Cell damage-inducing therapeutic agent

Abstract

A polypeptide complex showing cytotoxic activity against target cancer cells by T cells, the method for producing the polypeptide complex, and a cytotoxicity-inducing therapeutic agent containing the polypeptide complex as an active ingredient are provided. A pharmaceutical composition for treating or preventing various cancers containing the cytotoxicity-inducing therapeutic agent as an active ingredient or a treatment method using the pharmaceutical composition are provided. 【Solution means】(1) An antigen-binding domain, (2) a domain containing an Fc region with reduced binding activity to Fcγ receptor, and (3) a T cell receptor complex-binding domain, a polypeptide complex. A cell retaining a vector containing a polynucleotide encoding the polypeptide complex. A method for producing a polypeptide complex, which includes culturing the cell and recovering the polypeptide complex from the culture supernatant. A cytotoxicity-inducing therapeutic agent containing the polypeptide complex as an active ingredient, and a treatment method, which is characterized by administering to a patient in need of treatment. ​​​​​​

Description

[Technical Field]

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

[0002] To date, several therapeutic antibodies that have demonstrated excellent antitumor effects have been developed into pharmaceuticals for cancer treatment. These therapeutic antibodies target the signals required for cancer cell proliferation. signal inhibition, induction of cell death signals, or ADCC (Antibody Dependent Cell-mediated ed Cytotoxicity;antibody-dependent cytotoxicity), CDC(Complement Dependent Cytotoxicity; It is known that it exerts an antitumor effect on cancer cells through complement-dependent cytotoxicity. The Fc region of an antibody binds to effector cells such as NK cells and macrophages (Non-Patent Document 2). By binding to the Fc receptors present in the target cancer cells, the antibody binds to The cytotoxicity exerted by these effector cells is called ADCC. The complement complex binds to the binding site. The complement complex exists on the cell membrane of the cell to which the antibody binds. The complement components present in the cells form pores, which promote the inflow of water and ions into the cells. CDC is the cell damage that occurs when the nuclei are destroyed. Existing therapeutic antibodies have been shown to have excellent effects. However, the therapeutic outcomes 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 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 an antigen on the 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. 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 that contains a Fab that binds to a cancer antigen and a Fab that binds to the CD3 epsilon chain in each 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 including antibodies that bind to 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 an antigen on the 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. 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 that contains a Fab that binds to a cancer antigen and a Fab that binds to the CD3 epsilon chain in each 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

[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 that contains a Fab that binds to a cancer antigen and a Fab that binds to the CD3 epsilon chain in each 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 including antibodies that bind to 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 an antigen on the target cancer cells. 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. 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 with 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 that of various previously known TR antibodies (Non-Patent Documents 9 and 10). That is, BiTE exerts an antitumor effect at significantly lower concentrations 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, CD28 agonist antibodies, 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 medicinal effects clinically and has been approved as a therapeutic agent, and multiple BiTEs including blinatumomab exhibit strong antitumor effects, it is suggested that TR antibodies that recruit T cells as effector cells have extremely high potential as antitumor drugs compared to antibodies with the usual 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 very low dose of 5 μg / body was the maximum tolerated dose, and administration of higher doses has been 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 does not have a binding site for Fcγ receptors unlike catumaxomab, 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 an 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).

[0008] ​​​​​​​​​​​​​​In the clinical trials of Refs. 13 and 14, blinatumomab is administered by continuous intravenous infusion using a minipump. Such administration is not only extremely inconvenient for patients, but also potentially risks medical accidents due to equipment failures and the like, and thus 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 polypeptides Polypeptide complexes, methods for producing the polypeptide complexes, and methods for producing the polypeptide complexes The present invention aims to provide a therapeutic agent for inducing cell damage containing the compound as an active ingredient. A pharmaceutical composition for treating or preventing various cancers, which contains an activating therapeutic agent as an active ingredient. The object is to provide a method of treatment using the pharmaceutical composition. [Means for solving the problem]

[0011] The present inventors have demonstrated that BiTE has strong antitumor activity and suppresses cytokine storms in a cancer antigen-independent manner. It maintains the excellent safety profile of not inducing steroids, and has a long half-life in the blood. Furthermore, we have discovered a new polypeptide complex. By replacing In, it was found that the polypeptide aggregate causes cytotoxicity targeting various cells. Based on such a discovery, the inventors have clarified that the polypeptide aggregate according to the present invention damages cancer cells. Further, by introducing CH 1 / CL interface association control and Knob into Hole (KiH) modification into the polypeptide aggregate, it was found that more efficient cytotoxicity is brought about. In addition, the inventors have found that a cytotoxicity-inducing therapeutic agent containing the polypeptide aggregate according to the present invention as an active ingredient treats or prevents various cancers. That is, the present invention provides the following.

[0012] Namely, 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 Fcγ receptor, and (3) A T cell receptor complex-binding domain, A polypeptide aggregate comprising. [2] The polypeptide aggregate according to [1], wherein the T cell receptor complex-binding domain is a T cell receptor-binding domain. [3] The polypeptide aggregate according to [1], wherein the T cell receptor complex-binding domain is a CD3-binding domain. [4] The polypeptide aggregate according to any one of [1] to [3], wherein the antigen-binding domain is a bivalent antigen-binding domain. [5] The polypeptide aggregate according to [4], wherein the bivalent antigen-binding domain is a domain having an F(ab’)2 structure. [6] The polypeptide aggregate according to [5], wherein 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 conjugate according to 〔6〕, wherein the CD3-binding domain is linked to one or two CH3s constituting the Fc region. 〔8〕 The polypeptide conjugate according to 〔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 conjugate according to 〔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 conjugate according to 〔6〕, wherein the CD3-binding domain is linked to one or two CLs constituting F(ab’)2. 〔11〕 The polypeptide conjugate according to 〔6〕, wherein the CD3-binding domain is linked to one or two VHs constituting F(ab’)2. 〔12〕 The polypeptide conjugate according to 〔6〕, wherein the CD3-binding domain is linked to one or two VLs constituting F(ab’)2. 〔13〕 The polypeptide conjugate according to any one of 〔1〕 to 〔12〕, wherein the CD3-binding domain is Fv. 〔14〕 The polypeptide conjugate according to any one of 〔1〕 to 〔7〕 and 〔10〕 to 〔12〕, wherein the CD3-binding domain is Fab. 〔15〕 The polypeptide conjugate according to any one of 〔1〕 to 〔7〕 and 〔10〕 to 〔12〕, wherein the CD3-binding domain is scFv. 〔16〕 The polypeptide conjugate according to any one of 〔1〕 to 〔15〕, wherein the CD3-binding domain is monovalent. 〔17〕 The antigen-binding domain is monovalent scFv and monovalent Fab, and any one of 〔1〕 to 〔3〕 The polypeptide conjugate described in any one of the above. 〔18〕 A polypeptide conjugate in which a monovalent scFv is linked via an scFv constituting a CD3 binding domain to one polypeptide constituting an Fc region, and a monovalent Fab heavy chain Fv fragment is linked via a CH1 region to one polypeptide constituting an Fc region, and the light chain Fv fragment of the Fab is linked to a 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. 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. The polypeptide conjugate described in 〔17〕. 〔20〕 A polypeptide conjugate in which one polypeptide constituting an Fc region via a heavy chain Fv fragment constituting a CD3 binding domain of a monovalent scFv is linked to another polypeptide constituting an Fc region via a light chain Fv fragment constituting a CD3 binding domain of the other monovalent scFv. The polypeptide conjugate described in 〔19〕. The polypeptide conjugate described in 〔19〕. The polypeptide conjugate described in 〔19〕. 〔21〕 A polypeptide conjugate in which one polypeptide constituting an Fc region via an scFv constituting a CD3 binding domain of a monovalent scFv is linked to another polypeptide constituting an Fc region of the other monovalent scFv. The polypeptide conjugate described in 〔19〕. 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. The polypeptide conjugate described in 〔19〕. 〔23〕 A polypeptide conjugate in which the heavy chain Fv fragment of a monovalent Fab constituting an antigen-binding domain is linked via a CH1 region to one polypeptide constituting an Fc region, the light chain Fv fragment of the Fab is linked to a CL region, the heavy chain Fv fragment of a Fab constituting a T cell receptor binding domain is linked via a CH1 region to another polypeptide constituting an Fc region, and the light chain Fv fragment of the Fab is linked to a CL region. The polypeptide conjugate described in 〔22〕. The polypeptide conjugate described in 〔22〕. The polypeptide conjugate described in 〔22〕. The polypeptide conjugate described in 〔22〕. 〔24〕The heavy chain Fv fragment of a monovalent Fab constituting the antigen-binding domain is linked via the CH1 region to one polypeptide constituting the Fc region, and the light chain Fv fragment of the Fab is linked to the CL region. The light chain Fv fragment of 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 heavy chain Fv fragment of the Fab is linked to the CL region. The polypeptide aggregate according to 〔22〕 is as described. 〔25〕The heavy chain Fv fragment of a monovalent Fab constituting the antigen-binding domain is linked via the CH1 region to one polypeptide constituting the Fc region, and the light chain Fv fragment of the Fab is linked to the CL region. The heavy chain Fv fragment of the Fab constituting the T cell receptor-binding domain is linked via the CL region to the other polypeptide constituting the Fc region, and the light chain Fv fragment of the Fab is linked to the CH1 region. The polypeptide aggregate according to 〔22〕 is as described. 〔25〕The heavy chain Fv fragment of a monovalent Fab constituting the antigen-binding domain is linked via the CH1 region to one polypeptide constituting the Fc region, and the light chain Fv fragment of the Fab is linked to the CL region. The heavy chain Fv fragment of the Fab constituting the T cell receptor-binding domain is linked via the CL region to the other polypeptide constituting the Fc region, and the light chain Fv fragment of the Fab is linked to the CH1 region. The polypeptide aggregate according to 〔22〕 is as described. 〔26〕The heavy chain Fv fragment of a monovalent Fab constituting the T cell receptor-binding domain is linked via the CH1 region to one polypeptide constituting the Fc region, and the light chain Fv fragment of the Fab is linked to the CL region. The light chain Fv fragment of the Fab constituting the antigen-binding domain is linked via the CH1 region to the other polypeptide constituting the Fc region, and the heavy chain Fv fragment of the Fab is linked to the CL region. The polypeptide aggregate according to 〔22〕 is as described. 〔26〕The heavy chain Fv fragment of a monovalent Fab constituting the T cell receptor-binding domain is linked via the CH1 region to one polypeptide constituting the Fc region, and the light chain Fv fragment of the Fab is linked to the CL region. The light chain Fv fragment of the Fab constituting the antigen-binding domain is linked via the CH1 region to the other polypeptide constituting the Fc region, and the heavy chain Fv fragment of the Fab is linked to the CL region. The polypeptide aggregate according to 〔22〕 is as described. 〔27〕The heavy chain Fv fragment of a monovalent Fab constituting the T cell receptor-binding domain is linked via the CH1 region to one polypeptide constituting the Fc region, and the light chain Fv fragment of the Fab is linked to the CL region. The light chain Fv fragment of the Fab constituting the antigen-binding domain is linked via the CH1 region to the other polypeptide constituting the Fc region, and the heavy chain Fv fragment of the Fab is linked to the CL region. The polypeptide aggregate according to 〔22〕 is as described. 〔27〕The heavy chain Fv fragment of a monovalent Fab constituting the T cell receptor-binding domain is linked via the CH1 region to one polypeptide constituting the Fc region, and the light chain Fv fragment of the Fab is linked to the CL region. The heavy chain Fv fragment of the Fab constituting the antigen-binding domain is linked via the CL region to the other polypeptide constituting the Fc region, and the light chain Fv fragment of the Fab is linked to the CH1 region. The polypeptide aggregate according to 〔22〕 is as described. 〔28〕The heavy chain Fv fragment of a monovalent Fab constituting the T cell receptor-binding domain is linked via the CH1 region to one polypeptide constituting the Fc region, and the light chain Fv fragment of the Fab is linked to the CL region. The heavy chain Fv fragment of the Fab constituting the antigen-binding domain is linked via the CL region to the other polypeptide constituting the Fc region, and the light chain Fv fragment of the Fab is linked to the CH1 region. The polypeptide aggregate according to 〔22〕 is as described. The polypeptide complex described in 〔28〕(1) A monovalent Fab - structured heavy - chain Fv fragment that binds to an antigen is linked via a 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, resulting in an antigen - binding domain, and (2) A monovalent Fab - structured heavy - chain Fv fragment that binds to the T - cell receptor complex is linked via a 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, resulting in a T - cell receptor complex - binding domain, A polypeptide complex comprising the above, wherein the charges of the CH1 region and the CL region are controlled such that the heavy - chain Fv fragment in the antigen - binding domain associates with the light - chain Fv fragment in the antigen - binding domain or the heavy - chain Fv fragment in the T - cell receptor - binding domain associates with the light - chain Fv fragment in the T - cell receptor - binding domain. 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 the T cell receptor binding domain, the polypeptide aggregate according to any one of

[22] to

[33] .

[35] The T cell receptor binding domain is the 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 combinations 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 of the EU numbering, and the CL region An amino acid residue in the CL region, which is the amino acid residue at position 138 of the EU numbering (e) An amino acid residue in the CH1 region, which is the amino acid residue at position 147 of the EU numbering, and the CL region An amino acid residue in the CL region, which is the amino acid residue at position 123 of the EU numbering (f) An amino acid residue in the CH1 region, which is the amino acid residue at position 175 of the EU numbering, and the CL region An amino acid residue in the CL region, which is the amino acid residue at position 160 of the EU numbering selected from, and the amino acid residue in the CH1 region and the amino acid residue in the CL region have different charges from each other The polypeptide aggregate according to any one of

[32] or

[33] .

[37] Further, selected from the group consisting of the following sets of amino acid residues shown in (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 of the EU numbering, and the CL region An amino acid residue in the CL region, which is the amino acid residue at position 123 of the EU numbering

[38] The amino acid residue having the different charges is 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] , selected from the amino acid residues included in

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

[36] to [3 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 in the numbering are Glu, and the amino acid residues in the CL region are EU The amino acid residues at positions 180, 131, and 160 in the numbering are all Lys,

[36] The polypeptide complex according to any one of

[36] to

[38] . 〔41〕 Further, the amino acid residue in the CH1 region and the amino acid residue at position 213 in the EU numbering is Glu, and the amino acid residue in the CL region and the amino acid residue at position 123 in the EU numbering is Lys, the polypeptide complex according to

[40] . 〔42〕 The Fc region is one of FcγI, FcγIIA, FcγIIB, FcγIIIA, and / or FcγIIIB The Fc region with reduced binding activity to the Fcγ receptor, the polypeptide complex according to any one of [1] to

[41] any of the above. 〔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 described in SEQ ID NO: 26, and the amino acid constituting the Fc region is a mutated Fc region, the polypeptide complex according to any one of [1] to

[42] any of the above. 〔44〕 Any of the following amino acids specified according to the EU numbering among the amino acids constituting the Fc region; Any of the following; The Fc region in which 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 polypeptide complex according to

[43] any of the above. any of the above. 〔45〕 Any of the following amino acids specified according to the 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 Position 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298 , 299, 300, 325, 327, 328, 329, 330, 331, 332, A polypeptide complex according to

[43] , wherein the Fc region has mutations at the above positions.

[46] A polypeptide complex according to

[45] , wherein the amino acids constituting the Fc region are mutated from those of the Fc region set forth in SEQ ID NO: 23. The polypeptide complex according to

[45] , characterized in that the Fc region is a region in which the amino acids constituting the Fc region are mutated.

[47] Any one of the following amino acids identified 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 according to

[46] , characterized in that the Fc region is a region in which the amino acids constituting the Fc region are mutated.

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

[46] , characterized in that the Fc region is a region in which the amino acids are mutated.

[49] The polypeptide complex according to

[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. The polypeptide complex according to

[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.

[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 specified according to the EU numbering is cysteine, and the amino acid at position 366 is tryptophan; among the amino acid residues of the other polypeptide, according to the 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 mutated, and is a polypeptide complex according to any one of [1] to

[50] . 〔52〕One of the two polypeptides constituting the Fc region, wherein the amino acid residue at position 356 specified according to the EU numbering is lysine; among the amino acid residues of the other polypeptide, the amino acid at position 439 specified according to the EU numbering is glutamic acid, and the amino acid at position 435 specified according to the EU numbering among the amino acid residues of either one of the polypeptides is mutated to arginine, and is a polypeptide complex according to any one of [1] to

[50] . 〔53〕A 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〕A polypeptide complex according to any one of [1] to

[53] , wherein the antigen-binding domains bind to the same epitope. 〔55〕A polypeptide complex according to

[54] , wherein the same epitope is present in a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2. 〔56〕A polypeptide complex according to

[54] , wherein the same epitope is present in a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4. 〔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 a 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 a 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 a 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 the conjugate in the manufacture of a cytotoxic agent. The present invention also relates to the polypeptide conjugate of the present invention or the polypeptide conjugate produced by the production method of the present invention for use in the method of the present invention. Relates to the polypeptide conjugate of the present invention or the polypeptide conjugate 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 such as a cytokine storm are maintained, and a new polypeptide conjugate having a long blood half-life is provided. By replacing the antigen-binding domain in the polypeptide conjugate of the present invention, a cytotoxic agent containing the polypeptide conjugate as an active ingredient can cause 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 such as a cytokine storm are maintained, and a new polypeptide conjugate having a long blood half-life is provided. By replacing the antigen-binding domain in the polypeptide conjugate of the present invention, a cytotoxic agent containing the polypeptide conjugate as an active ingredient can cause 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 such as a cytokine storm are maintained, and a new polypeptide conjugate having a long blood half-life is provided. By replacing the antigen-binding domain in the polypeptide conjugate of the present invention, a cytotoxic agent containing the polypeptide conjugate as an active ingredient can cause 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 such as a cytokine storm are maintained, and a new polypeptide conjugate having a long blood half-life is provided. By replacing the antigen-binding domain in the polypeptide conjugate of the present invention, a cytotoxic agent containing the polypeptide conjugate as an active ingredient can cause 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 such as a cytokine storm are maintained, and a new polypeptide conjugate having a long blood half-life is provided. By replacing the antigen-binding domain in the polypeptide conjugate of the present invention, a cytotoxic agent containing the polypeptide conjugate as an active ingredient can cause 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 such as a cytokine storm are maintained, and a new polypeptide conjugate having a long blood half-life is provided. By replacing the antigen-binding domain in the polypeptide conjugate of the present invention, a cytotoxic agent containing the polypeptide conjugate as an active ingredient can cause 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 such as a cytokine storm are maintained, and a new polypeptide conjugate having a long blood half-life is provided. By replacing the antigen-binding domain in the polypeptide conjugate of the present invention, a cytotoxic agent containing the polypeptide conjugate as an active ingredient can cause 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]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Modes for Carrying Out the Invention

[0016] The following definitions are provided to facilitate the understanding of the 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 natural sources 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 obtained from natural sources such as plasma or serum in which they naturally exist, 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. It can be isolated from natural sources 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. include those obtained from natural sources such as plasma or serum in which they naturally exist, 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. Then, isotypes of immunoglobulins and subclasses of those isotypes 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 cells are screened by a normal screening method. ​​​​​​​​​​​​Hybrids producing anti-GPC3 antibodies were isolated by screening for null antibody-producing cells. A theme can be selected.

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

[0022] The purified GPC3 protein can be used as a sensitizing antigen for immunization of mammals. A partial peptide of GPC3 can also be used as a sensitizing antigen. It can also be obtained by chemical synthesis from the amino acid sequence of GPC3. Alternatively, it can be obtained by incorporating it into a vector and expressing it. It can also be obtained by 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. To produce 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 mammals immunized with the sensitizing antigen are not limited to specific animals, but are preferably mammals such as mice, rats, rabbits, guinea pigs, etc. are also specifically described in WO2003 / 000883, WO2004 / 022754, WO2006 / 0

[0024] The mammals immunized with the sensitizing antigen are not limited to specific animals, but It is preferably selected in consideration of compatibility with the parent cells used for cell fusion. Generally, mammals such as rodents, for example, mice, rats, hamsters, or rabbits, monkeys, etc. are preferably used.

[0025] The above animals are immunized with the sensitizing antigen according to a known method. For example, as a general method, immunization is carried out by administering the sensitizing antigen by injection into the abdominal cavity or subcutaneously of a mammal. Specifically, the sensitizing antigen diluted at an appropriate dilution ratio with PBS (Phosphate-Buffered Saline), physiological saline, etc. is mixed with a normal adjuvant, for example, Freund's complete adjuvant, emulsified, and then the sensitizing antigen is administered to the mammal several times every 4 to 21 days. Also, an appropriate carrier may be used during immunization with the sensitizing antigen. In particular, when a small molecular weight partial peptide is used as the sensitizing antigen, it is desirable to immunize the sensitizing antigen peptide conjugated with a carrier protein such as albumin or keyhole limpet hemocyanin in some cases.

[0026] 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 immunostimulation is given by expressing the gene encoding the antigen protein in the immunized animal, in the immunized animal to which vector DNA constructed in such a way that it can be expressed is administered, so that the sensitizing antigen is expressed in the living body of the immunized animal. Compared with the general immunization method in which a protein antigen is administered to an immunized animal, DNA immunization is expected to have the following advantages. - Immunostimulation can be given while maintaining the structure of a membrane protein 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 GPC3 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 individual immunized animal 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 binding to GPC3 in the serum is confirmed immunocytes are collected from the mammal and used for cell fusion. As the preferred immunocytes spleen cells can be particularly used

[0029] As the cells to be fused with the immunocytes, 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 phosphorothymidine sensitivity (hereinafter abbreviated as HAT sensitivity). Cells with HAT sensitivity cannot perform DNA synthesis in HAT selection medium and die, but when fused with normal cells, they can continue DNA synthesis using the salvage pathway of normal cells and thus proliferate 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 Immunology

[0030] (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.

[0031] 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 above 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 enhance 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 above cell fusion , for example, RPMI1640 culture solution, MEM culture solution, which are suitable for the growth of the above myeloma cell line, and other normal culture solutions used for this type of cell culture are used, and further, a serum supplement such as fetal calf serum (FCS ) can be preferably added.

[0034] Cell fusion is carried out by thoroughly mixing a predetermined amount of the above immunocytes and myeloma cells in the above 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). Culturing 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 using the above HAT culture medium to obtain. 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, cells that have successfully fused with normal cells can selectively proliferate in the HAT culture medium. Culturing using the above HAT culture medium is continued for a sufficient time for cells other than the desired hybridomas (non-fused cells) to die. Specifically, generally, the desired hybridomas can be selected by culturing for several days to several weeks. 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 preferably carried out by a screening method based on the known antigen-antibody reaction. For example, a monoclonal antibody that binds to GPC3 can bind to GPC3 expressed on the cell surface. Such a monoclonal antibody 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 producing 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.

[0038] 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.

[0039] is produced. When the monoclonal antibody is of mouse origin, the antibody bound to the cell can be detected by an anti-mouse immunoglobulin antibody. The hybridomas producing the desired antibody having the binding ability to the antigen selected by these screenings can be cloned by methods such as the limiting dilution method. The hybridomas producing the monoclonal antibodies thus prepared can be subcultured in a normal culture solution. Also, the hybridomas can be stored in liquid nitrogen over a long period. The desired monoclonal antibody can be obtained from the culture supernatant by culturing the hybridomas according to a normal method. Alternatively, the monoclonal antibody can be obtained from the ascites by administering the hybridomas to a compatible mammal for growth. The former method is suitable for obtaining a high-purity antibody. Antibodies encoded by 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). As described below, methods for producing recombinant antibodies are also known.

[0040] For example, from hybridoma cells producing an anti-GPC3 antibody, the variable region (V) of the anti-GPC3 antibody

[0041]

[0042]

[0043] ​​​​​​​​​​​​​cDNA encoding the (region) is obtained. For this purpose, usually, first total RN is extracted from the hybridoma. 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 QuickPrep mRNA Purification Kit (manufactured by GE Healthcare Biosciences), are also commercially available. Using such a kit, mRNA can be obtained from the hybridoma. cDNA encoding the antibody V region can be synthesized from the obtained mRNA using reverse transcriptase. The cDNA can be synthesized by, for example, AMV Reverse Transcriptase First - strand cDNA Synthesis Kit (manufactured by Seikagaku Corporation) or the like. Also, for the synthesis and amplification of cDNA, SMART RACE cDNA Amplification Kit (manufactured by Clontech) and 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 process of such cDNA synthesis.

[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 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 with 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 a 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 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, the 3'-side A primer that anneals to a portion corresponding to the constant region close to the variable region is used for the primer On the other hand, for the 5'-side primer, the primer attached to the 5’ RACE cDNA library preparation kit is used .

[0049] Using the PCR product amplified in this way, an immunoglobulin consisting of a combination 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 the purpose is to obtain an antibody against GPC3 , it is more preferable that the binding of the antibody to GPC3 is specific. An antibody that binds to GPC3 can be screened, for example, as follows: ; (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 GPC3-expressing cells and the antibody, and (3) A step of selecting an antibody that binds to GPC3-expressing cells.

[0050] Methods for detecting the binding between an antibody and GPC3-expressing cells are known. Specifically, the binding between an antibody and GPC3-expressing cells can be detected by methods such as the FACS described above. 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 the antibody gene is obtained as a library of heavy chain and light chain subclasses from a polyclonal antibody-expressing cell group, a phage vector is used . A screening method is advantageous. Genes encoding the variable regions of the heavy and light chains can be linked with a suitable 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, by recovering the phages bound to the antigen, DNA encoding the scFv having the desired binding activity can be recovered. By repeating this operation as necessary, the scFv having the desired binding activity can be concentrated.

[0052] After obtaining the cDNA encoding the V region of the target anti-GPC3 antibody, the cDNA is digested with a restriction enzyme that recognizes the 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, in order to insert one copy of the digested fragment into the vector in the correct direction, it is preferable to insert a restriction enzyme that gives sticky ends. By inserting the cDNA encoding the V region of the anti-GPC3 antibody digested as described above into a suitable 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, human-human homologous chimeric antibodies are also included in the chimeric antibodies in the present invention. By inserting the V region gene into an expression vector having a constant region in advance, a chimeric antibody is obtained. A MELA 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 retaining 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. Expression control regions for expressing an antibody include, for example, enhancers and promoters. 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 recombinant cell expressing DNA encoding an anti-GPC3 antibody can be obtained. For the expression of the antibody gene, DNAs encoding the antibody heavy chain (H chain) and light chain (L chain) are each incorporated into a separate expression vector. 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 can be obtained. .

[0054] For the expression of the antibody gene, DNAs encoding the antibody heavy chain (H chain) and light chain (L chain) are each incorporated into a separate expression vector. 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 can be obtained. Antibody molecules can be expressed. Alternatively, DNA encoding the H chain and L chain can be incorporated into a single expression vector to transform host cells (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, the following cells can be exemplified as animal 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. etc. (2) Amphibian cells: African clawed frog oocytes, etc. (3) Insect cells: sf9, sf21, Tn5, etc.

[0056] Alternatively, as plant cells, an antibody gene expression system 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. For the transformation of plant cells, callus-cultured cells can be appropriately used.

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

[0058] In addition, 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. 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 the 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. In addition, hormones can be administered to the transgenic goat to increase the amount of milk containing the desired antibody produced by the transgenic goat (Bio / Technology (1994), 12 (7), 699-702).

[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 the 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. In addition, hormones can be administered to the transgenic goat to increase the amount of milk containing the desired antibody produced by the transgenic goat (Bio / Technology (1994), 12 (7), 699-702). 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 the 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. In addition, hormones can be administered to the transgenic goat to increase the amount of milk containing the desired antibody produced by the transgenic goat (Bio / Technology (1994), 12 (7), 699-702). 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. In addition, hormones can be administered to the transgenic goat to increase the amount of milk containing the desired antibody produced by the transgenic goat (Bio / Technology (1994), 12 (7), 699-702). is obtained as a fusion protein with the milk protein. Also, hormones can be administered to the transgenic goat to increase the amount of milk containing the desired antibody produced by 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 As the antigen-binding domain in, for the purpose of reducing the heterologous antigenicity against humans, etc., An antigen-binding domain derived from a genetically engineered antibody artificially modified can be appropriately employed. 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 for creating 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. Humanized antibodies are also referred to as reshaped human antibodies. Specifically, humanized antibodies obtained by transplanting the CDRs of non-human animals, such as mouse antibodies, into human antibodies are known. General genetic recombination 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 Are used.

[0062] Humanized antibodies are also called reshaped human antibodies. Specifically, humanized antibodies obtained by transplanting the CDRs of non-human animals, such as mouse antibodies, into human antibodies are known. For example, humanized antibodies obtained by transplanting the CDRs of mouse antibodies into human antibodies are known. General genetic recombination 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 Are used. 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 the transplantation of 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 and is generally considered to be the case. That is, generally, it is preferable to use a human FR consisting of an amino acid sequence with a high identity 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 synthesized individually 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 CDRs 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 forming 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 that anneal to its 5'- and 3'-ends and add appropriate restriction enzyme recognition sequences.

[0064] By inserting the DNA obtained as described above and the DNA encoding the human antibody C-region into an expression vector in-frame so as to be fused, a vector for expressing a humanized antibody can be created. 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 produced by the cultured cells. ​​​​(See European Patent Publication EP 239400 and International Publication WO1996 / 002576).

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

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

[0067] Furthermore, there is also a technology to obtain human antibodies by panning using a human antibody library. For example, the V region of a human antibody is displayed as a single-chain antibody (scFv) on a phage. It is expressed on the surface of phages by the phage display method. Phages expressing scFv that binds to an antigen can be selected. By analyzing the genes of the selected phages, the DNA sequence encoding the V region of a 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 a 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 Publications WO 1992 / 001047, WO 1992 / 020791, WO 1993 / 006213, WO 1993 / 011236, WO 1993 / 019172, WO 1995 / 001438, WO 1995 / 015388). By analyzing the genes of the selected phages, the DNA sequence encoding the V region of a 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 a 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 Publications WO 1992 / 001047, WO 1992 / 020791, WO 1993 / 006213, WO 1993 / 011236, WO 1993 / 019172, WO 1995 / 001438, WO 1995 / 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 an antibody light chain variable region (VL) and an antibody heavy chain variable region (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 an antibody light chain variable region (VL) and an antibody heavy chain variable region (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 an antibody light chain variable region (VL) and an antibody heavy chain variable region (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. are preferably mentioned.

[0069] The antigen-binding domains in the polypeptide aggregates 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 aggregates of the present invention can bind to different epitopes from each other. Here, 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 antigen-binding domains in the polypeptide aggregates 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 aggregates of the present invention can bind to different epitopes from each other. Here, 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 antigen-binding domains in the polypeptide aggregates 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 aggregates of the present invention can bind to different epitopes from each other. Here, 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 antigen-binding domains in the polypeptide aggregates 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 aggregates of the present invention can bind to different epitopes from each other. Here, 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 antigen-binding domains in the polypeptide aggregates 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 aggregates of the present invention can bind to different epitopes from each other. Here, 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 antigen-binding domains in the polypeptide aggregates 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 aggregates of the present invention can bind to different epitopes from each other. Here, 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.

[0070] specific Specific means a state in which one of the molecules that specifically binds does not show any significant binding to molecules other than the one or more molecules to which it binds. Also, it is used when the antigen-binding domain is specific for a specific 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 aggregate having the antigen-binding domain can bind to various antigens containing the epitope. Specific means a state in which one of the molecules that specifically binds does not show any significant binding to molecules other than the one or more molecules to which it binds. Also, it is used when the antigen-binding domain is specific for a specific 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 aggregate having the antigen-binding domain can bind to various antigens containing the epitope. Specific means a state in which one of the molecules that specifically binds does not show any significant binding to molecules other than the one or more molecules to which it binds. Also, it is used when the antigen-binding domain is specific for a specific 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 aggregate having the antigen-binding domain can bind to various antigens containing the epitope. Specific means a state in which one of the molecules that specifically binds does not show any significant binding to molecules other than the one or more molecules to which it binds. Also, it is used when the antigen-binding domain is specific for a specific 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 aggregate having the antigen-binding domain can bind to various antigens containing the epitope. Specific means a state in which one of the molecules that specifically binds does not show any significant binding to molecules other than the one or more molecules to which it binds. Also, it is used when the antigen-binding domain is specific for a specific 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 aggregate having the antigen-binding domain can bind to various antigens containing the epitope. Specific means a state in which one of the molecules that specifically binds does not show any significant binding to molecules other than the one or more molecules to which it binds. Also, it is used when the antigen-binding domain is specific for a specific 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 aggregate having the antigen-binding domain can bind to various antigens containing the epitope.

[0071] antigen 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. 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. 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. 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. 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. Amily, tyrosine phosphatase type receptor family, adhesion factor family, hormone Receptors belonging to receptor families such as receptor families can be mentioned. Regarding the receptors belonging to these receptor families and their characteristics, there are a number of documents, 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)(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. Specific receptors belonging to the above receptor families include, for example, human or mouse ery

[0072] 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 is a mouse thrombopoietin (TPO) receptor (Proc Natl Acad Sci U S A. (1992) 89 (12 ), 5640-5644, EMBO J. (1993) 12(7), 2645-53), human or mouse insulin receptor (N ature (1985) 313 (6005), 756-761), human or mouse Flt-3 ligand receptor (Proc. Nat Acad. Sci. USA. (1994) 91 (2), 459-463), human or mouse platelet-derived growth factor (P DGF) receptor (Proc. Natl. Acad. Sci. USA. (1988) 85 (10) 3435-3439), human or mouse Mouse interferon (IFN)-α, β receptors (Cell (1990) 60 (2), 225-234. and Cell ( 1994) 77 (3), 391-400), human or mouse leptin receptor, human or mouse growth hormone human or mouse interleukin (GH) receptor, human or mouse interleukin (IL)-10 receptor, human or mouse interleukin (IL)-10 receptor insulin-like growth factor (IGF)-I receptor, human or mouse leukemia inhibitory factor (LIF) receptor, human Suitable examples include human or mouse ciliary neurotrophic factor (CNTF) receptors.

[0073] Cancer antigens are antigens that are expressed in association with the malignant transformation of cells, and are also called tumor-specific antigens. , abnormal sugar chains that appear on the cell surface or protein molecules when cells become cancerous are also cancer antigens, and are also called cancer carbohydrate antigens. Examples of cancer antigens include, for example, GPC3 that belongs to the GPI-anchored receptor family of the above receptors and is expressed in several cancers including liver cancer, (Int J Cancer. (2003) 103 (4), 455-6). EpCAM 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 as NM_002354.2 (SEQ ID NO: 3), and the polypeptide sequence is registered in RefSeq as NP_0 02345.2 (SEQ ID NO: 4)).), EGFR, CA19-9, CA15-3, sialyl SSEA-1 (SLX), etc. 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.

[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 specify the epitope by the amino acid residues constituting the epitope. Also, when the epitope is a sugar chain, it is possible to specify 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. A linear epitope typically contains 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. A conformational epitope encompasses a larger number of amino acids relative to a linear epitope.

[0077]

[0078] It may do so. Regarding the recognition of a conformational epitope, an antibody recognizes the three-dimensional structure of a peptide or a protein. For example, when a protein molecule folds to form a three-dimensional structure, certain amino acids and / or polypeptide main chains that form a conformational epitope become parallel, enabling the antibody to recognize the epitope. Methods for determining the conformational structure of an epitope include, for example, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-specific spin labeling and electron paramagnetic resonance spectroscopy. 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 a conformational epitope main chains become parallel, enabling the antibody to recognize the epitope. Methods for determining the conformational structure of an epitope include, for example, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-specific spin labeling and electron paramagnetic resonance spectroscopy. 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 a conformational epitope main chains become parallel, enabling the antibody to recognize the epitope. Methods for determining the conformational structure of an epitope include, for example, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-specific spin labeling and electron paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Volume 66, Morr is (ed.). 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. However, 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 exemplification. 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 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.

[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. 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. 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. 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. The binding activity of the test polypeptide complex containing a chid 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 clarified. By these tests, the binding activity of the polypeptide complex against the linear peptide can be clarified. 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 substantially does not bind to a linear peptide consisting of the amino acid sequence constituting the extracellular domain of immobilized GPC3. Here, substantially not 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] The binding activity of the test polypeptide complex containing an antigen-binding domain against GPC3 to GPC3-expressing cells is measured by, for example, the method described in Antibodies A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). ​​​That is, it can be evaluated by the principle of ELISA or FACS (fluorescence activated cell sor ting) using GPC3-expressing cells as an antigen.

[0083] In the ELISA format, the binding activity of the test polypeptide containing an antigen-binding domain against GPC3 to GPC3-expressing cells of the complex is quantitatively evaluated by comparing the signal levels generated by the enzyme reaction. That is, the test polypeptide complex is added to an ELISA plate immobilized with GPC3-expressing cells, and the test polypeptide complex bound to the cells is detected using an enzyme-labeled antibody that recognizes the test polypeptide complex. Alternatively, in FACS, a dilution series of the test polypeptide complex is prepared, and the binding activity of the test polypeptide complex against GPC3-expressing cells can be compared by determining the antibody-binding titer against

[0084] GPC3-expressing cells. The binding of the test polypeptide complex to an antigen expressed on the cell surface suspended in a buffer or the like can be detected by a flow cytometer. As for flow cytometers, for example, the following devices are known. FACSCanto TM II FACSAria TM FACSArray TM FACSVantage TM SE FACSCalibur TM (All 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 a 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, Stain with a FITC-labeled secondary antibody that recognizes the test polypeptide complex reacted with cells expressing GPC3. 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). This is reflected in the fluorescence intensity obtained, 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 a 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 can be detected by cross-blocking assays 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. 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. 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. 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. 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. 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. 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. 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.

[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. 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. 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. 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. 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. 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. 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] In a control test conducted in the absence of the candidate competing polypeptide complex, 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 for GPC3 compared to the binding activity obtained, the test polypeptide complex is In a control test conducted in the absence of the candidate competing polypeptide complex, 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 for GPC3 compared to the binding activity obtained, the test polypeptide complex is In a control test conducted in the absence of the candidate competing polypeptide complex, 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 for GPC3 compared to the binding activity obtained, the test polypeptide complex is In a control test conducted in the absence of the candidate competing polypeptide complex, 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 for GPC3 compared to the binding activity obtained, the test polypeptide complex is A polypeptide complex that binds to substantially the same epitope as, or competes with, the binding to the same epitope is a polypeptide complex.

[0090] When the structure of the epitope to which a test polypeptide complex containing an 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 and can be measured. For example, as a method for measuring such binding activity, 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 can be measured by comparison. As a method other than ELISA, the binding activity to the mutant peptide bound to a column can also be measured by quantifying the polypeptide complex eluted into the eluate after flowing the test polypeptide complex and the control polypeptide complex through the column. A method of adsorbing the mutant peptide to the column as a fusion peptide with, for example, GST is known. Furthermore, 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 into which a mutation has been 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 can be measured.

[0091] complex and the control polypeptide complex are used.

[0092] ​​​​​​​​​​The chido conjugate and the control polypeptide conjugate are added. Subsequently, to the cell suspension appropriately washed with a buffer, a FITC-labeled antibody capable of recognizing the test polypeptide conjugate and the control polypeptide conjugate is added. 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 desired concentrations by appropriately diluting with a suitable buffer. 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 the 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 cells expressing mutant GPC3 are stained with a 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, whereby the increase ratio of the fluorescence intensity due to the binding of the polypeptide conjugate is obtained. The test polypeptide conjugate and the control polypeptide conjugate are added to the cell suspension washed with a buffer as appropriate. Then, a FITC-labeled antibody capable of recognizing the test polypeptide conjugate and the control polypeptide conjugate is added. 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 desired concentrations by appropriately diluting with a suitable buffer. 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 the 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. The test polypeptide conjugate and the control polypeptide conjugate are added to the cell suspension washed with a buffer as appropriate. Then, a FITC-labeled antibody capable of recognizing the test polypeptide conjugate and the control polypeptide conjugate is added. 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 desired concentrations by appropriately diluting with a suitable buffer. 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 the 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. The test polypeptide conjugate and the control polypeptide conjugate are added to the cell suspension washed with a buffer as appropriate. Then, a FITC-labeled antibody capable of recognizing the test polypeptide conjugate and the control polypeptide conjugate is added. 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 desired concentrations by appropriately diluting with a suitable buffer. 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 the 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. The test polypeptide conjugate and the control polypeptide conjugate are added to the cell suspension washed with a buffer as appropriate. Then, a FITC-labeled antibody capable of recognizing the test polypeptide conjugate and the control polypeptide conjugate is added. 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 desired concentrations by appropriately diluting with a suitable buffer. 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 the 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. The test polypeptide conjugate and the control polypeptide conjugate are added to the cell suspension washed with a buffer as appropriate. Then, a FITC-labeled antibody capable of recognizing the test polypeptide conjugate and the control polypeptide conjugate is added. 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 desired concentrations by appropriately diluting with a suitable buffer. 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 the 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. The test polypeptide conjugate and the control polypeptide conjugate are added to the cell suspension washed with a buffer as appropriate. Then, a FITC-labeled antibody capable of recognizing the test polypeptide conjugate and the control polypeptide conjugate is added. 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 desired concentrations by appropriately diluting with a suitable buffer. 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 the 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. The test polypeptide conjugate and the control polypeptide conjugate are added to the cell suspension washed with a buffer as appropriate. Then, a FITC-labeled antibody capable of recognizing the test polypeptide conjugate and the control polypeptide conjugate is added. 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 desired concentrations by appropriately diluting with a suitable buffer. 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 the 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.

[0093] 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 cells expressing mutant GPC3 are stained with a 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, whereby the increase ratio of the fluorescence intensity due to the binding of the polypeptide conjugate is obtained. 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 cells expressing mutant GPC3 are stained with a 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, whereby the increase ratio of the fluorescence intensity due to the binding of the polypeptide conjugate is obtained. 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 cells expressing mutant GPC3 are stained with a 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, whereby the increase ratio of the fluorescence intensity due to the binding of the polypeptide conjugate is obtained. 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 cells expressing mutant GPC3 are stained with a 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, whereby 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 compared with the Geometric Mean comparison value (mutant GPC3 molecule ΔGeo-Mean value) reflected thereby. 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 advance to recognize an epitope in GPC3 is used as a control polypeptide aggregate.

[0096] If the ΔGeo-Mean comparison value of the test polypeptide aggregate to mutant GPC3-expressing cells is at least 80%, preferably 50%, more preferably 30%, particularly preferably less than 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 can be regarded as substantially the same by comparison, the

[0097] Fv (variable fragment) As used herein, the term "Fv (variable fragment)" refers to the variable region of the light chain of an antibody (VL (light chain variable region)) and the variable region of the heavy chain of the antibody (VH (heavy chain varia ble region)) and means the smallest unit of an antigen-binding domain derived from an antibody consisting of a pair. 19 In 1988, Skerra and Pluckthun inserted the antibody gene downstream of the signal sequence of bacteria By inducing the expression of the gene in Escherichia coli, it was found that it was prepared from the periplasmic fraction of Escherichia coli in a uniform and active state (Science (1988) 240 (4855), 1038-1041). The Fv prepared from the periplasmic fraction had VH and VL associated in a manner having binding to the antigen.

[0098] As used herein, examples of Fv include the following polypeptide aggregates; One polypeptide constituting the Fc region of a monovalent scFv among bivalent scFvs via a heavy chain Fv fragment constituting a CD3 binding domain, and the other monovalent scFv having a CD3 binding domain Linked to another polypeptide constituting the Fc region via a light chain Fv fragment, and the bivalent antigen-binding do The main chain is a bivalent scFv (1) a bivalent antigen-binding domain, (2) an Fc region composed of amino acids constituting the Fc region of IgG1, IgG2a, IgG3 or IgG4 and having no binding activity to the Fcγ receptor, and (3) at least a monovalent CD3 binding domain, In a polypeptide aggregate containing, etc., the light chain Fv fragment and the heavy chain Fv fragment are directed against CD3, which is an antigen. Also preferably included are a set of Fvs that associate in a manner having the bonds to be formed and constitute a CD3 binding domain.

[0099] scFv, single-chain antibody, or sc(Fv)2 As used herein, the terms "scFv", "single-chain antibody", or "sc(Fv)2" refer to an antibody fragment that contains variable regions derived from both a heavy chain and a light chain within a single polypeptide chain but lacks a constant region. Generally, a single-chain antibody further includes a polypeptide linker between a VH domain and a VL domain that enables the formation of a desired structure that is thought to allow for antigen binding. Single-chain antibodies are discussed in detail by Plückthun in The Pharmacology of Monoclonal Antibodies, Volume 113, Rosenberg, and Moore, eds., Springer-Verlag, New York, 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] 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 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 anti body as disclosed in Journal of Immunology (1994) 152 (11), 5368-5374, which recognizes two types of epitopes present in the same antigen, bispecific sc(Fv)2, is also preferred and is listed. sc(Fv)2 can be produced by methods known to those skilled in the art. For example, it can be produced by linking scFv 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, there are antibodies characterized in that 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, but the order of the two VHs and two VLs is not particularly limited to the above composition and 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. Based on these descriptions, those skilled in the art can appropriately produce the desired sc(Fv)2 for the preparation of the polypeptide aggregates disclosed in the present specification. ​​​

[0104] In addition, the polypeptide conjugate of the present invention may be conjugated with a carrier polymer such as PEG or an organic compound such as an anticancer agent. Further, 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 may be used, 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-(sulf ophosphosuccinimidooxycarbonyloxy)ethyl] sulfone (sulfo-BSOCOES), etc. There are, and 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 one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

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

[0111] "F(ab')2" is a protein consisting of two light chains and an interchain disulfide bond formed between two heavy chains. It comprises two heavy chains containing constant regions of the CH1 domain and a portion of the CH2 domain so as to form a The F(ab')2 constituting the polypeptide complexes disclosed herein binds to a desired antigen. A full-length monoclonal antibody having a binding domain is fragmented with a proteolytic enzyme such as pepsin. After digestion, the Fc fragment is removed by adsorption onto a protein A column, allowing for convenient extraction. Such protease can be obtained by appropriately setting the reaction conditions of the enzyme, such as pH. Any protease that can digest a full-length antibody to produce F(ab’)2 in a restricted manner is not particularly limited, and examples include pepsin and ficin. For example, pepsin, ficin, etc. can be exemplified.

[0112] Fc region The Fc region constituting the polypeptide complex 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, whereby it can be preferably obtained. Any protease that can digest an antibody such as a monoclonal antibody by appropriately setting the reaction conditions of the enzyme such as pH is not particularly limited, and examples include pepsin and ficin. For example, pepsin, ficin, etc. can be exemplified. is not particularly limited, and examples include pepsin and ficin. For example, pepsin, ficin, etc. can be exemplified.

[0113] The polypeptide complex 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 isotype of an antibody is determined by the structure of the constant region. The constant regions of each isotype 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.

[0114] The isotype of an antibody is determined by the structure of the constant region. The constant regions of each isotype 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 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 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. : 23, 24, 25, 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 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. is shown.

[0115] The Fc region consists of two light chains and two disulfide bonds formed between the heavy chains. Two heavy chains that include a portion of the constant region between the CH1 domain and the CH2 domain, as if refers to the region excluding (Fab’)2. The Fc region that constitutes the polypeptide complex disclosed in this specification can be preferably obtained by redissolving the fraction adsorbed to a protein A column after partially digesting IgG1, IgG2, IgG3, IgG4 monoclonal antibodies, etc. with a proteolytic enzyme such as pepsin. There is no particular limitation as long as it is an enzyme that can digest the full-length antibody so as to restrictively produce (Fab’)2 by appropriately setting the reaction conditions of the enzyme such as pH. Examples thereof include pepsin and ficin. By

[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 means 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 found in humans, mice, rats, rabbits, monkeys, dogs, cats, cows, horses, sheep, goats, pigs, chickens, ducks, geese, turkeys, quails, pigeons, doves, and any other mammals, and substantially means any member of the family of proteins encoded by the Fcγ receptor gene in these animals. and any undiscovered FcγRs or FcγR isoforms or allotypes in these animals are also included, but are not limited thereto. FcγRs are present in humans, mice, rats, rabbits, monkeys, dogs, cats, cows, horses, sheep, goats, pigs, chickens, ducks, geese, turkeys, quails, pigeons, doves, ​​​​Any biological origin, 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 FcγR isotypes or allotypes, but are not limited thereto. Suitable 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 respectively shown in SEQ ID NO: 13 (NM_000566.3) and 14 (NP_000557.1), the polynucleotide sequence and amino acid sequence of FcγIIA are respectively shown in SEQ ID NO: 15 (BC020823.1) and 16 (AAH20823.1), the polynucleotide sequence and amino acid sequence of FcγIIB are respectively shown in SEQ ID NO: 17 (BC146678.1) and 18 (AAI46679.1), the polynucleotide sequence and amino acid sequence of FcγIIIA are respectively shown in SEQ ID NO: 19 (BC033678.1) and 20 (AAH33678.1), and the polynucleotide sequence and amino acid sequence of FcγIIIB are respectively shown in SEQ ID NO: 21 (BC128562.1) and 22 (AAI28563.1) (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 Can be confirmed by 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, mannose - binding lectin, mannose receptor, Staphylococcus protein A, Staphylococcus 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 The fact that the binding activity of the Fc region to any of the Fcγ receptors FcγI, FcγIIA, FcγIIB, FcγIIIA, and / or FcγIIIB is reduced 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 BIACORE method using the surface plasmon resonance (SPR) phenomenon ( (Proc. Natl. Acad. Sci. USA (2006) 103 ( 11), 4005 - 4010) and 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] ALPHA Screen uses ALPHA technology, which uses two beads, a donor and an acceptor. The method is based on the following principle: molecules bound to donor beads are transferred to acceptor beads. When the two beads are in close proximity, they interact biologically with the molecules bound to the beads. Only the photon-emitting donor beads excited by the laser are detected. The sensitizer converts ambient oxygen into excited singlet oxygen, which is the donor. When it diffuses around the beads and reaches the nearby acceptor beads, it emits chemiluminescence within the beads. The donor bead is bound to an acceptor, which reacts with the donor bead and ultimately emits light. When the molecules bound to the beads do not interact, the singlet oxygen produced by the donor beads Since the acceptor beads are not reached, the chemiluminescence reaction does not occur.

[0120] For example, a biotin-labeled polypeptide complex is bound to a donor bead, and a biotin-labeled polypeptide complex is bound to an acceptor bead. The turbidibeads contain glutathione S-transferase (GST)-tagged Fcγ receptors. In the absence of a competing polypeptide complex with a mutant Fc region, the wild-type Fc region The polypeptide complex containing the 520-620 nm region interacts with the Fcγ receptor, producing a signal at 520-620 nm. Polypeptide complexes having untagged mutant Fc regions are similar to those having wild-type Fc regions. This competes with the interaction between the polypeptide complex and the Fcγ receptor. By quantifying the amount of the polypeptide, the relative binding affinity can be determined. It is known 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 a vector capable of expressing the fusion gene, and expressed in cells or the like, and a method of purifying using a glutathione column or the like can be appropriately employed. The obtained signal is preferably analyzed by fitting it to a one-site competition model that utilizes non-linear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego). One of the substances (ligand) for observing the interaction is immobilized 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, 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 immobilized 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, 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 immobilized 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, 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 immobilized 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, 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 ( One of the substances (ligand) for observing the interaction is immobilized 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, 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 immobilized 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, 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 immobilized 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, 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 immobilized 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, 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 ( The inhibition measurement method is also preferably used in the CORE method. An example of the inhibition measurement method is described in Proc. Natl. Acad. Sci. USA ( 2006) 103 (11), 4005 - 4010.

[0122] In this specification, the decrease in the binding activity to the Fcγ receptor means, for example, based on the above analysis method, the competitive activity of the test polypeptide complex is 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, 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 complex. This means showing a binding activity of.

[0123] As the control polypeptide complex, a polypeptide complex 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). Further, when using a polypeptide complex having a variant of the Fc region of an antibody of a specific isotype as a test substance, using a polypeptide complex having the Fc region of an antibody of the specific isotype as a control can verify the effect of the mutation in the variant on the binding activity to the Fcγ receptor. As described above, a polypeptide complex having a variant of the Fc region, for which it has been verified that the binding activity to the Fcγ receptor is decreased, is appropriately prepared. ​

[0124] Examples of such mutants include amino acid 23 as 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 position, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266 position, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 3 28 position, 329, 330, 331, 332 having a substituted Fc region of a polypeptide conjugate is preferably mentioned. The isotype of the antibody that is the origin of the Fc region is not particularly limited, and 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.

[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 (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 (d) C226S, C229S, E233P, L234V, L235A a polypeptide conjugate having an Fc region in which the amino acid sequence from position 231 to position 238 is deleted, or an Fc region in which the amino acid sequence from position 231 to position 238 is deleted may also be used as appropriate. A polypeptide conjugate having an Fc region in which the amino acid sequence from position 231 to position 238 is deleted, or an Fc region in which the amino acid sequence from position 231 to position 238 is deleted may also be used as appropriate.

[0127] In addition, among the amino acids constituting the Fc region of the IgG2 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); In addition, among the amino acids constituting the Fc region of the IgG2 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); 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); 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 A polypeptide conjugate having an Fc region in which the substitution has been carried out may also be used as appropriate.

[0128] In addition, among the amino acids constituting the Fc region of the IgG3 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); In addition, among the amino acids constituting the Fc region of the IgG3 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); 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); 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); (k)F241A (l) D265A (m)V264A Polypeptide complexes having an Fc domain modified with PEG can also be used as appropriate.

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

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

[0131] Other preferred examples include amino acids in the EU numbering system that constitute the Fc region of an IgG1 antibody. Any one or more of the following amino acids as specified in accordance with the coding sequence: positions 234, 235, 29 Polypeptide complexes having an Fc domain in which position 7 is substituted with another amino acid are preferably The type of amino acid present after substitution is not particularly limited, but may be any of the amino acids at positions 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254 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 another preferred example, among the amino acids constituting the Fc region of the IgG1 antibody, any of the following amino acids specified according to the EU numbering; an 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 a 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] In addition, 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 the 10 types of IgG. Furthermore, the desired combination ​​The secretion amount of the matched product also theoretically decreases significantly, so a large culture scale is required, and the manufacturing cost further increases. The manufacturing cost further increases.

[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")), so that the protrusion can be arranged in the void, promoting the formation of heterologous H chains and inhibiting 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 genes of this common L chain and both H chains 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 containing 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). Replace the amino acid side chain present in the CH3 region of one H chain with a larger side chain (knob (meaning "protrusion")), and replace the amino acid side chain present in the CH3 region of the other H chain with a smaller side chain (hole (meaning "void")), so that the protrusion can be arranged in the void, promoting the formation of heterologous H chains and inhibiting 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). This is a method of promoting the formation of heterologous H chains and inhibiting the formation of homologous H chains by enabling the protrusion to be arranged in the void. (WO1996 / 027011, Ridgway JB et al., Protein Engineering (1996) 9, 617-621, Merchant AM et al. Nature Biotechnology (1998) 16, 677-681). chain formation (WO1996 / 027011, Ridgway JB et al., Protein Engineering (1996) 9, 617-621, Merchant AM et al. Nature Biotechnology (1998) 16, 677-681).

[0136] 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 genes of this common L chain and both H chains 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 containing 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). When arbitrarily selecting two types of antibodies, the possibility of containing 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). (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 a bispecific antibody. 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 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 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 embodiment, 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, 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 the amino acid sequence of the polypeptide The 357th amino acid specified according to EU numbering is lysine Two polypeptides are preferably used, characterized in that

[0141] In yet another aspect, as the domain containing the Fc region according to the present invention, 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 the amino acid sequence of the polypeptide The 356th amino acid specified according to EU numbering is lysine Two polypeptides are preferably used, characterized in that

[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 Among them, 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, according to EU numbering the 399th amino acid specified is lysine, and the 357th amino acid is lysine, characterized in that two polypeptides (in this embodiment, it may be aspartic acid instead of the 370th glutamic acid, or it may be aspartic acid at the 392nd position instead of the 370th glutamic acid) ), Two polypeptides constituting the Fc region, wherein among the amino acid sequences of one of the polypeptides the 409th amino acid specified according to EU numbering is aspartic acid, and the 439th a mino acid is glutamic acid. Among the amino acid sequences of the other polypeptide, according to EU numbering the 399th amino acid specified is lysine, and the 356th amino acid is lysine, characterized in that two polypeptides (in this embodiment, it may be aspartic acid at the 360th position, aspartic acid at the 392nd position or aspartic acid at the 439th position instead of the 439th glutamic acid) ), Two polypeptides constituting the Fc region, wherein among the amino acid sequences of one of the polypeptides the 370th amino acid specified according to EU numbering is glutamic acid, and the 439th a mino acid is glutamic acid. Among the amino acid sequences of the other polypeptide, according to EU numbering the 357th amino acid specified is lysine, and the 356th amino acid is lysine, characterized in that two polypeptides, or Two polypeptides constituting the Fc region, wherein among the amino acid sequences of one of the polypeptides the 409th amino acid specified according to EU numbering is aspartic acid, and the 370th a ​The amino acid is glutamic acid, the amino acid at position 439 is glutamic acid, and the amino acid sequence of the other polypeptide, the amino acid at position 399 identified according to EU numbering is lysine, and the amino acid at position 357 is lysine, and the amino acid at position 356 is lysine. Two polypeptides characterized by this (in this embodiment, the amino acid at position 370 does not have to be substituted 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. are preferably used.

[0143] Furthermore, 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 356 identified according to EU numbering in the amino acid sequence of one of the polypeptides is lysine, and the amino acid at position 435 identified according to EU numbering in the amino acid sequence of the other polypeptide is arginine and the amino acid at position 439 is glutamic acid are also preferably used. By using two polypeptides constituting the Fc region originating from the above bispecific antibody as the domain containing the Fc region according to the present invention, the antigen-binding domain and / or the CD3-binding domain according to the present invention can be arranged in a desired combination.

[0144] In the present specification, as the Fc region having 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.

[0145] Fc region with improved C-terminal heterogeneity ​ 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, and an Fc region is provided.

[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 preferred 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 T cell receptor δ chain (SEQ ID NO: 71) of RefSeq accession number AAA61033.1. It can be cited.

[0149] CD3 binding domain In this specification, 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 from the variable domain of one or more antibodies. Preferably, the CD3 binding domain comprises the light chain variable region (VL) of the CD3 antibody and the heavy chain variable region (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 thereof.

[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 light chain variable region (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 heavy chain variable region (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 light chain variable region (VL) and the heavy chain variable region (VH) of various known CD3 antibodies are preferably used. In addition, 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 light chain variable region (VL) and the heavy chain variable region (VH) of various known CD3 antibodies 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. variable region (VH) are preferably used. Further, 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. A CD3 binding domain derived from a CD3 antibody having desired properties obtained by immunizing a desired animal with the γ-chain, δ-chain or ε-chain constituting human CD3 by the above method can be appropriately used. omain... As described above, the CD3 antibody serving as the main origin is appropriately a humanized antibody or a human antibody, which is appropriately used. The structures of the γ-chain, δ-chain, or ε-chain that constitutes CD3 are such that their polynucleotide sequences are set forth in SEQ ID NO: 27 (NM_000073.2), 29 (NM_000732.4), and 31 (NM_000733.3), and their polypeptide sequences are 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-described (1) antigen-binding domain, (2) domain containing an Fc region with reduced binding activity to the Fcγ receptor, and (3) T-cell receptor complex-binding domain, and its structure is not limited as long as it includes them. 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 (1) antigen-binding domain and the (2) domain containing the Fc region 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 other aspects, 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 an Fv, more preferably an 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 constituting 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 constituting 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 this binding occurs is efficiently obtained.

[0153] Also, 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 that make up 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 that makes up 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. 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 that make up 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 that makes up 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.

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

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

[0157] In another aspect, in order to bind a bivalent CD3 binding domain to the antibody structure, a monovalent CD3 binding domain can be bound to each N-terminus of the two heavy chain variable regions of the antibody structure via a peptide bond. 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 to the N-terminus of one of the two heavy 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 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 to the N-terminus of one of the two heavy chain variable regions via a peptide bond. 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. A monovalent CD3 binding domain can be bound to each N-terminus of the two heavy chain variable regions of the antibody structure via a peptide bond. In another aspect, in order to bind a bivalent CD3 binding domain to the antibody structure, a monovalent CD3 binding domain can be bound to each N-terminus of the two heavy chain variable regions of the antibody structure via a peptide bond. In another aspect, in order to bind a bivalent CD3 binding domain to the antibody structure, a bivalent scFv, i.e., sc(Fv)2, can be bound to the N-terminus of one of the two heavy 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 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. 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. In another aspect, in order to bind a bivalent CD3 binding domain to the antibody structure, a bivalent scFv, i.e., sc(Fv)2, can be bound to the N-terminus of one of the two heavy chain variable regions via a peptide bond. In another aspect, in order to bind a monovalent CD3 binding domain to the antibody structure, a monovalent scFv can be bound to the N-terminus of one of the two heavy chain variable regions via a peptide bond. A monovalent scFv can be bound to the N-terminus of one of the two heavy chain variable regions via a peptide bond. 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. 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 another aspect, in order to bind a monovalent CD3 binding domain to the antibody structure, a monovalent scFv can be bound to the N-terminus of one of the two heavy chain variable regions via a peptide bond.

[0158] In addition, when preparing the above polypeptide aggregate, each domain can be bound directly via a peptide bond, or each domain can be bound via a peptide bond through 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. Also, hydrogen bonds, disulfide bonds, covalent bonds, ionic interactions or the like 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. For example, linkers having peptide tags such as His tag, HA tag, myc tag, FLAG tag, etc. can also be appropriately used. In addition, when preparing the above polypeptide aggregate, each domain can be bound directly via a peptide bond, or each domain can be bound via a peptide bond through a peptide linker. The property of binding to each other by the combination of them can also be suitably utilized. For example, the affinity between CH1 and CL of an antibody is utilized, or the Fc region derived from the aforementioned bispecific antibody is used upon the association of the heterologous Fc regions. Furthermore, as described in the examples, the disulfide bonds formed between domains can also be suitably utilized. The affinity between CH1 and CL of an antibody is utilized, or the Fc region derived from the aforementioned bispecific antibody is used upon the association of the heterologous Fc regions. Furthermore, as described in the examples, the disulfide bonds formed between domains can also be suitably utilized. Another structure of the polypeptide aggregate according to the present invention includes, for example, (1) a structure in which the antigen-binding domain is a monovalent Fv and a monovalent Fab, which is also suitably used. In this case,

[0159] Another structure of the polypeptide aggregate according to the present invention includes, for example, (1) a structure in which the antigen-binding domain is a monovalent Fv and a monovalent Fab, which is also suitably used. In this case, the binding activity to the Fcγ receptor of (2) the polypeptide aggregate according to the present invention is reduced, and one of the two Fc regions is linked to the heavy chain CH1 region via a peptide bond. the binding activity to the Fcγ receptor of (2) the polypeptide aggregate according to the present invention is reduced, and one of the two Fc regions is linked to the heavy chain CH1 region via a peptide bond. Either the heavy chain Fv fragment (VH) or the 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. Either the heavy chain Fv fragment (VH) or the 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. By this, 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. By this, 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. At 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. At 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 aggregate having a structure in which the heavy chain CH1 region is linked to one of the two Fc regions constituting the polypeptide aggregate via a peptide bond, and sc(Fv)2 is linked to the other Fc region via a peptide bond can be produced. a polypeptide aggregate having a structure in which the heavy chain CH1 region is linked to one of the two Fc regions constituting the polypeptide aggregate via a peptide bond, and sc(Fv)2 is linked to the other Fc region via a peptide bond can be produced. When producing the above polypeptide aggregate, each In addition to being directly linked by a peptide bond, 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, linkers having peptide tags such as His tag, HA tag, myc tag, FLAG tag, etc. can also be appropriately used. In this case, as the linker to be 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. In addition to being directly linked by a peptide bond, 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, linkers having peptide tags such as His tag, HA tag, myc tag, FLAG tag, etc. can also be appropriately used. In addition to being directly linked by a peptide bond, 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, 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 aspect of such a structure, one of the bivalent scFvs is linked by a peptide bond to one of the two Fc regions having reduced binding activity to (2) the Fcγ receptor via 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 having reduced binding activity to (2) the Fcγ receptor via 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 polypeptide aggregate, in addition to being directly linked by a peptide bond, 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, 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 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 aspect of such a structure, one of the bivalent scFvs is linked by a peptide bond to one of the two Fc regions having reduced binding activity to (2) the Fcγ receptor via 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 having reduced binding activity to (2) the Fcγ receptor via 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 polypeptide aggregate, in addition to being directly linked by a peptide bond, 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, 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 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 aspect of such a structure, one of the bivalent scFvs is linked by a peptide bond to one of the two Fc regions having reduced binding activity to (2) the Fcγ receptor via 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 having reduced binding activity to (2) the Fcγ receptor via 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 polypeptide aggregate, in addition to being directly linked by a peptide bond, 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, 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 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 aspect of such a structure, one of the bivalent scFvs is linked by a peptide bond to one of the two Fc regions having reduced binding activity to (2) the Fcγ receptor via 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 having reduced binding activity to (2) the Fcγ receptor via 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 polypeptide aggregate, in addition to being directly linked by a peptide bond, 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, 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 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 aspect of such a structure, one of the bivalent scFvs is linked by a peptide bond to one of the two Fc regions having reduced binding activity to (2) the Fcγ receptor via 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 having reduced binding activity to (2) the Fcγ receptor via 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 polypeptide aggregate, in addition to being directly linked by a peptide bond, 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, 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 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 aspect of such a structure, one of the bivalent scFvs is linked by a peptide bond to one of the two Fc regions having reduced binding activity to (2) the Fcγ receptor via 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 having reduced binding activity to (2) the Fcγ receptor via 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 polypeptide aggregate, in addition to being directly linked by a peptide bond, 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, 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 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 aspect of such a structure, one of the bivalent scFvs is linked by a peptide bond to one of the two Fc regions having reduced binding activity to (2) the Fcγ receptor via 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 having reduced binding activity to (2) the Fcγ receptor via 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 polypeptide aggregate, in addition to being directly linked by a peptide bond, 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, linkers having peptide tags such as His tag, HA tag, myc tag, FLAG tag, etc. can also be appropriately used. In this case, it is also possible to use the Fc region derived from the above-mentioned bispecific antibody. When producing the above polypeptide aggregate, in addition to being directly linked by a peptide bond, 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, linkers having peptide tags such as His tag, HA tag, myc tag, FLAG tag, etc. can also be appropriately used. When producing the above polypeptide aggregate, in addition to being directly linked by a peptide bond, 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, linkers having peptide tags such as His tag, HA tag, myc tag, FLAG tag, etc. can also be appropriately used. When producing the above polypeptide aggregate, in addition to being directly linked by a peptide bond, 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, linkers having peptide tags such as His tag, HA tag, myc tag, FLAG tag, etc. can also be appropriately used. When producing the above polypeptide aggregate, in addition to being directly linked by a peptide bond, 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, linkers having peptide tags such as His tag, HA tag, myc tag, FLAG tag, etc. can also be appropriately used. When producing the above polypeptide aggregate, in addition to being directly linked by a peptide bond, 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, linkers having peptide tags such as His tag, HA tag, myc tag, FLAG tag, etc. can also be appropriately used.

[0161] (1) As another aspect 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 with reduced binding activity to the (2) Fcγ receptor via the scFv constituting the (3) CD3 binding domain and the other of the bivalent scFvs has a structure in which it is linked by a peptide bond to the other of the two Fc regions with reduced binding activity to the (2) Fcγ receptor A polypeptide conjugate can be prepared. In this case, by using the Fc region derived from the above bispecific antibody, one of the two Fc regions constituting the polypeptide conjugate has an scFv constituting an antigen-binding domain via an scFv constituting a CD3 binding domain, and the other Fc region has an scFv constituting an antigen-binding domain linked thereto via a peptide bond, respectively, to obtain a polypeptide conjugate having a structure. When preparing the above polypeptide conjugate, each domain is directly bound by a peptide bond, and each domain can also be bound 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, an HA tag, a myc tag, a FLAG tag, etc. can also be appropriately used. Another structure of the polypeptide conjugate according to the present invention includes, for example, a structure in which the antigen-binding domain and the T cell receptor complex-binding domain are each a monovalent Fab, which 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 A polypeptide conjugate having a structure in which the scFv constituting the antigen-binding domain is linked to the other Fc region via a peptide bond can be prepared. When preparing the above polypeptide conjugate, each domain is directly bound by a peptide bond, and each domain can also be bound 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, an HA tag, a myc tag, a FLAG tag, etc. can also be appropriately used. When preparing the polypeptide conjugate, each domain is directly bound by a peptide bond, and each domain can also be bound 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, an HA tag, a myc tag, a FLAG tag, etc. can also be appropriately used. 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, an HA tag, a myc tag, a FLAG tag, etc. can also be appropriately used. In addition to the linkers exemplified above, a linker having a peptide tag such as a His tag, an HA tag, a myc tag, a FLAG tag, etc. can also be appropriately 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.

[0162] Another structure of the polypeptide conjugate according to the present invention includes, for example, a structure in which the antigen-binding domain and the T cell receptor complex-binding domain are each a monovalent Fab, which 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. to the other polypeptide constituting the Fc region, and the light chain Fv fragment of the Fab is linked to the CL 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. Further, 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. A monovalent Fab heavy chain Fv fragment that constitutes a body-binding domain is linked to one polypeptide that constitutes the Fc region via the CH1 region, and the light chain Fv fragment of the Fab is linked to the CL region, and the antigen-binding domain The heavy chain Fv fragment of the Fab that constitutes the Fab is linked to the other polypeptide that constitutes the Fc region via the CL region A polypeptide assembly having a structure in which the light chain Fv fragment of the Fab is linked to the CH1 region can be produced.

[0165] 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 An antigen-binding domain in which the light chain Fv fragment of the Fab structure is linked to the CL region, 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 A T cell receptor complex-binding domain in which the light chain Fv fragment of the Fab structure is linked to the CL region, Including, 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 A polypeptide in which the charges of the CH1 region and the CL region are controlled so as to associate is preferably mentioned. In this embodiment, 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 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 If so, it is sufficient, 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 charge as each other. 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 charge as each other. A polypeptide conjugate can be prepared.

[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 charge as each other. 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 charge as each other. A polypeptide conjugate can be prepared.

[0168] As yet 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 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 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 charge as each other. 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 charge as each other. 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 charge as each other. 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 charge as each other. A polypeptide conjugate can be prepared.

[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 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 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 charge as each other. 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 charge as each other. ​​​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 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.

[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 in it and the light chain amino acid residues of the CL region linked to the Fv fragment have the same charge as each other, and the antigen-binding do The amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the main antigen-binding domain and the T cell receptor complex The amino acid residues of the CL region linked to the light chain Fv fragment in the binding domain have the same charge as each other And a polypeptide aggregate 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 antigen-binding domain both have different charges from each other.

[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 charge as each other, and the antigen-binding 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 antigen-binding The amino acid residues of the CH1 region linked to the heavy chain Fv fragment in the domain and the T cell receptor complex The amino acid residues of the CL region linked to the light chain Fv fragment in the binding 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 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, 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 charges from each other A polypeptide aggregate can be prepared.

[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, which is not the target, and the CL of the light chain of the antigen binding domain 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, which is not the target, and the CL of the light chain of the T cell receptor binding domain 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] 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, which is not the target, and the CL of the light chain of the antigen binding domain 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, which is not the target, and the CL of the light chain of the T cell receptor binding domain 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.

[0176] 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, which is not the target, and the CL of the light chain of the antigen binding domain 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, which is not the target, and the CL of the light chain of the T cell receptor binding domain 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. 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, which is not the target, and the CL of the light chain of the antigen binding domain 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, which is not the target, and the CL of the light chain of the T cell receptor binding domain 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. 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, which is not the target, and the CL of the light chain of the antigen binding domain 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, which is not the target, and the CL of the light chain of the T cell receptor binding domain 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. 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, which is not the target, and the CL of the light chain of the antigen binding domain 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, which is not the target, and the CL of the light chain of the T cell receptor binding domain 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. 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, which is not the target, and the CL of the light chain of the antigen binding domain 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, which is not the target, and the CL of the light chain of the T cell receptor binding domain 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. 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, which is not the target, and the CL of the light chain of the antigen binding domain 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, which is not the target, and the CL of the light chain of the T cell receptor binding domain 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. 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, which is not the target, and the CL of the light chain of the antigen binding domain 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, which is not the target, and the CL of the light chain of the T cell receptor binding domain 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. 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, which is not the target, and the CL of the light chain of the antigen binding domain 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, which is not the target, and the CL of the light chain of the T cell receptor binding domain 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. 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, which is not the target, and the CL of the light chain of the antigen binding domain 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, which is not the target, and the CL of the light chain of the T cell receptor binding domain 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. 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, which is not the target, and the CL of the light chain of the antigen binding domain is inhibited because both amino acid residues forming the interface are positively charged, and the association between the CH1 of the​ 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] Those 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, those skilled in the art can appropriately obtain sequences that can be used as CH1 or CL of an antibody in organisms such as humans, monkeys, mice, and rabbits by using public databases 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, at the interface of CH1 and CL 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 with each other ​​​​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 the EU numbering in the present invention are in accordance with 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 the EU numbering in the present invention are in accordance with EU numbering (Sequences of proteins of immunological interest, NIH Publication No. 91 - 3242). ​​​It is 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 an arbitrary 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". 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.

[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. As a result, 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. That is, 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. That is, 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. That is, the present invention provides a polypeptide aggregate in which the association of the heavy chain and the light chain is controlled, and one 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) have the same charge;

[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 one 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) have the same charge; (a) The amino acid residue contained in CH1 and corresponding to the amino acid residue at position 147 of EU numbering, and the amino acid residue contained in CL and corresponding to the amino acid residue at position 180 of EU numbering, (b) The amino acid residue contained in CH1 and corresponding to the amino acid residue at position 147 of EU numbering, and the amino acid residue contained in CL and corresponding to the amino acid residue at position 131 of EU numbering, (c) The amino acid residue contained in CH1 and corresponding to the amino acid residue at position 147 of EU numbering, and the amino acid residue contained in CL and corresponding to the amino acid residue at position 180 of EU numbering, (d) The amino acid residue contained in CH1 and corresponding to the amino acid residue at position 147 of EU numbering, and the amino acid residue contained in CL and corresponding to the amino acid residue at position 131 of EU numbering, (e) The amino acid residue contained in CH1 and corresponding to the amino acid residue at position 147 of EU numbering, and the amino acid residue contained in CL and corresponding to the amino acid residue at position 180 of EU numbering, (f) The amino acid residue contained in CH1 and corresponding to the amino acid residue at position 147 of EU numbering, and the amino acid residue contained in CL and corresponding to the amino acid residue at position 131 of EU numbering; (c) The amino acid residue contained in CH1 and corresponding to the amino acid residue at position 147 of EU numbering, and the amino acid residue contained in CL and corresponding to the amino acid residue at position 180 of EU numbering, an amino acid residue at position 164 in the EU numbering system; (d) an amino acid residue at position 147 (EU numbering) contained in CH1, and CL an amino acid residue at position 138 according to EU numbering, (e) an amino acid residue at position 147 (EU numbering) contained in CH1, and CL an amino acid residue at position 123 in the EU numbering system; (f) an amino acid residue at position 175 (EU numbering) contained in CH1, and CL The amino acid residue at position 160 in the EU numbering system is included in

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

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

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

[0187] In the above 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 present invention's association control method for modifying the amino acid residues of the amino acid residue sets 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] In the present invention, the amino acid residues to be "modified" are not limited to the amino acid residues of the above-described constant region. A person skilled in the art can find amino acid residues forming an interface for a polypeptide variant or a heteromultimer by homology modeling or the like using commercially available software, and can subject the amino acid residues at the site to modification so as to control the 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, as amino acid residues that come into contact at the interface between the heavy chain variable region (VH) and the light chain variable region (VL), for example, the 39th position of the FR2 of the heavy chain variable region (for example, WO2006 / 106905 the glutamine (Q) at position 39 in the amino acid sequence described as SEQ ID NO: 6 and the 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, the 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 the 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. 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). 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, by modifying the amino acid residues corresponding to the above amino acid residues the association of the variable regions of the antibody can be controlled.

[0191] More specifically, in an antibody comprising a heavy chain variable region and a light chain variable region, the following (1 ) and (2), or (3) and (4) may be mentioned as antibodies in which the amino acid residues have the same kind of charge; (1) an amino acid residue contained in the heavy chain variable region, which corresponds to position 39 in EU numbering

[0192] (1) an amino acid residue contained in the heavy chain variable region, which corresponds to the amino acid residue at position 39 ​ (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 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 those 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). Note that the types of the amino acid residues in the above (1) to (4) ​​​, not necessarily limited to the above amino acid residues, and other amino acids corresponding to the amino acids may also be used. For example, as the amino acid corresponding to position 38 of the EU numbering on the light chain variable region, in the case of human, for example, it may be histidine (H). Those skilled in the art can refer to known documents etc. (for example, J. Mol. Recognit. (2003) 16, 113-120) to know the types of amino acid residues corresponding to any position of the light chain, and can appropriately modify the amino acid residues (for example, substitute with charged amino acids). In the technology of modifying the amino acid residues forming the hydrophobic core existing at the interface between the heavy chain variable region and the light chain variable region into polar amino acids with a charge to suppress the association of the unwanted heavy chain and light chain, the amino acid residues that can form 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 position 45 on the heavy chain variable region and proline (P) at position 44 on the corresponding light chain variable region, which can be preferably exemplified. 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 on the outside, is the driving force for promoting the association of water-soluble polypeptides. is not necessarily limited to the above amino acid residues, and other amino acids corresponding to the amino acids may also be used. For example, as the amino acid corresponding to position 38 of the EU numbering on the light chain variable region, in the case of human, for example, it may be histidine (H). Those skilled in the art can refer to known documents etc. (for example, J. Mol. Recognit. (2003) 16, 113-120) to know the types of amino acid residues corresponding to any position of the light chain, and can appropriately modify the amino acid residues (for example, substitute with charged amino acids). is not necessarily limited to the above amino acid residues, and other amino acids corresponding to the amino acids may also be used. For example, as the amino acid corresponding to position 38 of the EU numbering on the light chain variable region, in the case of human, for example, it may be histidine (H). Those skilled in the art can refer to known documents etc. (for example, J. Mol. Recognit. (2003) 16, 113-120) to know the types of amino acid residues corresponding to any position of the light chain, and can appropriately modify the amino acid residues (for example, substitute with charged amino acids). is not necessarily limited to the above amino acid residues, and other amino acids corresponding to the amino acids may also be used. For example, as the amino acid corresponding to position 38 of the EU numbering on the light chain variable region, in the case of human, for example, it may be histidine (H). Those skilled in the art can refer to known documents etc. (for example, J. Mol. Recognit. (2003) 16, 113-120) to know the types of amino acid residues corresponding to any position of the light chain, and can appropriately modify the amino acid residues (for example, substitute with charged amino acids).

[0196] is not necessarily limited to the above amino acid residues, and other amino acids corresponding to the amino acids may also be used. For example, as the amino acid corresponding to position 38 of the EU numbering on the light chain variable region, in the case of human, for example, it may be histidine (H). Those skilled in the art can refer to known documents etc. (for example, J. Mol. Recognit. (2003) 16, 113-120) to know the types of amino acid residues corresponding to any position of the light chain, and can appropriately modify the amino acid residues (for example, substitute with charged amino acids). In the technology of modifying the amino acid residues forming the hydrophobic core existing at the interface between the heavy chain variable region and the light chain variable region into polar amino acids with a charge to suppress the association of the unwanted heavy chain and light chain, the amino acid residues that can form 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 position 45 on the heavy chain variable region and proline (P) at position 44 on the corresponding light chain variable region, which can be preferably exemplified. In the technology of modifying the amino acid residues forming the hydrophobic core existing at the interface between the heavy chain variable region and the light chain variable region into polar amino acids with a charge to suppress the association of the unwanted heavy chain and light chain, the amino acid residues that can form 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 position 45 on the heavy chain variable region and proline (P) at position 44 on the corresponding light chain variable region, which can be preferably exemplified. In the technology of modifying the amino acid residues forming the hydrophobic core existing at the interface between the heavy chain variable region and the light chain variable region into polar amino acids with a charge to suppress the association of the unwanted heavy chain and light chain, the amino acid residues that can form 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 position 45 on the heavy chain variable region and proline (P) at position 44 on the corresponding light chain variable region, which can be preferably exemplified. In the technology of modifying the amino acid residues forming the hydrophobic core existing at the interface between the heavy chain variable region and the light chain variable region into polar amino acids with a charge to suppress the association of the unwanted heavy chain and light chain, the amino acid residues that can form 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 position 45 on the heavy chain variable region and proline (P) at position 44 on the corresponding light chain variable region, which can be preferably exemplified.

[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 on the outside, is the 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 on the outside, is the 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 on the outside, is the 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 on the outside, is the 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 on the outside, is the 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 on the outside, is the driving force for promoting the association of water-soluble polypeptides. This results in the presence of hydrophobic amino acids on the molecular surface of two different domains, which are exposed to water molecules. This increases entropy and free energy. Therefore, in order to decrease free energy and achieve stabilization, the two domains associate with each other, and the hydrophobic amino acids at the interface become buried inside the molecule, forming a hydrophobic core. When the polypeptide association occurs, by modifying the hydrophobic amino acids that form the hydrophobic core to polar amino acids carrying a charge, the formation of the hydrophobic core is inhibited, and as a result, it is considered that the polypeptide association is inhibited. It is possible to apply other known techniques to the polypeptide aggregates of the present invention. For example, in addition to the "modification" of the present invention, in order to promote the association of the first VH (VH1) and the first VL (VL1), and / or the second VH (VH2) and the second VL (VL2), the amino acid side chains present in the variable region of one H chain are replaced with larger side chains (knob; protrusion), and the amino acid side chains present in the corresponding variable region of the other H chain are replaced with smaller side chains (hole; void), so that the protrusion can be arranged in the void to promote the association of VH1 and VL1, and / or VH2 and VL2, and as a result, further suppress the association between the 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).

[0198] When the polypeptide association occurs, by modifying the hydrophobic amino acids that form the hydrophobic core to polar amino acids carrying a charge, the formation of the hydrophobic core is inhibited, and as a result, the polypeptide association is inhibited. It is possible to apply other known techniques to the polypeptide aggregates of the present invention. For example, in addition to the "modification" of the present invention, in order to promote the association of the first VH (VH1) and the first VL (VL1), and / or the second VH (VH2) and the second VL (VL2), the amino acid side chains present in the variable region of one H chain are replaced with larger side chains (knob; protrusion), and the amino acid side chains present in the corresponding variable region of the other H chain are replaced with smaller side chains (hole; void), so that the protrusion can be arranged in the void to promote the association of VH1 and VL1, and / or VH2 and VL2, and as a result, further suppress the association between the 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). It is possible to apply other known techniques to the polypeptide aggregates of the present invention. For example, in addition to the "modification" of the present invention, in order to promote the association of the first VH (VH1) and the first VL (VL1), and / or the second VH (VH2) and the second VL (VL2), the amino acid side chains present in the variable region of one H chain are replaced with larger side chains (knob; protrusion), and the amino acid side chains present in the corresponding variable region of the other H chain are replaced with smaller side chains (hole; void), so that the protrusion can be arranged in the void to promote the association of VH1 and VL1, and / or VH2 and VL2, and as a result, further suppress the association between the 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).

[0199] Other known techniques can be further applied to the polypeptide aggregates of the present invention. For example, in addition to the "modification" of the present invention, in order to promote the association of the first VH (VH1) and the first VL (VL1), and / or the second VH (VH2) and the second VL (VL2), the amino acid side chains present in the variable region of one H chain are replaced with larger side chains (knob; protrusion), and the amino acid side chains present in the corresponding variable region of the other H chain are replaced with smaller side chains (hole; void), so that the protrusion can be arranged in the void to promote the association of VH1 and VL1, and / or VH2 and VL2, and as a result, further suppress the association between the 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). The amino acid side chains present in the variable region of one H chain are replaced with larger side chains (knob; protrusion), and the amino acid side chains present in the corresponding variable region of the other H chain are replaced with smaller side chains (hole; void), so that the protrusion can be arranged in the void to promote the association of VH1 and VL1, and / or VH2 and VL2, and as a result, further suppress the association between the 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). The protrusion can be arranged in the void to promote the association of VH1 and VL1, and / or VH2 and VL2, and as a result, further suppress the association between the 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). 9, 617 - 621, Merchant AM et al. Nature Biotechnology (1998) 16, 677 - 681).

[0200] When preparing the above-mentioned polypeptide complex, each domain is directly linked by a peptide bond In addition, 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, 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 the 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 .. .. .. ..

[0201] Examples of the polypeptide complex according to the present invention include the embodiments described in FIGS. 17, 19, and 24 ..

[0202] The polypeptide complex according to the present invention is prepared by the same method as the method for producing the recombinant antibody ..

[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 a suitable host with the expression vector it is possible to obtain an expression cell of the polypeptide complex. 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, the Cells harboring the vector, and the polypeptide complexes obtained by culturing the cells and recovering them from the culture supernatant These methods include, for example, producing polypeptide complexes using the recombinant It can be obtained by the same techniques as for antibodies.

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

Claims

1. The following domains: (1) A CD20-binding domain, (2) An Fc region containing amino acid mutations in the Fc region of a human IgG1 antibody, wherein among the amino acids constituting the Fc region of the human IgG1 antibody, leucine at position 234 specified according to EU numbering is substituted with valine, phenylalanine, or alanine, leucine at position 235 is substituted with glutamic acid or alanine, and aspartic acid at position 265 is substituted with alanine, and there are no other mutations in the CH2 region, and (3) A CD3-binding domain, A bispecific antibody comprising, wherein the CD20-binding domain and the CD3-binding domain are each a monovalent Fab, the heavy chain Fv fragment of the Fab constituting the CD20-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, the heavy chain Fv fragment of the Fab constituting the CD3-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. A bispecific antibody.

2. The bispecific antibody according to claim 1, wherein the binding activity to the Fcγ receptor is reduced as compared with a control bispecific antibody having the Fc region of a human IgG1 antibody without the amino acid mutations.

3. The bispecific antibody according to claim 2, wherein the Fc region is an Fc region having reduced binding activity to any one of the Fcγ receptors FcγI, FcγIIA, FcγIIB, FcγIIIA, and / or FcγIIIB.

4. The bispecific antibody according to any one of claims 1 to 3, having the CH1 region of a human IgG1 antibody.

5. The bispecific antibody according to any one of claims 1 to 4, having the hinge region of a human IgG1 antibody.

6. The bispecific antibody according to any one of claims 1 to 5, which is a chimeric antibody, a humanized antibody, or a human antibody.

7. The bispecific antibody according to any one of claims 1 to 6, which is a T cell engager.

8. A cancer therapeutic agent comprising the bispecific antibody according to any one of claims 1 to 7 as an active ingredient.

9. The cancer therapeutic agent according to claim 8, which does not induce or reduces the induction of a CD20-independent cytokine storm.

10. A pharmaceutical composition for treating or preventing cancer, comprising as an active ingredient the bispecific antibody according to any one of claims 1 to 7.

11. The pharmaceutical composition according to claim 10, for not inducing or reducing the induction of a CD20-independent cytokine storm.

Images