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 relates to a method for producing a cancer cell-specific T cell-specific cytotoxicity assay using a T cell-specific T cell. Polypeptide complex that enables cancer treatment through activity, and the polypeptide complex and a therapeutic agent for inducing cytotoxicity that contains the polypeptide complex as an active ingredient. The present invention also 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 symptoms of rheumatoid arthritis and a method of treatment using said pharmaceutical composition. [Background technology]

[0002] To date, several therapeutic antibodies that have demonstrated excellent antitumor effects have been developed into drugs for the treatment of cancer. These therapeutic antibodies are being developed to target the signal transduction pathway required for the proliferation of cancer cells (Non-Patent Document 1). 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. (Non-Patent Document 2) The Fc region of an antibody binds to effector cells such as NK cells and macrophages. By binding to the Fc receptors present on the target cancer cells, the antibody binds to the target cancer cells. The cytotoxicity exerted by these effector cells is called ADCC. Complement complexes bind to the binding sites. The complexes contain complement molecules that bind to the cell membrane of the cells to which the antibody is bound. The complement components present in the cells form pores, facilitating the inflow of water and ions into the cells. CDC is the cytotoxicity caused by the destruction of the nucleus. Existing therapeutic antibodies have been shown to have excellent effects. However, the therapeutic results obtained by administering such antibodies are still not satisfactory. Therefore, the development of therapeutic antibodies against cancer that exhibit more potent cytotoxic activity is desired. .

[0003] In addition to antibodies that utilize ADCC, which mobilizes the above-mentioned NK cells and macrophages as effector cells, as the mechanism of their antitumor effect, there are also cytotoxic antibodies that mobilize T cells as effector cells and use them as the mechanism of their antitumor effect. T cell-recruiting antibodies (T cell recruiting antibodies, TR antibodies) have also been known since the 1980s (Non-Patent Documents 3-5). TR antibodies are antibodies against any of the constituent subunits of the T cell receptor (TCR) complex on T cells, particularly antibodies that bind to the CD3 epsilon chain, and bispecific (dual-specificity) antibodies that include antibodies that bind to antigens on target cancer cells. When a TR antibody binds simultaneously to the CD3 epsilon chain and a cancer antigen, T cells approach the cancer cells. As a result, it is thought that the antitumor effect against cancer cells is exerted by the cytotoxic action of T cells. antibodies that bind to the CD3 epsilon chain, and bispecific (dual-specificity) antibodies that include antibodies that bind to antigens on target cancer cells. When a TR antibody binds simultaneously to the CD3 epsilon chain and a cancer antigen, T cells approach the cancer cells. As a result, it is thought that the antitumor effect against cancer cells is exerted by the cytotoxic action of T cells. T cells approach the cancer cells. As a result, it is thought that the antitumor effect against cancer cells is exerted by the cytotoxic action of T cells. T cells approach the cancer cells. As a result, it is thought that the antitumor effect against cancer cells is exerted by the cytotoxic action of T cells.

[0004] There is also an antibody called a trifunctional antibody known as one of the TR antibodies (Non-Patent Documents 6 , 7). This is a whole IgG-type bispecific antibody in which the Fab that binds to the cancer antigen and the Fab that binds to the CD3 epsilon chain are each contained in one arm. Administration of catumaxomab, a trifunctional antibody against EpCAM, into the peritoneal cavity of patients with malignant ascites having EpCAM-expressing positive cancer cells has shown an effect on the treatment of malignant ascites. In the EU, for the purpose of the above treatment has shown an effect on the treatment of malignant ascites. In the EU, for the purpose of the above treatment has shown an effect on the treatment of malignant ascites. In the EU, for the purpose of the above treatment The use of catumaxomab has been approved.

[0005] More recently, TR antibodies called BiTE (bispecific T-cell engager) have been known to exhibit strong antitumor activity (Non-Patent Documents 8 and 9). BiTE is a TR antibody having a molecular form in which the scFv of an antibody against a cancer antigen and the scFv of an antibody against the CD3 epsilon chain are linked via a short polypeptide linker. BiTE has been reported to have antitumor activity superior to various TR antibodies known so far (Non-Patent Documents 9 and 10). That is, BiTE exerts an antitumor effect at a significantly lower concentration and a lower effector cell: cancer cell ratio (ET ratio) compared to other known TR antibodies. It has also been shown that activation of effector cells with IL-2, a CD28 agonist antibody, etc. is not necessary for the expression of this effect. Blinatumomab (MT103), a BiTE against CD19, showed a more potent cytotoxic effect on cancer cells in vitro than rituxan, which is known to have excellent clinical effects. Furthermore, extremely excellent antitumor effects have been reported in recent phase I and phase II clinical trials (Non-Patent Document 11). (Non-Patent Document 11). Since catumaxomab has shown clinical efficacy and been approved as a therapeutic agent, and multiple BiTEs including blinatumomab exhibit strong antitumor effects, it has been suggested that TR antibodies that recruit T cells as effector cells have extremely high potential as antitumor drugs compared to antibodies that use normal ADCC as their mechanism of action.

[0006]

[0007] However, trifunctional antibodies bind to T cells, NK cells, macrophages, and other cells independent of cancer antigens, resulting in cross-linking of the receptors expressed on these cells, which is known to induce the expression of various cytokines independent of cancer antigens. Induction of such cytokine expression is thought to lead to cytokine storm-like side effects upon systemic administration of trifunctional antibodies. In fact, in a phase I clinical trial of systemic administration of catumaxomab to non-small cell lung cancer patients, a dose as low as 5 μg / body was the maximum tolerated dose, and administration of higher doses was reported to cause various severe side effects (Non-Patent Document 12). Administration of such a low dose of catumaxomab cannot reach its effective blood concentration. That is, the expected antitumor effect cannot be obtained by administration of such a low dose of catumaxomab. On the other hand, BiTE, unlike catumaxomab, does not have a binding site for the Fcγ receptor, so receptors expressed on T cells, NK cells, macrophages, etc. are not cross-linked independent of cancer antigens. Therefore, it has been shown that induction of cancer antigen-independent cytokines observed when catumaxomab is administered does not occur. However, since BiTE is a low-molecular-weight modified antibody molecule lacking the Fc region, there is a problem that the blood half-life of BiTE administered to patients is significantly shorter compared to IgG-type antibodies commonly used as therapeutic antibodies. In fact, it has been shown that the blood half-life of BiTE administered to the body is about several hours (Non-Patent Document 12). In the first-phase clinical trial of systemic administration of catumaxomab to non-small cell lung cancer patients, a dose as low as 5 μg / body was the maximum tolerated dose, and administration of higher doses was reported to cause various severe side effects (Non-Patent Document 12). Administration of such a low dose of catumaxomab cannot reach its effective blood concentration. That is, the expected antitumor effect cannot be obtained by administration of such a low dose of catumaxomab. By administering such a low dose of catumaxomab, its effective blood concentration cannot be reached at all. That is, the expected antitumor effect cannot be obtained by administering such a low dose of catumaxomab. By administering such a low dose of catumaxomab, its effective blood concentration cannot be reached at all. That is, the expected antitumor effect cannot be obtained by administering such a low dose of catumaxomab. By administering such a low dose of catumaxomab, the expected antitumor effect cannot be obtained.

[0008] On the other hand, since BiTE does not have a binding site for the Fcγ receptor unlike catumaxomab, receptors expressed on T cells, NK cells, macrophages, etc. are not cross-linked independent of cancer antigens. Therefore, it has been shown that induction of cancer antigen-independent cytokines observed when catumaxomab is administered does not occur. However, since BiTE is a low-molecular-weight modified antibody molecule lacking the Fc region, there is a problem that the blood half-life of BiTE administered to patients is significantly shorter compared to IgG-type antibodies commonly used as therapeutic antibodies. In fact, it has been shown that the blood half-life of BiTE administered to the body is about several hours (Non-Patent Document 12). However, since BiTE is a low-molecular-weight modified antibody molecule lacking the Fc region, there is a problem that the blood half-life of BiTE administered to patients is significantly shorter compared to IgG-type antibodies commonly used as therapeutic antibodies. In fact, it has been shown that the blood half-life of BiTE administered to the body is about several hours (Non-Patent Document 12). In the clinical trials of blinatumomab (References 13 and 14), blinatumomab has been administered by continuous intravenous infusion using a minipump. Such administration is not only extremely inconvenient for patients, but also poses a risk of medical accidents due to equipment failure, etc., and cannot be regarded as an ideal treatment method. This administration is not only extremely inconvenient for patients, but also poses a risk of medical accidents due to equipment failure, etc., and cannot be regarded as an ideal treatment method.

Prior Art Documents

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

[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 use of vesicles Polypeptide complexes, methods for producing the same, and methods for producing the same The present invention aims to provide a cytotoxicity-inducing therapeutic agent containing the compound as an active ingredient. A pharmaceutical composition for treating or preventing various cancers, comprising an agonist 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 storm in a cancer antigen-independent manner. It is a new compound that maintains the excellent safety profile of not inducing inflammatory bowel disorders and has a long half-life in the blood. Furthermore, the antigen-binding domain of the polypeptide complex was identified. By replacing In, it has been found that the polypeptide complex can target various cells to cause cytotoxicity We have found that the polypeptide complex according to the present invention can damage cancer cells. In addition, by introducing CH 1 / CL interface association control and Knob into Hole (KiH) modification into the polypeptide complex, it has been found that it can cause more efficient cytotoxicity. Further, the inventors have found that a cytotoxicity-inducing therapeutic agent containing the polypeptide complex according to the present invention as an active ingredient can treat or prevent various cancers .

[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 the Fcγ receptor, and (3) A T cell receptor complex-binding domain, A polypeptide complex containing. 〔2〕 The polypeptide complex according to 〔1〕, wherein the T cell receptor complex-binding domain is a T cell receptor-binding domain. 〔3〕 The polypeptide complex according to 〔1〕, wherein the T cell receptor complex-binding domain is a CD3-binding domain. 〔4〕 The polypeptide complex according to any one of 〔1〕 to 〔3〕, wherein the antigen-binding domain is a bivalent antigen-binding domain. 〔5〕 The polypeptide complex according to 〔4〕, wherein the bivalent antigen-binding domain is a domain having an F(ab’)2 structure. 〔6〕 The polypeptide complex according to 〔5〕, wherein the two polypeptides constituting the heavy chain constant region of the domain having an F(ab’)2 structure are each linked to each of the two polypeptides constituting the Fc region. ​ 〔7〕 The polypeptide complex described in 〔6〕, wherein the CD3 binding domain is linked to one or two CH3s constituting the Fc region. 〔8〕 The polypeptide complex described in 〔7〕, wherein the heavy chain Fv fragment constituting the CD3 binding domain is linked to one CH3 constituting the Fc region, and the light chain Fv fragment constituting the CD3 binding domain is linked to the other CH3 constituting the Fc region. 〔9〕 The polypeptide complex described in 〔8〕, wherein the CH1 domain of an antibody is linked to the heavy chain Fv fragment constituting the CD3 binding domain, and the CL domain of the antibody is linked to the light chain Fv fragment. 〔10〕 The polypeptide complex described in 〔6〕, wherein the CD3 binding domain is linked to one or two CLs constituting F(ab’)2. 〔11〕 The polypeptide complex described in 〔6〕, wherein the CD3 binding domain is linked to one or two VHs constituting F(ab’)2. 〔12〕 The polypeptide complex described in 〔6〕, wherein the CD3 binding domain is linked to one or two VLs constituting F(ab’)2. 〔13〕 The polypeptide complex according to any one of 〔1〕 to 〔12〕, wherein the CD3 binding domain is Fv. 〔14〕 The polypeptide complex according to any one of 〔1〕 to 〔7〕 and 〔10〕 to 〔12〕, wherein the CD3 binding domain is Fab. 〔15〕 The polypeptide complex according to any one of 〔1〕 to 〔7〕 and 〔10〕 to 〔12〕, wherein the CD3 binding domain is scFv. 〔16〕 The polypeptide complex according to any one of 〔1〕 to 〔15〕, wherein the CD3 binding domain is monovalent. 〔17〕 The antigen binding domain is a monovalent scFv and a monovalent Fab, and any one of 〔1〕 to 〔3〕 The polypeptide conjugate described in any one of them. 〔18〕 One polypeptide constituting the Fc region via the scFv constituting the CD3 binding domain by a monovalent scFv, and one polypeptide constituting the Fc region via the CH1 region by the heavy chain Fv fragment of a monovalent Fab are each linked to the polypeptide, and the light chain Fv fragment of the Fab is linked to the CL region. The polypeptide conjugate described in 〔17〕. 〔19〕 The polypeptide conjugate described in any one of 〔1〕 to 〔3〕, wherein the antigen binding domain is a bivalent scFv. 〔20〕 One polypeptide constituting the Fc region via the heavy chain Fv fragment constituting the CD3 binding domain by a monovalent scFv is linked to the other polypeptide constituting the Fc region via the light chain Fv fragment constituting the CD3 binding domain by the other monovalent scFv. The polypeptide conjugate described in 〔19〕. 〔21〕 One polypeptide constituting the Fc region via the scFv constituting the CD3 binding domain by a monovalent scFv is linked to the other polypeptide constituting the Fc region by the other monovalent scFv. The polypeptide conjugate described in 〔19〕. 〔22〕 The polypeptide conjugate described in any one of 〔1〕 to 〔3〕, wherein the antigen binding domain and the T cell receptor complex binding domain are each a monovalent Fab. 〔23〕 The heavy chain Fv fragment of the monovalent Fab constituting the antigen binding domain is linked to one polypeptide constituting the Fc region via the CH1 region, the light chain Fv fragment of the Fab is linked to the CL region, and the heavy chain Fv fragment of the Fab constituting the T cell receptor binding domain is linked to the other polypeptide constituting the Fc region via the CH1 region, and the light chain Fv fragment of the Fab is linked to the CL region. The polypeptide conjugate described in 〔22〕. 〔24〕 One polypeptide constituting the Fc region via the scFv constituting the CD3 binding domain by a monovalent scFv is linked to the other polypeptide constituting the Fc region by the other monovalent scFv. The polypeptide conjugate described in 〔19〕. 〔25〕 One polypeptide constituting the Fc region via the scFv constituting the CD3 binding domain by a monovalent scFv is linked to the other polypeptide constituting the Fc region by the other monovalent scFv. The polypeptide conjugate described in 〔19〕. 〔26〕 One polypeptide constituting the Fc region via the scFv constituting the CD3 binding domain by a monovalent scFv is linked to the other polypeptide constituting the Fc region by the other monovalent scFv. The polypeptide conjugate described in 〔19〕. 〔27〕 The polypeptide conjugate described in any one of 〔1〕 to 〔3〕, wherein the antigen binding domain and the T cell receptor complex binding domain are each a monovalent Fab. 〔28〕 The heavy chain Fv fragment of the monovalent Fab constituting the antigen binding domain is linked to one polypeptide constituting the Fc region via the CH1 region, the light chain Fv fragment of the Fab is linked to the CL region, and the heavy chain Fv fragment of the Fab constituting the T cell receptor binding domain is linked to the other polypeptide constituting the Fc region via the CH1 region, and the light chain Fv fragment of the Fab is linked to the CL region. The polypeptide conjugate described in 〔27〕. 〔29〕 One polypeptide constituting the Fc region via the scFv constituting the CD3 binding domain by a monovalent scFv is linked to the other polypeptide constituting the Fc region by the other monovalent scFv. The polypeptide conjugate described in 〔19〕. 〔30〕 One polypeptide constituting the Fc region via the scFv constituting the CD3 binding domain by a monovalent scFv is linked to the other polypeptide constituting the Fc region by the other monovalent scFv. The polypeptide conjugate described in 〔19〕. 〔31〕 The polypeptide conjugate described in any one of 〔1〕 to 〔3〕, wherein the antigen binding domain and the T cell receptor complex binding domain are each a monovalent Fab. 〔32〕 The heavy chain Fv fragment of the monovalent Fab constituting the antigen binding domain is linked to one polypeptide constituting the Fc region via the CH1 region, the light chain Fv fragment of the Fab is linked to the CL region, and the heavy chain Fv fragment of the Fab constituting the T cell receptor binding domain is linked to the other polypeptide constituting the Fc region via the CH1 region, and the light chain Fv fragment of the Fab is linked to the CL region. The polypeptide conjugate described in 〔31〕. 〔33〕 One polypeptide constituting the Fc region via the scFv constituting the CD3 binding domain by a monovalent scFv is linked to the other polypeptide constituting the Fc region by the other monovalent scFv. The polypeptide conjugate described in 〔19〕. 〔34〕 One polypeptide constituting the Fc region via the scFv constituting the CD3 binding domain by a monovalent scFv is linked to the other polypeptide constituting the Fc region by the other monovalent scFv. The polypeptide conjugate described in 〔19〕. 〔35〕 One polypeptide constituting the Fc region via the scFv constituting the CD3 binding domain by a monovalent scFv is linked to the other polypeptide constituting the Fc region by the other monovalent scFv. The polypeptide conjugate described in 〔19〕. 〔36〕 One polypeptide constituting the Fc region via the scFv constituting the CD3 binding domain by a monovalent scFv is linked to the other polypeptide constituting the Fc region by the other monovalent scFv. The polypeptide conjugate described in 〔19〕. 〔37〕 The polypeptide conjugate described in any one of 〔1〕 to 〔3〕, wherein the antigen binding domain and the T cell receptor complex binding domain are each a monovalent Fab. 〔24〕The heavy-chain Fv fragment of a monovalent Fab constituting the antigen-binding domain is linked to one polypeptide constituting the Fc region via the CH1 region, the light-chain Fv fragment of the Fab is linked to the CL region, and the light-chain Fv fragment of the Fab constituting the T-cell receptor-binding domain is linked to the other polypeptide constituting the Fc region via the CH1 region, and the heavy-chain Fv fragment of the Fab is linked to the CL region. The polypeptide conjugate described in 〔22〕 〔25〕The heavy-chain Fv fragment of a monovalent Fab constituting the antigen-binding domain is linked to one polypeptide constituting the Fc region via the CH1 region, the light-chain Fv fragment of the Fab is linked to the CL region, and the heavy-chain Fv fragment of the Fab constituting the T-cell receptor-binding domain is linked 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 region. The polypeptide conjugate described in 〔22〕 〔26〕The heavy-chain Fv fragment of a monovalent Fab constituting the T-cell receptor-binding domain is linked to one polypeptide constituting the Fc region via the CH1 region, the light-chain Fv fragment of the Fab is linked to the CL region, and the light-chain Fv fragment of the Fab constituting the antigen-binding domain is linked to the other polypeptide constituting the Fc region via the CH1 region, and the heavy-chain Fv fragment of the Fab is linked to the CL region. The polypeptide conjugate described in 〔22〕 〔27〕The heavy-chain Fv fragment of a monovalent Fab constituting the T-cell receptor-binding domain is linked to one polypeptide constituting the Fc region via the CH1 region, the light-chain Fv fragment of the Fab is linked to the CL region, and the heavy-chain Fv fragment of the Fab constituting the antigen-binding domain is linked 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 region. The polypeptide conjugate described in 〔22〕 ​​​​​​​​​​​​​​​The polypeptide complex described in [[ ]].

[28] (1) The heavy chain Fv fragment of a monovalent Fab structure that binds to an antigen is linked via the CH1 region to one polypeptide constituting the Fc region, and the light chain Fv fragment of the Fab structure is linked to the CL region An antigen-binding domain, and (2) The heavy chain Fv fragment of a monovalent Fab structure that binds to the T cell receptor complex is linked via the CH1 region to the other polypeptide constituting the Fc region, and the light chain Fv fragment of the Fab structure is linked to the CL region A T cell receptor complex-binding domain, A polypeptide complex comprising: the heavy chain Fv fragment in the antigen-binding domain and the light chain Fv fragment in the antigen-binding domain, or 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, wherein the charges of the CH1 region and the CL region are controlled so as to associate with each other. The polypeptide complex described in [

[22] ].

[29] 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 antigen-binding domain have the same type of charge as each other. The polypeptide complex described in [

[28] ].

[30] The amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen-binding domain and the amino acid residues of the CL region linked to the light chain Fv fragment in the T cell receptor complex-binding domain have the same type of charge as each other. The polypeptide complex described in [

[28] ].

[31] 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 antigen-binding domain have the same type of charge as each other, 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 light chain Fv fragment in the residue and the T cell receptor complex binding domain have the same charge as each other, the polypeptide aggregate according to

[28] .

[32] 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 have different charges from each other, the polypeptide aggregate according to

[29] or

[31] .

[33] The amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen binding domain and the amino acid residues of the CL region linked to the light chain Fv fragment in the antigen binding domain both have different charges from each other, the polypeptide aggregate according to

[30] or

[31] .

[34] The T cell receptor complex binding domain is a T cell receptor binding domain, the polypeptide aggregate according to any one of

[22] to

[33] .

[35] The T cell receptor binding domain is a CD3 binding domain, the polypeptide aggregate according to

[34] .

[36] The amino acid residues of the CH1 region and the amino acid residues of the CL region are one set or two or more sets of amino acid residue sets shown in the following (a) to (f); (a) The amino acid residue at position 147 of the EU numbering in the CH1 region, and the amino acid residue at position 180 of the EU numbering in the CL region (b) The amino acid residue at position 147 of the EU numbering in the CH1 region, and the amino acid residue at position 131 of the EU numbering in the CL region (c) The amino acid residue at position 147 of the EU numbering in the CH1 region, and the amino acid residue at position 164 of the EU numbering in the CL region ​​​​​(d) An amino acid residue in the CH1 region, which is the amino acid residue at position 147 in the EU numbering, and the CL region An amino acid residue in the CL region, which is the amino acid residue at position 138 in the EU numbering (e) An amino acid residue in the CH1 region, which is the amino acid residue at position 147 in the EU numbering, and the CL region An amino acid residue in the CL region, which is the amino acid residue at position 123 in the EU numbering (f) An amino acid residue in the CH1 region, which is the amino acid residue at position 175 in the EU numbering, and the CL region An amino acid residue in the CL region, which is the amino acid residue at position 160 in the EU numbering Selected from the amino acid residues having different charges from each other between the amino acid residue in the CH1 region and the amino acid residue in the CL region The polypeptide aggregate according to any one of

[32] or

[33] , which is an amino acid residue having different charges from each other between the amino acid residue in the CH1 region and the amino acid residue in the CL region.

[37] Further selected from the group consisting of sets of amino acid residues shown in the following (g): The polypeptide aggregate according to

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

[38] The polypeptide aggregate according to

[36] or

[37] , wherein the amino acid residue having different charges is selected from any one of the following (X) or (Y): group; (X) Glutamic acid (E), Aspartic acid (D); (Y) Lysine (K), Arginine (R), Histidine (H); The polypeptide aggregate according to

[36] or

[37] , which is selected from the amino acid residues included in the above.

[39] The amino acid residue having different charges is the amino acid residue in the CH1 region, which is Lys at position 175 in the EU numbering, and the amino acid residues in the CL region at positions 180, 131, and 160 in the EU numbering are all Glu. From

[36] to [3 The polypeptide aggregate according to

[36] , wherein the amino acid residue at position 175 in the EU numbering is Lys, and the amino acid residues at positions 180, 131, and 160 in the EU numbering in the CL region are all Glu. 7], The polypeptide complex according to any one of 〔8〕. 〔40〕 The amino acid residue having the different charge is an amino acid residue in the CH1 region and is EU The amino acid residues at 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〕 to the polypeptide complex according to any one of 〔38〕. 〔41〕 Further, the amino acid residue in the CH1 region and the amino acid residue at position 213 according to EU numbering is Glu, and the amino acid residue in the CL region and the amino acid residue at position 123 according to EU numbering is Lys, the polypeptide complex according to 〔40〕. 〔42〕 The Fc region is a Fc region with reduced binding activity to any one of the Fcγ receptors of FcγI, FcγIIA, FcγIIB, FcγIIIA, and / or FcγIIIB, any of 〔1〕 to 〔41〕 to the polypeptide complex according to any of them. The polypeptide complex according to any one of 〔1〕 to 〔41〕. 〔43〕 The Fc region is the Fc region described in SEQ ID NO: 23, the Fc region described in SEQ ID NO: 24, the Fc region described in SEQ ID NO: 25, or the Fc region characterized in that the amino acids constituting the Fc region described in SEQ ID NO: 26 are mutated, any of 〔1〕 to 〔42〕 The polypeptide complex according to any one of them. 〔44〕 Any of the following amino acids specified according to EU numbering among the amino acids constituting the Fc region; Any of the following; The amino acid sequence from position 118 to position 260 is the sequence described in SEQ ID NO: 24, and the amino acid sequence from position 261 to position 447 is the sequence described in SEQ ID NO: 26, the Fc region, the polypeptide dimer according to 〔43〕. The polypeptide complex according to 〔43〕. 〔45〕 Any of the following amino acids specified according to EU numbering among the amino acids constituting the Fc region; Any amino acid; Positions 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239 Positions 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298 , 299, 300, 325, 327, 328, 329, 330, 331, 332, The polypeptide complex described in

[43] , which is an Fc region in which the above are mutated.

[46] The Fc region in which the amino acids constituting the Fc region described in SEQ ID NO: 23 are mutated The polypeptide complex described in

[45] , characterized in that it is an Fc region.

[47] Any of the following amino acids specified according to EU numbering among the amino acids constituting the Fc region; Any amino acid; Positions 233, 234, 235, 236, 237, 327, 330, 331, The Fc region in which the EU numbering is substituted with the corresponding amino acid in the corresponding IgG2 or IgG4 The polypeptide complex described in

[46] , which is an Fc region.

[48] Any of the following amino acids specified according to EU numbering among the amino acids constituting the Fc region; Any amino acid; Positions 234, 235, 297, The polypeptide complex described in

[46] , characterized in that it is an Fc region in which the above are mutated.

[49] The polypeptide complex described in

[48] , characterized in that 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 mutated to alanine. Dimer.

[50] The polypeptide complex according to any one of

[43] to

[49] , characterized in that the sequences of the two polypeptides constituting the Fc region have different sequences from each other. ​〔51〕One of the two polypeptides constituting the Fc region, wherein the amino acid residue at position 349 identified according to EU numbering is cysteine, and the amino acid at position 366 is tryptophan; among the amino acid residues of the other polypeptide, according to EU numbering the amino acid at position 356 is cysteine, the amino acid at position 366 is serine, the amino acid at position 368 is alanine, and the amino acid at position 407 is valine, which is characterized in that it is a polypeptide complex as described in any one of 〔1〕 to 〔50〕. 〔52〕One of the two polypeptides constituting the Fc region, wherein the amino acid residue at position 356 identified according to EU numbering is lysine; among the amino acid residues of the other polypeptide, the amino acid at position 439 identified according to EU numbering is glutamic acid, and among the amino acid residues of either one of the polypeptides, the amino acid at position 435 identified according to EU numbering is arginine, which is characterized in that it is a polypeptide complex as described in any one of 〔1〕 to 〔50〕. 〔53〕The polypeptide complex according to 〔51〕 or 〔52〕, characterized in that the sequence GK present at the carboxy terminus of the two polypeptides constituting the Fc region is deleted. 〔54〕The polypeptide complex according to any one of 〔1〕 to 〔53〕, wherein the antigen-binding domains bind to the same epitope. 〔55〕The polypeptide complex according to 〔54〕, wherein the same epitope is present in the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2. 〔56〕The polypeptide complex according to 〔54〕, wherein the same epitope is present in the protein consisting of the amino acid sequence set forth in SEQ ID NO: 4. 〔57〕The polypeptide complex according to 〔54〕, wherein the same epitope is present in the protein consisting of the amino acid sequence set forth in SEQ ID NO: 6. 〔58〕The polypeptide complex according to 〔54〕, wherein the same epitope is present in the protein consisting of the amino acid sequence set forth in SEQ ID NO: 8. 〔59〕The polypeptide complex according to 〔54〕, wherein the same epitope is present in the protein consisting of the amino acid sequence set forth in SEQ ID NO: 10. 〔60〕The polypeptide complex according to 〔54〕, wherein the same epitope is present in the protein consisting of the amino acid sequence set forth in SEQ ID NO: 12. 〔61〕The polypeptide complex according to 〔54〕, wherein the same epitope is present in the protein consisting of the amino acid sequence set forth in SEQ ID NO: 14. 〔57〕A polypeptide complex according to any one of 〔1〕 to 〔53〕, wherein the antigen-binding domains bind to different epitopes. 〔58〕A polypeptide complex according to 〔57〕, wherein the different epitopes are present in the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2. 〔59〕A polypeptide complex according to 〔57〕, wherein the different epitopes are present in the protein consisting of the amino acid sequence set forth in SEQ ID NO: 4. 〔60〕A polynucleotide encoding a polypeptide complex according to any one of 〔1〕 to 〔59〕. 〔61〕A vector containing the polynucleotide according to 〔60〕. 〔62〕A cell harboring the vector according to 〔61〕. 〔63〕A method for producing a polypeptide complex, comprising culturing the cell according to 〔62〕 and recovering the polypeptide complex from the culture supernatant. 〔64〕A cytotoxin-inducing therapeutic agent comprising, as an active ingredient, a polypeptide complex according to any one of 〔1〕 to 〔59〕. 〔65〕The therapeutic agent according to 〔64〕, wherein the cytotoxin-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 or preventing cancer, comprising administering to a patient in need of treatment a polypeptide complex according to any one of 〔1〕 to 〔59〕. 〔68〕The method for treatment or prevention according to 〔67〕, wherein the cancer is liver cancer or lung cancer.

[0013] The present invention also relates to a kit for use in the method of the present invention, comprising the polypeptide complex of the present invention or the polypeptide complex produced by the production method of the present invention. The present invention also relates to a polypeptide complex of the present invention or a polypeptide produced by the production method of the present invention Relates to the use of an aggregate in the production of a cytotoxic induction therapeutic agent. The present invention also relates to the polypeptide aggregate of the present invention or the polypeptide aggregate produced by the production method of the present invention for use in the method of the present invention. Relates to the polypeptide aggregate of the present invention or the polypeptide aggregate produced by the production method of the present invention for use in the method of the present invention. Relates to the polypeptide aggregate of the present invention or the polypeptide aggregate produced by the production method of the present invention for use in the method of the present invention.

Advantages of the Invention

[0014] According to the present invention, the strong antitumor activity of BiTE and the excellent safety property of not inducing cytokines etc. independently of cancer antigens are maintained, and a new polypeptide aggregate having a long blood half-life is provided. By replacing the antigen-binding domain in the polypeptide aggregate of the present invention, a cytotoxic induction therapeutic agent containing the polypeptide aggregate as an active ingredient causes cytotoxicity targeting various cells including cancer cells, and can treat or prevent various cancers. For patients, not only is the safety high, but also a desirable treatment with less physical burden and high convenience can be achieved. According to the present invention, the strong antitumor activity of BiTE and the excellent safety property of not inducing cytokines etc. independently of cancer antigens are maintained, and a new polypeptide aggregate having a long blood half-life is provided. By replacing the antigen-binding domain in the polypeptide aggregate of the present invention, a cytotoxic induction therapeutic agent containing the polypeptide aggregate as an active ingredient causes cytotoxicity targeting various cells including cancer cells, and can treat or prevent various cancers. For patients, not only is the safety high, but also a desirable treatment with less physical burden and high convenience can be achieved. According to the present invention, the strong antitumor activity of BiTE and the excellent safety property of not inducing cytokines etc. independently of cancer antigens are maintained, and a new polypeptide aggregate having a long blood half-life is provided. By replacing the antigen-binding domain in the polypeptide aggregate of the present invention, a cytotoxic induction therapeutic agent containing the polypeptide aggregate as an active ingredient causes cytotoxicity targeting various cells including cancer cells, and can treat or prevent various cancers. For patients, not only is the safety high, but also a desirable treatment with less physical burden and high convenience can be achieved. According to the present invention, the strong antitumor activity of BiTE and the excellent safety property of not inducing cytokines etc. independently of cancer antigens are maintained, and a new polypeptide aggregate having a long blood half-life is provided. By replacing the antigen-binding domain in the polypeptide aggregate of the present invention, a cytotoxic induction therapeutic agent containing the polypeptide aggregate as an active ingredient causes cytotoxicity targeting various cells including cancer cells, and can treat or prevent various cancers. For patients, not only is the safety high, but also a desirable treatment with less physical burden and high convenience can be achieved. According to the present invention, the strong antitumor activity of BiTE and the excellent safety property of not inducing cytokines etc. independently of cancer antigens are maintained, and a new polypeptide aggregate having a long blood half-life is provided. By replacing the antigen-binding domain in the polypeptide aggregate of the present invention, a cytotoxic induction therapeutic agent containing the polypeptide aggregate as an active ingredient causes cytotoxicity targeting various cells including cancer cells, and can treat or prevent various cancers. For patients, not only is the safety high, but also a desirable treatment with less physical burden and high convenience can be achieved. According to the present invention, the strong antitumor activity of BiTE and the excellent safety property of not inducing cytokines etc. independently of cancer antigens are maintained, and a new polypeptide aggregate having a long blood half-life is provided. By replacing the antigen-binding domain in the polypeptide aggregate of the present invention, a cytotoxic induction therapeutic agent containing the polypeptide aggregate as an active ingredient causes cytotoxicity targeting various cells including cancer cells, and can treat or prevent various cancers. For patients, not only is the safety high, but also a desirable treatment with less physical burden and high convenience can be achieved. According to the present invention, the strong antitumor activity of BiTE and the excellent safety property of not inducing cytokines etc. independently of cancer antigens are maintained, and a new polypeptide aggregate having a long blood half-life is provided. By replacing the antigen-binding domain in the polypeptide aggregate of the present invention, a cytotoxic induction therapeutic agent containing the polypeptide aggregate as an active ingredient causes cytotoxicity targeting various cells including cancer cells, and can treat or prevent various cancers. For patients, not only is the safety high, but also a desirable treatment with less physical burden and high convenience can be achieved.

Brief Description of the Drawings

[0015]

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Mode for Carrying Out the Invention

[0016] The following definitions are provided to facilitate the understanding of the present invention described in this specification. 。

[0017] antibody In this specification, an antibody refers to an immunoglobulin that is natural or produced by partial or complete synthesis. An antibody can be isolated from a natural source such as plasma or serum in which it naturally exists or from the culture supernatant of hybridoma cells that produce the antibody, or can be partially or completely synthesized by using techniques such as genetic recombination. Examples of antibodies include those derived from natural sources such as plasma and serum where it exists naturally, or from the culture supernatant of hybridoma cells that produce the antibody. It can also be isolated or can be partially or completely synthesized by using techniques such as genetic recombination. Examples of antibodies include those derived from natural sources such as plasma and serum where it exists naturally, or from the culture supernatant of hybridoma cells that produce the antibody. It can also be isolated or can be partially or completely synthesized by using techniques such as genetic recombination. Examples of antibodies include those derived from natural sources such as plasma and serum where it exists naturally, or from the culture supernatant of hybridoma cells that produce the antibody. It can also be isolated or can be partially or completely synthesized by using techniques such as genetic recombination. Examples of antibodies Then, isotypes of immunoglobulins and their subclasses are preferably listed. As human immunoglobulins, nine classes (isotypes) of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM are known. The antibodies of the present invention may include IgG1, IgG2, IgG3, and IgG4 among these isotypes.

[0018] Methods for producing antibodies having a desired binding activity are known to those skilled in the art. Hereinafter, a method for producing an antibody (anti-GPC3 antibody) that binds to GPC3 (Int J Cancer. (2003) 103 (4), 455-65), which belongs to the GPI-anchor type receptor family, is exemplified. Antibodies that bind to antigens other than GPC3 can also be appropriately produced according to the following examples.

[0019] Anti-GPC3 antibodies can be obtained as polyclonal or monoclonal antibodies using known means. As anti-GPC3 antibodies, monoclonal antibodies derived from mammals are preferably produced. Monoclonal antibodies derived from mammals include those produced by hybridomas and those produced by host cells transformed with an expression vector containing an antibody gene by genetic engineering techniques.

[0020] Monoclonal antibody-producing hybridomas can be produced, for example, as follows by using known techniques. That is, using the GPC3 protein as an immunizing antigen, a mammal is immunized according to a normal immunization method. The obtained immune cells are fused with known parent cells by a normal cell fusion method. Next, monoclonal ​​​​​​​​​​​​Hybrids that produce anti-GPC3 antibodies can be selected by screening for naive antibody-producing cells. -mas can be selected.

[0021] Specifically, the production of monoclonal antibodies is carried out, for example, as shown below. First, Re fSeq accession number NM_001164617.1 (SEQ ID NO: 1) discloses the nucleotide sequence of GPC3 By expressing the gene, the GPC3 protein represented by RefSeq accession number NP _001158089.1 (SEQ ID NO: 2) can be obtained as the sensitizing antigen for antibody acquisition. That is, GP C3 is inserted into a known expression vector to transform an appropriate host cell The desired human GPC3 protein is purified from the host cell or the culture supernatant by a known method. To obtain soluble GPC3 from the culture supernatant, for example, among the GPC3 polypeptide sequences represented by SEQ ID NO: 2, 564-580 amino acids that constitute the hydrophobic region corresponding to the GPI anchor sequence used for GPC3 to be anchored on the cell membrane are deleted. The protein is expressed instead of the GPC3 protein represented by SEQ ID NO: 2. Also, the purified native GPC3 protein can be used as the sensitizing antigen in the same way. For example, in order to obtain soluble GPC3 from the culture supernatant, among the GPC3 polypeptide sequences represented by SEQ ID NO: 2, 564-580 amino acids that constitute the hydrophobic region corresponding to the GPI anchor sequence used for GPC3 to be anchored on the cell membrane are deleted. The protein is expressed instead of the GPC3 protein represented by SEQ ID NO: 2. Also, the purified native GPC3 protein can be used as the sensitizing antigen in the same way. Among the GPC3 polypeptide sequences represented by SEQ ID NO: 2, the 564-580 amino acids that constitute the hydrophobic region corresponding to the GPI anchor sequence used for GPC3 to be anchored on the cell membrane are deleted. The protein is expressed instead of the GPC3 protein represented by SEQ ID NO: 2. Also, the purified native GPC3 protein can be used as the sensitizing antigen in the same way. The 564-580 amino acids that constitute the hydrophobic region corresponding to the GPI anchor sequence used for GPC3 to be anchored on the cell membrane are deleted. The protein is expressed instead of the GPC3 protein represented by SEQ ID NO: 2. Also, the purified native GPC3 protein can be used as the sensitizing antigen in the same way. The protein is expressed instead of the GPC3 protein represented by SEQ ID NO: 2. Also, the purified native GPC3 protein can be used as the sensitizing antigen in the same way. The purified native GPC3 protein can also be used as the sensitizing antigen in the same way.

[0022] The purified GPC3 protein can be used as the sensitizing antigen for immunization of mammals. Partial peptides of GPC3 can also be used as sensitizing antigens. At this time, the partial peptides can also be obtained by chemical synthesis from the amino acid sequence of human GPC3. They can also be obtained by incorporating a part of the GPC3 gene into an expression vector and expressing it. Furthermore, by proteolysis They can also be obtained by incorporating a part of the GPC3 gene into an expression vector and expressing it. Furthermore, by proteolysis It can also be obtained by decomposing GPC3 protein using an enzyme, but the region and size of the GPC3 peptide used as a partial peptide are not particularly limited to any specific embodiment. Preferred regions may be an amino acid sequence corresponding to amino acids 564 - 580 in the amino acid sequence of SEQ ID NO:2, or any sequence can be selected. The number of amino acids constituting the peptide used as the sensitizing antigen is preferably at least 5 or more, for example 6 or more, or 7 or more. More specifically, peptides of 8 - 50, preferably 10 - 30 residues can be used as the sensitizing antigen.

[0023] In addition, a fusion protein in which a desired partial polypeptide or peptide of GPC3 protein is fused with a different polypeptide can be used as the sensitizing antigen. For producing the fusion protein used as the sensitizing antigen, for example, the Fc fragment of an antibody, a peptide tag, etc. can be suitably used obtained. The vector expressing the fusion protein can be prepared by fusing genes encoding two or more desired polypeptide fragments in-frame and inserting the fusion gene into the expression vector as described above. The method for producing the fusion protein is described in Molecular Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47 - 9.58(1989 ) Cold Spring Harbor Lab. press). The method for obtaining GPC3 used as the sensitizing antigen and the immunization method using the same are specifically described in WO2003 / 000883, WO2004 / 022754, WO2006 / 00669 3, etc. Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47 - 9.58(1989 ) Cold Spring Harbor Lab. press). The mammalian animal immunized with the sensitizing antigen is not limited to a specific animal, but is preferably a mouse, a rat, a rabbit, a guinea pig, a hamster, a dog, a cat, a monkey, etc. are specifically described in WO2003 / 000883, WO2004 / 022754, WO2006 / 006693, etc.

[0024] Although not limited to a specific animal, the mammalian animal immunized with the sensitizing antigen is preferably a mouse, a rat, a rabbit, a guinea pig, a hamster, a dog, a cat, a monkey, etc. It is preferably selected in consideration of compatibility with the parental cells to be used for cell fusion. Generally, rodents such as mice, rats, hamsters, or rabbits, monkeys, etc. are preferably used. The animals are immunized with the sensitizing antigen according to known methods. For example, as a general method, the sensitizing antigen is administered by injection into the peritoneal cavity or subcutaneously of a mammal to effect immunization. Specifically, the sensitizing antigen diluted at an appropriate dilution factor with PBS (Phosphate - Buffered Saline), physiological saline, etc. is mixed with a normal adjuvant such as Freund's complete adjuvant, emulsified, and then the sensitizing antigen is administered to the mammal several times every 4 to 21 days.

[0025] Also, an appropriate carrier may be used during immunization with the sensitizing antigen. Particularly when a small - molecular - weight partial peptide is used as the sensitizing antigen, it may be desirable to immunize the sensitizing antigen peptide conjugated with a carrier protein such as albumin, keyhole limpet hemocyanin. In addition, hybridomas that produce the desired antibody can also be prepared using DNA immunization as follows. DNA immunization is an immunization method in which vector DNA constructed in such a manner that the gene encoding the antigen protein can be expressed in the immunized animal is administered, and the sensitizing antigen is expressed in the living body of the immunized animal to provide an immune stimulus. Compared with the general immunization method in which a protein antigen is administered to an immunized animal, the following advantages are expected for DNA immunization. - Immune stimulation can be provided while maintaining the structure of membrane proteins such as GPC3. It can be expected to provide immune stimulation while maintaining the structure of membrane proteins such as GPC3. It can be expected to provide immune stimulation while maintaining the structure of membrane proteins such as GPC3. It can be expected to provide immune stimulation while maintaining the structure of membrane proteins such as GPC3. It can be expected to provide immune stimulation while maintaining the structure of membrane proteins such as GPC3. It can be expected to provide immune stimulation while maintaining the structure of membrane proteins such as GPC3.

[0026] Moreover, hybridomas that produce the desired antibody can be prepared using DNA immunization as follows. DNA immunization refers to an immunization method in which vector DNA constructed in such a way that the gene encoding the antigen protein can be expressed in the immunized animal is administered, and the sensitizing antigen is expressed in the living body of the immunized animal to provide an immune stimulus. Compared with the general immunization method in which a protein antigen is administered to an immunized animal, the following advantages are expected for DNA immunization. - Immune stimulation can be provided while maintaining the structure of membrane proteins such as GPC3. Compared with the general immunization method in which a protein antigen is administered to an immunized animal, the following advantages are expected for DNA immunization. - Immune stimulation can be provided while maintaining the structure of membrane proteins such as GPC3. - Immune stimulation can be provided while maintaining the structure of membrane proteins such as GPC3. - There is no need to purify the immunizing antigen

[0027] To obtain the monoclonal antibody of the present invention by DNA immunization, first, the GPC3 protein is The DNA encoding it is administered to the immunized animal. The DNA encoding GPC3 can be synthesized by known methods such as PCR The obtained DNA is inserted into an appropriate expression vector and administered to the immunized animal As the expression vector, commercially available expression vectors such as pcDNA3.1 can be preferably used As a method for administering the vector to a living body, generally used methods can be utilized For example, DNA immunization is performed by introducing gold particles adsorbed with the expression vector into the cells of an immunized animal individual with a gene gun Furthermore, the antibody recognizing GPC3 can also be produced using the method described in International Publication WO2003 / 104453

[0028] After the mammal is immunized in this way and an increase in the antibody titer that binds to GPC3 in the serum is confirmed Immune cells are collected from the mammal and used for cell fusion. As the preferred immune cells Spleen cells can be particularly used

[0029] As the cells to be fused with the immune cells, mammalian myeloma cells are used. The myeloma cells preferably have an appropriate selection marker for screening The selection marker refers to a trait that can (or cannot) survive under specific culture conditions Known selection markers include hypoxanthine-guanine phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency), or thymidine kinase deficiency (hereinafter abbreviated as TK deficiency), etc Cells having a deficiency of HGPRT or TK are hypoxanthine-aminopterin has a sensitivity to hypoxanthine-aminopterin-thymidine (hereinafter abbreviated as HAT sensitivity). HAT-sensitive cells cannot carry out DNA synthesis in HAT selection medium and die, but when fused with normal cells, they can utilize the salvage pathway of normal cells to continue DNA synthesis and thus grow in HAT selection medium. Cells lacking HGPRT or TK can be selected in media containing 6-thioguanine, 8-azaguanine (hereinafter abbreviated as 8AG), or 5-bromodeoxyuridine, respectively. Normal cells that incorporate these pyrimidine analogs into DNA die. On the other hand, cells lacking these enzymes that cannot incorporate these pyrimidine analogs can survive in the selection medium. In addition, a selection marker called G418 resistance confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) by the neomycin resistance gene. Various myeloma cells suitable for cell fusion are known. As such myeloma cells, for example, P3 (P3x63Ag8.653) (J. Immunol. (1979 ) 123 (4), 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunolo

[0030] 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. has a sensitivity to hypoxanthine-aminopterin-thymidine (hereinafter abbreviated as HAT sensitivity). HAT-sensitive cells cannot carry out DNA synthesis in HAT selection medium and die, but when fused with normal cells, they can utilize the salvage pathway of normal cells to continue DNA synthesis and thus grow in HAT selection medium. Cells lacking HGPRT or TK can be selected in media containing 6-thioguanine, 8-azaguanine (hereinafter abbreviated as 8AG), or 5-bromodeoxyuridine, respectively. Normal cells that incorporate these pyrimidine analogs into DNA die. On the other hand, cells lacking these enzymes that cannot incorporate these pyrimidine analogs can survive in the selection medium. In addition, a selection marker called G418 resistance confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) by the neomycin resistance gene. Various myeloma cells suitable for cell fusion are known. As such myeloma cells, 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 (

[0031] has a sensitivity to hypoxanthine-aminopterin-thymidine (hereinafter abbreviated as HAT sensitivity). HAT-sensitive cells cannot carry out DNA synthesis in HAT selection medium and die, but when fused with normal cells, they can utilize the salvage pathway of normal cells to continue DNA synthesis and thus grow in HAT selection medium. ) 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 to obtain.

[0032] Basically, according to known methods, for example, the method of Keller and Milstein et al. (Methods Enzymo l. (1981) 73, 3-46), etc., cell fusion of the immunocytes and myeloma cells is carried out . More specifically, for example, in the presence of a cell fusion promoter in a normal nutrient culture solution, the cell fusion can be carried out. As the cell fusion promoter, for example, polyethylene glycol (PEG), Sendai virus (HVJ), etc. are used, and if desired, an auxiliary agent such as dimethyl sulfoxide is added to increase the fusion efficiency and used.

[0033] The usage ratio of immunocytes and myeloma cells can be arbitrarily set. For example, it is preferable to make the immunocytes 1 to 10 times that of the myeloma cells. As the culture solution used for the cell fusion , for example, RPMI1640 culture solution, MEM culture solution, which are suitable for the growth of the myeloma cell line, and other normal culture solutions used for this type of cell culture are used, and furthermore, a serum supplement such as fetal calf serum (FCS ) can be preferably added.

[0034] For cell fusion, a predetermined amount of the immunocytes and myeloma cells are well mixed in the culture solution, and a PEG solution (for example, with an average molecular weight of about 1000 to 6000) pre-warmed to about 37°C is usually added at a concentration of 30 to 6 0% (w / v). By gently mixing the mixture, the desired fused cells ​​​Hybridomas are formed. Then, the appropriate culture medium mentioned above is sequentially added , and by repeating the operation of centrifuging to remove the supernatant, cell fusion agents and the like that are not favorable for the growth of hybridomas can be removed .

[0035] The hybridomas thus obtained can be selected by culturing them in a normal selection culture medium, for example, HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). The culture using the above-mentioned HAT culture medium is continued for a sufficient time ( usually, such a sufficient time is several days to several weeks) for cells other than the desired hybridomas (non-fused cells) to die . Then, screening and single cloning of hybridomas that produce the desired antibody are carried out by the usual limiting dilution method .

[0036] The hybridomas thus obtained can be selected by using a selection culture medium corresponding to the selection marker possessed by the myeloma used for cell fusion. For example, cells lacking HGPRT or TK can be selected by culturing them in HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, in the HAT culture medium, cells that have successfully fused with normal cells can selectively proliferate . The culture using the above-mentioned HAT culture medium is continued for a sufficient time for cells other than the desired hybridomas (non-fused cells) to die. Specifically, generally, by culturing for several days to several weeks , the desired hybridomas can be selected. Then, screening and single cloning of hybridomas that produce the desired antibody can be carried out by the usual limiting dilution method . . ​​​​

[0037] Screening and single cloning of the desired antibody can be suitably carried out by a screening method based on a known antigen-antibody reaction. For example, monoclonal antibodies that bind to GPC3 can bind to GPC3 expressed on the cell surface. Such monoclonal antibodies can be screened, for example, by FACS (fluorescence activated cell sorting). FACS is a system that enables the measurement of antibody binding to the cell surface by analyzing cells contacted with a fluorescent antibody with a laser beam and measuring the fluorescence emitted by individual cells. To screen for hybridomas that produce the monoclonal antibody of the present invention by FACS, first, cells expressing GPC3 are prepared. Preferred cells for screening are mammalian cells that overexpress GPC3. By using untransformed mammalian cells used as host cells as a control, the binding activity of the antibody to GPC3 on the cell surface can be selectively detected. That is, by selecting hybridomas that produce antibodies that do not bind to host cells but bind to GPC3 overexpressing cells, hybridomas that produce GPC3 monoclonal antibodies can be obtained. Alternatively, the binding activity of the antibody to immobilized GPC3-expressing cells can be evaluated based on the principle of ELISA. For example, GPC3-expressing cells are immobilized in the wells of an ELISA plate. The culture supernatant of the hybridoma is contacted with the immobilized cells in the well, and the antibody that binds to the immobilized cells is detected.

[0038]

[0039] ​​​​​​​​​​​It is produced. When the monoclonal antibody is derived from a mouse, the antibody bound to the cell can be detected by an anti-mouse immunoglobulin antibody. The hybridomas that produce the desired antibody having the ability to bind to the antigen, selected by these screenings, can be cloned by methods such as limiting dilution. The hybridomas that produce the monoclonal antibodies thus produced can be subcultured in a normal culture medium. Also, the hybridomas can be stored in liquid nitrogen over a long period. The hybridomas that produce the desired antibody having the ability to bind to the antigen, selected by these screenings, can be cloned by methods such as limiting dilution. The hybridomas that produce the monoclonal antibodies thus produced can be subcultured in a normal culture medium. Also, the hybridomas can be stored in liquid nitrogen over a long period.

[0040] The hybridomas that produce the monoclonal antibodies thus produced can be subcultured in a normal culture medium. Also, the hybridomas can be stored in liquid nitrogen over a long period. The hybridomas that produce the monoclonal antibodies thus produced can be subcultured in a normal culture medium. Also, the hybridomas can be stored in liquid nitrogen over a long period. The hybridomas that produce the monoclonal antibodies thus produced can be subcultured in a normal culture medium. Also, the hybridomas can be stored in liquid nitrogen over a long period.

[0041] Culturing the hybridomas according to a normal method, the desired monoclonal antibody can be obtained from the culture supernatant. Alternatively, the hybridomas can be administered to a compatible mammal to proliferate, and the monoclonal antibody can be obtained from the ascites. The former method is suitable for obtaining a high-purity antibody. Culturing the hybridomas according to a normal method, the desired monoclonal antibody can be obtained from the culture supernatant. Alternatively, the hybridomas can be administered to a compatible mammal to proliferate, and the monoclonal antibody can be obtained from the ascites. The former method is suitable for obtaining a high-purity antibody. Culturing the hybridomas according to a normal method, the desired monoclonal antibody can be obtained from the culture supernatant. Alternatively, the hybridomas can be administered to a compatible mammal to proliferate, and the monoclonal antibody can be obtained from the ascites. The former method is suitable for obtaining a high-purity antibody. Culturing the hybridomas according to a normal method, the desired monoclonal antibody can be obtained from the culture supernatant. Alternatively, the hybridomas can be administered to a compatible mammal to proliferate, and the monoclonal antibody can be obtained from the ascites. The former method is suitable for obtaining a high-purity antibody.

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

[0043] For example, from hybridoma cells that produce an anti-GPC3 antibody, the variable region (V) of the anti-GPC3 antibody cDNA encoding the (domain) is obtained. For this purpose, usually, first total RN is extracted from the hybridoma. A is extracted. As a method for extracting mRNA from cells, for example, the following methods can be used. - Guanidine ultracentrifugation method (Biochemistry (1979) 18 (24), 5294 - 5299) - AGPC method (Anal. Biochem. (1987) 162 (1), 156 - 159)

[0044] The extracted mRNA can be purified using an mRNA Purification Kit (manufactured by GE Healthcare Biosciences) or the like. Alternatively, kits for directly extracting total mRNA from cells, such as the QuickPrep mRNA Purification Kit (manufactured by GE Healthcare Bio sciences), are also commercially available. Using such a kit, mRNA can be obtained from the hybridoma. The obtained mRNA can be used to synthesize cDNA encoding the antibody V region using reverse transcriptase. The cDNA can be synthesized by AMV Reverse Transcriptase First - strand cDNA Synthesis Kit (manufactured by Seikagaku Corporation) or the like. In addition, for the synthesis and amplification of cDNA, the SMART RACE cDNA amplification kit ( manufactured by 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 appropriately used. Furthermore, appropriate restriction enzyme sites described below can be introduced at both ends of the cDNA during the synthesis of such cDNA.

[0045] ​​The target cDNA fragment is purified from the obtained PCR product and then ligated to vector DNA. After the recombinant vector is thus prepared, introduced into Escherichia coli or the like, and colonies are selected, the desired recombinant vector can be prepared from the Escherichia coli that formed the colonies. Then, whether the recombinant vector has the nucleotide sequence of the target cDNA or not is confirmed by a known method, for example, the dideoxynucleotide chain termination method or the like.

[0046] To obtain a gene encoding a variable region, it is convenient to use the 5'-RACE method using primers for amplifying the variable region gene. First, cDNA is synthesized using RNA extracted from hybridoma cells as a template, and a 5'-RACE cDNA library is obtained. For the synthesis of the 5'-RACE cDNA library, commercially available kits such as the SMART RACE cDNA amplification kit are appropriately used.

[0047] Using the obtained 5'-RACE cDNA library as a template, the antibody gene is amplified by the PCR method. Primers for amplifying the mouse antibody gene can be designed based on the known antibody gene sequence. These primers have different nucleotide sequences for each immunoglobulin subclass. Therefore, it is desirable to determine the subclass in advance using a commercially available kit such as the Iso Strip Mouse Monoclonal Antibody Isotyping Kit (Roche Diagnostics).

[0048] Specifically, for example, when aiming to obtain a gene encoding mouse IgG, it is possible to amplify the genes encoding γ1, γ2a, γ2b, γ3 as the heavy chain and κ chain and λ chain as the light chain. No primer can be used. To amplify the variable region gene of IgG, generally, a primer that anneals to a portion corresponding to the constant region close to the variable region is used for the 3'-side primer. On the other hand, for the 5'-side primer, the primer attached to the 5’ RACE cDNA library preparation kit is used. Using the PCR product amplified in this way, immunoglobulins composed of combinations of heavy and light chains can be reconstituted. Using the binding activity of the reconstituted immunoglobulin to GPC3 as an index, the desired antibody can be screened. For example, when aiming to obtain an antibody against GPC3, it is more preferable that the binding of the antibody to GPC3 is specific. Antibodies that bind to GPC3 can be screened, for example, as follows:

[0049] (1) A step of contacting an antibody containing a V region encoded by cDNA obtained from a hybridoma with GPC3-expressing cells, (2) A step of detecting the binding between the GPC3-expressing cells and the antibody, and (3) A step of selecting an antibody that binds to the GPC3-expressing cells. Methods for detecting the binding between an antibody and GPC3-expressing cells are known. Specifically, the binding between the antibody and GPC3-expressing cells can be detected by methods such as the FACS mentioned above. A fixed specimen of GPC3-expressing cells can be appropriately used to evaluate the binding activity of the antibody. As a method for screening an antibody using the binding activity as an index, the panning method using a phage vector is also preferably used. When obtaining antibody genes as a library of subclasses of heavy and light chains from a polyclonal antibody-expressing cell group, a phage vector is used. (1) A step of contacting an antibody containing a V region encoded by cDNA obtained from a hybridoma with GPC3-expressing cells, (2) A step of detecting the binding between the GPC3-expressing cells and the antibody, and (3) A step of selecting an antibody that binds to the GPC3-expressing cells. (3) A step of selecting an antibody that binds to the GPC3-expressing cells.

[0050] Methods for detecting the binding between an antibody and GPC3-expressing cells are known. Specifically, the binding between the antibody and GPC3-expressing cells can be detected by methods such as the FACS mentioned above. A fixed specimen of GPC3-expressing cells can be appropriately used to evaluate the binding activity of the antibody. As a method for screening an antibody using the binding activity as an index, the panning method using a phage vector is also preferably used. When obtaining antibody genes as a library of subclasses of heavy and light chains from a polyclonal antibody-expressing cell group, a phage vector is used. A fixed specimen of GPC3-expressing cells can be appropriately used to evaluate the binding activity of the antibody.

[0051] As a method for screening an antibody using the binding activity as an index, the panning method using a phage vector is also preferably used. When obtaining antibody genes as a library of subclasses of heavy and light chains from a polyclonal antibody-expressing cell group, a phage vector is used. When obtaining antibody genes as a library of subclasses of heavy and light chains from a polyclonal antibody-expressing cell group, a phage vector is used. When obtaining antibody genes as a library of subclasses of heavy and light chains from a polyclonal antibody-expressing cell group, a phage vector is used. The following screening method is advantageous. Genes encoding the variable regions of the heavy and light chains can be ligated with an appropriate linker sequence to form a single-chain Fv (scFv). By inserting the gene encoding the scFv into a phage vector, phages expressing the scFv on the surface can be obtained. After contact between this phage and the desired antigen, DNA encoding an scFv having the desired binding activity can be recovered by recovering the phage bound to the antigen. By repeating this operation as necessary, an scFv having the desired binding activity can be concentrated. After obtaining cDNA encoding the V region of the target anti-GPC3 antibody, the cDNA is digested with a restriction enzyme that recognizes the restriction enzyme sites inserted at both ends of the cDNA. A preferred restriction enzyme recognizes and digests a nucleotide sequence that appears with a low frequency in the nucleotide sequence constituting the antibody gene. Further, for inserting one copy of the digested fragment into the vector in the correct direction, insertion of a restriction enzyme that provides cohesive ends is preferred. By inserting the cDNA encoding the V region of the anti-GPC3 antibody digested as described above into an appropriate expression vector, an antibody expression vector can be obtained. At this time, if the gene encoding the antibody constant region (C region) and the gene encoding the V region are fused in-frame, a chimeric antibody is obtained. Here, a chimeric antibody means that the origin of the constant region and the variable region is different. Therefore, in addition to heterologous chimeric antibodies such as mouse-human chimeric antibodies, human-human homologous chimeric antibodies are also included in the chimeric antibodies of the present invention. By inserting the V region gene into an expression vector having a constant region in advance, a chimeric antibody can be obtained. After contact between this phage and the desired antigen, DNA encoding an scFv having the desired binding activity can be recovered by recovering the phage bound to the antigen. By repeating this operation as necessary, an scFv having the desired binding activity can be concentrated. After obtaining cDNA encoding the V region of the target anti-GPC3 antibody, the cDNA is digested with a restriction enzyme that recognizes the restriction enzyme sites inserted at both ends of the cDNA. A preferred restriction enzyme recognizes and digests a nucleotide sequence that appears with a low frequency in the nucleotide sequence constituting the antibody gene.

[0052] Further, for inserting one copy of the digested fragment into the vector in the correct direction, insertion of a restriction enzyme that provides cohesive ends is preferred. The cDNA encoding the V region of the anti-GPC3 antibody digested as described above is inserted into an appropriate expression vector to obtain an antibody expression vector. At this time, if the gene encoding the antibody constant region (C region) and the gene encoding the V region are fused in-frame, a chimeric antibody is obtained. Here, a chimeric antibody means that the origin of the constant region and the variable region is different. Therefore, in addition to heterologous chimeric antibodies such as mouse-human chimeric antibodies, human-human homologous chimeric antibodies are also included in the chimeric antibodies of the present invention. By inserting the V region gene into an expression vector having a constant region in advance, a chimeric antibody can be obtained. At this time, if the gene encoding the antibody constant region (C region) and the gene encoding the V region are fused in-frame, a chimeric antibody is obtained. Here, a chimeric antibody means that the origin of the constant region and the variable region is different. Therefore, in addition to heterologous chimeric antibodies such as mouse-human chimeric antibodies, human-human homologous chimeric antibodies are also included in the chimeric antibodies of the present invention. By inserting the V region gene into an expression vector having a constant region in advance, a chimeric antibody can be obtained. At this time, if the gene encoding the antibody constant region (C region) and the gene encoding the V region are fused in-frame, a chimeric antibody is obtained. A chimeric antibody expression vector can be constructed. Specifically, for example, a restriction enzyme recognition sequence of a restriction enzyme that digests the V region gene is appropriately arranged on the 5' side of an expression vector that holds DNA encoding a desired antibody constant region (C region). By digesting both with the same combination of restriction enzymes and fusing them in-frame, a chimeric antibody expression vector is constructed . . .

[0053] To produce an anti-GPC3 monoclonal antibody, the antibody gene is incorporated into an expression vector so as to be expressed under the control of an expression control region. An expression control region for expressing an antibody includes, for example, an enhancer and a promoter. Also, an appropriate signal sequence can be added to the amino terminus so that the expressed antibody is secreted extracellularly. In the examples described later , a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 72) is used as the signal sequence, but other suitable signal sequences can also be added. The expressed polypeptide is cleaved at the carboxyl terminal portion of the above sequence, and the cleaved polypeptide can be secreted extracellularly as a mature polypeptide. Then, by transforming an appropriate host cell with this expression vector, a transformed cell that expresses DNA encoding an anti-GPC3 antibody can be obtained. For the expression of the antibody gene, DNA encoding the antibody heavy chain (H chain) and light chain (L chain) is incorporated into separate expression vectors. By co-transfecting the same host cell with the vectors incorporating the H chain and L chain, an H chain and an L chain are provided . .

[0054] For the expression of the antibody gene, DNA encoding the antibody heavy chain (H chain) and light chain (L chain) is incorporated into separate expression vectors. By co-transfecting the same host cell with the vectors incorporating the H chain and L chain, a cell equipped with the H chain and L chain is obtained . Antibody molecules can be expressed. Alternatively, the host cell can be transformed by incorporating the DNA encoding the H chain and the L chain into a single expression vector (see International Publication WO 94 / 11523 ).

[0055] Many combinations of host cells and expression vectors for producing antibodies by introducing an isolated antibody gene into an appropriate host are known. Any of these expression systems can be applied to isolate the antigen-binding domain or CD3-binding domain of the present invention. When eukaryotic cells are used as host cells, animal cells, plant cells, or fungal cells can be appropriately used . 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 laevis oocytes, etc. (3) Insect cells: sf9, sf21, Tn5, etc. (3) Insect cells: sf9, sf21, Tn5, etc. (3) Insect cells: sf9, sf21, Tn5, etc.

[0056] Alternatively, as plant cells, an expression system of an antibody gene using cells derived from the genus Nicotiana such as Nicotiana tabacum is known. For the transformation of plant cells, callus-cultured cells can be appropriately used . (3) Insect cells: sf9, sf21, Tn5, etc.

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

[0058] Also, an expression system for antibody genes using prokaryotic cells is also known. For example, when using bacterial cells, bacterial cells such as Escherichia coli (E. coli) and Bacillus subtilis can be appropriately used. An expression vector containing the target antibody gene is introduced into these cells by transformation. By culturing the transformed cells in vitro, the desired antibody can be obtained from the culture of the transformed cells. When using, bacterial cells such as Escherichia coli and Bacillus subtilis can be appropriately used. An expression vector containing the target antibody gene is introduced into these cells by transformation. By culturing the transformed cells in vitro, the desired antibody can be obtained from the culture of the transformed cells.

[0059] In addition to the above host cells, transgenic animals can also be used for the production of 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, the antibody gene can be constructed as a fusion gene by inserting it in-frame into the gene encoding a protein that is specifically produced in milk. As a protein secreted in milk, for example, goat β-casein can be used. A DNA fragment containing the fusion gene into which the antibody gene has been inserted is injected into a goat embryo, and the injected embryo is introduced into a female goat. The desired antibody can be obtained as a fusion protein with the milk protein from the milk produced by the transgenic goat (or its offspring) born from the goat that received the embryo. Also, to increase the amount of milk containing the desired antibody produced by the transgenic goat, hormones can be administered to the transgenic goat (Bio / Technology (1994), 12 (7), 699 - 702). That is, the antibody can be obtained from an animal into which a gene encoding the desired antibody has been introduced. For example, the antibody gene can be constructed as a fusion gene by inserting it in-frame into the gene encoding a protein that is specifically produced in milk. As a protein secreted in milk, for example, goat β-casein can be used. A DNA fragment containing the fusion gene into which the antibody gene has been inserted is injected into a goat embryo, and the injected embryo is introduced into a female goat. The desired antibody can be obtained as a fusion protein with the milk protein from the milk produced by the transgenic goat (or its offspring) born from the goat that received the embryo. Also, to increase the amount of milk containing the desired antibody produced by the transgenic goat, hormones can be administered to the transgenic goat (Bio / Technology (1994), 12 (7), 699 - 702). Technology (1994), 12 (7), 699 - 702).

[0060] When the polypeptide complex described in this specification is administered to humans, the complex may appropriately employ an antigen-binding domain derived from a genetically engineered antibody artificially modified for the purpose of, for example, reducing heterologous antigenicity against humans as the antigen-binding domain in it. Genetically engineered antibodies include, for example, humanized antibodies and the like. These modified antibodies are appropriately produced using known methods.

[0061] The variable region of the antibody used to produce the antigen-binding domain in the polypeptide complex described in this specification is usually composed of three complementarity-determining regions (CDRs) sandwiched between four framework regions (FRs). CDR is a region that substantially determines the binding specificity of the antibody. The amino acid sequence of CDR is rich in diversity. On the other hand, the amino acid sequences constituting FR often show high identity even among antibodies with different binding specificities. Therefore, generally, it is said that the binding specificity of one antibody can be transplanted to another antibody by transplanting CDR.

[0062] Humanized antibodies are also referred to as reshaped human antibodies. Specifically, humanized antibodies obtained by transplanting the CDRs of antibodies from non-human animals, such as mouse antibodies, into human antibodies, are known. General genetic engineering techniques for obtaining humanized antibodies are also known. Specifically, as a method for transplanting the CDRs of mouse antibodies into human FRs, for example, Overlap Extension PCR is known. In Overlap Extension PCR, primers for synthesizing human antibody FR A nucleotide sequence encoding the CDR of the mouse antibody to be transplanted is added. Four primers are prepared for each of the four FRs. Generally, in transplanting mouse CDRs into human FRs, selecting a human FR with a high identity to the mouse FR is advantageous for maintaining the function of the CDR is considered. That is, generally, it is preferable to use a human FR consisting of an amino acid sequence highly identical to the amino acid sequence of the FR adjacent to the mouse CDR to be transplanted.

[0063] Also, the linked nucleotide sequences are designed to be connected in-frame with each other. Each human FR is individually synthesized by each primer. As a result, a product is obtained in which DNA encoding the mouse CDR is added to each FR. The nucleotide sequences encoding the mouse CDR of each product are designed to overlap with each other. Subsequently, the overlapping CDR portions of the products synthesized using the human antibody gene as a template are annealed to each other to perform a complementary strand formation reaction. By this reaction, the human FRs are linked via the sequences of the mouse CDRs. Finally, the V-region gene in which three CDRs and four FRs are linked is amplified in its entirety by primers annealed to its 5'-end and 3'-end and having appropriate restriction enzyme recognition sequences added thereto.

[0064] By inserting the DNA obtained as described above and the DNA encoding the human antibody C-region into an expression vector so as to be fused in-frame, a vector for expressing a humanized antibody can be prepared. After introducing the integration vector into a host to establish recombinant cells, the recombinant cells are cultured and the DNA encoding the humanized antibody is expressed, whereby the humanized antibody is obtained from the cultured cells. (See European Patent Publication EP 239400 and International Publication WO 1996 / 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 may 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 has the base sequence The binding activity of the mutant antibody with the amino acid substitution to the antigen is 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 carrying the entire repertoire of human antibody genes (International Open WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, WO1 996 / 033735) can be used as immunized animals to obtain the desired human antibodies by DNA immunization.

[0067] In addition, 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 expressed as a single chain antibody (scFv) by phage display. It is expressed on the surface of the phage by the phage display method. Phages expressing scFv that binds to an antigen can be selected. By analyzing the gene of the selected phage, the DNA sequence encoding the V region of the human antibody that binds to the antigen can be determined. After determining the DNA sequence of the scFv that binds to the antigen, the V region sequence is fused in-frame with the sequence of the desired human antibody C region and then inserted into an appropriate expression vector to produce an expression vector. The expression vector is introduced into suitable expression cells as described above, and the gene encoding the human antibody is expressed to obtain the human antibody. These methods are already known (see International Publication WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, WO1995 / 015388). By analyzing the gene of the selected phage, the DNA sequence encoding the V region of the human antibody that binds to the antigen can be determined. After determining the DNA sequence of the scFv that binds to the antigen, the V region sequence is fused in-frame with the sequence of the desired human antibody C region and then inserted into an appropriate expression vector to produce an expression vector. The expression vector is introduced into suitable expression cells as described above, and the gene encoding the human antibody is expressed to obtain the human antibody. These methods are already known (see International Publication WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, WO1995 / 015388). 1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, WO1995 / 015388).

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

[0069] The antigen-binding domains in the polypeptide complex of the present invention can bind to the same epitope. Here, the same epitope can be present in a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4. Alternatively, the antigen-binding domains in the polypeptide complex of the present invention can bind to different epitopes from each other. Here, the different epitopes can be present in a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4. The term "specific" refers to a state in which one of the molecules that specifically binds does not show any significant binding to molecules other than one or more of its binding partner molecules. Also, it is used when the antigen-binding domain is specific for a particular epitope among a plurality of epitopes contained in a certain antigen. Further, when the epitopes to which the antigen-binding domain binds are contained in a plurality of different antigens, the polypeptide complex having the antigen-binding domain can bind to various antigens containing the epitope. In the present specification, the antigen is not particularly limited and can be any antigen except CD3. Examples of antigens include, for example, receptors, cancer antigens, MHC antigens, differentiation antigens, etc. Examples of receptors include, for example, the hematopoietic factor receptor family, the cytokine receptor family, the tyrosine kinase type receptor family, the serine / threonine kinase type receptor family, the TNF receptor family, the G protein-coupled receptor family, the GPI anchor type receptor family.

[0070] specific

[0071] antigen ​​​​​​​​​​​​​​Amily, tyrosine phosphatase type receptor family, adhesion factor family, hormone Examples thereof include receptors belonging to receptor families such as receptor families, etc. Regarding the receptors belonging to these receptor families and their characteristics, there are numerous literatures, for example, 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 supervised by Masayuki Miyasaka, Cell Engineering Separate Volume Handbook Series "Adhesion Factor Handbook" "(1994) (Shujunsha, Tokyo, Japan), etc. In addition to reviews, Patthy (Cell (1990) 61 (1), 13- 14), Ullrich et al. (Cell (1990) 61 (2), 203-212), Massague (e has an acute accent 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 . Rev. Biochem. (1993), 62, 453-481), Smith et al. (Cell (1994) 76 (6) 959-962), Flower DR. Biochim. Biophys. Acta, Flower (Biochim. Biophys. Acta (1999) 1422 (3) 207-234, etc. are described.

[0072] Specific receptors belonging to the above receptor family include, for example, human or mouse ery ​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 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 l. 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 interferon (IFN)-α, β receptor (Cell (1990) 60 (2), 225-234. and Cell ( 1994) 77 (3), 391-400), human or mouse leptin receptor, human or mouse growth hormone ( GH) receptor, human or mouse interleukin (IL)-10 receptor, human or mouse insulin-like growth factor (IGF)-I receptor, human or mouse leukemia inhibitory factor (LIF) receptor, human or mouse ciliary neurotrophic factor (CNTF) receptor, etc. are preferably exemplified. Cancer antigens are antigens that are expressed with the malignant transformation of cells and are also called tumor-specific antigens. Also

[0073] ​, abnormal sugar chains that appear on the cell surface or protein molecules when cells become cancerous are also cancer antigens, and are also called cancer glycan antigens. Examples of cancer antigens include, for example, GPC3 that belongs to the GPI-anchor type receptor family as the above receptor and is expressed in several cancers including liver cancer (Int J Cancer. (2003) 103 (4), 455-65), EpCAM that is expressed in multiple cancers including lung cancer (Proc Natl Acad Sci U S A. (1989) 86 (1), 27-31) (its polynucleotide sequence is registered in RefSeq accession number NM_002354.2 (SEQ ID NO: 3), and the polypeptide sequence is registered in RefSeq accession number NP_0 02345.2 (SEQ ID NO: 4) respectively.), EGFR, CA19-9, CA15-3, sialyl SSEA-1 (SLX), etc. are preferably mentioned. efSeq accession number NM_002354.2(SEQ ID NO: 3) for the polynucleotide sequence, and RefSeq accession number NP_0 02345.2(SEQ ID NO: 4) for the polypeptide sequence, respectively.), EGFR, CA19-9, CA15-3, sialyl SSEA-1 (SLX), etc. are preferably mentioned. are preferably mentioned.

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

[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 may be included.

[0076] epitope An epitope, which means an antigenic determinant present in an antigen, is the site on the antigen to which the antigen-binding domain in the polypeptide complex disclosed herein binds. Thus, for example, an epitope can be defined by its structure. Also, the epitope can be defined by the binding activity of the polypeptide complex recognizing the epitope to the antigen. When the antigen is a peptide or polypeptide, it is also possible to identify the epitope by the amino acid residues constituting the epitope. Also, when the epitope is a sugar chain, it is possible to identify the epitope by a specific sugar chain structure. A linear epitope is an epitope that includes an epitope whose amino acid primary sequence is recognized. Linear epitopes typically contain at least 3, and most commonly at least 5, for example about 8 to about 10, 6 to 20 amino acids in a unique sequence. In contrast to a linear epitope, a conformational epitope is an epitope (e.g., an epitope whose amino acid primary sequence is not necessarily recognized by an antibody that defines the epitope) in which the primary sequence of the amino acids containing the epitope is not a single defining component of the recognized epitope. Conformational epitopes include a greater number of amino acids relative to linear epitopes.

[0077]

[0078] It may do so. Regarding the recognition of conformational epitopes, antibodies recognize the three-dimensional structure of peptides or proteins. For example, when a protein molecule folds to form a three-dimensional structure, certain amino acids and / or polypeptide main chains that form conformational epitopes become parallel, enabling the antibody to recognize the epitope. Methods for determining the conformational structure of an epitope include, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, as well as site-specific spin labeling and electron paramagnetic resonance spectroscopy, but are not limited thereto. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Volume 66, Morr is (ed.). For example, when a protein molecule folds to form a three-dimensional structure certain amino acids and / or polypeptides that form conformational epitopes main chains become parallel, enabling the antibody to recognize the epitope. The conformational structure of the epitope can be determined by methods such as X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-specific spin labeling and electron paramagnetic resonance spectroscopy, among others, but are not limited to these. For example spin labeling and electron paramagnetic resonance spectroscopy are included, but not limited to these. For example see Epitope Mapping Protocols in Methods in Molecular Biology (1996), Volume 66, Morr is (ed.).

[0079] A method for confirming the binding of a test polypeptide aggregate containing an antigen-binding domain to GPC3 to an epitope is exemplified below, but a method for confirming the binding of a test polypeptide aggregate containing an antigen-binding domain to an antigen other than GPC3 to an epitope can also be appropriately carried out according to the following example. For example, that a test polypeptide aggregate containing an antigen-binding domain to GPC3 recognizes a linear epitope present in the GPC3 molecule can be confirmed, for example, as follows. For this purpose, a linear peptide consisting of the amino acid sequence constituting the extracellular domain of GPC3 is synthesized. The peptide can be chemically synthesized. Alternatively, it can be obtained by genetic engineering techniques using the region encoding the amino acid sequence corresponding to the extracellular domain in the cDNA of GPC3. Next, a linear peptide consisting of the amino acid sequence constituting the extracellular domain

[0080] For example, that a test polypeptide aggregate containing an antigen-binding domain to GPC3 recognizes a linear epitope present in the GPC3 molecule can be confirmed, for example, as follows. For example, that a test polypeptide aggregate containing an antigen-binding domain to GPC3 recognizes a linear epitope present in the GPC3 molecule can be confirmed, for example, as follows. For the above purpose, a linear peptide consisting of the amino acid sequence constituting the extracellular domain of GPC3 is synthesized. The peptide can be chemically synthesized. Alternatively, it can be obtained by genetic engineering techniques using the region encoding the amino acid sequence corresponding to the extracellular domain in the cDNA of GPC3. Next, a linear peptide consisting of the amino acid sequence constituting the extracellular domain is synthesized. The binding activity of the test polypeptide complex containing a chide and an antigen-binding domain against GPC3 is evaluated. For example, the binding activity of the polypeptide complex against the peptide can be evaluated by ELISA using an immobilized linear peptide as an antigen. Alternatively, based on the level of inhibition by the linear peptide in the binding of the polypeptide complex to GPC3-expressing cells, the binding activity against the linear peptide can be revealed. Through these tests, the binding activity of the polypeptide complex against the linear peptide can be revealed. Also, that the test polypeptide complex containing an antigen-binding domain against GPC3 recognizes a conformational epitope can be confirmed as follows. For the above purpose, cells expressing GPC3 are prepared.

[0081] When the test polypeptide complex containing an antigen-binding domain against GPC3 contacts GPC3-expressing cells, it strongly binds to the cells, while the polypeptide complex does not substantially bind to a linear peptide consisting of the amino acid sequence constituting the extracellular domain of immobilized GPC3. Here, not substantially binding means a binding activity of 80% or less, usually 50% or less, preferably 30% or less, particularly preferably 15% or less of the binding activity against human GPC3-expressing cells. Examples of the method for measuring the binding activity of the test polypeptide complex containing an antigen-binding domain against GPC3 to GPC3-expressing cells include the method described in Antibodies A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420).

[0082] (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420) can be mentioned. That is, ELISA and FACS (fluorescence activated cell sorting) using GPC3 expressing cells as antigens are used. This can be evaluated according to the principle of ting.

[0083] In an ELISA format, a test polypeptide comprising 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 enzyme reaction. The results are quantitatively evaluated by comparing the results 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 test polypeptide complex is detected by FACS. In this study, a dilution series of the test polypeptide complex was prepared and the antibody binding to GPC3-expressing cells was assayed. The activity of the test polypeptide complex against GPC3-expressing cells was determined by determining the titer. Binding activities can be compared.

[0084] The antibody binds to a test polypeptide complex that binds to 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 of these are product 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 product names of Beckman Coulter)

[0085] For example, for the antigen of the test polypeptide complex containing an antigen-binding domain against GPC3 As an example of a preferred method for measuring the binding activity, the following method can be mentioned. First, the test polypeptide complex reacted with cells expressing GPC3 is stained with an FITC-labeled secondary antibody that recognizes it. By appropriately diluting the test polypeptide complex with a suitable buffer, the complex is prepared and used at a desired concentration. For example, it can be used at any concentration between 10 μg / ml and 10 ng / ml Next, the fluorescence intensity and the number of cells are measured by FACSCalibur (BD) The amount of antibody bound to the cells is analyzed using CELL QUEST Software (BD) and is reflected in the fluorescence intensity obtained thereby, that is, the value of Geometric Mean. That is, by obtaining the value of the Geometric Mean the binding activity of the test polypeptide complex represented by the amount of binding of the test polypeptide complex can be measured.

[0086] That the test polypeptide complex containing an antigen-binding domain against GPC3 shares an epitope with a certain polypeptide complex can be confirmed by competition for the same epitope between the two. The competition between polypeptide complexes is detected by cross-blocking assay or the like For example, a competitive ELISA assay is a preferred cross-blocking assay.

[0087] ​​Specifically, in the cross-blocking assay, the GPC3 protein coated on the well of the microtiter plate is pre-incubated in the presence or absence of the candidate competing polypeptide complex, and then the test polypeptide complex is added. The amount of the test polypeptide complex bound to the GPC3 protein in the well is indirectly correlated with the binding ability of the candidate competing polypeptide complex that competes with the binding to the same epitope. That is, the greater the affinity of the competing polypeptide complex for the same epitope, the lower the binding activity of the test polypeptide complex to the well coated with the GPC3 protein.

[0088] The amount of the test polypeptide complex bound to the well via the GPC3 protein can be easily measured by pre-labeling the polypeptide complex. For example, a biotin-labeled polypeptide complex is measured by using an avidin peroxidase conjugate and an appropriate substrate. The cross-blocking assay using an enzyme label such as peroxidase is particularly called a competitive ELISA assay. The polypeptide complex can be labeled with other labeling substances that can be detected or measured. Specifically, radiolabels or fluorescent labels are known.

[0089] If the competing polypeptide complex can block at least 20%, preferably at least 20 - 50%, more preferably at least 50% of the binding of the test polypeptide complex containing the antigen-binding domain to GPC3, compared to the binding activity obtained in the control test conducted in the absence of the candidate competing polypeptide complex, then the test polypeptide complex ​​​​​​​​​​​​​​​​ A polypeptide complex that binds to substantially the same epitope as, or competes with, the competing polypeptide complex for binding to the same epitope. is a polypeptide complex.

[0090] When the structure of the epitope to which the test polypeptide complex containing the antigen-binding domain for GPC3 binds has been identified, whether the test polypeptide complex and the control polypeptide complex share the epitope can be evaluated by comparing the binding activities of both polypeptide complexes to a peptide in which an amino acid mutation has been introduced into the peptide constituting the epitope. introducing an amino acid mutation It can be measured by comparing the binding activities of both polypeptide complexes to the peptide.

[0091] As a method for measuring such binding activity, for example, it can be measured by comparing the binding activities of the test polypeptide complex and the control polypeptide complex to a linear peptide into which a mutation has been introduced in the above-mentioned ELISA format. As a method other than ELISA, it can also be measured by quantifying the polypeptide complex eluted in the eluate after flowing down the test polypeptide complex and the control polypeptide complex through a column bound with the mutant peptide. A method of adsorbing the mutant peptide to a column as a fusion peptide with, for example, GST is known. to the mutant peptide

[0092] When the identified epitope is a conformational epitope, whether the test polypeptide complex and the control polypeptide complex share the epitope can be evaluated by the following method. First, cells expressing GPC3 and cells expressing GPC3 with a mutation introduced into the epitope are prepared. For a cell suspension in which these cells are suspended in an appropriate buffer such as PBS, the test polypeptide ​​​​​​​​​​The chimeric conjugate and the control polypeptide conjugate are added. Subsequently, the FITC-labeled antibody capable of recognizing the test polypeptide conjugate and the control polypeptide conjugate is added to the cell suspension appropriately washed with a buffer solution. The fluorescence intensity and the number of cells of the cells stained with the labeled antibody are measured by FACSCalibur (BD). The concentrations of the test polypeptide conjugate and the control polypeptide conjugate are adjusted to the desired concentrations by appropriately diluting with a suitable buffer solution. For example, they are used at any concentration between 10 μg / ml and 10 ng / ml. The amount of the labeled antibody bound to the cells is reflected in the fluorescence intensity, i.e., the value of Geometric Mean, obtained by analyzing using CELL QUEST Software (BD). That is, by obtaining the value of Geometric Mean, the binding activity of the test polypeptide conjugate and the control polypeptide conjugate represented by the amount of the labeled antibody bound can be measured. In this method, for example, "substantially not binding to mutant GPC3-expressing cells" can be determined by the following method. First, the test polypeptide conjugate and the control polypeptide conjugate bound to the cells expressing mutant GPC3 are stained with the labeled antibody. Subsequently, the fluorescence intensity of the cells is detected. When FACSCalibur is used as flow cytometry for fluorescence detection, the obtained fluorescence intensity can be analyzed using CELL QUEST Software. From the values of Geometric Mean in the presence and absence of the polypeptide conjugate, this comparative value (ΔGeo-Mean) is calculated based on the following formula to determine the increase ratio of the fluorescence intensity due to the binding of the polypeptide conjugate. The FITC-labeled antibody capable of recognizing the test polypeptide conjugate and the control polypeptide conjugate is added to the cell suspension appropriately washed with a buffer solution. The fluorescence intensity and the number of cells of the cells stained with the labeled antibody are measured by FACSCalibur (BD). The concentrations of the test polypeptide conjugate and the control polypeptide conjugate are adjusted to the desired concentrations by appropriately diluting with a suitable buffer solution. For example, they are used at any concentration between 10 μg / ml and 10 ng / ml. The amount of the labeled antibody bound to the cells is reflected in the fluorescence intensity, i.e., the value of Geometric Mean, obtained by analyzing using CELL QUEST Software (BD). That is, by obtaining the value of Geometric Mean, the binding activity of the test polypeptide conjugate and the control polypeptide conjugate represented by the amount of the labeled antibody bound can be measured. In this method, for example, "substantially not binding to mutant GPC3-expressing cells" can be determined by the following method. First, the test polypeptide conjugate and the control polypeptide conjugate bound to the cells expressing mutant GPC3 are stained with the labeled antibody.

[0093] First, the test polypeptide conjugate and the control polypeptide conjugate bound to the cells expressing mutant GPC3 are stained with the labeled antibody. Subsequently, the fluorescence intensity of the cells is detected. When FACSCalibur is used as flow cytometry for fluorescence detection, the obtained fluorescence intensity can be analyzed using CELL QUEST Software. From the values of Geometric Mean in the presence and absence of the polypeptide conjugate, this comparative value (ΔGeo-Mean) is calculated based on the following formula to determine the increase ratio of the fluorescence intensity due to the binding of the polypeptide conjugate. The sum can be obtained.

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

[0095] The binding amount of the test polypeptide aggregate obtained by analysis to mutant GPC3-expressing cells is reflected by the Geometric Mean comparison value (mutant GPC3 molecule ΔGeo-Mean value), which is compared with the ΔGeo-Mean comparison value reflected by the binding amount of the test polypeptide aggregate to GPC3-expressing cells. In this case, when determining the ΔGeo-Mean comparison values for mutant GPC3-expressing cells and GPC3-expressing cells, the concentrations of the test polypeptide aggregates used are adjusted to be the same or substantially the same as each other, which is particularly preferred. A polypeptide aggregate that has been confirmed in

[0096] advance to recognize an epitope in GPC3 is used as a control polypeptide aggregate. If the ΔGeo-Mean comparison value of the test polypeptide aggregate to mutant GPC3-expressing cells is less than at least 80%, preferably 50%, more preferably 30%, and particularly preferably 15% of the ΔGeo-Mean comparison value of the test polypeptide aggregate to GPC3-expressing cells, it is considered that the test polypeptide aggregate "substantially does not bind to mutant GPC3-expressing cells". The calculation formula for obtaining the Geo-Mean value (Geometric Mean) is described in the CE L L QUEST Software User’s Guide (BD biosciences). If the comparison values

[0097] Fv (variable fragment) As used herein, the term "Fv (variable fragment)" refers to the minimum unit of an antigen-binding domain derived from an antibody, which consists of a pair of the variable region of the light chain (VL (light chain variable region)) and the variable region of the heavy chain (VH (heavy chain variable region)) of the antibody. In 1988, Skerra and Pluckthun inserted the antibody gene downstream of the signal sequence of bacteria and induced the expression of the gene in Escherichia coli, and found that it could be prepared from the periplasmic fraction of Escherichia coli in a homogeneous and active state (Science (1988) 240 (4855), 1038-1041). The Fv prepared from the periplasmic fraction had VH and VL associated in a manner that had binding to the antigen.

[0098] As used herein, examples of Fv include the following polypeptide aggregates: A bivalent antigen-binding domain in which one monovalent scFv of a bivalent scFv is linked to one polypeptide constituting the Fc region via a heavy chain Fv fragment constituting the CD3-binding domain, and the other monovalent scFv is linked to the other polypeptide constituting the Fc region via a light chain Fv fragment constituting the CD3-binding domain, (1) a bivalent antigen-binding domain, (2) a domain containing an amino acid constituting the Fc region of IgG1, IgG2a, IgG3 or IgG4 that does not have binding activity to the Fcγ receptor, and (3) a polypeptide aggregate containing at least a monovalent CD3-binding domain, etc., in which the light chain Fv fragment and the heavy chain Fv fragment are specific for CD3, an antigen. ​​​​​​Also preferably included are a set of Fvs that associate in a manner having the bonds to be formed and constitute the 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 an antibody fragment that contains variable regions derived from both the heavy and light chains but lacks the constant regions within a single polypeptide chain. Generally, a single-chain antibody further includes a polypeptide linker between the VH and VL domains that enables the formation of a desired structure that is thought to allow antigen binding. Single-chain antibodies are described in detail by Plückthun in The Pharmacology of Monoclonal Antibodies, Volume 113, Rosenburg, and Moore, eds., Springer-Verlag, New York, pages 269-315 (1994). See also International Patent Application Publication WO1988 / 001649 and U.S. Patent Nos. 4,946,778 and 5,260,203. In certain embodiments, the single-chain antibody can also be bispecific and / or humanized.

[0100]

[0101] An scFv is an antigen-binding domain in which the VH and VL that constitute the Fv are linked by a peptide linker (Proc. Natl. Acad. Sci. U.S.A. (1988) 85 (16), 5879-5883). The VH and VL can be held in close proximity by the peptide linker.

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

[0102] As the composition of the antigen-binding domains constituting sc(Fv)2 in the present specification, two VHs and two VLs are arranged in the order of VH, VL, VH, VL ([VH] linker -[VL] linker [VH] linker [VL]) starting from the N-terminal side of the single-chain polypeptide. Antibodies characterized by this are mentioned, but the order of the two VHs and two VLs is not particularly limited to the above composition, and they can be arranged in any order. For example, the following order compositions can also be mentioned. [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. Those skilled in the art can, based on these descriptions, appropriately produce the desired sc(Fv)2 for the production of the polypeptide aggregates disclosed in the present specification. ​

[0104] In addition, the polypeptide complex of the present invention may be conjugated with a carrier polymer such as PEG or an organic compound such as an anticancer agent. Also, a sugar chain addition sequence may be inserted and preferably added for the purpose of obtaining a desired effect with the sugar chain. As the linker that binds to the variable region of the antibody, any peptide linker that can be introduced by genetic engineering or a linker disclosed in a synthetic compound linker (for example, see Protein Engineering, 9 (3), 299-305, 1996), etc. can be used. In the present invention, a peptide linker is preferred. The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art according to the purpose. However, the preferred length is 5 amino acids or more (the upper limit is not particularly limited, but usually 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. When three peptide linkers are included in sc(Fv)2, peptide linkers of the same length or peptide linkers of different lengths may be used.

[0105]

[0106] For example, in the case of 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 (SEQ ID NO: 12) (Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 7))n (Ser·Gly·Gly·Gly·Gly (SEQ ID NO: 8))n [n is an integer of 1 or more], etc. can be mentioned. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art according to the purpose.

[0107] Synthetic chemical linkers (chemical crosslinking agents) are crosslinking agents commonly used for crosslinking peptides, for example, N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis (sulfosuccinimidyl) suberate (BS3), dithiobis (succinimidyl prop ionate) (DSP), dithiobis (sulfosuccinimidyl propionate) (DTSSP) , ethylene glycol bis (succinimidyl succinate) (EGS), ethylene glycol bis (sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(s uccinimidooxycarbonyloxy)ethyl] sulfone (BSOCOES), bis[2-(sul fosuccinimidooxycarbonyloxy)ethyl] sulfone (sulfo-BSOCOES), etc. These crosslinking agents are commercially available.

[0108] When binding four antibody variable regions, usually three linkers are required, but all the same linker may be used, or different linkers may be used.

[0109] Fab, F(ab’)2, or Fab’ "Fab" consists of a single light chain, as well as the CH1 region and the variable region of a single heavy chain. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0110] "F(ab’)2" and "Fab’" are antibody fragments produced by treating an immunoglobulin (monoclonal antibody) with a proteolytic enzyme such as pepsin or papain, and digested before and after the disulfide bond existing between two H chains in the hinge region. For example, by treating IgG with papain, it is cleaved upstream of the disulfide bond existing between two H chains in the hinge region, resulting in an L chain consisting of VL (L chain variable region) and CL (L chain constant region), and two identical antibody fragments in which the H chain fragment consisting of VH (H chain variable region) and CHγ1 (γ1 region in the H chain constant region) is bound by a disulfide bond at the C-terminal region. These two identical antibody fragments are each referred to as Fab'.

[0111] "F(ab’)2" includes two light chains, as well as two heavy chains including a part of the constant region of the CH1 domain and the CH2 domain such that disulfide bonds between the chains are formed between the two heavy chains. The F(ab’)2 constituting the polypeptide aggregate disclosed in this specification can be preferably obtained by partially digesting a full-length monoclonal antibody or the like having a desired antigen-binding domain with a proteolytic enzyme such as pepsin, and then removing the Fc fragment by adsorbing it to a protein A column. As such a proteolytic enzyme, the reaction conditions of the enzyme such as pH are appropriately set. ​​​​​​​​​There is no particular limitation as long as the full-length antibody can be digested to produce F(ab’)2 in a restricted manner. For example, pepsin, ficin, etc. can be exemplified. There is no particular limitation as long as the full-length antibody can be digested to produce F(ab’)2 in a restricted manner. For example, pepsin, ficin, etc. can be exemplified.

[0112] Fc region The Fc region constituting the polypeptide aggregate disclosed in this specification is obtained by partially digesting an antibody such as a monoclonal antibody with a proteolytic enzyme such as pepsin, and then adsorbing the fragment to a protein A column or a protein G column, and then eluting it with an appropriate elution buffer or the like. Such a proteolytic enzyme is not particularly limited as long as it can digest an antibody such as a monoclonal antibody by appropriately setting the reaction conditions of the enzyme such as pH. For example, pepsin, ficin, etc. can be exemplified. The Fc region constituting the polypeptide aggregate disclosed in this specification is obtained by partially digesting an antibody such as a monoclonal antibody with a proteolytic enzyme such as pepsin, and then adsorbing the fragment to a protein A column or a protein G column, and then eluting it with an appropriate elution buffer or the like. Such a proteolytic enzyme is not particularly limited as long as it can digest an antibody such as a monoclonal antibody by appropriately setting the reaction conditions of the enzyme such as pH. For example, pepsin, ficin, etc. can be exemplified. The Fc region constituting the polypeptide aggregate disclosed in this specification is obtained by partially digesting an antibody such as a monoclonal antibody with a proteolytic enzyme such as pepsin, and then adsorbing the fragment to a protein A column or a protein G column, and then eluting it with an appropriate elution buffer or the like. Such a proteolytic enzyme is not particularly limited as long as it can digest an antibody such as a monoclonal antibody by appropriately setting the reaction conditions of the enzyme such as pH. For example, pepsin, ficin, etc. can be exemplified. The Fc region constituting the polypeptide aggregate disclosed in this specification is obtained by partially digesting an antibody such as a monoclonal antibody with a proteolytic enzyme such as pepsin, and then adsorbing the fragment to a protein A column or a protein G column, and then eluting it with an appropriate elution buffer or the like. Such a proteolytic enzyme is not particularly limited as long as it can digest an antibody such as a monoclonal antibody by appropriately setting the reaction conditions of the enzyme such as pH. For example, pepsin, ficin, etc. can be exemplified. The Fc region constituting the polypeptide aggregate disclosed in this specification is obtained by partially digesting an antibody such as a monoclonal antibody with a proteolytic enzyme such as pepsin, and then adsorbing the fragment to a protein A column or a protein G column, and then eluting it with an appropriate elution buffer or the like. Such a proteolytic enzyme is not particularly limited as long as it can digest an antibody such as a monoclonal antibody by appropriately setting the reaction conditions of the enzyme such as pH. For example, pepsin, ficin, etc. can be exemplified. The Fc region constituting the polypeptide aggregate disclosed in this specification is obtained by partially digesting an antibody such as a monoclonal antibody with a proteolytic enzyme such as pepsin, and then adsorbing the fragment to a protein A column or a protein G column, and then eluting it with an appropriate elution buffer or the like. Such a proteolytic enzyme is not particularly limited as long as it can digest an antibody such as a monoclonal antibody by appropriately setting the reaction conditions of the enzyme such as pH. For example, pepsin, ficin, etc. can be exemplified.

[0113] The polypeptide aggregate described in this specification includes an Fc region in which the binding activity to the Fcγ receptor is reduced among the amino acids constituting the Fc region of IgG1, IgG2, IgG3 or IgG4. The polypeptide aggregate described in this specification includes an Fc region in which the binding activity to the Fcγ receptor is reduced among the amino acids constituting the Fc region of IgG1, IgG2, IgG3 or IgG4.

[0114] The isotype of an antibody is determined by the structure of the constant region. The constant regions of the respective isotypes of IgG1, IgG2, IgG3, and IgG4 are called Cγ1, Cγ2, Cγ3, and Cγ4, respectively. The amino acid sequences of the polypeptides constituting the Fc regions of human Cγ1, Cγ2, Cγ3, and Cγ4 are exemplified in SEQ ID NOs: 23, 24, 25, and 26. The relationship between the amino acid residues constituting each amino acid sequence and the EU numbering of Kabat (also called EU INDEX in this specification) is shown in FIG. 18. The isotype of an antibody is determined by the structure of the constant region. The constant regions of the respective isotypes of IgG1, IgG2, IgG3, and IgG4 are called Cγ1, Cγ2, Cγ3, and Cγ4, respectively. The amino acid sequences of the polypeptides constituting the Fc regions of human Cγ1, Cγ2, Cγ3, and Cγ4 are exemplified in SEQ ID NOs: 23, 24, 25, and 26. The relationship between the amino acid residues constituting each amino acid sequence and the EU numbering of Kabat (also called EU INDEX in this specification) is shown in FIG. 18. The isotype of an antibody is determined by the structure of the constant region. The constant regions of the respective isotypes of IgG1, IgG2, IgG3, and IgG4 are called Cγ1, Cγ2, Cγ3, and Cγ4, respectively. The amino acid sequences of the polypeptides constituting the Fc regions of human Cγ1, Cγ2, Cγ3, and Cγ4 are exemplified in SEQ ID NOs: 23, 24, 25, and 26. The relationship between the amino acid residues constituting each amino acid sequence and the EU numbering of Kabat (also called EU INDEX in this specification) is shown in FIG. 18. The isotype of an antibody is determined by the structure of the constant region. The constant regions of the respective isotypes of IgG1, IgG2, IgG3, and IgG4 are called Cγ1, Cγ2, Cγ3, and Cγ4, respectively. The amino acid sequences of the polypeptides constituting the Fc regions of human Cγ1, Cγ2, Cγ3, and Cγ4 are exemplified in SEQ ID NOs: 23, 24, 25, and 26. The relationship between the amino acid residues constituting each amino acid sequence and the EU numbering of Kabat (also called EU INDEX in this specification) is shown in FIG. 18. The isotype of an antibody is determined by the structure of the constant region. The constant regions of the respective isotypes of IgG1, IgG2, IgG3, and IgG4 are called Cγ1, Cγ2, Cγ3, and Cγ4, respectively. The amino acid sequences of the polypeptides constituting the Fc regions of human Cγ1, Cγ2, Cγ3, and Cγ4 are exemplified in SEQ ID NOs: 23, 24, 25, and 26. The relationship between the amino acid residues constituting each amino acid sequence and the EU numbering of Kabat (also called EU INDEX in this specification) is shown in FIG. 18. The isotype of an antibody is determined by the structure of the constant region. The constant regions of the respective isotypes of IgG1, IgG2, IgG3, and IgG4 are called Cγ1, Cγ2, Cγ3, and Cγ4, respectively. The amino acid sequences of the polypeptides constituting the Fc regions of human Cγ1, Cγ2, Cγ3, and Cγ4 are exemplified in SEQ ID NOs: 23, 24, 25, and 26. The relationship between the amino acid residues constituting each amino acid sequence and the EU numbering of Kabat (also called EU INDEX in this specification) is shown in FIG. 18.

[0115] The Fc region consists of two light chains and two disulfide bonds formed between the heavy chains. two heavy chains containing a portion of the constant region between the CH1 domain and the CH2 domain, such that the F( region excluding (ab’)2 is referred to. The Fc region constituting the polypeptide aggregate disclosed herein is obtained by partially digesting IgG1, IgG2, IgG3, IgG4 monoclonal antibodies, etc. with a proteolytic enzyme such as pepsin and then redissolving the fraction adsorbed to a protein A column. Any protease that can digest the full-length antibody to produce F(ab’)2 restrictively by appropriately setting the reaction conditions of the enzyme such as pH can be used, and examples include pepsin and ficin. and the like. By appropriately setting the reaction conditions of the enzyme such as pH, any protease that can digest the full-length antibody to produce F(ab’)2 restrictively can be used, and there is no particular limitation. For example, pepsin, ficin, etc. can be exemplified. Fcγ receptor refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, IgG4 monoclonal antibodies, and substantially refers to any member of the family of proteins encoded by the Fcγ receptor gene. In humans, this family includes FcγRI (CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32) including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any undiscovered human FcγRs or FcγR isoforms or allotypes, but is not limited thereto. FcγRs are present in humans, mice, rats, etc. etc.

[0116] Fcγ receptor Fcγ receptor refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, IgG4 monoclonal antibodies, and substantially refers to any member of the family of proteins encoded by the Fcγ receptor gene. In humans, this family includes FcγRI (CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32) including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any undiscovered human FcγRs or FcγR isoforms or allotypes, but is not limited thereto. FcγRI (CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32) including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any undiscovered human FcγRs or FcγR isoforms or allotypes, but is not limited thereto. FcγRs are present in humans, mice, rats, etc. FcγRI (CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32) including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any undiscovered human FcγRs or FcγR isoforms or allotypes, but is not limited thereto. FcγRI (CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32) including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any undiscovered human FcγRs or FcγR isoforms or allotypes, but is not limited thereto. FcγRs are present in humans, mice, rats, etc. FcγRI (CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32) including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any undiscovered human FcγRs or FcγR isoforms or allotypes, but is not limited thereto. FcγRI (CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32) including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any undiscovered human FcγRs or FcγR isoforms or allotypes, but is not limited thereto. FcγRs are present in humans, mice, rats, etc. FcγRI (CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32) including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any undiscovered human FcγRs or FcγR isoforms or allotypes, but is not limited thereto. FcγRI (CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32) including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any undiscovered human FcγRs or FcγR isoforms or allotypes, but is not limited thereto. FcγRs are present in humans, mice, rats, etc. Any organism-derived, including but not limited to rats, rabbits, and monkeys is also acceptable. Mouse FcγRs include FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRs or Fcγ receptor isotypes or allotypes, but are not limited thereto. Preferred examples of such Fcγ receptors include human FcγI (CD64), FcγIIA (CD32), FcγIIB (CD32), FcγIIIA (CD1 6) and / or FcγIIIB (CD16). The polynucleotide sequence and amino acid sequence of FcγI are shown in SEQ ID NO: 13 (NM_000566.3) and 14 (NP_000557.1) respectively, and those of FcγIIA are shown in SEQ ID NO: 15 (BC020823.1) and 16 (AAH20823.1) respectively, those of FcγIIB are shown in SEQ ID NO: 17 (BC146678.1) and 18 (AAI46679.1) respectively, those of FcγIIIA are shown in SEQ ID NO: 19 (BC033678.1) and 20 (AAH33678.1) respectively, and those of FcγIIIB are shown in SEQ ID NO: 21 ( BC128562.1) and 22 (AAI28563.1) respectively (the numbers in parentheses indicate RefSeq accession numbers). Whether the Fcγ receptor has binding activity to the Fc region of IgG1, IgG2, IgG3, IgG4 monoclonal antibodies can be determined by methods such as FACS and ELISA formats described above, as well as ALPHA screen ( Amplified Luminescent Proximity Homogeneous Assay) and surface plasmon resonance (SPR) assay in addition to those described above. Whether the Fcγ receptor has binding activity to the Fc region of IgG1, IgG2, IgG3, IgG4 monoclonal antibodies can be determined by methods such as FACS and ELISA formats described above, as well as ALPHA screen ( Amplified Luminescent Proximity Homogeneous Assay) and surface plasmon resonance (SPR) assay in addition to those described above. Whether the Fcγ receptor has binding activity to the Fc region of IgG1, IgG2, IgG3, IgG4 monoclonal antibodies can be determined by methods such as FACS and ELISA formats described above, as well as ALPHA screen ( Amplified Luminescent Proximity Homogeneous Assay) and surface plasmon resonance (SPR) Can be confirmed by methods such as the BIACORE method using an elephant (Proc. Natl. Acad. Sci. USA (2006) 103 ( 11), 4005 - 4010).

[0117] Also, "Fc ligand" or "effector ligand" refers to a molecule, preferably a polypeptide, derived from any organism that binds to the Fc region of an antibody to form an Fc / Fc ligand complex. The binding of the Fc ligand to Fc preferably induces one or more effector functions. Fc ligands include, but are not limited to, Fc receptors, FcγR, FcαR, FcεR, FcRn, C1q, C3, mannan - binding lectin, mannose receptor, Staphylococcus protein A, Staphylococcus coccus protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRH) (Davis et al., (2002) Immunological Reviews 190, 123 - 136), which are a family of Fc receptors homologous to FcγR. Fc ligands may also include undiscovered molecules that bind to Fc. (Davis et al.,(2002)Immunological Reviews 190, 123 - 136) are also included. Fc ligands may also include undiscovered molecules that bind to Fc.

[0118] binding activity to Fcγ receptor A decrease in the binding activity of the Fc region to any of the Fcγ receptors FcγI, FcγIIA, FcγIIB, FcγIIIA, and / or FcγIIIB can be confirmed by, in addition to the FACS and ELISA formats described above, methods such as ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) and the BIACORE method using the surface plasmon resonance (SPR) phenomenon ( (Proc. Natl. Acad. Sci. USA (2006) 103 ( 11), 4005 - 4010) and the BIACORE method using the surface plasmon resonance (SPR) phenomenon ( (Proc. Natl. Acad. Sci. USA (2006) 103 ( Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0119] The AlphaScreen is carried out based on the following principle by the Alpha technology that uses two beads, a donor bead and an acceptor bead. When the molecule bound to the donor bead biologically interacts with the molecule bound to the acceptor bead, a luminescence signal is detected only when the two beads are in proximity. The photosensitizer in the donor bead excited by a laser converts the surrounding oxygen into singlet oxygen in an excited state. The singlet oxygen diffuses around the donor bead and, when it reaches the nearby acceptor bead, causes a chemiluminescence reaction in the bead, and finally light is emitted. When the molecule bound to the donor bead and the molecule bound to the acceptor bead do not interact, the singlet oxygen produced by the donor bead does not reach the acceptor bead, so the chemiluminescence reaction does not occur. For example, a biotin-labeled polypeptide conjugate is bound to the donor bead, and an Fcγ receptor tagged with glutathione S-transferase (GST) is bound to the acceptor bead. In the absence of a polypeptide conjugate with a competing mutant Fc region, the polypeptide conjugate with a wild-type Fc region interacts with the Fcγ receptor and generates a signal at 520 - 620 nm. A polypeptide conjugate with a non-tagged mutant Fc region competes with the interaction between the polypeptide conjugate with a wild-type Fc region and the Fcγ receptor. The relative binding affinity can be determined by quantifying the decrease in fluorescence resulting from the competition.

[0120] ​​​​​​​​​​​​​​It is known to biotinylate the conjugate using Sulfo-NHS-biotin or the like. As a method of tagging the Fcγ receptor with GS T, a fusion gene in which a polynucleotide encoding the Fcγ receptor and a polynucleotide encoding GST are fused in-frame is held in an expression vector, and the fusion gene is expressed in cells or the like, and a method of purifying using a glutathione column or the like can be appropriately adopted tained. The obtained signal is preferably analyzed by fitting it to a one-site competition model using non-linear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego). One of the substances (ligand) for observing the interaction is fixed on the gold thin film of the sensor chip, and when light is applied from the back side of the sensor chip so as to totally reflect at the interface between the gold thin film and the glass, a portion where the reflection intensity decreases (SPR signal) is formed in a part of the reflected light. When the other substance (analyte) for observing the interaction is flowed on the surface of the sensor chip and the ligand and the analyte bind to each other, the mass of the immobilized ligand molecules increases, and the refractive index of the solvent on the surface of the sensor chip changes. Due to this change in the refractive index, the position of the SPR signal shifts (conversely, when the binding dissociates, the position of the signal returns). The Biacore system takes the amount of the above shift, that is, the mass change on the surface of the sensor chip as the vertical axis, and displays the time change of the mass as measurement data (sensorgram). From the curve of the sensorgram, the kinetic constants: the association rate constant ( ka) and the dissociation rate constant (kd) are obtained, and the affinity (KD) is obtained from the ratio of the constants. BIA

[0121] One of the substances (ligand) for observing the interaction is fixed on the gold thin film of the sensor chip, and when light is applied from the back side of the sensor chip so as to totally reflect at the interface between the gold thin film and the glass, a portion where the reflection intensity decreases (SPR signal) is formed in a part of the reflected light. When the other substance (analyte) for observing the interaction is flowed on the surface of the sensor chip and the ligand and the analyte bind to each other, the mass of the immobilized ligand molecules increases, and the refractive index of the solvent on the surface of the sensor chip changes. Due to this change in the refractive index, the position of the SPR signal shifts (conversely, when the binding dissociates, the position of the signal returns). The Biacore system takes the amount of the above shift, that is, the mass change on the surface of the sensor chip as the vertical axis, and displays the time change of the mass as measurement data (sensorgram). From the curve of the sensorgram, the kinetic constants: the association rate constant ( ka) and the dissociation rate constant (kd) are obtained, and the affinity (KD) is obtained from the ratio of the constants. BIA One of the substances (ligand) for observing the interaction is fixed on the gold thin film of the sensor chip, and when light is applied from the back side of the sensor chip so as to totally reflect at the interface between the gold thin film and the glass, a portion where the reflection intensity decreases (SPR signal) is formed in a part of the reflected light. When the other substance (analyte) for observing the interaction is flowed on the surface of the sensor chip and the ligand and the analyte bind to each other, the mass of the immobilized ligand molecules increases, and the refractive index of the solvent on the surface of the sensor chip changes. Due to this change in the refractive index, the position of the SPR signal shifts (conversely, when the binding dissociates, the position of the signal returns). The Biacore system takes the amount of the above shift, that is, the mass change on the surface of the sensor chip as the vertical axis, and displays the time change of the mass as measurement data (sensorgram). From the curve of the sensorgram, the kinetic constants: the association rate constant ( ka) and the dissociation rate constant (kd) are obtained, and the affinity (KD) is obtained from the ratio of the constants. BIA One of the substances (ligand) for observing the interaction is fixed on the gold thin film of the sensor chip, and when light is applied from the back side of the sensor chip so as to totally reflect at the interface between the gold thin film and the glass, a portion where the reflection intensity decreases (SPR signal) is formed in a part of the reflected light. When the other substance (analyte) for observing the interaction is flowed on the surface of the sensor chip and the ligand and the analyte bind to each other, the mass of the immobilized ligand molecules increases, and the refractive index of the solvent on the surface of the sensor chip changes. Due to this change in the refractive index, the position of the SPR signal shifts (conversely, when the binding dissociates, the position of the signal returns). The Biacore system takes the amount of the above shift, that is, the mass change on the surface of the sensor chip as the vertical axis, and displays the time change of the mass as measurement data (sensorgram). From the curve of the sensorgram, the kinetic constants: the association rate constant ( ka) and the dissociation rate constant (kd) are obtained, and the affinity (KD) is obtained from the ratio of the constants. BIA ka) and the dissociation rate constant (kd) are obtained from the ratio of the constants, and the affinity (KD) is obtained from the ratio of the constants. BIA ​In the CORE method, an inhibition measurement method is also preferably used. An example of the inhibition measurement method is described in Proc. Natl. Acad. Sci. USA ( 2006) 103 (11), 4005 - 4010.

[0122] In this specification, a decrease in the binding activity to the Fcγ receptor means, for example, that based on the above analysis method, the competitive activity of the test polypeptide aggregate is 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, 15% or less, particularly preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less compared to the competitive activity of the control polypeptide aggregate. This indicates a binding activity.

[0123] As the control polypeptide aggregate, a polypeptide aggregate having the Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be appropriately used. The structure of the Fc region is shown in SEQ ID No.: 23 (A added to the N-terminus of RefSeq accession number AAC82527.1), 24 (A added to the N-terminus of RefSeq accession number AAB59393 .1), 25 (A added to the N-terminus of RefSeq accession number CAA27268.1), 26 (A added to the N-terminus of RefSeq accession number AAB59394.1). Also, when using a polypeptide aggregate having a mutant of the Fc region of an antibody of a specific isotype as a test substance, using a polypeptide aggregate having the Fc region of an antibody of the specific isotype as a control can verify the effect of the mutation of the mutant on the binding activity to the Fcγ receptor. As described above, a polypeptide aggregate having a mutant of the Fc region for which a decrease in the binding activity to the Fcγ receptor has been verified is appropriately prepared. ​

[0124] Examples of such mutants include amino acid 23 specified 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 29S, E233P, L234V, L235A (Blood (2007) 109, 1185 - 1192), etc. are known mutants.

[0125] That is, among the amino acids constituting the Fc region of an antibody of a specific isotype, any of the following amino acids specified according to EU numbering; positions 220, 226, 229, 231, 232 Positions, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266 Positions, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 3 28 positions, 329, 330, 331, 332, a polypeptide conjugate having an Fc region in which these are substituted is preferably mentioned. The isotype of the antibody that is the origin of the Fc region is not particularly limited, An Fc region derived from an IgG1, IgG2, IgG3 or IgG4 monoclonal antibody can be appropriately used, but An Fc region derived from an IgG1 antibody is preferably used. For example, among the amino acids constituting the Fc region of an IgG1 antibody, any of the following substitutions specified according to EU numbering (the numbers are the positions of amino acid residues specified according to EU numbering ), are specified

[0126] For example, among the amino acids constituting the Fc region of an IgG1 antibody, any of the following substitutions specified according to EU numbering One of the following substitutions (the numbers are the positions of amino acid residues specified according to EU numbering The single-letter amino acid symbol located before the number represents the amino acid residue before substitution, and the single-letter amino acid symbol located after the number represents the amino acid residue before substitution, respectively); (a) L234F, L235E, P331S, (b) C226S, C229S, P238S, (c) C226S, C229S, (d) C226S, C229S, E233P, L234V, L235A Fc region that has been subjected to, or an Fc region having an amino acid sequence deleted from positions 231 to 238 The polypeptide aggregate can also be appropriately used.

[0127] In addition, among the amino acids constituting the Fc region of the IgG2 antibody, any of the following substitutions specified according to the EU numbering (the position of the amino acid residue specified according to the EU numbering is the number , the single-letter amino acid symbol located before the number represents the amino acid residue before substitution, and the single-letter amino acid symbol located after the number represents the amino acid residue before substitution, respectively); (e) H268Q, V309L, A330S, P331S (f) V234A (g) G237A (h) V234A, G237A (i) A235E, G237A (j) V234A, A235E, G237A The polypeptide aggregate having an Fc region that has been subjected to can also be appropriately used.

[0128] In addition, among the amino acids constituting the Fc region of the IgG3 antibody, any of the following substitutions specified according to the EU numbering (the position of the amino acid residue specified according to the EU numbering is the number , the single-letter amino acid symbol located before the number represents the amino acid residue before substitution, and the single-letter amino acid symbol located after the number represents the amino acid residue before substitution, respectively); ​​ (k) F241A (l) D265A (m) V264A A polypeptide complex having an Fc region to which the following has been applied may also be used as appropriate.

[0129] Also, among the amino acids constituting the Fc region of an IgG4 antibody, any of the following substitutions specified according to EU numbering (the position of the amino acid residue specified according to EU numbering, the single-letter amino acid symbol located before the number represents the amino acid residue before substitution, and the single-letter amino acid symbol located after the number represents the amino acid residue before substitution respectively); (n) L235A, G237A, E318A (o) L235E (p) F234A, L235A (n) L235A, G237A, E318A (o) L235E (p) F234A, L235A A polypeptide complex having an Fc region to which the following has been applied may also be used as appropriate.

[0130] As other preferred examples, among the amino acids constituting the Fc region of an IgG1 antibody, any of the following amino acids specified according to EU numbering; positions 233, 234, 235, 236, 237, 327, 330, 331, which are substituted with the corresponding amino acids in the corresponding IgG2 or IgG4 according to their EU numbering, a polypeptide complex having an Fc region is mentioned. (o) Positions 233, 234, 235, 236, 237, 327, 330, 331 In the corresponding IgG2 or IgG4, the EU numbering is substituted with the corresponding amino acids, and a polypeptide complex having an Fc region is mentioned. (p) A polypeptide complex having an Fc region in which any one or more of the following amino acids specified according to EU numbering among the amino acids constituting the Fc region of an IgG1 antibody; positions 234, 235, 297 are substituted with other amino acids is preferably mentioned.

[0131] As other preferred examples, among the amino acids constituting the Fc region of an IgG1 antibody, any one or more of the following amino acids specified according to EU numbering; positions 234, 235, 297 are substituted with other amino acids, a polypeptide complex having an Fc region is preferably mentioned. The type of amino acid present after substitution is not particularly limited, but positions 234, 235, 297 (q) Positions 234, 235, 297 A polypeptide complex having an Fc region in which the following has been substituted with other amino acids is preferably mentioned. (r) The type of amino acid present after substitution is not particularly limited, but positions 234, 235, 297 Having an Fc region in which any one or more of the 7 amino acids are substituted with alanine A polypeptide aggregate is particularly preferred.

[0132] As other preferred examples, among the amino acids constituting the Fc region of the IgG1 antibody, any of the following amino acids specified according to the EU numbering; the Fc region in which the 265th amino acid is substituted with another amino acid A polypeptide aggregate having is preferably exemplified. The type of amino acid present after substitution is not particularly limited, but a polypeptide aggregate having an Fc region in which the 265th amino acid is substituted with alanine is particularly preferred.

[0133] Fc region derived from bispecific antibody In the present specification, as an Fc region having reduced binding activity to the Fcγ receptor, an Fc region derived from a bispecific antibody is also appropriately used. A bispecific antibody is an antibody having two different specificities. An IgG-type bispecific antibody can be secreted by a hybrid hybridoma (quadroma) generated by fusing two types of hybridomas that produce IgG antibodies (Milstein C et al. Nature (1983) 305, 537-540).

[0134] Also, an IgG-type bispecific antibody is secreted by introducing a total of four genes, the L-chain and H-chain genes constituting the two types of IgG of interest, into cells and co-expressing them. However, the combination of the H-chain and L-chain of IgG produced by these methods theoretically amounts to 10 types. It is difficult to purify IgG consisting of the H-chain and L-chain of the desired combination from 10 types of IgG. Furthermore, the desired set ​​​​​​​​Since the secretion amount of the conjugate is also significantly reduced theoretically, a large-scale culture is required, and the manufacturing cost will further increase. The manufacturing cost will further increase.

[0135] At this time, by performing appropriate amino acid substitutions on the CH3 region constituting the Fc region of the H chain, heterologous combinations of IgG can be preferentially secreted with respect to the H chain. Specifically, the amino acid side chain present in the CH3 region of one H chain is replaced with a larger side chain (knob (meaning "protrusion")), and the amino acid side chain present in the CH3 region of the other H chain is replaced with a smaller side chain (hole (meaning "void")). By doing so, the protrusion can be arranged within the void to promote the formation of heterologous H chains and inhibit the formation of homologous H chains (WO1996 / 027011, Ridgway JB et al., Protein Engineering (1996) 9, 617-621, Merchant AM et al. Nature Biotechnology (1998) 16, 677-681). Regarding the L chain, since the diversity of the L chain variable region is lower than that of the H chain variable region, it is expected that a common L chain capable of binding to both H chains can be obtained. By introducing the common L chain and both H chain genes into cells to express IgG, efficient expression of bispecific IgG becomes possible (Nature Biotechnology (1998) 16, 677-681). However, when arbitrarily selecting two types of antibodies, the possibility of including the same L chain is low, and it is difficult to implement the above idea. A method of selecting a common L chain that corresponds to any different H chain and exhibits high binding ability has also been proposed (WO2004 / 065611). The amino acid side chain present in the CH3 region of one H chain is replaced with a larger side chain (knob (meaning "protrusion")), and the amino acid side chain present in the CH3 region of the other H chain is replaced with a smaller side chain (hole (meaning "void")). By replacing the amino acid side chain present in the CH3 region of the other H chain with a smaller side chain (hole (meaning "void")), the protrusion can be arranged within the void to promote the formation of heterologous H chains and inhibit the formation of homologous H chains. This is a method of promoting the formation of heterologous H chains and inhibiting the formation of homologous H chains so that the protrusion can be arranged within the void. (WO1996 / 027011, Ridgway JB et al., Protein Engineering (1996) 9, 617-621, Merchant AM et al. Nature Biotechnology (1998) 16, 677-681). Engineering (1996) 9, 617-621, Merchant AM et al. Nature Biotechnology (1998) 16, 677-681).

[0136] Also, regarding the L chain, since the diversity of the L chain variable region is lower than that of the H chain variable region, it is expected that a common L chain capable of binding to both H chains can be obtained. By introducing the common L chain and both H chain genes into cells to express IgG, efficient expression of bispecific IgG becomes possible. (Nature Biotechnology (1998) 16, 677-681). However, when arbitrarily selecting two types of antibodies, the possibility of including the same L chain is low, and it is difficult to implement the above idea. A method of selecting a common L chain that corresponds to any different H chain and exhibits high binding ability has also been proposed. When arbitrarily selecting two types of antibodies, the possibility of including the same L chain is low, and it is difficult to implement the above idea. A method of selecting a common L chain that corresponds to any different H chain and exhibits high binding ability has also been proposed. (WO2004 / 065611).

[0137] In addition, a method for controlling the association of polypeptides or the association of heteromultimers composed of polypeptides is utilized for the association of two polypeptides constituting the Fc region to produce bispecific antibodies. That is, by modifying the amino acid residues forming the interface within the two polypeptides constituting the Fc region, the association of polypeptides constituting the Fc region having the same sequence is inhibited, and a method for controlling the formation of a polypeptide complex composed of two Fc regions with different sequences can be adopted for the production of bispecific antibodies (WO20 06 / 106905).

[0138] As the domain containing the Fc region according to the present invention, two polypeptides constituting the Fc region derived from the above-mentioned bispecific antibody can be appropriately used. More specifically, two polypeptides constituting the Fc region, wherein the amino acid at position 349 specified according to EU numbering in the amino acid sequence of one of the polypeptides is cysteine, the amino acid at position 366 is tryptophan, and in the amino acid sequence of the other polypeptide, the amino acid at position 356 specified according to EU numbering is cysteine, the amino acid at position 366 is serine, the amino acid at position 368 is alanine, and the amino acid at position 407 is valine. These two polypeptides are preferably used.

[0139] In another aspect, as the domain containing the Fc region according to the present invention, two polypeptides constituting the Fc region, wherein the amino acid at position 409 specified according to EU numbering in the amino acid sequence of one of the polypeptides is aspartic acid, and the other polypeptide The 399th amino acid specified according to EU numbering in the amino acid sequence of the peptide is lysine Two polypeptides are preferably used, characterized in that. In the above aspect, the 40 9th amino acid may be glutamic acid instead of aspartic acid, and the 399th amino acid may be arginine instead of lysine In addition to the lysine at position 399, aspartic acid at position 360 or Aspartic acid at position 392 may also be preferably added

[0140] In another aspect, as the domain containing the Fc region according to the present invention, there are two polypeptides constituting the Fc region, wherein the 370th amino acid specified according to EU numbering in the amino acid sequence of one of the polypeptides is glutamic acid, and the 357th amino acid specified according to EU numbering in the amino acid sequence of the other polypeptide is lysine Two polypeptides are preferably used, characterized in that In another aspect, as the domain containing the Fc region according to the present invention, there are two polypeptides constituting the Fc region, wherein the 439th amino acid specified according to EU numbering in the amino acid sequence of one of the polypeptides is glutamic acid, and the 356th amino acid specified according to EU numbering in the amino acid sequence of the other polypeptide is lysine Two polypeptides are preferably used, characterized in that In yet another aspect, as the domain containing the Fc region according to the present invention, there are two polypeptides constituting the Fc region, wherein the 439th amino acid specified according to EU numbering in the amino acid sequence of one of the polypeptides is glutamic acid, and the 356th amino acid specified according to EU numbering in the amino acid sequence of the other polypeptide is lysine

[0141] Furthermore, as the domain containing the Fc region according to the present invention, there are embodiments in which these are combined Two polypeptides constituting the Fc region, wherein the amino acid sequence of one of the polypeptides The 439th amino acid specified according to EU numbering is glutamic acid, and the 356th amino acid specified according to EU numbering in the amino acid sequence of the other polypeptide Is lysine, and two polypeptides are preferably used In yet another aspect, as the domain containing the Fc region according to the present invention, there are two polypeptides constituting the Fc region, wherein the 439th amino acid specified according to EU numbering in the amino acid sequence of one of the polypeptides is glutamic acid, and the 356th amino acid specified according to EU numbering in the amino acid sequence of the other polypeptide is lysine

[0142] Furthermore, as the domain containing the Fc region according to the present invention, there are embodiments in which these are combined ; Two polypeptides constituting the Fc region, wherein the amino acid sequence of one of the polypeptides Of these, the 409th amino acid specified according to EU numbering is aspartic acid, and the 370th a mino acid is glutamic acid. Among the amino acid sequences of the other polypeptide, the 399th amino acid specified according to EU numbering is lysine, and the 357th amino acid is lysine. Characterized by two polypeptides (in this embodiment, it may be aspartic acid instead of glutamic acid at the 370th position, or it may be aspartic acid at the 392nd position instead of glutamic acid at the 370th position), Two polypeptides constituting the Fc region. Among the amino acid sequences of one of the polypep tides, the 409th amino acid specified according to EU numbering is aspartic acid, and the 439th amino acid is glutamic acid. Among the amino acid sequences of the other polypeptide, the 399th amino acid specified according to EU numbering is lysine, and the 356th amino acid is lysine. Characterized by two polypeptides (in this embodiment, instead of glutamic acid at the 439th pos ition, it may be aspartic acid at the 360th position, aspartic acid at the 392nd position, or aspartic acid at the 439th position), Two polypeptides constituting the Fc region. Among the amino acid sequences of one of the polypep tides, the 370th amino acid specified according to EU numbering is glutamic acid, and the 439th amino acid is glutamic acid. Among the amino acid sequences of the other polypeptide, the 357th amino acid specified according to EU numbering is lysine, and the 356th amino acid is lysine. Characterized by two polypeptides, or Two polypeptides constituting the Fc region. Among the amino acid sequences of one of the polypep The amino acid is glutamic acid, the amino acid at position 439 is glutamic acid, and for the other polypeptide in the amino acid sequence, the amino acid at position 399 specified according to EU numbering is lysine, and at position 357 the amino acid is lysine, and the amino acid at position 356 is lysine. Two polypeptides (in this embodiment, it is not necessary to substitute the amino acid at position 370 with glutamic acid, and further, without substituting the amino acid at position 370 with glutamic acid, it may be asparagine instead of the glutamic acid at position 439 or asparagine at position 392 instead of the glutamic acid at position 439) are preferably used.

[0143] Furthermore, in another aspect, as the domain containing the Fc region according to the present invention, there are two polypeptides constituting the Fc region. Among the amino acid sequences of one of the polypeptides, the amino acid at position 356 specified according to EU numbering is lysine, and for the other polypeptide in the amino acid sequence, the amino acid at position 435 specified according to EU numbering is arginine , and the amino acid at position 439 is glutamic acid. Two polypeptides characterized by this are also preferably used.

[0144] By using the two polypeptides constituting the Fc region originating from the bispecific antibody as the domain containing the Fc region according to the present invention, it becomes possible to arrange the antigen-binding domain and / or the CD3-binding domain according to the present invention in a desired combination.

[0145] Fc region with improved C-terminal heterogeneity In the present specification, as the Fc region with reduced binding activity to the Fcγ receptor, in addition to the above characteristics, an Fc region with improved heterogeneity at the C-terminus of the Fc region can be appropriately used. It is. More specifically, glycine at position 446 and lysine at position 447, which are specified according to EU numbering among the amino acid sequences of the two polypeptides constituting the Fc region originating from IgG1, IgG2, IgG3 or IgG4, are deleted to provide an Fc region.

[0146] T cell receptor complex binding domain In the present specification, the "T cell receptor complex binding domain" refers to a part of the T cell receptor complex antibody that specifically binds to a part or all of the T cell receptor complex and is complementary, and includes a region that is complementary thereto. The T cell receptor complex may be the T cell receptor itself, or an adapter molecule that constitutes the T cell receptor complex together with the T cell receptor. A suitable adapter is CD3.

[0147] T cell receptor binding domain In the present specification, the "T cell receptor binding domain" refers to a part of the T cell receptor antibody that specifically binds to a part or all of the T cell receptor and is complementary, and includes a region that is complementary thereto.

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

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

[0150] The CD3 binding domain according to the present invention can bind to any epitope as long as it is an epitope present in the γ-chain, δ-chain or ε-chain sequence constituting human CD3. In the present invention, preferably, a CD3 binding domain comprising the variable region of the light chain (VL) of a CD3 antibody that binds to an epitope present in the extracellular region of the ε-chain of the human CD3 complex and the variable region of the heavy chain (VH) of the CD3 antibody is preferably used . Examples of such CD3 binding domains include the OKT3 antibody (Proc. Natl. Acad. Sci. USA (1980) 77, 4914-4917) and CD3 binding domains comprising the variable region of the light chain (VL) of various known CD3 antibodies and the variable region of the heavy chain (VH) of the CD3 antibody are preferably used. Also, a CD3 binding domain derived from a CD3 antibody having desired properties obtained by immunizing an animal with the γ-chain, δ-chain or ε-chain constituting human CD3 by the above method can be appropriately used. The CD3 binding domain (1980) 77, 4914-4917) and CD3 binding domains comprising the variable region of the light chain (VL) of various known CD3 antibodies and the variable region of the heavy chain (VH) of the CD3 antibody are preferably used. Also, a CD3 binding domain derived from a CD3 antibody having desired properties obtained by immunizing an animal with the γ-chain, δ-chain or ε-chain constituting human CD3 by the above method can be appropriately used. The CD3 binding domain ​​​​ As described above, the CD3 antibody serving as the main origin may be appropriately a humanized antibody or a human antibody. The structures of the γ-chain, δ-chain, or ε-chain constituting CD3 are such that their polynucleotide sequences are as set forth in SEQ ID NO: 27 (NM_000073.2), 29 (NM_000732.4), and 31 (NM_000733.3), and their polypeptide sequences are as set forth in SEQ ID NO: 28 (NP_000064.1), 30 (NP_000723.1), and 32 ( NP_000724.1) (the numbers in parentheses indicate RefSeq accession numbers).

[0151] polypeptide aggregate The polypeptide conjugate according to the present invention may include the above-mentioned (1) antigen-binding domain, (2) a domain containing an Fc region with reduced binding activity to the Fcγ receptor, and (3) a T cell receptor complex-binding domain, and its structure is not limited as long as it contains these. In the present invention, the T cell receptor complex binding domain is preferably a T cell receptor-binding domain or a CD3-binding domain. Each of the above domains can be directly linked by a peptide bond. For example, when using F(ab')2 as the (1) antigen binding domain and using these Fc regions as the (2) domain containing an Fc region with reduced binding activity to the Fcγ receptor, when the antigen-binding domain described in (1) and the domain containing the Fc region described in (2) are linked by a peptide bond, the linked polypeptide forms the structure of an antibody. To produce such an antibody, in addition to purifying from the culture solution of the aforementioned hybridoma, the antibody can also be purified from the culture solution of a desired host cell in which the polynucleotide encoding the polypeptide constituting the antibody is stably retained. ​

[0152] When the (3) CD3 binding domain binds to the antibody structure, the CD3 binding domain can be bound via a peptide bond to the C-terminus of the constant region of the antibody structure. As another aspect, the CD3 binding domain can be bound via a peptide bond to the N-terminus of the heavy chain variable region or the light chain variable region of the antibody structure. As another aspect, the CD3 binding domain can be bound via a peptide bond to the C-terminus of the light chain constant region of the antibody structure. The CD3 binding domain to be bound can employ a CD3 binding domain having a desired structure, but preferably Fv, more preferably scFv, is appropriately used. The valence of the CD3 binding domain that binds to the antibody structure is not limited. To bind a bivalent CD3 binding domain to the antibody structure, a monovalent CD3 binding domain can be bound via a peptide bond to the C-terminus of each of the two Fc regions that make up the constant region of the antibody structure. Also, to bind a bivalent CD3 binding domain to the antibody structure, a bivalent scFv, i.e., sc(Fv)2, can be bound via a peptide bond to the C-terminus of one of the two Fc regions. In this case, by using the Fc region derived from the above-mentioned bispecific antibody, a polypeptide aggregate in which the bivalent scFv, i.e., sc(Fv)2, binds only to the C-terminus of one of the two Fc regions that make up the constant region of the antibody structure is efficiently obtained. Also, to bind a monovalent CD3 binding domain to the antibody structure, a monovalent scFv can be bound via a peptide bond to the C-terminus of one of the two Fc regions. In this case, by using the Fc region derived from the above-mentioned bispecific antibody, the antibody structure ​​​​​​​​​​​​​​​​A monovalent scFv binds only to the C-terminus of one of the two Fc regions that make up the constant region. The polypeptide conjugate according to the present invention in which binding occurs is efficiently obtained.

[0153] In addition, when the CD3-binding domain can be bound via a peptide bond to the C-terminus of the constant region of the antibody structure, a polypeptide conjugate in which the heavy-chain Fv fragment constituting the CD3-binding domain is linked to the C-terminus (CH3 domain) of one of the constant regions constituting the Fc region, and the light-chain Fv fragment constituting the CD3-binding domain is linked to the C-terminus (CH3 domain) of the other constant region constituting the Fc region is also appropriately used. In this case, when linking the heavy-chain Fv fragment or the light-chain Fv fragment to the C-terminus (CH3 domain) of the constant region, a linker such as Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 7) is appropriately inserted. The number of repeats of the linker is not limited and is selected from 1 to 10, preferably 2 to 8, more preferably 2 to 6, that is, a linker such as Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 7) consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 repeats can be appropriately inserted.

[0154] Furthermore, when a polypeptide conjugate in which the heavy-chain Fv fragment constituting the CD3-binding domain is linked to the C-terminus (CH3 domain) of one of the constant regions constituting the Fc region, and the light-chain Fv fragment constituting the CD3-binding domain is linked to the C-terminus (CH3 domain) of the other constant region constituting the Fc region is produced, in order to strengthen the association between the heavy-chain Fv fragment and the light-chain Fv fragment, modification of amino acid residues to form a disulfide bond between the heavy-chain Fv fragment and the light-chain Fv fragment can also be appropriately carried out.

[0155] ​​​​​​In another aspect, while one of the constant regions constituting the Fc region is linked to the C-terminus (CH3 domain) of the heavy chain Fv fragment constituting the CD3 binding domain, the light chain Fv fragment constituting the CD3 binding domain is linked to the C-terminus (CH3 domain) of the other constant region constituting the Fc region. When a polypeptide aggregate is prepared, the CH1 domain and the CL domain of an antibody may be linked to each of the heavy chain Fv fragment and the light chain Fv fragment to strengthen the association between the heavy chain Fv fragment and the light chain Fv fragment. In another aspect, to bind a bivalent CD3 binding domain to the antibody structure, a monovalent CD3 binding domain may be bound to each C-terminus of the two light chain constant regions or each N-terminus of the light chain variable regions of the antibody structure via a peptide bond. Also, to bind a bivalent CD3 binding domain to the antibody structure, a bivalent scFv, i.e., sc(Fv)2, may be bound to each C-terminus of the two light chain constant regions or each N-terminus of the light chain variable regions via a peptide bond. In this case, by using the Fc region derived from the above-mentioned bispecific antibody, a polypeptide aggregate in which a bivalent scFv, i.e., sc(Fv)2, binds to the C-terminus or N-terminus of one of the two light chain variable regions of the antibody structure is efficiently obtained. Also, to bind a monovalent CD3 binding domain to the antibody structure, a monovalent scFv may be bound to the C-terminus or N-terminus of one of the two light chain variable regions via a peptide bond. In this case, by using the Fc region derived from the above-mentioned bispecific antibody, a polypeptide aggregate according to the present invention in which a monovalent scFv binds to the N-terminus or C-terminus of one of the two light chain variable regions of the antibody structure is efficiently obtained. In another aspect, when a polypeptide aggregate is prepared in which one of the constant regions constituting the Fc region is linked to the C-terminus (CH3 domain) of the heavy chain Fv fragment constituting the CD3 binding domain, and the light chain Fv fragment constituting the CD3 binding domain is linked to the C-terminus (CH3 domain) of the other constant region constituting the Fc region, the CH1 domain and the CL domain of an antibody may be linked to each of the heavy chain Fv fragment and the light chain Fv fragment to strengthen the association between the heavy chain Fv fragment and the light chain Fv fragment. In another aspect, to bind a bivalent CD3 binding domain to the antibody structure, a monovalent CD3 binding domain may be bound to each C-terminus of the two light chain constant regions or each N-terminus of the light chain variable regions of the antibody structure via a peptide bond. Also, to bind a bivalent CD3 binding domain to the antibody structure, a bivalent scFv, i.e., sc(Fv)2, may be bound to each C-terminus of the two light chain constant regions or each N-terminus of the light chain variable regions via a peptide bond. In this case, by using the Fc region derived from the above-mentioned bispecific antibody, a polypeptide aggregate in which a bivalent scFv, i.e., sc(Fv)2, binds to the C-terminus or N-terminus of one of the two light chain variable regions of the antibody structure is efficiently obtained.

[0156] In yet another aspect, to bind a bivalent CD3 binding domain to the antibody structure, a monovalent CD3 binding domain may be bound to each C-terminus of the two light chain constant regions or each N-terminus of the light chain variable regions of the antibody structure via a peptide bond. Also, to bind a bivalent CD3 binding domain to the antibody structure, a bivalent scFv, i.e., sc(Fv)2, may be bound to each C-terminus of the two light chain constant regions or each N-terminus of the light chain variable regions via a peptide bond. In this case, by using the Fc region derived from the above-mentioned bispecific antibody, a polypeptide aggregate in which a bivalent scFv, i.e., sc(Fv)2, binds to the C-terminus or N-terminus of one of the two light chain variable regions of the antibody structure is efficiently obtained. Also, to bind a monovalent CD3 binding domain to the antibody structure, a monovalent scFv may be bound to the C-terminus or N-terminus of one of the two light chain variable regions via a peptide bond. In this case, by using the Fc region derived from the above-mentioned bispecific antibody, a polypeptide aggregate according to the present invention in which a monovalent scFv binds to the N-terminus or C-terminus of one of the two light chain variable regions of the antibody structure is efficiently obtained. In another aspect, when a polypeptide aggregate is prepared in which one of the constant regions constituting the Fc region is linked to the C-terminus (CH3 domain) of the heavy chain Fv fragment constituting the CD3 binding domain, and the light chain Fv fragment constituting the CD3 binding domain is linked to the C-terminus (CH3 domain) of the other constant region constituting the Fc region, the CH1 domain and the CL domain of an antibody may be linked to each of the heavy chain Fv fragment and the light chain Fv fragment to strengthen the association between the heavy chain Fv fragment and the light chain Fv fragment. In another aspect, to bind a bivalent CD3 binding domain to the antibody structure, a monovalent CD3 binding domain may be bound to each C-terminus of the two light chain constant regions or each N-terminus of the light chain variable regions of the antibody structure via a peptide bond. Also, to bind a bivalent CD3 binding domain to the antibody structure, a bivalent scFv, i.e., sc(Fv)2, may be bound to each C-terminus of the two light chain constant regions or each N-terminus of the light chain variable regions via a peptide bond. In this case, by using the Fc region derived from the above-mentioned bispecific antibody, a polypeptide aggregate in which a bivalent scFv, i.e., sc(Fv)2, binds to the C-terminus or N-terminus of one of the two light chain variable regions of the antibody structure is efficiently obtained. Also, to bind a monovalent CD3 binding domain to the antibody structure, a monovalent scFv may be bound to the C-terminus or N-terminus of one of the two light chain variable regions via a peptide bond. In this case, by using the Fc region derived from the above-mentioned bispecific antibody, a polypeptide aggregate according to the present invention in which a monovalent scFv binds to the N-terminus or C-terminus of one of the two light chain variable regions of the antibody structure is efficiently obtained. In another aspect, when a polypeptide aggregate is prepared in which one of the constant regions constituting the Fc region is linked to the C-terminus (CH3 domain) of the heavy chain Fv fragment constituting the CD3 binding domain, and the light chain Fv fragment constituting the CD3 binding domain is linked to the C-terminus (CH3 domain) of the other constant region constituting the Fc region, the CH1 domain and the CL domain of an antibody may be linked to each of the heavy chain Fv fragment and the light chain Fv fragment to strengthen the association between the heavy chain Fv fragment and the light chain Fv fragment. In another aspect, to bind a bivalent CD3 binding domain to the antibody structure, a monovalent CD3 binding domain may be bound to each C-terminus of the two light chain constant regions or each N-terminus of the light chain variable regions of the antibody structure via a peptide bond.

[0157] In another aspect, in order to bind a bivalent CD3 binding domain to the antibody structure, a monovalent CD3 binding domain can be bound via a peptide bond to the N-terminus of each of the two heavy chain variable regions of the antibody structure. Also, in order to bind a bivalent CD3 binding domain to the antibody structure, a bivalent scFv, i.e., sc(Fv)2, can be bound via a peptide bond to the N-terminus of one of the two heavy chain variable regions. In this case, by using the Fc region derived from the above-mentioned bispecific antibody, a polypeptide aggregate in which the bivalent scFv, i.e., sc(Fv)2, binds only to the N-terminus of one of the two heavy chain variable regions of the antibody structure can be efficiently obtained. Also, in order to bind a monovalent CD3 binding domain to the antibody structure, a monovalent scFv can be bound via a peptide bond to the N-terminus of one of the two heavy chain variable regions. In this case, by using the Fc region derived from the above-mentioned bispecific antibody, the polypeptide aggregate according to the present invention in which the monovalent scFv binds to the N-terminus of one of the two heavy chain variable regions of the antibody structure can be efficiently obtained. antibody structure can be bound to each N-terminus of the two heavy chain variable regions via a peptide bond. In addition, in order to bind a bivalent CD3 binding domain to the antibody structure, a bivalent scFv, i.e., sc(Fv)2, can be bound via a peptide bond to the N-terminus of one of the two heavy chain variable regions. In this case, by using the Fc region derived from the above-mentioned bispecific antibody, a polypeptide aggregate in which the bivalent scFv, i.e., sc(Fv)2, binds only to the N-terminus of one of the two heavy chain variable regions of the antibody structure can be efficiently obtained. Also, in order to bind a monovalent CD3 binding domain to the antibody structure, a monovalent scFv can be bound via a peptide bond to the N-terminus of one of the two heavy chain variable regions. In this case, by using the Fc region derived from the above-mentioned bispecific antibody, the polypeptide aggregate according to the present invention in which the monovalent scFv binds to the N-terminus of one of the two heavy chain variable regions of the antibody structure can be efficiently obtained. In this case, by using the Fc region derived from the above-mentioned bispecific antibody, a polypeptide aggregate in which the monovalent scFv binds to the N-terminus of one of the two heavy chain variable regions of the antibody structure can be efficiently obtained. In addition, when preparing the above polypeptide aggregate, each domain can be bound not only directly by a peptide bond but also by a peptide bond via a peptide linker.

[0158] In this case, as the linker to be adopted, in addition to the linkers exemplified above, a linker having a peptide tag such as a His tag, HA tag, myc tag, FLAG tag, etc. can also be appropriately used. Also, hydrogen bonds, disulfide bonds, covalent bonds, ionic interactions or the like In addition, when preparing the above polypeptide aggregate, each domain can be bound not only directly by a peptide bond but also by a peptide bond via a peptide linker. In this case, as the linker to be adopted, in addition to the linkers exemplified above, a linker having a peptide tag such as a His tag, HA tag, myc tag, FLAG tag, etc. can also be appropriately used. For example, a linker having a peptide tag such as a His tag, HA tag, myc tag, FLAG tag, etc. can also be appropriately used. In addition, hydrogen bonds, disulfide bonds, covalent bonds, ionic interactions or the like The property of binding to each other by the combination of these can also be suitably utilized. For example, the affinity between CH1 and CL of an antibody is utilized, or an Fc region derived from the aforementioned bispecific antibody is used in the association of the heterologous Fc region. Further, as described in the examples, the disulfide bond formed between domains can also be suitably utilized. The affinity between CH1 and CL of an antibody is utilized, or an Fc region derived from the aforementioned bispecific antibody is used in the association of the heterologous Fc region. Furthermore, as described in the examples, the disulfide bond formed between domains can also be suitably utilized. Another structure of the polypeptide conjugate according to the present invention is, for example, (1) a structure in which a monovalent Fv and a monovalent Fab are used as antigen-binding domains. In this case,

[0159] (2) a heavy chain CH1 region linked via a peptide bond to one of the two Fc regions with reduced binding activity to the Fcγ receptor constituting the polypeptide conjugate according to the present invention, and a heavy chain Fv fragment (VH) or a light chain Fv fragment (VL) of the monovalent Fv is linked via a peptide bond to the heavy chain CH1 region, and the other VL or VH fragment of the monovalent Fv is linked via a peptide bond to the light chain CH region bound to the heavy chain CH1 region via a disulfide bond, so that a structure in which VH and VL bound to the ends of the heavy chain CH1 region and the light chain CL region form an antibody-binding domain is used. To the N-terminus of the other different Fc region of the two Fc regions, (1) an antibody-binding domain and (3) an sc(Fv)2 forming a CD3-binding domain can be linked via a peptide bond. In this case, by using the Fc region derived from the aforementioned bispecific antibody, a polypeptide conjugate having a structure in which a heavy chain CH1 region is linked via a peptide bond to one of the two Fc regions constituting the polypeptide conjugate, and sc(Fv)2 is linked via a peptide bond to the other Fc region can be produced. When producing the above polypeptide conjugate, each The affinity between CH1 and CL of an antibody is utilized, or an Fc region derived from the aforementioned bispecific antibody is used in the association of the heterologous Fc region. Furthermore, as described in the examples, the disulfide bond formed between domains can also be suitably utilized. Another structure of the polypeptide conjugate according to the present invention is, for example, (1) a structure in which a monovalent Fv and a monovalent Fab are used as antigen-binding domains. In this case, (2) a heavy chain CH1 region linked via a peptide bond to one of the two Fc regions with reduced binding activity to the Fcγ receptor constituting the polypeptide conjugate according to the present invention, and a heavy chain Fv fragment (VH) or a light chain Fv fragment (VL) of the monovalent Fv is linked via a peptide bond​ The domains are directly linked by peptide bonds. Additionally, each domain can be linked by a peptide bond via a peptide linker. In this case, as the linker to be adopted, in addition to the linkers exemplified above, linkers having peptide tags such as His tag, HA tag, myc tag, FLAG tag, etc. can also be appropriately used.

[0160] As another structure of the polypeptide aggregate according to the present invention, for example, (1) a structure in which a bivalent scFv is used as an antigen-binding domain is also preferably used. As an embodiment of such a structure, one of the bivalent scFvs is linked by a peptide bond to one of the two Fc regions with reduced binding activity to the (2) Fcγ receptor via the VH constituting the (3) CD3-binding domain, and the other of the bivalent scFvs is linked by a peptide bond to the other of the two Fc regions with reduced binding activity to the (2) Fcγ receptor via the VL constituting the (3) CD3-binding domain, thereby producing a polypeptide aggregate having such a structure. In this case, it is also possible to use the Fc region derived from the above-mentioned bispecific antibody. When producing the above-mentioned polypeptide aggregate, each domain is directly linked by a peptide bond. Additionally, each domain can be linked by a peptide bond via a peptide linker. In this case, as the linker to be adopted, in addition to the linkers exemplified above, linkers having peptide tags such as His tag, HA tag, myc tag, FLAG tag, etc. can also be appropriately used. As another embodiment of the structure in which a bivalent scFv is used as the (1) antigen-binding domain,

[0161] (1) As another embodiment of the structure in which a bivalent scFv is used as the antigen-binding domain, One of the bivalent scFvs is linked by a peptide bond to one of two Fc regions in which the binding activity to the (2) Fcγ receptor is reduced via the scFv constituting the (3) CD3 binding domain and the other of the bivalent scFvs has a structure in which the other of the two Fc regions in which the binding activity to the (2) Fcγ receptor is reduced is linked by a peptide bond. A polypeptide conjugate can be produced. In this case, by using the Fc region derived from the above-mentioned bispecific antibody, one of the two Fc regions constituting the polypeptide conjugate has a scFv constituting an antigen-binding domain via a scFv constituting a CD3 binding domain, and the other Fc region has a scFv constituting an antigen-binding domain via a peptide bond. A polypeptide conjugate having a structure in which each is linked can be produced. When producing the above polypeptide conjugate, each domain is directly linked by a peptide bond, and each domain can also be linked by a peptide bond via a peptide linker. In this case, as the linker employed, in addition to the linkers exemplified above, linkers having peptide tags such as His tag, HA tag, myc tag, FLAG tag, etc. can also be appropriately used. As another structure of the polypeptide conjugate according to the present invention, for example, a structure in which the antigen-binding domain and the T cell receptor complex-binding domain are each monovalent Fab is also preferably used. As an aspect of such a structure, the heavy chain Fv fragment of the monovalent Fab constituting the antigen-binding domain is linked to one polypeptide constituting the Fc region via the CH1 region, and the light chain Fv fragment of the Fab is linked to the CL region. The heavy chain Fv fragment of the Fab constituting the T cell receptor binding domain is linked via the CH1 region to the other polypeptide constituting the Fc region, and the light chain Fv fragment of the Fab is linked to the CL region. When producing the above polypeptide conjugate, each domain is directly linked by a peptide bond, and each domain can also be linked by a peptide bond via a peptide linker. In this case, as the linker employed, in addition to the linkers exemplified above, linkers having peptide tags such as His tag, HA tag, myc tag, FLAG tag, etc. can also be appropriately used.

[0162] As another structure of the polypeptide conjugate according to the present invention, for example, a structure in which the antigen-binding domain and the T cell receptor complex-binding domain are each monovalent Fab is also preferably used. As an aspect of such a structure, the heavy chain Fv fragment of the monovalent Fab constituting the antigen-binding domain is linked to one polypeptide constituting the Fc region via the CH1 region, and the light chain Fv fragment of the Fab is linked to the CL region. This structure is also preferably used. As an aspect of such a structure, the heavy chain Fv fragment of the monovalent Fab constituting the antigen-binding domain is linked to one polypeptide constituting the Fc region via the CH1 region, and the light chain Fv fragment of the Fab is linked to the CL region. One polypeptide constituting the Fc region is linked, and the light chain Fv fragment of the Fab is linked to the CL region. The heavy chain Fv fragment of the Fab constituting the T cell receptor binding domain is linked via the CH1 region​ linked to the other polypeptide constituting the Fc region, and a polypeptide aggregate having a structure in which the light chain Fv fragment of the Fab is linked to the CL region can be produced.

[0163] As another aspect of such a structure, the heavy chain Fv fragment of a monovalent Fab constituting the antigen-binding domain is linked to one polypeptide constituting the Fc region via the CH1 region, and the light chain Fv fragment of the Fab is linked to the CL region, and the light chain Fv fragment of the Fab constituting the T cell receptor-binding domain is linked to the other polypeptide constituting the Fc region via the CH1 region, and a polypeptide aggregate having a structure in which the heavy chain Fv fragment of the Fab is linked to the CL region can be produced. Also, the heavy chain Fv fragment of a monovalent Fab constituting the T cell receptor-binding domain is linked to one polypeptide constituting the Fc region via the CH1 region, the light chain Fv fragment of the Fab is linked to the CL region, and the light chain Fv fragment of the Fab constituting the antigen-binding domain is linked to the other polypeptide constituting the Fc region via the CH1 region, and a polypeptide aggregate having a structure in which the heavy chain Fv fragment of the Fab is linked to the CL region can also be produced.

[0164] Furthermore, as another aspect of such a structure, the heavy chain Fv fragment of a monovalent Fab constituting the antigen-binding domain is linked to one polypeptide constituting the Fc region via the CH1 region, the light chain Fv fragment of the Fab is linked to the CL region, and the heavy chain Fv fragment of the Fab constituting the T cell receptor-binding domain is linked to the other polypeptide constituting the Fc region via the CL region, and a polypeptide aggregate having a structure in which the light chain Fv fragment of the Fab is linked to the CH1 region can be produced. Also, ​​​​​​​​​​​​​​The heavy chain Fv fragment of a monovalent Fab that constitutes the body-binding domain is linked to one polypeptide that constitutes the Fc region via the CH1 region, the light chain Fv fragment of the Fab is linked to the CL region, and the heavy chain Fv fragment of the Fab that constitutes the antigen-binding domain is linked to the other polypeptide that constitutes the Fc region via the CL region, and the light chain Fv fragment of the Fab is linked to the CH1 region, and a polypeptide assembly having such a structure can be produced.

[0165] As another structure of the polypeptide assembly according to the present invention, as one aspect of the structure in which the antigen-binding domain and the T cell receptor complex-binding domain are each a monovalent Fab, (1) The heavy chain Fv fragment of the monovalent Fab structure that binds to the antigen is linked to one polypeptide that constitutes the Fc region via the CH1 region, and the light chain Fv fragment of the Fab structure is linked to the CL region, and the antigen 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 other polypeptide that constitutes the Fc region via the CH1 region, and the light chain Fv fragment of the Fab structure is linked to the CL region, and the T cell receptor complex-binding domain, including, and the heavy chain Fv fragment in the antigen-binding domain and the light chain Fv fragment in the antigen-binding domain or 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, and a polypeptide in which the charges of the CH1 region and the CL region are controlled is preferably mentioned. In this aspect, it is only necessary that the charges of the CH1 region and the CL region are controlled so that the heavy chain Fv fragment in the antigen-binding domain and the light chain Fv fragment in the antigen-binding domain or 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, and the polypeptide ​​​​​​​The structure of the conjugate (conjugation control structure) is not limited to a specific structure.

[0166] As one aspect of the conjugation control structure, a polypeptide conjugate can be prepared in which 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 antigen binding domain have the same type of charge as each other. And the amino acid residues of the CL region linked to the light chain Fv fragment in the antigen binding domain have the same type of charge as each other. A polypeptide conjugate can be prepared in which the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen binding domain and the amino acid residues of the CL region linked to the light chain Fv fragment in the T cell receptor complex binding domain have the same type of charge as each other. Can be obtained.

[0167] As another aspect of the conjugation control structure, a polypeptide conjugate can be prepared in which the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen binding domain and the amino acid residues of the CL region linked to the light chain Fv fragment in the T cell receptor complex binding domain have the same type of charge as each other. And the amino acid residues of the CL region linked to the light chain Fv fragment in the T cell receptor complex binding domain have the same type of charge as each other. A polypeptide conjugate can be prepared in which the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen binding domain and the amino acid residues of the CL region linked to the light chain Fv fragment in the T cell receptor complex binding domain have the same type of charge as each other. Can be obtained.

[0168] As a further aspect of the conjugation control structure, a polypeptide conjugate can be prepared in which 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 antigen binding domain have the same type of charge as each other, and the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen binding domain and the amino acid residues of the CL region linked to the light chain Fv fragment in the T cell receptor complex binding domain have the same type of charge as each other. And the amino acid residues of the CL region linked to the light chain Fv fragment in the antigen binding domain have the same type of charge as each other, and the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen binding domain and the amino acid residues of the CL region linked to the light chain Fv fragment in the T cell receptor complex binding domain have the same type of charge as each other. Have the same type of charge as each other, and the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen binding domain and the amino acid residues of the CL region linked to the light chain Fv fragment in the T cell receptor complex binding domain have the same type of charge as each other. And the amino acid residues of the CL region linked to the light chain Fv fragment in the T cell receptor complex binding domain have the same type of charge as each other. A polypeptide conjugate can be prepared in which the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen binding domain and the amino acid residues of the CL region linked to the light chain Fv fragment in the T cell receptor complex binding domain have the same type of charge as each other. Can be obtained.

[0169] Also, as one aspect of the conjugation control structure, 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 antigen binding domain have the same type of charge as each other, and the T cell receptor complex And the amino acid residues of the CL region linked to the light chain Fv fragment in the antigen binding domain have the same type of charge as each other, and the T cell receptor complex Have the same type of charge as each other, and the T cell receptor complex The amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the body-binding domain and the T cell receptor The amino acid residues of the CL region linked to the light chain Fv fragment in the body-binding domain have different charges from each other A polypeptide aggregate having can be created.

[0170] Also, as another aspect of the association control structure, 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 light chain F in the antigen binding domain The amino acid residues of the CL region linked to the v fragment have the same charge as each other, and the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen binding domain and the T cell receptor complex binding The amino acid residues of the CL region linked to the v fragment in the domain have the same charge as each other, and 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 T cell receptor binding The amino acid residues of the CL region linked to the light chain Fv fragment in the domain have different charges from each other, and a polypeptide aggregate having such can be produced. The amino acid residues of the CL region linked to the light chain Fv fragment in the domain have the same charge as each other, and 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 T cell receptor binding The amino acid residues of the CL region linked to the light chain Fv fragment in the domain have different charges from each other, and a polypeptide aggregate having such can be produced. The amino acid residues of the CL region linked to the light chain Fv fragment in the domain have different charges from each other, and a polypeptide aggregate having such can be produced. The amino acid residues of the CL region linked to the light chain Fv fragment in the domain have different charges from each other, and a polypeptide aggregate having such can be produced.

[0171] Furthermore, as one aspect of the association control structure, the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen binding domain 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 as each other, and the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen binding domain and the antigen binding domain The amino acid residues of the CL region linked to the light chain Fv fragment in the domain have the same charge as each other, and the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen binding domain and the light chain The amino acid residues of the CL region linked to the Fv fragment in the domain both have different charges, and a polypeptide aggregate having such can be produced. The amino acid residues of the CL region linked to the Fv fragment in the domain both have different charges, and a polypeptide aggregate having such can be produced. The amino acid residues of the CL region linked to the Fv fragment in the domain both have different charges, and a polypeptide aggregate having such can be produced.

[0172] Furthermore, as another aspect 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 therein and the light chain amino acid residues of the CL region linked to the Fv fragment in the antigen-binding domain have the same kind of charge as each other, and the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen-binding domain and the amino acid residues of the CL region linked to the light chain Fv fragment in the T cell receptor complex binding domain have the same kind of charge as each other, and the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen-binding domain and the amino acid residues of the CL region linked to the light chain Fv fragment in the antigen-binding domain both have different kinds of

[0173] charges, and a polypeptide aggregate can be produced.

[0173] In addition, as a different aspect of the association control structure, 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 antigen-binding domain have the same kind of charge as each other, and the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen-binding domain and the amino acid residues of the CL region linked to the light chain Fv fragment in the T cell receptor complex binding domain have the same kind of charge as each other, and 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 have different kinds of charges as each other, and the amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the antigen-binding domain and the amino acid residues of the CL region linked to the light chain Fv fragment in the antigen-binding domain have different kinds of charges as each other, and a polypeptide aggregate can be produced.

[0174] control of charges of CH1 region and CL region The heavy chain and light chain of the T cell receptor binding domain recognize the epitope of the T cell receptor binding domain, and also recognize the epitope of the antigen by the heavy chain and light chain of the antigen binding domain. When obtaining a bispecific polypeptide conjugate, when producing the polypeptide conjugate, theoretically, 10 types of polypeptide conjugate molecules may be produced when expressing the four respective chains. In this case, for example, if the association between the heavy chain of the T cell receptor binding domain and the light chain of the antigen binding domain and / or the heavy chain of the antigen binding domain and the light chain of the T cell receptor binding domain is inhibited, it is possible to preferentially obtain the desired polypeptide conjugate molecule. For example, an example can be given in which the amino acid residues forming the interface between the CH1 of the heavy chain of the T cell receptor binding domain and the CL of the light chain of the antigen binding domain are modified to amino acid residues having a positive charge, and the amino acid residues forming the interface between the CH1 of the heavy chain of the antigen binding domain and the CL of the light chain of the T cell receptor binding domain are modified to amino acid residues having a negative charge. By this modification, the association between the CH1 of the heavy chain of the T cell receptor binding domain and the CL of the light chain of the antigen binding domain, which is not the target, is inhibited because both amino acid residues forming the interface are positively charged, and the association between the CH1 of the heavy chain of the antigen binding domain and the CL of the light chain of the T cell receptor binding domain, which is not the target, is inhibited because both amino acid residues forming the interface are negatively charged. As a result, the association between the CH1 of the heavy chain of the target T cell receptor binding domain and the CL of the light chain of the T cell receptor binding domain and the association between the CH1 of the heavy chain of the target antigen binding domain and the CL of the light chain of the antigen binding domain occur, and the polypeptide conjugate of the present invention is effective.

[0175]

[0176] ​​​​​​​​​​​​​​ can be efficiently obtained. Preferably, the association between the heavy chain of the target T cell receptor binding domain and the light chain of the T cell receptor binding domain is promoted because the amino acid residues forming the interface have different charges from each other, and the association between the heavy chain of the target antigen binding domain and the light chain of the antigen binding domain is also promoted because the amino acid residues forming the interface have different charges from each other. As a result, the polypeptide complex of the present invention in which the target association has occurred can be efficiently obtained.

[0177] In addition, by utilizing the association control of the present invention, it is also possible to suppress the association between CH1s (the heavy chain of the T cell receptor binding domain and the heavy chain of the antigen binding domain) or between CLs (the light chain of the T cell receptor binding domain and the light chain of the antigen binding domain).

[0178] A person skilled in the art can appropriately know the types of amino acid residues approaching at the interface of CH1 and CL when the desired polypeptide complex whose association is to be controlled by the present invention associates.

[0179] In addition, in organisms such as humans, monkeys, mice, and rabbits, a person skilled in the art can appropriately obtain sequences that can be used as CH1 or CL of an antibody by using a public database or the like. More specifically, the amino acid sequence information of CH1 or CL can be obtained by the means described in the examples below.

[0180] For example, as shown in the examples below, when CH1 and CL that are respectively linked to VH and VL constituting the T cell receptor binding domain or the antigen binding domain associate, the interface between CH1 and CL Specific examples of amino acid residues that approach (relatively or in contact) include the following combinations as follows · Lysine (K) at position 147 of the EU numbering of CH1 (e.g., position 147 in the amino acid sequence set forth in SEQ ID NO: 1) and threonine (T) at position 180 of the EU numbering of the relative (contacting) CL · Lysine (K) at position 147 of the EU numbering of CH1 and serine (S) at position 131 of the EU numbering of the relative (contacting) CL · Lysine (K) at position 147 of the EU numbering of CH1 and threonine (T) at position 164 of the EU numbering of the relative (contacting) CL · Lysine (K) at position 147 of the EU numbering of CH1 and asparagine (N) at position 138 of the EU numbering of the relative (contacting) CL · Lysine (K) at position 147 of the EU numbering of CH1 and glutamic acid (E) at position 123 of the EU numbering of the relative (contacting) CL · Glutamine (Q) at position 175 of the EU numbering of CH1 and glutamine (Q) at position 160 of the EU numbering of the relative (contacting) CL · Lysine (K) at position 213 of the EU numbering of CH1 and glutamic acid (E) at position 123 of the EU numbering of the relative (contacting) CL Note that for the numbering of these sites, reference is made to the literature of Kabat et al. (Kabat EA et al. 1991. Sequence of Proteins of Immunological Interest. NIH) Also, the numbers described as EU numbering in the present invention follow EU numbering (Sequences of proteins of immunological interest, NIH Publication No. 91 - 3242) In addition, the numbers described as EU numbering in the present invention follow EU numbering (Sequences of proteins of immunological interest, NIH Publication No.91 - 3242) Note that for the numbering of these sites, reference is made to the literature of Kabat et al. (Kabat EA et al. 1991. Sequence of Proteins of Immunological Interest. NIH) Also, the numbers described as EU numbering in the present invention follow EU numbering (Sequences of proteins of immunological interest, NIH Publication No.91 - 3242) ​It is as described. In the present invention, "the amino acid residue at position X of EU numbering", " the amino acid at position X of EU numbering" (X is any number) can also be read as "the amino acid residue corresponding to position X of EU numbering", " "the amino acid corresponding to position X of EU numbering".

[0181] As shown in the examples described later, by modifying these amino acid residues and implementing the method of the present invention, the desired polypeptide aggregate can be preferentially obtained.

[0182] Since these amino acid residues are known to be highly conserved in humans and mice (J. Mol. Recognit. (2003) 16, 113 - 120), for the association of CH1 and CL other than the polypeptide aggregates shown in the examples, by modifying the amino acid residues corresponding to the above amino acid residues, the association of the constant region of the polypeptide aggregate of the present invention can be controlled.

[0183] That is, the present invention provides a polypeptide aggregate in which the association of the heavy chain and the light chain is controlled, and is composed of one set or two or more sets of amino acid residues selected from the group consisting of the sets of amino acid residues shown in the following (a) to (f), wherein the amino acid residues have the same charge; (a) The amino acid residue contained in CH1 and being the amino acid residue at position 147 of EU numbering, and the amino acid residue contained in CL and being the amino acid residue at position 180 of EU numbering, (b) The amino acid residue contained in CH1 and being the amino acid residue at position 147 of EU numbering, and the amino acid residue contained in CL and being the amino acid residue at position 131 of EU numbering, (c) The amino acid residue contained in CH1 and being the amino acid residue at position 147 of EU numbering, and the amino acid residue contained in CL ​​an amino acid residue at position 164 (EU numbering) (d) an amino acid residue at position 147 (EU numbering) contained in CH1, and a CL an amino acid residue at position 138 according to the EU numbering system; (e) an amino acid residue at position 147 (EU numbering) contained in CH1, and a CL an amino acid residue at position 123 (EU numbering) contained in (f) an amino acid residue at position 175 (EU numbering) contained in CH1, and a CL The amino acid residue at position 160 according to the EU numbering system.

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

[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 use commercially available software to find the desired CH1 or CL. By homology modeling using the above, the amino acid residues (a) to (g) are identified. It is possible to find a site corresponding to the desired sequence and modify the amino acid residue at that site as appropriate. It 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 polypeptide complex, "having the same charge" means, for example, that any of two or more amino acid residues has an amino acid residue included in any one of the above groups (X) or (Y). "Having opposite charges" means, for example, that when at least one of two or more amino acid residues has an amino acid residue included in any one of the above groups (X) or (Y), the remaining amino acid residues have amino acid residues included in different groups.

[0188] Also, the method for producing the above polypeptide complex and the method for controlling association of the present invention for modifying the amino acid residues of the set of amino acid residues shown in the above (a) to (g) so as to be amino acid residues having the same charge are also preferred embodiments of the present invention.

[0189] The amino acid residues to be "modified" in the present invention are not limited to the amino acid residues of the above-described constant region. Those skilled in the art can find amino acid residues forming an interface by homology modeling or the like using commercially available software for polypeptide variants or heteromultimers, and can subject the amino acid residues at the site to modification so as to control association.

[0190] In a technique for introducing a charge repulsion at the interface between the heavy chain variable region and the light chain variable region to suppress the association of an unwanted heavy chain and light chain, examples of the amino acid residues that contact at the interface between the heavy chain variable region (VH) and the light chain variable region (VL) include, for example, position 39 of the FR2 of the heavy chain variable region (for example, WO2006 / 106905 In the amino acid sequence described as SEQ ID NO: 6, glutamine (Q) at position 39 and glutamine (Q) at position 38 of the light chain variable region FR2 that is in contact therewith (for example, position 44 in the amino acid sequence described as SEQ ID NO: 8 in WO2006 / 106905) may be mentioned. Furthermore, leucine (L) at position 45 of the heavy chain variable region FR2 (for example, position 45 in the amino acid sequence described as SEQ ID NO: 6 in WO2006 / 106905) and proline (P) at position 44 of the light chain variable region FR2 that is opposite thereto (for example, position 50 in the amino acid sequence described in SEQ ID NO: 8 in WO2006 / 106905) may be preferably exemplified. Regarding the numbering of these sites, the literature of Kabat et al. (Kabat EA et al. 1991. Sequence of Proteins of Immunological Interest. NIH) is referred to. Since these amino acid residues are known to be highly conserved in humans and mice (J. Mol. Recognit. (2003) 16, 113 - 120), for the association of VH and VL other than the polypeptide complex shown in the examples, the association of the variable regions of the antibody can be controlled by modifying the amino acid residues corresponding to the above amino acid residues.

[0191]

[0192] More specifically, in an antibody containing a heavy chain variable region and a light chain variable region, the antibody may be such that the amino acid residues of (1) and (2), or (3) and (4) below have the same kind of charge; (1) An amino acid residue contained in the heavy chain variable region, which corresponds to position 39 in the EU numbering amino acid residue,​​​​​​​​​​​ (2) An amino acid residue contained in the light chain variable region, corresponding to position 38 in the EU numbering, amino acid residue, (3) An amino acid residue contained in the heavy chain variable region, corresponding to position 45 in the EU numbering, amino acid residue, (4) An amino acid residue contained in the light chain variable region, corresponding to position 44 in the EU numbering, amino acid residue.

[0193] When the respective amino acid residues described in the above (1) and (2), (3) and (4) associate, they are close to each other. Those skilled in the art can find the sites corresponding to the amino acid residues described in the above (1) to (4) by homology modeling or the like using commercially available software for a desired heavy chain variable region or light chain variable region, and can appropriately subject the amino acid residues at these sites to modification.

[0194] In the above antibody, the "amino acid residue having a charge" is preferably selected from amino acid residues contained in any one of the following groups (X) or (Y ); (X) Glutamic acid (E), aspartic acid (D), (Y) Lysine (K), arginine (R), histidine (H).

[0195] The amino acid residues described in the above (1) to (4) are usually each (1) Glutamine (Q), (2) Glutamine (Q), (3) Leucine (L), (4) Proline (P) in humans and mice. Therefore, in a preferred embodiment of the present invention, these amino acid residues are subjected to modification (for example, substitution with charged amino acids). Incidentally, the types of the amino acid residues in the above (1) to (4) are ​​​, and is not necessarily limited to the above amino acid residues, and other amino acids corresponding to the amino acids may be used. For example, as the amino acid corresponding to the 38th position of the EU numbering on the light chain variable region, in the case of human, for example, histidine (H) may be used. Those skilled in the art can know the type of amino acid residue corresponding to that position for any position on the light chain by referring to known literature etc. (for example, J. Mol. Recognit. (2003) 16, 113-120), and can appropriately modify the amino acid residue (for example, substitute with a charged amino acid). In the technique of suppressing the association of unwanted heavy and light chains by modifying the amino acid residues forming the hydrophobic core present at the interface between the heavy chain variable region and the light chain variable region to polar amino acids having a charge, the amino acid residues capable of forming a hydrophobic core at the interface between the heavy chain variable region (VH) and the light chain variable region (VL) include, for example, leucine (L) at the 45th position on the heavy chain variable region and proline (P) at the 44th position on the opposing light chain variable region. Generally, the "hydrophobic core" refers to the part formed by the aggregation of the side chains of hydrophobic amino acids inside the associated polypeptide. Hydrophobic amino acids include, for example, alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, etc. Also, amino acid residues other than hydrophobic amino acids (for example, tyrosine) may be involved in the formation of the hydrophobic core. This hydrophobic core, together with the hydrophilic surface where the side chains of hydrophilic amino acids are exposed to the outside, is a driving force for promoting the association of water-soluble polypeptides. It is not necessarily limited to the above amino acid residues, and other amino acids corresponding to the amino acids may be used. For example, as the amino acid corresponding to the 38th position of the EU numbering on the light chain variable region, in the case of human, for example, histidine (H) may be used. Those skilled in the art can know the type of amino acid residue corresponding to that position for any position on the light chain by referring to known literature etc. (for example, J. Mol. Recognit. (2003) 16, 113-120), and can appropriately modify the amino acid residue (for example, substitute with a charged amino acid). It is not necessarily limited to the above amino acid residues, and other amino acids corresponding to the amino acids may be used. For example, as the amino acid corresponding to the 38th position of the EU numbering on the light chain variable region, in the case of human, for example, histidine (H) may be used. Those skilled in the art can know the type of amino acid residue corresponding to that position for any position on the light chain by referring to known literature etc. (for example, J. Mol. Recognit. (2003) 16, 113-120), and can appropriately modify the amino acid residue (for example, substitute with a charged amino acid). It is not necessarily limited to the above amino acid residues, and other amino acids corresponding to the amino acids may be used. For example, as the amino acid corresponding to the 38th position of the EU numbering on the light chain variable region, in the case of human, for example, histidine (H) may be used. Those skilled in the art can know the type of amino acid residue corresponding to that position for any position on the light chain by referring to known literature etc. (for example, J. Mol. Recognit. (2003) 16, 113-120), and can appropriately modify the amino acid residue (for example, substitute with a charged amino acid).

[0196] In the technique of suppressing the association of unwanted heavy and light chains by modifying the amino acid residues forming the hydrophobic core present at the interface between the heavy chain variable region and the light chain variable region to polar amino acids having a charge, the amino acid residues capable of forming a hydrophobic core at the interface between the heavy chain variable region (VH) and the light chain variable region (VL) include, for example, leucine (L) at the 45th position on the heavy chain variable region and proline (P) at the 44th position on the opposing light chain variable region. In the technique of suppressing the association of unwanted heavy and light chains by modifying the amino acid residues forming the hydrophobic core present at the interface between the heavy chain variable region and the light chain variable region to polar amino acids having a charge, the amino acid residues capable of forming a hydrophobic core at the interface between the heavy chain variable region (VH) and the light chain variable region (VL) include, for example, leucine (L) at the 45th position on the heavy chain variable region and proline (P) at the 44th position on the opposing light chain variable region. In the technique of suppressing the association of unwanted heavy and light chains by modifying the amino acid residues forming the hydrophobic core present at the interface between the heavy chain variable region and the light chain variable region to polar amino acids having a charge, the amino acid residues capable of forming a hydrophobic core at the interface between the heavy chain variable region (VH) and the light chain variable region (VL) include, for example, leucine (L) at the 45th position on the heavy chain variable region and proline (P) at the 44th position on the opposing light chain variable region. In the technique of suppressing the association of unwanted heavy and light chains by modifying the amino acid residues forming the hydrophobic core present at the interface between the heavy chain variable region and the light chain variable region to polar amino acids having a charge, the amino acid residues capable of forming a hydrophobic core at the interface between the heavy chain variable region (VH) and the light chain variable region (VL) include, for example, leucine (L) at the 45th position on the heavy chain variable region and proline (P) at the 44th position on the opposing light chain variable region. In the technique of suppressing the association of unwanted heavy and light chains by modifying the amino acid residues forming the hydrophobic core present at the interface between the heavy chain variable region and the light chain variable region to polar amino acids having a charge, the amino acid residues capable of forming a hydrophobic core at the interface between the heavy chain variable region (VH) and the light chain variable region (VL) include, for example, leucine (L) at the 45th position on the heavy chain variable region and proline (P) at the 44th position on the opposing light chain variable region.

[0197] Generally, the "hydrophobic core" refers to the part formed by the aggregation of the side chains of hydrophobic amino acids inside the associated polypeptide. Hydrophobic amino acids include, for example, alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, etc. Also, amino acid residues other than hydrophobic amino acids (for example, tyrosine) may be involved in the formation of the hydrophobic core. This hydrophobic core, together with the hydrophilic surface where the side chains of hydrophilic amino acids are exposed to the outside, is a driving force for promoting the association of water-soluble polypeptides. Generally, the "hydrophobic core" refers to the part formed by the aggregation of the side chains of hydrophobic amino acids inside the associated polypeptide. Hydrophobic amino acids include, for example, alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, etc. Also, amino acid residues other than hydrophobic amino acids (for example, tyrosine) may be involved in the formation of the hydrophobic core. This hydrophobic core, together with the hydrophilic surface where the side chains of hydrophilic amino acids are exposed to the outside, is a driving force for promoting the association of water-soluble polypeptides. Generally, the "hydrophobic core" refers to the part formed by the aggregation of the side chains of hydrophobic amino acids inside the associated polypeptide. Hydrophobic amino acids include, for example, alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, etc. Also, amino acid residues other than hydrophobic amino acids (for example, tyrosine) may be involved in the formation of the hydrophobic core. This hydrophobic core, together with the hydrophilic surface where the side chains of hydrophilic amino acids are exposed to the outside, is a driving force for promoting the association of water-soluble polypeptides. Generally, the "hydrophobic core" refers to the part formed by the aggregation of the side chains of hydrophobic amino acids inside the associated polypeptide. Hydrophobic amino acids include, for example, alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, etc. Also, amino acid residues other than hydrophobic amino acids (for example, tyrosine) may be involved in the formation of the hydrophobic core. This hydrophobic core, together with the hydrophilic surface where the side chains of hydrophilic amino acids are exposed to the outside, is a driving force for promoting the association of water-soluble polypeptides. Generally, the "hydrophobic core" refers to the part formed by the aggregation of the side chains of hydrophobic amino acids inside the associated polypeptide. Hydrophobic amino acids include, for example, alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, etc. Also, amino acid residues other than hydrophobic amino acids (for example, tyrosine) may be involved in the formation of the hydrophobic core. This hydrophobic core, together with the hydrophilic surface where the side chains of hydrophilic amino acids are exposed to the outside, is a driving force for promoting the association of water-soluble polypeptides. Generally, the "hydrophobic core" refers to the part formed by the aggregation of the side chains of hydrophobic amino acids inside the associated polypeptide. Hydrophobic amino acids include, for example, alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, etc. Also, amino acid residues other than hydrophobic amino acids (for example, tyrosine) may be involved in the formation of the hydrophobic core. This hydrophobic core, together with the hydrophilic surface where the side chains of hydrophilic amino acids are exposed to the outside, is a driving force for promoting the association of water-soluble polypeptides. become. Hydrophobic amino acids in two different domains are present on the molecular surface and are exposed to water molecules and entropy increases and free energy increases. Therefore, the two domains are self- In order to reduce free energy and stabilize, they associate with each other, and the hydrophobic amino acids at the interface are buried inside the molecule and form a hydrophobic core.

[0198] When the polypeptide association occurs, by modifying the hydrophobic amino acids that form the hydrophobic core to polar amino acids with a charge, the formation of the hydrophobic core is inhibited, and as a result, it is considered that the association of the polypeptide is inhibited.

[0199] Other known techniques can be further applied to the polypeptide aggregates of the present invention. For example by substituting the amino acid side chains present in the variable region of one H chain with larger side chains (knob; protrusions), and the relative variable region of the other H chain In addition to the "modification" of the present invention, so that the association of the first VH (VH1) and the first VL (VL1), and / or the second VH (VH2) and the second VL (VL2 is promoted, and the amino acid side chains present in the variable region are substituted with smaller side chains (hole; voids), so that the protrusions can be arranged in the voids to promote the association of VH1 and VL1, and / or VH2 and VL2 and as a result, further suppress the association between polypeptides of VH1 and VL2, and / or VH2 and VL1 (WO1996 / 027011, Ridgway JB et al., Protein Engineering (1996) 9, 617-621, Merchant AM et al. Nature Biotechnology (1998) 16, 677-681). 9, 617-621, Merchant AM et al. Nature Biotechnology (1998) 16, 677-681).

[0200] When producing the above-mentioned polypeptide complex, each domain is directly linked by a peptide bond Alternatively, each domain can be linked by a peptide bond via a peptide linker. In this case, as the linker to be employed, in addition to the linkers exemplified above, for example, a linker having a peptide tag such as His tag, HA tag, myc tag, FLAG tag, etc. can also be appropriately used . Also, the property of binding to each other by hydrogen bond, disulfide bond, covalent bond, ionic interaction or a combination of these bonds can also be suitably utilized. For example, the affinity between CH1 and CL of an antibody is utilized, or an Fc region derived from the aforementioned bispecific antibody is used when the heterologous Fc regions associate . Furthermore, as described in the examples, the disulfide bonds formed between domains can also be suitably utilized . Examples of the polypeptide complex according to the present invention include the embodiments described in FIGS. 17, 19 and 24 . . The polypeptide complex according to the present invention is produced by the same method as the method for producing the recombinant antibody .

[0201] . The present invention also relates to a polynucleotide encoding the polypeptide complex of the present invention .

[0202] . The polypeptide complex according to the present invention can be incorporated into any expression vector. By transforming an appropriate host with the expression vector, an expression cell of the polypeptide complex can be obtained. By culturing the expression cell of the polypeptide complex and recovering the expression product from the culture supernatant, the polypeptide complex encoded by the polynucleotide can be obtained. That is, the present invention relates to a vector containing a polynucleotide encoding the polypeptide complex of the present invention, and the .

[0203] . The present invention also relates to a polynucleotide encoding the polypeptide complex of the present invention . The polypeptide complex of the present invention can be incorporated into any expression vector. By transforming an appropriate host with the expression vector, an expression cell of the polypeptide complex can be obtained. By culturing the expression cell of the polypeptide complex and recovering the expression product from the culture supernatant, the polypeptide complex encoded by the polynucleotide can be obtained. That is, the present invention relates to a vector containing a polynucleotide encoding the polypeptide complex of the present invention, and the . By culturing the expression cell of the polypeptide complex and recovering the expression product from the culture supernatant, the polypeptide complex encoded by the polynucleotide can be obtained. That is, the present invention relates to a vector containing a polynucleotide encoding the polypeptide complex of the present invention, and the . By culturing the expression cell of the polypeptide complex and recovering the expression product from the culture supernatant, the polypeptide complex encoded by the polynucleotide can be obtained. That is, the present invention relates to a vector containing a polynucleotide encoding the polypeptide complex of the present invention, and the . That is, the present invention relates to a vector containing a polynucleotide encoding the polypeptide complex of the present invention, and the . That is, the present invention relates to a vector containing a polynucleotide encoding the polypeptide complex of the present invention, and the A method for producing a polypeptide complex, comprising culturing a cell that retains a vector and recovering the polypeptide complex from the culture supernatant. These can be obtained, for example, by the same method as the above recombinant antibody.

[0204] pharmaceutical composition From another aspect, the present invention provides a pharmaceutical composition containing, as an active ingredient, a polypeptide complex comprising (1) an antigen-binding domain, (2) a domain containing an Fc region with reduced binding activity to an Fcγ receptor, and (3) a CD3-binding domain. Further, the present invention relates to a therapeutic agent for inducing cytotoxicity (cytotoxicity-inducing therapeutic agent), a cell proliferation inhibitor, and an anticancer agent containing the complex as an active ingredient. The pharmaceutical composition of the present invention can also be used as a cancer therapeutic agent or a cancer preventive agent. The cytotoxicity-inducing therapeutic agent, cell proliferation inhibitor, and anticancer agent of the present invention are preferably administered to a subject suffering from cancer or a subject at risk of recurrence.

[0205] Also, 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) A CD3-binding domain, A method for preventing or treating cancer, which comprises administering the polypeptide complex to a subject, or the use of the polypeptide complex in the production of a cytotoxicity-inducing therapeutic agent, a cell proliferation inhibitor, and an anticancer agent, can also be expressed as a cytotoxicity-inducing therapeutic agent, a cell proliferation inhibitor, and an anticancer agent containing, as an active ingredient, a polypeptide complex comprising the above.

[0206] In the present invention, "(1) an antigen-binding domain, (2) a domain containing an Fc region with reduced binding activity to an Fcγ receptor,​​​​​​​​​​​ A polypeptide complex comprising: (1) a domain containing an Fc region, and (3) a CD3 binding domain "Containing the polypeptide complex as an active ingredient" means containing the polypeptide complex as a main active ingredient, and does not limit the content rate of the polypeptide complex.

[0207] Furthermore, in the pharmaceutical composition, cell injury-inducing therapeutic agent, cell growth inhibitor, and anticancer agent of the present invention, a plurality of types of polypeptide complexes may be blended as necessary. For example, by making a cocktail of a plurality of polypeptide complexes of the present invention that bind to the same antigen, there is a possibility of enhancing the cytotoxic effect on cells expressing the antigen. Alternatively, in addition to the polypeptide complex of the present invention containing an antigen-binding domain for one antigen, by blending a polypeptide complex of the present invention containing an antigen-binding domain that binds to another cancer antigen, the therapeutic effect can be enhanced. In addition, if necessary, the polypeptide complex of the present invention may be encapsulated in microcapsules (such as microcapsules of hydroxymethylcellulose, gelatin, poly [methyl methacrylic acid], etc.),

[0208] and may be made into a colloidal drug delivery system (such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) ("Remington’s Pharmaceutical Science 16 edition", Oslo Ed. (1980), etc.). Furthermore, a method of making the drug into a sustained-release drug is also known, and the method can be applied to the polypeptide complex of the present invention (J. Biomed.Mater.Res. (1981) 15, 267-277, Chemtech. (1982) 12, 98-105, U.S. Patent No. 377 edition", Oslo Ed. (1980), etc.). Furthermore, a method of making the drug into a sustained-release drug th edition", Oslo Ed. (1980), etc.). Furthermore, a method of making the drug into a sustained-release drug is also known, and the method can be applied to the polypeptide complex of the present invention (J. Biomed.Mater.Res. (1981) 15, 267-277, Chemtech. (1982) 12, 98-105, U.S. Patent No. 377 No. 3719, European Patent Publication No. EP58481 / EP133988, Biopolymers (1983) 22, 547-556) .

[0209] The pharmaceutical composition, cell proliferation inhibitor and anticancer agent of the present invention may be administered orally or parenterally. The compound can be administered to a patient by any of the following methods, preferably parenteral. Specific examples of the administration method include injection, intranasal 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 and anticancer drugs can be administered systemically or locally. The administration method can be appropriately selected depending on the patient. The dosage is, for example, about 100 mg / kg per administration. The dosage can be selected in the range of 0.0001 mg to 1000 mg per kg of patient weight. The dosage may 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 in accordance with conventional methods (for example, Remington's Pharmaceutical armaceutical Science, latest edition, Mark Publishing Company, Easton, USA), The composition may also contain pharma- ceutically acceptable carriers and additives. For example, surfactants, excipients, etc. Vehicle, coloring agent, flavoring agent, preservative, stabilizer, buffer, suspending agent, isotonicity agent, binder, disintegrating agent, Lubricants, flow enhancers, flavoring agents, etc. are also included, but are not limited to these. A carrier can be used as appropriate. Specifically, light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinyl pyrrolidone, gelatin, medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, etc. can be mentioned as carriers.

[0211] In addition, the present invention provides a method for causing damage to cancer antigen-expressing cells or a method for suppressing cell proliferation by contacting a cell expressing a certain cancer antigen with the polypeptide aggregate of the present invention that binds to the cancer antigen. The monoclonal antibody that binds to the cancer antigen is the polypeptide aggregate of the present invention that binds to the cancer antigen contained in the cell damage-inducing therapeutic agent, cell proliferation inhibitor, and anticancer agent of the present invention as described above. The cells to which the polypeptide aggregate of the present invention that binds to the cancer antigen binds are not particularly limited as long as they are cells expressing the cancer antigen. Preferred cancer antigen-expressing cells in the present invention are specifically ovarian cancer, prostate cancer, breast cancer, uterine cancer, liver cancer, lung cancer, pancreatic cancer, gastric cancer, bladder cancer, and colorectal cancer cells, etc. are preferably mentioned. When the cancer antigen is GPC3, there is no limitation as long as it is a cancer cell expressing GPC3, but hepatocellular carcinoma, lung cancer, ovarian cancer, etc. are preferably mentioned as suitable cancer cells. In the present invention, "contact" is carried out, for example, by adding the polypeptide aggregate of the present invention that binds to the cancer antigen to the culture solution of cancer antigen-expressing cells cultured in a test tube. In the present invention, "contact" is carried out, for example, by adding the polypeptide aggregate of the present invention that binds to the cancer antigen to the culture solution of cancer antigen-expressing cells cultured in a test tube. In the present invention, "contact" is carried out, for example, by adding the polypeptide aggregate of the present invention that binds to the cancer antigen to the culture solution of cancer antigen-expressing cells cultured in a test tube. In the present invention, "contact" is carried out, for example, by adding the polypeptide aggregate of the present invention that binds to the cancer antigen to the culture solution of cancer antigen-expressing cells cultured in a test tube. In the present invention, "contact" is carried out, for example, by adding the polypeptide aggregate of the present invention that binds to the cancer antigen to the culture solution of cancer antigen-expressing cells cultured in a test tube.

[0212] In the present invention, "contact" is carried out, for example, by adding the polypeptide aggregate of the present invention that binds to the cancer antigen to the culture solution of cancer antigen-expressing cells cultured in a test tube. In the present invention, "contact" is carried out, for example, by adding the polypeptide aggregate of the present invention that binds to the cancer antigen to the culture solution of cancer antigen-expressing cells cultured in a test tube. In this case, as the shape of the added polypeptide aggregate, shapes such as a solution or a solid obtained by lyophilization etc. can be appropriately used. When added as an aqueous solution it can be an aqueous solution containing purely only the polypeptide aggregate of the present invention, or for example, a solution containing the above-described surfactant, excipient, coloring agent, flavoring agent, preservative, stabilizer, buffer, suspending agent, isotonic agent , binder, disintegrant, lubricant, fluidity promoter, flavoring agent, etc. The concentration to be added is not particularly limited, but as the final concentration in the culture solution, it is preferably in the range of 1 pg / ml to 1 g / ml , more preferably in the range of 1 ng / ml to 1 mg / ml, and even more preferably 1 μg / ml to 1 mg / ml can be suitably used.

[0213] In the present invention, "contact" further, in another aspect, is also carried out by transplanting cancer antigen-expressing cells into the body of a non-human animal or an animal having cancer cells that inherently express the cancer antigen, and administering thereto. The administration method can be carried out by either oral or parenteral administration. Particularly preferred is the administration method by parenteral administration, and specific examples of such administration methods include injection administration, nasal administration, pulmonary administration, transdermal administration, etc. Examples of injection administration include, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc. For example, by injection administration, the pharmaceutical composition of the present invention, or a cytocidal induction therapeutic agent, a cell growth inhibitor, and an anticancer agent can be administered systemically or locally. Also, the administration method can be appropriately selected according to the age and symptoms of the test animal. When administered as an aqueous solution, it may be an aqueous solution containing purely only the polypeptide aggregate of the present invention, or for example, a solution containing the above-described surfactant, excipient, coloring agent, flavoring agent, etc. It may be a solution containing spices, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, fluidity promoters, flavor correctors, etc. As the dosage, for example, the dosage can be selected in the range of 0.0001 mg to 1000 mg per 1 kg of body weight per single administration. Alternatively, for example, the dosage can be selected in the range of 0.001 to 100000 mg / body per patient. However, the dosage of the polypeptide aggregate of the present invention is not limited to these dosages. It may be a solution containing spices, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, fluidity promoters, flavor correctors, etc. As the dosage, for example, the dosage can be selected in the range of 0.0001 mg to 1000 mg per 1 kg of body weight per single administration. Alternatively, for example , the dosage can be selected in the range of 0.001 to 100000 mg / body per patient. However, the dosage of the polypeptide aggregate of the present invention is not limited to these dosages.

[0214] As a method for evaluating or measuring the cytotoxicity caused in cells expressing an antigen to which the antigen-binding domain constituting the polypeptide aggregate of the present invention binds by contact with the polypeptide aggregate of the present invention, the following method is preferably used. As a method for evaluating or measuring the cytotoxic activity in vitro, measurement methods such as cytotoxic T cell activity can be mentioned. Whether the polypeptide aggregate of the present invention has T cell-mediated cytotoxic activity can be measured by a known method (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 the activity, a polypeptide aggregate that binds to an antigen different from the antigen to which the antigen-binding domain of the present invention binds and that is not expressed by the cells used in the test is used as a control in the same manner as the polypeptide aggregate of the present invention, and the polypeptide aggregate of the present invention shows stronger cytotoxic activity than the polypeptide aggregate used as a control, whereby the activity can be determined. (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 the activity, a polypeptide aggregate that binds to an antigen different from the antigen to which the antigen-binding domain of the present invention binds and that is not expressed by the cells used in the test is used as a control in the same manner as the polypeptide aggregate of the present invention, and the polypeptide aggregate of the present invention shows stronger cytotoxic activity than the polypeptide aggregate used as a control, whereby the activity can be determined. shows stronger cytotoxic activity than the polypeptide aggregate used as a control, whereby the activity can be determined.

[0215] ​ In addition, in order to evaluate or measure cytotoxic activity in vivo, for example, the polypeptide of the present invention A cell expressing an antigen to which the antigen-binding domain constituting the conjugate binds is used as a non-human test animal After transplantation into the dermis or subcutaneous tissue, the test polypeptide conjugate is administered daily or at intervals of several days starting from the same day or the next day It is administered intravenously or intraperitoneally. By measuring the size of the tumor over time, the difference in the change in the size of the tumor Can be defined as cytotoxic activity. Similar to the evaluation in vitro, a control The polypeptide conjugate is administered, and the size of the tumor in the administration group of the polypeptide conjugate of the present invention Is significantly smaller than the size of the tumor in the administration group of the control polypeptide conjugate It can be determined to have cytotoxic activity.

[0216] Regarding the suppression effect on the growth of cells expressing the antigen to which the antigen-binding domain constituting the polypeptide conjugate of the present invention binds due to the contact with the polypeptide conjugate of the present invention As a method for evaluating or measuring, measurement of the uptake of isotope-labeled thymidine into cells and the MTT method are preferably used Moreover, as a method for evaluating or measuring cell growth inhibitory activity in vivo, the same method as the method for evaluating or measuring cytotoxic activity in vivo described above can be preferably used 。 。

[0217] The present invention also provides a kit for use in the method of the present invention, which contains the polypeptide conjugate of the present invention or the polypeptide conjugate produced by the production method of the present invention In addition, the kit may be packaged with other pharmaceutically acceptable carriers, media, instructions describing the method of use, etc. 。 The present invention also relates to the polypeptide conjugate of the present invention or the present invention for use in the method of the present invention Relates to a polypeptide aggregate produced by the production method of the invention.

[0218] All prior art documents cited in this specification are incorporated herein by reference. Included.

Examples

[0219] The present invention will be described in more detail below with reference to examples, but these examples do not limit the scope of the present invention. Not limited.

[0220] 〔Example 1〕Preparation and examination of GPC3 ERY2 (1) Overview As a method for extending the blood half-life of a protein administered in vivo, a method of adding the Fc domain of an antibody to the target protein and utilizing the recycling function via FcRn is well known. However, at this time, when natural Fc is added to BiTE, one molecule binds to T cells via the anti-CD3 scFv of the BiTE part, and at the same time, binds to FcgR (Fcγ receptor) on the cell membranes of cells such as NK cells and macrophages via the Fc part, which may activate these cells by cross-linking them in a cancer antigen-independent manner and lead to the induction of various cytokines. Therefore, a molecule, ERY2, was prepared by linking a silent Fc region with reduced binding activity to the Fcγ receptor via a polypeptide linker to BiTE, and its activity was verified by comparison with BiTE. The scFv of an antibody against Glypican 3 (GPC3), a GPI-anchored protein known to be highly expressed in liver cancer cells, and the scFv of an antibody against CD3 epsilon were linked by a short peptide linker to obtain BiTE against GPC3. 3 scFv binds to T cells via the anti-CD 3 scFv, and at the same time binds to FcgR (Fcγ receptor) on the cell membranes of cells such as NK cells and macrophages via the Fc part, which may activate these cells by cross-linking them in a cancer antigen-independent manner and lead to the induction of various cytokines. Therefore, a molecule, ERY2, was prepared by linking a silent Fc region with reduced binding activity to the Fcγ receptor via a polypeptide linker to BiTE, and its activity was verified by comparison with BiTE. The scFv of an antibody against Glypican 3 (GPC3), a GPI-anchored protein known to be highly expressed in liver cancer cells, and the scFv of an antibody against CD3 epsilon were linked by a short peptide linker to obtain BiTE against GPC3. Therefore, a molecule, ERY2, was prepared by linking a silent Fc region with reduced binding activity to the Fcγ receptor via a polypeptide linker to BiTE, and its activity was verified by comparison with BiTE. The scFv of an antibody against Glypican 3 (GPC3), a GPI-anchored protein known to be highly expressed in liver cancer cells, and the scFv of an antibody against CD3 epsilon were linked by a short peptide linker to obtain BiTE against GPC3. Therefore, a molecule, ERY2, was prepared by linking a silent Fc region with reduced binding activity to the Fcγ receptor via a polypeptide linker to BiTE, and its activity was verified by comparison with BiTE. The scFv of an antibody against Glypican 3 (GPC3), a GPI-anchored protein known to be highly expressed in liver cancer cells, and the scFv of an antibody against CD3 epsilon were linked by a short peptide linker to obtain BiTE against GPC3. Therefore, a molecule, ERY2, was prepared by linking a silent Fc region with reduced binding activity to the Fcγ receptor via a polypeptide linker to BiTE, and its activity was verified by comparison with BiTE. The scFv of an antibody against Glypican 3 (GPC3), a GPI-anchored protein known to be highly expressed in liver cancer cells, and the scFv of an antibody against CD3 epsilon were linked by a short peptide linker to obtain BiTE against GPC3. The scFv of an antibody against Glypican 3 (GPC3), a GPI-anchored protein known to be highly expressed in liver cancer cells, and the scFv of an antibody against CD3 epsilon were linked by a short peptide linker to obtain BiTE against GPC3. By linking the scFv of an antibody against Glypican 3 (GPC3), a GPI-anchored protein known to be highly expressed in liver cancer cells, and the scFv of an antibody against CD3 epsilon with a short peptide linker, BiTE against GPC3 was obtained. (GPC3 BiTE) was prepared (Figure 17A). Subsequently, GPC3 with a silent type Fc linked thereto ERY2 against GPC3 (GPC3 ERY2) was prepared (Figure 17C). Also, as a comparison, a normal IgG-type anti-G PC3 antibody was prepared. At this time, the IgG-type anti-GPC3 antibody is known to have enhanced ADCC activity An antibody with reduced fucose content in the sugar chain portion, that is, a low-fucose type antibody was prepared as.

[0221] (2) Preparation of GPC3 BiTE By using the expression vector of the anti-GPC3 antibody as a template, the H chain variable region (anti-GPC3 VH) and the cDNA encoding the L chain variable region (anti-GPC3 VL) were each obtained. Using primers with appropriate sequences and the cDNA as a template, by PCR method, anti-GPC3 VH and anti-GPC3 VL are linked by a linker consisting of a sequence repeating Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 7) three times, and a cDNA fragment encoding anti-GPC3 sc Fv having the amino acid sequence was prepared.

[0222] Also, a series of oligonucleotides having a nucleotide sequence encoding the partial sequences of the H chain variable region (M12 VH) and the L chain variable region (M12 VL) of the anti-CD3 antibody (M12), and having a complementary sequence at its terminal sequence were prepared. By polymerase reaction, these series of oligonucleotides are linked through their complementary sequence portions, and are designed to synthesize polynucleotides corresponding to the H chain variable region (M12 VH) and the L chain variable region (M12 VL). The oligonucleotide After mixing the oligonucleotides, the oligonucleotides are ligated by PCR to produce the individual variable regions. Two cDNAs encoding the amino acid sequences of were obtained. Using these cDNAs as templates, PCR was performed to confirm that M12 VL and M12 VH 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 M12 scFv having the amino acid sequence was generated.

[0223] Next, primers with appropriate sequences and anti-GPC3 scFv and M12 scFv were encoded. Anti-GPC3 scFv and M12 scFv were synthesized by PCR using the cDNA fragment encoding the Gly·Gl The linker consists 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 ends as described in SEQ ID NO:33). A cDNA fragment encoding the amino acid sequence (excluding amino acids) was generated.

[0224] The primers were prepared by adding the appropriate sequence and the amino-terminal 19 amino acid sequence described in SEQ ID NO:33. The cDNA fragment encoding the amino acid sequence excluding the amino acid was used as a template for PCR. On the 5' side of the A fragment, there is a base sequence encoding an EcoRI cleavage sequence, a kozac sequence, and a secretion signal sequence. A cDNA fragment was created in which a Not I cleavage sequence was added to the 3' end of the cDNA. The NA fragment was digested with EcoRI and NotI and inserted into a mammalian cell expression vector. , 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 gene encoding the ribozyme (not included in the above) was obtained.

[0225] When the vector was introduced into the CHO cell DG44 strain by electroporation . By culturing the cells introduced with the gene in the presence of 1 mg / mL Geneticine after limiting dilution , a drug-resistant cell line was isolated. By performing Western blot analysis on the culture supernatant of the obtained cell line using an antibody against the His tag , a cell line expressing GPC3 BiTE was selected.

[0226] The culture supernatant obtained by culturing the above cell line in large quantities was added to an SP Sepharose FF column (GE Healthcare). After washing the column, the fraction containing GPC3 BiTE was eluted by a concentration gradient of NaCl . Further, the fraction was added to a HisTrap HP column (GE Healthcare) . After washing the column, the fraction containing GPC3 BiTE was eluted by a concentration gradient of imidazole . After the fraction was concentrated with an ultrafiltration membrane, the concentrated solution was added to a Superdex 200 column (GE Healthcare). By recovering only the monomeric GPC3 BiTE fraction , purified GPC3 BiTE was obtained.

[0227] (3) Preparation of GPC3 ERY2 Using a PCR method with primers to which appropriate sequences were added in the same manner as the method described above, and methods using the QuikC hange Site-Directed Mutagenesis Kit (Stratagene), etc., known methods for those skilled in the art , GPC3 ERY2_Hk (SEQ ID NO: 34, the 19 amino acids at the amino terminus which are the signal sequence are not included in the mature sequence), and GPC3 ERY2_Hh (SEQ ID NO: 35, the signal ​ The polynucleotides encoding the amino-terminal 19 amino acids, which are the signal sequences and are not included in the mature sequence, were inserted respectively. An expression vector was prepared by inserting the polynucleotides encoding the amino-terminal 19 amino acids, which are the signal sequences and are not included in the mature sequence, respectively.

[0228] These expression vectors were co-introduced into FreeStyle293-F cells (Invitrogen), and GPC3 ERY2 was transiently expressed. The obtained culture supernatant was added 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 added to a HisTrap HP column (GE Healthcare). After washing the column, elution was performed with a concentration gradient of imidazole. The fraction containing GPC3 ERY2 was added to a HisTrap HP column (GE Healthcare). After washing the column, elution was performed with a concentration gradient of imidazole. After washing the column, elution was performed with a concentration gradient of imidazole. The fraction containing GPC3 ERY2 was concentrated using an ultrafiltration membrane, and then the concentrated solution was added to a Superdex 200 column (GE Healthcare). After washing the column, elution was performed with a concentration gradient of imidazole. The fraction containing GPC3 ERY2 was concentrated using an ultrafiltration membrane, and then the concentrated solution was added to a Superdex 200 column (GE Healthcare). Purified GPC3 ERY2 was obtained by collecting only the monomeric GPC3 ERY2 fraction of the eluate. Purified GPC3 ERY2 was obtained by collecting only the monomeric GPC3 ERY2 fraction of the eluate.

[0229] (4) Preparation of afucosylated anti-GPC3 antibody The expression vector of the anti-GPC3 antibody (described as the humanized GC33 antibody in WO2006 / 006693) was introduced into GDP-fucose knockout CHO cell line DXB11 (Cancer Sci. (2010) 101(10), 2227-33) by electroporation. The expression vector of the anti-GPC3 antibody (described as the humanized GC33 antibody in WO2006 / 006693) was introduced into GDP-fucose knockout CHO cell line DXB11 (Cancer Sci. (2010) 101(10), 2227-33) by electroporation. After limiting dilution, drug-resistant strains were selected by culturing in the presence of 0.5 mg / mL Geneticin, and afucosylated anti-GPC3 antibody-expressing strains were obtained. After limiting dilution, drug-resistant strains were selected by culturing in the presence of 0.5 mg / mL Geneticin, and afucosylated anti-GPC3 antibody-expressing strains were obtained. From the culture supernatant prepared by culturing these cells, Hitrap (R) ProteinA (Phar An antibody fraction was prepared by ordinary affinity purification using (Macia). Next, the antibody fraction was subjected to gel filtration purification using Superdex 200 26 / 60 (Pharmacia), and a low-fucose type GPC3 antibody was obtained by fractionating the molecular weight fraction of the eluate. The antibody fraction was subjected to gel filtration purification using Superdex 200 26 / 60 (Pharmacia), and a low-fucose type GPC3 antibody was obtained by fractionating the molecular weight fraction of the eluate. The antibody fraction was subjected to gel filtration purification using Superdex 200 26 / 60 (Pharmacia), and a low-fucose type GPC3 antibody was obtained by fractionating the molecular weight fraction of the eluate.

[0230] (5) Measurement of cytotoxic activity using human peripheral blood mononuclear cells (5-1) Preparation of human peripheral blood mononuclear cell (PBMC) solution Using a syringe pre-injected with 1,000 units / mL of heparin solution (Novolin Heparin Injection 5,000 units, Novo Nordisk), 50 mL of peripheral blood was collected from healthy adult volunteers. After diluting it 2-fold with PBS(-), the quartered peripheral blood was added to a Leucosep lymphocyte separation tube (Cat. No. 227290, Greiner bio-one) pre-injected with 15 mL of Ficoll-Paque PLUS and centrifuged. After centrifuging the separation tube (2,150 rpm, 10 minutes, room temperature), the mononuclear cell fraction layer was collected. The cells of the mononuclear cell fraction were washed once with Dulbecco's Modified Eagle's Medium containing 10% FBS (SIGMA, hereinafter 10% FBS / D-MEM), and then the cells were adjusted to a cell density of 4×10 / mL using 10% FBS / D-MEM. The cell solution thus prepared was used as the human PBMC solution in subsequent tests. Using a syringe pre-injected with 1,000 units / mL of heparin solution (Novolin Heparin Injection 5,000 units, Novo Nordisk), 50 mL of peripheral blood was collected from healthy adult volunteers. After diluting it 2-fold with PBS(-), the quartered peripheral blood was added to a Leucosep lymphocyte separation tube (Cat. No. 227290, Greiner bio-one) pre-injected with 15 mL of Ficoll-Paque PLUS and centrifuged. After centrifuging the separation tube (2,150 rpm, 10 minutes, room temperature), the mononuclear cell fraction layer was collected. The cells of the mononuclear cell fraction were washed once with Dulbecco's Modified Eagle's Medium containing 10% FBS (SIGMA, hereinafter 10% FBS / D-MEM), and then the cells were adjusted to a cell density of 4×10 / mL using 10% FBS / D-MEM. The cell solution thus prepared was used as the human PBMC solution in subsequent tests. Using a syringe pre-injected with 1,000 units / mL of heparin solution (Novolin Heparin Injection 5,000 units, Novo Nordisk), 50 mL of peripheral blood was collected from healthy adult volunteers. After diluting it 2-fold with PBS(-), the quartered peripheral blood was added to a Leucosep lymphocyte separation tube (Cat. No. 227290, Greiner bio-one) pre-injected with 15 mL of Ficoll-Paque PLUS and centrifuged. After centrifuging the separation tube (2,150 rpm, 10 minutes, room temperature), the mononuclear cell fraction layer was collected. The cells of the mononuclear cell fraction were washed once with Dulbecco's Modified Eagle's Medium containing 10% FBS (SIGMA, hereinafter 10% FBS / D-MEM), and then the cells were adjusted to a cell density of 4×10 / mL using 10% FBS / D-MEM. The cell solution thus prepared was used as the human PBMC solution in subsequent tests. Using a syringe pre-injected with 1,000 units / mL of heparin solution (Novolin Heparin Injection 5,000 units, Novo Nordisk), 50 mL of peripheral blood was collected from healthy adult volunteers. After diluting it 2-fold with PBS(-), the quartered peripheral blood was added to a Leucosep lymphocyte separation tube (Cat. No. 227290, Greiner bio-one) pre-injected with 15 mL of Ficoll-Paque PLUS and centrifuged. After centrifuging the separation tube (2,150 rpm, 10 minutes, room temperature), the mononuclear cell fraction layer was collected. The cells of the mononuclear cell fraction were washed once with Dulbecco's Modified Eagle's Medium containing 10% FBS (SIGMA, hereinafter 10% FBS / D-MEM), and then the cells were adjusted to a cell density of 4×10 / mL using 10% FBS / D-MEM. The cell solution thus prepared was used as the human PBMC solution in subsequent tests. Using a syringe pre-injected with 1,000 units / mL of heparin solution (Novolin Heparin Injection 5,000 units, Novo Nordisk), 50 mL of peripheral blood was collected from healthy adult volunteers. After diluting it 2-fold with PBS(-), the quartered peripheral blood was added to a Leucosep lymphocyte separation tube (Cat. No. 227290, Greiner bio-one) pre-injected with 15 mL of Ficoll-Paque PLUS and centrifuged. After centrifuging the separation tube (2,150 rpm, 10 minutes, room temperature), the mononuclear cell fraction layer was collected. The cells of the mononuclear cell fr...

Claims

1. The following domains: (1) an antigen-binding domain, (2) a domain including an Fc region having reduced binding activity to an Fcγ receptor; and (3) a 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 assembly.

4. 4. The method according to 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 constituting the heavy chain constant region of the domain having the structure F(ab')2 are Fc domains. The polypeptide polypeptide of claim 5, which is linked to each of the two polypeptides constituting the domain. 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 that constitutes the CD3 binding domain is the CH1 domain of an antibody, and the light chain Fv fragment is the antibody The polypeptide complex of claim 8, wherein the CL domain of

10. The method 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. 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 according to 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 according to 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. A monovalent scFv is a single polypeptide that constitutes 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 the CH1 domain to form an Fc domain. and the light chain Fv fragment of said 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. A monovalent scFv is a single molecule that constitutes an Fc region via a heavy chain Fv fragment that constitutes a CD3-binding domain. The other monovalent scFv binds to the Fc polypeptide via a 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. A monovalent scFv is a single polypeptide that constitutes the Fc region via the scFv that constitutes the CD3 binding domain. and the other monovalent scFv is 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 a 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 a CL region, and the light chain Fv fragment of the Fab is linked to a T cell receptor region. The heavy chain Fv fragment of Fab, which constitutes the antibody-binding domain, binds to the other fragment constituting the Fc domain via the CH1 domain.

23. The method of claim 22, wherein the light chain Fv fragment of the Fab is linked to a CL domain of the polypeptide. 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 a CL region, and the light chain Fv fragment of the Fab is linked to a T cell receptor region. The light chain Fv fragment of Fab, which constitutes the Fab-binding domain, binds to the other fragment constituting 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 CL domain of the polypeptide. 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 a CL region, and the light chain Fv fragment of the Fab is linked to a T cell receptor region. The heavy chain Fv fragment of Fab, which constitutes the antibody-binding domain, binds to the other polypeptide, 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 CH1 domain. 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 domain via the CH1 domain. and the light chain Fv fragment of said 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 domain via the CH1 domain. and the light chain Fv fragment of said Fab is linked to a CL region, The heavy chain Fv fragment of Fab constituting the antigen-binding domain is bound to the other constituting 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. an original binding domain; and (2) A monovalent Fab heavy chain Fv fragment that binds to the T cell receptor complex is fused to the Fc 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; wherein the heavy chain Fv fragment and the antigen-binding domain are A light chain Fv fragment in the cytoplasm or a heavy chain Fv fragment and a T cell receptor binding domain in the cytoplasm The charges of the CH1 region and the CL region are controlled so that the light chain Fv fragment in the antibody associates 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 of 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 identical 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 have 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 amino acid sequence and the amino acid sequence are heterogeneous.

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 claims 3 to 4.

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 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 an amino acid residue at position 147 (EU numbering) in the CL region which is the amino acid residue at position 131 according to the EU numbering system (c) an amino acid residue at position 147 (EU numbering) in the CH1 region, and a CL region which is the amino acid residue at position 164 according to the EU numbering system (d) an amino acid residue at position 147 (EU numbering) in the CH1 region, and a CL region which is the amino acid residue at position 138 according to the EU numbering system (e) an amino acid residue at position 147 (EU numbering) in the CH1 region, and a CL region which is the amino acid residue at position 123 according to the EU numbering system (f) an amino acid residue at position 175 (EU numbering) in the CH1 region, and a CL region which is the amino acid residue at position 160 according to 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 set of amino acid residues shown in (g) below. The polypeptide complex of 6. (g) an amino acid residue at position 213 (EU numbering) in the CH1 region, and a CL region which is the amino acid residue at position 123 according to the EU numbering system

38. The amino acid residues having different electric 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 residue having a different charge is an amino acid residue in the CH1 region, and is represented by the EU numbering The amino acid residue at position 175 in the CL domain 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 above.

40. The amino acid residue having a different charge is an amino acid residue in the CH1 region, and is represented by the EU numbering The amino acid residues at positions 147 and 175 of the nucleotide sequence are Glu and CL domain amino acid residues according to EU numbering. 36 to 37, wherein the amino acid residues at positions 180, 131 and 160 are all Lys. 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 a reduced binding activity to a receptor. Polypeptide complex.

43. The Fc region is an Fc region of SEQ ID NO: 23, an Fc region of SEQ ID NO: 24, an Fc region of SEQ ID NO: SEQ ID NO: 25 or the amino acid constituting the Fc region of SEQ ID NO: 26 is mutated. A polypeptide according to any one of claims 1 to 42, characterized in that it is an Fc region Do-aggregate.

44. Any of the following amino acids that compose the Fc region, as specified according to the EU numbering system: amino acid; The amino acid sequence of positions 118 to 260 is the sequence set forth in SEQ ID NO: 24; 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 assembly.

45. Any of the following amino acids that compose the Fc region, as specified according to the EU numbering system: 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 positions 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 compose the Fc region, as specified according to the EU numbering system: amino acid; 233rd, 234th, 235th, 236th, 237th, 327th, 330th, 331st, is replaced by the corresponding amino acid 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 the EU numbering system: 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 association 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. Among the amino acid residues of one of the two polypeptides constituting the Fc region, The amino acid at position 349 is cysteine ​​and the amino acid at position 366 is tripeptide. The amino acid residues of the other polypeptide are identified according to the EU numbering system. The 356th amino acid is cysteine, the 366th amino acid is serine, and the 368th amino acid is aryl.

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

52. Among the amino acid residues of one of the two polypeptides constituting the Fc region, The amino acid at position 356, specified according to the numbering, is a lysine, and the amino acid of the other polypeptide is an amino acid. The amino acid residue at position 439, as specified by the EU numbering system, 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. 54. 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 4.

58. Different epitopes are present 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 are present 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. A vector comprising the polynucleotide of claim 60.

62. A cell carrying the vector described in claim 61.

63. The method comprises 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 cell-injured patient a polypeptide complex of any one of claims 1 to 59 as an active ingredient. Harm-inducing therapeutic agents.

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

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

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