Cytotoxicity inducing therapeutic agents

Abstract

To provide a polypeptide complex that exhibits cytotoxicity to a target cancer cell by T cells, a method for preparing the polypeptide, a cytotoxicity-inducing therapeutic agent comprising the polypeptide complex as an effective ingredient, as well as a pharmaceutical composition including the cytotoxicity-inducing therapeutic agent as an effective ingredient for treating or preventing various cancers or a therapeutic method using the pharmaceutical composition.SOLUTION: The invention provides a polypeptide complex which comprises: (1) an antigen-binding domain; (2) a domain comprising an Fc region with reduced Fcγ receptor-binding activity; and (3) a T cell receptor complex-binding domain. The invention also provides: a cell comprising the vector comprising the polynucleotide encoding the polypeptide complex; a method for preparing the polypeptide complex, comprising culturing the cell to harvest the polypeptide complex from the culture supernatant; a cytotoxicity-inducing therapeutic agent comprising the polypeptide complex as an effective ingredient; and a treating method comprising administering the therapeutic agent to a patient in need thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention provides a method for bringing T cells into close proximity with target cancer cells, thereby enabling the T cells to activate cytotoxicity against the target cancer cells. Polypeptide complex that enables cancer treatment through sexual activity, and the polypeptide complex and a therapeutic agent for inducing cytotoxicity containing the polypeptide complex as an active ingredient. Furthermore, the present invention relates to a method for treating or preventing various cancers, which comprises the cytotoxicity-inducing therapeutic agent as an active ingredient. The present invention relates to a pharmaceutical composition for treating the condition, or a method of treatment using the pharmaceutical composition. [Background technology]

[0002] To date, several therapeutic antibodies that have demonstrated excellent antitumor effects have been developed into pharmaceuticals for cancer treatment. These therapeutic antibodies target the signals required for cancer cell proliferation. signal inhibition, induction of cell death signals, or ADCC (Antibody Dependent Cell-mediated ed Cytotoxicity;antibody-dependent cytotoxicity), CDC(Complement Dependent Cytotoxicity; It is known that it exerts an antitumor effect on cancer cells through complement-dependent cytotoxicity. The Fc region of an antibody binds to effector cells such as NK cells and macrophages (Non-Patent Document 2). By binding to the Fc receptors present in the target cancer cells, the antibody binds to The cytotoxicity exerted by these effector cells is called ADCC. The complement complex binds to the binding site. The complement complex exists on the cell membrane of the cell to which the antibody binds. The complement components present in the cells form pores, which promote the inflow of water and ions into the cells. CDC is the cell damage that occurs when the nuclei are destroyed. Existing therapeutic antibodies have been shown to have excellent effects. However, the therapeutic results obtained by administering these antibodies are still not satisfactory. Therefore, there is a need to develop therapeutic antibodies against cancer that have even stronger cytotoxic activity. .

[0003] The ADCC that recruits the above-mentioned NK cells and macrophages as effector cells is considered to be the key to its antitumor effect. In addition to antibodies as the effective mechanism, cytotoxicity that recruits T cells as effector cells is also T cell recruiting antibodies (T cell recruiting antibodies) are antibodies whose antitumor effect is mediated by T cell receptors. ng antibody, TR antibody) have also been known since the 1980s (Non-patent Documents 3-5). Antibodies against any of the constituent subunits of the T cell receptor (TCR) complex, especially CD 3 Bi-spec antibody containing an antibody that binds to the epsilon chain and an antibody that binds to an antigen on the target cancer cell TR antibodies bind to both the CD3 epsilon chain and cancer antigens simultaneously. This allows T cells to approach cancer cells, resulting in the cytotoxicity of T cells. It is believed to have an antitumor effect.

[0004] One type of TR antibody known as a trifunctional antibody is also known (Non-patent Document 6 , 7). This is because the Fab that binds to the cancer antigen and the Fab that binds to the CD3 epsilon chain are each attached to one arm. It is a whole IgG type bi-specific antibody that contains EpCAM. It is a trifunctional antibody against EpCAM. Intraperitoneal administration of catumaxomab to patients with malignant ascites harboring EpCAM-positive cancer cells The effectiveness of this drug in treating malignant ascites has been demonstrated. Catumaxomab is approved for use in this setting.

[0005] More recently, a TR antibody called BiTE (bispecific T-cell engager) has been shown to have strong anti-tumor properties. BiTE has been shown to have anti-tumor activity against cancer antigens (Non-patent Documents 8 and 9). The scFv of the antibody and the scFv of the antibody against the CD3 epsilon chain are linked via a short polypeptide linker. BiTE is a TR antibody with a molecular structure that is bound to the antibody. It has been reported that all of these compounds have excellent antitumor effects (Non-Patent Documents 9 and 10). TE inhibited the effector cell:cancer cell ratio at significantly lower concentrations than other TR antibodies. It exerts an antitumor effect under the ET ratio. It has also been shown that there is no need to activate the cells with IL-2 or CD28 agonist antibodies. Much stronger in vitro than Rituxan, which is known to have excellent clinical efficacy. Blinatumomab (MT103), a BiTE targeting CD19, demonstrated cytotoxic activity against cancer cells. Furthermore, recent Phase I and II clinical trials have shown extremely excellent antitumor effects. It has been reported that this was demonstrated (Non-Patent Document 11).

[0006] Catumaxomab has demonstrated clinical efficacy and has been approved as a therapeutic agent for blinatumoma. Since multiple BiTEs, including b, exert strong antitumor effects, T cells are considered to be effective cells. TR antibodies that recruit cells have a much higher activity than antibodies that use conventional ADCC as their mechanism of action. It was suggested that it has potential as an antitumor drug.

[0007] However, trifunctional antibodies can activate T cells, NK cells, and macrophages independently of cancer antigens. As a result of simultaneous binding to cells such as IL-1, receptors expressed on these cells are cross-linked. It is known that these compounds induce the expression of various cytokines independent of cancer antigens. Induction of cytokine expression was confirmed by the cytokine storage induced by systemic administration of trifunctional antibodies. In fact, catu In a Phase 1 clinical trial of systemic maxomab, an extremely low dose of 5 μg / body was used. This is the maximum tolerated dose, and administration of higher doses may cause various serious side effects. It has been reported (Non-patent Document 12). Administration of such low doses of catumaxomab In other words, at such low doses of catumaxomab, the effective blood concentration is not reached. The expected antitumor effect is not achieved by administration.

[0008] On the other hand, unlike catumaxomab, BiTE does not have a binding site for Fcγ receptors, and therefore, Antigen-independent cross-linking of receptors expressed on T cells and NK cells, macrophages, etc. Therefore, the cancer antigen-independent site observed when catumaxomab was administered is not However, BiTE is a low-molecular-weight protein lacking the Fc region. Because it is a molecular-weight engineered antibody molecule, it is more effective than IgG antibodies, which are commonly used as therapeutic antibodies. In comparison, there is a problem in that the half-life of BiTE administered to patients is significantly shorter. It has been shown that the blood half-life of BiTE administered to a living body is about several hours (Non-Patent Document References 13, 14) In clinical trials of blinatumomab, continuous intravenous administration was performed using a minipump. This administration is extremely convenient for patients. Not only is this an ineffective administration method, but there is also the potential risk of medical accidents due to equipment failure, etc. It cannot be said to be a desirable treatment. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Clin Cancer Res. (2010) 16 (1), 11-20 [Non-patent document 2] Drug Des Devel Ther (2009) 3, 7-16 [Non-patent document 3] Nature (1985) 314 (6012), 628-31 [Non-patent document 4] Int J Cancer (1988) 41 (4), 609-15. [Non-Patent Document 5] Proc Natl Acad Sci USA (1986) 83 (5), 1453-7 [Non-patent document 6] Cancer Treat Rev. (2010) 36 (6), 458-67 [Non-Patent Document 7] Expert Opin Biol Ther (2010) 10 (8), 1259-69 [Non-patent document 8] Proc Natl Acad Sci USA. (1995) 92 (15), 7021-5 [Non-Patent Document 9] Drug Discov Today (2005), 10 (18), 1237-44 [Non-Patent Document 10] Trends Biotechnol (2004) 22 (5), 238-44 [Non-Patent Document 11] Science (2008), 321 (5891), 974-7 [Non-Patent Document 12] Cancer Immunol Immunother (2007) 56 (10), 1637-44 [Non-Patent Document 13] Cancer Immunol Immunother. (2006) 55(5), 503-14 [Non-Patent Document 14] Cancer Immunol Immunother. (2009) 58(1), 95-109 Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention has been made in view of the above circumstances, and provides a method for treating cancer by bringing T cells into close proximity with target cancer cells. Polypeptides that can treat cancer through cytotoxic activity against target cancer cells by the polypeptides Polypeptide complexes, methods for producing the polypeptide complexes, and methods for producing the polypeptide complexes The present invention aims to provide a therapeutic agent for inducing cell damage containing the compound as an active ingredient. A pharmaceutical composition for treating or preventing various cancers, which contains an activating therapeutic agent as an active ingredient. The object is to provide a method of treatment using the pharmaceutical composition. [Means for solving the problem]

[0011] The present inventors have demonstrated that BiTE has strong antitumor activity and suppresses cytokine storms in a cancer antigen-independent manner. It maintains the excellent safety characteristics of not inducing steroids, etc., and has a long half-life in the blood. Furthermore, we have discovered a new polypeptide complex. By substituting the cytosine, the polypeptide complex targets various cells and induces cytotoxicity. Based on this discovery, the present inventors have found that the polypeptide according to the present invention We also found that polypeptide complexes can damage cancer cells. By introducing 1 / CL interface aggregation control and Knob into Hole (KiH) modification, Furthermore, the present inventors have found that the polypeptide of the present invention causes cytotoxicity. We have found that a cytotoxicity-inducing therapeutic agent containing the aggregate as an active ingredient can treat or prevent various cancers. did.

[0012] That is, the present invention provides the following. [1] The following domains: (1) an antigen-binding domain, (2) a domain containing an Fc region with reduced binding activity to an Fcγ receptor; and (3) T cell receptor complex binding domain, A polypeptide complex comprising the [2] The T cell receptor complex-binding domain is a T cell receptor-binding domain, The polypeptide complex described above. [3] The polypeptide of [1], wherein the T cell receptor complex-binding domain is a CD3-binding domain. Peptide aggregates. [4] Any of [1] to [3], wherein the antigen-binding domain is a bivalent antigen-binding domain. The polypeptide complex of any one of the preceding claims. [5] The antibody according to [4], wherein the bivalent antigen-binding domain has a F(ab')2 structure. The polypeptide complex described above. [6] Two polypeptides constituting the heavy chain constant region of a domain having the structure F(ab')2 The polypeptide of [5], wherein Do-aggregate. [7] [6], in which the CD3-binding domain is linked to one or two CH3s constituting the Fc region. The polypeptide complex described above. [8] A heavy chain Fv fragment constituting the CD3-binding domain is linked to one CH3 constituting the Fc region. The light chain Fv fragment constituting the CD3-binding domain is linked to the other CH3 constituting the Fc region. [8] The polypeptide complex of [7]. [9] A heavy chain Fv fragment constituting a CD3-binding domain, a CH1 domain of an antibody, and a light chain Fv fragment The polypeptide complex of [8], in which an antibody CL domain is linked to

[10] A CD3-binding domain linked to one or two CLs constituting F(ab')2, [6

[0023] The polypeptide complex of

[0024]

[11] A CD3-binding domain linked to one or two VHs constituting F(ab')2.

[0023] The polypeptide complex of

[0024]

[12] A CD3-binding domain linked to one or two VLs constituting F(ab')2. ] The polypeptide complex of

[13] The polypeptide of any one of [1] to

[12] , wherein the CD3-binding domain is an Fv. Peptide aggregate.

[14] The antibodies according to [1] to [7] and

[10] to

[12] , wherein the CD3-binding domain is Fab. The polypeptide complex of any one of the above.

[15] [1] to [7] and

[10] to

[12] , wherein the CD3-binding domain is an scFv. The polypeptide complex of any one of the preceding claims.

[16] The polypeptide of any one of [1] to

[15] , wherein the CD3-binding domain is monovalent. Peptide aggregates.

[17] Any of [1] to [3], wherein the antigen-binding domain is a monovalent scFv and a monovalent Fab. The polypeptide complex described in any one of the above.

[18] A monovalent scFv constitutes an Fc region via an scFv that constitutes a CD3-binding domain. The polypeptide is a monovalent Fab heavy chain Fv fragment bound to one polypeptide constituting an Fc region via a CH1 region. and the light chain Fv fragment of the Fab is linked to a CL region. The polypeptide complex described above.

[19] The antibody of any one of [1] to [3], wherein the antigen-binding domain is a bivalent scFv. Polypeptide complex.

[20] Monovalent scFv constructs an Fc region via a heavy chain Fv fragment that constitutes the CD3-binding domain. One polypeptide contains a light chain Fv fragment that constitutes the CD3-binding domain, and the other monovalent scFv contains a light chain Fv fragment that constitutes the CD3-binding domain. The polypeptide of

[19] , linked to another polypeptide constituting an Fc region via a Peptide aggregates.

[21] A monovalent scFv constitutes the CD3-binding domain and constitutes the Fc region via the scFv. The other monovalent scFv is linked to another polypeptide constituting the Fc region. The polypeptide complex of

[19] , wherein the polypeptide complex is ligated.

[22] The antigen-binding domain and the T cell receptor complex-binding domain are each monovalent Fab. The polypeptide complex of any one of [1] to [3],

[23] A monovalent Fab heavy chain Fv fragment constituting the antigen-binding domain is connected to the Fc region via the CH1 region. The light chain Fv fragment of the Fab is linked to one of the polypeptides constituting the Fab, and the light chain Fv fragment of the Fab is linked to the CL region, The heavy chain Fv fragment of Fab, which constitutes the cell receptor binding domain, constitutes the Fc region via the CH1 region

[22] linked to another polypeptide, and the light chain Fv fragment of the Fab is linked to the CL region. The polypeptide complex of any one of claims 1 to 3.

[24] A monovalent Fab heavy chain Fv fragment constituting the antigen-binding domain is connected to the Fc region via the CH1 region. The light chain Fv fragment of the Fab is linked to one of the polypeptides constituting the Fab, and the light chain Fv fragment of the Fab is linked to the CL region, The light chain Fv fragment of Fab, which constitutes the cell receptor binding domain, constitutes the Fc region via the CH1 region

[22] linked to another polypeptide, and the heavy chain Fv fragment of the Fab is linked to the CL region. The polypeptide complex of any one of claims 1 to 3.

[25] A monovalent Fab heavy chain Fv fragment constituting the antigen-binding domain is connected to the Fc region via the CH1 region. The light chain Fv fragment of the Fab is linked to one of the polypeptides constituting the Fab, and the light chain Fv fragment of the Fab is linked to the CL region, The heavy chain Fv fragment of Fab, which constitutes the cell receptor binding domain, constitutes the Fc region via the CL region

[22] linked to another polypeptide, and the light chain Fv fragment of the Fab is linked to the CH1 region. The polypeptide complex of any one of claims 1 to 3.

[26] The heavy chain Fv fragment of the monovalent Fab, which constitutes the T cell receptor binding domain, binds to the T cell receptor via the CH1 region. The light chain Fv fragment of the Fab is linked to one of the polypeptides constituting the Fc region, and the light chain Fv fragment of the Fab is linked to the CL region. The light chain Fv fragment of Fab, which constitutes the antigen-binding domain, is bound to the Fc region via the CH1 region. the heavy chain Fv fragment of the Fab is linked to the CL region of the other polypeptide, ] The polypeptide complex of

[27] The heavy chain Fv fragment of the monovalent Fab, which constitutes the T cell receptor binding domain, binds to the T cell receptor via the CH1 region. The light chain Fv fragment of the Fab is linked to one of the polypeptides constituting the Fc region, and the light chain Fv fragment of the Fab is linked to the CL region. The heavy chain Fv fragment of Fab, which constitutes the antigen-binding domain, is bound to the Fc region via the CL region. the light chain Fv fragment of the Fab is linked to the CH1 region of the other polypeptide, ] The polypeptide complex of

[28] (1) A monovalent Fab heavy chain Fv fragment that binds to an antigen is linked to the Fc domain via the CH1 domain. The light chain Fv fragment of the Fab structure is linked to one of the polypeptides constituting the CL region. an antigen-binding domain, and (2) The monovalent Fab heavy chain Fv fragment that binds to the T cell receptor complex binds to the Fc domain via the CH1 region. The light chain Fv fragment of the Fab structure is linked to the other polypeptide constituting the CL region. a bound T cell receptor complex binding domain, a heavy chain Fv fragment in the antigen-binding domain and a polypeptide complex comprising the heavy chain Fv fragment in the antigen-binding domain; The light chain Fv fragment in the Fv domain or the heavy chain Fv fragment and the T cell receptor binding domain in the T cell receptor binding domain The charges of the CH1 and CL regions are controlled so that the light chain Fv fragments in the lignin assemble together. ] The polypeptide complex of

[29] Amino acid sequence of the CH1 region linked to the heavy chain Fv fragment in the T cell receptor complex binding domain The amino acid residues in the CL region linked to the light chain Fv fragment in the antigen-binding domain are The polypeptide complex of

[28] , wherein

[30] Amino acid residues in the CH1 region linked to the heavy chain Fv fragment in the antigen-binding domain and T The amino acid residues of the CL region linked to the light chain Fv fragment in the cell receptor complex binding domain are The polypeptide complex of

[28] , wherein

[31] Amino acid sequence of the CH1 region linked to the heavy chain Fv fragment in the T cell receptor complex binding domain The amino acid residues in the CL region linked to the light chain Fv fragment in the antigen-binding domain are and the amino acid of the CH1 region linked to the heavy chain Fv fragment in the antigen-binding domain. residues and amino acids of the CL region linked to the light chain Fv fragment in the T cell receptor complex binding domain The polypeptide complex of

[28] , wherein the acid residues have the same electric charge.

[32] Amino acid sequence of the CH1 region linked to the heavy chain Fv fragment in the T cell receptor complex binding domain and amino acid residues in the CL region linked to the light chain Fv fragment in the T cell receptor binding domain. The polypeptide complex of

[29] or

[31] , wherein the groups have opposite charges.

[33] Amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen-binding domain, and The amino acid residues in the CL region linked to the light chain Fv fragment in the antigen-binding domain are of different charges. The polypeptide complex of

[30] or

[31] , comprising:

[34] The T cell receptor complex binding domain is a T cell receptor binding domain.

[22] The polypeptide complex of any one of

[33] to

[33] .

[35] The polypeptide of

[34] , wherein the T cell receptor-binding domain is a CD3-binding domain. Peptide aggregate.

[36] The amino acid residues in the CH1 region and the amino acid residues in the CL region are selected from the following (a) to (f) a group consisting of one or more pairs of amino acid residues shown in (a) the amino acid residue at position 147 (EU numbering) in the CH1 region, and the amino acid residue at position 147 in the CL region the amino acid residue at position 180 according to EU numbering; (b) the amino acid residue at position 147 (EU numbering) in the CH1 region, and the amino acid residue at position 147 in the CL region the amino acid residue at position 131 according to EU numbering (c) an amino acid residue at position 147 (EU numbering) in the CH1 region, and an amino acid residue at position 147 in the CL region the amino acid residue at position 164 in the EU numbering system (d) an amino acid residue at position 147 (EU numbering) in the CH1 region, and an amino acid residue at position 147 in the CL region the amino acid residue at position 138 in the EU numbering system (e) an amino acid residue at position 147 (EU numbering) in the CH1 region, and an amino acid residue at position 147 in the CL region the amino acid residue at position 123 in the EU numbering system (f) an amino acid residue at position 175 (EU numbering) in the CH1 region, and a CL region the amino acid residue at position 160 in the EU numbering system The amino acid residues in the CH1 region and the amino acid residues in the CL region have opposite charges to each other. The polypeptide association of either

[32] or

[33] , wherein the amino acid residues are body.

[37] Further, the amino acid sequence may be selected from the group consisting of the following amino acid residues (g): The polypeptide complex of

[36] . (g) an amino acid residue at position 213 (EU numbering) in the CH1 region, and an amino acid residue at position 213 in the CL region the amino acid residue at position 123 in the EU numbering system

[38] The amino acid residue having a different charge is either (X) or (Y) below: group of; (X) glutamic acid (E), aspartic acid (D); (Y) lysine (K), arginine (R), histidine (H);

[36] or

[37] , wherein the amino acid residue is selected from the amino acid residues contained in Merge.

[39] The amino acid residue having a different charge is an amino acid residue in the CH1 region, and The amino acid residue at position 175 is Lys, an amino acid residue in the CL region, and is an EU numbering The amino acid residues at positions 180, 131, and 160 of the amino acid sequence are all Glu. 8).

[40] The amino acid residue having a different charge is an amino acid residue in the CH1 region, and The amino acid residues at numbering positions 147 and 175 are Glu, and the amino acid residues in the CL region are EU The amino acid residues at positions 180, 131, and 160 are all Lys.

[36] The polypeptide complex of any one of

[38] to

[39] .

[41] Furthermore, the amino acid residue at position 213 (EU numbering) in the CH1 region is Glu, and the amino acid residue at position 123 (EU numbering) in the CL region is The polypeptide complex of

[40] , wherein the amino acid sequence is Lys.

[42] The Fc region is any one of FcγI, FcγIIA, FcγIIB, FcγIIIA, and / or FcγIIIB. any of [1] to

[41] , which is an Fc region having reduced binding activity to Fcγ receptors; The polypeptide complex described in any one of the above.

[43] the Fc region is selected from the group consisting of the Fc region set forth in SEQ ID NO: 23, the Fc region set forth in SEQ ID NO: 24, Amino acids constituting the Fc region of SEQ ID NO: 25 or the Fc region of SEQ ID NO: 26

[42] The Fc region according to any one of [1] to

[42] , wherein Polypeptide complex.

[44] Among the amino acids that constitute the Fc region, the following amino acids are identified according to EU numbering: Any amino acid; The amino acid sequence of positions 118 to 260 is the sequence set forth in SEQ ID NO: 24, and the amino acid sequence of positions 261 to 447 is the sequence set forth in SEQ ID NO: 25. The polypeptide of

[43] , which is an Fc region whose amino acid sequence is the sequence set forth in SEQ ID NO: 26. Do-aggregate.

[45] Among the amino acids that make up the Fc region, the following amino acids are identified according to EU numbering: Any amino acid; 220th, 226th, 229th, 231st, 232nd, 233rd, 234th, 235th, 236th, 237th, 238th, 239 240th, 264th, 265th, 266th, 267th, 269th, 270th, 295th, 296th, 297th, 298th , 299th, 300th, 325th, 327th, 328th, 329th, 330th, 331st, 332nd, The polypeptide complex of

[43] , wherein the Fc domain is mutated.

[46] Fc domain comprising the amino acid sequence set forth in SEQ ID NO: 23 mutated The polypeptide complex of

[45] , wherein the polypeptide complex is a region.

[47] Among the amino acids that constitute the Fc region, the following amino acids are identified according to EU numbering: Any amino acid; 233rd, 234th, 235th, 236th, 237th, 327th, 330th, 331st, is replaced by the corresponding amino acid in the EU numbering in the corresponding IgG2 or IgG4 The polypeptide complex of

[46] , which is an Fc domain.

[48] Among the amino acids that constitute the Fc region, the following amino acids are identified according to EU numbering: Any amino acid; 234th place, 235th place, 297th place, The polypeptide complex of

[46] , wherein the Fc domain is mutated.

[49] The amino acid at position 234 is alanine, the amino acid at position 235 is alanine, and / or The polypeptide of

[48] , wherein the amino acid at position 1 is mutated to alanine. Do-aggregate.

[50] The sequences of the two polypeptides constituting the Fc region are different from each other. The polypeptide complex of any one of

[43] to

[49] ,

[51] Amino acid residues of one of the two polypeptides constituting the Fc region The amino acid at position 349, identified according to the EU numbering system, is cysteine, and the amino acid at position 366 is cysteine. is tryptophan, and the amino acid residues of the other polypeptide are The amino acid at position 356 is identified as cysteine, the amino acid at position 366 is identified as serine, and the amino acid at position 368 is identified as cysteine. [1] characterized in that the amino acid at position 405 is mutated to alanine and the amino acid at position 407 is mutated to valine. The polypeptide complex of any one of

[50] to

[51] .

[52] Amino acid residues of one of the two polypeptides constituting the Fc region The amino acid at position 356, as specified by the EU numbering system, is lysine, and the other polypeptide Among the amino acid residues of the α-glutamyl group, the 439th amino acid, as specified by the EU numbering system, is glutamic acid. The amino acid residues of either polypeptide are mutated to amino acids according to EU numbering. [1], characterized in that the amino acid at position 435, identified by the above formula, is mutated to arginine. The polypeptide complex of any one of

[50] to

[51] .

[53] The GK sequence present at the carboxy terminus of the two polypeptides that make up the Fc region is missing. The polypeptide complex of

[51] or

[52] , wherein the polypeptide complex is missing a sequence identical to that of

[53] or

[54] .

[54] Any of [1] to

[53] , wherein the antigen-binding domains bind to the same epitope. The polypeptide complex described in any one of the above.

[55] The same epitope is present in a protein comprising the amino acid sequence set forth in SEQ ID NO: 2. The polypeptide complex of

[54] , present in

[56] The same epitope is present in a protein comprising the amino acid sequence set forth in SEQ ID NO: 4. The polypeptide complex of

[54] , present in

[57] [1] to

[53] , each of which has an antigen-binding domain that binds to a different epitope. The polypeptide complex of any one of the preceding claims.

[58] A protein in which different epitopes are present, the protein comprising the amino acid sequence set forth in SEQ ID NO: 2. The polypeptide complex of

[57] , present in

[59] A protein in which different epitopes consist of the amino acid sequence set forth in SEQ ID NO: 4. The polypeptide complex of

[57] , present in

[60] A polypeptide encoding the polypeptide complex of any one of [1] to

[59] . nucleotide.

[61] A vector comprising the polynucleotide according to

[60] .

[62] A cell harboring the vector described in

[61] .

[63] Culturing the cell of

[62] and collecting the polypeptide complex from the culture supernatant. and (b) producing a polypeptide complex comprising:

[64] A pharmaceutical composition comprising the polypeptide complex of any one of [1] to

[59] as an active ingredient. A cytotoxicity-inducing therapeutic agent comprising:

[65] The therapeutic agent according to

[64] , wherein the cytotoxicity-inducing therapeutic agent is a cancer therapeutic agent.

[66] The therapeutic agent according to

[65] , wherein the cancer is liver cancer or lung cancer.

[67] A method for treating a patient in need of treatment with the polypeptide complex of any one of [1] to

[59] . A method for treating or preventing cancer, comprising administering to a subject a compound according to the present invention.

[68] The method for treating or preventing cancer according to

[67] , wherein the cancer is liver cancer or lung cancer.

[0013] The present invention also provides polypeptide complexes of the present invention or polypeptide complexes produced by the production methods of the present invention. The present invention also relates to kits for use in the methods of the present invention, which contain polypeptide complexes. a polypeptide complex of the present invention or a polypeptide produced by a production method of the present invention The present invention also relates to the use of the aggregate in the production of a therapeutic agent for inducing cytotoxicity. a polypeptide complex of the present invention or a polypeptide complex produced by a production method of the present invention for use in a method for producing a polypeptide complex of the present invention; The present invention relates to a polypeptide complex. [Effects of the Invention]

[0014] The present invention demonstrates the potent antitumor activity of BiTE and its ability to induce cytokine storage independent of cancer antigens. It maintains its excellent safety profile by not inducing steroids, etc., and has a long half-life in the blood. Novel polypeptide complexes are provided. Antigen binding in the polypeptide complexes of the present invention By substituting the domain, a cytotoxic agent containing the polypeptide complex as an active ingredient can be obtained. The induced therapeutic agent targets various cells, including cancer cells, causing cytotoxicity and treating various cancers. For patients, it is not only safe but also reduces the physical burden. This will enable desirable treatment that is both cost-effective and convenient. [Brief explanation of the drawings]

[0015] [Figure 1] 1 is a graph comparing the cytotoxic activity of GPC3 ERY1 (GPC3 BiTE), GPC3 ERY2, and an IgG-type GPC3 antibody. The black squares (■) represent the cytotoxic activity of GPC3 ERY1 (GPC3 BiTE), the black triangles (▲) represent the cytotoxic activity of GPC3 ERY2, and the white squares (□) represent the cytotoxic activity of an IgG-type GPC3 antibody. [Figure 2] 1 is a graph showing a comparison of the cytotoxic activities of GPC3 BiTE and GPC3 ERY5. Black squares (■) represent the cytotoxic activity of GPC3 BiTE, and white circles (○) represent the cytotoxic activity of GPC3 ERY5. [Figure 3] 1 is a graph showing a comparison of the cytotoxic activities of GPC3 BiTE and GPC3 ERY6. Black squares (■) represent the cytotoxic activity of GPC3 BiTE, and black triangles (▲) represent the cytotoxic activity of GPC3 ERY6. [Figure 4] 1 is a graph showing a comparison of the cytotoxic activities of GPC3 BiTE and GPC3 ERY7. Black squares (■) represent the cytotoxic activity of GPC3 BiTE, and black diamonds (◆) represent the cytotoxic activity of GPC3 ERY7. [Figure 5] 1 is a graph comparing the cytotoxic activities of GPC3 BiTE, GPC3 ERY8-2, GPC3 ERY9-1, and GPC3 ERY10-1. The closed squares (■) represent the cytotoxic activities of GPC3 BiTE, the closed triangles (▲) represent the cytotoxic activities of GPC3 ERY8-2, the open circles (○) represent the cytotoxic activities of GPC3 ERY9-1, and the open squares (□) represent the cytotoxic activities of GPC3 ERY10-1. [Figure 6] This is a graph showing the in vivo antitumor effect of GPC3 ERY8-2 in a PC-10 pre-mix model. Open squares (□) represent changes in tumor volume in the GPC3 ERY7-administered group. Closed diamonds (◆) represent changes in tumor volume in the control group (administered PBS). [Figure 7] Figure 1 shows a graph depicting the in vivo antitumor effect of GPC3 ERY10-1 in a PC-10 pre-mix model. Open squares (□) represent changes in tumor volume in the GPC3 ERY10-1 administration group. Closed diamonds (◆) represent changes in tumor volume in the control group (PBS administration). [Figure 8] Figure 1 shows a graph depicting the in vivo antitumor effect of GPC3 ERY10-1 in a PC-10 T cell transfer model. Open squares (□) represent changes in tumor volume in the GPC3 ERY10-1 administration group. Closed diamonds (◆) represent changes in tumor volume in the control group (PBS administration). [Figure 9] 1 is a graph showing the time courses of plasma concentrations of GPC3 ERY9-1 and GPC3 ERY10-1 measured using GPC3-expressing Ba / F3 cells. Black diamonds (◆) represent the time courses of GPC3 ERY9-1 and white squares (□) represent the time courses of GPC3 ERY10-1. [Figure 10] 1 is a graph showing the time courses of plasma concentrations of GPC3 ERY9-1 and GPC3 ERY10-1 measured using CD3-expressing Ba / F3 cells. Black diamonds (◆) represent the time courses of plasma concentrations of GPC3 ERY9-1, and white squares (□) represent the time courses of plasma concentrations of GPC3 ERY10-1. [Figure 11] Fig. 10 is a graph showing an evaluation of the cancer antigen-independent cytokine induction ability of GPC3 BiTE, GPC3 ERY9-1, GPC3 ERY10-1, GPC3 ERY15-1, and catumaxomab. [Figure 12] Figure 1 shows a graph showing the in vitro cytotoxic activity of GPC3 ERY18 L1, GPC3 ERY18L2, GPC3 ERY18L3, GPC3 ERY18L4, and GPC3 ERY18S1. Black triangles (▲) represent the cytotoxic activity of GPC3 ERY18 L1, black circles (●) represent GPC3 ERY18 L2, black squares (■) represent GPC3 ERY18 L3, open squares (□) represent GPC3 ERY18 L4, and open diamonds (◇) represent GPC3 ERY18 S1. [Figure 13] 1 is a graph comparing the in vitro cytotoxic activity of GPC3 ERY18 L3 and GPC3 ERY10-1. Black squares (■) represent the cytotoxic activity of GPC3 ERY18 L3, and white squares (□) represent the cytotoxic activity of GPC3 ERY10-1. [Figure 14] 1 is a graph comparing the in vitro cytotoxic activity of GPC3 ERY19-3 and GPC3 BiTE. Open squares (□) represent the cytotoxic activity of GPC3 ERY19-3, and closed squares (■) represent the cytotoxic activity of GPC3 BiTE. [Figure 15] A. Chromatograms showing the results of size-exclusion chromatography analysis of CM expressing NTA1L / NTA1R / GC33-k0. B. Chromatograms showing the results of size-exclusion chromatography analysis of CM expressing NTA2L / NTA2R / GC33-k0. [Figure 16]FIG. 1 shows the domains constituting the polypeptide complexes 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 described in the Examples herein. Domains represented by crossed lines represent heavy-chain variable regions of anti-cancer antigen (GPC3, EpCAM, EGFR) antibodies; domains represented by diagonal lines represent light-chain variable regions of anti-cancer antigen (GPC3, EpCAM, EGFR) antibodies; domains represented by dotted lines represent heavy-chain variable regions of anti-CD3 antibodies; domains represented by solid black represent light-chain variable regions of anti-CD3 antibodies; domains represented by open white represent antibody constant regions; crosses represent silent Fc mutations; and asterisks represent mutations that allow heterotypic Fc assembly. [Figure 17] A: Schematic diagram of GPC3 BiTE, B: Schematic diagram of GPC3 ERY10, C: Schematic diagram of GPC3 ERY2, D: Schematic diagram of GPC3 ERY5, E: Schematic diagram of GPC3 ERY6, F: Schematic diagram of GPC3 ERY7, G: Schematic diagram of GPC3 ERY8-2, H: Schematic diagram of GPC3 ERY9-1, I: Schematic diagram of GPC3 ERY10-1, J: Schematic diagram of GPC3 ERY15, K: Schematic diagram of GPC3 ERY18, and L: Schematic diagram of GPC3 ERY19-3. [Figure 18] The relationship between the amino acid residues constituting the Fc regions of IgG1, IgG2, IgG3, and IgG4 and the EU numbering system of Kabat (also referred to as EU INDEX herein) is shown. [Figure 19]FIG. 1 shows the domains constituting the polypeptide complexes GPC3 ERY17-2, GPC3 ERY17-3, EpCAM ERY17-2, and EpCAM ERY17-3 described in the Examples of the present specification. Domains represented by crossed lines represent heavy-chain variable regions of anti-cancer antigen (GPC3, EpCAM, EGFR) antibodies; domains represented by diagonal lines represent light-chain variable regions of anti-cancer antigen (GPC3, EpCAM, EGFR) antibodies; domains represented by dotted lines represent heavy-chain variable regions of anti-CD3 antibodies; domains represented by solid black represent light-chain variable regions of anti-CD3 antibodies; domains represented by open white represent antibody constant regions; crosses represent silent Fc mutations; and asterisks represent mutations that allow hetero-Fc aggregation. [Figure 20] 1 is a graph comparing the cytotoxic activities of GPC3 BiTE, GPC3 ERY17-2, GPC3 ERY17-3, and GPC3 ERY10-1. Black squares (■) represent the cytotoxic activity of GPC3 BiTE, black triangles (▲) represent the cytotoxic activity of GPC3 ERY17-2, white circles (○) represent the cytotoxic activity of GPC3 ERY17-3, and white squares (□) represent the cytotoxic activity of GPC3 ERY10-1. [Figure 21] Figure 1 shows a graph depicting the in vivo antitumor effect of GPC3 ERY17-2 in a PC-10 T cell transfer model. Open squares (□) represent changes in tumor volume in the GPC3 ERY17-2 administration group. Closed diamonds (◆) represent changes in tumor volume in the control group (PBS administration). [Figure 22] 1 is a graph comparing the cytotoxic activities of GPC3 ERY17-2 and GPC3 ERY17-2-M20. Black triangles (▲) and white circles (○) represent the cytotoxic activity of GPC3 ERY17-2 and GPC3 ERY17-2-M20, respectively. [Figure 23] 1 is a graph showing a comparison of the cytotoxic activity of EpCAM ERY17-2 and EpCAM ERY17-3. Black triangles (▲) and white squares (□) represent the cytotoxic activity of EpCAM ERY17-2 and EpCAM ERY17-3, respectively. [Figure 24]Figure 1 shows the domains constituting the polypeptide complexes GM1, GM2, and GM0 described in the Examples of the present specification. A denotes a polypeptide complex containing both a regulated CH1 / CL interface association and a Knob-into-Hole (KiH) modification, while B denotes a polypeptide complex without either regulated CH1 / CL interface association or KiH modification. The domains indicated by crossed lines are the heavy-chain variable region of an anti-cancer antigen (GPC3, EpCAM) antibody, the domains indicated by diagonal lines are the light-chain variable region of an anti-cancer antigen (GPC3, EpCAM) antibody, the domains indicated by dotted lines are the heavy-chain variable region of an anti-CD3 antibody, the domains indicated by black solid lines are the light-chain variable region of an anti-CD3 antibody, the domains indicated by white solid lines are the antibody constant regions, crosses represent silent Fc mutations, asterisks represent mutations that induce heterotypic Fc association, and hollow circles represent mutations that regulate CH1 / CL interface association. [Figure 25] This is a graph showing a comparison of the cytotoxic activity of GM1, GM2, and GM0. Black triangles (▲) represent the cytotoxic activity of GM1, white squares (□) represent the cytotoxic activity of GM2, and white circles (○) represent the cytotoxic activity of GM0. [Figure 26] 1 is a graph showing the cytotoxic activity of EGFR ERY17-2. Black triangles (▲) represent the cytotoxic activity of EGFR ERY17-2. DETAILED DESCRIPTION OF THE INVENTION

[0016] The following definitions are provided to facilitate understanding of the invention described herein. .

[0017] antibody As used herein, antibodies are antibodies that may be natural or partially or wholly synthetic. Antibodies are immunoglobulins produced from natural sources such as plasma and serum in which they occur. The antibody can be isolated from the culture supernatant of hybridoma cells that produce the antibody, or can be isolated from a genetically engineered Antibodies can be partially or completely synthesized using techniques such as recombinant DNA technology. Immunoglobulin isotypes and their isotype subclasses are preferably mentioned. Human immunoglobulins include IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, and IgA1. The antibodies of the present invention contain nine classes (isotypes): IgE, IgM, and IgM. Isotypes may include IgG1, IgG2, IgG3, and IgG4.

[0018] Methods for producing antibodies with desired binding activity are known to those skilled in the art. GPC3, which belongs to the I-anchored receptor family (Int J Cancer. (2003) 103 (4), 455-65 For example, a method for producing an antibody (anti-GPC3 antibody) that binds to an antigen other than GPC3 is used. Such antibodies can also be prepared appropriately according to the examples below.

[0019] Anti-GPC3 antibodies are prepared as polyclonal or monoclonal antibodies using known methods. As the anti-GPC3 antibody, a monoclonal antibody derived from a mammal is preferably produced. Mammalian-derived monoclonal antibodies include those produced by hybridomas, and host cells transformed by genetic engineering techniques with an expression vector containing an antibody gene. This includes those produced by the

[0020] Monoclonal antibody-producing hybridomas can be isolated by known techniques, e.g. It can be prepared as follows: GPC3 protein is used as a sensitizing antigen. The mammal is immunized according to the immunization method described above. The obtained immune cells are then subjected to a conventional cell fusion method. The clones are then fused with known parent cells. Hybrids producing anti-GPC3 antibodies were isolated by screening for null antibody-producing cells. A theme can be selected.

[0021] Specifically, monoclonal antibodies are produced, for example, as follows. GPC3, the nucleotide sequence of which is disclosed in fSeq accession number NM_001164617.1 (SEQ ID NO: 1). The gene is expressed and used as a sensitizing antigen for antibody production (RefSeq accession number NP). The GPC3 protein represented by SEQ ID NO: 2 can be obtained. The gene sequence encoding C3 is inserted into a known expression vector to produce the gene in a suitable host cell. The desired human GPC3 protein is then extracted from the host cells or the culture supernatant. To obtain soluble GPC3 from the culture supernatant, for example, a soluble GPC3 having the sequence Among the GPC3 polypeptide sequences represented by No. 2, the sequence used for anchoring GPC3 on the cell membrane is The amino acids 564-580 constituting the hydrophobic region corresponding to the GPI anchor sequence were deleted. The protein is expressed in place of the GPC3 protein shown in SEQ ID NO: 2. Naturally occurring GPC3 protein can also be used as a sensitizing antigen.

[0022] The purified GPC3 protein can be used as a sensitizing antigen for immunization of mammals. A partial peptide of GPC3 can also be used as a sensitizing antigen. It can also be obtained by chemical synthesis from the amino acid sequence of GPC3. Alternatively, it can be obtained by incorporating it into a vector and expressing it. It can also be obtained by degrading the GPC3 protein using an enzyme, but partial peptides The region and size of the GPC3 peptide used as a target protein are not particularly limited to a particular embodiment. The region is an amino acid sequence corresponding to amino acids 564-580 in the amino acid sequence of SEQ ID NO: 2. Any sequence can be selected from the above. The number of amino acids constituting the peptide to be used as a sensitizing antigen is small. It is preferable that both of them are 5 or more, for example, 6 or more, or 7 or more. More specifically, it is 8 to 50, preferably Preferably, a peptide of 10 to 30 residues can be used as a sensitizing antigen.

[0023] In addition, a desired partial polypeptide or peptide of the GPC3 protein can be fused with a different polypeptide. The fusion protein can be used as a sensitizing antigen. For example, Fc fragments of antibodies and peptide tags are preferably used to produce proteins. The vector expressing the fusion protein contains two or more desired polypeptides. The genes encoding the fragments are fused in frame, and the fused gene is expressed as described above. Fusion proteins can be produced by inserting them into a vector. Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58(1989 ) Cold Spring Harbor Lab. Press). GPC3 used as a sensitizing antigen The method for obtaining the above and the immunization method using the same are described in WO2003 / 000883, WO2004 / 022754, WO2006 / 00669 3 is also specifically described.

[0024] The mammals to be immunized with the sensitizing antigen are not limited to specific animals, but include: It is preferable to select the cells in consideration of their compatibility with the parent cells used in cell fusion. Rodents, such as mice, rats, hamsters, rabbits, monkeys, etc., are preferably used. It is used.

[0025] The above animals are immunized with a sensitizing antigen according to a known method. As a method, a sensitizing antigen is administered to a mammal by intraperitoneal or subcutaneous injection. Immunization is carried out. Specifically, PBS (Phosphate-Buffered Saline) or physiological saline is used appropriately. The sensitizing antigen diluted at an appropriate dilution ratio is mixed with a conventional adjuvant, such as furoin, if desired. After being mixed and emulsified with a complete adjuvant, the sensitizing antigen is administered to the mammal every 4 to 21 days. In addition, a suitable carrier can be used when immunizing with a sensitizing antigen. When small partial peptides of the above are used as sensitizing antigens, albumin, keyhole lipase, Immunization with the sensitizing antigen peptide bound to a carrier protein such as amputee hemocyanin. may be desirable.

[0026] Alternatively, hybridomas producing the desired antibodies can be isolated using DNA immunization as follows: DNA immunization is the process of expressing a gene encoding an antigen protein in an immunized animal. In the immunized animal to which the vector DNA constructed in such a manner as to be able to be administered, the sensitizing antigen The method of immunization is characterized in that the expression of the antibody in vivo provides immune stimulation to the immunized animal. Compared with the general immunization method in which protein antigens are administered to immunized animals, DNA immunization has the following advantages: Such advantages are expected. -Maintaining the structure of membrane proteins such as GPC3 can provide immune stimulation -No need to purify the immunogen

[0027] To obtain the monoclonal antibody of the present invention by DNA immunization, first, GPC3 protein is The DNA encoding GPC3 is then administered to the immunized animal. The resulting DNA is inserted into an appropriate expression vector and administered to an animal to be immunized. As the expression vector, a commercially available expression vector such as pcDNA3.1 can be suitably used. A commonly used method can be used to administer the vector to the living body. For example, gold particles carrying an expression vector can be injected into the cells of an animal immunized with a gene gun. DNA immunization is achieved by introducing GPC3 into the host. It can also be produced using the method described in International Publication WO2003 / 104453.

[0028] In this way, mammals were immunized and an increase in antibody titers that bind to GPC3 was confirmed in the serum. After the incubation, immune cells are collected from the mammal and subjected to cell fusion. As the cell, in particular, splenocytes can be used.

[0029] Mammalian myeloma cells are used as the cells to be fused with the immune cells. Preferably, the roma cells are equipped with an appropriate selectable marker for screening. A selectable marker is a trait that allows a plant to survive (or not survive) under specific culture conditions. The selectable marker is a hypoxanthine-guanine-phosphoribosyltransferase deficiency gene. deficiency (hereafter abbreviated as HGPRT deficiency), or thymidine kinase deficiency (hereafter abbreviated as TK deficiency). It is known that cells lacking HGPRT or TK are hypoxanthine-aminopeptide-dependent. HAT-sensitive cells are HAT-selected. In selective medium, they cannot synthesize DNA and die, but when they fuse with normal cells, they They can continue to synthesize DNA using the salvage cycle, allowing them to grow even in HAT selective medium. You will start to do this.

[0030] HGPRT-deficient and TK-deficient cells express 6-thioguanine and 8-azaguanine (hereafter abbreviated as 8AG), respectively. These pyrimidines can be selected on a medium containing 5' bromodeoxyuridine or 5' bromodeoxyuridine. Normal cells that incorporate pyrimidine analogs into their DNA die. Cells lacking these enzymes, which cannot incorporate the log, are unable to survive in selective media. Another selectable marker called G418 resistance is the neomycin resistance gene. Conferring resistance to oxystreptamine antibiotics (gentamicin analogues). Various suitable myeloma cells are known.

[0031] Such myeloma cells include, for example, P3 (P3x63Ag8.653) (J. Immunol. (1979) )123 (4), 1548-1550), P3x63Ag8U.1(Current Topics in Microbiology and Immunolo gy(1978)81, 1-7), NS-1(C. Eur. J. Immunol.(1976)6 (7), 511-519), MPC-11( Cell(1976)8 (3), 405-415), SP2 / 0(Nature(1978)276 (5685), 269-270), FO(J. Immunol. Methods(1980)35 (1-2), 1-21), S194 / 5.XX0.BU.1(J. Exp. Med.(1978) 148 (1), 313-323), R210 (Nature (1979) 277 (5692), 131-133), etc. are preferably used. obtain.

[0032] Basically, the method is carried out by known methods, for example, the method of Kohler and Milstein et al. (Methods Enzyme (1981) 73, 3-46) and the like, cell fusion between the immune cells and myeloma cells was carried out. do. More specifically, the cell fusion can be carried out in a normal nutrient culture medium in the presence of a cell fusion promoter. Fusion promoters include, for example, polyethylene glycol (PEG), sen Divirus (HVJ) or the like is used, and dimethylsulfoxide may be used if desired to further increase the fusion efficiency. When used, an adjuvant such as sulfoxide is added.

[0033] The ratio of immune cells to myeloma cells can be set arbitrarily. It is preferable that the number of immune cells is 1 to 10 times that of cells. Examples of suitable medium for growing the myeloma cell line include RPMI1640 culture medium, MEM culture medium, and the like. In addition, the usual culture medium used for this type of cell culture is used, and fetal calf serum (FCS) ) or other serum replacement fluids may be suitably added.

[0034] The cell fusion is carried out by thoroughly mixing predetermined amounts of the immune cells and myeloma cells in the culture medium; PEG solution (average molecular weight: 1000 to 6000) preheated to about 37°C is usually added in 30 to 6 The mixture is gently mixed to obtain the desired fused cells. Then, the appropriate culture medium listed above is added sequentially to form hybridoma cells. By repeating the procedure of centrifugation and removal of the supernatant, the cells that are not favorable for hybridoma growth can be isolated. Unwanted cell fusion agents and the like can be removed.

[0035] The hybridomas thus obtained are cultured in a conventional selective culture medium, such as HAT culture medium (HAT medium). Selection by culturing in a medium containing thrombin, aminopterin, and thymidine The time required for the cells other than the desired hybridoma (non-fused cells) to die ( (Usually, this period is sufficient, from several days to several weeks.) Cultivation using the above HAT culture medium is continued. Then, hybridomas producing the desired antibodies are screened by the conventional limiting dilution method. Removal and single cloning are performed.

[0036] The hybridomas thus obtained contain the same nucleotide sequence as the myelomas used in the cell fusion. Selection can be achieved by using a selective medium depending on the selectable marker. For example, HGPRT and T K-deficient cells were grown in HAT medium (containing hypoxanthine, aminopterin, and thymidine). In other words, HAT-sensitive myeloma cells can be selected by culturing them in a medium containing When cells are used for cell fusion, cells that have successfully fused with normal cells are selected in the HAT culture medium. The cells other than the desired hybridoma (non-fused cells) can be selectively grown. The culture is continued in the HAT medium for a suitable period of time. Specifically, the period is generally from several days to several weeks. The desired hybridomas can then be selected by culturing for a period of time. Therefore, screening and single cloning of hybridomas producing the desired antibodies is performed. can be implemented.

[0037] Screening and single cloning of desired antibodies is based on known antigen-antibody reactions. This can be suitably carried out by a screening method. For example, a monoclonal antibody that binds to GPC3 can be used. Such monoclonal antibodies can bind to GPC3 expressed on the cell surface. Antibodies can be screened, for example, by FACS (fluorescence activated cell sorting). FACS analyzes cells that have been brought into contact with fluorescent antibodies using laser light, allowing individual cells to be identified. Allows measurement of antibody binding to cell surfaces by measuring the emitted fluorescence It is a system.

[0038] Screening hybridomas producing the monoclonal antibodies of the present invention by FACS To screen for GPC3, first prepare cells expressing GPC3. The cells were mammalian cells in which GPC3 was forcibly expressed. By using mammalian cells without GPC3 as a control, the expression of antibodies against GPC3 on the cell surface was confirmed. The binding activity can be selectively detected. That is, the binding activity does not bind to host cells, but to GPC3-expressing cells. By selecting hybridomas that produce antibodies that bind to GPC3, Hybridomas producing the antibodies can be obtained.

[0039] Alternatively, the binding activity of antibodies to immobilized GPC3-expressing cells can be evaluated based on the principles of ELISA. For example, GPC3-expressing cells can be immobilized on the wells of an ELISA plate. The culture supernatant of the lysoma was brought into contact with the immobilized cells in the wells, and antibodies that bind to the immobilized cells were detected. If the monoclonal antibody is of mouse origin, the antibody bound to the cells will be anti-mouse antibody. These screening tests can detect immunoglobulin antibodies. Hybridomas that produce the desired antibodies capable of binding to the antigen can be isolated by limiting dilution or the like. It can be cloned by

[0040] The hybridomas producing the monoclonal antibodies thus produced are cultured in a conventional manner. The hybridomas can be subcultured in liquid nitrogen. It is possible.

[0041] The hybridoma is cultured according to a conventional method, and the desired monoclonal antibody is isolated from the culture supernatant. Alternatively, the hybridoma can be administered to a compatible mammal to obtain a human antibody. The former method allows the production of highly purified monoclonal antibodies. The antibody is suitable for obtaining the above antibody.

[0042] The antibody gene is cloned from the antibody-producing cell such as the hybridoma. The cloned antibody gene can be incorporated into an appropriate vector. By incorporating the gene into a host and introducing it into the host, the antibody encoded by the gene is expressed. Methods for isolating antibody genes, introducing them into vectors, and transforming host cells are described, for example, in For example, it has already been established by Vandamme et al. (Eur. J. Biochem. (1990) 192 (3), 767- 775). Methods for producing recombinant antibodies are also known, as described below.

[0043] For example, the variable region (V) of the anti-GPC3 antibody can be isolated from hybridoma cells that produce the anti-GPC3 antibody. To do this, the entire RNA is first extracted from the hybridoma. A is extracted. As a method for extracting mRNA from cells, for example, the following method is used. It can be used. -Guanidine ultracentrifugation (Biochemistry (1979) 18 (24), 5294-5299) -AGPC method (Anal. Biochem. (1987) 162 (1), 156-159)

[0044] The extracted mRNA was purified using an mRNA Purification Kit (GE Healthcare Biosciences). Alternatively, the mRNA can be purified using the QuickPrep mRNA Purification Kit (GE Healthcare Biosciences). Kits for extracting total mRNA directly from cells are also commercially available, such as those manufactured by Biosciences. Using such a kit, mRNA can be obtained from hybridomas. cDNA encoding the antibody V region can be synthesized from the mRNA using reverse transcriptase. Using a Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation), etc. Alternatively, the SMART RACE cDNA Amplification Kit ( Clontech) and the 5'-RACE method using PCR (Proc. Natl. Acad. Sci. USA (1988) 85 (2 3), 8998-9002, Nucleic Acids Res. (1989) 17 (8), 2919-2932) can be used as appropriate. Furthermore, during the synthesis of such cDNA, appropriate restriction enzyme sites (described later) are inserted at both ends of the cDNA. It can be introduced.

[0045] The desired cDNA fragment is purified from the resulting PCR product and then ligated with vector DNA. In this way, a recombinant vector is created, introduced into E. coli, etc., and colonies are selected. The desired recombinant vector can be prepared from the colonized E. coli. Whether or not the recombinant vector contains the desired cDNA base sequence can be determined by known methods, e.g. For example, it can be confirmed by the dideoxynucleotide chain termination method.

[0046] To obtain the gene encoding the variable region, primers for amplifying the variable region gene are used. It is easy to use the 5'-RACE method using the cDNA is synthesized using RNA as a template, and a 5'-RACE cDNA library is obtained. A library can be synthesized using a commercially available kit such as the SMART RACE cDNA Amplification Kit. do.

[0047] The antibody gene was amplified by PCR using the obtained 5'-RACE cDNA library as a template. Primers for amplifying mouse antibody genes are designed based on known antibody gene sequences. These primers have different base sequences for each immunoglobulin subclass. Therefore, the subclasses were determined in advance by Iso Strip mouse monoclonal antibody isotypes. Determine this using a commercially available kit such as the ELISA Kit (Roche Diagnostics) It is desirable.

[0048] Specifically, when the objective is to obtain a gene encoding mouse IgG, It is possible to amplify genes encoding γ1, γ2a, γ2b, and γ3 as chains, and κ and λ as light chains. To amplify the IgG variable region gene, a suitable primer can be used. The primers used are those that anneal to the constant region close to the variable region. On the other hand, the 5' primer is the one included in the 5' RACE cDNA library construction kit. A primer is used.

[0049] The amplified PCR products are used to generate immunoglobulins consisting of a combination of heavy and light chains. The binding activity of the reconstituted immunoglobulin to GPC3 is used as an index. For example, in order to obtain an antibody against GPC3, In this case, it is more preferable that the antibody binds to GPC3 specifically. Antibodies that possess the antibody can be screened, for example, as follows: (1) Antibodies containing V regions encoded by cDNA obtained from hybridomas expressing GPC3 contacting the cells with the (2) detecting the binding of the antibody to GPC3-expressing cells; and (3) A step of selecting an antibody that binds to GPC3-expressing cells.

[0050] Methods for detecting the binding of an antibody to GPC3-expressing cells are known. The binding of the antibody to GPC3-expressing cells can be detected by techniques such as CS. For the evaluation, fixed specimens of GPC3-expressing cells can be appropriately used.

[0051] As a method for screening antibodies using binding activity as an index, a phage vector was used. Panning is also suitable. When the subclasses of the nucleotides and light chains are obtained as libraries, they are expressed using phage vectors. The genes encoding the heavy and light chain variable regions are preferably By linking the Fvs with a suitable linker sequence, single-chain Fv (scFv) can be formed. By inserting the gene encoding the scFv into a phage vector, the scFv can be attached to the surface. After contacting the phage with the desired antigen, the phage can be obtained. The combined phages are collected to obtain DNA encoding scFv with the desired binding activity. By repeating this procedure as necessary, a protein having the desired binding activity can be recovered. The scFv can be concentrated.

[0052] After obtaining the cDNA encoding the V region of the target anti-GPC3 antibody, The cDNA is digested with a restriction enzyme that recognizes the inserted restriction enzyme site. The enzyme recognizes and digests base sequences that appear infrequently in the base sequences that make up antibody genes. Furthermore, to insert one copy of the digested fragment into the vector in the correct orientation, it is necessary to provide sticky ends. It is preferable to insert a restriction enzyme capable of cleaving the V region of the anti-GPC3 antibody digested as described above. An antibody expression vector can be obtained by inserting the cDNA into an appropriate expression vector. At this time, the gene encoding the antibody constant region (C region) and the gene encoding the V region are If the antibody is fused in-frame, a chimeric antibody is obtained. The origin of the constant region and variable region is different. In addition to chimeric antibodies, human-human allogeneic chimeric antibodies are also included in the chimeric antibodies of the present invention. The V region gene is inserted into an expression vector that already contains a constant region. A vector for expressing a human antibody can be constructed. Specifically, for example, a vector encoding a desired antibody constant region (C The V region gene is inserted into the 5' end of an expression vector carrying DNA encoding the V region The restriction enzyme recognition sequences of the restriction enzymes can be appropriately arranged. The two are fused in frame to construct a chimeric antibody expression vector. .

[0053] To produce anti-GPC3 monoclonal antibodies, the antibody gene is regulated by the expression control region. The expression control region for expressing the antibody is incorporated into an expression vector so that it can be expressed under The expression vector includes, for example, an enhancer and a promoter. An appropriate signal sequence can be added to the amino terminus so that the desired signal sequence can be obtained. has the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 72) as a signal sequence Although peptides have been used, other suitable signal sequences may be added. The resulting polypeptide is truncated at the carboxyl terminal portion of the sequence, and the truncated polypeptide The mature polypeptide can then be secreted outside the cell. By transforming an appropriate host cell, a recombinant vector capable of expressing DNA encoding an anti-GPC3 antibody can be produced. Recombinant cells can be obtained.

[0054] For the expression of antibody genes, DNA encoding the antibody heavy chain (H chain) and light chain (L chain) is Each is incorporated into a separate expression vector. By co-transfecting the same host cell, Alternatively, the DNA encoding the heavy and light chains may be expressed in a single expression vector. host cells can be transformed by incorporating the vector into the vector (see International Publication WO 94 / 11523). (This refers to

[0055] A host for producing antibodies by introducing the isolated antibody genes into a suitable host. Many combinations of cells and expression vectors are known. This method can be applied to isolate specific antigen-binding domains and CD3-binding domains. When cells are used, animal cells, plant cells, or fungal cells can be used as appropriate. Specifically, examples of animal cells include the following cells: (1) Mammalian cells: CHO, COS, myeloma, BHK (baby hamster kidney), Hela, Vero etc. (2) Amphibian cells: Xenopus oocytes, etc. (3) Insect cells: sf9, sf21, Tn5, etc.

[0056] Alternatively, plant cells such as Nicotiana tabacum may be used. An antibody gene expression system using cells derived from the genus Nicotiana is known. For cell transformation, callus cultured cells can be appropriately used.

[0057] Furthermore, the following fungal cells can be used: - Yeast: Saccharomyces such as Saccharomyces cerevisiae Saccharomyces genus, Pichia genus such as Pichia pastoris -Filamentous fungi: Aspergillus, such as Aspergillus niger gillus) genus

[0058] Furthermore, antibody gene expression systems using prokaryotic cells are also known. When the cells are used, bacterial cells such as E. coli and Bacillus subtilis can be appropriately used. An expression vector containing the desired antibody gene is introduced into the cells by transformation. The transformed cells are cultured in vitro to produce the desired product from the culture of the transformed cells. Antibodies can be obtained.

[0059] In addition to the above host cells, transgenic animals can also be used to produce recombinant antibodies. That is, the antibody can be obtained from an animal into which a gene encoding the desired antibody has been introduced. For example, antibody genes are genes that encode proteins that are specifically produced in milk. It can be constructed as a fusion gene by inserting it in-frame within a gene. For example, goat β-casein can be used as a protein secreted into the body. The DNA fragment containing the inserted fusion gene is injected into a goat embryo, and the injected embryo The embryo is transferred to a female goat. The transgenic goat (or goat) born from the recipient goat is From the milk produced by the mouse (or its progeny), the desired antibody is synthesized as a fusion protein with a milk protein. In addition, milk containing the desired antibody produced from the transgenic goat can be obtained. Hormones can be administered to transgenic goats to increase the amount of Technology (1994), 12 (7), 699-702).

[0060] When the polypeptide complexes described herein are administered to humans, the complexes The purpose of this is to reduce heterologous antigenicity to humans as an antigen-binding domain in Therefore, an antigen-binding domain derived from an artificially modified recombinant antibody can be appropriately used. Genetically recombinant antibodies include, for example, humanized antibodies. The mutant antibody is suitably produced using known methods.

[0061] Producing the antigen-binding domain of the polypeptide complex described herein The variable region of an antibody used for this purpose is usually surrounded by four framework regions (FR). It consists of three complementarity-determining regions (CDRs). The CDRs are essentially the regions that determine the binding specificity of the antibody. On the other hand, the amino acid sequences that make up the FR are highly diverse among antibodies with different binding specificities. However, they often show high identity. Therefore, in general, CDR grafting can be used to It is believed that the binding specificity of the antibody can be transferred to other antibodies.

[0062] Humanized antibodies are also called reshaped human antibodies. For example, humanized antibodies in which the CDRs of a mouse antibody are grafted onto a human antibody are known. General genetic recombination techniques for obtaining antibody clones are also known. As a method for grafting CDRs of a target gene to human FRs, for example, overlap extension PCR is known. In overlap extension PCR, the primers for synthesizing the FR of the human antibody are The base sequence encoding the CDR of the mouse antibody to be grafted is added to the primer. Generally, in grafting mouse CDRs to human FRs, Selecting human FRs that are highly identical to mouse FRs is advantageous in maintaining CDR function. In other words, the amino acid sequence of the FR adjacent to the mouse CDR to be transplanted is generally It is preferable to use a human FR consisting of an amino acid sequence highly identical to the sequence.

[0063] The nucleotide sequences to be linked are designed to be connected in-frame with each other. Each primer synthesizes a human FR individually, resulting in the addition of mouse CDRs to each FR. The resulting product contains the DNA encoding the mouse CDR. The human antibody genes are then templated. The overlapping CDR portions of the synthesized products are annealed to each other to form complementary strands. This reaction links the human FRs to the mouse CDRs via their sequences.

[0064] The V region gene, in which three CDRs and four FRs are finally linked, has annealing sites at its 5' and 3' ends. The full length of the fragment is amplified using primers containing appropriate restriction enzyme recognition sequences. The DNA obtained as described above is fused in frame with DNA encoding the C region of a human antibody. By inserting the fragment into an expression vector as described above, a vector for expressing a human antibody can be prepared. After the integration vector is introduced into a host to establish a recombinant cell, the recombinant cell is cultured. and expressing the DNA encoding the humanized antibody, thereby producing the humanized antibody in the cultured cells. (See European Patent Publication EP 239400 and International Publication WO1996 / 002576).

[0065] The antigen-binding activity of the humanized antibody prepared as described above is qualitatively or quantitatively measured, By evaluating whether or not the CDRs form a good antigen-binding site when linked via the CDRs, If necessary, the CDRs of the reshaped human antibody can be appropriately selected. The amino acid residues of the FR can be substituted to form a suitable antigen-binding site. For example, The PCR method used to graft mouse CDRs onto human FRs was applied to introduce amino acid sequence mutations into the FRs. Specifically, a partial base sequence mutation can be added to the primer annealing to the FR. The FR synthesized by such a primer contains the base sequence The antigen-binding activity of the mutant antibody with the amino acid substitution is then determined by the above method. By measuring and evaluating the properties, mutant FR sequences having desired properties can be selected (Sato, K. et al. al., Cancer Res, 1993, 53, 851-856).

[0066] In addition, transgenic animals with a full repertoire of human antibody genes (International Publication) Open WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, WO1 996 / 033735) can be used as immunized animals to obtain desired human antibodies by DNA immunization.

[0067] Furthermore, there is also a technology to obtain human antibodies by panning using a human antibody library. For example, the V region of a human antibody is displayed as a single-chain antibody (scFv) on a phage. The scFv is expressed on the surface of the phage by the Ray method. Phages expressing scFv that bind to the antigen are selected. By analyzing the genes of the selected phages, human antibodies that bind to the antigen can be identified. The DNA sequence encoding the V region of the antibody can be determined. The DNA sequence of the scFv that binds to the antigen was determined. Then, the V region sequence is fused in frame with the sequence of the C region of a desired human antibody, and then the V region sequence is subjected to an appropriate An expression vector can be prepared by inserting the vector into an expression vector. The gene encoding the human antibody is expressed by introducing it into a suitable expression cell such as those listed above. These methods are already known (International Publication WO 2007 / 023106). 1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, see WO1995 / 015388).

[0068] antigen-binding domain As used herein, the term "antigen-binding domain" refers to a domain that specifically binds to a part or all of an antigen. When the molecular weight of the antigen is large, the antibody can only bind to a specific part of the antigen, which is called an epitope. The antigen-binding domain may be provided by one or more antibody variable domains. The antigen-binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Examples of such antigen-binding domains include "scFv (single chain Fv)" and "single chain antibody (single "F(ab')", "Fv", "scFv2 (single chain Fv 2)", "Fab" or "F(ab' )2" are preferred examples.

[0069] The antigen-binding domains in the polypeptide complexes of the present invention bind to the same epitope. The same epitope can be found in SEQ ID NO: 2 or SEQ ID NO: 4. Alternatively, the polypeptide of the present invention may be present in a protein consisting of the amino acid sequence of The antigen-binding domains in the peptide assembly can bind to different epitopes. Here, the different epitopes can be the amino acid sequences set forth in SEQ ID NO: 2 or SEQ ID NO: 4. It can be present in a protein consisting of the sequence

[0070] specific Specificity means that one molecule of a molecule that specifically binds to one or more of its binding partners. This refers to a state in which the antigen-binding domain does not show any significant binding to any molecules other than the antigen-binding domain. The antibody is specific to a particular epitope among multiple epitopes contained in an antigen. It is also used when the antigen-binding domain binds to multiple different epitopes. When the polypeptide complex contains the antigen-binding domain of a target antigen, the polypeptide complex containing the antigen-binding domain is It can bind to a variety of antigens, including pitopes.

[0071] antigen In the present specification, the antigen is not particularly limited, and may be any antigen except for CD3. Suitable examples include receptors, cancer antigens, MHC antigens, differentiation antigens, etc. Examples of the receptor family include the hematopoietic factor receptor family, the cytokine receptor family, Tyrosine kinase receptor family, serine / threonine kinase receptor family , TNF receptor family, G protein-coupled receptor family, GPI-anchored receptor family family, tyrosine phosphatase receptor family, adhesion factor family, hormone Examples of receptors that belong to the receptor family include the α- and β-glucan receptor family, ... and β-glucan receptor family. The receptors belonging to these receptor families and their characteristics are described in many publications, e.g. Cooke BA., King RJB., van der Molen HJ. ed. New Comprehesive Biochemistry Vol.18 B "Hormones and their Actions Part II"pp.1-46 (1988) Elsevier Science Publishers BV, or Masayuki Miyasaka, Cell Engineering Special Edition Handbook Series "Adhesion Factor Handbook" " (1994) (Shujunsha, Tokyo, Japan), and other reviews. 14), Ullrich et al. (Cell (1990) 61 (2), 203-212), Massague (e (Cent symbol) (Cell (1992) 69 (6), 1067-1070), Miyajima et al. (Annu. Rev. Immu nol. (1992) 10, 295-331), Taga et al. (FASEB J. (1992) 6, 3387-3396), Fantl et al. (Annu Rev. Biochem. (1993), 62, 453-481), Smith et al. (Cell (1994) 76 (6) 959-962), Fl ower DR. Biochim. Biophys. Acta, Flower(Biochim. Biophys. Acta (1999) 1422 (3) 207-234, etc.

[0072] Specific receptors belonging to the above receptor family include, for example, human or mouse E1 receptors. Thrombopoietin (EPO) receptor (Blood (1990) 76 (1), 31-35, Cell (1989) 57 (2), 277-2 85), human or mouse granulocyte colony-stimulating factor (G-CSF) receptor (Proc. Natl. Acad. Sci USA. (1990) 87 (22), 8702-8706, mG-CSFR, Cell (1990) 61 (2), 341-350), human or is a mouse thrombopoietin (TPO) receptor (Proc Natl Acad Sci U S A. (1992) 89 (12 ), 5640-5644, EMBO J. (1993) 12(7), 2645-53), human or mouse insulin receptor (N ature (1985) 313 (6005), 756-761), human or mouse Flt-3 ligand receptor (Proc. Nat Acad. Sci. USA. (1994) 91 (2), 459-463), human or mouse platelet-derived growth factor (P DGF) receptor (Proc. Natl. Acad. Sci. USA. (1988) 85 (10) 3435-3439), human or mouse Mouse interferon (IFN)-α, β receptors (Cell (1990) 60 (2), 225-234. and Cell ( 1994) 77 (3), 391-400), human or mouse leptin receptor, human or mouse growth hormone human or mouse interleukin (GH) receptor, human or mouse interleukin (IL)-10 receptor, human or mouse interleukin (IL)-10 receptor insulin-like growth factor (IGF)-I receptor, human or mouse leukemia inhibitory factor (LIF) receptor, human Suitable examples include human or mouse ciliary neurotrophic factor (CNTF) receptors.

[0073] Cancer antigens are antigens that are expressed in association with the malignant transformation of cells, and are also called tumor-specific antigens. Abnormal sugar chains that appear on the cell surface or protein molecules when cells become cancerous are also cancer antigens. They are also called cancer carbohydrate antigens. Examples of cancer antigens include the GPI-anchored receptors mentioned above. GPC3, a member of the GPC-type receptor family, is expressed in several cancers, including hepatocellular carcinoma (HCC) (Int J Cancer. (2003) 103 (4), 455-65), EpCA, which is expressed in several cancers including lung cancer. M (Proc Natl Acad Sci U S A. (1989) 86 (1), 27-31) (the polynucleotide sequence is The RefSeq accession number is NM_002354.2 (SEQ ID NO: 3), and the polypeptide sequence is RefSeq accession number is NP_0 02345.2 (SEQ ID NO: 4), EGFR, CA19-9, CA15-3, and silico. Suitable examples include AlSSEA-1 (SLX).

[0074] MHC antigens are mainly classified into MHC class I antigens and MHC class II antigens. MHC class II antigens include HLA-A, -B, -C, -E, -F, -G, and -H, and MHC class II antigens include HLA-DR, -DQ, and -D. Contains P.

[0075] Differentiation antigens include CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD 13, CD14, CD15s, CD16, CD18, CD19, CD20, CD21, CD23, CD25, CD28, CD29, CD30, CD3 2, CD33, CD34, CD35, CD38, CD40, CD41a, CD41b, CD42a, CD42b, CD43, CD44, CD45, C D45RO, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, CD54, CD55, CD56, C D57, CD58, CD61, CD62E, CD62L, CD62P, CD64, CD69, CD71, CD73, CD95, CD102, CD106 , CD122, CD126, CDw130.

[0076] epitope An epitope, which means an antigenic determinant present in an antigen, is a polypeptide as disclosed herein. It refers to the site on the antigen to which the antigen-binding domain in the polypeptide complex binds. For example, an epitope can be defined by its structure. The epitope can also be defined by the antigen-binding activity of the polypeptide complex. When the antigen is a peptide or polypeptide, the amino acid residues that constitute the epitope It is also possible to identify the epitope by It is also possible to identify epitopes based on specific sugar chain structures.

[0077] A linear epitope is an epitope that contains a recognized primary amino acid sequence. Linear epitopes typically have at least three, and most usually at least Five, for example, about 8 to about 10, 6 to 20 amino acids are included in the unique sequence.

[0078] Conformational epitopes, in contrast to linear epitopes, are those that are formed by the sequence of amino acids that comprise the epitope. Epitopes whose primary sequence is not a single defined component of the recognized epitope (e.g., amino acid sequence). (An epitope in which the primary sequence of amino acids is not necessarily recognized by the antibody that defines the epitope) Conformational epitopes encompass an increased number of amino acids relative to linear epitopes. With regard to the recognition of conformational epitopes, antibodies may bind to peptides or proteins. Recognizes the three-dimensional structure of proteins. For example, protein molecules fold to form three-dimensional structures. In this case, certain amino acids and / or polypeptides that form conformational epitopes The main chains are juxtaposed, allowing the antibody to recognize the epitope. Methods for determining structure include, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-specific These include, but are not limited to, spin labeling and electromagnetic paramagnetic resonance spectroscopy. , Epitope Mapping Protocols in Methods in Molecular Biology (1996), Volume 66, Morr See is(ed.).

[0079] The epitopes of test polypeptide complexes containing the GPC3 antigen-binding domain are shown below. Examples of methods for confirming binding to antigens other than GPC3 include methods for confirming binding to antigens other than GPC3. The binding of the experimental polypeptide complex to the epitope can also be confirmed by the following appropriate method. It can be applied.

[0080] For example, a test polypeptide complex containing a GPC3 antigen-binding domain is The recognition of a linear epitope present in the For the above purpose, a linear polypeptide consisting of the amino acid sequence constituting the extracellular domain of GPC3 can be prepared. The peptide is synthesized. The peptide can be chemically synthesized. Alternatively, the GPC3 cDNA can be used. The region encoding the amino acid sequence corresponding to the extracellular domain is used for genetic engineering. Next, a linear peptide consisting of the amino acid sequence that constitutes the extracellular domain is obtained. The binding activity of the peptide to a test polypeptide complex containing a GPC3 antigen-binding domain was evaluated. For example, the peptide can be detected by ELISA using an immobilized linear peptide as an antigen. Alternatively, the binding activity of the polypeptide complex to GPC3-expressing cells can be assessed. The level of inhibition by the linear peptide of the binding of the polypeptide complex to cells Based on these results, binding activity to linear peptides can be determined. The binding activity of the polypeptide complex toward a linear peptide can be determined.

[0081] Furthermore, test polypeptide complexes containing a GPC3 antigen-binding domain are For the above purpose, recognition of the GPC3-expressing gene can be confirmed as follows. Cells are prepared that express a test polypeptide complex containing a GPC3 antigen-binding domain. Upon contact with GPC3-expressing cells, the polypeptide complex binds strongly to the cells. For the immobilized linear peptide consisting of the amino acid sequence constituting the extracellular domain of GPC3, Here, "not substantially binding" refers to cases where the binding is not substantial and the binding is not substantial. The binding activity to the expressing cells is 80% or less, usually 50% or less, preferably 30% or less, and particularly preferably This refers to a binding activity of 15% or less.

[0082] Binding of a test polypeptide complex containing a GPC3 antigen-binding domain to GPC3-expressing cells The binding activity can be measured, for example, by the method described in Antibodies A Laboratory Manual. (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420) That is, ELISA and FACS (fluorescence activated cell sorting) using GPC3-expressing cells as antigens are used. It can be evaluated by the principle of (unclear)

[0083] In an ELISA format, a test polypeptide containing an antigen-binding domain for GPC3 The binding activity of the aggregates to GPC3-expressing cells was evaluated by measuring the signal level generated by the enzymatic reaction. This can be quantitatively evaluated by comparing the results with those of ELISA using fixed GPC3-expressing cells. A test polypeptide complex was added to the plate, and the test polypeptide complex bound to the cells was The test polypeptide complex is detected using an enzyme-labeled antibody that recognizes the test polypeptide complex. Alternatively, the complex is detected by FACS. In this study, a dilution series of the test polypeptide complex was prepared, and antibody binding to GPC3-expressing cells was assessed. The activity of the test polypeptide complex on GPC3-expressing cells was determined by determining the titer. Binding activities can be compared.

[0084] The test polypeptide complex reacts with an antigen expressed on the surface of cells suspended in a buffer solution or the like. Binding can be detected by a flow cytometer. For example, the following device is known. FACSCanto TM II FACSAria TM FACSArray TM FACSVantage TM SE FACSCalibur TM (All are trade names of BD Biosciences) EPICS ALTRA HyperSort Cytomics FC 500 EPICS XL-MCL ADC EPICS XL ADC Cell Lab Quanta / Cell Lab Quanta SC (both are trade names of Beckman Coulter)

[0085] For example, the binding of a test polypeptide complex containing a GPC3 antigen-binding domain to an antigen A suitable example of a method for measuring binding activity is as follows. First, The cells are reacted with a test polypeptide complex and then stained with an FITC-labeled secondary antibody that recognizes the polypeptide complex. The test polypeptide complex is diluted appropriately with a suitable buffer solution to obtain the complex. The desired concentration is prepared and used. For example, the concentration is between 10 μg / ml and 10 ng / ml. Then, the fluorescence intensity and cell number were measured using a FACSCalibur (BD). The amount of antibody binding to the cells is analyzed using CELL QUEST Software (BD). This is reflected in the fluorescence intensity obtained by the above process, i.e., the value of the Geometric Mean. The Geometric Mean value is obtained to determine the amount of binding of the test polypeptide complex. The binding activity of the test polypeptide complex can then be measured.

[0086] A test polypeptide complex containing a GPC3 antigen-binding domain is The fact that the two proteins share an epitope is confirmed by their competition for the same epitope. Competition between polypeptide complexes can be detected by cross-blocking assays, etc. For example, a competitive ELISA assay is a preferred cross-blocking assay.

[0087] Specifically, in the cross-blocking assay, the wells of a microtiter plate GPC3 protein coated on a membrane was detected in the presence of a candidate competitor polypeptide complex. After preincubation in the absence of ATP, the test polypeptide complex is added. The amount of test polypeptide complex bound to GPC3 protein in the well was determined by the amount of the test polypeptide complex bound to the same epitope. This indirectly correlates with the binding ability of candidate competitor polypeptide complexes. In other words, the greater the affinity of a competing polypeptide complex for the same epitope, the better. The greater the number of nuclei, the lower the binding activity of the test polypeptide complex to wells coated with GPC3 protein. do.

[0088] The amount of test polypeptide complex bound to the wells via the GPC3 protein was determined by measuring the amount of the polypeptide complex in advance. By labeling the peptide aggregate, it can be easily measured. For example, biotin labeling The recognized polypeptide complex is then incubated with an avidin-peroxidase conjugate and an appropriate substrate. It is measured by using a cross-blocking assay using an enzyme label such as peroxidase. The binding assay is particularly called a competitive ELISA assay. Alternatively, it may be labeled with other measurable labeling substances, such as radiolabels or fluorescent labels. Signs and the like are well known.

[0089] The results obtained in a control experiment conducted in the absence of a candidate competitor polypeptide complex The binding activity of the competitor polypeptide complex is compared with that of the GPC3 antigen-binding domain. at least 20%, preferably at least 20-50%, of the binding of a test polypeptide complex containing More preferably, the test polypeptide complex is capable of blocking at least 50%. Binds to substantially the same epitope as the competitor polypeptide complex, or binds to the same epitope It is a polypeptide complex that competes for binding of

[0090] The epitope to which a test polypeptide complex containing a GPC3 antigen-binding domain binds When the structure has been identified, the test polypeptide complex and the control polypeptide complex are Sharing an epitope involves introducing amino acid mutations into the peptide that constitutes the epitope. The binding activity of both polypeptide complexes against the selected peptide was compared. It can be done.

[0091] As a method for measuring such binding activity, for example, the above-mentioned ELISA format Test polypeptide complexes and control polypeptides for linear peptides into which mutations have been introduced The binding activity of the aggregates can be measured by comparing them. The binding activity of the mutant peptide bound to the column was determined by applying the test polypeptide to the column. Polypeptide complexes eluted in the eluate after the combined and control polypeptide complexes were allowed to flow down The mutant peptide can also be measured by quantifying the amount of the fusion peptide with GST. The method of adsorbing the compound onto a column as a soluble solid is well known.

[0092] Furthermore, if the identified epitope is a conformational epitope, the test polypeptide complex Whether a control polypeptide complex shares an epitope with a target polypeptide complex can be assessed by the following method. Cells expressing GPC3 and cells expressing GPC3 with mutations introduced into the epitope were prepared. These cells are suspended in an appropriate buffer solution such as PBS, and the test polypeptide is then reacted with the cell suspension. Then, the cells are washed with an appropriate buffer solution and the control polypeptide complex is added. The test polypeptide complex and the control polypeptide complex were recognized in the cell suspension. FITC-labeled antibodies are added. The fluorescence intensity of the cells stained by the labeled antibodies is The cell number was measured using a FACSCalibur (BD). The concentration of the peptide aggregate can be adjusted to a desired concentration by diluting it appropriately with a suitable buffer solution. For example, it may be used at a concentration between 10 μg / ml and 10 ng / ml. The amount of labeled antibody binding to the cells was analyzed using CELL QUEST Software (BD). This is reflected in the fluorescence intensity obtained by the measurement, i.e., the Geometric Mean value. By obtaining the value of the Geometric Mean, the test point represented by the amount of binding of the labeled antibody can be determined. The binding activity of the polypeptide complex and the control polypeptide complex can then be measured.

[0093] In this method, for example, "not substantially binding to mutant GPC3-expressing cells" can be achieved by the following methods: First, the test compound bound to cells expressing mutant GPC3 was The polypeptide complex and the control polypeptide complex are stained with a labeled antibody. Detect light intensity. When FACSCalibur is used as a flow cytometer for fluorescence detection, The obtained fluorescence intensity can be analyzed using CELL QUEST Software. From the Geometric Mean values in the presence and absence of ions, this comparison value (ΔGeo-Mean) was calculated as follows: The percentage increase in fluorescence intensity due to binding of the polypeptide complex was calculated based on the formula. It is possible to find a match.

[0094] ΔGeo-Mean = Geo-Mean (in the presence of polypeptide complex) / Geo-Mean (in the presence of polypeptide complex) (in the absence)

[0095] The binding amount of the test polypeptide complex to mutant GPC3-expressing cells obtained by the analysis is expressed as a function of time. The Geometric Mean comparison value (ΔGeo-Mean value of mutant GPC3 molecule) was used to compare the measured polypeptide association. The value is compared with the ΔGeo-Mean comparison value, which reflects the amount of binding to GPC3-expressing cells in the body. Used to calculate the ΔGeo-Mean comparison value for mutant GPC3 expressing cells and GPC3 expressing cells. The concentrations of the test polypeptide complexes are adjusted to be the same or substantially the same. It is particularly preferable to use a polypeptide that has been previously confirmed to recognize an epitope in GPC3. The polypeptide complex is used as a control polypeptide complex.

[0096] The ΔGeo-Mean value of the test polypeptide complex compared to mutant GPC3-expressing cells was At least 80%, preferably 80%, of the ΔGeo-Mean comparison value of the peptide assembly against GPC3-expressing cells If the percentage is less than 50%, more preferably 30%, and particularly preferably 15%, it is considered to be "mutated GPC3-expressing cells." The formula for calculating the Geo-Mean value is CE The results are described in the LL QUEST Software User's Guide (BD Biosciences). If the results are substantially equivalent by comparison, the test polypeptide complex is The epitope of the control polypeptide complex can be assessed to be identical.

[0097] Fv (variable fragment) As used herein, the term "Fv (variable fragment)" refers to the light chain variable region of an antibody. (VL (light chain variable region)) and antibody heavy chain variable region (VH (heavy chain variable The term "antibody-derived antigen-binding domain" refers to the smallest unit of an antibody-derived antigen-binding domain consisting of a pair of a nucleotide sequence and a nucleotide sequence. In 1988, Skerra and Pluckthun inserted an antibody gene downstream of a bacterial signal sequence. By inducing the expression of the gene in E. coli, the gene is expressed uniformly and in an active state. It was found that it was prepared from the periplasmic fraction of Escherichia coli (Science (1988) 240 (4855), 1038-1041). Fv prepared from the periplasmic fraction binds to the antigen in a manner that VH and VL were meeting.

[0098] As used herein, Fv includes, for example, the following polypeptide complexes: Among the bivalent scFvs, the monovalent scFv binds the Fc region via the heavy chain Fv fragment that constitutes the CD3-binding domain. One polypeptide comprises a light chain Fv, and the other monovalent scFv comprises a CD3-binding domain. A bivalent antigen-binding domain linked via a fragment to another polypeptide constituting the Fc region. The main antibody is a bivalent scFv (1) with a bivalent antigen-binding domain, (2) with an IgG1, IgG2a, IgG3, or is the amino acid that constitutes the Fc region of IgG4, but does not have binding activity to Fcγ receptors. and (3) at least a monovalent CD3 binding domain; In a polypeptide complex comprising the above compound, a light chain Fv fragment and a heavy chain Fv fragment bind to the antigen CD3. Also preferably included are a pair of Fvs that associate in a manner that allows for corresponding binding to form a CD3-binding domain.

[0099] scFv, single chain antibody, or sc(Fv)2 As used herein, the terms "scFv," "single chain antibody," or "sc(Fv)2" refer to a single Within the polypeptide chain of the Generally, single-chain antibodies are antibody fragments that lack the required amino acid sequence to bind to an antigen. A polypeptide linker between the VH and VL domains that enables it to form the desired structure. Single chain antibodies are described in The Pharmacology of Monoclonal Antibodies, Vol. 113, In Plu, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994) Also, International Patent Application Publication No. WO 1988 / 001649 and See U.S. Patent Nos. 4,946,778 and 5,260,203. In certain embodiments, single chain antibodies are It may also be bispecific and / or humanized.

[0100] scFv is an antigen-binding domain in which the VH and VL constituting the Fv are linked by a peptide linker. (Proc. Natl. Acad. Sci. USA (1988) 85 (16), 5879-5883). The VH and VL can be held in close proximity by a linker.

[0101] sc(Fv)2 consists of four variable regions, two VL and two VH, linked together by a linker such as a peptide linker. It is a single-chain antibody in which the fragments are linked together to form a single chain (J Immunol. Methods (1999) 231 (1-2), 1 77-189). The two VH and VL may be derived from different monoclonal antibodies. For example, the same antibodies as disclosed in Journal of Immunology (1994) 152 (11), 5368-5374 Bispecific sc(Fv)2 that recognizes two different epitopes present in the antigen is also suitable. sc(Fv)2 can be produced by methods known to those skilled in the art. For example, It can be produced by connecting with a linker such as a peptide linker.

[0102] The antigen-binding domain constituting the sc(Fv)2 herein comprises two VH and Two VLs are linked together by a VH, VL, VH, VL ([VH] linker) with the N-terminal end of the single-chain polypeptide as the base point. [VL] linker [VH] linker [VL] However, the order of the two VHs and two VLs is not limited to the above configuration, and any order may be used. For example, the following order configurations are also possible. [VL] linker [VH] linker [VH] linker [VL] [VH] linker [VL] linker [VL] linker [VH] [VH] linker [VH] linker [VL] linker [VL] [VL] linker [VL] linker [VH] linker [VH] [VL] linker [VH] linker [VL] linker [VH]

[0103] The molecular form of sc(Fv)2 is also described in detail in WO2006 / 132352, and those skilled in the art will be able to Based on these descriptions, the polypeptide complexes disclosed herein can be produced using the methods described above. Therefore, it is possible to prepare the desired sc(Fv)2 as needed.

[0104] Furthermore, the polypeptide complexes of the present invention can be used in combination with carrier polymers such as PEG and organoglycoside-containing anticancer drugs. Alternatively, a glycosylation sequence may be inserted to conjugate the glycosylation to achieve the desired effect. It can be suitably added for the purpose of:

[0105] As a linker for connecting the variable regions of an antibody, any peptide that can be introduced by genetic engineering can be used. a tido linker, or a synthetic compound linker (e.g., Protein Engineering, 9 (3), 299-30 5, 1996) can be used. The length of the peptide linker is not particularly limited, and can be determined by those skilled in the art depending on the purpose. The length can be selected appropriately by the person skilled in the art, but the preferred length is 5 amino acids or more (there is no particular upper limit). Although it is not necessary to use a polynucleotide containing 30 or less amino acids, it is usually 30 or less amino acids, preferably 20 or less amino acids. When sc(Fv)2 contains three peptide linkers, all of them have the same length. Peptide linkers of different lengths may be used. .

[0106] For example, for a peptide linker: Ser 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·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 is an integer of 1 or more], etc. However, depending on the length of the peptide linker and The sequence can be appropriately selected by those skilled in the art depending on the purpose.

[0107] Synthetic chemical linkers (chemical crosslinkers) are crosslinkers commonly used for crosslinking peptides. For example, N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), Bis(sulfosuccinimidyl) suberate (BS3), dithiobis(succinimidyl protease) dithiobis(sulfosuccinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP) , ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol Sulfosuccinimidyl bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl Disulfosuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(sulfo- succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), bis[2-(sulfonyloxy)ethyl]sulfone Phosuccinimideoxycarbonyloxy)ethyl] sulfone (sulfo-BSOCOES), etc. These crosslinkers are commercially available.

[0108] When linking four antibody variable regions, three linkers are usually required. The same linker may be used, or different linkers may be used.

[0109] Fab, F(ab')2, or Fab' "Fab" consists of one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0110] "F(ab')2" and "Fab'" are immunoglobulins (monoclonal antibodies) that bind to proteins. It is produced by treating it with the protein-degrading enzymes pepsin or papain, and the hinge The antibody fragments are generated by digestion before and after the disulfide bond between the two heavy chains in the region. For example, by treating IgG with papain, the two amino acids in the hinge region are separated. It is cleaved upstream of the disulfide bond between the H chains to form VL (Variable Region of Light Chain) and CL (Constant Region of Light Chain). The L chain consists of a VH (heavy chain variable region) and a CHγ1 (γ1 region in the heavy chain constant region). Two homologous antibodies in which the heavy chain fragments consisting of the following are linked by disulfide bonds at the C-terminal regions: These two homologous antibody fragments can be produced, each of which is called Fab'. It can be done.

[0111] "F(ab')2" is a protein consisting of two light chains and an interchain disulfide bond formed between two heavy chains. It comprises two heavy chains containing constant regions of the CH1 domain and a portion of the CH2 domain so as to form a The F(ab')2 constituting the polypeptide complexes disclosed herein binds to a desired antigen. A full-length monoclonal antibody having a binding domain is fragmented with a proteolytic enzyme such as pepsin. After digestion, the Fc fragment is removed by adsorption onto a protein A column, allowing for convenient extraction. Such protease can be obtained by appropriately setting the reaction conditions of the enzyme, such as pH. There are no particular limitations as long as it can digest a full-length antibody to generate F(ab')2 in a limited manner. Examples of suitable anti-inflammatory agents include pepsin and ficin.

[0112] Fc area The Fc domain of the polypeptide complexes disclosed herein is a monoclonal antibody. After partially digesting antibodies such as antibodies with a protease such as pepsin, the fragments are separated into protein A fragments. After adsorption onto a column or protein G column, the sample is eluted with an appropriate elution buffer. Such proteolytic enzymes can be suitably obtained by releasing the enzymes, such as those affected by pH. If the reaction conditions are appropriately set, antibodies such as monoclonal antibodies can be digested. There is no particular limitation on the type of enzyme used, and examples thereof include pepsin and ficin.

[0113] The polypeptide complexes described herein contain an IgG1, IgG2, IgG3, or IgG4 Fc domain. The amino acids comprising the Fc region include an Fc region with reduced binding activity to Fcγ receptors.

[0114] The antibody isotype is determined by the structure of the constant region: IgG1, IgG2, IgG3, IgG4 The constant regions of each isotype are called Cγ1, Cγ2, Cγ3, and Cγ4, respectively. The amino acid sequences of polypeptides constituting the Fc region of Cγ1, Cγ2, Cγ3, and Cγ4 are shown in SEQ ID NO: : 23, 24, 25, 26. The amino acid residues constituting each amino acid sequence and ka The relationship of the bat to the EU numbering (also referred to herein as the EU INDEX) is shown in FIG. It has been done.

[0115] The Fc region consists of two light chains and interchain disulfide bonds formed between two heavy chains. F( ab')2. The Fc region, which is composed of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, is a protein that reacts with pepsin and other proteins. After partial digestion with enzymes, the fraction adsorbed to the Protein A column was re-eluted. Therefore, such a protease can be obtained by appropriately adjusting the reaction conditions of the enzyme, such as pH. If the full-length antibody can be digested to generate F(ab')2 only by setting There is no particular limitation on the type of enzyme used, and examples thereof include pepsin and ficin.

[0116] Fcγ receptor Fcγ receptors are receptors that can bind to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies. It refers to any member of a family of proteins essentially encoded by Fcγ receptor genes. In humans, this family includes the isoforms FcγRIa, Fc FcγRI (CD64), which includes γRIb and FcγRIc; the isoform FcγRIIa (allotype H131 and and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc FcγRII (CD32); and isoform FcγRIIIa (including allotypes V158 and F158) ) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) III (CD16), and any undiscovered human FcγRs or FcγR isoforms or antigens. FcγRs include, but are not limited to, human, mouse, and human allotypes. of any biological origin, including but not limited to rats, rabbits and monkeys. Mouse FcγRs include FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and and FcγRIII-2 (CD16-2), as well as any unidentified mouse FcγRs or FcγR isoforms. These Fcγ receptor preferences include, but are not limited to, the form or allotype. Suitable examples include human FcγI (CD64), FcγIIA (CD32), FcγIIB (CD32), and FcγIIIA (CD1 The polynucleotide sequences and amino acid sequences of FcγI and FcγIIIB (CD16) are also included. The sequences of FcγIIA and FcγIIB are shown in SEQ ID NOs: 13 (NM_000566.3) and 14 (NP_000557.1), respectively. The polynucleotide and amino acid sequences of SEQ ID NO: 15 (BC020823.1) and SEQ ID NO: 20 (BC020823.1), respectively. The polynucleotide sequence and amino acid sequence of FcγIIB are located in AAH20823.1. Sequence numbers 17 (BC146678.1) and 18 (AAI46679.1) contain the polynucleotide sequences for FcγIIIA. The sequence and amino acid sequence are set forth in SEQ ID NOs: 19 (BC033678.1) and 20 (AAH33678.1), respectively. The polynucleotide and amino acid sequences of FcγIIIB are set forth in SEQ ID NO: 21( BC128562.1) and 22 (AAI28563.1) (RefSeq accession numbers are in parentheses). Fcγ receptors bind to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies. Whether or not a gene has this property can be determined by the FACS and ELISA formats described above, as well as the ALPHA screen ( Amplified Luminescent Proximity Homogeneous Assay (ALU) and Surface Plasmon Resonance (SPR) This can be confirmed by the BIACORE method using an electron microscope (Proc. Natl. Acad. Sci. USA (2006) 103 ( 11), 4005-4010).

[0117] An "Fc ligand" or "effector ligand" is a molecule that binds to the Fc region of an antibody and binds to the Fc region. A molecule, preferably a polypeptide, derived from any organism that forms a Fc / Fc ligand complex. Binding of an Fc ligand to Fc preferably results in the activation of one or more effectors. Fc ligands include Fc receptors, FcγR, FcαR, FcεR, FcRn, C1q, C3, and mannose-binding lectin, mannose receptor, Staphylococcus protein A, Staphylococcus These include, but are not limited to, B. lococcus protein G and viral FcγR. Fc ligands include Fc receptor homologs (FcRHs), a family of Fc receptors homologous to FcγRs. (Davis et al., (2002) Immunological Reviews 190, 123-136) The domain may also include undiscovered molecules that bind to Fc.

[0118] Fcγ receptor binding activity The Fc region is an Fcγ receptor selected from FcγI, FcγIIA, FcγIIB, FcγIIIA, and / or FcγIIIB. The decreased binding activity to the receptor can be confirmed by FACS and ELISA formats described above. In addition to the ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), This can be confirmed by the BIACORE method, which utilizes the surface plasmon resonance (SPR) phenomenon. Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0119] ALPHA Screen uses ALPHA technology, which uses two beads, a donor and an acceptor. The method is based on the following principle: molecules bound to donor beads are transferred to acceptor beads. When the two beads are in close proximity, they interact biologically with the molecules bound to the beads. Only the photon-emitting donor beads excited by the laser are detected. The sensitizer converts ambient oxygen into excited singlet oxygen, which is the donor. When it diffuses around the beads and reaches the nearby acceptor beads, it emits chemiluminescence within the beads. The donor bead is bound to an acceptor, which reacts with the donor bead and ultimately emits light. When the molecules bound to the beads do not interact, the singlet oxygen produced by the donor beads Since the acceptor beads are not reached, the chemiluminescence reaction does not occur.

[0120] For example, a biotin-labeled polypeptide complex is bound to a donor bead, and a biotin-labeled polypeptide complex is bound to an acceptor bead. The turbidibeads contain glutathione S-transferase (GST)-tagged Fcγ receptors. In the absence of a competing polypeptide complex with a mutant Fc region, the wild-type Fc region The polypeptide complex containing the domain interacts with the Fcγ receptor, producing a signal at 520-620 nm. Polypeptide complexes having untagged mutant Fc regions are similar to those having wild-type Fc regions. This competes with the interaction between the polypeptide complex and the Fcγ receptor. By quantifying the amount of the polypeptide, the relative binding affinity can be determined. It is known that the conjugate can be biotinylated using Sulfo-NHS-biotin or the like. The method of tagging with T involves combining a polynucleotide encoding an Fcγ receptor with a polynucleotide encoding GST. A fusion gene in which a polynucleotide corresponding to the gene is fused in frame is contained in an expression vector. The method of expressing the protein in cells containing the protein and purifying it using a glutathione column can be appropriately adopted. The obtained signals are analyzed using software such as GRAPHPAD PRISM (GraphPad, San Diego). A one-site competition model was applied using nonlinear regression analysis with software. The analysis is preferably performed by combining the two.

[0121] One of the substances (ligands) whose interactions are to be observed is immobilized on the gold thin film of the sensor chip. When light is shone from the back of the sensor chip so that it is totally reflected at the interface between the gold thin film and the glass, the reflection A part of the light has a reduced reflection intensity (SPR signal). Observe the interaction. The other substance (analyte) is poured onto the surface of the sensor chip, and the ligand and analyte bind to each other. When the immobilized ligand molecule is absorbed, the mass of the immobilized ligand molecule increases, and the refractive index of the solvent on the sensor chip surface decreases. This change in refractive index causes a shift in the position of the SPR signal (as opposed to the binding (The signal position returns to normal upon dissociation.) The Biacore system uses the amount of shift mentioned above, i.e. The vertical axis represents the change in mass on the sensor chip surface, and the time change in mass is displayed as measurement data. (sensorgram). Kinetics: binding rate constant ( The affinity (KD) is calculated from the ratio of the dissociation rate constant (kA) and the dissociation rate constant (kD). In the CORE method, an inhibition assay is also preferably used. An example of an inhibition assay is described in Proc. Natl. Acad. Sci. USA ( 2006) 103 (11), 4005-4010.

[0122] As used herein, the term "having reduced binding activity to an Fcγ receptor" refers to, for example, the above-mentioned Based on the analytical method, the competitive activity of the test polypeptide is compared with that of the control polypeptide complex. The competitive activity of the tide complex is 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less. Preferably, the ratio is 20% or less, 15% or less, more preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less. The term "below" refers to a binding activity of 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less.

[0123] As a control polypeptide complex, IgG1, IgG2, IgG3, or IgG4 monoclonal antibody was used. A polypeptide complex having an Fc domain of the same type can be used as appropriate. The structure of the Fc domain is No.: 23 (RefSeq accession number AAC82527.1 with an A at the N-terminus), 24 (RefSeq accession number AAB59393 .1), 25 (RefSeq accession number CAA27268.1), 26 (RefSeq accession number The antibody of a certain isotype is described in the following. When a polypeptide complex containing an Fc domain mutant is used as a test substance, Use a polypeptide complex containing the Fc region of an antibody of a specific isotype as a control. The effect of the mutations in the mutants on the binding activity to Fcγ receptors was verified. As described above, it was verified that the binding activity to Fcγ receptors was reduced. Polypeptide complexes containing Fc domain mutants are appropriately prepared.

[0124] Examples of such variants include 23 amino acids identified according to EU numbering. Deletion of 1A-238S (WO 2009 / 011941), C226S, C229S, P238S, (C220S) (J. Rheumatol (2007) ) 34, 11), C226S, C229S(Hum.Antibod.Hybridomas (1990) 1(1), 47-54), C226S, C2 Mutations such as 29S, E233P, L234V, and L235A (Blood (2007) 109, 1185-1192) are known.

[0125] That is, among the amino acids that make up the Fc region of a specific isotype antibody, the EU number Any of the following amino acids identified according to the coding sequence: 220, 226, 229, 231, 232 233rd, 234th, 235th, 236th, 237th, 238th, 239th, 240th, 264th, 265th, 266th , 267th, 269th, 270th, 295th, 296th, 297th, 298th, 299th, 300th, 325th, 327th, 3 Polypeptide complexes comprising an Fc domain with substitutions at positions 28, 329, 330, 331, or 332 The isotype of the antibody from which the Fc region is derived is not particularly limited, and examples thereof include: Fc regions derived from IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies can be used as appropriate. Preferably, an Fc region derived from an IgG1 antibody is used.

[0126] For example, among the amino acids that make up the Fc region of an IgG1 antibody, the amino acids identified according to EU numbering are: Any of the following substitutions (numbers indicate amino acid residue positions according to EU numbering): The one-letter amino acid code before the number indicates the amino acid residue before substitution, and the one-letter code after the number indicates the amino acid residue before substitution. Each single-letter amino acid symbol represents the amino acid residue before substitution); (a) L234F, L235E, P331S, (b) C226S, C229S, P238S, (c) C226S, C229S, (d)C226S, C229S, E233P, L234V, L235A or an Fc region in which the amino acid sequence at positions 231 to 238 has been deleted. Polypeptide complexes can also be used as appropriate.

[0127] In addition, among the amino acids that make up the Fc region of IgG2 antibodies, those identified according to EU numbering are Any of the following substitutions (numbers indicate amino acid residue positions according to EU numbering): The single-letter amino acid code before the number indicates the amino acid residue before substitution, and the single-letter code after the number indicates the amino acid residue before substitution. Each single-letter amino acid symbol represents the amino acid residue before substitution); (e)H268Q, V309L, A330S, P331S (f)V234A (g)G237A (h) V234A, G237A (i) A235E, G237A (j) V234A, A235E, G237A Polypeptide complexes having an Fc domain modified with PEG can also be used as appropriate.

[0128] In addition, among the amino acids that make up the Fc region of IgG3 antibodies, those identified according to EU numbering are Any of the following substitutions (numbers indicate amino acid residue positions according to EU numbering): The single-letter amino acid code before the number indicates the amino acid residue before substitution, and the single-letter code after the number indicates the amino acid residue before substitution. Each single-letter amino acid symbol represents the amino acid residue before substitution); (k)F241A (l) D265A (m)V264A Polypeptide complexes having an Fc domain modified with PEG can also be used as appropriate.

[0129] In addition, among the amino acids that make up the Fc region of IgG4 antibodies, those identified according to EU numbering are Any of the following substitutions (numbers indicate amino acid residue positions according to EU numbering): The single-letter amino acid code before the number indicates the amino acid residue before substitution, and the single-letter code after the number indicates the amino acid residue before substitution. Each single-letter amino acid symbol represents the amino acid residue before substitution); (n) L235A, G237A, E318A (o)L235E (p)F234A, L235A Polypeptide complexes having an Fc domain modified with PEG can also be used as appropriate.

[0130] Other preferred examples include amino acids in the EU numbering system that constitute the Fc region of an IgG1 antibody. Any of the following amino acids identified according to the coding sequence: 233, 234, 235, 236, 237 The positions 327, 330, and 331 correspond to the corresponding EU numbering in the corresponding IgG2 or IgG4. Examples of polypeptide complexes include those having an Fc domain substituted with amino acids corresponding to the amino acid sequence of the polypeptide complex.

[0131] Other preferred examples include amino acids in the EU numbering system that constitute the Fc region of an IgG1 antibody. Any one or more of the following amino acids as specified in accordance with the coding sequence: positions 234, 235, 29 Polypeptide complexes having an Fc domain in which position 7 is substituted with another amino acid are preferably The type of amino acid present after substitution is not particularly limited, but may be any of the amino acids at positions 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254 An Fc region in which one or more amino acids at position 7 are substituted with alanine Polypeptide complexes are particularly preferred.

[0132] Other preferred examples include amino acids in the EU numbering system that constitute the Fc region of an IgG1 antibody. Any of the following amino acids specified according to the coding sequence; position 265 is substituted by another amino acid: Preferred examples of such polypeptide complexes include those having an Fc domain containing the amino acid sequence of the amino acid sequence. The type of amino acid is not particularly limited, but an Fc domain in which the amino acid at position 265 is substituted with alanine is Polypeptide complexes having a nucleotide sequence similar to that of the nucleotide sequence ...

[0133] Fc regions derived from bispecific antibodies As used herein, an Fc region having reduced binding activity to an Fcγ receptor is Fc regions derived from bispecific antibodies may also be used as appropriate. IgG type bispecific antibodies are antibodies that produce IgG antibodies. Hybrid hybridoma (quadroma) is produced by fusing two types of hybridomas. It can be secreted by the ATPase (Milstein C et al. Nature (1983) 305, 537-540).

[0134] In addition, IgG-type bispecific antibodies are made by combining the genes of the L and H chains that make up the two types of IgG of interest, It is secreted by introducing four types of genes into cells and co-expressing them. Theoretically, there are 10 different combinations of H and L chains in the IgG produced by these methods. Therefore, it is difficult to purify IgG consisting of the desired combination of H and L chains. Theoretically, the secretion amount of the combination will also be significantly reduced, so a large scale of cultivation will be required, and production The construction costs will increase further.

[0135] In this case, appropriate amino acid substitutions are made in the CH3 region that constitutes the Fc region of the H chain to Specifically, the CH3 region of one H chain may be preferentially secreted. The amino acid side chains present in the The amino acid side chains in the CH3 region of the H chain are replaced with smaller side chains (holes). By replacing the protrusions, the protrusions can be positioned in the cavity, promoting heterologous H chain formation and homologous H chain formation. This method causes inhibition of chain formation (WO1996 / 027011, Ridgway JB et al., Protein En gineering (1996) 9, 617-621, Merchant AM et al. Nature Biotechnology (1998) 16, 677-681).

[0136] In addition, the L chain variable region has less diversity than the H chain variable region, so It is expected that a common light chain that can confer binding ability to both heavy chains will be obtained. Efficient expression of bispecific IgG by expressing IgG by introducing the vector into cells (Nature Biotechnology (1998) 16, 677-681). However, two kinds of antibodies can be used arbitrarily. If you select the same L chain, it is unlikely that it will contain the same L chain, making it difficult to implement the above idea. A method for selecting a common L chain that exhibits high binding ability corresponding to any different H chain has also been proposed. (WO2004 / 065611).

[0137] Also, the association of polypeptides or the association of heteromultimers composed of polypeptides By utilizing the control method of the two polypeptides that make up the Fc region, Techniques for producing specific antibodies are also known. By modifying the amino acid residues that form the interface between the Fc region and the Fc domain, an Fc region having the same sequence as the Fc region can be constructed. The association of the polypeptides that make up the two Fc regions with different sequences is inhibited, and A method for controlling the formation of a bispecific antibody aggregate can be employed to prepare a bispecific antibody (WO20 06 / 106905).

[0138] The domain containing the Fc region according to the present invention includes the Fc domain derived from the bispecific antibody. Two polypeptides constituting the Fc region can be used as appropriate. Two polypeptides corresponding to each other, wherein the amino acid sequence of one of the polypeptides is EU number The amino acid at position 349 is cysteine and the amino acid at position 366 is tryptophan, as specified by the barring. The amino acid sequence of the other polypeptide is identified according to EU numbering. The amino acid at position 356 is cysteine, the amino acid at position 366 is serine, and the amino acid at position 368 is adenine. Two polypeptides are preferred, characterized in that the amino acid at position 407 is valine and the amino acid at position 408 is lanine. It can be used appropriately.

[0139] In another embodiment, the domain comprising an Fc region according to the present invention is a domain comprising an Fc region. Two polypeptides consisting of two amino acid sequences, one of which has a EU amino acid sequence The amino acid at position 409, as determined by the numbering, is aspartic acid, and the other polypeptide The amino acid at position 399 of the amino acid sequence of the peptide, as specified by EU numbering, is a lysine. In the above embodiment, two polypeptides are preferably used. The amino acid at position 9 is replaced with glutamic acid instead of aspartic acid, and the amino acid at position 399 is replaced with lysine. In addition to lysine at position 399, aspartic acid or An aspartic acid at position 392 may also be suitably added.

[0140] In another embodiment, the domain comprising an Fc region according to the present invention is a domain comprising an Fc region. Two polypeptides, one of which has an amino acid sequence with EU numbering The amino acid at position 370, as specified by the coding sequence, is glutamic acid, and the amino acid at position 370 of the other polypeptide is The amino acid at position 357 in the amino acid sequence, as specified by EU numbering, is lysine. Two polypeptides characterized by the above are preferably used.

[0141] In yet another embodiment, the domain comprising an Fc region according to the present invention is a domain comprising an Fc region. Two polypeptides corresponding to each other, wherein the amino acid sequence of one of the polypeptides is EU number The amino acid at position 439, as specified by Barring, is glutamic acid, and the other polypeptide The amino acid at position 356 in the amino acid sequence of the nucleotide sequence, as specified by EU numbering, is lysine. Two polypeptides characterized by the above-mentioned features are preferably used.

[0142] Furthermore, the domain containing the Fc region according to the present invention may be a combination of these. ; The amino acid sequence of one of the two polypeptides that make up the Fc region The amino acid at position 409, identified according to the EU numbering system, is aspartic acid, and the amino acid at position 370 is The amino acid is glutamic acid, and the amino acid sequence of the other polypeptide is The amino acid at position 399 is lysine and the amino acid at position 357 is lysine, as determined according to the method of Two polypeptides (in this embodiment, asparagine instead of glutamic acid at position 370) are used. Alternatively, glutamic acid at position 370 may be replaced by aspartic acid at position 392. stomach), The amino acid sequence of one of the two polypeptides that make up the Fc region The amino acid at position 409, identified according to the EU numbering system, is aspartic acid, and the amino acid at position 439 is The amino acid is glutamic acid, and the amino acid sequence of the other polypeptide is The amino acid at position 399 is lysine and the amino acid at position 356 is lysine, as determined according to the method of Two polypeptides (in this embodiment, an aspartate at position 360 is substituted for glutamic acid at position 439) aspartic acid at position 392 or aspartic acid at position 439), The amino acid sequence of one of the two polypeptides that make up the Fc region The amino acid at position 370, identified according to the EU numbering system, is glutamic acid, and the amino acid at position 439 is amino acid. The amino acid in the other polypeptide is glutamic acid, and the amino acid in the other polypeptide is Therefore, the amino acid at position 357 is lysine, and the amino acid at position 356 is lysine. or two polypeptides The amino acid sequence of one of the two polypeptides that make up the Fc region The amino acid at position 409, identified according to the EU numbering system, is aspartic acid, and the amino acid at position 370 is The amino acid at position 439 is glutamic acid, and the amino acid at position 439 is glutamic acid. In the amino acid sequence, the 399th amino acid, identified according to EU numbering, is lysine, and the 357th amino acid is The amino acid at position 1 is lysine, and the amino acid at position 356 is lysine. In this embodiment, the amino acid at position 370 may not be substituted with glutamic acid, and The amino acid at position 439 is replaced with asparagine, while the amino acid at position 439 is not replaced with glutamic acid. (an aspartic acid at position 392 may be substituted for glutamic acid at position 439, or an aspartic acid at position 392 may be substituted for glutamic acid at position 439); is preferably used.

[0143] Furthermore, in another embodiment, the domain containing an Fc region according to the present invention includes an Fc region. Two polypeptides constituting the polypeptide, one of which has an amino acid sequence that is EU The amino acid at position 356, as specified by the numbering, is lysine, and the other polypeptide The amino acid at position 435 in the amino acid sequence specified by EU numbering is arginine. and two polypeptides characterized in that the amino acid at position 439 is glutamic acid. It is used.

[0144] The Fc region of the present invention is an Fc region derived from the bispecific antibody. By using two polypeptides constituting the antigen-binding domain and It is possible to arrange the CD3-binding domain and / or the CD3-binding domain in any desired combination.

[0145] C-terminal heterogeneity-reduced Fc region As used herein, the Fc region having reduced binding activity to Fcγ receptors includes the above-mentioned In addition to the characteristics, an Fc region with improved C-terminal heterogeneity of the Fc region can be used as appropriate. More specifically, the two polypeptides constituting the Fc region originating from IgG1, IgG2, IgG3, or IgG4 are glycine at position 446 as specified according to EU numbering in the amino acid sequence of the polypeptide; and an Fc region lacking lysine at position 447.

[0146] T cell receptor complex binding domain As used herein, the term "T cell receptor complex binding domain" refers to a domain that is part of the T cell receptor complex. T cell receptor complex antibodies comprising a region that specifically binds to and is complementary to part or all of The T cell receptor complex may be the T cell receptor itself or a part of the body that is composed of the T cell receptor and the T cell receptor. The adaptor molecule may also be an adaptor molecule that also constitutes the T cell receptor complex. The most common is CD3.

[0147] T cell receptor binding domain As used herein, the term "T cell receptor binding domain" refers to a part or the whole of a T cell receptor. The term "T cell receptor" refers to the portion of an antibody that comprises the region that specifically binds to and is complementary to a T cell receptor.

[0148] The T cell receptor may be a variable region or a constant region, but a preferred CD3 binding domain is The epitope to which the main binds is present in the constant region. For example, the T cell receptor α chain (SEQ ID NO: 67) of RefSeq accession number CAA26636.1, T cell receptor β chain (SEQ ID NO: 68) of accession number C25777, T cell receptor β chain (RefSeq accession number A26659) T cell receptor γ1 chain (SEQ ID NO: 69), T cell receptor γ2 chain (SEQ ID NO: 10) of RefSeq accession number AAB63312.1 No. 70), and the sequence of the T cell receptor δ chain (SEQ ID NO: 71) of RefSeq accession number AAA61033.1. Some examples include:

[0149] CD3-binding domain As used herein, the term "CD3-binding domain" refers to a domain that specifically binds to a part or all of CD3 and The CD3-binding domain refers to the portion of a CD3 antibody comprising a region complementary to one or more Preferably, the CD3 binding domain is provided by the variable domain of the light chain of a CD3 antibody. The variable region (VL) and heavy chain variable region (VH) of a CD3 antibody are included. Examples of such CD3-binding domains include: Examples include "scFv (single chain Fv)", "single chain antibody", Fv," "scFv2 (single chain Fv 2)," "Fab," or "F(ab')2." do.

[0150] The CD3-binding domain of the present invention is present in the γ chain, δ chain, or ε chain sequence constituting human CD3. In the present invention, the antibody may bind to any epitope as long as it is an epitope that can be expressed by the antibody. Preferably, the CD3 antibody binds to an epitope present in the extracellular region of the ε chain of the human CD3 complex. A CD3 binding domain containing a light chain variable region (VL) and a heavy chain variable region (VH) of a CD3 antibody is preferably used. Such CD3-binding domains include the OKT3 antibody (Proc. Natl. Acad. Sci. USA (1980) 77, 4914-4917) and the light chain variable region (VL) and heavy chain variable region (VL) of various known CD3 antibodies. A CD3-binding domain containing a variable region (VH) and a γ domain constituting human CD3 is preferably used. The δ chain, ε chain or δ chain is obtained by immunizing a desired animal using the above method. Any CD3-binding domain derived from a CD3 antibody having the desired properties can be used. As mentioned above, the CD3 antibody that is the main source may be a humanized antibody or a human antibody. The structure of the γ chain, δ chain, or ε chain that constitutes CD3 is determined by the polynucleotide sequence of the Column numbers: 27 (NM_000073.2), 29 (NM_000732.4) and 31 (NM_000733.3) The polypeptide sequences are shown in SEQ ID NOs: 28 (NP_000064.1), 30 (NP_000723.1) and 32 ( NP_000724.1) (RefSeq accession numbers are shown in parentheses).

[0151] Polypeptide complex The polypeptide complex of the present invention comprises the above-described (1) an antigen-binding domain, (2) a domain containing an Fc region with reduced binding activity to an Fcγ receptor; and (3) T cell receptor complex binding domain, The structure is not limited as long as it contains the T cell receptor complex. The combined binding domain is preferably a T cell receptor binding domain or a CD3 binding domain. The above domains can be directly linked by peptide bonds. For example, (1) antigen (2) F(ab')2 is used as the binding domain, and the binding activity to Fcγ receptors is reduced. When these Fc regions are used as domains containing the c region, the antigen binding described in (1) When the domain and the domain containing the Fc region described in (2) are linked by a peptide bond, The linked polypeptides form the structure of an antibody. To produce such an antibody, In addition to purifying the antibody from the culture medium of the hybridoma, the polypeptide constituting the antibody is also The antibody is purified from the culture medium of the desired host cells in which the polynucleotide encoding the antibody is stably maintained. You can also do this.

[0152] When (3) a CD3-binding domain is attached to the antibody structure, the CD3-binding domain is attached to the antibody. In another embodiment, the antibody can be linked to the C-terminus of the constant region of the antibody structure via a peptide bond. The CD3 binding domain is a peptide at the N-terminus of the heavy chain variable region or the light chain variable region of the antibody structure. In another embodiment, the CD3 binding domain can be linked via a bond. The CD3-binding domain can be linked to the C-terminus of the light chain constant region of the CD3-binding domain via a peptide bond. As the domain, a CD3-binding domain having a desired structure can be used, preferably an Fv, more preferably a Alternatively, scFv may be used as appropriate. The valency of the CD3-binding domain bound to the antibody structure is limited. In order to bind a bivalent CD3-binding domain to the antibody structure, the antibody structure must be defined. The two Fc regions that make up the common region each have a monovalent CD3-binding domain at their C-termini via a peptide bond. In addition, to attach a bivalent CD3-binding domain to the antibody structure, A bivalent scFv, i.e., sc(Fv), is attached to the C-terminus of one of the two Fc regions via a peptide bond. In this case, the Fc region derived from the bispecific antibody can be used. By this, one of the two Fc regions constituting the constant region of the antibody structure Polypeptide complexes in which a bivalent scFv, i.e., sc(Fv)2, is bound only to the C-terminus can be efficiently obtained. In addition, in order to bind a monovalent CD3-binding domain to the antibody structure, two Fc regions A monovalent scFv can be linked to the C-terminus of one of the Fc regions via a peptide bond. In the present invention, by using an Fc region derived from the bispecific antibody, the antibody structure A monovalent scFv binds to the C-terminus of only one of the two Fc regions that make up the constant region of Thus, the polypeptide complexes of the present invention can be efficiently obtained.

[0153] (3) The CD3-binding domain is attached to the C-terminus of the constant region of the antibody structure via a peptide bond. When the heavy chain Fv fragment that constitutes the CD3-binding domain constitutes the Fc region, the heavy chain Fv fragment that constitutes the CD3-binding domain constitutes the Fc region. A light chain Fv fragment that is linked to the C-terminus (CH3 domain) of the constant region of The polypeptide is linked to the C-terminus (CH3 domain) of the other constant region that constitutes the Fc region. In this case, a heavy chain Fv fragment or a light chain Fv fragment is used as a constant region. When linking to the C-terminus (CH3 domain), a linker such as Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 7) is used. The number of repeats of the linker is not limited, and may be 1 to 10, preferably 2 to 10. 8, and further selected from the numbers 2 to 6, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 or A linker such as Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 7) consisting of 10 repeats may be used as appropriate. It can be inserted.

[0154] Furthermore, the heavy chain Fv fragment constituting the CD3-binding domain is a C-terminal fragment of one of the constant regions constituting the Fc region. The Fc region is composed of the light chain Fv fragment, which is linked to the end (CH3 domain) and constitutes the CD3-binding domain A polypeptide complex was constructed in which the constant region of the corresponding polypeptide was linked to the C-terminus (CH3 domain) of the other polypeptide. When the heavy chain Fv fragment is used, the heavy chain Fv fragment is used to enhance the association between the heavy chain Fv fragment and the light chain Fv fragment. The amino acid residues may also be appropriately modified to form disulfide bonds between the Fv fragment and the light chain. It is possible.

[0155] In another embodiment, the heavy chain Fv fragment comprising the CD3 binding domain is a The light chain Fv fragment, which is linked to the C-terminus of the constant region (CH3 domain) and constitutes the CD3-binding domain, is F A polypeptide linked to the C-terminus (CH3 domain) of the other constant region that constitutes the c region When an aggregate is produced, the heavy chain Fv fragment and the light chain Fv fragment are bound to each other in order to enhance the association. The heavy chain Fv fragment and the light chain Fv fragment can be linked to the CH1 domain and the CL domain of an antibody, respectively. do.

[0156] In yet another embodiment, to attach a bivalent CD3 binding domain to the antibody structure, Peptide bonds are attached to the C-terminus of each of the two light chain constant regions or the N-terminus of each of the light chain variable regions of the antibody structure. Each monovalent CD3-binding domain can be linked via a divalent CD3 binding domain. To combine the binding domains, the C-terminus of each of the two light chain constant regions or the N-terminus of each of the light chain variable regions is A divalent scFv, i.e., sc(Fv)2, can be linked to the end via a peptide bond. In this case, By using the Fc region derived from the bispecific antibody, two of the antibody structures can be A bivalent scFv, i.e., sc(Fv)2, is bound to the C-terminus or N-terminus of one of the light chain variable regions. Furthermore, polypeptide complexes that bind to monovalent CD3 can be efficiently obtained. To combine the domains, the C-terminus or C-terminal of one of the two light chain variable regions is A monovalent scFv can be bound to the N-terminus of the above-mentioned By using an Fc region derived from a bispecific antibody, the two light chains of the antibody structure can be The present invention relates to a method for producing a light chain antibody having a monovalent scFv bound to the N-terminus or C-terminus of one of the light chain variable regions. Polypeptide complexes can be obtained efficiently.

[0157] In another embodiment, to attach a bivalent CD3 binding domain to the antibody structure, The antibody structure has two heavy chain variable regions, each with a monovalent CD3-binding domain attached via a peptide bond to the N-terminus of the heavy chain variable region. In addition, to attach a bivalent CD3-binding domain to the antibody structure, The bivalent s cFv, i.e., sc(Fv)2, can be bound. In this case, the bispecific antibody By using the Fc region, one of the two heavy chain variable regions of the antibody structure Polypeptide complexes in which a bivalent scFv, i.e., sc(Fv)2, binds only to the N-terminus of the domain are efficiently obtained. In addition, in order to attach a monovalent CD3-binding domain to the antibody structure, two heavy chains are A monovalent scFv is bound to the N-terminus of one of the heavy chain variable regions via a peptide bond. In this case, by using the Fc region derived from the bispecific antibody, Therefore, a monovalent scF is attached to the N-terminus of one of the two heavy chain variable regions of the antibody structure. Polypeptide complexes of the present invention to which v is linked can be efficiently obtained.

[0158] Furthermore, when preparing the above polypeptide complex, each domain is directly bound by a peptide bond. In addition to being linked by a peptide bond, each domain can be linked by a peptide bond via a peptide linker. In this case, the linker to be used may be any of the linkers exemplified above, as well as For example, linkers having peptide tags such as His tag, HA tag, myc tag, and FLAG tag can also be used as appropriate. In addition, hydrogen bonds, disulfide bonds, covalent bonds, ionic interactions or the like may be used. The property of binding to each other by a combination of these bonds can also be suitably utilized. For example, The affinity between CH1 and CL is utilized, and the hetero Fc region is used to form the bispecific antibody. Furthermore, as described in the Examples, the Fc region derived from the domain Disulfide bonds formed between them can also be suitably utilized.

[0159] Other examples of the structure of the polypeptide complex of the present invention include: (1) an antigen-binding domain; Monovalent Fv and monovalent Fab structures are also preferably used as the domains. (2) a polypeptide complex of the present invention having reduced Fcγ receptor-binding activity The heavy chain CH1 region is linked via a peptide bond to one of the two Fc regions. The heavy chain Fv fragment (VH) or light chain Fv fragment (VL) of the monovalent Fv is linked to the and a light chain CH region bound to the heavy chain CH1 region via a disulfide bond, wherein the light chain CH region is a monovalent Fv. The other VL or VH fragment is linked via a peptide bond to form a heavy chain CH1 region and A structure in which VH and VL bound to the ends of the light chain CL region and the VH and VL regions form an antibody binding domain is used. The other of the two Fc regions has an N-terminus containing (1) an antibody-binding domain. and (3) sc(Fv)2 forming the CD3-binding domain can be linked via a peptide bond. In this case, by using the Fc region derived from the bispecific antibody, and one of the two Fc domains constituting the polypeptide complex contains a heavy chain CH1 domain. The other Fc region is linked to sc(Fv)2 via a peptide bond. When producing the above polypeptide complexes, The domains are connected by direct peptide bonds, or by peptide linkers. In this case, the linker to be used may be any of the above-described linkers. In addition to the exemplified linkers, peptide tags such as His tag, HA tag, myc tag, and FLAG tag can also be used. A linker having the following structure may also be used as appropriate.

[0160] Other examples of the structure of the polypeptide complex of the present invention include: (1) an antigen-binding domain; A structure in which the scFv is bivalent as the domain is also preferably used. One of the two scFvs binds to the Fcγ receptor (2) via the VH that constitutes the CD3-binding domain (3). Linked by peptide bond to one of two Fc regions with reduced binding activity to The other of the bivalent scFvs binds to (2)F via the VL that constitutes (3) the CD3-binding domain. The other of the two Fc regions has reduced binding activity to the cγ receptor. In this case, a polypeptide complex having a structure in which the polypeptides are linked by a tide bond can be produced. In the above-mentioned method, the Fc region derived from the bispecific antibody can also be used. When preparing a peptide complex, each domain is directly linked by a peptide bond, or Each domain can be linked by a peptide bond via a peptide linker. The linker to be used may be any of the linkers exemplified above, as well as, for example, His tag, HA Linkers having peptide tags such as tag, myc tag, and FLAG tag can also be used as appropriate.

[0161] (1) Another embodiment of the structure in which a bivalent scFv is used as the antigen-binding domain is: One of the bivalent scFvs binds to the Fcγ receptor via the scFv that comprises the CD3-binding domain (3). The Fc region is linked by a peptide bond to one of the two Fc regions that have reduced binding activity to the The other of the bivalent scFvs has reduced binding activity to the Fcγ receptor (2). A polynucleotide having a structure in which the other of the two Fc regions is linked by a peptide bond. In this case, a peptide complex originating from the bispecific antibody can be produced. By using an Fc domain, one of the two Fc domains constituting the polypeptide complex can be expressed as The Fc region of one scFv contains a CD3-binding domain, and the other scFv contains an antigen-binding domain. The scFv, which constitutes the antigen-binding domain in the other Fc region, binds to the scFv via peptide bonds. A polypeptide complex having a structure in which the polypeptide complexes are linked together can be produced. When constructing the antibody, each domain is directly linked by a peptide bond, and each domain is also linked by a peptide bond. The linker may be a peptide bond. Examples of linkers include the above-mentioned linkers as examples, as well as His tags, HA tags, myc tags, and FLAG tags. Linkers having peptide tags such as tags may also be used as appropriate.

[0162] Other examples of the structure of the polypeptide complexes of the present invention include those having an antigen-binding domain and A structure in which the T cell receptor complex binding domain and the T cell receptor complex binding domain are each monovalent Fab is also preferably used. In one embodiment of this structure, the heavy chain Fv fragment of a monovalent Fab constituting the antigen-binding domain is CH1. The light chain Fv fragment of the Fab is linked to one of the polypeptides constituting the Fc region via the C The heavy chain Fv fragment of Fab, which is linked to the L region and constitutes the T cell receptor binding domain, binds to the Fab via the CH1 region. The light chain Fv fragment of the Fab is linked to the other polypeptide constituting the Fc region, and the light chain Fv fragment of the Fab is linked to the CL region. A polypeptide complex having a structure in which the polypeptide complex is bound can be produced.

[0163] Another embodiment of such a structure is the heavy chain Fv fragment of a monovalent Fab that constitutes the antigen-binding domain. is linked to one of the polypeptides constituting the Fc region via the CH1 region, and the light chain Fv fragment of the Fab The light chain Fv fragment of Fab, which constitutes the T cell receptor binding domain, is linked to the CL region. The heavy chain Fv fragment of the Fab is linked to the other polypeptide constituting the Fc region via a nucleotide sequence. Polypeptide complexes having a structure in which the polypeptide is linked to a T cell receptor-binding domain can be produced. The heavy chain Fv fragment of the monovalent Fab constituting the binding domain is connected via the CH1 region to one of the two constituting the Fc region. a polypeptide, and the light chain Fv fragment of the Fab is linked to the CL region, forming an antigen-binding domain The light chain Fv fragment of Fab constituting the Fc region is linked via the CH1 region to the other polypeptide constituting the Fc region. and a polypeptide complex having a structure in which the heavy chain Fv fragment of the Fab is linked to the CL domain. It can also be made.

[0164] In another embodiment of such a structure, the heavy chain F of a monovalent Fab constituting the antigen-binding domain is The Fab fragment is linked to one of the polypeptides constituting the Fc region via the CH1 region, and The heavy chain Fv fragment of Fab, which constitutes the T cell receptor binding domain, is linked to the CL region. The light chain Fv fragment of the Fab is linked to the other polypeptide constituting the Fc region via the C Polypeptide complexes having a structure in which the H1 domain is linked to the T cell receptor agonist can be produced. The heavy chain Fv fragment of the monovalent Fab that constitutes the antibody-binding domain is connected via the CH1 region to form the Fc region. The light chain Fv fragment of the Fab is linked to the CL region, forming an antigen-binding domain. The heavy chain Fv fragment of Fab constituting the nucleotide sequence is coupled to the other polypeptide constituting the Fc region via the CL region. and the light chain Fv fragment of the Fab is linked to the CH1 domain. A body can be created.

[0165] Another structure of the polypeptide complex of the present invention is an antigen-binding domain and a T cell receptor domain. In one embodiment, each receptor complex binding domain is a monovalent Fab, (1) A monovalent Fab heavy chain Fv fragment that binds to an antigen constitutes the Fc region via the CH1 region. The light chain Fv fragment of the Fab structure is linked to the CL region of the antigen-binding domain. a binding domain, and (2) The heavy chain Fv fragment of the monovalent Fab structure that binds to the T cell receptor complex binds to the Fc domain via the CH1 domain. The light chain Fv fragment of the Fab structure is linked to the other polypeptide constituting the CL region. a T cell receptor complex binding domain, a heavy chain Fv fragment in the antigen-binding domain and a light chain Fv fragment in the antigen-binding domain or a T cell The heavy chain Fv fragment in the T cell receptor binding domain and the light chain Fv fragment in the T cell receptor binding domain associate Suitable examples include polypeptides in which the charges of the CH1 and CL regions are controlled so that the polypeptides have the same structure. In this embodiment, a heavy chain Fv fragment in the antigen-binding domain and a light chain Fv fragment in the antigen-binding domain or a heavy chain Fv fragment in the T cell receptor binding domain and a light chain Fv fragment in the T cell receptor binding domain The charges of the CH1 and CL regions must be controlled so that the fragments can assemble together. The structure of the aggregate (aggregation-controlling structure) is not limited to a specific structure.

[0166] As one embodiment of the association-regulating structure, a heavy chain Fv fragment in the T cell receptor complex binding domain is and the amino acid residues of the CH1 region linked to the light chain Fv fragment in the antigen-binding domain. A polypeptide complex is created in which the amino acid residues in the linked CL domain have the same electric charge. It is possible.

[0167] In another embodiment of the association-controlling structure, the heavy chain Fv fragment in the antigen-binding domain is linked to The amino acid residues of the CH1 region and the light chain Fv fragment in the T cell receptor complex binding domain are linked to the A polypeptide complex is created in which the amino acid residues in the CL domain have the same charge. obtain.

[0168] In a further embodiment of the association-regulating structure, Amino acid residues of the CH1 region linked to the heavy chain Fv fragment and the light chain Fv in the antigen-binding domain The amino acid residues in the CL region linked to the fragments have the same charge, and Amino acid residues of the CH1 region linked to the heavy chain Fv fragment and the T cell receptor complex binding domain of The amino acid residues of the CL region linked to the light chain Fv fragment in the polypeptide have the same charge. Tide aggregates can be produced.

[0169] In addition, as one embodiment of the association-regulating structure, a heavy chain in the T cell receptor complex-binding domain The amino acid residues of the CH1 region linked to the light chain Fv fragment and the light chain Fv fragment in the antigen-binding domain The amino acid residues in the CL region linked to the fragment have the same charge, and the T cell receptor complex Amino acid residues in the CH1 region linked to the heavy chain Fv fragment in the T cell binding domain and the T cell receptor The amino acid residues in the CL region linked to the light chain Fv fragment in the antibody-binding domain have opposite charges. A polypeptide complex having the above structure can be produced.

[0170] In another embodiment of the association-regulating structure, the T cell receptor complex-binding domain The amino acid residues of the CH1 region linked to the heavy chain Fv fragment of the antigen-binding domain of the light chain F The amino acid residues of the CL region linked to the v fragment have the same charge as each other, and the antigen-binding domain The amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the DNA and the T cell receptor complex The amino acid residues of the CL region linked to the light chain Fv fragment in the binding domain have the same charge. the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the T cell receptor complex binding domain; and the amino acid residues of the CL region linked to the light chain Fv fragment in the T cell receptor binding domain. Polypeptide complexes in which the groups have opposite charges can be produced.

[0171] Furthermore, as one embodiment of the association-regulating structure, The amino acid residues of the CH1 region and the light chain Fv fragment in the T cell receptor complex binding domain The amino acid residues of the CL region linked to the fragment have the same charge, and the antigen-binding domain and the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen-binding domain. The amino acid residues in the CL region linked to the Fv fragment of each chain have opposite charges. Coalescence can be created.

[0172] Furthermore, as another embodiment of the association-regulating structure, the T cell receptor complex-binding domain and the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen-binding domain. The amino acid residues of the CL region linked to the chain Fv fragment have the same charge, and the antigen-binding domain Amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the T cell receptor complex The amino acid residues in the CL region linked to the light chain Fv fragment in the binding domain have the same charge. The amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen-binding domain and the corresponding The amino acid residues of the CL region linked to the light chain Fv fragment in the antigen-binding domain are heterologous. Polypeptide complexes bearing a specific amino acid sequence can be produced.

[0173] In addition, as a different embodiment of the association control structure, the T cell receptor complex binding domain The amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the The amino acid residues in the CL region linked to the light chain Fv fragment have the same charge, and Amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the domain and the T cell receptor complex The amino acid residues in the CL region linked to the light chain Fv fragment in the antibody-binding domain have the same charge. and an amino acid sequence of the CH1 region linked to the heavy chain Fv fragment in the T cell receptor complex binding domain. and the amino acid residues of the CL region linked to the light chain Fv fragment in the T cell receptor binding domain. The acid residues have opposite charges to each other, and the CH1 region is linked to the heavy chain Fv fragment in the antigen-binding domain. amino acid residues in the CL region linked to the light chain Fv fragment in the antigen-binding domain Polypeptide complexes in which residues have opposite charges can be produced.

[0174] Controlling the charge of the CH1 and CL regions The heavy and light chains of the T cell receptor binding domain bind to the epitope of the T cell receptor binding domain. and recognizes an epitope of an antigen by the heavy and light chains of the antigen-binding domain. When a bispecific polypeptide complex is desired to be obtained, the following steps are required for the production of the polypeptide complex: If each of the four chains is expressed, theoretically, 10 types of polypeptide complex molecules will be produced. There is a possibility that this may occur.

[0175] In this case, for example, a heavy chain of a T cell receptor binding domain and a light chain of an antigen binding domain and and / or inhibiting the association between the heavy chain of the antigen-binding domain and the light chain of the T cell receptor-binding domain. By controlling the expression of the polypeptide complexes in this way, it is possible to preferentially obtain the desired polypeptide complex molecules. do.

[0176] For example, the interface between the heavy chain CH1 of the T cell receptor binding domain and the light chain CL of the antigen binding domain is formed. The amino acid residues forming the heavy chain of the antigen-binding domain are modified to positively charged amino acid residues. The amino acid residues that form the interface between CH1 and the light chain CL of the T cell receptor binding domain are negatively charged. For example, an amino acid residue can be modified to one that is related to the target T. The association of the heavy chain CH1 of the cell receptor binding domain with the light chain CL of the antigen binding domain forms an interface. Both of the amino acid residues involved are positively charged, which inhibits the formation of unintended antigen-binding domains. The association between the heavy chain CH1 and the light chain CL of the T cell receptor binding domain involves determining which amino acid residues form an interface. Both are negatively charged and are therefore inhibited. The association of the heavy chain CH1 with the light chain CL of the T cell receptor binding domain and the target antigen binding domain The polypeptide complex of the present invention in which the heavy chain CH1 and the antigen-binding domain of the light chain CL are associated is effective. Preferably, the heavy chain of the T cell receptor binding domain of interest and the T cell The association of the cellular receptor binding domain with the light chain occurs because the amino acid residues forming the interface are of different electrical conductivity. The heavy chain of the antigen-binding domain of interest and the light chain of the antigen-binding domain are promoted by having a charge. Association with the chain is also facilitated by the opposite charges of the amino acid residues that form the interface. As a result, polypeptide complexes of the present invention in which the desired assembly has occurred can be efficiently obtained. .

[0177] Furthermore, by utilizing the association regulation of the present invention, CH1s (T cell receptor binding domains) can be easily linked together. heavy chain and heavy chain of antigen-binding domain), or CLs (light chain and T cell receptor-binding domain) It is also possible to inhibit the association of the antigen-binding domain (light chain).

[0178] Those skilled in the art will readily understand the mechanisms underlying the synthesis of desired polypeptide complexes whose association is to be controlled by the present invention. It is possible to appropriately determine the types of amino acid residues that approach each other at the interface between CH1 and CL upon association. It is Noh.

[0179] It can also be used as CH1 or CL of antibodies in organisms such as humans, monkeys, mice, and rabbits. Those skilled in the art can appropriately obtain such sequences by using public databases, etc. More specifically, amino acid sequence information of CH1 or CL is obtained by the means described in the Examples below. It is possible.

[0180] For example, as shown in the Examples below, a T cell receptor binding domain or an antigen binding domain At the interface between CH1 and CL when CH1 and CL, which are linked to VH and VL, respectively, constituting the main Specific examples of amino acid residues that are close (facing or contacting) in the above sequence include the following combinations: can be. Position 147 (EU numbering) of CH1 (for example, 147 in the amino acid sequence set forth in SEQ ID NO: 1) Lysine (K) at position 180 of the CL (EU numbering) and threonine (T) at position 180 of the CL (EU numbering) Lysine (K) at position 147 (EU numbering) of CH1 and lysine (K) at position 131 (EU numbering) of CL, which faces (contacts) Serine(S) Lysine (K) at position 147 (EU numbering) of CH1 and lysine (K) at position 164 (EU numbering) of CL, which faces (contacts) Threonine (T) Lysine (K) at position 147 (EU numbering) of CH1 and lysine (K) at position 138 (EU numbering) of CL, which faces (contacts) Asparagine (N) Lysine (K) at position 147 (EU numbering) of CH1 and lysine (K) at position 123 (EU numbering) of CL, which faces (contacts) Glutamic acid (E) The glutamine (Q) at position 175 in the EU numbering of CH1 and the corresponding (contacting) glutamine (Q) at position 175 in the EU numbering of CL Glutamine (Q) at 160th place Lysine (K) at position 213 (EU numbering) of CH1 and lysine (K) at position 123 (EU numbering) of CL, which faces (contacts) Glutamic acid (E) The numbering of these regions is based on the reference by Kabat et al. (Kabat EA et al. 1991. Sequence of Proteins of Immunological Interest (NIH) was used as a reference. In addition, the numbers described as EU numbering in the present invention are EU numbering (Sequences of proteins of immunological interest, NIH Publication No. 91-3242 In the present invention, "amino acid residue at position X in EU numbering" and "Amino acid at position X in EU numbering" (X is an arbitrary number) is "an amino acid corresponding to position X in EU numbering" It can also be read as "amino acid residue" or "amino acid corresponding to position X in EU numbering." do.

[0181] As will be shown in the Examples below, by modifying these amino acid residues and carrying out the method of the present invention, In this way, the desired polypeptide complex can be preferentially obtained.

[0182] These amino acid residues are known to be highly conserved in humans and mice. (J. Mol. Recognit. (2003) 16, 113-120) Therefore, the polypeptide association shown in the examples For the association of CH1 and CL other than the combination, the amino acid residues corresponding to the above amino acid residues were modified. By doing so, the association of the constant region of the polypeptide complex of the present invention can be regulated.

[0183] That is, the present invention provides a polypeptide complex in which heavy chain and light chain association is controlled, comprising the following: One or more pairs of amino acid residues selected from the group consisting of pairs of amino acid residues shown in (a) to (f) providing a polypeptide complex in which amino acid residues have the same electric charge; (a) an amino acid residue at position 147 (EU numbering) contained in CH1, and a CL an amino acid residue at position 180 in the EU numbering system; (b) an amino acid residue at position 147 (EU numbering) contained in CH1, and CL an amino acid residue at position 131 according to EU numbering, (c) an amino acid residue at position 147 (EU numbering) contained in CH1, and CL an amino acid residue at position 164 in the EU numbering system; (d) an amino acid residue at position 147 (EU numbering) contained in CH1, and CL an amino acid residue at position 138 according to EU numbering, (e) an amino acid residue at position 147 (EU numbering) contained in CH1, and CL an amino acid residue at position 123 in the EU numbering system; (f) an amino acid residue at position 175 (EU numbering) contained in CH1, and CL The amino acid residue at position 160 in the EU numbering system is included in

[0184] In another embodiment of the present invention, the amino acid residues of the set of amino acid residues shown in (g) below are further included. providing antibodies in which the groups are of like charge; (g) an amino acid residue at position 213 (EU numbering) contained in CH1, and CL The amino acid residue at position 123 in the EU numbering system is included in

[0185] As shown in the examples below, each of the amino acid residues in the above combinations can be associated with Those skilled in the art can easily use commercially available software to find the desired CH1 or CL. By homology modeling using the above, the amino acid residues (a) to (g) It is possible to find a site corresponding to the amino acid sequence and modify the amino acid residue at that site as appropriate. is.

[0186] In the above-mentioned antibody, the "charged amino acid residue" is, for example, the following (X) or ( Y) is preferably selected from the amino acid residues included in any one of the groups; (X) glutamic acid (E), aspartic acid (D), (Y) Lysine (K), Arginine (R), Histidine (H).

[0187] In the above-described polypeptide complex, "having the same type of charge" means, for example, that two or more Any of the amino acid residues is an amino acid included in either group (X) or (Y) above. "Having opposite charges" means, for example, that two or more amino groups At least one amino acid residue among the amino acid residues is either (X) or (Y) above. If the amino acid residues are in one group, the remaining amino acid residues are in a different group. It means that the amino acid residues are

[0188] Furthermore, the method for producing the above-mentioned polypeptide complex and the amino acid sequences shown in (a) to (g) above The amino acid residues of the pair of amino acid residues are modified to have the same charge. The method for controlling association of the present invention is also a preferred embodiment of the present invention.

[0189] In the present invention, the amino acid residues to be "modified" include the amino acids in the constant region described above. Those skilled in the art will appreciate that polypeptide variants or heteromultimers are not limited to the amino acid residues. The amino acid residues that form the interface were identified by homology modeling using commercially available software. and modifying the amino acid residues at the site to control the association. is possible.

[0190] Introducing charge repulsion at the interface between the heavy chain variable region and the light chain variable region prevents unwanted heavy and light chain In this technology, the binding between the heavy chain variable region (VH) and the light chain variable region (VL) is suppressed. The amino acid residue that contacts the heavy chain variable region FR2 is, for example, position 39 (see, for example, WO2006 / 106905 Glutamine (Q) at position 39 in the amino acid sequence set forth as SEQ ID NO: 6 in and position 38 of the opposing (contacting) light chain variable region FR2 (for example, SEQ ID NO: 1 in WO2006 / 106905). :8) may be mentioned. Furthermore, position 45 of the heavy chain variable region FR2 (for example, the sequence shown in WO2006 / 106905 as SEQ ID NO: 6) The leucine (L) at position 45 in the amino acid sequence shown in Fig. 1 corresponds to the 44 position (for example, the 50th position in the amino acid sequence set forth in SEQ ID NO: 8 in WO2006 / 106905) A suitable example is proline (P) at the position K. Kabat et al. (1991. Sequence of Proteins of Immunological Interests) This is based on the National Institutes of Health (NIH).

[0191] These amino acid residues are known to be highly conserved in humans and mice. (J. Mol. Recognit. (2003) 16, 113-120) Therefore, the polypeptide association shown in the examples For the association of VH and VL other than the combination, the amino acid residues corresponding to the above amino acid residues may be modified. By doing so, the association of the antibody variable regions can be controlled.

[0192] More specifically, in an antibody comprising a heavy chain variable region and a light chain variable region, the following (1) ) and (2), or (3) and (4) are amino acid residues with the same charge The antibody may be an antibody which is (1) An amino acid residue contained in the heavy chain variable region, which corresponds to position 39 in the EU numbering system amino acid residues, (2) an amino acid residue contained in the light chain variable region, the amino acid corresponding to position 38 in the EU numbering system; amino acid residues, (3) an amino acid residue contained in the heavy chain variable region, the amino acid corresponding to position 45 in the EU numbering system; amino acid residues, (4) an amino acid residue in the light chain variable region corresponding to position 44 in the EU numbering system; amino acid residues.

[0193] The amino acid residues described in (1) and (2), (3) and (4) above are, when associated, Those skilled in the art can easily find a commercially available heavy chain variable region or light chain variable region of interest. By homology modeling using commercially available software, etc., the above (1) to (4) A site corresponding to the amino acid residue can be found, and the amino acid residue at that site can be appropriately modified. It is possible to provide

[0194] In the above-described antibody, the "charged amino acid residue" is, for example, the following (X) or (Y ) is preferably selected from amino acid residues included in any one of the groups: (X) glutamic acid (E), aspartic acid (D), (Y) Lysine (K), Arginine (R), Histidine (H).

[0195] The amino acid residues described in (1) to (4) above are generally expressed in humans and mice. Each (1) Glutamine (Q), (2) glutamine (Q), (3) leucine (L), (4) Proline (P) Therefore, in a preferred embodiment of the present invention, these amino acid residues are modified (e.g., The types of amino acid residues (1) to (4) above are The amino acid residues are not necessarily limited to those mentioned above, and may be other amino acids corresponding to those amino acids. For example, the amino acid corresponding to position 38 in the EU numbering system in the light chain variable region may be human. In the case of α-amino acid, it may be, for example, histidine (H). Those skilled in the art can refer to known literature (for example, J. Mol. Recognit. (2003) 16, 113-120) for any position in the light chain. It is possible to know the type of amino acid residue corresponding to that position, and the amino acid residue corresponding to that position can be appropriately determined. Acid residues can be modified (eg, substituted with charged amino acids).

[0196] The amino acid residues that form the hydrophobic core at the interface between the heavy chain variable region and the light chain variable region are In this technology, undesired association of heavy and light chains is suppressed by modifying the heavy and light chains to polar amino acids with a heavy charge. In this case, amino acids that can form a hydrophobic core at the interface between the heavy chain variable region (VH) and the light chain variable region (VL) are The acid residues include, for example, leucine (L) at position 45 in the heavy chain variable region and the corresponding leucine (L) at position 46 in the light chain variable region. A suitable example is proline (P) at position 44 on the amino acid sequence.

[0197] Generally, the "hydrophobic core" refers to the interior of an assembled polypeptide. It refers to the portion formed by the aggregation of side chains of hydrophobic amino acids. Nine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan The hydrophobic core is formed by amino acids other than hydrophobic amino acids. This hydrophobic core is flanked by hydrophilic amino acids. The hydrophilic surface of the chains, together with the externally exposed hydrophilic surface, is the driving force for the association of water-soluble polypeptides. The hydrophobic amino acids in the two different domains are located on the molecular surface and are exposed to water molecules. As the entropy increases, the free energy also increases. To reduce the free energy and stabilize the molecules, they associate with each other, and the hydrophobic amino acids at the interface are separated. The molecules are embedded inside the molecule, forming a hydrophobic core.

[0198] When polypeptides assemble, charges are transferred from the hydrophobic amino acids that form the hydrophobic core. By modifying the amino acids to polar amino acids, the formation of the hydrophobic core is inhibited, resulting in the formation of a poly It is believed that the association of the peptides is inhibited.

[0199] Other known techniques can also be applied to the polypeptide complexes of the present invention. For example, a first VH (VH1) and a first VL (VL1), and / or a second VH (VH2) and a second VL (VL2) In addition to the "modification" of the present invention, a nucleotide sequence present in the variable region of one of the H chains may be added to promote the association of the two H chains. The amino acid side chain of the other H chain is replaced with a larger side chain (knob), and the corresponding By replacing the amino acid side chains in the variable region with smaller side chains (holes), The protrusions can be arranged in the gap to promote the association of VH1 and VL1, and / or VH2 and VL2. As a result, the association between the VH1 and VL2 polypeptides and / or the VH2 and VL1 polypeptides is further inhibited. It is possible to achieve this (WO1996 / 027011, Ridgway JB et al., Protein Engineering (1996) 9, 617-621, Merchant AM et al. Nature Biotechnology (1998) 16, 677-681).

[0200] When preparing the above polypeptide complex, each domain is directly linked via a peptide bond. Alternatively, each domain can be linked by a peptide bond via a peptide linker. In this case, the linker to be used may be any of the linkers exemplified above, as well as, for example, His tag. Linkers having peptide tags such as HA tag, myc tag, and FLAG tag can also be used as appropriate. Also, hydrogen bonds, disulfide bonds, covalent bonds, ionic interactions, or these bonds For example, the CH1 and CL of an antibody can be used in combination to bind to each other. The affinity between the two Fc regions is utilized, and the aforementioned bispecific antibodies originate from the association of hetero Fc regions. Furthermore, as described in the Examples, an Fc region formed between domains may be used. Disulfide bonds that are bonded to the hydroxyl group can also be suitably used.

[0201] Examples of polypeptide complexes of the present invention include those shown in Figures 17, 19, and 24. Examples of such embodiments include:

[0202] The polypeptide complexes of the present invention are produced by the same method as the recombinant antibody production method described above. can be.

[0203] The present invention also relates to polynucleotides encoding the polypeptide complexes of the present invention. The polypeptide complexes of the present invention can be incorporated into any expression vector. A suitable host can be transformed with the vector to produce cells that express the polypeptide complex. The polypeptide complex can be isolated by culturing cells expressing the polypeptide complex and recovering the expression product from the culture supernatant. Thus, a polypeptide complex encoded by the nucleotide of the present invention can be obtained. The present invention also provides a vector comprising a polynucleotide encoding the polypeptide complex of the present invention, Cells harboring the vector, and the polypeptide complexes obtained by culturing the cells and recovering them from the culture supernatant These methods include, for example, producing polypeptide complexes using the recombinant It can be obtained by the same techniques as for antibodies.

[0204] Pharmaceutical Composition In another aspect, the present invention provides an antibody comprising: (1) an antigen-binding domain; (2) a binding domain to an Fcγ receptor; (3) a domain containing an Fc region with reduced binding activity, and a CD3-binding domain. The present invention also provides a pharmaceutical composition containing a peptide complex as an active ingredient. A therapeutic agent that induces cell damage (a therapeutic agent that induces cell damage) containing the body as an active ingredient, The pharmaceutical composition of the present invention is used as a cancer treatment agent or a cancer prevention agent. The cytotoxicity-inducing therapeutic agent, cell proliferation inhibitor and anticancer agent of the present invention can also be used as It is preferably administered to a subject who has cancer or is at risk of recurrence.

[0205] In addition, in the present invention, (1) an antigen-binding domain, (2) a domain containing an Fc region with reduced binding activity to an Fcγ receptor, and (3) CD3 binding domain, A therapeutic agent for inducing cytotoxicity, a therapeutic agent for inhibiting cell proliferation, and a therapeutic agent for inhibiting cell proliferation comprising a polypeptide complex comprising the compound as an active ingredient. The anticancer agent and anticancer agent are used to prevent or treat cancer, which comprises administering the polypeptide complex to a subject. or in the manufacture of a cytotoxicity-inducing therapeutic agent, a cell growth inhibitor, or an anticancer agent. This can also be expressed as use of the polypeptide complex in a therapeutic agent.

[0206] In the present invention, "(1) an antigen-binding domain, (2) a domain having low binding activity to an Fcγ receptor" a polypeptide chain comprising a domain containing an Fc region, and (3) a CD3-binding domain; "Containing the polypeptide complex as an active ingredient" means that the polypeptide complex is used as the main active ingredient. This does not limit the percentage of the polypeptide complex contained.

[0207] Furthermore, the pharmaceutical composition, cytotoxicity-inducing therapeutic agent, cell proliferation inhibitor, and If necessary, multiple types of polypeptide complexes can be formulated into the anticancer drug. By preparing a cocktail of multiple polypeptide complexes of the present invention that bind to a single antigen, It may be possible to enhance the cytotoxic effect against cells expressing the antigen. In addition to the polypeptide complexes of the present invention containing antigen-binding domains for one antigen, By incorporating a polypeptide complex of the present invention containing an antigen-binding domain that binds to an antigen, This enhances the therapeutic effect.

[0208] If necessary, the polypeptide complexes of the present invention can be packaged in microcapsules (e.g., hydroxymethyl Encapsulated in microcapsules made of cellulose, gelatin, poly(methyl methacrylate), etc. , colloidal drug delivery systems (liposomes, albumin microspheres, microspheres) (Remington's Pharmaceuticals, Inc., 2004) Optical Science 16 th (See, for example, "Oslo Ed. (1980)"). Methods for preparing the polypeptide complexes of the present invention as preparations are also known, and these methods can be applied to the polypeptide complexes of the present invention (J. Biomed. Mater. Res. (1981) 15, 267-277, Chemtech. (1982) 12, 98-105, U.S. Pat. No. 377 No. 3719, European Patent Publication No. EP58481 / EP133988, Biopolymers (1983) 22, 547-556) .

[0209] The pharmaceutical composition, cell growth inhibitor and anticancer agent of the present invention may be administered orally or parenterally. The compound can be administered to a patient by either oral or parenteral administration, preferably parenteral administration. Specific methods include injection, nasal administration, pulmonary administration, and transdermal administration. Injection may be, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc. For example, the pharmaceutical composition of the present invention, the cytotoxicity-inducing therapeutic agent, the cell Antiproliferative agents and anticancer agents can be administered systemically or locally. The dosage can be selected appropriately depending on the patient. The dosage can be selected from the range of 0.0001 mg to 1000 mg per kg of body weight. The dosage can be selected within the range of 0.001 mg / body to 100,000 mg / body. However, the dosage of the pharmaceutical composition of the present invention is not limited to these amounts.

[0210] The pharmaceutical composition of the present invention can be formulated according to conventional methods (for example, Remington's Ph.D. armaceutical Science, latest edition, Mark Publishing Company, Easton, USA), Pharmaceutically acceptable carriers and additives may also be included. For example, surfactants, excipients, etc. Vehicle, coloring agent, flavoring agent, preservative, stabilizer, buffer, suspending agent, isotonic agent, binder, disintegrating agent, Examples of the additives include lubricants, flow enhancers, and flavoring agents. A carrier can be appropriately used. Specifically, light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, etc. starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethyl cellulose Aminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride, poly Oxyethylene hydrogenated castor oil 60, white sugar, carboxymethyl cellulose, corn starch, Examples of the carrier include inorganic salts.

[0211] The present invention also relates to a polypeptide of the present invention that binds to a cell expressing a cancer antigen and the cancer antigen. A method for injuring cells expressing the cancer antigen by contacting the cells with the aggregate, or The monoclonal antibody that binds to the cancer antigen is a compound of the present invention. The present invention relates to a compound that binds to the cancer antigen and is contained in a clear cytotoxicity-inducing therapeutic agent, a cell growth inhibitor, and an anticancer agent. The polypeptide complexes of the present invention that bind to the cancer antigen are as described above. The cells to which the polypeptide complex binds are particularly those expressing the cancer antigen. In the present invention, preferred cancer antigen-expressing cells are specifically those of ovarian cancer, Suitable cancer cells include prostate cancer, breast cancer, uterine cancer, liver cancer, lung cancer, pancreatic cancer, stomach cancer, bladder cancer, and colon cancer cells. When the cancer antigen is GPC3, the cancer cells are not limited to those expressing GPC3. Although not limited to these, preferred cancer cells include hepatocellular carcinoma, lung cancer, ovarian cancer, and the like.

[0212] In the present invention, "contact" refers to, for example, contact with a culture medium of cancer antigen-expressing cells cultured in a test tube. This is carried out by adding a polypeptide complex of the present invention that binds to the cancer antigen to the tumor. In this case, the polypeptide complex to be added may be in the form of a solution or lyophilized. When the compound is added as an aqueous solution, it may be in the form of a solid or the like obtained by the above method. The aqueous solution may be an aqueous solution containing only the polypeptide complex of the present invention, or may be, for example, an aqueous solution containing only the polypeptide complex described above. Surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffers, suspending agents, and isotonicity agents It may also be a solution containing a binder, a disintegrant, a lubricant, a flow enhancer, a flavoring agent, etc. The concentration is not particularly limited, but the final concentration in the culture medium is preferably 1 pg / ml to 1 g / ml. More preferably, the range is from 1 ng / ml to 1 mg / ml, and even more preferably from 1 μg / ml to 1 mg / ml may be suitably used.

[0213] In another embodiment of the present invention, "contact" refers to transplanting cancer antigen-expressing cells into the body. By administering the antigen to non-human animals or animals that have cancer cells that endogenously express the cancer antigen, The administration method can be either oral or parenteral. Or parenteral administration, and specific examples of such administration methods include injection administration, Examples of administration by injection include intranasal administration, pulmonary administration, and transdermal administration. For example, injection administration of the present invention may be performed by injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection. The pharmaceutical composition, or the cytotoxicity-inducing therapeutic agent, the cell proliferation inhibitor and the anticancer agent are administered systemically or The administration method should be selected appropriately depending on the age and symptoms of the test animal. When administered as an aqueous solution, the polypeptide complex of the present invention can be administered purely. The aqueous solution may contain only the surfactant, excipient, colorant, etc. Flavoring agents, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, fluidity It may be a solution containing an enhancer, a flavoring agent, etc. The dosage may be, for example, The dosage can be chosen in the range of 0.0001 mg to 1000 mg per kg of body weight. The dose can be selected within the range of 0.001 to 100,000 mg / body per patient. The dosage of the polypeptide complex of the invention is not limited to these dosages.

[0214] Contact of the polypeptide complex of the present invention with the antigen-binding protein that constitutes the polypeptide complex The cytotoxicity caused to cells expressing the antigen to which the binding domain binds is evaluated or measured. The following methods are preferably used: Examples of methods for measuring the activity of cytotoxic T cells include those for measuring the activity of cytotoxic T cells. Whether or not the polypeptide complex of the present invention has T cell cytotoxicity can be determined by known methods. (See, for example, Current protocols in Immunology, Chapter 7. Immunologic studies in humans, Editor, John E, Coligan et al., John Wiley & Sons, Inc.,(1993 ) etc.) When measuring activity, the antigen to which the antigen-binding domain binds may be different from that of the present invention. A polypeptide complex that binds to an antigen that is not expressed by the cells used in the test. was used as a control in the same manner as for the polypeptide complex of the present invention, and The complex exhibited stronger cytotoxic activity than the polypeptide complex used as a control. The activity can be determined.

[0215] Furthermore, in order to evaluate or measure cytotoxic activity in vivo, for example, the polypeptide of the present invention can be used. Cells expressing the antigen to which the antigen-binding domain constituting the aggregate binds are cultured in a non-human test animal. After intradermal or subcutaneous implantation, the test polypeptide complex is administered daily or at intervals of several days from the day of or the following day. It is administered intravenously or intraperitoneally. The size of the tumor is measured over time to determine its size. The difference in the change in size can be defined as the cytotoxic activity. The polypeptide complex of the present invention was administered, and tumor size was reduced in the group administered the polypeptide complex of the present invention. The size of the tumors was significantly smaller than that in the control polypeptide complex-treated group. It can be determined to have cytotoxic activity.

[0216] Antigen binding of the polypeptide complex of the present invention due to contact of the polypeptide complex Method for evaluating or measuring the inhibitory effect on the proliferation of cells expressing an antigen to which the domain binds The preferred methods for measuring the uptake of isotope-labeled thymidine into cells and the MTT method are Furthermore, as a method for evaluating or measuring the cell proliferation inhibitory activity in vivo, the above-described biological The same methods as those for evaluating or measuring cytotoxic activity in vivo can be suitably used. .

[0217] The present invention also provides polypeptide complexes of the present invention or polypeptide complexes produced by the production methods of the present invention. A kit for use in the method of the present invention is provided, which comprises a polypeptide complex. The package also contains other pharmaceutically acceptable carriers, media, instructions for use, etc. You can keep it that way. The present invention also provides a polypeptide complex of the present invention or a polypeptide complex of the present invention for use in a method of the present invention. The present invention relates to polypeptide complexes produced by the production methods of the present invention.

[0218] All prior art documents cited in this specification are hereby incorporated by reference. It can be put in. [Example]

[0219] The present invention will be described in more detail below with reference to examples, but these examples are not intended to limit the scope of the present invention. It is not a restriction.

[0220] Example 1: Preparation and analysis of GPC3 ERY2 (1) Overview One way to extend the blood half-life of a protein administered to the body is to use a A well-known method is to add the Fc domain of an antibody and utilize the recycling function via FcRn. However, when native Fc is added to BiTE, one molecule contains the anti-CD40 of the BiTE portion. 3 Binds to T cells via scFv and simultaneously binds to NK cells, macrophages, etc. via the Fc portion By binding to Fcγ receptors (Fcγ receptors) on the cell membrane of cancer cells, It may activate the IL-1 receptor by cross-linking, leading to the induction of various cytokines. Therefore, we considered that the binding of BiTE to Fcγ receptors via a polypeptide linker A molecule called ERY2 was created by linking an Fc region with reduced activity (silent Fc). The activity of BiTE was compared with that of BiTE. The scFv of an antibody against Glypican 3 (GPC3), a GPI-anchored protein, and CD3 epsilon The scFv of the antibody against GPC3 was linked with a short peptide linker to form a BiTE against GPC3. GPC3 BiTE was then constructed (Figure 17A). For comparison, a GPC3 ERY2 (GPC3 ERY2) antibody was generated against the normal IgG anti-GPC3 antibody (Figure 17C). The IgG type anti-GPC3 antibody was known to have enhanced ADCC activity. Antibodies with reduced fucose content in the glycan moiety, i.e., low-fucose antibodies, was prepared as.

[0221] (2) Preparation of GPC3 BiTE Using the anti-GPC3 antibody expression vector as a template, H was amplified by PCR. The cDNAs encoding the chain variable region (anti-GPC3 VH) and the L chain variable region (anti-GPC3 VL) were The cDNA was then used as a template for PCR amplification. As a result, anti-GPC3 VH and anti-GPC3 VL were linked together with 3 Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 7). Anti-GPC3 sc having an amino acid sequence linked by a linker consisting of a sequence repeated 10 times A cDNA fragment encoding the Fv was generated.

[0222] In addition, the heavy chain variable region (M12 VH) and the light chain variable region (M12 VL) of the anti-CD3 antibody (M12) A series of oligonucleotides having a base sequence encoding the sequence and having complementary sequences at the ends A polymerase reaction was then carried out to create a series of oligonucleotides. The H chain variable region (M12 VH) and the L chain variable region (M12 The oligonucleotide was designed to synthesize a polynucleotide corresponding to the VL. After mixing the oligonucleotides, these oligonucleotides are ligated by PCR to generate the individual variable regions. Two cDNAs encoding the amino acid sequence of the nucleotide sequence were obtained. Using these cDNAs as templates, PCR was performed to identify M12 VL and M12 VH, which were Gly·Gly·Gly· Amino acids linked by a linker consisting of three repeats of Gly·Ser (SEQ ID NO: 7) A cDNA fragment encoding the M12 scFv having the amino acid sequence was generated.

[0223] Next, primers containing the appropriate sequences and anti-GPC3 scFv and M12 scFv were used to encode the respective scFvs. Anti-GPC3 scFv and M12 scFv were isolated by PCR using the corresponding cDNA fragment as a template. linked by a linker consisting of the sequence y·Gly·Gly·Ser (SEQ ID NO: 7), and A His tag (8 His) was added to the end (19 amino-terminal regions as set forth in SEQ ID NO: 33). A cDNA fragment encoding the amino acid sequence (excluding the amino acids) was generated.

[0224] The primers were prepared by adding the appropriate sequence and the amino-terminal 19 amino acids set forth in SEQ ID NO: 33. The cDNA fragment encoding the amino acid sequence excluding the amino acid was used as a template for PCR. The nucleotide sequence encoding the EcoRI cleavage sequence, the kozac sequence, and the secretion signal sequence on the 5' side of the A fragment A cDNA fragment was prepared by adding a Not I cleavage sequence to the 3' end of the cDNA fragment. The NA fragment was digested with EcoRI and NotI and inserted into an expression vector for mammalian cells. , GPC3 BiTE (SEQ ID NO: 33, the amino-terminal 19 amino acids, which are the signal sequence, are the mature sequence The expression vector for the vector (not included in the above) was obtained.

[0225] The vector was introduced into the CHO cell line DG44 by electroporation. After limiting dilution, the transfected cells were cultured in the presence of 1 mg / mL Geneticine. Drug-resistant cell lines were isolated using an antibody against the His tag. Western blot analysis of the supernatants confirmed that GPC3 BiTE-expressing cell lines was selected.

[0226] The culture supernatant obtained by mass culturing the cell line was then passed through an SP Sepharose FF column. After washing the column, the fraction containing GPC3 BiTE was added to a 1000 ml column of 1000 ml of NaCl. The fraction was eluted by a concentration gradient. The fraction was then passed through a HisTrap HP column (GE Healthcare). After washing the column, the fraction containing GPC3 BiTE was added to the imidazole gradient. The fraction was concentrated using an ultrafiltration membrane, and the concentrate was then filtered through a Superdex 200 column. The GPC3 BiTE fraction was collected using a GPC3 ELISA kit (GE Healthcare). Purified GPC3 BiTE was obtained.

[0227] (3) Preparation of GPC3 ERY2 PCR using primers with appropriate sequences added as in the above method, and QuikC Those skilled in the art can easily perform the method using the Hane Site-Directed Mutagenesis Kit (Stratagene). GPC3 ERY2_Hk (SEQ ID NO: 34, signal sequence: amino-terminal 19 amino acids not included in the mature sequence), and GPC3 ERY2_Hh (SEQ ID NO: 35, signature The amino-terminal 19 amino acids, which are null sequences, are not included in the mature sequence. An expression vector was constructed into which the polynucleotide was inserted.

[0228] These expression vectors were co-transfected into FreeStyle293-F cells (Invitrogen), and the GPC3 ERY2 was expressed in the culture supernatant, which was then applied to an Anti-FLAG M2 column (Sigma). After washing the column, elution was performed with 0.1 mg / mL FLAG peptide (Sigma). The fraction containing GPC3 ERY2 was applied to a HisTrap HP column (GE Healthcare). After washing, elution was performed with an imidazole gradient. After the concentrate was concentrated using an ultrafiltration membrane, the concentrate was applied to a Superdex 200 column (GE Healthcare). Purified GPC3 ERY2 was obtained by collecting only the monomeric GPC3 ERY2 fraction from the eluate. was made.

[0229] (4) Preparation of low-fucose anti-GPC3 antibodies Expression vector of anti-GPC3 antibody (described as humanized GPC3 antibody in WO2006 / 006693) The vector was electroporated to produce GDP-fucose knockout CHO cell line DXB11 (C Ancer Sci. (2010) 101(10), 2227-33). After limiting dilution, 0.5 mg / mL Ge Drug-resistant cells were selected by culturing in the presence of neticine, and low-fucose anti-GPC3 antibodies were produced. The present strain was obtained. The culture supernatant prepared by culturing this cell line was purified by Hitrap (R) Protein A (Phar The antibody fraction was prepared by conventional affinity purification using a chromatographic method (Macia). The soluble fraction was purified by gel filtration using Superdex 20026 / 60 (Pharmacia). The low-fucose GPC3 antibody was obtained by fractionating the monomeric fraction.

[0230] (5) Measurement of cytotoxic activity using human peripheral blood mononuclear cells (5-1) Preparation of human peripheral blood mononuclear cell (PBMC) solution A 1,000 unit / mL heparin solution (Novo Heparin Injection 5,000 units, Novo Nordisk) was used. Using a syringe prefilled with 100 μL, 50 mL of peripheral blood was collected from healthy adult volunteers. After diluting 2-fold with PBS(-), the peripheral blood was divided into four equal parts and placed in 15 mL of Ficoll-Paque PLUS pre-filled and centrifuged Leucosep lymphocyte separation tubes (Cat. No. 227 The tube was centrifuged (2,150 rpm, 10 min, After incubation at room temperature, the mononuclear cell fraction was separated. The mononuclear cell fraction was washed once with Medium (SIGMA, hereinafter referred to as 10% FBS / D-MEM), and then the cells The cells were cultured in 10% FBS / D-MEM at a cell density of 4 × 10 6 / mL. The cell solution was used as a human PBMC solution in the subsequent tests.

[0231] (5-2) Measurement of cytotoxic activity Cytotoxic activity was measured using the xCELLigence Real-Time Cell Analyzer (Roche Diagnostics, Inc.). The target cells were the SK-HEP-1 cell line and human erythropoietin. The SK-pca13a cell line, established by forced expression of GPC3, was used. Peel off from the 1×10 4 Plate 96 cells / well on an E-Plate (Roche Diagnostics) The cells were plated at 100 μL / well on a plate (Cell Signaling Technology, Inc.) and analyzed using the xCELLigence Real-Time Cell Analyzer. The next day, the xCELLigence Real-Time Cell Analyzer was used to measure live cells. The plate was then filled with 50 ml of each antibody at various concentrations (0.004, 0.04, 0.4, 4 nM). After incubation at room temperature for 15 minutes, 5 μL of the human PBMC solution prepared in (5-1) was added. 0 μL (2 × 10 5 cells / well) were added and subjected to the xCELLigence Real-Time Cell Analyzer. The plate was reset to start the measurement of viable cells. The experiment was carried out at 37°C, and cell proliferation was calculated from the Cell Index value 72 hours after the addition of human PBMCs using the following formula: The cell index value used in the calculation was the cell index immediately before the addition of the antibody. The values were used after normalization so that the index value was 1.

[0232] Cell proliferation inhibition rate (%) = (AB) × 100 / (A-1)

[0233] A is the average Cell Index value in wells to which no antibody was added (target cells and human PBMCs) B indicates the average Cell Index value for each well. It was said.

[0234] PBMCs (Peripheral Blood Mononuclear Cells) prepared from human blood were used as effector cells. The cytotoxic activity of GPC3 BiTE, GPC3 ERY2, and IgG-type GPC3 antibody was measured. However, GPC3 BiTE showed extremely strong activity (Figure 1). This activity was due to the low-fucose anti-GPC3 It is much stronger than antibodies, and GPC3 BiTE is a superior cancer treatment drug that surpasses IgG antibodies. On the other hand, GPC3 ERY2 showed activity greater than that of IgG anti-GPC3 antibodies. However, the activity was not as high as that of GPC3 BiTE. It was thought that the desired molecule could not be created by this alone.

[0235] Example 2: Preparation and examination of GPC3 ERY5, GPC3 ERY6, and GPC3 ERY7 Next, by making the binding domain to the cancer antigen (GPC3) bivalent, it is possible to more effectively target cancer cells. We attempted to improve the specific activity by increasing the binding activity of GPC3 to ERY2. GPC3 ERY5 (Fig. 17D) has another scFv binding domain for GPC3. GPC3 ERY7 (Fig. 17F) was produced using Fab instead of scFv. We also constructed GPC3 ERY6 (Fig. 17E), in which the anti-CD3 epsilon scFv of PC3 ERY5 was separated into both arms. was done.

[0236] That is, PCR method using primers with appropriate sequences added similar to the above method, etc. GPC3 ERY5_Hh, GPC3 ERY6_Hk, GPC3 ERY6_Hh, and GPC3 A series of expression vectors containing polynucleotides encoding ERY7_Hh, GPC3, and ERY7_L were inserted. A vector was created.

[0237] The following combinations of expression vectors were transfected into FreeStyle293-F cells to express each target molecule. It was expressed transiently.

[0238] A. Target molecule: GPC3 ERY5 Polypeptide encoded by the polynucleotide inserted into the expression vector: GPC3 E RY5_Hh (SEQ ID NO: 36; the amino-terminal 19 amino acids, which are a signal sequence, are not included in the mature sequence) (Not common), GPC3 ERY2_Hk

[0239] B. Target molecule: GPC3 ERY6 Polypeptide encoded by the polynucleotide inserted into the expression vector: GPC3 E RY6_Hk (SEQ ID NO: 37; the amino-terminal 19 amino acids, which are the signal sequence, are not included in the mature sequence) rare), GPC3 ERY6_Hh (SEQ ID NO: 38, the amino-terminal 19 amino acids are the signal sequence), (The acid is not included in the mature sequence)

[0240] C. Target molecule: GPC3 ERY7 Polypeptide encoded by the polynucleotide inserted into the expression vector: GPC3 E RY7_Hh (SEQ ID NO: 39; the amino-terminal 19 amino acids, which are a signal sequence, are not included in the mature sequence) GPC3 ERY7_L (SEQ ID NO: 40, the amino-terminal 19 amino acids are the signal sequence) acid is not included in the mature sequence), GPC3 ERY2_Hk

[0241] The obtained culture supernatant was applied to an Anti-FLAG M2 column (Sigma), and the column was washed. Then, elution was performed with 0.1 mg / mL FLAG peptide (Sigma). The fraction containing the target molecule was It was added to an isTrap HP column (GE Healthcare), and after washing the column, imidazole was added. The fraction containing the target molecule was concentrated using an ultrafiltration membrane, and then eluted with a concentration gradient of The fraction was applied to a Superdex 200 column (GE Healthcare), and the monomer fraction of the eluate was analyzed. Only the purified target molecules were obtained by collecting the purified target molecules.

[0242] The cytotoxic activities of these polypeptide complexes were compared with those of GPC3 BiTE. As a result, the cytotoxic activity of these polypeptide complexes was not comparable to that of GPC3 BiTE. This revealed that the structure of BiTE or its mimics could be used to The desired structure cannot be achieved by simply adding Fc to the structure and further binding bivalently to the cancer antigen. It was thought that they were unable to produce children.

[0243] Example 3: Preparation and examination of GPC3 ERY8-2, GPC3 ERY9-1, and GPC3 ERY10-1 (1) Preparation of GPC3 ERY8-2, GPC3 ERY9-1, and GPC3 ERY10-1 Next, we attempted to create a molecule that has the desired activity without the BiTE structure. A molecule was created using an IgG against CD3 epsilon as the base skeleton, to which an scFv against CD3 epsilon was added. In this case, the Fc of the IgG used as the base skeleton was FcgR (Fc A silenced Fc with reduced binding to CD3 epsilon (γ receptor) was used. GPC3 ERY8-2 (Figure 17G) has a cFv attached to the N-terminus of the H chain of the anti-GPC3 antibody IgG. GPC3 ERY10-1 (Fig. 17I) was attached to the C-terminus of the L chain, and GPC3 ERY9-1 (Fig. 17H) was attached to the C-terminus of the L chain. It was created.

[0244] That is, PCR method using primers with appropriate sequences added as in the above method, etc. GPC3 ERY8-2_Hk (SEQ ID NO: 41, signal sequence: the amino-terminal 19 amino acids are not included in the mature sequence), GPC3 ERY8-2_Hh (SEQ ID NO: 4 2. The mature sequence does not contain the amino-terminal 19 amino acids that form the signal sequence. 9-1_H (SEQ ID NO: 43, the amino-terminal 19 amino acids, which are the signal sequence, are not included in the mature sequence) GPC3 ERY9-1_L-His (SEQ ID NO: 44, amino terminal 19 amino acids not included in the mature sequence), GPC3 ERY9-1_L-FLAG (SEQ ID NO: 45, signal The amino-terminal 19 amino acids, which are the amino acid sequence, are not included in the mature sequence), GPC3 ERY10-1_Hh( SEQ ID NO:46, the amino terminal 19 amino acids which are the signal sequence are not included in the mature sequence A series of expression vectors were constructed, each containing a polynucleotide encoding .

[0245] The following combinations of expression vectors were transfected into FreeStyle293-F cells to express each target molecule. It was expressed transiently.

[0246] D. Target molecule: GPC3 ERY8-2 Polypeptide encoded by the polynucleotide inserted into the expression vector: GPC3 E RY8-2_Hk (SEQ ID NO: 41, the amino-terminal 19 amino acids, which are the signal sequence, are not included in the mature sequence) GPC3 ERY8-2_Hh (SEQ ID NO: 42, not including the amino terminal 19 amino acids which are signal sequences) amino acids are not included in the mature sequence), GPC3 ERY7_L

[0247] E. Target molecule: GPC3 ERY9-1 Polypeptide encoded by the polynucleotide inserted into the expression vector: GPC3 E RY9-1_H (SEQ ID NO: 43, the amino-terminal 19 amino acids, which are the signal sequence, are not included in the mature sequence) GPC3 ERY9-1_L-His (SEQ ID NO: 44, signal sequence, amino terminal 1 9 amino acids not included in the mature sequence), GPC3 ERY9-1_L-FLAG (SEQ ID NO: 45, signal The amino-terminal 19 amino acids, which are the amino acid sequence, are not included in the mature sequence.

[0248] F. Target molecule: GPC3 ERY10-1 Polypeptide encoded by the polynucleotide inserted into the expression vector: GPC3 E RY10-1_Hh (SEQ ID NO: 46, the amino-terminal 19 amino acids, which are the signal sequence, are in the mature sequence) does not include), GPC3 ERY8-2_Hk, GPC3 ERY7_L

[0249] The obtained culture supernatant was applied to an Anti-FLAG M2 column (Sigma), and the column was washed. Afterwards, elution was carried out with 0.1 mg / mL FLAG peptide (Sigma). was applied to a HisTrap HP column (GE Healthcare), and after washing the column, imidazoline was added. The fraction containing the target molecule was concentrated by ultrafiltration. After this, the fraction was loaded onto a Superdex 200 column (GE Healthcare) and the monomer content of the eluate was By collecting only the total fraction, each target molecule was purified.

[0250] The in vitro cytotoxic activity of these molecules was examined, and all of them inhibited GPC3 B. It was revealed that GPC3 ERY9-1 showed cytotoxic activity equivalent to or greater than that of iTE (Figure 5). However, it was found that GPC3 ERY10-1 clearly had a cytotoxic activity superior to that of GPC3 BiTE. In addition, molecules that combine anti-cancer antigen IgG with anti-CD3 epsilon scFv also exhibited the same properties as BiTE. In particular, GPC3 ERY9-1 and GPC3 ER As in Y10-1, the distance between the cancer antigen-binding domain and the CD3 epsilon-binding domain is The unexpected finding was that the larger molecule exhibited significantly stronger cytotoxic activity than BiTE. It was a success.

[0251] (2) Evaluation of the in vivo efficacy of GPC3 ERY8-2 and GPC3 ERY10-1: In the in vitro assay described in (1), cytotoxic activity equivalent to or greater than that of GPC3 BiTE was observed. The in vivo efficacy of the GPC3 ERY8-2 and GPC3 ERY10-1 clones was evaluated. PC-10, a human lung cancer cell line that expresses leukemia, was mixed with human PBMCs and transplanted into NOD scid mice. The mice were treated with GPC3 ERY8-2 or GPC3 ERY10-1. (This is referred to as the pre-mix model).

[0252] That is, in the efficacy test of GPC3 ERY8-2 using the PC-10 pre-mix model, the following results were obtained: A study was conducted to identify CD4+ cells from PBMCs isolated from blood collected from healthy volunteers. NK cells were depleted using 56 MicroBeads, human (MCAS Miltenyi biotec). Squamous cell carcinoma cell line PC-10 (Immunobiological Research Institute) 5 x 10 6 cells and NK cell-depleted human PBM C 4.5×10 6 The cells and Matrigel basement membrane matrix (BD) were mixed, and NOD scid cells were The mice were transplanted subcutaneously into the groin of a mouse (Japan CLEA, female, 7 weeks old). The day of transplantation was designated as day 0. The day before transplantation, the mice were intraperitoneally administered with anti-asialo GM1 antibody (Wako Pure Chemical Industries, Ltd.) at 0.2 mg / mouse. Two hours after transplantation, GPC ERY8-2 was intraperitoneally administered at 30 μg / mouse. It has been held a total of five times so far.

[0253] In addition, in efficacy tests using the PC-10 pre-mix model of GPC3 ERY10-1, the following results were obtained: A study was conducted to identify CD56 markers in PBMCs isolated from blood collected from healthy volunteers. NK cells were depleted using MicroBeads, human (MACS Miltenyi Biotec). Squamous cell carcinoma cell line PC-10 (Immuno-Biological Research Institute) 5 x 10 6 cells and NK cell-depleted human PBMCs 4.5×10 6 The cells and Matrigel basement membrane matrix (BD) were mixed, and NOD scid cells were The mice were transplanted subcutaneously into the groin of a mouse (Japan CLEA, female, 7 weeks old). The day of transplantation was designated as day 0. The day before transplantation, the mice were intraperitoneally administered with anti-asialo GM1 antibody (Wako Pure Chemical Industries, Ltd.) at 0.2 mg / mouse. Two hours after transplantation, GPC ERY10-1 was intraperitoneally administered at 30 μg / mouse. 4. The experiment was conducted 13 times in total between days 7-11 and 14-16.

[0254] As a result, in the GPC3 ERY8-2 or GPC3 ERY10-1 administration groups, It was revealed that tumor growth was clearly suppressed compared to the vehicle (PBS) administration group ( Figures 6 and 7).

[0255] The in vivo efficacy of GPC3 ERY10-1 was also evaluated in a different model. In other words, in vitro tumor formation was confirmed in NOD scid mice in which transplanted PC-10 cells were used. T cells expanded by culturing human PBMC were transferred to the mice. Treatment was performed by administering ERY10-1 (referred to as the T cell transfer model).

[0256] That is, in the efficacy test using the PC-10 T cell transfer model of GPC3 ERY10-1, the following Such a test was carried out. PBMC and T cells isolated from blood collected from healthy volunteers were used. T cell expansion using the cell activation / expansion kit / human (MACS Miltenyi Biotec) Large-scale culture was performed. Human lung squamous cell carcinoma cell line PC-10 (Immuno-Biological Research Institute) 1×10 7 Cells and The cells were mixed with Matrigel basement membrane matrix (BD), and cultured in NOD scid mice (CLEA Japan, female). The day of transplantation was designated as day 0. The mice were given a 24-hour period before transplantation. On days 6, 8, 12, 16, and 20, anti-asialo GM1 antibody (Wako Pure Chemical Industries, Ltd.) was administered intraperitoneally at 0.2 mg / animal. Six days after transplantation, the mice were divided into groups according to tumor size and body weight, and then the expansion culture was T cells obtained by 7 GPC E cells were transplanted into the abdominal cavity at 100x the dose of ... RY10-1 was intraperitoneally administered at 30 μg / mouse. GPC ERY10-1 was administered on days 7, 8, 12, 16, and 17. It was held a total of five times.

[0257] As a result, in this model, the GPC3 ERY10-1 administration group showed a significant improvement compared to the vehicle administration group. A clear antitumor effect was observed (Fig. 8).

[0258] Based on the above, we have developed a method to develop a method for the development of ... A series of molecules containing one scFv of the antibody exhibited clear in vivo antitumor effects. It was shown that this is the case.

[0259] (3) Evaluation of plasma retention A series of molecules such as GPC3 ERY8-2, GPC3 ERY9-1, and GPC3 ERY10-1 are more prominent than GPC3 BiTE. To verify whether the tumor cells have a significantly longer plasma half-life, we used NOD mice without cancer cells. The plasma concentrations of GPC3 ERY9-1 and GPC3 ERY10-1 administered at 30 μg / animal to scid mice were Measured over time.

[0260] Specifically, the following PK analysis test was carried out in NOD scid mice (CLEA Japan, female, GPC3 ERY9-1 and GPC3 ERY10-1 were intraperitoneally administered at 30 μg / mouse at 8 weeks (W8). At each time point, 1 day, 2 days, and 7 days, hematocrit capillaries (Terumo) were collected from the buccal vein of the mice. ) and plasma was prepared.

[0261] GPC3-expressing Ba / F3 cells (GPC3 / BaF) and human CD3 epsilon-expressing Ba / F3 cells (GPC3 / BaF) Appropriately diluted GPC3 ERY9-1 or GPC3 ERY10-1 was added to the cells (CD3 / BaF). GPC3 ERY10-1 was reacted with GPC3 / BaF or CD3 / BaF. After washing these cells, FITC A labeled secondary antibody was added and allowed to react further. After washing the cells, The fluorescently labeled cells were analyzed using an Epics XL flow cytometer (Beckman Coulter). Light intensity was measured and a standard curve was generated for each antibody.

[0262] Prepared from blood collected serially from mice administered GPC3 ERY9-1 or GPC3 ERY10-1 The plasma was diluted appropriately. The antibody reacts with CD3 / BaF and binds to GPC3 ERY9-1 and GPC3 ERY10-1 cells present in the plasma. The total amount was measured. Using the measured value and the standard curve, the concentration of each antibody in the plasma was calculated. was done.

[0263] As a result, both GPC3 ERY9-1 and GPC3 ERY10-1 showed activity of 10 nM or more two days after administration. It was clear that the blood concentration of GPC3 was maintained (Figures 9 and 10). The plasma half-lives of a series of molecules, such as ERY9-1 and GPC3 ERY10-1, are significantly improved compared to BiTEs. It was shown that this is the case.

[0264] (4) Effect of silent Fc on tumor antigen-independent cytokine induction (4-1) Generation of GPC3 ERY15-1 with FcgR-binding Fc A series of molecules such as GPC3 ERY8-2, GPC3 ERY9-1, and GPC3 ERY10-1 are cancer antigen-independent cytokines. To verify whether FcγR-binding Fc induces IL-1 expression, we used GPC3 ERY15-1 (Figure 17) which has FcγR-binding Fc. J) was produced.

[0265] That is, similar to the above method, PCR using primers with appropriate sequences added, and and methods using QuikChange Site-Directed Mutagenesis Kit (Stratagene) GPC3 ERY15-1_Hh (SEQ ID NO: 47, signal sequence: The amino-terminal 19 amino acids are not included in the mature sequence), GPC3 ERY15-1_Hk (SEQ ID NO: 4 8, and the amino-terminal 19 amino acids that are the signal sequence are not included in the mature sequence) An expression vector was constructed into which the encoding polynucleotide was inserted.

[0266] GPC3 ERY15-1_Hh (SEQ ID NO: 47, the amino-terminal 19 amino acids, which are the signal sequence, are not synthesized) GPC3 ERY15-1_Hk (SEQ ID NO: 48, signal sequence not included in the mature sequence), The terminal 19 amino acids are not included in the mature sequence), and the expression vector for GPC3 ERY7_L Both were transfected into FreeStyle293-F cells, causing transient expression of GPC3 ERY15-1. The supernatant was applied to an Anti-FLAG M2 column (Sigma), and after washing the column, 0.1 mg / mL F Elution was performed with LAG peptide (Sigma). Fractions containing GPC3 ERY15-1 were purified by HisTrap HP chromatography. After washing the column, a concentration gradient of imidazole was added. The fraction containing GPC3 ERY15-1 was concentrated by ultrafiltration, and then eluted with HCl. The fraction was applied to a Superdex 200 column (GE Healthcare), and the eluate was purified by GPC3 Purified GPC3 ERY15-1 was obtained by collecting only the ERY15-1 fraction.

[0267] (4-2) Measurement of tumor antigen-independent cytokine induction The tumor antigen-independent cytokine induction ability of GPC3 ERY15-1 was confirmed by GPC3 BiTE, GPC3 ERY9-1, and GP The results were compared with those of C3 ERY10-1 and catumaxomab. Blood samples were collected from healthy volunteers. PBMCs were prepared from the solution by the method described above. 50 μL (2 × 10 5 cells / well) 50 μL of each antibody adjusted to 40 nM was added to the plate, and 100 μL of 10% FBS / D-MEM was further added. The reaction mixture was incubated under 5% carbon dioxide gas at 37°C. After 72 hours of incubation, the culture supernatant was collected. The collected cells were analyzed by Cytometric Beads Array (CBA) using the Human Th1 / Th2 / Th17 Kit (BD). The cytokines secreted into the culture supernatant were quantified using the measurement method according to the attached protocol. The test was carried out in triplicate.

[0268] As a result, GPC3 ERY15-1 and catumaxomab, which have FcgR-binding Fc, showed clear cytokine responses. Induction was observed in GPC3 BiTE without Fc and GPC3 with silent Fc. No cytokine induction was observed in ERY9-1 or GPC3 ERY10-1 (Figure 11). A series of molecules such as GPC3 ERY8-2, GPC3 ERY9-1, and GPC3 ERY10-1, which have an inelastic Fc, are non-cancer antigens. It is believed to be a molecule with extremely high safety, without causing cytokine-dependent induction. It was.

[0269] Example 4: Preparation and examination of GPC3 ERY18 L1, L2, L3, L4, and S1 Molecules with a CD3-binding domain different from the scFv structure were investigated. G is a molecule in which the VH and VL regions of the CD3 antibody are bound to the C-terminus of two IgG H chains. PC3 ERY18 (Figure 17K) was constructed using a linker (Gly·Gly·Gly·Gly·Ser) between the two. A series of molecules (GPC3 ERY18 L1-L4) were prepared in which the number of α- and β-terminal amino acids was varied from 1 to 4. A molecule that allows the introduction of disulfide bonds by substituting Cys at the appropriate amino acid position. (GPC3 ERY18 S1) was also produced at the same time.

[0270] That is, in the same manner as the above method, the relevant method such as PCR using primers with appropriate sequences added thereto can be used. GPC3 ERY18 L1_Hh (SEQ ID NO: 49, signal sequence) was synthesized by methods known to those skilled in the art. the amino-terminal 19 amino acids are not included in the mature sequence), GPC3 ERY18 L1_Hk (SEQ ID NO: 50, the amino-terminal 19 amino acids that serve as a signal sequence are not included in the mature sequence), GPC3 E RY18 L2_Hh (SEQ ID NO: 51, the amino-terminal 19 amino acids, which are the signal sequence, are the same as in the mature sequence) GPC3 ERY18 L2_Hk (SEQ ID NO: 52, signal sequence: amino terminal 1 9 amino acids not included in the mature sequence), GPC3 ERY18 L3_Hh (SEQ ID NO: 53, signal The amino-terminal 19 amino acids are not included in the mature sequence), GPC3 ERY18 L3_Hk (sequence Sequence number: 54, the amino-terminal 19 amino acids, which are the signal sequence, are not included in the mature sequence) GPC3 ERY18 L4_Hh (SEQ ID NO: 55, the amino-terminal 19 amino acids, which are the signal sequence, are not sequenced) GPC3 ERY18 L4_Hk (SEQ ID NO: 56, signal sequence is not included in the mature sequence), the terminal 19 amino acids are not included in the mature sequence), GPC3 ERY18 S1_Hh (SEQ ID NO: 57, The mature sequence does not contain the amino-terminal 19 amino acids that form the signal sequence), GPC3 ERY18 S 1_Hk (SEQ ID NO: 58, the amino-terminal 19 amino acids, which are the signal sequence, are not included in the mature sequence) A series of expression vectors into which polynucleotides encoding each of the was done.

[0271] The following combinations of expression vectors were transfected into FreeStyle293-F cells to express each target molecule. It was expressed transiently.

[0272] G. Target molecule: GPC3 ERY18 L1 Expression vector: GPC3 ERY18 L1_Hh (SEQ ID NO: 49, signal sequence: amino-terminal 1 9 amino acids not included in the mature sequence), GPC3 ERY18 L1_Hk (SEQ ID NO: 50, signal The amino-terminal 19 amino acids of the GPC3 ERY7 L

[0273] H. Target molecule: GPC3 ERY18 L2 Expression vector: GPC3 ERY18 L2_Hh (SEQ ID NO: 51, signal sequence: amino-terminal 1 9 amino acids not included in the mature sequence), GPC3 ERY18 L2_Hk (SEQ ID NO: 52, signal The amino-terminal 19 amino acids of the GPC3 ERY7 L

[0274] I. Target molecule: GPC3 ERY18 L3 Expression vector: GPC3 ERY18 L3_Hh (SEQ ID NO: 53, signal sequence: amino-terminal 1 9 amino acids not included in the mature sequence), GPC3 ERY18 L3_Hk (SEQ ID NO: 54, signal The amino-terminal 19 amino acids of the GPC3 ERY7 L

[0275] J. Target molecule: GPC3 ERY18 L4 Expression vector: GPC3 ERY18 L4_Hh (SEQ ID NO: 55, signal sequence: amino-terminal 1 9 amino acids not included in the mature sequence), GPC3 ERY18 L4_Hk (SEQ ID NO: 56, signal The amino-terminal 19 amino acids of the GPC3 ERY7 L

[0276] K. Target molecule: GPC3 ERY18 S1 Expression vector: GPC3 ERY18 S1_Hh (SEQ ID NO: 57, signal sequence: amino-terminal 1 9 amino acids not included in the mature sequence), GPC3 ERY18 S1_Hk (SEQ ID NO: 58, signal The amino-terminal 19 amino acids of the GPC3 ERY7 L

[0277] The obtained culture supernatant was applied to an Anti-FLAG M2 column (Sigma), and the column was washed. Afterwards, elution was carried out with 0.1 mg / mL FLAG peptide (Sigma). was applied to a HisTrap HP column (GE Healthcare), and after washing the column, imidazoline was added. The fraction containing the target molecule was concentrated by ultrafiltration. After this, the fraction was loaded onto a Superdex 200 column (GE Healthcare) and the monomer content of the eluate was By collecting only the total fraction, each target molecule was purified.

[0278] GPC3 ERY18 L1, GPC3 ERY18L2, GPC3 ERY18L3, GPC3 ERY18L4, and GPC3 ERY18S1 molecules The in vitro cytotoxic activity of GPC3 ERY18 L was evaluated (Figures 12 and 13). All of the molecules except for 1 were found to have activity equivalent to that of GPC3 ERY10-1. It was shown that molecules with different structures have equivalent cytotoxic activity. The VH and VL regions of the CD3 antibody are bound to the C-terminus of the two H chains of the corresponding IgG. These are expected to contribute to the stabilization of the polypeptide complex molecules of the present invention.

[0279] Example 5: Preparation and analysis of GPC3 ERY19-3 Next, we investigated molecules with Fab-like structures in the CD3-binding domain. The C-terminus of the two H chains of the IgG antibody is connected to the VH and CH1 regions of the CD3 antibody, and the VL and CL regions of the CD3 antibody. A molecular form in which the domains were linked, GPC3 ERY19-3, was constructed (Fig. 17L). Similarly, methods known to those skilled in the art, such as PCR using primers with appropriate sequences added, GPC3 ERY19-3_Hh (SEQ ID NO: 59, amino terminal 19 amino acids, signal sequence) was isolated by the method described above. amino acids are not included in the mature sequence), GPC3 ERY19-3_Hk (SEQ ID NO: 60, signal sequence The amino-terminal 19 amino acids, which are not included in the mature sequence, are encoded by polynucleotides An expression vector was constructed into which the nucleotide was inserted.

[0280] GPC3 ERY19-3_Hh (SEQ ID NO: 59, the amino-terminal 19 amino acids, which are the signal sequence, are not included) GPC3 ERY19-3_Hk (SEQ ID NO: 60, signal sequence not included in the mature sequence), ... The terminal 19 amino acids are not included in the mature sequence), and the expression vector for GPC3 ERY7_L Both were transfected into FreeStyle293-F cells to transiently express GPC3 ERY19-3. The supernatant was applied to a HiTrap rProtein A FF column (GE Healthcare), and the column was washed. Afterwards, acid elution was carried out. The fraction containing GPC3 ERY19-3 was concentrated by ultrafiltration. After that, the fraction was applied to a Superdex 200 column (GE Healthcare) and the monomer of the eluate was analyzed. Purified GPC3 ERY19-3 was obtained by collecting only the GPC3 ERY19-3 fraction.

[0281] The in vitro cytotoxic activity of the GPC3 ERY19-3 molecule was evaluated. It was shown that the CD3-binding domain has an Fab-like structure and has the same activity as the Fab-binding domain (Fig. 14). This is expected to contribute to the stabilization of the polypeptide complex molecules of the present invention.

[0282] [Example 6] Introduction of mutations into the CH3 domain of GPC3 ERY 10-1 to improve the protein A purification process Preparation of polypeptide complexes using (1) Overview In GPC3 ERY10-1 prepared in Example 3, the CH3 domain has a knobs-into-hole structure. Each heavy chain has a His tag and a FLAG tag attached to its C-terminus. By performing two types of affinity purification using these tags, the two types of target proteins were isolated. The GPC3 ERY10-1 molecule, in which the heavy chains are hetero-associated, was purified. When producing GPC3 ERY10-1, protein A chromatography was performed on the culture supernatant of cells expressing GPC3 ERY10-1. The polypeptide complex containing the Fc domain is first purified using a His Tag affinity chromatography and FLAG tag affinity chromatography A purification process using two different chromatograms is required, which increases the cost of the purification process. Therefore, in this example, a protein was synthesized without using a His tag and a FLAG tag. By using only PEG-A chromatography, the two types of H chains of interest were hetero-associated. Molecular modifications that would enable the purified GPC3 ERY10-1 molecule were investigated.

[0283] Specifically, we are investigating modifications to eliminate the binding of one of the two H chains to Protein A. This modification resulted in homo-associated heavy chains that no longer bind to Protein A. The molecule cannot bind to Protein A and therefore passes Protein A chromatography. On the other hand, the H chains that have lost their binding to Protein A and the H chains that retain their binding to Protein A The molecule is a hetero-associated molecule of heavy chains and a homo-associated molecule of heavy chains that retains binding to Protein A. By utilizing the difference in binding ability between molecules and Protein A, Protein A chromatography It was thought that these molecules could be separated by fluorography. In this study, the binding sites of Protein A and FcRn, which are important for the plasma retention of antibodies, do not overlap. Therefore, it selectively binds to Protein A while maintaining its binding to FcRn. Such a modification involves replacing His at position 435 in the EU numbering system with Arg. In addition to this mutation, a modification that promotes hetero-association of two types of H chains was found. The mutation described in WO2006 / 106905 (replacement of Asp at position 356 (EU numbering) in one H chain with Lys) and Lys at position 439 (EU numbering) of the other H chain is replaced with Glu) By using Protein A chromatography alone, it is possible to identify polypeptide associations such as GPC3 ERY10-1. It was then examined whether the combined molecule could be purified.

[0284] (2) Construction of antibody gene expression vectors and expression of each antibody The antibody H-chain variable region was GC33(2)H (anti-human Glypican-3 antibody H-chain variable region, SEQ ID NO: 61, the mature sequence does not contain the amino-terminal 19 amino acids that act as a signal sequence The gene encoding the antibody L chain was constructed by a method known to those skilled in the art. 3-k0 (anti-human Glypican-3 antibody L chain, SEQ ID NO: 62, amino-terminal 19:1 signal sequence) The gene encoding the amino acid sequence (amino acids not included in the mature sequence) can be prepared by methods known to those skilled in the art. Next, the following genes were used as antibody H chain constant regions, and are publicly known to those skilled in the art. It was created by the method of knowledge.

[0285] L. Target molecule: LALA-G1d In the IgG1 sequence, Leu at positions 234 and 235 (EU numbering) was substituted with Ala, and LALA-G1d (sequence number LALA-G1d) was prepared by introducing a mutation in which Asn was replaced with Ala at the C-terminus and Gly and Lys were removed. Sequence number: 63, the amino-terminal 19 amino acids, which are the signal sequence, are not included in the mature sequence)

[0286] M. Target molecule: LALA-G1d-CD3 LALA-G1d (SEQ ID NO: 63, the amino-terminal 19 amino acids, which are the signal sequence, are the mature sequence The CD3 scFv (anti-human CD3 antibody H chain variable region and anti-human CD3 antibody L chain variable region) LALA-G1d-CD3 in which the C-terminal domains are linked via a polypeptide linker (SEQ ID NO: 64, the amino terminal 19 amino acids, which are the signal sequence, are not included in the mature sequence) stomach)

[0287] N. Target molecule: LALA-G3S3E-G1d LALA-G1d (SEQ ID NO: 63, the amino-terminal 19 amino acids, which are the signal sequence, are the same as in the mature sequence) A mutation that substitutes His at position 435 (EU numbering) with Arg in the sequence of LALA-G3S3E-G1d (SEQ ID NO: 6) was treated with a mutant LALA-G3S3E-G1d (SEQ ID NO: 6) in which a mutation was introduced to replace Lys at position 439 with Glu. 5. The mature sequence does not contain the amino-terminal 19 amino acids that serve as a signal sequence.

[0288] O. Target molecule: LALA-S3K-G1d-CD3 LALA-G1d-CD3 (SEQ ID NO: 64, the amino-terminal 19 amino acids, which are the signal sequence, are the mature sequence) A mutation was introduced to replace Asp at position 356 (EU numbering) with Lys in the sequence of The amino-terminal 19 amino acids of the LALA-S3K-G1d-CD3 (SEQ ID NO: 66) are the signal sequence. is not included in the mature sequence).

[0289] By linking LALA-G1d-CD3 or LALA-G1d downstream of GC33(2) H, the anti-human GPC3 antibody H The chain gene NTA1L or NTA1R was constructed. By linking LALA-G3S3E-G1d, the anti-human GPC3 antibody H chain gene NTA2L or NTA2R was created. was manufactured.

[0290] The genes for NTA1L, NTA1R, NTA2L, and NTA2R (heavy chain) and GC33-k0 (light chain) were expressed in mammalian cells. The expression vector for the gene was constructed by incorporating it into a vector. These vectors were then transfected into FreeStyle293 cells (Invitrogen) using methods known to those skilled in the art. The following polypeptide complexes were transiently expressed by introducing the vector into the mouse model. As shown below, the combination of the introduced genes is in the order of first H chain / second H chain / L chain. The name of the polypeptide complex is indicated by the symbol NTA1L / NTA1R / GC33-k0 NTA2L / NTA2R / GC33-k0

[0291] (3) Purification of expression samples and evaluation of hetero-assembly formation The culture supernatant of FreeStyle293 cells containing the polypeptide complex shown below (hereinafter referred to as CM) (which can be seen in the figure) was used as a sample. NTA1L / NTA1R / GC33-k0 NTA2L / NTA2R / GC33-k0

[0292] A 0.22 mm diameter rProtein A Sepharose Fast Flow column (GE Healthcare) was equilibrated with D-PBS. The CM filtered through a μm filter was loaded and washed with buffers 1, 2, and 3 listed in Table 1. Each step of elution 1 was performed. The amount of antibody loaded was adjusted to 20 mg / mL resine. The loading amount was adjusted. Size exclusion chromatography analysis of the collected elution fractions revealed The components contained in the eluted fractions were identified.

[0293] [Table 1]

[0294] The results of size exclusion chromatography analysis of each elution fraction are shown in Figure 15 and Table 2. The area of the elution peak is expressed as a percentage. In CM expressing 2R / GC33-k0, both homoantibodies against CD3 (NTA1L / GC33-k0, NTA2L / GC 33-k0) was barely detected. was detected in approximately 76% of CM expressing NTA1L / NTA1R / GC33-k0, whereas NTA2L / N Only about 2% of the TA2R / GC33-k0-expressing CM was detected. In addition to the mutation of His at position 435 of the ring to Arg, the heterodimer of each H chain was efficiently formed. To achieve this, Asp at position 356 (EU numbering) in the polypeptide sequence of one of the H chains was replaced with Lys. and a mutation to change Lys at position 439 (EU numbering) to Glu in the polypeptide sequence of the other H chain. By introducing a mutation that replaces the target The target GPC3 ERY10-1 was synthesized to produce hetero-assembled polypeptide complexes with a molecular structure similar to that of the target GPC3 ERY10-1 at a rate of 98% or more. It was found that efficient purification to the above purity was possible.

[0295] [Table 2]

[0296] Example 7: Preparation and examination of GPC3 ERY 17-2 and GPC3 ERY 17-3 (1) Preparation of GPC3 ERY 17-2 and GPC3 ERY 17-3 Next, we used IgG against the cancer antigen (GPC3) as the base skeleton, and one Fab against CD3 epsilon. The Fc of the IgG base structure was replaced with the binding domain. As mentioned above, the silent type with reduced binding to FcgR (Fcγ receptor) The Fc domain was used as the binding domain for CD3 epsilon. GPC3 ERY17-2 (Fig. 19A) in which the H and VL domains were replaced, and GPC3 ERY17-2 (Fig. 19B) in which the CH1 and CL domains were replaced GPC3 ERY17-3 (Figure 19B) was constructed by replacing the GPC3 with GPC3.

[0297] That is, PCR method using primers with appropriate sequences added as in the above method, etc. ERY17-2_Hh (SEQ ID NO: 73, signal sequence A) was synthesized by a method known to those skilled in the art. the amino-terminal 19 amino acids are not included in the mature sequence), ERY17-2_L (SEQ ID NO: 74, signal The amino-terminal 19 amino acids, which are null sequences, are not included in the mature sequence), ERY17-3_Hh (sequence No. 75, the amino-terminal 19 amino acids that are the signal sequence are not included in the mature sequence), ERY17-3_L (SEQ ID NO: 76, the amino-terminal 19 amino acids, which are the signal sequence, are the same as in the mature sequence) A series of expression vectors into which polynucleotides encoding each of the was created.

[0298] The following combinations of expression vectors were transfected into FreeStyle293-F cells to express each target molecule. It was expressed transiently.

[0299] P. Target molecule: GPC3 ERY17-2 Polypeptide encoded by the polynucleotide inserted into the expression vector: GPC3 E RY8-2_Hk, GPC3 ERY7_L, ERY17-2_Hh (SEQ ID NO: 73, signal sequence is amino-terminal 19 amino acids not included in the mature sequence), ERY17-2_L (SEQ ID NO: 74, signal sequence The amino-terminal 19 amino acids are not included in the mature sequence.

[0300] Q. Target molecule: GPC3 ERY17-3 Polypeptide encoded by the polynucleotide inserted into the expression vector: GPC3 E RY8-2_Hk, GPC3 ERY7_L, ERY17-3_Hh (SEQ ID NO: 75, signal sequence is amino terminal 19 amino acids not included in the mature sequence), ERY17-3_L (SEQ ID NO: 76, signal sequence The amino-terminal 19 amino acids are not included in the mature sequence.

[0301] (2) Purification of GPC3 ERY 17-2 and GPC3 ERY 17-3 The obtained culture supernatant was applied to an Anti-FLAG M2 column (Sigma), and the column was washed. Afterwards, elution was carried out with 0.1 mg / mL FLAG peptide (Sigma). was applied to a HisTrap HP column (GE Healthcare), and after washing the column, imidazoline was added. The fraction containing the target molecule was concentrated by ultrafiltration. After this, the fraction was loaded onto a Superdex 200 column (GE Healthcare) and the monomer content of the eluate was By collecting only the total fraction, each target molecule was purified.

[0302] (3) Cytotoxic activity of GPC3 ERY 17-2 and GPC3 ERY 17-3 The in vitro cytotoxic activity of GPC3 ERY 17-2 and GPC3 ERY 17-3 was examined (Figure 2 0) As a result, both molecules clearly exhibited cytotoxic activity superior to that of GPC3 BiTE. In the present invention, IgG against a cancer antigen is used as the base skeleton, and one Fab is linked to CD3 ep Molecules with silon-binding domains replaced with silon-binding domains also exhibit cytotoxic activity equivalent to or greater than that of BiTEs. This became clear for the first time.

[0303] (4) Efficacy study of GPC3 ERY17-2 using a PC-10 T cell transfer model GPC3 ERY17-2 demonstrated cytotoxic activity equivalent to or greater than that of GPC3 BiTE in in vitro assays The in vivo efficacy of GPC3 E was evaluated using a PC-10 T cell transfer model. In the efficacy test using the RY17-2 PC-10 T cell transfer model, the following tests were conducted: PBMCs and T cell activation / e T cell expansion was performed using the expansion kit / human (MACS Miltenyi Biotec). Human lung squamous cell carcinoma cell line PC-10 (Immuno-Biological Research Institute) 1×10 7 Cells and Matrigel basement membrane Matrix (BD) was mixed and applied to the groin skin of NOD scid mice (Japan CLEA, female, 7W). The day of transplantation was designated as day 0. Mice were treated with 100 mg / kg of cerebrospinal fluid on the day before transplantation and on days 13, 17, and 21. On the 25th day after transplantation, anti-asialo GM1 antibody (Wako Pure Chemical Industries, Ltd.) was intraperitoneally administered at 0.2 mg / mouse. The mice were divided into groups according to tumor size and body weight, and on the 14th day after transplantation, the mice were cultured for 14 days. The number of T cells was 3 × 10 7 Two hours later, GPC ERY17-2 cells were injected into the abdominal cavity of the mice at a dose of 300 mg / mouse. GPC ERY17-2 was administered intravenously at a dose of μg / animal. GPC ERY17-2 was administered five times on days 14, 15, 16, 17, and 18. It was said.

[0304] As a result, in this model, the GPC3 ERY17-2 administration group showed a significant improvement compared to the vehicle administration group. A clear antitumor effect was observed (FIG. 21).

[0305] Based on the above, we have developed a method to develop a IgG antibody against a cancer antigen as a base, with one Fab antibody directed against CD3 epsilon. The molecule in which the binding domain was replaced with a corresponding one exhibited clear antitumor effects in vivo. was shown.

[0306] Example 8: Preparation and study of GPC3 ERY17-2-M20 (1) Preparation of GPC3 ERY17-2-M20 Next, we investigated the molecular structure of the CD3 epsilon-binding domain that retains the desired activity even when the sequence of the domain is changed. The creation of a GPC3 ERY17-2 GPC3 was attempted by altering the sequence of the CD3 epsilon-binding domain of the GPC3 ERY17-2 GPC3. 3 ERY17-2-M20 (Figure 19A) was constructed. That is, the expression vector for the CD3 antibody (M20) PCR was performed using primers with appropriate sequences added as templates, similar to the method described above. ERY17-2-M20_Hh (SEQ ID NO: 77, signal The amino-terminal 19 amino acids are not included in the mature sequence), ERY17-2-M20_L (sequence No. 78, the amino-terminal 19 amino acids that are the signal sequence are not included in the mature sequence) A series of expression vectors were constructed, each with an encoding polynucleotide inserted.

[0307] (2) Purification of GPC3 ERY17-2-M20 GPC3 ERY8-2_Hk, GPC3 ERY7_L, ERY17-2-M20_Hh (SEQ ID NO: 77), and ERY17-2-M 20_L (SEQ ID NO: 78) expression vector was introduced into FreeStyle293-F cells, and transient GPC3 ERY17-2-M20 was expressed in the culture supernatant. The resulting culture supernatant was filtered through a 0.22 μm filter. After the elution, the eluate was loaded onto an equilibrated rProtein A Sepharose Fast Flow column (GE Healthcare). Washing steps 1, 2, and elution 1 were performed using the buffers shown in Table 3. Purified GPC3 ERY17-2-M20 was obtained.

[0308] [Table 3]

[0309] (3) Cytotoxic activity of GPC3 ERY17-2-M20 The in vitro cytotoxic activity of GPC3 ERY17-2-M20 was examined and found to be nearly equivalent to that of GPC3 ERY17-2. This indicates that the binding domain for CD3 epsilon is It was revealed that molecules with altered amino acid sequences also have equivalent cytotoxic activity.

[0310] [Example 9] Preparation and examination of EpCAM ERY17-2 and EpCAM ERY17-3 (1) Preparation of EpCAM ERY17-2 and EpCAM ERY17-3 Next, attempts were made to create a molecule that would retain the desired activity even when the target cancer antigen changed. EpCAM ERY17-2 (Figure 19A), in which the Fab against GPC3 of ERY17-2 was replaced with the Fab against EpCAM; EpCAM ERY17-3, in which the anti-GPC3 Fab of GPC3 ERY17-3 was replaced with the anti-EpCAM Fab (Figure 19B) The expression vector for the EpCAM antibody was used as a template, and the above Those skilled in the art can use methods such as PCR using primers with appropriate sequences added, similar to the method described above. EpCAM ERY17_Hk (SEQ ID NO: 79, signal sequence: amino terminal 1) was synthesized by a known method. 9 amino acids not included in the mature sequence), EpCAM ERY17_L (SEQ ID NO: 80, signal sequence The amino-terminal 19 amino acids, which are not included in the mature sequence, are encoded by the polynucleotides A series of expression vectors with inserted nucleotides were constructed.

[0311] The following combinations of expression vectors were transfected into FreeStyle293-F cells to express each target molecule. It was expressed transiently.

[0312] R. Target molecule: EpCAM ERY17-2 Polypeptide encoded by the polynucleotide inserted into the expression vector: EpCAM ERY17_Hk (SEQ ID NO: 79, the amino-terminal 19 amino acids, which are the signal sequence, are not included in the mature sequence) EpCAM ERY17_L (SEQ ID NO: 80, the amino terminal 19 amino acids are not included in the signal sequence) amino acids are not included in the mature sequence), ERY17-2_Hh, ERY17-2_L

[0313] S. Target molecule: EpCAM ERY17-3 Polypeptide encoded by the polynucleotide inserted into the expression vector: EpCAM ERY17_Hk, EpCAM ERY17_L, ERY17-3_Hh, ERY17-3_L

[0314] (2) Purification of EpCAM ERY17-2 and EpCAM ERY17-3 The obtained culture supernatant was applied to an Anti-FLAG M2 column (Sigma), and the column was washed. Afterwards, elution was carried out with 0.1 mg / mL FLAG peptide (Sigma). was applied to a HisTrap HP column (GE Healthcare), and after washing the column, imidazoline was added. The fraction containing the target molecule was concentrated by ultrafiltration. After this, the fraction was loaded onto a Superdex 200 column (GE Healthcare) and the monomer content of the eluate was By collecting only the total fraction, each target molecule was purified.

[0315] (3) Cytotoxic activity of EpCAM ERY17-2 and EpCAM ERY17-3 The in vitro cytotoxic activity of EpCAM ERY17-2 and EpCAM ERY17-3 was examined. In the present invention, strong cytotoxic activity was observed in the tumor antigens (Fig. 23). A molecule based on IgG, with one Fab replaced with a CD3 epsilon-binding domain It was revealed that the cytotoxic activity was maintained even when the type of cancer antigen was changed.

[0316] [Example 10] Preparation and examination of bispecific antibodies with regulated CH1 / CL interface association (1) Design of bispecific antibodies Mutations were introduced into the CH1 and CL domains of the bispecific antibody to alter the charge balance at the CH1 / CL interface. By utilizing the repulsion between the H and L chains, we were able to control the CH1 / CL interface association. Only the H chain and L chain for CD3 can specifically associate with each other. It was thought that the CH1 / CL interface association was controlled by utilizing the charge repulsion. or the amino acid residue in the C1 of the L chain is replaced by Lys, which is positively charged, or Glu, which is negatively charged. Ta.

[0317] (2) Construction of antibody gene expression vectors and expression of each antibody Anti-CD3 antibody M12 (H chain, SEQ ID NO: 81 and L chain, SEQ ID NO: 82) and Anti-G PC3 antibody GC33(2) (H chain, SEQ ID NO: 83 and L chain, SEQ ID NO: 84) was synthesized using the CH1 / CL interface. Furthermore, to prevent the association of H chains, a knob-into-hole (KiH) (WO1996 / 0 27011, Ridgway JB et al. (Protein Engineering (1996) 9, 617-621), Merchant AM et al. (N at. Biotechnol. (1998) 16, 677-681)) have been introduced into the bispecific antibodies. As a control, neither CH1 / CL interface association control nor Knob-into-Hole (KiH) modification was introduced. A bispecific antibody was also produced (Figure 24B). Specifically, the H chain of M12 (SEQ ID NO: 81) M12_TH2h (SEQ ID NO: 85) in which several amino acids in CH1 of the L chain were substituted with Lys, and M12_TL17 (SEQ ID NO: 86) in which several amino acids of CL are substituted with Glu An expression vector containing an inserted oligonucleotide was prepared by a method known to those skilled in the art. Similarly, GC33(2)_, in which several amino acids in CH1 of the H chain of GC33(2) (SEQ ID NO: 83) are substituted with Glu, TH13k (SEQ ID NO: 87), GC33(2)_TH15k (SEQ ID NO: 88), L chain (SEQ ID NO: 84) CL GC33(2)_TL16 (SEQ ID NO: 89), GC33(2)_TL19 (SEQ ID NO: 90) are inserted into expression vectors that are readily accessible to those skilled in the art. It was prepared by a known method.

[0318] The combination of expression vectors encoding the sequences shown below was transfected into FreeStyle293-F cells. Each target molecule was transiently expressed.

[0319] T. Target molecule: GM1 Expression vectors: M12_TH2h (SEQ ID NO: 85), M12_TL17 (SEQ ID NO: 86), GC33(2)_TH13 k (SEQ ID NO: 87), and GC33(2)_TL16 (SEQ ID NO: 89)

[0320] U. Target molecule: GM2 Expression vectors: M12_TH2h (SEQ ID NO: 85), M12_TL17 (SEQ ID NO: 86), GC33(2)_TH15 k (SEQ ID NO: 88), and GC33(2)_TL19 (SEQ ID NO: 90)

[0321] V. Target molecule: GM0 Expression vector: M12 H chain (SEQ ID NO: 81), M12 L chain (SEQ ID NO: 82), GC33(2) H chain chain (SEQ ID NO: 83), and the L chain of GC33(2) (SEQ ID NO: 84)

[0322] The resulting culture supernatant was purified using rProtein A Sepharose™ Fast Flow (GE Healthcare). The antibodies were purified by methods known to those skilled in the art.

[0323] (3)GM1, GM2, GM0 cytotoxic activity The in vitro cytotoxic activity of each polypeptide complex of GM1, GM2, and GM0 was examined. It was found that GM0 and GM2 exhibited equivalent cytotoxic activity, and the activity was clearly superior to that of GM0. In the present invention, the introduction of CH1 / CL interface regulation and It was revealed that combining KiH modifications enabled efficient production of bispecific antibodies. It became like this.

[0324] [Example 11] Construction and study of EGFR ERY17-2 (1) Generation of EGFR ERY17-2 Furthermore, attempts were made to create molecules with the desired activity targeting other cancer antigens. The Fab against GPC3 in ERY-2 was replaced with the Fab against EGFR, creating EGFR ERY17-2 (Figure 19A). That is, an expression vector for an EGFR antibody is used as a template, and the expression vector is then appropriately transfected in the same manner as described above. EGFR is isolated by a method known to those skilled in the art, such as PCR using primers containing a sequence similar to that of the EGFR gene. ERY17_Hk (SEQ ID NO: 91, the amino-terminal 19 amino acids, which are the signal sequence, are not included in the mature sequence) EGFR ERY17_L (SEQ ID NO: 92, signal sequence is the amino-terminal 19 amino acid sequence), The polynucleotides encoding each of the amino acids (the amino acids not included in the mature sequence) are inserted into the A series of expression vectors were constructed.

[0325] The following combinations of expression vectors were transfected into FreeStyle293-F cells to express each target molecule. It was expressed transiently.

[0326] W. Target molecule: EGFR ERY17-2 Polypeptide encoded by the polynucleotide inserted into the expression vector: EGFR E RY17_Hk (SEQ ID NO: 91, the amino-terminal 19 amino acids, which are the signal sequence, are not included in the mature sequence) EGFR ERY17_L (SEQ ID NO: 92, signal sequence is the amino-terminal 19 amino acid sequence), (The amino acid is not included in the mature sequence), ERY17-2_Hh, ERY17-2_L

[0327] (2) Purification of EGFR ERY17-2 The obtained culture supernatant was applied to an Anti-FLAG M2 column (Sigma), and the column was washed. Afterwards, elution was carried out with 0.1 mg / mL FLAG peptide (Sigma). was applied to a HisTrap HP column (GE Healthcare), and after washing the column, imidazoline was added. The fraction containing the target molecule was concentrated by ultrafiltration. After this, the fraction was loaded onto a Superdex 200 column (GE Healthcare) and the monomer content of the eluate was By collecting only the total fraction, each target molecule was purified.

[0328] (3) Cytotoxic activity of EGFR ERY17-2 When the in vitro cytotoxic activity of EGFR ERY17-2 was examined, strong cytotoxic activity was observed. In the present invention, IgG against a cancer antigen is used as the basic framework, and one Fab is linked to CD3 ep The molecule in which the binding domain for silon was replaced, not only GPC3 and EpCAM but also cancer antigens It was revealed that cytotoxic activity was maintained even when the type of antibody was further changed. [Industrial Applicability]

[0329] The present invention demonstrates the potent antitumor activity of BiTE and its ability to induce cytokine storage independent of cancer antigens. It maintains its excellent safety profile by not inducing steroids, etc., and has a long half-life in the blood. Novel polypeptide complexes are provided. Antigen binding in the polypeptide complexes of the present invention By substituting the domain, a cytotoxic agent containing the polypeptide complex as an active ingredient can be obtained. The induced therapeutic agent targets various cells, including cancer cells, causing cytotoxicity and treating various cancers. For patients, it is not only safe but also reduces the physical burden. This will enable desirable treatment that is both cost-effective and convenient.

Claims

1. The following domains: (1) an antigen-binding domain, (2) a domain containing an Fc region with reduced binding activity to an Fcγ receptor; and (3) T cell receptor complex binding domain, A polypeptide complex comprising the

2. The T cell receptor complex binding domain of claim 1 is a T cell receptor binding domain. Polypeptide complex.

3. The polypeptide of claim 1, wherein the T cell receptor complex binding domain is a CD3 binding domain. Chid aggregates.

4. 4. The method of claim 1, wherein the antigen-binding domain is a bivalent antigen-binding domain. Polypeptide complex of

5. The antibody according to claim 4, wherein the bivalent antigen-binding domain has a F(ab')2 structure. Polypeptide complex.

6. The two polypeptides that make up the heavy chain constant region of the domain with the structure F(ab')2 are Fc domains. The polypeptide chain of claim 5, linked to each of the two polypeptides constituting the region. Merge.

7. The antibody according to claim 6, wherein the CD3 binding domain is linked to one or two CH3 domains constituting the Fc region. Polypeptide complex of

8. The heavy chain Fv fragment constituting the CD3 binding domain is linked to one of the CH3 constituting the Fc region, and CD3 The light chain Fv fragment constituting the binding domain is linked to the other CH3 constituting the Fc region. The polypeptide complex of claim 7.

9. The heavy chain Fv fragment, which constitutes the CD3 binding domain, contains the CH1 domain of the antibody, and the light chain Fv fragment contains the antibody The polypeptide complex of claim 8, wherein the CL domain of

10. The antibody according to claim 6, wherein the CD3 binding domain is linked to one or two CLs constituting the F(ab')2. The polypeptide complex described above.

11. The antibody according to claim 6, wherein the CD3-binding domain is linked to one or two VHs constituting the F(ab')2. The polypeptide complex described above.

12. 7. The antibody according to claim 6, wherein the CD3-binding domain is linked to one or two VLs constituting the F(ab')2. The polypeptide complex described above.

13. The polypeptide association of any one of claims 1 to 12, wherein the CD3-binding domain is an Fv. body.

14. The polypeptide of any one of claims 1 to 7 and 10 to 12, wherein the CD3 binding domain is a Fab. Lipeptide complex.

15. 13. The method of claim 1, wherein the CD3 binding domain is an scFv. Polypeptide complex.

16. The polypeptide of any one of claims 1 to 15, wherein the CD3 binding domain is monovalent. Merge.

17. 4. The method according to claim 1, wherein the antigen-binding domain is a monovalent scFv and a monovalent Fab. The polypeptide complex described above.

18. The monovalent scFv binds to a single polypeptide constituting the Fc region via the scFv that constitutes the CD3-binding domain. The peptide is a polypeptide in which the heavy chain Fv fragment of a monovalent Fab is bound to a single polypeptide constituting an Fc region via the CH1 region.

18. The polypeptide of claim 17, wherein the light chain Fv fragment of the Fab is linked to a CL region. Lipeptide complex.

19. The polypeptide of any one of claims 1 to 3, wherein the antigen-binding domain is a bivalent scFv. Do-aggregate.

20. The monovalent scFv comprises a single Fc region via a heavy chain Fv fragment that constitutes the CD3-binding domain. The other monovalent scFv binds to the Fc polypeptide via the light chain Fv fragment that constitutes the CD3-binding domain. The polypeptide of claim 19 linked to another polypeptide constituting the domain. Do-aggregate.

21. The monovalent scFv binds to a single polypeptide constituting the Fc region via the scFv that constitutes the CD3-binding domain. and another monovalent scFv linked to another polypeptide constituting the Fc region. The polypeptide complex of claim 19.

22. The antigen-binding domain and the T cell receptor complex-binding domain are each monovalent Fab. The polypeptide complex of any one of claims 1 to 3.

23. The heavy chain Fv fragment of the monovalent Fab that constitutes the antigen-binding domain constitutes the Fc region via the CH1 region. The light chain Fv fragment of the Fab is linked to the CL region of one of the polypeptides, and the light chain Fv fragment of the Fab is linked to the CL region of the other ... The heavy chain Fv fragment of Fab, which constitutes the antibody-binding domain, binds to the other fragment, which constitutes the Fc region, via the CH1 region.

23. The method of claim 22, wherein the light chain Fv fragment of the Fab is linked to a polypeptide and the light chain Fv fragment of the Fab is linked to a CL region. Polypeptide complex of

24. The heavy chain Fv fragment of the monovalent Fab that constitutes the antigen-binding domain constitutes the Fc region via the CH1 region. The light chain Fv fragment of the Fab is linked to the CL region of one of the polypeptides, and the light chain Fv fragment of the Fab is linked to the CL region of the other ... The light chain Fv fragment of Fab, which constitutes the antibody-binding domain, binds to the other fragment that constitutes the Fc region via the CH1 region.

23. The method of claim 22, wherein the heavy chain Fv fragment of the Fab is linked to a polypeptide and the heavy chain Fv fragment of the Fab is linked to a CL region. Polypeptide complex of

25. The heavy chain Fv fragment of the monovalent Fab that constitutes the antigen-binding domain constitutes the Fc region via the CH1 region. The light chain Fv fragment of the Fab is linked to the CL region of one of the polypeptides, and the light chain Fv fragment of the Fab is linked to the CL region of the other ... The heavy chain Fv fragment of Fab, which constitutes the antibody-binding domain, binds to the other fragment, which constitutes the Fc region, via the CL region.

23. The method of claim 22, wherein the light chain Fv fragment of the Fab is linked to a CH1 domain of the Fab. Polypeptide complex of

26. The heavy chain Fv fragment of the monovalent Fab, which constitutes the T cell receptor binding domain, binds to the Fc region via the CH1 region. and the light chain Fv fragment of the Fab is linked to a CL region, The light chain Fv fragment of Fab, which constitutes the antigen-binding domain, is bound to the other, which constitutes the Fc domain, via the CH1 domain.

23. The method of claim 22, wherein the heavy chain Fv fragment of the Fab is linked to a polypeptide, and the heavy chain Fv fragment of the Fab is linked to a CL region. The polypeptide complex described above.

27. The heavy chain Fv fragment of the monovalent Fab, which constitutes the T cell receptor binding domain, binds to the Fc region via the CH1 region. and the light chain Fv fragment of the Fab is linked to a CL region, The heavy chain Fv fragment of Fab, which constitutes the antigen-binding domain, is bound to the other, which constitutes the Fc domain, via the CL domain.

23. The method of claim 22, wherein the light chain Fv fragment of the Fab is linked to a polypeptide, and the light chain Fv fragment of the Fab is linked to a CH1 domain. The polypeptide complex described above.

28. (1) A monovalent heavy chain Fv fragment of an antigen-binding Fab structure constitutes the Fc domain via the CH1 domain. The light chain Fv fragment of the Fab structure is linked to a CL region of the antibody. a protease-binding domain, and (2) The heavy chain Fv fragment of the monovalent Fab structure that binds to the T cell receptor complex is linked to the Fc via the CH1 region. The light chain Fv fragment of the Fab structure is linked to the other polypeptide constituting the CL region. a bound T cell receptor complex binding domain, a heavy chain Fv fragment in the antigen-binding domain and a polypeptide complex comprising the heavy chain Fv fragment in the antigen-binding domain; The light chain Fv fragment in the Fv domain or the heavy chain Fv fragment and the T cell receptor binding domain in the T cell receptor binding domain The charges of the CH1 region and the CL region are controlled so that the light chain Fv fragments in the molecule associate with each other.

23. The polypeptide complex of 22.

29. Amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the T cell receptor complex binding domain and and the amino acid residues of the CL region linked to the light chain Fv fragment in the antigen-binding domain are identical to each other. The polypeptide complex of claim 28, which has a charge.

30. Amino acid residues in the CH1 region linked to the heavy chain Fv fragment in the antigen-binding domain and T cell reception The amino acid residues of the CL region linked to the light chain Fv fragment in the antibody complex binding domain are homologous to each other. The polypeptide complex of claim 28, which has a charge.

31. Amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the T cell receptor complex binding domain and and the amino acid residues of the CL region linked to the light chain Fv fragment in the antigen-binding domain are identical to each other. Amino acid residues in the CH1 region that are charged and linked to the heavy chain Fv fragment in the antigen-binding domain and and the amino acid residues of the CL region linked to the light chain Fv fragment in the T cell receptor complex binding domain. The polypeptide complex of claim 28, which has the same electric charge as each other.

32. Amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the T cell receptor complex binding domain and and the amino acid residues of the CL region linked to the light chain Fv fragment in the T cell receptor binding domain are The polypeptide complex of claim 29 or 31, wherein the and have charges opposite to each other.

33. Amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen-binding domain and antigen binding The amino acid residues in the CL region linked to the light chain Fv fragment in the domain both have opposite charges The polypeptide complex of claim 30 or 31.

34. 22 to 3, wherein the T cell receptor complex binding domain is a T cell receptor binding domain. The polypeptide complex of any one of 3.

35. The polypeptide of claim 34, wherein the T cell receptor binding domain is a CD3 binding domain. Association.

36. The amino acid residues in the CH1 region and the CL region are shown in (a) to (f) below. a group consisting of one or more pairs of amino acid residues represented by the formula (I) (a) an amino acid residue at position 147 (EU numbering) in the CH1 region, and C an amino acid residue at position 180 (EU numbering) in the L region; (b) an amino acid residue at position 147 (EU numbering) in the CH1 region, and a CL region the amino acid residue at position 131 according to EU numbering (c) an amino acid residue at position 147 (EU numbering) in the CH1 region, and a CL region the amino acid residue at position 164 in the EU numbering system (d) an amino acid residue at position 147 (EU numbering) in the CH1 region, and a CL region the amino acid residue at position 138 in the EU numbering system (e) an amino acid residue at position 147 (EU numbering) in the CH1 region, and a CL region the amino acid residue at position 123 in the EU numbering system (f) an amino acid residue at position 175 (EU numbering) in the CH1 region, and a CL region the amino acid residue at position 160 in the EU numbering system The amino acid residues in the CH1 region and the amino acid residues in the CL region are selected from the following: The polypeptide complex of claim 32 or 33, wherein the amino acid residue is a charged amino acid residue.

37. Further, the amino acid residues are selected from the group consisting of the following set of amino acid residues (g):

7. The polypeptide complex of 6. (g) an amino acid residue at position 213 (EU numbering) in the CH1 region, and a CL region the amino acid residue at position 123 in the EU numbering system

38. The amino acid residues having opposite charges are either of the following groups (X) or (Y): (X) glutamic acid (E), aspartic acid (D); (Y) lysine (K), arginine (R), histidine (H); The polypeptide association of claim 36 or 37 is selected from the amino acid residues contained in body.

39. The amino acid residues having a different charge are amino acid residues in the CH1 region and are represented by EU numbering. The amino acid residue at position 175 in the CL region is Lys, and the amino acid residue at position 180 in the EU numbering 39. Any of claims 36 to 38, wherein the amino acid residues at positions 131 and 160 are both Glu. The polypeptide complex of any one of the preceding claims.

40. The amino acid residues having a different charge are amino acid residues in the CH1 region and are represented by EU numbering. The amino acid residues at positions 147 and 175 of the nucleotide sequence are Glu and CL region amino acid residues, and are EU numbered 36 to 37, wherein the amino acid residues at positions 180, 131 and 160 are all Lys.

9. The polypeptide complex of any one of 8.

41. Furthermore, the amino acid residue at position 213 (EU numbering) in the CH1 region is Glu. and the amino acid residue at position 123 (EU numbering) in the CL region is Lys. The polypeptide complex of claim 40.

42. The Fc region is an Fcγ receptor selected from FcγI, FcγIIA, FcγIIB, FcγIIIA, and / or FcγIIIB. The Fc region according to any one of claims 1 to 41, wherein the Fc region has reduced binding activity to a receptor. Polypeptide complex.

43. The Fc region is an Fc region set forth in SEQ ID NO: 23, an Fc region set forth in SEQ ID NO: 24, an Fc region set forth in SEQ ID NO: The amino acids constituting the Fc region of SEQ ID NO: 25 or the Fc region of SEQ ID NO: 26 are mutated.

43. The polypeptide according to any one of claims 1 to 42, characterized in that it is an Fc region containing Do-aggregate.

44. Any of the following amino acids that make up the Fc region, identified according to EU numbering: amino acid; The amino acid sequence of positions 118 to 260 is the sequence set forth in SEQ ID NO: 24, and the amino acid sequence of positions 261 to 447 is the sequence set forth in SEQ ID NO:

25.

44. The polypeptide of claim 43, wherein the Fc region has the sequence set forth in SEQ ID NO:

26. Chid aggregates.

45. Any of the following amino acids that make up the Fc region, identified according to EU numbering: amino acid; 220th, 226th, 229th, 231st, 232nd, 233rd, 234th, 235th, 236th, 237th, 238th, 239 240th, 264th, 265th, 266th, 267th, 269th, 270th, 295th, 296th, 297th, 298th , 299th, 300th, 325th, 327th, 328th, 329th, 330th, 331st, 332nd, are mutated F The polypeptide complex of claim 43, which is the c domain.

46. The Fc region is an Fc region in which the amino acids constituting the Fc region set forth in SEQ ID NO: 23 have been mutated. The polypeptide complex of claim 45.

47. Any of the following amino acids that make up the Fc region, identified according to EU numbering: amino acid; 233rd, 234th, 235th, 236th, 237th, 327th, 330th, 331st, is replaced by the corresponding amino acid in the EU numbering in the corresponding IgG2 or IgG4 The polypeptide complex of claim 46, which is an Fc domain.

48. Any of the following amino acids that make up the Fc region, as specified according to EU numbering: amino acids; 234th place, 235th place, 297th place, the polypeptide complex of claim 46, wherein the Fc domain is mutated 。

49. The amino acid at position 234 is alanine, the amino acid at position 235 is alanine, and / or the amino acid at position 297 is alanine.

49. The polypeptide of claim 48, wherein the amino acid is mutated to alanine. body.

50. The Fc region is characterized in that the sequences of the two polypeptides constituting the Fc region are different from each other. The polypeptide complex of any one of claims 43 to 49.

51. The amino acid residues of one of the two polypeptides that make up the Fc region are EU-NA. The amino acid at position 349 is cysteine and the amino acid at position 366 is tripeptide, as identified according to the numbering. The amino acid residues of the other polypeptide are identified according to EU numbering. The amino acid at position 356 is cysteine, the amino acid at position 366 is serine, and the amino acid at position 368 is arabinopeptide.

50. The method of claim 1, wherein the amino acid at position 407 is mutated to valine. The polypeptide complex of any one of the preceding claims.

52. The amino acid residues of one of the two polypeptides that make up the Fc region are EU-NA. The amino acid at position 356, specified according to the numbering, is lysine, and the amino acid at position 356 is lysine. The amino acid residue at position 439, as specified by EU numbering, is changed to glutamic acid. The amino acid residues of either polypeptide are identified according to EU numbering.

50. The method of claim 1, wherein the amino acid at position 435 is mutated to arginine. The polypeptide complex of any one of the preceding claims.

53. The GK sequence at the carboxy terminus of the two polypeptides that make up the Fc region is deleted. The polypeptide complex of claim 51 or 52.

54. 54. The method of claim 1, wherein the antigen-binding domains bind to the same epitope. Polypeptide complex of

55. The same epitope is present in a protein consisting of the amino acid sequence set forth in SEQ ID NO:

2. The polypeptide complex of claim 54.

56. The same epitope is present in a protein consisting of the amino acid sequence set forth in SEQ ID NO:

4. The polypeptide complex of claim 54.

57. Any of claims 1 to 53, wherein the antigen-binding domains bind to different epitopes. The polypeptide complex of any one of claims 1 to 3.

58. Different epitopes exist in the protein consisting of the amino acid sequence set forth in SEQ ID NO:

2. The polypeptide complex of claim 57.

59. Different epitopes exist in the protein consisting of the amino acid sequence set forth in SEQ ID NO:

4. The polypeptide complex of claim 57.

60. A polynucleotide encoding the polypeptide complex of any one of claims 1 to 59. Do.

61. 61. A vector comprising the polynucleotide of claim 60.

62. A cell harboring the vector of claim 61.

63. 63. A method for producing a polypeptide complex comprising culturing the cell of claim 62 and recovering the polypeptide complex from the culture supernatant. A method for producing a polypeptide complex comprising the steps of:

64. A method for treating cell injury comprising administering to a patient a therapeutically effective amount of the polypeptide complex of any one of claims 1 to 59 as an active ingredient. Harm-inducing treatment agent.

65. The therapeutic agent according to claim 64, wherein the cytotoxicity-inducing therapeutic agent is a cancer therapeutic agent.

66. The therapeutic agent according to claim 65, wherein the cancer is liver cancer or lung cancer.

67. A method for administering the polypeptide complex of any one of claims 1 to 59 to a patient in need of treatment. A method for treating or preventing cancer, comprising:

68. 68. The method for treatment or prevention according to claim 67, wherein the cancer is liver cancer or lung cancer.

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