numbering) in both the first and second constant regions of the heavy chain. 33. The bispecific antibody of claim 1, wherein one or more substitutions comprise a substitution at position 297 (according to EU numbering) in both the first and second constant regions of the heavy chain. 34. The bispecific antibody of claim 1, wherein the residues at positions 234 and 235 (according to EU numbering) are alanine in both the first and second constant regions of the heavy chain. 35. The bispecific antibody of claim 1, wherein the residues at positions 234, 235 and 297 (according to EU numbering) are all alanine in both the first and second constant regions of the heavy chain. 36. The bispecific antibody of claim 1, wherein positions 446 and 447 (according to EU numbering) are deleted in the sequence of each of the first and second constant regions of the heavy chain. 37. The bispecific antibody of claim 1, wherein the only substitution(s) in the amino acid sequence of one or both of the first and second constant regions of the heavy chain compared to the amino acid sequence of SEQ ID NO: 23 are at a position(s) independently selected from the following positions according to EU numbering: 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331 and 332. 38. The bispecific antibody of claim 1, wherein the only substitution(s) in the amino acid sequence of one or both of the first and second constant regions of the heavy chain compared to the amino acid sequence of SEQ ID NO: 24 are at a position(s) independently selected from the following positions according to EU numbering: 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331, and 332. 39. The bispecific antibody of claim 1, wherein the only substitution(s) in the amino acid sequence of one or both of the first and second constant regions of the heavy chain compared to the amino acid sequence of SEQ ID NO: 25 are at a position(s) independently selected from the following positions according to EU numbering: 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331 and 332. 40. The bispecific antibody of claim 1, wherein the only substitution(s) in the amino acid sequence of one or both of the first and second constant regions of the heavy chain compared to the amino acid sequence of SEQ ID NO: 26 are at a position(s) independently selected from the following positions according to EU numbering: 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331 and 332. 41. The bispecific antibody of claim 1, wherein the amino acid sequence of one or both of the first and second constant regions of the heavy chain has a substitution compared to the amino acid sequence of SEQ ID NO: 23 at a position(s) independently selected from the following positions according to EU numbering: 229, 239, 240, 264, 265, 267, 297, 298, 299, 329, 331 and 332. 42. The bispecific antibody of claim 1, wherein the amino acid sequence of one or both of the first and second constant regions of the heavy chain has a substitution compared to the amino acid sequence of SEQ ID NO: 24 at a position(s) independently selected from the following positions according to EU numbering: 229, 239, 240, 264, 265, 267, 297, 298, 299, 329, 331 and 332. 43. The bispecific antibody of claim 1, wherein the amino acid sequence of one or both of the first and second constant regions of the heavy chain has a substitution compared to the amino acid sequence of SEQ ID NO: 25 at a position(s) independently selected from the following positions according to EU numbering: 229, 239, 240, 264, 265, 267, 297, 298, 299, 329, 331 and 332. 44. The bispecific antibody of claim 1, wherein the amino acid sequence of one or both of the first and second constant regions of the heavy chain has a substitution compared to the amino acid sequence of SEQ ID NO: 26 at a position(s) independently selected from the following positions according to EU numbering: 229, 239, 240, 264, 265, 267, 297, 298, 299, 329 and 332. 45. The bispecific antibody of claim 1, wherein the bispecific antibody is a human or humanized antibody. 46. The bispecific antibody of claim 1, wherein the isotype of the bispecific antibody is human IgG1, human IgG2, human IgG3 or human IgG4. 47. The bispecific antibody of claim 1, wherein the isotype of each first and second constant region of the heavy chain is independently selected from human IgG1, human IgG2, human IgG3 and human IgG4. 48. A pharmaceutical composition for treating cancer, wherein the pharmaceutical composition contains as an active ingredient a bispecific antibody according to any one of claims 1-47.