Antibody cleavage site binding molecules

Abstract

The present disclosure provides a pharmaceutical composition that is to be used in combination with administration of an antigen-binding molecule capable of binding to a target antigen, and that contains a cell which expresses an antibody having ADCC activity, a T cell-redirecting antibody, or a chimera receptor. The antigen-binding molecule as a primary molecule contains a linker which is to be cleaved by a protease. The antigen-binding molecule in which the linker is cleaved has the ability to bind to the target antigen. A variable domain of the antibody having ADCC activity or the T cell-redirecting antibody, and an extracellular binding domain of the chimera receptor, bind to a cell that expresses the target antigen, through binding to the antigen-binding molecule, in which the linker is cleaved, obtained after cleavage of the cleaved linker.

Description

[Technical Field] 【0001】 This disclosure relates to antibodies having ADCC activity, T cell redirecting antibodies, chimeric receptors, cells expressing chimeric receptors, and methods for treating diseases using such cells or antibodies, particularly therapies utilizing the ADCC activity of antibodies, CAR-T therapy using such cells, and T cell redirecting antibody therapy. [Background technology] 【0002】 Antibody drugs are pharmaceuticals whose main component is immunoglobulin or its analogues, which are part of the body's immune system (Non-Patent Literature 1 and Non-Patent Literature 2). Compared to conventional small molecule compounds, antibody drugs have a larger molecular weight and are capable of complex molecular recognition, resulting in high specificity of target recognition and fewer unexpected side effects. Furthermore, while foreign substances in the blood are generally taken up and broken down by cells through endocytosis, antibodies have a recovery mechanism via a specific receptor FcRn and antibody Fc region, resulting in longer retention in the bloodstream and the characteristic of providing long-lasting therapeutic effects with a single administration. In addition, since antibody drugs are prepared as recombinant proteins, their function can be modified using genetic engineering. 【0003】 For example, antibody-dependent cell-mediated cytotoxicity (ADCC), which is induced by the binding of the constant region of an antibody to FcγR on NK cells or macrophages, is known to be more strongly induced when an antibody with a modified constant region that enhances binding to FcγR is used (Non-Patent Literature 3). 【0004】 Furthermore, while ordinary antibodies only recognize and bind to one epitope of an antigen, by improving naturally occurring IgG-type antibodies, antibodies that can bind to two or more antigens with a single molecule (called bispecific antibodies) have been developed (Non-Patent Literature 11), making it possible to bind proteins expressed on T cells (CD3 epsilon and TCR) and proteins expressed on cancer cells (cancer antigens). As one type of bispecific antibody, T cell-redirecting antibodies, which employ cytotoxicity that recruits T cells as effector cells as a mechanism for their antitumor effect, have been known since the 1980s (Non-Patent Literature 12, 13, 14). Unlike antibodies that utilize ADCC, which recruits NK cells or macrophages as effector cells, as the mechanism of their antitumor effect, T-cell redirecting antibodies can bind to any of the constituent subunits of the T cell receptor (TCR) complex, particularly the domain that binds to the CD3 epsilon chain, and to the antigen on the target cancer cell. This cross-links T cells and cancer antigen-expressing cells, inducing strong cytotoxicity (T-cell dependent cellular cytotoxicity; TDCC) against cancer antigen-expressing cells by using T cells as effector cells (Non-patent Literature 4, 12, 13, 14). 【0005】 Furthermore, in recent years, antitumor therapies that utilize the high specificity of antibodies for antigen recognition by effector cells have been developed, showing remarkable efficacy in several types of cancer. In this treatment method, called chimeric antigen receptor (CAR) adoptive immunotherapy, an intracellular signaling domain is artificially fused to an extracellular domain with antigen-binding ability, such as scFv derived from an antibody, to form a CAR, which is then expressed in effector cells such as T cells. When these CAR-expressing T cells (hereinafter also called "CAR-T cells") are transferred to a cancer patient, and tumor antigens are recognized by the CAR, the intracellular domain is activated, inducing cytotoxic activity in effector cells, thereby damaging tumor cells and exerting therapeutic effects (Non-Patent Literature 5). Clinical trials have been conducted on cancer immunotherapy using CAR-T cells (Non-Patent Literature 10), and results have shown that CAR-T cell immunotherapy is effective in hematopoietic malignancies such as leukemia and lymphoma. In 2017, Kymriah® (Novartis, tisagenlecleucel, CTL-019, CD3 zeta-CD137) and Yescarta® (KiTE, axicabtagene ciloleucel, CD3 zeta-CD28), which use CD19 as an antigen, were approved as pharmaceuticals in the United States, and in 2019, they were approved in Japan. 【0006】 In addition, methods have been proposed that utilize single-domain antibodies derived from camelid animals to target a variety of antigens and to simplify antibody preparation. Overall, antibody drugs have numerous advantages and are therefore applied to a wide range of diseases, including tumors, autoimmune diseases, and infectious diseases (Non-Patent Literature 6). 【0007】 On the other hand, limitations of antibody drugs have also been pointed out. One of these is the issue of antigen site specificity. Surface antigens targeted by antibodies may also be expressed in normal tissues other than the lesion site, and although the expression level of the antigen is lower than in the lesion site, the antibody may act on the normal tissue expressing the antigen, potentially causing side effects. 【0008】 A potential solution to this problem is the identification of lesion sites by targeting lesion-specific protease activity. For example, it has been shown that lesion sites can be identified by protease activity by administering a protease substrate with a fluorescent dye attached to a synthetic peptide chain containing a protease cleavage site that is activated at the lesion site, and measuring the change in fluorescence associated with cleavage (Non-Patent Literature 7). 【0009】 Studies have been reported in which lesion sites were detected by recognizing protease cleavage products generated at the lesion site with antibodies (Non-Patent Literature 8). In this study, it was reported that cleavage products by IdeS protease expressed by actinomycetes could be specifically recognized in vivo using antibodies that specifically bind to these cleavage products (Non-Patent Literature 8). In the field of chronic diseases, with the aim of developing reagents to detect lesions in osteoarthritis of the knee, antibodies that recognize collagen II cleaved by MMPs (matrix metalloproteinases) activated at the site of osteoarthritis of the knee have been developed (Non-patent Literature 9). 【0010】 An example of applying existing protease-activated antibody technology to therapy is the "Probody® technology," which expands tissue specificity and the therapeutic window by conferring sensitivity to proteases whose expression and activation levels increase in lesion sites such as cancerous tissue and inflammatory tissue to antibodies (Figure 1). 【0011】 "Probody®" is a molecule formed by linking an antibody with a mask peptide that masks the antigen-binding site of the antibody, which is then cleaved by a protease expressed at the lesion site (Non-Patent Literature 15). When the peptide sequence is not cleaved, the antigen-binding site of the antibody is masked by the mask peptide and cannot bind to the antigen. When the cleaved peptide sequence of Probody® is cleaved by a protease expressed at the target disease site, the mask peptide dissociates, generating an antibody molecule with antigen-binding activity, which can then bind to the target disease tissue-specific antigen. Because antigen-antibody binding is inhibited in non-lesion sites where proteases are not present, Probody® can be administered in larger quantities than conventional antibodies, and is expected to expand the therapeutic window. [Prior art documents] [Patent Documents] 【0012】 [Patent Document 1] WO2009 / 025846 【Patent document 2】 WO2017 / 143094 【Patent document 3】 WO2018 / 097307 【Non-licensed literature】 【0013】 【Non-licensed literature 1】 Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Monoclonal antibody successes in the clinic., Nat. Biotechnol. (2005) 23, 1073 - 1078 [Non-licensed document 2] Pavlou AK, Belsey MJ., The therapeutic antibodies market to 2008., Eur J Pharm Biopharm. (2005) 59 (3), 389-396 [Non-licensed document 3] The impact of Fc engineering on an anti-CD19 antibody: increased Fcgamma receptor affinity enhances B-cell clearing in nonhuman primates. Zalevsky J, Leung IW, Karki S, Chu SY, Zhukovsky EA, Desjarlais JR, Carmichael DF, Lawrence CE. Blood. 2009 Apr 16;113(16):3735-43. 【Non-licensed Document 4】 Advances in bispecific biotherapeutics for the treatment of cancer Biochem Pharmacol. 2012 Nov 1;84(9):1105-12 【Non-licensed Document 5】 Chimeric Antigen Receptor Therapy N Engl J Med 2018; 379:64-73 [Non-Patent Document 6] Single-domain antibodies for biomedical applications. Immunopharmacol Immunotoxicol. 2016;38(1):21-8 [Non-Patent Document 7] Shedding light onto live molecular targets Nat Med. 2003 Jan;9(1):123-8 [Non-Patent Document 8] Structure and specificity of an antibody targeting a proteolytically cleaved IgG hinge Malia TJ1, Teplyakov A, Brezski RJ, Luo J, Kinder M, Sweet RW, Almagro JC, Jordan RE, Gilliland GL. Proteins. 2014 Aug;82(8):1656-67 [Non-Patent Document 9] Development of a novel immunoassay for the measurement of type II collagen neoepitope generated by collagenase cleavage Clin Chim Acta. 2012 Oct 9;413(19-20):1591-9. [Non-Patent Document 10] Grupp et al. 2013 N Engl J Med 368(16): 1509- 18. [Non-Patent Document 11] Kontermann, mAbs 2012;4:182-197. [Non-Patent Document 12] Mezzanzanica et al., International journal of cancer 1988;41:609-615. [Non-Patent Document 13] Staerz and Bevan, Proceedings of the National Academy of Sciences of the United States of America 1986;83:1453-1457. [Non-Patent Document 14] Staerz et al., Nature 1985;314:628-631. [Non-Patent Document 15] Desnoyers LR et al., Sci Transl Med. 2013 Oct 16;5(207):207ra144. [Overview of the project] [Problems that the invention aims to solve] 【0014】 Probody® exhibits cytotoxic activity even in normal tissues due to its high blood retention rate in activated Probody® and its antigen-binding activity even in an inactivated state that is not cleaved by proteases. Furthermore, while high cytotoxic activity can be obtained in T-cell redirecting antibody therapy and CAR-T therapy, this activity is also known to occur in normal cells, causing serious side effects, and improvements in the safety of these therapies have been sought. Furthermore, since a single tumor antigen is not universally expressed in all cancers, the antigen recognition site for these therapies needs to be constructed for each targeted tumor antigen, which presents significant challenges in terms of economic costs and labor involved in this process. Moreover, the targeted tumor antigen may decrease in expression or mutate in response to treatment, leading to immune evasion and resulting in reduced or complete loss of therapeutic effect. While research is being conducted on CAR-T cells and T-cell redirecting antibodies that can change the tumor antigen they recognize depending on the treatment stage, and on therapeutic methods utilizing these, there is a need for safer and more affordable treatments that have sufficient therapeutic efficacy and high safety when administered to patients, as well as technologies that can be used universally. [Means for solving the problem] 【0015】 To solve these problems, the inventors conducted extensive research and discovered that antibodies, T-cell redirecting antibodies, or CAR-T cells possessing ADCC activity that bind to a newly generated antigen-binding domain after cleavage by a protease selectively act on therapeutic target cells and are effective in treatment, thus completing the present invention. In one aspect of this disclosure, we disclose a group of versatile therapeutic molecules in which molecules containing an antigen-binding domain generated by a protease have a short blood half-life and do not exert pharmacological effects if cleavage by a protease does not occur. 【0016】 For example, this disclosure provides a pharmaceutical composition comprising a molecule having antigen-binding ability and a molecule having effector-activating ability, wherein the two molecules associate by being cleaved by a protease specifically expressed in a target tissue, thereby bridging the relationship between antigen-expressing target cells and effector cells and activating effector cells; a pharmaceutical composition for use in the treatment of diseases caused by a target tissue; and a molecule having antigen-binding ability and a molecule having effector-activating ability used as active ingredients of the pharmaceutical composition. Furthermore, a method for producing the pharmaceutical composition and the molecule having antigen-binding ability and the molecule having effector-activating ability used as active ingredients is also provided. 【0017】 Furthermore, this disclosure makes it possible to select the optimal therapy from multiple therapies, including CAR-T therapy, bispecific antibody therapy, and antibody therapy with ADCC activity, based on the patient's treatment suitability, or to modify or add therapies according to the treatment status, by using molecules that bind to tumor antigens as a common feature. 【0018】 Furthermore, this disclosure makes it possible to use multiple molecules that bind to tumor antigens, and by using them as a common element, to select the optimal therapy from among multiple therapies such as CAR-T therapy, bispecific antibody therapy, and antibody therapy with ADCC activity, according to the patient's treatment suitability, or to change or add therapies according to the treatment status. 【0019】 One aspect of this disclosure relates to an antibody, bispecific antibody, or CAR-T cell having ADCC activity against target cells expressing a target antigen, wherein the CAR-T cell or the bispecific antibody or the antibody having ADCC activity binds to the target cell via binding to an antigen-binding molecule. The antigen-binding molecule comprises a linker that is cleaved by a protease and has the ability to bind to the target antigen after cleavage of the linker by the protease. This disclosure relates to the CAR-T cell or the bispecific antibody or the antibody having ADCC activity. 【0020】 Further aspects of this disclosure relate to isolated nucleic acid molecules encoding ADCC-active antibodies, bispecific antibodies, and / or CARs, which can be used to target cells expressing a target antigen of interest. 【0021】 Further aspects of this disclosure relate to vectors containing antibodies having ADCC activity, bispecific antibodies, and / or isolated nucleic acid molecules encoding CARs, which can be used to target cells expressing a target antigen of interest. 【0022】 Further aspects of this disclosure relate to cells expressing the CARs of this disclosure, or cells transfected or transduced with the nucleic acid molecules or vectors of this disclosure. 【0023】 More specifically, in one aspect of this disclosure, the following invention is provided. 【0024】 [1] A pharmaceutical composition comprising cells expressing a chimeric receptor for use in combination with the administration of an antigen-binding molecule, The antigen-binding molecule contains a linker that is cleaved by a protease, and after the linker is cleaved, it has the ability to bind to the target antigen. A pharmaceutical composition comprising a chimeric receptor comprising an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular binding domain has the ability to bind to an antigen-binding molecule after linker cleavage, and can bind to cells expressing the target antigen via binding to the antigen-binding molecule after linker cleavage. 【0025】 [2] A pharmaceutical composition comprising an antigen-binding molecule for use in combination with the administration of cells expressing a chimeric receptor, The antigen-binding molecule contains a linker that is cleaved by a protease, and after the linker is cleaved, it has the ability to bind to the target antigen. A pharmaceutical composition comprising a chimeric receptor comprising an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular binding domain has the ability to bind to an antigen-binding molecule after linker cleavage, and can bind to cells expressing the target antigen via binding to the antigen-binding molecule after linker cleavage. 【0026】 [3] A pharmaceutical composition comprising a bispecific antibody for use in combination with the administration of an antigen-binding molecule, The antigen-binding molecule contains a linker that is cleaved by a protease, and after the linker is cleaved, it has the ability to bind to the target antigen. A bispecific antibody comprises an antibody variable region that has binding activity to antigen-binding molecules after the linker has been cleaved by a protease, and an antibody variable region that has binding activity to molecules expressed on the surface of T cells. A bispecific antibody is a pharmaceutical composition that can bind to cells expressing a target antigen via binding to an antigen-binding molecule after linker cleavage. 【0027】 [4] A pharmaceutical composition comprising an antigen-binding molecule for use in combination with the administration of a bispecific antibody, The antigen-binding molecule contains a linker that is cleaved by a protease, and after the linker is cleaved, it has the ability to bind to the target antigen. A bispecific antibody comprises an antibody variable region that has binding activity to antigen-binding molecules after the linker has been cleaved by a protease, and an antibody variable region that has binding activity to molecules expressed on the surface of T cells. A bispecific antibody is a pharmaceutical composition that can bind to cells expressing a target antigen via binding to an antigen-binding molecule after linker cleavage. 【0028】 [5] A pharmaceutical composition comprising an IgG antibody, characterized in that its antibody-dependent cell-mediated cytotoxicity is enhanced, for use in combination with the administration of an antigen-binding molecule, The antigen-binding molecule contains a linker that is cleaved by a protease, and has binding activity to the antigen expressed on the surface of target cells after the linker is cleaved by the protease. The IgG antibody comprises an antibody variable region having binding activity to the antigen-binding molecule after the linker has been cleaved by the protease. An IgG antibody is a pharmaceutical composition that can bind to target cells via binding to an antigen-binding molecule after linker cleavage. 【0029】 [6] A pharmaceutical composition comprising an antigen-binding molecule for use in combination with the administration of an IgG antibody characterized by enhanced antibody-dependent cytotoxicity, The antigen-binding molecule contains a linker that is cleaved by a protease, and after the linker is cleaved by the protease, it has binding activity to the antigen expressed on the surface of the target cell. IgG contains an antibody variable region that has binding activity to the antigen-binding molecule after the linker has been cleaved by a protease. An IgG antibody is a pharmaceutical composition that can bind to target cells via binding to an antigen-binding molecule after linker cleavage. 【0030】 [7] K of the antigen-binding molecule before linker cleavageD K for the antigen of the antigen after linker cleavage relative to the value D Ratio of values ​​(K D (After cutting) / K D A pharmaceutical composition according to any one of [1] to [6], wherein (before cleavage) is 0.1 or 0.01 or less. 【0031】 [8] The pharmaceutical composition according to any one of [1] to [7], wherein the antigen-binding molecule is an IgG antibody, an IgG antibody-like molecule, a heavy chain antibody, or a monodomain antibody. 【0032】 [9] The pharmaceutical composition according to any one of [1] to [8], wherein the antigen-binding molecule comprises a variable region and a constant region of an antibody, and a linker that is cleaved by a protease, wherein the antibody is selected from an IgG antibody, an IgG antibody-like molecule, or a heavy chain antibody, and the antigen-binding molecule whose linker is cleaved by a protease comprises an antigen-binding domain and a portion of the cleaved linker. 【0033】

[10] The pharmaceutical composition according to any one of [1] to [9], wherein the linker that is cleaved by the protease of the antigen-binding molecule is located near the boundary between the variable region and the constant region or near the boundary between CH1 and CH2 within the constant region. 【0034】

[11] The pharmaceutical composition according to any one of [1] to

[10] , wherein the antigen-binding molecule is an antibody or IgG antibody-like molecule containing a linker that is cleaved by a protease, and the antigen-binding molecule after linker cleavage is the VL, VH, VHH of the antibody or its antigen-binding fragment. 【0035】

[12] The pharmaceutical composition according to any one of [1] to

[11] , wherein the antigen-binding molecule is a single-domain antibody containing a linker that is cleaved by a protease, and the antigen-binding molecule after linker cleavage is a part of the antigen-binding domain and linker of the single-domain antibody. 【0036】

[13] A pharmaceutical composition according to any one of [1] to

[12] , wherein the linker cleaved by the protease comprises a protease cleavage sequence.

[14] A pharmaceutical composition according to any one of [1] to

[12] , wherein the linker cleaved by the protease is a peptide having one of the protease cleavage sequences of SEQ ID NOs. 1 to 725. 【0037】

[15] A pharmaceutical composition according to any one of [1] to

[14] for use in the treatment or prevention of cancer. 【0038】 [A1-1] A pharmaceutical composition comprising cells expressing a chimeric receptor for use in combination with the administration of an antigen-binding molecule, The antigen-binding molecule contains a linker that is cleaved by a protease, and after the linker is cleaved, it has the ability to bind to the target antigen. A pharmaceutical composition comprising a chimeric receptor comprising an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular binding domain has the ability to bind to an antigen-binding molecule after linker cleavage, and can bind to cells expressing the target antigen via binding to the antigen-binding molecule after linker cleavage. 【0039】 [A1-2] A pharmaceutical composition comprising an antigen-binding molecule for use in combination with the administration of cells expressing a chimeric receptor, The antigen-binding molecule contains a linker that is cleaved by a protease, and after the linker is cleaved, it has the ability to bind to the target antigen. A pharmaceutical composition comprising a chimeric receptor comprising an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular binding domain has the ability to bind to an antigen-binding molecule after linker cleavage, and can bind to cells expressing the target antigen via binding to the antigen-binding molecule after linker cleavage. 【0040】 [A1-3] K of the antigen-binding molecule before linker cleavage D K for the antigen of the antigen after linker cleavage relative to the value D Ratio of values ​​(K D (After cutting) / K DThe pharmaceutical composition according to [A1-1] or [A1-2], wherein (before cutting) is 0.1 or less or 0.01 or less. 【0041】 [A1-4] The pharmaceutical composition according to any one of [A1-1] to [A1-3], wherein the antigen-binding molecule is an IgG antibody, an IgG antibody-like molecule, a heavy chain antibody, or a monodomain antibody. 【0042】 [A1-5] A pharmaceutical composition according to any one of [A1-1] to [A1-4], wherein the antigen-binding molecule comprises a variable region and a constant region of an antibody, and a linker that is cleaved by a protease, wherein the antibody is selected from an IgG antibody, an IgG antibody-like molecule, or a heavy chain antibody, and the antigen-binding molecule whose linker is cleaved by the protease comprises an antigen-binding domain and a portion of the cleaved linker. 【0043】 [A1-6] A pharmaceutical composition according to any one of [A1-1] to [A1-4], wherein the antigen-binding molecule comprises the VHH of a single-domain antibody and a linker that is cleaved by a protease, and the antigen-binding molecule whose linker has been cleaved by the protease comprises an antigen-binding domain and a portion of the cleaved linker. 【0044】 [A1-7] A pharmaceutical composition according to any one of [A1-1] to [A1-6], wherein the linker cleaved by the protease of the antigen-binding molecule is located near the boundary between the variable region and the constant region, or near the boundary between CH1 and CH2 within the constant region. 【0045】 [A1-8] A pharmaceutical composition according to any one of [A1-1] to [A1-7], wherein the linker that is cleaved by the protease of the antigen-binding molecule is located near the hinge region. 【0046】 [A1-9] The pharmaceutical composition according to any one of [A1-1] to [A1-8], wherein the antigen-binding molecule is an antibody or IgG antibody-like molecule containing a linker that is cleaved by a protease, and the antigen-binding molecule after linker cleavage is the VL, VH, VHH, or antigen-binding fragment of the antibody. 【0047】 [A1-10] The pharmaceutical composition according to any one of [A1-1] to [A1-9], wherein the antigen-binding molecule is a heavy-chain antibody or a single-domain antibody containing a linker cleaved by a protease, and the antigen-binding molecule after linker cleavage is a VHH of the antigen-binding molecule or a part thereof containing an antigen-binding domain. 【0048】 [A1-11] The pharmaceutical composition according to any one of [A1-1] to [A1-10], wherein the antigen-binding molecule after linker cleavage is scFv, Fv, Fab, Fab’, F(ab’)2, VH or VHH. 【0049】 [A1-12] The pharmaceutical composition according to any one of [A1-1] to [A1-11], wherein the extracellular binding domain of the chimeric receptor recognizes a cleaved linker, a part of the linker, or a part containing the linker. 【0050】 [A1-13] The K D value for the antigen-binding molecule before linker cleavage of the extracellular binding domain of the chimeric receptor to the K D value for the antigen-binding molecule after linker cleavage of the linker, and the ratio of the K D (after cleavage) / K D (before cleavage)) is 0.1 or less or 0.01 or less, and the pharmaceutical composition according to any one of [A1-1] to [A1-12]. 【0051】 [A1-14] The pharmaceutical composition according to any one of [A1-1] to [A1-13], wherein the linker cleaved by a protease contains a protease cleavage sequence. 【0052】 [A1-15] The pharmaceutical composition according to any one of [A1-1] to [A1-14], wherein the linker cleaved by a protease contains a peptide having any one of the protease cleavage sequences of SEQ ID NOs: 1 to 725. 【0053】 [A1-16] The pharmaceutical composition according to any one of [A1-1] to [A1-15], wherein the linker cleaved by a protease further contains a flexible linker. 【0054】 [A1-17] The pharmaceutical composition according to any one of [A1-1] to [A1-16], wherein the protease is a protease that is specifically expressed in the target tissue. 【0055】 [A1-18] A pharmaceutical composition according to any one of [A1-1] to [A1-17], wherein the target cell is a tumor cell and the protease is a tumor protease. 【0056】 [A1-19] A pharmaceutical composition according to any of [A1-1] to [A1-18] for use in the treatment or prevention of cancer. 【0057】 [A1-20] The pharmaceutical composition according to [A1-19], wherein the cancer is selected from the group consisting of carcinoma, lymphoma, sarcoma, blastoma, and leukemia. 【0058】 [A1-21] The pharmaceutical composition according to [A1-19], wherein the cancer is selected from the group consisting of B-cell lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell non-Hodgkin lymphoma, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin lymphoma. 【0059】 [A1-22] A pharmaceutical composition according to any of [A1-1] to [A1-21] for use in CAR-T therapy. 【0060】 [A2-1] A chimeric receptor comprising an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular binding domain binds to the antigen-binding molecule after the linker has been cleaved by the protease, and the extracellular binding domain binds to the antigen-expressing cell via binding to the antigen-binding molecule after the linker has been cleaved. 【0061】 [A2-2] The chimeric receptor according to [A2-1], wherein the extracellular binding domain recognizes a linker cleaved by a protease, a part of the linker, or a part containing the linker. [A2-3] A chimeric receptor as described in [A2-1] or [A2-2], wherein the transmembrane domain contains CD28. [A2-4] A chimeric receptor according to any of [A2-1] to [A2-3], further comprising one or more costimulatory molecules located between the transmembrane domain and the intracellular signaling domain. 【0062】 [A2-5] The chimeric receptor according to [A2-4], wherein the costimulatory molecule is CD3 zeta, CD28, 4-1BB, 4-1BBL, ICOS, or OX40. [A2-6] A chimeric receptor according to any one of [A2-1] to [A2-5], wherein the intracellular signaling domain includes CD3 zeta. 【0063】 [A2-7] A chimeric receptor according to any one of [A2-1] to [A2-6], wherein the linker cleaved by the protease comprises a peptide having one of the protease cleavage sequences of SEQ ID NOs: 1 to 725. A nucleic acid encoding a chimeric receptor as described in any of [A2-8], [A2-1], or [A2-7]. 【0064】 A vector containing the nucleic acids described in [A2-9] and [A2-8]. Cells containing the vectors described in [A2-10] and [A2-8]. [A2-11] The cell described in [A2-9], wherein the cell is a T cell. [A2-12] The T cells are CD4 + or CD8 + The cells described in [A2-11] are T cells. 【0065】 [A2-13] The cell according to [A2-11], wherein the T cell is a regulatory T cell (Treg) or a follicular regulatory T cell (TFR). 【0066】 [A3-1] An antigen-binding molecule containing a linker that is cleaved by a protease, An antigen-binding molecule in which the antigen-binding molecule after linker cleavage has the ability to bind to an antigen, and the extracellular binding domain of the chimeric receptor can bind to target cells expressing the antigen via binding to the antigen-binding molecule after linker cleavage. 【0067】 [A3-2] The antigen-binding molecule according to [A3-1], comprising a peptide having one of the protease cleavage sequences of SEQ ID NOs: 1 to 725, which is cleaved by the protease. 【0068】 [B1-1] A pharmaceutical composition comprising a bispecific antibody for use in combination with the administration of an antigen-binding molecule, The antigen-binding molecule contains a linker that is cleaved by a protease, and after the linker is cleaved, it has the ability to bind to the target antigen. A bispecific antibody comprises an antibody variable region that has binding activity to antigen-binding molecules after the linker has been cleaved by a protease, and an antibody variable region that has binding activity to molecules expressed on the surface of T cells. A bispecific antibody is a pharmaceutical composition that can bind to cells expressing a target antigen via binding to an antigen-binding molecule after linker cleavage. 【0069】 [B1-2] A pharmaceutical composition containing an antigen-binding molecule for use in combination with the administration of a bispecific antibody, The antigen-binding molecule contains a linker that is cleaved by a protease, and after the linker is cleaved, it has the ability to bind to the target antigen. A bispecific antibody comprises an antibody variable region that has binding activity to antigen-binding molecules after the linker has been cleaved by a protease, and an antibody variable region that has binding activity to molecules expressed on the surface of T cells. A bispecific antibody is a pharmaceutical composition that can bind to cells expressing a target antigen via binding to an antigen-binding molecule after linker cleavage. 【0070】 [B1-3] K of the antigen-binding molecule before linker cleavageD K for the antigen of the antigen after linker cleavage relative to the value D Ratio of values ​​(K D (After cutting) / K D The pharmaceutical composition according to [B1-1] or [B1-2], wherein (before cleavage) is 0.1 or less or 0.01 or less. 【0071】 [B1-4] The pharmaceutical composition according to any one of [B1-1] to [B1-3], wherein the antigen-binding molecule is an IgG antibody or a heavy chain antibody. 【0072】 [B1-5] A pharmaceutical composition according to any one of [B1-1] to [B1-4], wherein the antigen-binding molecule comprises a variable region and a constant region of an antibody, and a linker that is cleaved by a protease, and the antigen-binding molecule from which the linker has been cleaved by the protease comprises the variable region or the antigen-binding fragment thereof. 【0073】 [B1-6] A pharmaceutical composition according to any one of [B1-1] to [B1-4], wherein the antigen-binding molecule comprises a variable region and a constant region of the antibody, and a linker that is cleaved by a protease, and the linker is located near the boundary between the variable region and the constant region or near the boundary between CH1 and CH2 within the constant region. 【0074】 [B1-7] The pharmaceutical composition according to any one of [B1-1] to [B1-6], wherein the antibody contains a linker whose antigen-binding molecule is cleaved by a protease, and the antigen-binding molecule after linker cleavage is the VL, VH, or antigen-binding fragment thereof of the antibody. 【0075】 [B1-8] The pharmaceutical composition according to any one of [B1-1] to [B1-6], wherein the antigen-binding molecule is a heavy chain antibody containing a linker that is cleaved by a protease, and the antigen-binding molecule after linker cleavage is the VHH of the heavy chain antibody. 【0076】 [B1-9] A pharmaceutical composition according to any one of [B1-1] to [B1-8], wherein the antigen-binding molecule after linker cleavage is scFv, Fv, Fab, Fab', F(ab')2, VH, or VHH. 【0077】 [B1-10] A pharmaceutical composition according to any one of [B1-1] to [B1-9] wherein a bispecific antibody recognizes a cleaved linker, a portion of the linker, or a portion containing the linker. 【0078】 [B1-11] K for antigen-binding molecules before linker cleavage of bispecific antibodies D K for the antigen-binding molecule after linker cleavage relative to the value D Ratio of values ​​(K D (After cutting) / K D A pharmaceutical composition according to any of [B1-1] to [B1-10], wherein (before cutting) is 0.1 or less or 0.01 or less. 【0079】 [B1-12] A pharmaceutical composition according to any one of [B1-1] to [B1-11], wherein the linker cleaved by the protease comprises a protease cleavage sequence. 【0080】 [B1-13] The pharmaceutical composition according to [B1-1] to [B1-12], wherein the linker cleaved by the protease comprises a peptide having one of the protease cleavage sequences of SEQ ID NOs: 1 to 725. 【0081】 [B1-14] A pharmaceutical composition according to any one of [B1-1] to [B1-13], wherein the linker cleaved by the protease further comprises a movable linker. 【0082】 [B1-15] The pharmaceutical composition according to any one of [B1-1] to [B1-14], wherein the protease is a protease that is specifically expressed in the target tissue. 【0083】 [B1-16] A pharmaceutical composition according to any one of [B1-1] to [B1-15], wherein the target cell is a tumor cell and the protease is a tumor protease. 【0084】 [B1-17] A pharmaceutical composition according to any of [B1-1] to [B1-16] for use in the treatment or prevention of cancer. 【0085】 [B1-18] The pharmaceutical composition according to [B1-17], wherein the cancer is selected from the group consisting of carcinoma, lymphoma, sarcoma, blastoma, and leukemia. 【0086】 [B1-19] The pharmaceutical composition according to [B1-17], wherein the cancer is selected from the group consisting of B-cell lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell non-Hodgkin lymphoma, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin lymphoma. 【0087】 [B1-20] A pharmaceutical composition according to any of [B1-1] to [B1-19] for use in bispecific antibody therapy. 【0088】 [B2-1] A bispecific antibody comprising: 1) a first antibody variable region having binding activity to a molecule expressed on the surface of a T cell; and 2) a second antibody variable region having binding activity to an antigen-binding molecule containing a linker that is cleaved by a protease, after the linker has been cleaved by a protease; A bispecific antibody is a bispecific antibody that has binding activity to antigens expressed on the surface of target cells after the linker has been cleaved by a protease, and can bind to target cells via binding to the antigen-binding molecule after the linker has been cleaved. 【0089】 [B2-2] A bispecific antibody as described in [B2-1], wherein the molecule expressed on the surface of T cells is CD3. [B2-3] A bispecific antibody as described in [B2-1], wherein the antibody variable region, which has binding activity to molecules expressed on the surface of T cells, binds to CD3 epsilon. 【0090】 [B2-4] A bispecific antibody according to [B2-1] to [B2-3] that recognizes a linker cleaved by the protease, a part of the linker, or a part containing the linker. [B2-5] A bispecific antibody as described in any of [B2-1] to [B2-4], which contains an Fc region in which binding activity to the Fc gamma receptor is reduced. 【0091】 [B2-6] A bispecific antibody according to any one of [B1-1] to [B1-5], wherein the linker cleaved by the protease contains a peptide having one of the protease cleavage sequences. 【0092】 [B2-7] A bispecific antibody according to any one of [B1-1] to [B1-5], comprising a peptide in which the linker cleaved by the protease has one of the protease cleavage sequences of SEQ ID NOs: 1 to 725. 【0093】 [B2-8] A bispecific antibody according to any one of [B1-1] to [B1-7], wherein the linker cleaved by the protease further comprises a movable linker. 【0094】 [B2-9]IgG antibody, a bispecific antibody as described in any of [B2-1] to [B2-8]. A nucleic acid encoding a bispecific antibody as described in any of [B3-1], [B2-1], or [B2-9]. A vector containing the nucleic acids described in [B3-2] and [B3-1]. Cells containing the vectors described in [B3-3] and [B3-2]. A method for producing a bispecific antibody, comprising culturing the cells described in [B3-4] and [B3-3] and recovering the bispecific antibody from the culture supernatant. 【0095】 [C1-1] A pharmaceutical composition comprising an IgG antibody characterized by enhanced antibody-dependent cellular cytotoxicity (ADCC) for use in combination with the administration of an antigen-binding molecule, The antigen-binding molecule contains a linker that is cleaved by a protease, and has binding activity to the antigen expressed on the surface of target cells after the linker is cleaved by the protease. The IgG antibody comprises an antibody variable region having binding activity to the antigen-binding molecule after the linker has been cleaved by the protease. An IgG antibody is a pharmaceutical composition that can bind to target cells via binding to an antigen-binding molecule after linker cleavage. 【0096】 [C1-2] A pharmaceutical composition comprising an antigen-binding molecule for use in combination with the administration of an IgG antibody characterized by enhanced antibody-dependent cellular cytotoxicity (ADCC), The antigen-binding molecule contains a linker that is cleaved by a protease, and after the linker is cleaved by the protease, it has binding activity to the antigen expressed on the surface of the target cell. IgG contains an antibody variable region that has binding activity to the antigen-binding molecule after the linker has been cleaved by a protease. An IgG antibody is a pharmaceutical composition that can bind to target cells via binding to an antigen-binding molecule after linker cleavage. 【0097】 [C1-3] K of the antigen-binding molecule before linker cleavage D K for the antigen of the antigen after linker cleavage relative to the value D Ratio of values ​​(K D (After cutting) / K D The pharmaceutical composition according to [C1-1] or [C1-2], wherein (before cleavage) is 0.1 or 0.01 or less. 【0098】 [C1-4] The pharmaceutical composition according to any one of [C1-1] to [C1-3], wherein the antigen-binding molecule is an IgG antibody, an IgG antibody-like molecule, a heavy chain antibody, or a monodomain antibody. 【0099】 [C1-5] A pharmaceutical composition according to any one of [C1-1] to [C1-4], wherein the antigen-binding molecule comprises a variable region and a constant region of an antibody, and a linker that is cleaved by a protease, and the antigen-binding molecule from which the linker has been cleaved by the protease comprises the variable region or the antigen-binding fragment thereof. 【0100】 [C1-6] A pharmaceutical composition according to any one of [C1-1] to [C1-4], wherein the antigen-binding molecule comprises a variable region and a constant region of the antibody, and a linker that is cleaved by a protease, and the linker is located near the boundary between CH1 and CH2 within the constant region. 【0101】 [C1-7] The pharmaceutical composition according to any one of [C1-1] to [C1-6], wherein the antibody contains a linker whose antigen-binding molecule is cleaved by a protease, and the antigen-binding molecule after linker cleavage is the VL, VH, or antigen-binding fragment thereof of the antibody. 【0102】 [C1-8] The pharmaceutical composition according to any one of [C1-1] to [C1-6], wherein the antigen-binding molecule is a heavy chain antibody containing a linker that is cleaved by a protease, and the antigen-binding molecule after linker cleavage is the VHH of the heavy chain antibody. 【0103】 [C1-9] A pharmaceutical composition according to any one of [C1-1] to [C1-8], wherein the antigen-binding molecule after linker cleavage is scFv, Fv, Fab, Fab', F(ab')2, VH, or VHH. 【0104】 A pharmaceutical composition according to any one of [C1-1] to [C1-9] wherein the [C1-10]IgG antibody recognizes a cleaved linker, a portion of the linker, or a portion containing the linker. 【0105】 [C1-11] K for antigen-binding molecule before linker cleavage of IgG antibody D K for the antigen-binding molecule after linker cleavage relative to the value D Ratio of values ​​(K D (After cutting) / K D A pharmaceutical composition according to any of [C1-1] to [C1-10], wherein (before cleavage) is 0.1 or 0.01 or less. 【0106】 [C1-12] A pharmaceutical composition according to any one of [C1-1] to [C1-11], wherein the linker cleaved by the protease comprises a protease cleavage sequence. 【0107】 [C1-1] The pharmaceutical composition according to [C1-1] to [C1-12], wherein the linker cleaved by the protease comprises a peptide having one of the protease cleavage sequences of SEQ ID NOs: 1 to 725. 【0108】 [C1-14] A pharmaceutical composition according to any one of [C1-1] to [C1-13], wherein the linker cleaved by the protease further comprises a movable linker. 【0109】 [C1-1] A pharmaceutical composition according to any one of [C1-1] to [C1-13], wherein the [C1-14] protease is a protease that is specifically expressed in the target tissue. 【0110】 [C1-15] A pharmaceutical composition according to any one of [C1-1] to [C1-14], wherein the target cell is a tumor cell and the protease is a tumor protease. 【0111】 [C1-16] A pharmaceutical composition according to any of [C1-1] to [C1-15] for use in the treatment or prevention of antibody-dependent cell cytotoxicity (ADCC) or antibody-dependent cell phagocytosis (ADCP). [C1-17] A pharmaceutical composition according to any of [C1-1] to [C1-16] for use in the treatment or prevention of cancer. 【0112】 [C1-18] The pharmaceutical composition according to [C1-17], wherein cancer is selected from the group consisting of carcinoma, lymphoma, sarcoma, blastoma, and leukemia. 【0113】 [C1-19] The pharmaceutical composition according to [C1-17], wherein the cancer is selected from the group consisting of B-cell lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell non-Hodgkin lymphoma, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin lymphoma. [C1-20] A pharmaceutical composition according to any of [C1-1] to [C1-19] for use in IgG antibody therapy. 【0114】 [C2-1] An antigen-binding molecule containing a linker that is cleaved by a protease, wherein the IgG antibody contains an antibody variable region having binding activity to the antigen-binding molecule after the linker has been cleaved by the protease; Antigen-binding molecules have binding activity to antigens expressed on the surface of target cells after the linker is cleaved by a protease, and IgG antibodies can bind to target cells by binding to the antigen-binding molecule after the linker is cleaved. 【0115】 [C2-2] An IgG antibody as described in [C2-1], which exhibits enhanced antibody-dependent cellular cytotoxicity (ADCC). 【0116】 [C2-3] An IgG antibody as described in [C2-1] or [C2-2], comprising an Fc region in which binding activity to the Fc gamma receptor is increased. 【0117】 [C2-4] A pharmaceutical composition according to any one of [C1-1] to [C1-3], wherein the linker cleaved by the protease comprises a protease cleavage sequence. 【0118】 [C2-5] A pharmaceutical composition according to any one of [C1-1] to [C1-4], comprising a peptide having one of the protease cleavage sequences of SEQ ID NOs: 1 to 725, wherein the linker cleaved by the protease is [C2-5]. 【0119】 A pharmaceutical composition according to any one of [C1-1] to [C1-5], wherein the linker cleaved by the [C2-6] protease further comprises a movable linker. 【0120】 A nucleic acid encoding an IgG antibody as described in any of [C3-1], [C2-1], or [C2-6]. A vector containing the nucleic acids described in [C3-2] and [C3-1]. Cells containing the vectors described in [C3-3] and [C3-2]. A method for producing IgG antibodies, comprising culturing the cells described in [C3-4] and [C3-3] and recovering IgG antibodies from the culture supernatant. 【0121】 [D1-1] A pharmaceutical composition comprising a secondary molecule for use in combination with the administration of a primary molecule, The primary molecule contains a linker that is cleaved by a protease, and after the linker is cleaved, it has the ability to bind to the target antigen. A pharmaceutical composition in which a secondary molecule has the ability to bind to a primary molecule after linker cleavage, and can bind to cells expressing the target antigen via binding to the primary molecule after linker cleavage. 【0122】 [D1-2] A pharmaceutical composition comprising a primary molecule for use in combination with the administration of a secondary molecule, The primary molecule contains a linker that is cleaved by a protease, and after the linker is cleaved, it has the ability to bind to the target antigen. A pharmaceutical composition in which a secondary molecule has the ability to bind to a primary molecule after linker cleavage, and can bind to cells expressing the target antigen via binding to the primary molecule after linker cleavage. 【0123】 [D1-3] The pharmaceutical composition according to [D1-1] or [D1-2], wherein the primary molecule is an antigen-binding molecule, and the secondary molecule is a bispecific antibody, a chimeric receptor, or an IgG antibody characterized by enhanced antibody-dependent cytotoxicity. 【0124】 A pharmaceutical composition described in any of [D1-4], [A1-1] to [A1-21], [B1-1] to [B1-20], and [C1-1] to [C1-20], which is a pharmaceutical composition described in any of [D1-1] to [D1-3]. [Brief explanation of the drawing] 【0125】 [Figure 1]Figure 1 illustrates the concept of antibody technology (Probody®) that expands tissue specificity and therapeutic window by conferring sensitivity to proteases whose expression levels are elevated in lesion sites such as cancerous tissue and inflammatory tissue. [Figure 2] Figure 2 is a schematic diagram illustrating the induction of TDCC activity by antibodies that specifically recognize antigens generated by protease-mediated linker cleavage. [Figure 3] Figure 3 is a schematic diagram illustrating the induction of ADCC activity by antibodies that specifically recognize antigens generated by protease-mediated linker cleavage. [Figure 4] Figure 4 is a schematic diagram illustrating the induction of TDCC activity by antibodies that specifically recognize antigens generated by protease-mediated linker cleavage. [Figure 5] Figure 5 is a schematic diagram illustrating the induction of ADCC activity by antibodies that specifically recognize antigens generated by protease-mediated linker cleavage. [Figure 6-1] Figure 6-1 is a schematic diagram illustrating the induction of cytotoxic activity by CAR-T receptors that specifically recognize antigen-binding molecules cleaved by proteases. [Figure 6-2] Figure 6-2 is a schematic diagram illustrating the induction of cytotoxic activity by CAR-T receptors that specifically recognize antigens exposed by cleavage by proteases. [Figure 7] Figure 7 shows the in vitro cleavage of an antibody with an inserted protease cleavage sequence (collagen II partial sequence) by the protease (MMP13). The lanes from left to right correspond to wells 1-5. Well 1 shows the MWM, wells 2 and 3 show the antigen-binding molecule before and after the reaction without the cleavage sequence, respectively, and wells 4 and 5 show the antigen-binding molecule containing the cleavage sequence before and after the reaction when MMP13 is added. [Figure 8]Figure 8 shows the in vitro cleavage of an antibody that recognizes a tumor antigen by a protease (IdeS). The leftmost lane corresponds to wells 6-10. Well 6 shows the MWM, wells 7 and 8 show the antigen-binding molecule before and after the reaction without the cleavage sequence, respectively, and wells 9 and 10 show the antigen-binding molecule containing the cleavage sequence before and after the reaction when IdeS is added, respectively. [Figure 9] Figure 9 (left) shows the results of treating tumor cell lines with an antigen-binding molecule (antibody) into which a protease-cleaved sequence (collagen II subsequence) was inserted. Figure 9 (right) shows the results of treating tumor cell lines with an antigen-binding molecule without the protease-cleaved sequence (collagen II subsequence) inserted. [Figure 10] Figure 10 shows the results of Biacore measurement, demonstrating that an anti-cleavage linker anti-CD3 bispecific antibody binds to an antigen-binding molecule whose linker (collagen II subsequence) has been cleaved by a protease. [Figure 11] Figure 11 shows the results of Biacore measurement, demonstrating that an anti-cleavage linker anti-CD3 bispecific antibody binds to an antigen-binding molecule (IgG1) cleaved by a protease. [Figure 12] Figure 12 shows the results of Biacore measurement, demonstrating that the ADCC activity-enhancing antibody binds to the antigen-binding molecule whose linker (collagen II subsequence) has been cleaved by the protease. [Figure 13] Figure 13 shows the results of Biacore measurement, demonstrating that the ADCC activity-enhancing antibody binds to the antigen-binding molecule (IgG1) cleaved by the protease. [Figure 14] Figure 14 shows the results of a Jurkat reporter gene assay using antigen-binding molecules (anti-GPC3 antibody, IgG1) and an anti-linker anti-CD3 bispecific antibody, in which the linker (collagen II partial sequence) was cleaved by a protease. [Figure 15]Figure 15 shows the results of a Jurkat reporter gene assay using antigen-binding molecules (anti-GPC3 antibody, IgG1) and an anti-linker anti-CD3 bispecific antibody, in which the linker (collagen II partial sequence) was cleaved by a protease. [Figure 16] Figure 16 shows the results of a Jurkat reporter gene assay using ADCC activity enhancement against antigen-binding molecules whose linker (collagen II subsequence) has been cleaved by a protease. [Figure 17] Figure 17 shows the results of a Jurkat reporter gene assay using ADCC activity enhancement against antigen-binding molecules whose linker (collagen II subsequence) has been cleaved by a protease. [Figure 18] Figure 18 shows the results of a cytotoxic assay using human PBMCs with an anti-linker anti-CD3 bispecific antibody that recognizes antigen-binding molecules whose linker (collagen II subsequence) has been cleaved by protease, and an anti-GPC3 antibody with an inserted protease cleavage sequence. [Figure 19] Figure 19 is a schematic diagram showing the vector construct and the arrangement order of frame-unit components from the 5' end to the 3' end. [Figure 20] Figure 20 shows the evaluation results of the cytotoxic activity of PC-10, which can cleave MMP linkers. The percentage of residual cancer cells was calculated as the percentage of CD45- fraction cells in the viable cells. The horizontal axis shows the concentration of the added antigen-binding molecule. [Figure 21] Figure 21 shows the evaluation results of KYSE70 cytotoxic activity, which is largely incapable of cleaving MMP linkers. The percentage of residual cancer cells was calculated as the percentage of CD45- fraction cells among the viable cells. The horizontal axis shows the concentration of the added antigen-binding molecule. [Modes for carrying out the invention] 【0126】 Other features and advantages of this disclosure will become apparent from the following detailed description. However, since various changes and modifications within the spirit and scope of this disclosure will become obvious to those skilled in the art from this detailed description, the detailed description and specific examples illustrating preferred embodiments of this disclosure should be understood to be provided for illustrative purposes only. The embodiments of this disclosure will be described below with reference to the drawings. 【0127】 The terms “substantially,” “about,” or “approximately” mean a reasonable deviation of the modified word such that it does not significantly alter the final result, i.e., within the acceptable margin of error of a particular value as determined by those skilled in the art. For example, “about” may mean the acceptable standard deviation according to the practice of the art. Alternatively, “about” may mean up to ±20%, preferably up to ±10%, more preferably up to ±5%, and even more preferably up to ±1% of a given value. Or, particularly in biological systems or processes, the term may mean within one order of magnitude, preferably up to twice, of a given value. When a particular value is described herein and in the claims, unless otherwise specified, the term “about” is implied to mean within the acceptable margin of error of that particular value. 【0128】 In English translations of this specification and the claims, the singular forms “a,” “an,” and “the” include plural demonstratives unless explicitly stated otherwise. Furthermore, the term “or” is generally used within its scope of meaning, including “and / or,” unless explicitly stated otherwise. 【0129】 In this disclosure, an enumeration of numerical ranges by endpoint includes all numbers and fractions contained within that range (for example, 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It should also be understood that all numbers and fractions will be modified with the term “approximately”. However, if it is clear that the numbers indicated by a numerical range are integers, the numerical range should be understood as a restrictive enumeration of the integers contained within the range. In such cases, for example, 1 to 5, or 1 through 5, should be understood as a restrictive enumeration of 1, 2, 3, 4, and 5. 【0130】 Furthermore, the definitions and embodiments described in certain sections are intended to apply to other embodiments of this Specification where they are described as preferred, as will be understood by those skilled in the art. For example, the following passages provide more detailed definitions of various aspects of this Disclosure. Each of these aspects may be combined with any one or more other aspects unless otherwise explicitly stated. In particular, any feature shown as preferred or advantageous may be combined with any one or more other features shown as preferred or advantageous. 【0131】 In this disclosure, an antigen-binding molecule containing a region that binds to an antigen expressed on target cells ("antigen-binding domain") and a linker that is cleaved by a protease may be referred to as a "primary molecule." The primary molecule releases an antigen-binding fragment that binds to an antigen expressed on target cells or lesion cells ("target antigen") when the linker is cleaved by a protease. One of the antigen-binding molecules produced when the linker is cleaved by a protease is called a "linker-cleaved antigen-binding molecule" or "antigen-binding molecule after linker cleavage," and includes the antigen-binding domain and a portion of the cleaved linker. A polypeptide that cross-links target cells and effector cells to induce cytotoxic activity may be referred to as a "secondary molecule." Examples of secondary molecules include an antibody with ADCC activity that has an antibody variable region capable of binding to the linker-cleaved antigen-binding molecule, a T-cell redirecting antibody that has an antibody variable region capable of binding to the linker-cleaved antigen-binding molecule and an antibody variable region capable of binding to a T-cell receptor complex, or a chimeric receptor that has an extracellular domain capable of binding to the linker-cleaved antigen-binding molecule. The linker contained in the antigen-binding molecule contains a protease cleavage sequence and has a cleavage site that is cleaved by a protease. A linker consisting of a peptide having a protease cleavage sequence is sometimes called a protease cleavage linker. 【0132】 In one embodiment, the antigen-binding molecule (primary molecule) containing a linker cleaved by a protease is an antibody, more specifically an IgG antibody or a heavy chain antibody containing a linker cleaved by a protease, and more preferably an IgG1 antibody, a camelid heavy chain antibody (hcIgG), or a shark heavy chain antibody (IgNAR). 【0133】 In one embodiment, antigen-binding molecules produced by the cleavage of the linker by a protease include Fv, Fab, Fab', Fab'-SH, F(ab')2, mini-bodies, single-chain antibody molecules (e.g., scFv), VHH, and VH, and more specifically, Fab, scFv, VHH, and VH. 【0134】 In the present invention, polypeptides typically refer to peptides and proteins having a length of about 4 amino acids or more. While polypeptides in the present invention are typically polypeptides consisting of artificially designed sequences, they are not particularly limited and may, for example, be polypeptides of biological origin. They may also be natural polypeptides, synthetic polypeptides, recombinant polypeptides, etc. Furthermore, fragments of the above-mentioned polypeptides are also included in the polypeptides of the present invention. 【0135】 In this specification, amino acids are represented by single-letter codes, three-letter codes, or both, for example, Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, and Val / V. When representing an amino acid at a specific position, a representation that includes both a number indicating the specific position and the single-letter or three-letter code of the amino acid may be used as appropriate. For example, the amino acid 37V, which is contained in a single-domain antibody, represents Val, which is located at position 37 in Kabat numbering. 【0136】 For modifying amino acids in the amino acid sequences of polypeptides such as antibodies, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately employed. Furthermore, several known methods for modifying amino acids by substituting them with non-natural amino acids can also be employed (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing tRNA in which a non-natural amino acid is bound to a complementary amber suppressor tRNA of the UAG codon (amber codon), one of the stop codons, is suitably used. In this specification, substitution is mentioned as a modification, but is not limited to this. 【0137】 In this specification, the term "and / or" used to describe the modification sites of amino acids includes any combination of "and" and "or" as appropriate. Specifically, for example, "amino acids 37, 45, and / or 47 are substituted" includes the following variations of amino acid modification sites: (a) 37, (b) 45, (c) 47, (d) 37 and 45, (e) 37 and 47, (f) 45 and 47, and (g) 37, 45, and 47. 【0138】 In this specification, expressions that include a number representing a specific position followed by a one-letter or three-letter code of the amino acid before and after the modification may be used as appropriate to represent amino acid modifications. For example, the modification F37V or Phe37Val, used when making amino acid substitutions in the antibody variable region or single-domain antibody, represents the substitution of Phe at position 37, as represented by Kabat numbering, to Val. That is, the number represents the position of the amino acid as represented by Kabat numbering, the one-letter or three-letter code of the amino acid listed before it represents the amino acid before substitution, and the one-letter or three-letter code of the amino acid listed after it represents the amino acid after substitution. Similarly, the modification P238A or Pro238Ala, used when making amino acid substitutions in the Fc region included in the antibody constant region, represents the substitution of Pro at position 238, as represented by EU numbering, to Ala. In other words, the numbers represent the position of the amino acid as expressed in EU numbering, the one-letter or three-letter code of the amino acid listed before it represents the amino acid before substitution, and the one-letter or three-letter code of the amino acid listed after it represents the amino acid after substitution. 【0139】 In this specification, the term “antibody” is used in its broadest sense and is not limited to any antibody that exhibits the desired antigen-binding activity, but encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-domain antibodies, and antibody fragments. 【0140】 An "antibody fragment" refers to a molecule other than the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments are not limited to these, but include Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments. 【0141】 The terms "full-length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a native antibody, or having a heavy chain containing an Fc region as defined herein. 【0142】 The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of an antibody (VH and VL, respectively) typically have a similar structure, with each domain containing four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. 【0143】 As used herein, the terms “complementarity-determining region” or “CDR” refer to the regions of the variable domain of an antibody that are hypervariable in sequence and / or form structurally defined loops (“hypervariable loops”) and / or antigen contact residues (“antigen contacts”). Typically, an antibody contains six CDRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Illustrative CDRs as used herein include: (a) Hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and, (d) A combination of (a), (b), and / or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). 【0144】 Unless otherwise indicated, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein in accordance with Kabat et al. 【0145】 The "framework" or "FR" refers to variable domain residues other than complementarity-determining region (CDR) residues. The variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the sequences of the CDR and FR usually appear in the VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. 【0146】 In this specification, the term “constant region” or “constant domain” refers to the portion of an antibody other than the variable region. For example, an IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons, composed of two identical disulfide-linked light chains and two identical heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by a heavy chain constant region (CH) containing the CH1 domain, hinge region, CH2 domain, and CH3 domain. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chains of native antibodies may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. 【0147】 In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes both the native sequence Fc region and mutant Fc regions. In one embodiment, for human IgG1, the heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain, provided that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region is present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991. 【0148】 The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. 【0149】 In this specification, the term "antigen-binding domain" is limited solely to domains that bind to the target antigen. Any domain structure may be used as the antigen-binding domain, as long as it binds to the target antigen. Examples of such domains, though not limited to these, include, for example, the heavy chain variable region (VH) and light chain variable region (VL) of antibodies, single-domain antibodies (sdAb), a module called the A domain of about 35 amino acids contained in Avimer, a cell membrane protein present in living organisms (International Publication WO2004 / 044011, WO2005 / 040229), Adnectin containing the 10Fn3 domain, a protein-binding domain in fibronectin, a glycoprotein expressed on the cell membrane (International Publication WO2002 / 032925), Affibody (International Publication WO1995 / 001937), which uses an IgG-binding domain as a scaffold to form a bundle of three helices consisting of 58 amino acids of Protein A, and DARPins (Designed Examples include Ankyrin Repeat proteins (International Publication WO2002 / 020565), Anticalin (International Publication WO2003 / 029462), which consists of four loop regions supporting one side of a barrel structure twisted towards the center by eight highly conserved antiparallel strands in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL), and recessed regions of parallel sheet structures within a horseshoe-shaped structure formed by repeatedly stacked leucine-rich repeat (LRR) modules of variable lymphocyte receptors (VLRs) that do not possess the structure of immunoglobulins, as part of the acquired immune system of jawless fish such as lampreys and hagfish (International Publication WO2008 / 016854). 【0150】 Suitable examples of the antigen-binding domain of the present invention include an antigen-binding domain that can exhibit antigen-binding function in a molecule composed solely of the antigen-binding domain, and an antigen-binding domain that can exhibit antigen-binding function independently after being released from other linked peptides. Examples of such antigen-binding domains, though not limited to these, include single-domain antibodies, scFv, Fv, Fab, Fab', F(ab')2, and the like. 【0151】 One preferred example of the antigen-binding domain of the present invention is an antigen-binding domain with a molecular weight of 60 kDa or less. Examples of such antigen-binding domains, but not limited to them, include single-domain antibodies, scFv, Fab, and Fab'. Antigen-binding domains with a molecular weight of 60 kDa or less are usually highly likely to be cleared by the kidney when they exist in the blood as monomers (see J Biol Chem. 1988 Oct 15;263(29):15064-70). 【0152】 From another perspective, one preferred example of the antigen-binding domain of the present invention is an antigen-binding domain with a blood half-life of 12 hours or less. Examples of such antigen-binding domains, but not limited to them, include single-domain antibodies, scFv, Fab, Fab', etc. 【0153】 One preferred example of the antigen-binding domain of the present invention is a single-domain antibody (sdAb). 【0154】 In this specification, the term "monodomain antibody" is used without regard to its structure, as long as the domain alone can exhibit antigen-binding activity. While conventional antibodies, such as IgG antibodies, exhibit antigen-binding activity when a variable region is formed by the pairing of VH and VL domains, monodomain antibodies are known to exhibit antigen-binding activity solely through their own domain structure, without pairing with other domains. Monodomain antibodies usually have a relatively low molecular weight and exist in monomeric form. 【0155】 Examples of monodomain antibodies include, but are not limited to, antigen-binding molecules that congenitally lack a light chain, such as VHH from camelid animals or VNAR from sharks, or antibody fragments containing all or part of the VH domain or all or part of the VL domain of an antibody. Examples of monodomain antibodies that are antibody fragments containing all or part of the VH / VL domain of an antibody include, but are not limited to, monodomain antibodies artificially produced starting from human antibody VH or human antibody VL, as described in, for example, U.S. Patent No. 6,248,516B1. In some embodiments of the present invention, one monodomain antibody has three CDRs (CDR1, CDR2, and CDR3). 【0156】 If a single-domain antibody is a VHH antibody, the CDR of the single-domain antibody may include, exemplarily, the following: 【0157】 (a) Hypervariable loops occurring at amino acid residues 26-32 (CDR1), 53-55 (CDR2), and 96-101 (CDR3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 31-35b (CDR1), 50-65 (CDR2), and 95-102 (CDR3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact occurring at amino acid residues 30-35b (CDR1), 47-58 (CDR2), and 93-101 (CDR3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and, (d) A combination of (a), (b), and / or (c) containing CDR amino acid residues 26-35 (CDR1), 26-35b (CDR1), 49-65 (CDR2), 93-102 (CDR3), or 94-102 (CDR3). 【0158】 If a single-domain antibody is a single-domain VL antibody, the CDR of the single-domain antibody may include, as an example, the following: 【0159】 (a) Hypervariable loops occurring at amino acid residues 26-32 (CDR1), 50-52 (CDR2), and 91-96 (CDR3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact occurring at amino acid residues 27c-36 (CDR1), 46-55 (CDR2), and 89-96 (CDR3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and, (d) A combination of (a), (b), and / or (c) comprising CDR amino acid residues 46-56 (CDR2), 47-56 (CDR2), 48-56 (CDR2), or 49-56 (CDR2). Unless otherwise indicated, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al. above. 【0160】 Monodomain antibodies can be obtained from animals capable of producing monodomain antibodies, or by immunizing animals capable of producing monodomain antibodies. Examples of animals capable of producing monodomain antibodies include, but are not limited to, camelids and transgenic animals into which a gene capable of producing monodomain antibodies has been introduced. Camelids include camels, llamas, alpacas, dromedaries, and guanacos. Examples of transgenic animals into which a gene capable of producing monodomain antibodies has been introduced include, but are not limited to, the transgenic animals described in International Publication WO2015 / 143414 and U.S. Patent Publication US2011 / 0123527A1. Humanized monodomain antibodies can also be obtained by using a human germline sequence or a similar sequence as the framework sequence of a monodomain antibody obtained from an animal. Humanized monodomain antibodies (e.g., humanized VHH) are also one embodiment of the monodomain antibody of the present invention. 【0161】 Furthermore, single-domain antibodies can be obtained from polypeptide libraries containing single-domain antibodies by methods such as ELISA and panning. Examples of polypeptide libraries containing single-domain antibodies include, but are not limited to, naive antibody libraries obtained from various animals or humans (e.g., Methods in Molecular Biology 2012 911 (65-78), Biochimica et Biophysica Acta - Proteins and Proteomics 2006 1764:8 (1307-1319)), antibody libraries obtained by immunizing various animals (e.g., Journal of Applied Microbiology 2014 117:2 (528-536)), or synthetic antibody libraries created from antibody genes of various animals or humans (e.g., Journal of Biomolecular Screening 2016 21:1 (35-43), Journal of Biological Chemistry 2016 291:24 (12641-12657), AIDS 2016 30:11). (1691-1701) is one example. 【0162】 In this specification, “antigen” is limited only to comprising an epitope to which an antigen-binding domain binds. Preferred examples of antigens, but are not limited to, include, for example, animal or human peptides, polypeptides, and proteins. In the present invention, a target antigen is an antigen used to treat a disease caused by a target tissue, and preferred examples, but are not limited to, include, for example, molecules expressed on the surface of target cells (e.g., cancer cells, inflammatory cells), molecules expressed on the surface of other cells in the tissue containing the target cells, molecules expressed on the surface of cells that have an immunological role to the target cells and the tissue containing the target cells, and macromolecules present in the stroma of the tissue containing the target cells. The following antigens can be given as examples of target antigens. 【0163】 Antigens include the following molecules: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Adresin, aFGF, ALCAM, ALK, ALK-1, ALK-7, Alpha-1-Antitrypsin, Alpha-V / Beta-1 Antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, A RC, ART, Artemin, Anti-Id, ASPARTIC, Atrial Natriuretic Factor, av / b3 Integrin, Axl, b2M, B7-1, B7-2, B7-H, B-Lymphocyte-Stimulating Factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2, BMP-2a, BMP-3, Osteogenin, BMP-4, BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, β-NGF, BOK, Bombecin, Bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, Complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, Calcitonin, cAMP, Carcinoembryonic antigen (CEA), Cancer-associated antigen, Cathepsin A, Cathepsin B, Cathepsin C / DPPI, Cathepsin D, Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O, Cathepsin S, Cathepsin V, Cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD 8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD3 3 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, PD1, PDL1, LAG3, TIM3, galectin-9, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, Cytokine-related antigen, DAN, DCC, DCR3, DC-SIGN, Complement-accelerating factor (Decay accelerating)factor), des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, DNase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / E phB4, EPO, ERCC, E-selectin, ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX, Fibroblast-activating protein (FAP), Fas, FcR1, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Follicle-stimulating hormone, Fractal In, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (Myostatin), GD F-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-Alpha 1, GFR-Alpha 2, GFR-Alpha 3, GITR, Glucagon, Glut4, Glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, Growth Hormone Releasing Factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFGPEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2 IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-21, IL-23, IL-27, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like proliferation Factor 1, Integrin Alpha 2, Integrin Alpha 3, Integrin Alpha 4, Integrin Alpha 4 / Beta 1, Integrin Alpha 4 / Beta 7, Integrin Alpha 5 (Alpha V), Integrin Alpha 5 / Beta 1, Integrin Alpha 5 / Beta 3, Integrin Alpha 6, Integrin Beta 1, Integrin Beta 2, Interferon Gamma, IP-10, I-TAC, JE, Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12, Kallikrein 14, Kallikrein 15, Kallikrein L1, Kallikrein L2, Kallikrein L3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), Laminin 5, LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Müllerian duct inhibitor, Mug, MuSK, NAIP, NAP, NCAD, NC adherin, NCA 90, NCAM, NCAM, Neprilysin, Neurotrophin-3, -4, or -6, Neuroturin, Nerve Growth Factor (NGF), NGFR, NGF-Beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, Parathyroid Hormone, PARC, PARP, PBR, PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDK-1, P ECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A chain, relaxin B chain, renin, polynuclear respiratory virus (RSV) F, RSVFgp, Ret, Rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, Thrombin, Thymus Ck-1, Thyroid-stimulating hormone, Tie, TIMP, TIQ, Tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha-beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B(OPG OCIF, TR1), TNFRSF12(TWEAK R FN14), TNFRSF13B(TACI), TNFRSF13C(BAFF R), TNFRSF14(HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17(BCMA), TNFRSF18(GITR AITR), TNFRSF19(TROY TAJ, TRADE), TNFRSF19L(RELT), TNFRSF1A(TNF RI CD120a, p55-60), TNFRSF1B(TNF RIICD120b, p75-80), TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF5(CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2 TNFRH2), TNFRST23(DcTRAIL R1 TNFRH1), TNFRSF25(DR3) Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-α connectin, DIF, TNFSF2), TNFSF1B (TNF-β LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand) gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TLR (Toll-likereceptor)1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expression Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1(flt-1), VEGF, VEGFR, VEGFR-3(flt-4), VEGI, VIM, Viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aβ, CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp90, IL-17 / IL-17R, IL-20 / IL-20R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B, tau, VAP1, polymeric kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, factor H, properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factorExamples include Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, Syndecan-1, Syndecan-2, Syndecan-3, Syndecan-4, LPA, S1P, and receptors for hormones and growth factors. 【0164】 While the above examples of antigens include receptors, these receptors can also be used as antigens to which the antigen-binding domain of the present invention binds, even when they exist in a soluble form in biological fluids. One non-limiting embodiment of such a soluble receptor is, for example, a protein that is soluble IL-6R, as described by Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968). 【0165】 The above examples of antigens include membrane-bound molecules expressed on cell membranes and soluble molecules secreted from cells into the extracellular space. When the antigen-binding domain of the present invention binds to a soluble molecule secreted from a cell, it is preferable that the antigen-binding domain has neutralizing activity. 【0166】 The soluble molecule is not limited to the solution in which it exists; it can be present in any biological fluid, i.e., any fluid that fills the spaces between blood vessels or tissues and cells within a living organism. In one non-limiting embodiment, the soluble molecule to which the antigen-binding domain of the present invention binds can be present in extracellular fluid. Extracellular fluid refers to the general term for components of bone and cartilage such as plasma, interstitial fluid, lymph, dense connective tissue, cerebrospinal fluid, cerebrospinal fluid, puncture fluid, or synovial fluid, as well as cellular permeable fluids such as alveolar fluid (bronchial alveolar lavage fluid), ascites, pleural fluid, pericardial fluid, cystic fluid, or aqueous humor (aqueous humor) (fluids in various glandular lumens resulting from the active transport and secretory activity of cells, and fluids in the gastrointestinal tract and other body cavities). 【0167】 The term "tumor antigen" refers to an antigen expressed on cancer cells, meaning an antigenic biomolecule whose expression becomes recognized in relation to malignant changes in the cell. Tumor antigens in this disclosure include tumor-specific antigens (antigens present only on tumor cells and not on other normal cells) and tumor-associated antigens (antigens present in other organs and tissues or in heterogeneous and allogeneic normal cells, or antigens expressed during development and / or differentiation). Abnormal glycans that appear on the cell surface or on protein molecules when cells become cancerous are also tumor antigens, and are also called cancer glycan antigens. In one embodiment of the present invention, the target antigen is a tumor antigen. 【0168】 Examples of tumor antigens include, for example, GPC3 (Int J Cancer. (2003) 103 (4), 455-65), which belongs to the GPI-anchored receptor family as a receptor and is expressed in several cancers, including liver cancer, and EpCAM (Proc Natl Acad Sci US A. (1989) 86 (1)), which is expressed in multiple cancers, including lung cancer. 27-31) (The polynucleotide sequence is described in RefSeq registry number NM_002354.2, and the polypeptide sequence is described in RefSeq registry number NP_002345.2.), EGFR, CA19-9, CA15-3, serial SSEA-1 (SLX), Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), tumor antigen-125 (CA-125), calretinin, MUC-1, MUC-16, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), chromogranin, cytokeratin, desmin, glial fiber acidic protein (GFAP), gross cystic disease fluid protein Protein (GCDFP-15), HMB-45 antigen, Protein Melan-A (Melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, Muscle-specific actin (MSA), Neurofilament, Nerve-specific enolase (NSE), Placental alkaline phosphatase, Synaptophysis, Thyroglobulin, Thyroid transcription factor-1, Dimeric form of pyruvate kinase isoenzyme type M2 (Tumor M2-PK), GD2 (Ganglioside G2), EGFRvIII (Epidermal Growth Factor Variant III), Sperm Protein 17 (Sp17), mesoserine, PAP (prostatic acid phosphatase), prostain, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAP1 (six-transmembrane epithelial antigen of prostate 1), TROP-2, Claudin6, RNF43a, abnormal ras protein, or abnormal p53 protein, integrin alpha v beta 3 (CD61), galectin, K-Ras (V-Ki-ras2 kirsten rat sarcoma virus oncogene), or Ral-B are preferred examples. 【0169】 Furthermore, thyroid-stimulating hormone receptor (TSHR); CD171; CS-1 (CD2 subset 1, CRACC, SLAMF7, CD319 and 19A24); C-type lectin-like molecule-1 (CLL-1); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); Tn antigen (Tn Ag); T antigen (T Ag); Fms-like tyrosine kinase 3 (FLT3); CD38; CD44v6; B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2); interleukin-11 receptor alpha (IL-11Ra); interleukin-2 receptor alpha (IL-2Ra); prostate stem cell antigen (PSCA); protease serine 21 (PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2) Lewis (Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); Neuronal cell adhesion molecule (NCAM); Carbonic anhydrase IX (CAIX); Proteasome (prosome, macropain) subunit, beta type, 9 (LMP2); Ephrin type A receptor 2 (EphA2); Fucosyl GM1; Sialyl Lewis adhesion molecule (sLe); Ganglioside GM3 (aNeu5Ac(2-3)bDGal p(1-4)bDGlcp(1-1)Cer;TGS5;High molecular weight melanoma-associated antigen (HMWMAA);o-acetyl-GD2 ganglioside (OAcGD2);folate receptor beta;tumor endothelial marker 1 (TEM1 / CD248);tumor endothelial marker 7-associated (TEM7R);claudin 6 (CLDN6);G protein-coupled receptor class C group 5, member D (GPRC5D);chromosome X open reading frame 61 (CXORF61);CD97 CD179a; Anaplastic lymphoma kinase (ALK); Polysialic acid; Placenta-specific receptor 1 (PLAC1); Hexasaccharide moiety of globoH glycoceramide (GloboH); Mammary gland differentiation antigen (NY-BR-1); Uloplakin 2 (UPK2); Hepatitis A virus cytotoxic receptor 1 (HAVCR1); Adrenergic receptor beta 3 (ADRB3); Pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); Lymphocyte antigen 6 complex, locus K9 (LY6K);Olfactory receptor 51E2 (OR51E2); TCR gamma-selective leading frame protein (TARP); Wilms tumor protein (WT1); ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); Melanoma carcinoma testicular antigen-1 (MAD-CT-1); Melanoma carcinoma testicular antigen-2 (MAD-CT-2); Fos-related antigen 1; p53 variant; Human telomerase reverse transcriptase (hTERT); Sarcoma translocation breakpoint; Apoptotic melanoma inhibitor (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyl-transferase V (NA17); Paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin B1; v-myc myeloma virus oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); Cytochrome P450 1B1 (CYP1B1); CCCTC binding factor (zinc finger protein)-like (BORIS); squamous cell tumor antigen recognized by T cell 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein p32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X-section 2 (SSX2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); IgA receptor Other examples include body Fc fragments (FCAR); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1), etc. 【0170】 MHC antigens are gene products of the major histocompatibility complex (MHC), and among them, glycoproteins expressed on the cell membrane 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, and -H, while MHC class II antigens include HLA-DR, -DQ, and -DP. In addition, tumor antigen-derived peptides presented by these MHC antigens are also included. Tumor antigens such as GP100, MART-1, and MAGE-1, or complexes with MHC that present mutated regions such as RAS and p53, can also be considered as tumor antigens. 【0171】 "Differentiation antigens" are a general term for cell surface molecules that change during the differentiation of macrophages, T cells, B cells, etc., from bone marrow stem cells. Differentiation antigens include CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15s, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD29, CD30, CD32, CD33, CD34, CD35, CD38, CD40, CD41a, CD41b, CD42a, CD42b, and CD4 3, CD44, CD45, CD45RO, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, CD54, CD55, CD56, CD57, CD58, CD61, CD62E, CD62L, CD62P, CD64, CD69, CD70, CD71, CD73, CD95, CD99, CD102, CD106, CD117, CD122, CD126, CDw130 may be included. 【0172】 The term “tumor” generally refers to any growth that develops on or inside the body, which may be palpable as a mass or have a discolored appearance. Tumors are classified into malignant tumors, which have three characteristics: autonomous growth, invasion and metastasis, and cachexia, and benign tumors, which are characterized only by autonomous growth. Malignant tumors, or “cancer,” refer to diseases characterized by the uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Various examples of cancers described in this disclosure include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and related cancers. The terms “tumor” and “cancer” are used synonymously in this disclosure, and for example, both terms encompass solid and liquid tumors, such as diffuse or circulating tumors. When used in this disclosure, the terms “cancer” or “tumor” encompass precancerous, as well as malignant, cancers and tumors. 【0173】 The cancers treated with the anticancer drugs disclosed herein and the cancer treatment methods described later include adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma, skin cancer, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, uterine cancer, liver cancer, kidney cancer, pancreatic cancer, spleen cancer, lung cancer, tracheal cancer, bronchial cancer, colon cancer, small intestine cancer, stomach cancer, esophageal cancer, gallbladder cancer, testicular cancer, ovarian cancer, and other cancers, as well as cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue, and hematopoietic tissue. Additionally, sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcoma, as well as blastomas such as hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreaticoblastoma, pleuropulmonary blastoma, and retinoblastoma, germ cell tumors, lymphoma, and leukemia. 【0174】 In one embodiment, in relation to cancer type, the tumor antigen is a marker expressed by both normal and cancer cells, e.g., a lineage marker, e.g., CD19 on B cells. In certain embodiments, the tumor antigens of this disclosure are derived from cancer, including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinoma, e.g., breast cancer, prostate cancer, ovarian cancer, pancreatic cancer and similar cancers. In one embodiment, the tumor antigen is an antigen common to a particular proliferative disorder. In one embodiment, the cancer-associated antigen is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, e.g., 1x expression, 2x overexpression, 3x overexpression or more compared to normal cells. In some embodiments, the cancer-associated antigen is a cell surface molecule that is improperly synthesized in cancer cells, e.g., a molecule containing deletions, additions or mutations compared to a molecule expressed in normal cells. In one embodiment, cancer-associated antigens are expressed exclusively on the cell surface of cancer cells, either whole or as fragments (e.g., MHC / peptide), and are neither synthesized nor expressed on the surface of normal cells. In some embodiments, the chimeric receptors and TRABs of this disclosure comprise CARs and TRABs containing an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to a peptide presented by an MHC. Typically, peptides derived from endogenous proteins fill the pocket of a major histocompatibility complex (MHC) class I molecule, and CD8 +It is recognized by the T cell receptor (TCR) on T lymphocytes. MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent a unique class of cell surface targets for immunotherapy. TCR-like antibodies targeting viral or tumor antigen-derived peptides in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described [see, for example, Sastry et al., J Virol. 2011 85(5):1935-1942; Sergeeva et al., Bood, 2011 117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther 2001 8(21):1601-1608; Dao et al., Sci Transl Med 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 2012 19(2):84-100]. For example, TCR-like antibodies can be identified by screening libraries such as human scFv phage-presenting libraries. 【0175】 An epitope, meaning an antigenic determinant present in an antigen, refers to a site on the antigen to which an antigen-binding domain, as disclosed herein, binds. Therefore, for example, an epitope can be defined by its structure. Alternatively, an epitope can be defined by the binding activity of the antigen-binding domain that recognizes it. If the antigen is a peptide or polypeptide, the epitope can also be identified by the amino acid residues that constitute it. Furthermore, if the epitope is a glycan, it can also be identified by a specific glycan structure. 【0176】 A linear epitope is an epitope that contains an epitope whose amino acid primary sequence has been recognized. A linear epitope typically contains at least three, and most commonly at least five, amino acids, e.g., about eight to about ten, or six to twenty, in a specific sequence. 【0177】 A stereoepitope, in contrast to a linear epitope, is an epitope in which the primary amino acid sequence containing the epitope is not a single defining component of the recognized epitope (for example, an epitope whose primary amino acid sequence is not necessarily recognized by the antibody defining the epitope). A stereoepitope may contain a larger number of amino acids than a linear epitope. In relation to the recognition of a stereoepitope, the antigen-binding domain recognizes the three-dimensional structure of the peptide or protein. For example, if a protein molecule folds to form a three-dimensional structure, certain amino acids and / or polypeptide backbone that form the stereoepitope are parallel, allowing the antibody to recognize the epitope. Methods for determining the three-dimensional structure of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-specific spin labeling and electromagnetic paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.). 【0178】 The structure of an antigen-binding domain that binds to an epitope is called a paratope. The epitope and paratope bind stably due to hydrogen bonds, electrostatic forces, van der Waals forces, hydrophobic bonds, etc., acting between them. This binding force between the epitope and paratope is called affinity. The sum of the binding forces when multiple antigens and multiple antigen-binding domains bind is called avidity. When antibodies containing multiple antigen-binding domains (i.e., polyvalent antibodies) bind to multiple epitopes, the binding forces (affinity) work synergistically, resulting in avidity that is higher than affinity. 【0179】 In certain embodiments, the antigen-binding domains provided herein are ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (for example, 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 It has a dissociation constant (Kd) of M. 【0180】 The following methods for confirming the binding of an antigen-binding domain to an epitope, or an antigen-binding molecule containing an antigen-binding domain, can be appropriately performed in accordance with the following examples. 【0181】 For example, the recognition of a linear epitope present in an antigen molecule by an antigen-binding domain can be confirmed, for instance, as follows: A linear peptide consisting of the amino acid sequence constituting the extracellular domain of an antigen is synthesized for the purpose described above. This peptide can be synthesized chemically, or obtained by genetic engineering using the region of the antigen's cDNA that codes for the amino acid sequence corresponding to the extracellular domain. Next, the binding activity between the linear peptide, consisting of the amino acid sequence constituting the extracellular domain, and the antigen-binding domain for the antigen is evaluated. For example, the binding activity of the antigen-binding domain to the immobilized linear peptide can be evaluated by ELISA using the immobilized linear peptide as the antigen. Alternatively, the binding activity to the linear peptide can be determined based on the level of inhibition by the linear peptide in the binding of the antigen-binding domain to an antigen-expressing cell. These tests can reveal the binding activity of the antigen-binding domain to the linear peptide. 【0182】 Furthermore, the recognition of a stereoepitope by an antigen-binding domain for a certain antigen can be confirmed as follows. For the above purpose, cells expressing a certain antigen are prepared. When the antigen-binding domain for a certain antigen comes into contact with a cell expressing that antigen, it binds strongly to the cell, but the antigen-binding domain does not substantially bind to a linear peptide consisting of amino acid sequences constituting the extracellular domain of an immobilized antigen, or to a linear peptide consisting of amino acid sequences constituting the extracellular domain of an antigen that has been denatured using a common denaturing agent such as guanidine. Here, substantially non-binding means a binding activity of 80% or less, usually 50% or less, preferably 30% or less, and particularly preferably 15% or less of the binding activity to a human antigen-expressing cell. 【0183】 Another method for confirming the antigen-binding activity of an antigen-binding domain is to measure the Kd value using, for example, a radiolabeled antigen-binding assay (RIA). In one embodiment, the RIA is performed using the antigen-binding domain of interest and its antigen. For example, the solution-bound affinity of an antigen-binding domain to an antigen is measured by equilibrating the antigen-binding domain with a minimum concentration of (125I)-labeled antigen in the presence of an increasing dose series of unlabeled antigens, and then capturing the bound antigen with a plate coated with the antigen-binding domain. (See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999)). 【0184】 In another embodiment, Kd is measured by surface plasmon resonance using BIACORE®. For example, the measurement method using BIACORE®-2000 or BIACORE®-3000 (BIACORE, Inc., Piscataway, NJ) is performed at 25°C using a CM5 chip immobilized with approximately 10 response units (RUs) of antigen. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8 before being injected at a flow rate of 5 μl / min to achieve binding of approximately 10 response units (RUs) of protein. After antigen injection, 1M ethanolamine is injected to block unreacted groups. For kinetics measurement, two-fold serial dilutions (0.78 nM to 500 nM) of the antigen-binding domain in PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20®) surfactant are injected at 25°C and a flow rate of approximately 25 μl / min. The binding rate (kon) and dissociation rate (koff) are calculated by simultaneously fitting binding and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® evaluation software version 3.2). The equilibrium dissociation constant (Kd) is calculated as the koff / kon ratio. Furthermore, the apparent dissociation constant (Kd) can also be determined using equilibrium analysis. Refer to the protocols included with BIACORE® for these methods. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999) and Methods Enzymol. 2000;323:325-40. Furthermore, in surface plasmon resonance assays, the amount of protein immobilized, the amount of protein used in the reaction, the temperature, and the solution composition can be varied as desired by those skilled in the art.The on-velocity was determined to be 10 by the surface plasmon resonance assay described above. 6 M -1 s -1 If it exceeds this, the ON rate can be determined by measuring the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass 16 nm) at 25°C in the presence of gradually increasing concentrations of antigen using a spectrometer (e.g., a stop-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO® spectrophotometer (ThermoSpectronic) using a stirred cuvette). 【0185】 Furthermore, the antigen-binding activity of the antigen-binding domain can also be measured using known methods for measuring intermolecular interactions, such as electrochemiluminescence. 【0186】 One method for measuring the binding activity of an antigen-binding domain to cells expressing a particular antigen is 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 using the principles of ELISA or FACS (fluorescence activated cell sorting) with cells expressing the antigen as the antigen. 【0187】 In the ELISA format, the binding activity of an antigen-binding domain to cells expressing a particular antigen is quantitatively evaluated by comparing the signal levels produced by the enzymatic reaction. Specifically, the test antigen-binding domain is added to an ELISA plate immobilized with cells expressing a particular antigen, and the antigen-binding domain bound to the cells is detected using an enzyme-labeled antibody that recognizes the test antigen-binding domain. Alternatively, in FACS, the binding activity of the test antigen-binding domain to cells expressing a particular antigen can be compared by creating a dilution series of the test antigen-binding domain and determining the antibody binding titer to cells expressing a particular antigen. 【0188】 The binding of the target antigen-binding domain to an antigen expressed on the cell surface suspended in a buffer solution can be detected by a flow cytometer. Examples of known flow cytometers include the following: FACSCanto™ II FACSAria™ FACSArray™ FACSVantage™ SE FACSCalibur™ (both 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). 【0189】 For example, the following method is a suitable method for measuring the binding activity of an antigen-binding domain to a given antigen. First, cells expressing a given antigen are stained with a FITC-labeled secondary antibody that recognizes the test antigen-binding domain. The test antigen-binding domain is then diluted with a suitable buffer to prepare it to the desired concentration. For example, it can be used at a concentration between 10 μg / ml and 10 ng / ml. Next, the fluorescence intensity and cell count are measured using FACSCalibur (BD). The amount of antigen-binding domain bound to the cells is reflected in the fluorescence intensity, i.e., the Geometric Mean value, obtained by analysis using CELL QUEST Software (BD). That is, by obtaining the Geometric Mean value, the binding activity of the test antigen-binding domain, expressed by the amount of binding, can be measured. 【0190】 The sharing of an epitope between an antigen-binding domain and another antigen-binding domain can be confirmed by competition between the two domains for the same epitope. Competition between antigen-binding domains can be detected by cross-blocking assays, for example. A competitive ELISA assay is a preferred cross-blocking assay. 【0191】 Specifically, in a cross-blocking assay, an antigen protein coated on a well of a microtiter plate is pre-incubated in the presence or absence of a candidate competitive antigen-binding domain, after which the test antigen-binding domain is added. The amount of the test antigen-binding domain bound to the antigen protein in the well is indirectly correlated with the binding ability of the candidate competitive antigen-binding domains that compete for binding to the same epitope. In other words, the greater the affinity of the competitive antigen-binding domain for the same epitope, the lower the binding activity of the test antigen-binding domain to the well coated with the antigen protein. 【0192】 The amount of antigen-binding domain bound to a well via a certain antigen protein can be easily measured by pre-labeling the antigen-binding domain. For example, biotin-labeled antigen-binding domains can be measured using an avidin peroxidase conjugate and an appropriate substrate. Cross-blocking assays utilizing enzymatic labeling such as peroxidase are specifically called competitive ELISA assays. Antigen-binding domains can be labeled with other detectable or measurable labeling substances. Specifically, radiolabeling and fluorescent labeling are well known. 【0193】 If, compared to the binding activity obtained in a control test performed in the absence of a candidate competing antigen-binding domain aggregate, a competing antigen-binding domain can block the binding of an antigen-binding domain to a given antigen by at least 20%, preferably at least 20-50%, and more preferably at least 50%, then the test antigen-binding domain is either substantially binding to the same epitope as the competing antigen-binding domain, or is an antigen-binding domain that competes for binding to the same epitope. 【0194】 If the structure of an epitope to which an antigen-binding domain binds to a particular antigen has been identified, the sharing of the epitope between the test antigen-binding domain and the control antigen-binding domain can be evaluated by comparing the binding activity of the two antigen-binding domains to a peptide or polypeptide into which amino acid mutations have been introduced into the peptide constituting the epitope. 【0195】 One method for measuring such binding activity is to compare the binding activity of the test antigen-binding domain and the control antigen-binding domain to a linear peptide into which a mutation has been introduced in the aforementioned ELISA format. Alternatively, the binding activity to the mutated peptide bound to a column can be measured by quantitatively determining the antigen-binding domain eluted into the eluate after the test antigen-binding domain and the control antigen-binding domain have been passed down the column. Methods for adsorbing the mutated peptide onto a column as a fusion peptide with, for example, GST, are well known. 【0196】 Furthermore, if the identified epitope is a stereoepitope, the sharing of the epitope between the test antigen-binding domain and the control antigen-binding domain can be evaluated by the following method. First, cells expressing a certain antigen and cells expressing a certain antigen with a mutation introduced into the epitope are prepared. The test antigen-binding domain and the control antigen-binding domain are added to the cell suspension, in which these cells are suspended in a suitable buffer such as PBS. Next, a FITC-labeled antibody capable of recognizing the test antigen-binding domain and the control antigen-binding domain is added to the cell suspension, which has been washed with a buffer as appropriate. The fluorescence intensity and cell count of the cells stained with the labeled antibody are measured using FACSCalibur (BD). The concentrations of the test antigen-binding domain and the control antigen-binding domain are adjusted to the desired concentration by appropriately diluting them with a suitable buffer. For example, concentrations between 10 μg / ml and 10 ng / ml are used. The amount of labeled antibody bound to the cells is reflected in the fluorescence intensity, i.e., the Geometric Mean value, obtained by analysis using CELL QUEST Software (BD). In other words, by obtaining the Geometric Mean value, the binding activity of the test antigen-binding domain and the control antigen-binding domain, which is represented by the amount of labeled antibody bound, can be measured. 【0197】 Furthermore, to confirm competition between antigen-binding domains for the same epitope as other antigen-binding domains, methods other than ELISA and FACS, such as radiolabeled antigen binding assay (RIA), BIACORE® surface plasmon resonance assay, and electrochemiluminescence, can also be used. 【0198】 The Geometric Mean comparison value (molecular ΔGeo-Mean value of a mutated antigen) obtained by analysis, which reflects the amount of binding of the test antigen-binding domain to cells expressing a certain antigen, is compared with the ΔGeo-Mean comparison value which reflects the amount of binding of the test antigen-binding domain to cells expressing a certain antigen. In this case, it is particularly preferable that the concentrations of the test antigen-binding domain used when determining the ΔGeo-Mean comparison values ​​for cells expressing a certain mutated antigen and cells expressing a certain antigen are prepared at the same or substantially the same concentration. An antigen-binding domain that has been confirmed to recognize an epitope in a certain antigen in advance is used as a control antigen-binding domain. 【0199】 If the ΔGeo-Mean comparison value of the test antigen-binding domain to cells expressing a mutated antigen is less than at least 80%, preferably 50%, more preferably 30%, and particularly preferably 15% of the ΔGeo-Mean comparison value of the test antigen-binding domain to cells expressing an antigen with the test antigen-binding domain, it is considered that the domain "substantially does not bind to cells expressing a mutated antigen." The formula for calculating the Geo-Mean value (Geometric Mean) is described in the CELL QUEST Software User's Guide (BD biosciences). If the comparison values ​​are substantially equivalent, the epitopes of the test antigen-binding domain and the control antigen-binding domain can be considered identical. 【0200】 In this specification, the term "transport portion" refers to the portion of an antigen-binding molecule other than the antigen-binding domain. The transport portion of the present invention is typically a peptide or polypeptide composed of amino acids, and in one specific embodiment, the transport portion in the antigen-binding molecule is linked to the antigen-binding domain via a cleavage site. The transport portion of the present invention may be a series of peptides or polypeptides linked by amide bonds, or a complex in which multiple peptides or polypeptides are formed by covalent bonds such as disulfide bonds or non-covalent bonds such as hydrogen bonds or hydrophobic interactions. 【0201】 In some embodiments of the present invention, the antigen-binding molecule after linker cleavage exhibits higher antigen-binding activity compared to the antigen-binding molecule before linker cleavage. In other words, the antigen-binding activity of the antigen-binding domain of the antigen-binding molecule is suppressed by the inhibitory domain due to the portion removed by linker cleavage. Methods to confirm that the antigen-binding activity of the antigen-binding domain is suppressed by the inhibitory domain include FACS (fluorescence activated cell sorting), ELISA (enzyme-linked immunosorbent assay), ECL (electrogenerated chemiluminescence), SPR (Surface Plasmon Resonance) (Biacore), and BLI (Bio-Layer Interferometry) (Octet). In some embodiments of the present invention, the antigen-binding activity of the antigen-binding molecule after linker cleavage is 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 90, 1000, 2000, or 3000 times greater than the antigen-binding activity of the antigen-binding molecule before linker cleavage. In some more specific embodiments of the present invention, when the antigen-binding activity of the antigen-binding domain is measured by one of the methods selected from the above methods, no binding between the antigen-binding domain and the antigen is observed. 【0202】 In some embodiments of the present invention, the comparison of antigen-binding activity can be performed by comparing the antigen-binding activity before and after linker cleavage. That is, the antigen-binding activity measured using the antigen-binding molecule after linker cleavage is 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 90, 1000, 2000, or 3000 times greater than the antigen-binding activity measured using the antigen-binding molecule before linker cleavage. 【0203】 In some embodiments of the present invention, the linker of the antigen-binding molecule is cleaved by a protease, so the comparison of antigen-binding activity in such embodiments can be performed by comparing the antigen-binding activity of the antigen-binding molecule before and after protease treatment. That is, the antigen-binding activity measured using an antigen-binding molecule after protease treatment is 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 90, 1000, 2000, or 3000 times or more than the antigen-binding activity measured using an antigen-binding molecule that has not been protease-treated. In some more specific embodiments, when the antigen-binding activity of an untreated protease-containing antigen-binding molecule is measured by one of the methods selected from the above, no binding between the antigen-binding domain and the antigen is observed. 【0204】 In the present invention, the antigen-binding molecule before the linker is cleaved has a longer plasma half-life compared to the antigen-binding molecule after cleavage. In order to make the half-life of the antigen-binding molecule longer, in some embodiments of the present invention, the antigen-binding molecule before the linker is cleaved is designed to have a longer plasma half-life. Examples of embodiments for extending the plasma half-life include, but are not limited to, the antigen-binding molecule before the linker is cleaved having a large molecular weight, or having FcRn-binding property, or having albumin-binding property, or being PEGylated. 【0205】 In the present invention, for the half-life comparison, it is preferable to compare the plasma half-life in humans. When it is difficult to measure the plasma half-life in humans, based on the plasma half-life in mice (e.g., normal mice, human antigen-expressing transgenic mice, human FcRn-expressing transgenic mice, etc.) or monkeys (e.g., cynomolgus monkeys, etc.), the plasma half-life in humans can be predicted. 【0206】 As one embodiment for extending the plasma half-life of the antigen-binding molecule, it includes imparting FcRn-binding property to the antigen-binding molecule before the linker is cleaved. To confer FcRn-binding property, usually, there is a method of providing an FcRn-binding region in the antigen-binding molecule before the linker is cleaved. The FcRn-binding region refers to a region having binding property to FcRn, and any structure can be used as long as it has binding property to FcRn. 【0207】 The presence of an FcRn binding domain allows IgG molecules to be taken up into cells via the FcRn salvage pathway and then returned to the plasma. For example, the relatively long retention time (slow disappearance) of IgG molecules in plasma is due to the function of FcRn, which is known as a salvage receptor for IgG molecules. IgG molecules taken up into endosomes by pinocytosis bind to FcRn expressed in endosomes under acidic conditions. IgG molecules that do not bind to FcRn proceed to lysosomes and are degraded there, but IgG molecules that do bind to FcRn migrate to the cell surface and dissociate from FcRn under neutral conditions in plasma, returning to the plasma. 【0208】 The FcRn binding region is preferably a region that directly binds to FcRn. A preferred example of an FcRn binding region is the Fc region of an antibody. However, regions that can bind to polypeptides that have the ability to bind to FcRn, such as albumin and IgG, can indirectly bind to FcRn via albumin or IgG, so the FcRn binding region in the present invention may be a region that binds to such polypeptides that have the ability to bind to FcRn. 【0209】 The binding activity of the FcRn binding domain in the present invention to FcRn, particularly human FcRn, can be measured by methods known to those skilled in the art, as described in the section on binding activity, and the conditions can be appropriately determined by those skilled in the art. The binding activity to human FcRn can be evaluated as KD (Dissociation constant), apparent KD (Apparent dissociation constant), dissociation rate (kd), or apparent dissociation rate (kd). These can be measured by methods known to those skilled in the art. For example, Biacore (GE Healthcare), scatchard plots, flow cytometers, etc., can be used. 【0210】 The conditions for measuring the binding activity of the FcRn binding region to FcRn can be appropriately selected by those skilled in the art and are not particularly limited. For example, it can be measured under conditions of MES buffer and 37°C, as described in WO2009 / 125825. Furthermore, the measurement of the binding activity of the FcRn binding region of the present invention to FcRn can be performed by methods known to those skilled in the art, for example, by using Biacore (GE Healthcare). 【0211】 The binding affinity between the FcRn binding region and FcRn may be evaluated at any pH between pH 4.0 and pH 6.5 as the pH used for measurement conditions. Preferably, a pH between pH 5.8 and pH 6.0, which is close to the pH in early endosomes in vivo, is used to determine the binding affinity between the FcRn binding region and human FcRn. The binding affinity between the FcRn binding region and FcRn may be evaluated at any temperature between 10°C and 50°C as the temperature used for measurement conditions. Preferably, a temperature between 15°C and 40°C is used to determine the binding affinity between the FcRn binding region and human FcRn. More preferably, any temperature between 20°C and 35°C, such as any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C, is also used to determine the binding affinity between the FcRn binding region and FcRn. The temperature of 25°C is a non-limiting example of an embodiment of the present invention. 【0212】 One example of an FcRn binding region, though not limited to this, is the Fc region of an IgG antibody. When using the Fc region of an IgG antibody, the type is not limited, and it is possible to use Fc regions of IgG1, IgG2, IgG3, IgG4, etc. 【0213】 Furthermore, not only the Fc region of natural IgG antibodies, but also modified Fc regions with one or more amino acid substitutions can be used, as long as they retain FcRn binding ability. For example, EU numberings 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, and 314 in the IgG antibody Fc region. It is possible to use a modified Fc region containing an amino acid sequence in which at least one amino acid selected from positions 315, 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 is substituted with another amino acid. 【0214】 More specifically, EU numbering in the Fc region of IgG antibodies An amino acid substitution in which Gly at position 237 is replaced with Met. An amino acid substitution where the 238th Pro is replaced with Ala. An amino acid substitution where Ser at position 239 is replaced with Lys. An amino acid substitution where Lys at position 248 is replaced with Ile. Amino acid substitutions that replace the 250th Thr with Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr. Amino acid substitutions that replace Met at position 252 with Phe, Trp, or Tyr. An amino acid substitution where Ser at position 254 is replaced with Thr. Amino acid substitution where Arg at position 255 is replaced with Glu, Amino acid substitutions that replace the 256th Thr with Asp, Glu, or Gln. Amino acid substitutions that replace the 257th Pro with Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val. An amino acid substitution in which Glu at position 258 is replaced with His. An amino acid substitution in which Asp at position 265 is replaced with Ala. An amino acid substitution where Asp at position 270 is replaced with Phe. Amino acid substitutions that replace the 286th Asn with Ala or Glu. An amino acid substitution where the 289th Thr is replaced with His, An amino acid substitution in which the 297th Asn is replaced with Ala. An amino acid substitution in which Ser at position 298 is replaced with Gly. An amino acid substitution in which the 303rd Val is replaced with Ala. An amino acid substitution in which the 305th Val is replaced with Ala. Amino acid substitutions that replace the 307th Thr with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr. Amino acid substitutions that replace the 308th Val with Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr. Amino acid substitutions that replace the 309th Leu or Val with Ala, Asp, Glu, Pro, or Arg. Amino acid substitutions that replace the 311th Gln with Ala, His, or Ile. Amino acid substitutions that replace Asp at position 312 with Ala or His. Amino acid substitutions that replace the 314th Leu with Lys or Arg. Amino acid substitutions that replace the 315th Asn with Ala or His, An amino acid substitution in which Lys at position 317 is replaced with Ala. Amino acid substitution where Asn at position 325 is replaced with Gly. Amino acid substitution where Ile at position 332 is replaced with Val. An amino acid substitution where Lys at position 334 is replaced with Leu. An amino acid substitution where the 360th Lys is replaced with His. An amino acid substitution in which Asp at position 376 is replaced with Ala. An amino acid substitution where Glu at position 380 is replaced with Ala. An amino acid substitution that replaces the 382nd Glu with Ala, An amino acid substitution that replaces the 384th Asn or Ser with Ala, An amino acid substitution that replaces the 385th Gly with Asp or His, An amino acid substitution that replaces the 386th Gln with Pro, An amino acid substitution that replaces the 387th Pro with Glu, An amino acid substitution that replaces the 389th Asn with Ala or Ser, An amino acid substitution that replaces the 424th Ser with Ala, An amino acid substitution that replaces the 428th Met with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr, An amino acid substitution that replaces the 433rd His with Lys, An amino acid substitution that replaces the 434th Asn with Ala, Phe, His, Ser, Trp, or Tyr, and An amino acid substitution that replaces the 436th Tyr or Phe with His It is possible to use a modified Fc region comprising at least one amino acid substitution selected from the above. 【0215】 From another perspective, in the Fc region of an IgG antibody, according to the EU numbering Met at the 237th amino acid, Ala at the 238th amino acid, Lys at the 239th amino acid, Ile at the 248th amino acid, Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr at the 250th amino acid, Phe, Trp, or Tyr at the 252nd amino acid, Thr at the 254th amino acid, Glu at the 255th amino acid, Asp, Glu, or Gln at the 256th amino acid, Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val at amino acid position 257 His at the 258th amino acid, Ala at the 265th amino acid, Phe at amino acid 270, Ala or Glu at the 286th amino acid, His at the 289th amino acid, Ala at the 297th amino acid, Gly at the 298th amino acid, Ala at amino acid position 303, Ala at amino acid position 305, Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr at amino acid position 307, Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr at amino acid position 308, Ala, Asp, Glu, Pro, or Arg at amino acid position 309, Ala, His, or Ile at the 311th amino acid, Ala or His at the 312th amino acid, Lys or Arg at the 314th amino acid, Ala or His at the 315th amino acid, Ala at amino acid position 317, Gly at amino acid position 325, Val at amino acid position 332, Leu at amino acid position 334, His at amino acid position 360, Ala at amino acid position 376, Ala at amino acid position 380, Ala at amino acid position 382, Ala at amino acid position 384, Asp or His at the 385th amino acid, Pro at amino acid 386, Glu at amino acid 387, Ala or Ser at the 389th amino acid, Ala at amino acid position 424, Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr at amino acid position 428, Lys at amino acid position 433, Ala, Phe, His, Ser, Trp, or Tyr at amino acid position 434, and His at amino acid position 436 It is possible to use an Fc region containing at least one amino acid selected from the following. 【0216】 The antigen-binding molecule may have FcRn binding ability in its antigen-binding domain. As an embodiment to make the blood half-life of the antigen-binding molecule before linker cleavage longer than the blood half-life of the antigen-binding domain, the antigen-binding domain may not have FcRn binding ability, or even if the antigen-binding domain has FcRn binding ability, it may have weaker FcRn binding ability than the antigen-binding molecule before linker cleavage. 【0217】 Another method to extend the half-life in the blood is to bind the antigen-binding molecule to albumin before linker cleavage. Albumin is not excreted by the kidneys and has FcRn-binding properties, resulting in a long half-life of 17-19 days (J Clin Invest. 1953 Aug; 32(8): 746-768). Therefore, the protein bound to albumin becomes bulkier and can indirectly bind to FcRn, which has been reported to increase the half-life in the blood (Antibodies 2015, 4(3), 141-156). 【0218】 Furthermore, one embodiment of extending the half-life in the blood is a method of PEGylation of the antigen-binding molecule before linker cleavage. It is thought that PEGylation of the protein increases its bulk and simultaneously suppresses its degradation by proteases in the blood, thereby extending the half-life of the protein in the blood (J Pharm Sci. 2008 Oct;97(10):4167-83.). 【0219】 In some embodiments of the present invention, the antigen-binding molecule before linker cleavage includes the antibody Fc region. In one specific embodiment, the antigen-binding molecule before linker cleavage includes the CH2 and CH3 domains of a human IgG antibody. In another specific embodiment, the antigen-binding molecule before linker cleavage includes a portion extending from Cys226 or Pro230 of the human IgG1 antibody heavy chain to the carboxyl terminus of the heavy chain. However, the lysine (Lys447) or glycine-lysine (Gly446-Lys447) at the C-terminus of the Fc region may or may not be present. 【0220】 In some embodiments of the present invention, the antigen-binding molecule before linker cleavage includes an antibody constant region. In a more preferred embodiment, the antigen-binding molecule before linker cleavage includes an IgG antibody constant region. In a more preferred embodiment, the antigen-binding molecule before linker cleavage includes a human IgG antibody constant region. 【0221】 In some further embodiments of the present invention, the antigen-binding molecule before linker cleavage includes a region having a structure substantially similar to that of the antibody light chain, and a region having a structure substantially similar to that of the antibody light chain, which is linked to the said region by covalent bonds such as disulfide bonds or non-covalent bonds such as hydrogen bonds or hydrophobic interactions. 【0222】 The linker of the antigen-binding molecule is broken down by proteases to approximately 0.001-1500 × 10⁻¹⁰ 4 M -1 S -1Or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500 × 10 4 M -1 S -1 It is specifically cut at this speed. 【0223】 In this specification, the term “protease” means an enzyme such as an endopeptidase or exopeptidase that hydrolyzes peptide bonds, usually an endopeptidase. The proteases used in this disclosure are limited only to those that can cleave protease cleavage sequences, and their type is not particularly limited. In some embodiments, target tissue-specific proteases are used. Target tissue-specific proteases are, for example, (1) Proteases that are expressed at higher levels in target tissue than in normal tissue, (2) Proteases that have higher activity in target tissue than in normal tissue, (3) Proteases expressed at higher levels in target cells than in normal cells, (4) This can refer to any of the following: a protease that has higher activity in target cells than in normal cells. 【0224】 In a more specific embodiment, a cancer tissue-specific protease or an inflammatory tissue-specific protease is used. In this specification, the term “target tissue” means tissue containing at least one target cell. In some embodiments of the present invention, the target tissue is cancerous tissue. In some embodiments of the present invention, the target tissue is inflammatory tissue. 【0225】 The term "cancer tissue" means tissue containing at least one cancer cell. Therefore, it refers to all cell types that contribute to the formation of a tumor mass, including cancer cells and endothelial cells, such as cancer tissue containing cancer cells and blood vessels. In this specification, "tumor" means a foci of tumor tissue. The term "tumor" is generally used to mean either a benign or malignant neoplasm. 【0226】 In this specification, "inflammatory tissue" includes, for example, the following: Joints in rheumatoid arthritis and osteoarthritis • Lungs (alveoli) in bronchial asthma and COPD • Digestive organs in inflammatory bowel disease, Crohn's disease, and ulcerative colitis • Fibrotic tissue in fibrosis of the liver, kidneys, and lungs • Tissues that are being rejected in organ transplants • Blood vessels and heart (myocardium) in arteriosclerosis and heart failure • Visceral fat in metabolic syndrome • Skin tissue in atopic dermatitis and other skin inflammations • Spinal nerves in herniated discs and chronic lower back pain 【0227】 In some types of target tissues, proteases that are specifically expressed or specifically activated, or proteases that are thought to be associated with the disease state of the target tissue (target tissue-specific proteases), are known. For example, proteases that are specifically expressed in cancer tissue are disclosed in international publications WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846, among others. Furthermore, proteases thought to be associated with inflammation have been disclosed in J Inflamm (Lond). 2010; 7: 45., Nat Rev Immunol. 2006 Jul;6(7):541-50., Nat Rev Drug Discov. 2014 Dec;13(12):904-27., Respir Res. 2016 Mar 4;17:23., Dis Model Mech. 2014 Feb;7(2):193-203., and Biochim Biophys Acta. 2012 Jan;1824(1):133-45. 【0228】 In addition to proteases that are specifically expressed in target tissues, there are also proteases that are specifically activated in target tissues. For example, proteases may be expressed in an inactive form and then become active, and in many tissues, substances that inhibit the active protease exist, and the activity is controlled by the activation process and the presence of inhibitors (Nat Rev Cancer. 2003 Jul;3(7):489-501.). In target tissues, the active protease may escape inhibition and be specifically activated. Active proteases can be measured using methods that employ antibodies that recognize active proteases (PNAS 2013 Jan 2; 110(1): 93-98.) or by fluorescently labeling the peptide recognized by the protease, quenching it before cleavage, and then emitting light after cleavage (Nat Rev Drug Discov. 2010 Sep;9(9):690-701. doi: 10.1038 / nrd3053.). 【0229】 From one perspective, the term "target tissue-specific protease" is, (i) Proteases expressed at higher levels in target tissue than in normal tissue, (ii) Proteases that have higher activity in target tissue than in normal tissue, (iii) Proteases expressed at higher levels in target cells than in normal cells, (iv) Proteases that have higher activity in target cells than in normal cells, It can refer to any of the following: 【0230】 While not meant to be interpreted restrictively, specific proteases include cysteine ​​proteases (including cathepsin family B, L, S, etc.), aspartyl proteases (cathepsin D, E, K, O, etc.), serine proteases (including matryptase (MT-SP1), cathepsin A and G, thrombin, plasmin, urokinase (uPA), tissue plasminogen activator (tPA), elastase, proteinase 3, thrombin, kallikrein, tryptase, chymase), metalloproteases (including both membrane-bound (MMP14-17 and MMP24-25) and secreted (MMP1-13, MMP18-23, and MMP26-28) metalloproteases (MMP1-28), protease A disintegrin and metalloproteases (A disintegrin and metalloproteinase (ADAM), metalloproteinases with a disintegrin or thrombospondin motif (ADAM proteases with thrombospondin motif;ADAMTS), meprin (meprin alpha, meprin beta), CD10 (CALLA), as well as prostate-specific antigen (PSA), regmine, TMPRSS3, TMPRSS4, neutrophil elastase (HNE), beta-secretase (BACE), fibroblast-activating protein alpha (FAP), granzyme B, Guanidinobenzoate (GB), hepsin, neprilysin, NS3 / 4A, HCV-NS3 / 4, calpain, ADAMDEC1, renin, cathepsin C, cathepsin V / L2, cathepsin X / Z / P, cruzipain, otsubine 2, kallikrein-related peptidases (KLKs (KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14)), bone morphogenetic protein 1 (BMP-1), activated protein C, blood coagulation-related proteases (Factor VIIa, Factor IXa, Factor Xa, Factor XIa, Factor Examples include XIIa), HtrA1, lactoferrin, malapsin, PACE4, DESC1, dipeptidyl peptidase 4 (DPP-4), TMPRSS2, cathepsin F, cathepsin H, cathepsin L2, cathepsin O, cathepsin S, granzyme A, gepsin calpain 2, glutamate carboxypeptidase 2, AMSH-Like Proteases, AMSH, gamma secretase, A anti-plasmin cleavage enzyme (APCE), Decysin 1, N-Acetylated Alpha-Linked Acidic Dipeptidase-Like 1 (NAALADL1), and furin. 【0231】 From another perspective, target tissue-specific proteases can refer to cancer tissue-specific proteases or inflammatory tissue-specific proteases. Examples of cancer tissue-specific proteases include those disclosed in international publications WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846, which are specifically expressed in cancer tissue. 【0232】 The type of cancer tissue-specific protease that exhibits high specificity in the target cancer tissue yields a greater reduction in side effects. It is preferable that the concentration of the cancer tissue-specific protease in cancer tissue is at least five times higher than that in normal tissue, more preferably at least ten times higher, even more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. Furthermore, it is preferable that the activity of the cancer tissue-specific protease in cancer tissue is at least twice as high as that in normal tissue, more preferably at least three times higher, four times higher, five times higher, ten times higher, even more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. 【0233】 Furthermore, cancer tissue-specific proteases may be bound to the cell membrane of cancer cells, or they may not be bound to the cell membrane and may be secreted extracellularly. If cancer tissue-specific proteases are not bound to the cell membrane of cancer cells, it is preferable that the cancer tissue-specific protease is located inside or near the cancer tissue in order for the cytotoxicity by immune cells to be specific to cancer cells. In this specification, "near the cancer tissue" means the range in which the cancer tissue-specific protease cleavage sequence is cleaved and the antigen-binding domain exhibits antigen-binding activity. However, it is preferable that this range does not damage normal cells as much as possible. 【0234】 From another perspective, cancer tissue-specific proteases are, (i) Proteases expressed at higher levels in cancer tissue than in normal tissue, (ii) Proteases that have higher activity in cancer tissue than in normal tissue, (iii) Proteases expressed at higher levels in cancer cells than in normal cells, (iv) Proteases that have higher activity in cancer cells than in normal cells, It is one of the following: 【0235】 Cancer tissue-specific proteases may be used individually or in combination of two or more. The number of types of cancer tissue-specific proteases can be appropriately determined by a person skilled in the art, taking into consideration the type of cancer being treated. 【0236】 From the above viewpoint, among the proteases exemplified above, serine proteases and metalloproteases are preferred as cancer tissue-specific proteases, matryptase (including MT-SP1), urokinase (uPA), and metalloproteases are more preferred, and MT-SP1, uPA, MMP2, and MMP9 are even more preferred. 【0237】 The type of inflammation-specific protease that exhibits high specificity in the inflammatory tissue being treated is preferable to achieve a reduction in side effects. It is preferable that the concentration of the inflammation-specific protease in inflammatory tissue is at least five times higher than that in normal tissue, more preferably at least ten times higher, even more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. Furthermore, it is preferable that the activity of the inflammation-specific protease in inflammatory tissue is at least twice as high as that in normal tissue, more preferably at least three times, four times, five times, ten times, even more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. 【0238】 Furthermore, the inflammation tissue-specific protease may be bound to the cell membrane of inflammatory cells, or it may not be bound to the cell membrane and may be secreted extracellularly. If the inflammation tissue-specific protease is not bound to the cell membrane of inflammatory cells, it is preferable that the inflammation tissue-specific protease is located inside or near the inflammatory tissue in order for the cytotoxicity by immune cells to be specific to inflammatory cells. In this specification, "near the inflammatory tissue" means the range in which the inflammation tissue-specific protease cleavage sequence is cleaved and the antigen-binding domain exerts antigen-binding activity. However, it is preferable that this range does not damage normal cells as much as possible. 【0239】 From another perspective, inflammation-specific proteases are, (i) Proteases expressed at higher levels in inflammatory tissue than in normal tissue, (ii) Proteases that have higher activity in inflammatory tissue than in normal tissue, (iii) Proteases expressed at higher levels in inflammatory cells than in normal cells, (iv) Proteases that have higher activity in inflammatory cells than in normal cells, It is one of the following: 【0240】 Inflammatory tissue-specific proteases may be used individually or in combination of two or more types. The number of types of inflammatory tissue-specific proteases can be appropriately determined by those skilled in the art, taking into consideration the disease condition being treated. 【0241】 From the above perspective, among the proteases exemplified above, metalloproteases are preferred as inflammatory tissue-specific proteases, and among metalloproteases, ADAMTS5, MMP2, MMP7, MMP9, and MMP13 are more preferred. 【0242】 A protease cleavage sequence is a specific amino acid sequence that is specifically recognized by a target tissue-specific protease when an antigen-binding molecule is hydrolyzed by that protease in an aqueous solution. 【0243】 From the standpoint of reducing side effects, the protease cleavage sequence is preferably an amino acid sequence that is hydrolyzed with high specificity by a target tissue-specific protease that is more specifically expressed in or more specifically activated in the target tissue / cells being treated. 【0244】 Specific examples of protease cleavage sequences include, for example, target sequences that are specifically hydrolyzed by proteases specifically expressed in cancer tissue, inflammatory tissue-specific proteases, etc., as disclosed in International Publications WO2013 / 128194, WO2010 / 081173, WO2009 / 025846, etc. Artificially modified sequences, such as those obtained by introducing appropriate amino acid mutations into target sequences specifically hydrolyzed by known proteases, can also be used. Furthermore, protease cleavage sequences identified by methods known to those skilled in the art, as described in Nature Biotechnology 19, 661 - 667 (2001), may also be used. 【0245】 Furthermore, naturally occurring protease cleavage sequences may also be used. For example, just as TGFβ changes to its latent form upon protease cleavage, sequences in proteins that undergo protease cleavage to change their molecular shape can also be used. 【0246】 Examples of protease cleavage sequences, but not limited to these, include International Publications WO2015 / 116933, WO2015 / 048329, WO2016 / 118629, WO2016 / 179257, WO2016 / 179285, WO2016 / 179335, WO2016 / 179003, WO2016 / 046778, WO2016 / 014974, Japanese Patent Application No. 2019-105464, U.S. Patent Publication US2016 / 0289324, U.S. Patent Publication US2016 / 0311903, PNAS (2000) 97: 7754-7759, and Biochemical Journal. The sequences shown in (2010) 426: 219-228. and Beilstein J Nanotechnol. (2016) 7: 364-373. can be used. 【0247】 As described above, the protease cleavage sequence is more preferably an amino acid sequence that is specifically hydrolyzed by a suitable target tissue-specific protease. Among amino acid sequences that are specifically hydrolyzed by a target tissue-specific protease, sequences containing the following amino acid sequences are preferred. LSGRSDNH (can be cut using MT-SP1, uPA) PLALAG (can be cut using MMP2 and MMP9) VPLSLTMG (can be cut using MMP7) The following sequences can also be used as protease cleavage sequences. TSTSGRSANPRG (can be cut using MT-SP1 and uPA) ISSGLLSGRSDNH (can be cut with MT-SP1, uPA) AVGLLAPPGGLSGRSDNH (can be cut by MT-SP1, uPA) GAGVPMSMRGGAG (Can be cut by MMP1) GAGIPVSLRSGAG ​​(can be disconnected via MMP2) GPLGIAGQ (can be disconnected via MMP2) GGPLGMLSQS (can be disconnected via MMP2) PLGLWA (can be disconnected via MMP2) GAGRPFSMIMGAG (can be cut using MMP3) GAGVPLSLTMGAG (Can be cut using MMP7) GAGVPLSLYSGAG (can be cut by MMP9) AANLRN (can be disconnected via MMP11) AQAYVK (can be disconnected via MMP11) AANYMR (can be disconnected via MMP11) AAALTR (can be cut by MMP11) AQNLMR (can be disconnected via MMP11) AANYTK (can be disconnected via MMP11) GAGPQGLAGQRGIVAG (Can be cut using MMP13) PRFKIIGG (can be cleaved by pro-urokinase) PRFRIIGG (can be cleaved by pro-urokinase) GAGSGRSAG (can be disconnected via uPA) SGRSA (can be disconnected by uPA) GSGRSA (can be cut by uPA) SGKSA (can be cut by uPA) SGRSS (can be cut by uPA) SGRRA (can be cut by uPA) SGRNA (can be cleaved by uPA) SGRKA (can be cut by uPA) QRGRSA (can be cleaved by tPA) GAGSLLKSRMVPNFNAG (Can be disconnected by cathepsin B) TQGAAA (can be cleaved by cathepsin B) GAAAAA (Can be cleaved by cathepsin B) GAGAAG (can be cleaved by cathepsin B) AAAAAG (can be cleaved by cathepsin B) LCGAAI (can be cleaved by cathepsin B) FAQALG (Can be cleaved by cathepsin B) LLQANP (can be cleaved by cathepsin B) LAAANP (can be cleaved by cathepsin B) LYGAQF (can be cleaved by cathepsin B) LSQAQG (Can be cleaved by cathepsin B) ASAASG (can be cleaved by cathepsin B) FLGASL (can be cleaved by cathepsin B) AYGATG (can be cleaved by cathepsin B) LAQATG (can be cleaved by cathepsin B) GAGSGVVIATVIVITAG (Can be cut by cathepsin L) APMAEGGG (can be cut by Meprin α and Meprin β) EAQGDKII (can be cut by Meprin α and Meprin β) LAFSDAGP (can be cut by Meprin α and Meprin β) YVADAPK (can be cut by Meprin α and Meprin β) RRRRR (Can be cut by Fuhrin) RRRRRR (Can be cut by Fuhrin) GQSSRHRRAL (Can be cut by Fulin) SSRHRRALD (Can be cut by TGFβ) RKSSIIIRMRDVVL (Can be cut with plasminogen) SSSFDKGKYKKGDDA (Can be cleaved by staphylokinase) SSSFDKGKYKRGDDA (Can be cleaved by staphylokinase) IEGR (Can be cut using Factor Xa) IDGR (Can be cut using Factor Xa) GGSIDGR (Can be disconnected by Factor Xa) GPQGIAGQ (Can be cleaved by collagenase) GPQGLLGA (Can be cut by collagenase) GIAGQ (can be cut by collagenase) GPLGIAG (can be cut by collagenase) GPEGLRVG (can be truncated by Collagenase) YGAGLGVV (Can be cut by collagenase) AGLGVVER (Can be cut by collagenase) AGLGISST (Can be cut by collagenase) EPQALAMS (can be cleaved by collagenase) QALAMSAI (can be cut by collagenase) AAYHLVSQ (can be cut by collagenase) MDAFLESS (can be cut by collagenase) ESLPVVAV (Can be cut by collagenase) SAPAVESE (Can be cut by collagenase) DVAQFVLT (Can be cut by collagenase) VAQFVLTE (Can be disconnected by Collagenase) AQFVLTEG (Can be cut by collagenase) PVQPIGPQ (Can be cleaved by collagenase) LVPRGS (can be cut by Thrombin). 【0248】 In some embodiments, the protease-cleaved sequence can be cleaved by at least a cysteine ​​protease. In some embodiments, the protease-cleaved sequence can be cleaved by at least a metalloprotease. In some embodiments, the protease-cleaved sequence can be cleaved by at least a matryptase. In some embodiments, the protease-cleaved sequence can be cleaved by at least MT-SP1. In some embodiments, the protease-cleaved sequence can be cleaved by at least uPA. In some embodiments, the protease-cleaved sequence can be cleaved by at least a matryptase and uPA. In some embodiments, the protease-cleaved sequence can be cleaved by at least MT-SP1 and uPA. 【0249】 In one embodiment, the protease cleavage sequence is selected from the group consisting of PLALAG, VPLSLTMG, GAGVPMSMRGGAG, GAGIPVSLRSGAG, GPLGIAGQ, GGPLGMLSQS, PLGLWA, GAGRPFSMIMGAG, GAGVPLSLTMGAG, GAGVPLSLYSGAG, AANLRN, AQAYVK, AANYMR, AAALTR, AQNLMR, AANYTK, and GAGPQGLAGQRGIVAG, which can be cleaved by MMP. 【0250】 In one embodiment, the protease cleavage sequence is selected from the group consisting of GPQGIAGQ, GPQGLLGA, GIAGQ, GPLGIAG, GPEGLRVG, YGAGLGVV, AGLGVVER, AGLGISST, EPQALAMS, QALAMSAI, AAYHLVSQ, MDAFLESS, ESLPVVAV, SAPAVESE, DVAQFVLT, VAQFVLTE, AQFVLTEG, and PVQPIGPQ, which are cleavable by collagenase. 【0251】 The sequences shown in sequence numbers 1 to 725 can also be used as protease cleavage sequences. 【0252】 The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 In this case, X1 to X8 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and X7 is an amino acid selected from Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. 【0253】 The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 In this context, X1 to X8 each represent a single amino acid, where X1 is an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is A, X8 is an amino acid selected from D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. 【0254】 The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 In this case, X1 to X8 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is A, D, E, X8 is an amino acid selected from F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. 【0255】 The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 In this context, X1 to X8 each represent a single amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is A, X8 is an amino acid selected from D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. 【0256】 The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 In this context, X1 to X8 each represent a single amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, G, H, I, K, L, M, N, Q, R, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is A, X8 is an amino acid selected from D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. 【0257】 The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 In this case, X1 to X8 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from E, F, K, M, N, P, Q, R, S, and W; X7 is A, D, E, F, G, X8 is an amino acid selected from H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. 【0258】 The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 In this case, X1 to X8 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and X7 is an amino acid selected from Y; X8 is an amino acid selected from A, D, F, G, L, M, P, Q, V, and W; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. 【0259】 The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 In this case, X1 to X8 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and X7 is an amino acid selected from Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, I, K, N, T and W. 【0260】 The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 In this sequence, X1 through X8 each represent a single amino acid. X1 is an amino acid selected from A, G, I, P, Q, S, and Y; X2 is an amino acid selected from K or T; X3 is G; X4 is R; X5 is S; X6 is A; X7 is an amino acid selected from H, I, and V; and X8 is an amino acid selected from H, V, and Y. 【0261】 The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 In this sequence, X1 through X8 each represent a single amino acid: X1 is Y; X2 is an amino acid selected from S and T; X3 is G; X4 is R; X5 is S; X6 is an amino acid selected from A and E; and X8 is an amino acid selected from H, P, V, and Y. 【0262】 The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 In this case, X1 to X9 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and X7 is an amino acid selected from Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from R and G. 【0263】 The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 In this case, X1 to X9 each represent one amino acid, where X1 is an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is A, X8 is an amino acid selected from D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from R and G. 【0264】 The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 In this context, X1 to X9 each represent a single amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is A, D, E, F X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from R and G. 【0265】 The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 In this context, X1 to X9 each represent a single amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is A, X8 is an amino acid selected from D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from R and G. 【0266】 The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 In this context, X1 to X9 each represent a single amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, G, H, I, K, L, M, N, Q, R, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is A, X8 is an amino acid selected from D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from R and G. 【0267】 The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 In this case, X1 to X9 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from E, F, K, M, N, P, Q, R, S, and W; X7 is A, D, E, F, G, X8 is an amino acid selected from H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from R and G. 【0268】 The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 In this case, X1 to X9 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and X7 is an amino acid selected from Y; X8 is an amino acid selected from A, D, F, G, L, M, P, Q, V, and W; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from R and G. 【0269】 The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 In this case, X1 to X9 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and X7 is an amino acid selected from Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X9 is an amino acid selected from R and G. 【0270】 The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 In this sequence, X1 through X9 each represent a single amino acid. X1 is an amino acid selected from A, G, I, P, Q, S, and Y; X2 is an amino acid selected from K or T; X3 is G; X4 is R; X5 is S; X6 is A; X7 is an amino acid selected from H, I, and V; X8 is an amino acid selected from H, V, and Y; and X9 is an amino acid selected from R and G. 【0271】 The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 In this sequence, X1 through X9 each represent a single amino acid: X1 is Y; X2 is an amino acid selected from S and T; X3 is G; X4 is R; X5 is S; X6 is an amino acid selected from A and E; X8 is an amino acid selected from H, P, V and Y; and X9 is an amino acid selected from R and G. 【0272】 In addition to using the above-mentioned protease cleavage sequences, new protease cleavage sequences may be obtained through screening. For example, new protease cleavage sequences can be searched for by changing the interaction between the cleavage sequence and the active / recognized residues of the enzyme based on the results of crystal structure analysis of known protease cleavage sequences. Furthermore, new protease cleavage sequences can be searched for by modifying the amino acids in known protease cleavage sequences and confirming their interaction with the protease. As another example, sequences cleaved by proteases can be searched for by displaying a peptide library using in vitro display methods such as phage display or ribosome display, or by confirming the interaction with the protease using a peptide array immobilized on a chip or beads. The interaction between the protease cleavage sequence and the protease can be confirmed by confirming protease cleavage in vitro or in vivo. 【0273】 The protease-cleaved sequences of this disclosure are cleaved by the protease to approximately 0.001 to 1500 × 10⁻¹⁴. 4 M -1 S -1 Or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500 × 10 4 M -1 S -1 It can be specifically modified (cut) at a certain speed. 【0274】 Method for confirming protease cleavage An example of a method for evaluating the cleavage of a protease substrate or protease cleavage sequence by a protease described herein is the method described in Mol Cell Proteomics. 2014 Jun;13(6):1585-97. doi: 10.1074 / mcp.M113.033308. Epub 2014 Apr 4. 【0275】 The type and concentration of protease used for evaluation, as well as the processing temperature and processing time, can be selected as appropriate. For example, PBS containing 1000 nM human uPA, PBS containing 1000 nM mouse uPA, PBS containing 500 nM human MT-SP1, or PBS containing 500 nM mouse MT-SP1 can be used, and the treatment can be carried out at 37°C for 1 hour. 【0276】 Alternatively, instead of using a protease-containing solution, the peptide array may be processed using serum (including human serum and mouse serum), and the fluorescence values ​​measured from the chip may be used. 【0277】 The type and concentration of serum used for evaluation, as well as the processing temperature and processing time, can be selected as appropriate. For example, human serum diluted to 80% concentration can be used as the processing solution and processed overnight at 37°C. 【0278】 To qualitatively confirm whether a protease-cleaved sequence contained in a polypeptide has been cleaved by a protease, a solution containing the polypeptide with the protease-cleaved sequence can be subjected to SDS-PAGE (polyacrylamide gel electrophoresis), and the molecular weight of the fragment can be measured. Alternatively, this can be confirmed by comparing the molecular weight of an untreated polypeptide with that of a polypeptide treated with a protease. 【0279】 In this specification, the term "cleaved" refers to a state in which a polypeptide is fragmented after modification of the protease cleavage sequence by a protease and / or reduction of the cysteine-cysteine ​​disulfide bond of the protease cleavage sequence. In this specification, the term "uncleaved" refers to a state in which the portions on both sides of the protease cleavage sequence in a polypeptide are linked together in the absence of cleavage of the protease cleavage sequence by a protease and / or the absence of reduction of the cysteine-cysteine ​​disulfide bond of the protease cleavage sequence. 【0280】 Furthermore, by quantifying the amount of cleavage fragments after protease treatment separated by electrophoresis methods such as SDS-PAGE, it is possible to evaluate the protease cleavage sequence and the cleavage rate of molecules into which the protease cleavage sequence has been introduced. The following is one non-limiting embodiment of a method for evaluating the cleavage rate of molecules into which the protease cleavage sequence has been introduced. For example, when evaluating the cleavage rate of an antibody variant into which a protease cleavage sequence has been introduced using recombinant human u-Plasminogen Activator / Urokinase (human uPA, huPA) (R&D Systems; 1310-SE-010) or recombinant human Matriptase / ST14 Catalytic Domain (human MT-SP1, hMT-SP1) (R&D Systems; 3946-SE-010), the mixture is reacted for 1 hour under conditions of huPA 40 nM or hMT-SP1 3 nM, antibody variant 100 μg / mL, PBS, and 37°C, and then subjected to a capillary electrophoresis immunoassay. Wes (Protein Simple) can be used for the capillary electrophoresis immunoassay, but is not limited to this method. Alternatively, the sample may be separated by SDS-PAGE or similar methods and then detected by Western blotting. For detecting light chains before and after cleavage, an anti-human lambda chain HRP-labeled antibody (abcam; ab9007) can be used, but any antibody capable of detecting cleavage fragments can be used. By outputting the area of ​​each peak obtained after protease treatment using Wes-specific software (Compass for SW; Protein Simple), the cleavage rate (%) of the antibody modifier can be calculated using the formula (cleavage light chain peak area) * 100 / (cleavage light chain peak area + uncleaved light chain peak area). By calculating the cleavage rate using the method described above, it is possible to compare the in vivo cleavage rates of antibody modifiers with different cleavage sequences, and also to compare the cleavage rates of the same antibody modifier between different animal models, such as normal mouse models and tumor-transplanted mouse models. 【0281】 For example, antigen-binding molecules containing the protease cleavage sequences exemplified in Sequence ID Nos. 1 to 725 as linkers are all useful as protease substrates that are hydrolyzed by the action of proteases. That is, in the present invention, linkers that serve as protease substrates as exemplified herein can be used. Such linkers can be used, for example, as a library for selecting linkers with properties appropriate to the purpose when incorporating them into antigen-binding molecules. Specifically, in order to selectively cleave the antigen-binding molecule with a protease localized in the lesion, the protease sensitivity can be evaluated. Antigen-binding molecules containing linkers may reach the lesion after being administered to a living organism, after coming into contact with various proteases. Therefore, it is desirable to have sensitivity to proteases localized in the lesion while having as high a tolerance as possible to other proteases. In order to select a desirable protease cleavage sequence according to the purpose, protease resistance can be determined by comprehensively analyzing the sensitivity of each protease substrate to various proteases in advance. Based on the obtained protease resistance spectrum, protease cleavage sequences with the required sensitivity and resistance can be identified. 【0282】 Alternatively, antigen-binding molecules incorporating protease cleavage sequences reach the lesion not only through enzymatic action by proteases, but also through various environmental stresses such as pH changes, temperature, and redox stress. Even in response to such external factors, it is possible to select a protease cleavage sequence with desirable properties for the purpose based on information comparing the resistance of various protease substrates. 【0283】 In one embodiment of the present invention, a movable linker is further added to either one or both ends of the protease cleavage sequence. The movable linker at one end of the protease cleavage sequence may be referred to as the first movable linker, and the movable linker at the other end may be referred to as the second movable linker. In a particular embodiment, the protease cleavage sequence and the movable linker include one of the following formulas. 【0284】 (Protease cleavage sequence) (First movable linker)-(Protease cleavage sequence) (Protease cleavage sequence)-(Second movable linker) (First movable linker) - (Protease cleavage sequence) - (Second movable linker) 【0285】 In this embodiment, the movable linker is preferably a peptide linker. The first movable linker and the second movable linker are independently and optionally present, and are identical or different movable linkers containing at least one flexible amino acid (such as Gly). For example, the protease cleavage sequence contains a sufficient number of residues to obtain the desired protease accessibility (amino acids optionally selected from Arg, Ile, Gln, Glu, Cys, Tyr, Trp, Thr, Val, His, Phe, Pro, Met, Lys, Gly, Ser, Asp, Asn, Ala, etc., particularly Gly, Ser, Asp, Asn, Ala, especially Gly and Ser, especially Gly, etc.). 【0286】 A movable linker suitable for use at both ends of a protease cleavage sequence typically improves protease access to the protease cleavage sequence, thereby increasing the efficiency of protease cleavage. Suitable movable linkers are readily selectable, and suitable options can be chosen from a variety of lengths, including 3 to 12 amino acids, as well as 1 to 20 amino acids (e.g., Gly), 2 to 15 amino acids, 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids. In some embodiments of the present invention, the movable linker is a peptide linker with 1 to 7 amino acids. 【0287】 Examples of movable linkers, though not limited to these, include, for example, glycine polymer (G)n, glycine-serine polymer (e.g., (GS) n (GSGGS) n and (GGGS) nExamples include glycine-alanine polymers, alanine-serine polymers, and other movable linkers known in the prior art (where n is at least an integer of 1). 【0288】 Among these, glycine and glycine-serine polymers are attracting attention because these amino acids are relatively unstructured and easily function as neutral tethers between components. 【0289】 Examples of movable linkers made of glycine-serine polymers include, but are not limited to, the following: Ser Gly·Ser(GS) Ser·Gly(SG) Gly·Gly·Ser(GGS) Gly·Ser·Gly (GSG) Ser·Gly·Gly (SGG) Gly·Ser·Ser (GSS) Ser·Ser·Gly (SSG) Ser·Gly·Ser(SGS) Gly·Gly·Gly·Ser(GGGS) Gly·Gly·Ser·Gly (GGSG) Gly·Ser·Gly·Gly(GSGG) Ser·Gly·Gly·Gly(SGGG) Gly·Ser·Ser·Gly(GSSG) Gly·Gly·Gly·Gly·Ser(GGGGS) Gly·Gly·Gly·Ser·Gly(GGGSG) Gly·Gly·Ser·Gly·Gly(GGSGG) Gly·Ser·Gly·Gly·Gly(GSGGG) Gly·Ser·Gly·Gly·Ser(GSGGS) Ser·Gly·Gly·Gly·Gly(SGGGG) Gly·Ser·Ser·Gly·Gly(GSSGG) Gly・Ser・Gly・Ser・Gly(GSGSG) Ser·Gly·Gly·Ser·Gly(SGGSG) Gly・Ser・Ser・Ser・Gly(GSSSG) Gly·Gly·Gly·Gly·Gly·Ser(GGGGGS) Ser·Gly·Gly·Gly·Gly·Gly(SGGGGG) Gly·Gly·Gly·Gly·Gly·Gly·Ser(GGGGGGS) Ser·Gly·Gly·Gly·Gly·Gly·Gly(SGGGGGG) (Gly·Gly·Gly·Gly·Ser(GGGGS)) n (Ser·Gly·Gly·Gly·Gly(SGGGG)) n These are some examples. 【0290】 In this specification, "association" can be rephrased as referring to a state in which, for example, two or more polypeptide regions interact. Generally, hydrophobic bonds, hydrogen bonds, ionic bonds, etc., are formed between the target polypeptide regions to create an aggregate. One common example of association is in antibodies, such as natural antibodies, where the heavy chain variable region (VH) and the light chain variable region (VL) are known to maintain a paired structure through non-covalent bonds between them. 【0291】 In this specification, "interface" usually refers to the association surface during association (interaction), and the amino acid residues forming the interface usually refer to one or more amino acid residues contained in the polypeptide region that are subject to the association, more preferably amino acid residues that approach each other during the association and participate in the interaction. Specifically, this interaction includes non-covalent bonds such as hydrogen bonds, electrostatic interactions, and salt bridges formed between amino acid residues that approach each other during the association. 【0292】 In this specification, "interface-forming amino acid residues" refers, more specifically, to amino acid residues contained within a polypeptide region that constitutes an interface. An interface-forming polypeptide region, for example, refers to a polypeptide region in antibodies, ligands, receptors, substrates, etc., that is responsible for selective intramolecular or intermolecular binding. Examples of interface-forming amino acid residues, though not limited to these, include amino acid residues that come into close proximity during association. These amino acid residues can be identified, for example, by analyzing the three-dimensional structure of a polypeptide and examining the amino acid sequence of the polypeptide region that forms the interface during the polypeptide's association. 【0293】 In some embodiments of the present invention, the antigen-binding domain VHH is associated with the repressive domain VL. Examples of amino acid residues in VHH that are involved in association with VL include those that form the interface between VHH and VL. Examples of amino acid residues in VHH that are involved in association with VL include, but are not limited to, those at positions 37, 44, 45, and 47 (J. Mol. Biol. (2005) 350, 112-125). The association between VHH and VL is promoted, thereby suppressing the activity of VHH. At the same time, examples of amino acid residues in VL that are involved in association with VHH include those that form the interface between VHH and VL. 【0294】 To facilitate the association of VHH and VL, the amino acid residues in VHH that are involved in the association with VL can be modified. Examples of such amino acid substitutions, but not limited to, include F37V, Y37V, E44G, Q44G, R45L, H45L, G47W, F47W, L47W, T47W, or / and S47W. Furthermore, it is also possible to use VHH that has the amino acid residues 37V, 44G, 45L, or / and 47W from the beginning without modifying any residues in VHH. 【0295】 Furthermore, to the extent that the objective of promoting the association of VHH and VL is achieved, it is possible to modify the amino acid residues in VL that are involved in the association with VHH, rather than the amino acids in VHH, and it is also possible to introduce amino acid modifications to both VHH and VL. 【0296】 For modifying amino acids in the amino acid sequence of polypeptides, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately employed. In addition, several known methods can be used for modifying amino acids by substituting them with amino acids other than natural ones (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing tRNA in which a non-natural amino acid is bound to the complementary amber suppressor tRNA of the UAG codon (amber codon), one of the stop codons, is also suitably used. 【0297】 In some further embodiments of the present invention, VHH can be used as the antigen-binding domain and VH or VHH can be used as the repression domain, and the antigen-binding domain and the repression domain can be associated. To promote the association between the antigen-binding domain VHH and the repression domain VH or VHH, amino acid residues in the antigen-binding domain VHH that are involved in the association with the repression domain VH or VHH can be identified and these amino acid residues can be modified. Alternatively, amino acid residues in the repression domain VH or VHH that are involved in the association with the antigen-binding domain VHH can be identified and these amino acid residues can be modified. 【0298】 Furthermore, when using a single-domain antibody other than VHH as the antigen-binding domain, the amino acid residues involved in association within the antigen-binding domain or repressive domain can be identified and modified in the same way. 【0299】 In a particular embodiment, the protease cleavage sequence is located within the antibody constant region of the antigen-binding molecule. In this case, the protease cleavage sequence should be located within the antibody constant region so that the antigen-binding domain can be released when cleaved by the protease. In a specific embodiment, the protease cleavage sequence is located within the antibody heavy chain constant region of the antigen-binding molecule, more specifically, on the antigen-binding domain side of amino acid 140 (EU numbering) in the antibody heavy chain constant region, preferably on the antigen-binding domain side of amino acid 122 (EU numbering) in the antibody heavy chain constant region. In another specific embodiment, the protease cleavage sequence is located within the antibody light chain constant region of the antigen-binding molecule, more specifically, on the antigen-binding domain side of amino acid 130 (Kabat numbering) in the antibody light chain constant region, preferably on the antigen-binding domain side of amino acid 113 (Kabat numbering) in the antibody light chain constant region. 【0300】 In certain embodiments, the linker cleaved by the protease is located near the boundary between the variable region and the constant region, or near the boundary between CH1 and CH2 within the constant region. The area near the boundary between the variable region and the constant region refers to the area before and after the site where VH and CH1 are linked, or before and after the site where VL and CL are linked, which does not significantly affect the secondary structure of the antigen-binding domain, and includes the elbow hinge region (109 (EU numbering) to 140 (EU numbering)). The area near the CH1 and CH2 boundary refers to the area before and after the site where CH1 and CH2 are linked, which does not significantly affect the secondary structure of the antigen-binding domain, and includes the upper hinge region (215 (EU numbering) to 220 (EU numbering)) and the lower hinge region (221 (EU numbering) to 230 (EU numbering)). 【0301】 In a more specific embodiment, the linker cleaved by the protease is located near the boundary between the antigen-binding domain and the antibody constant region within the antigen-binding molecule. The boundary between the antigen-binding domain and the antibody constant region can refer to the boundary between the antigen-binding domain and the antibody heavy chain constant region, or the boundary between the antigen-binding domain and the antibody light chain constant region. When the antigen-binding domain is a single-domain antibody or VHH created from VH and is linked to the antibody heavy chain constant region, the boundary between the antigen-binding domain and the antibody constant region can refer to the area between amino acid 101 (Kabat numbering) of the single-domain antibody and amino acid 140 (EU numbering) of the antibody heavy chain constant region, preferably between amino acid 109 (Kabat numbering) of the single-domain antibody and amino acid 122 (EU numbering) of the antibody heavy chain constant region. When the antigen-binding domain is a single-domain antibody or VHH created from VH and linked to the constant region of the antibody light chain, the area near the boundary between the antigen-binding domain and the constant region of the antibody light chain can refer to the region between amino acid 101 (Kabat numbering) of the single-domain antibody and amino acid 130 (Kabat numbering) of the constant region of the antibody light chain, preferably between amino acid 109 (Kabat numbering) of the single-domain antibody and amino acid 113 (Kabat numbering) of the constant region of the antibody light chain. When the antigen-binding domain is a single-domain antibody created from VL, the area near the boundary between the antigen-binding domain and the constant region refers to the region before and after the site where VHH and CH2 are linked, which does not significantly affect the secondary structure of the antigen-binding domain, and includes the hinge (lower hinge) region, starting from single-domain antibody 96 (Kabat numbering), preferably from single-domain antibody 104 (Kabat numbering). 【0302】 In another embodiment of the present invention, the cleavage site / protease cleavage sequence is located on the variable region side of amino acid 140 (EU numbering) in the constant region of the antibody heavy chain, preferably on the variable region side of amino acid 122 (EU numbering) in the constant region of the antibody heavy chain. In some specific embodiments, the cleavage site / protease cleavage sequence is introduced at any position in the sequence from amino acid 118 (EU numbering) to amino acid 140 (EU numbering) in the constant region of the antibody heavy chain. In another, more specific embodiment, the cleavage site / protease cleavage sequence is located on the variable region side of amino acid 130 (Kabat numbering) in the constant region of the antibody light chain, preferably on the variable region side of amino acid 113 (Kabat numbering) and on the variable region side of amino acid 112 (Kabat numbering) in the constant region of the antibody light chain. In some specific embodiments, the cleavage site / protease cleavage sequence is introduced at any position within the sequence from amino acid 108 (Kabat numbering) to amino acid 131 (Kabat numbering) of the antibody light chain constant region. 【0303】 In one embodiment, the cleavage site / protease cleavage sequence is located near the boundary between the antibody VL and the antibody constant region. The area near the boundary between the antibody VL and the antibody light chain constant region can refer to the area between amino acid 96 (Kabat numbering) of the antibody VL and amino acid 130 (EU numbering) (Kabat numbering 130) of the antibody light chain constant region, preferably between amino acid 104 (Kabat numbering) of the antibody VL and amino acid 113 (EU numbering) (Kabat numbering 113) of the antibody light chain constant region, or between amino acid 105 (Kabat numbering) of the antibody VL and amino acid 112 (EU numbering) (Kabat numbering 112) of the antibody light chain constant region. When the antibody VL and the antibody heavy chain constant region are linked, the area near the boundary between the antibody VL and the antibody heavy chain constant region can refer to the area between amino acid 96 (Kabat numbering) of the antibody VL and amino acid 140 (EU numbering) of the antibody heavy chain constant region, preferably between amino acid 104 (Kabat numbering) of the antibody VL and amino acid 122 (EU numbering) of the antibody heavy chain constant region, or between amino acid 105 (Kabat numbering) of the antibody VL and amino acid 122 (EU numbering) of the antibody heavy chain constant region. 【0304】 In one embodiment, the cleavage site / protease cleavage sequence is introduced near the CH2 / CH3 interface in the constant region of the antibody heavy chain. Here, the region near the CH2 / CH3 interface is the region from EU numbering 335 to 345. 【0305】 Multiple cleavage sites / protease cleavage sequences can be provided within the ligand-binding molecule, for example, at multiple locations selected from within the antibody constant region, within antibody VH, within antibody VL, near the boundary between antibody VH and the antibody constant region, and near the boundary between antibody VL and the antibody constant region. Furthermore, a person skilled in the art familiar with the present invention can change the shape of the molecule containing antibody VH, antibody VL, and the antibody constant region, such as by swapping antibody VH and antibody VL, and such molecular shape does not deviate from the scope of the present invention. 【0306】 As used herein, the term "IgG antibody-like molecule" is used to define a molecule having a substantially similar structure to an IgG antibody, specifically a portion substantially similar to the structure of a constant domain or constant region, and a portion substantially similar to the structure of an IgG antibody, specifically a variable domain or variable region, and having a substantially similar three-dimensional structure to an IgG antibody. However, as used herein, an "IgG antibody-like molecule" is not limited to exhibiting antigen-binding activity while maintaining a structure similar to an IgG antibody. 【0307】 When the antigen-binding molecule is an IgG antibody-like molecule, an embodiment in which antigen-binding domains are provided in the regions corresponding to the two variable regions of the IgG antibody would be understandable to a person skilled in the art who is familiar with the present invention. Whether the antigen-binding domains incorporated in both arms have similar or different antigen-binding specificities, this is an embodiment that would be naturally understandable to a person skilled in the art who is familiar with the present invention, and it is clear that this does not deviate from the scope of the present invention. 【0308】 In this specification, the term "specificity" refers to the property that one molecule of a specifically binding molecule does not substantially bind to any molecule other than the one or more target molecules it binds to. It is also used when an antigen-binding domain has specificity to an epitope contained in a particular antigen. It is also used when an antigen-binding domain has specificity to a particular epitope among several epitopes contained in a certain antigen. Here, "substantially not binding" is determined according to the method described in the section on binding activity, and means that the binding activity of the specific binding molecule to molecules other than the target molecules is 80% or less, usually 50% or less, preferably 30% or less, and particularly preferably 15% or less of the binding activity to the target molecules. 【0309】 As used herein, “treatment” (and its grammatical derivatives, e.g., “to treat,” “to treat,” etc.) means a clinical intervention intended to modify the natural course of the individual being treated, and may be carried out for preventive purposes or during the course of a clinical condition. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, reduction of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction of the rate of disease progression, recovery or mitigation of the disease state, and remission or improved prognosis. In some embodiments, the pharmaceutical compositions of the present invention are used to delay the onset of disease or to slow the progression of disease. 【0310】 In this invention, a pharmaceutical composition generally refers to an agent used for the treatment or prevention of a disease, or for examination and diagnosis. In this invention, when a pharmaceutical composition is used in combination with the administration of other components, the pharmaceutical composition may be administered simultaneously, separately, or consecutively with the other components. This pharmaceutical composition may contain other components as components. 【0311】 The pharmaceutical compositions of the present invention can be formulated using methods known to those skilled in the art. For example, they can be administered parenterally in the form of sterile solutions with water or other pharmaceutically acceptable liquids, or as injectable suspensions. For example, they can be formulated by mixing them with pharmacokinetically acceptable carriers or media, specifically sterile water or saline solution, vegetable oil, emulsifiers, suspensions, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, etc., in a unit dose form generally required for pharmaceutical practice. The amount of active ingredient in these formulations is set to obtain an appropriate volume within the indicated range. 【0312】 Sterile compositions for injection can be formulated in accordance with standard formulation procedures using a vehicle such as distilled water for injection. Examples of aqueous solutions for injection include physiological saline, glucose, and isotonic solutions containing other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). Appropriate solubilizers, such as alcohols (ethanol, etc.), polyalcohols (propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (polysorbate 80™, HCO-50, etc.), may be used in combination. 【0313】 Examples of oily solutions include sesame oil and soybean oil, and benzyl benzoate and / or benzyl alcohol may also be used as solubilizers. Furthermore, buffers (e.g., phosphate buffer and sodium acetate buffer), analgesics (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants may be added. The prepared injection solution is usually filled into appropriate ampoules. 【0314】 The pharmaceutical composition of the present invention is preferably administered by parenteral administration. For example, compositions in the form of injection, nasal administration, pulmonary administration, or transdermal administration may be administered. For example, it may be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc. 【0315】 The method of administration may be appropriately selected depending on the patient's age and symptoms. The dosage of the pharmaceutical composition of the present invention may be set, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight per dose. Alternatively, for example, a dosage of 0.001 to 100,000 mg per patient may be set, but the present invention is not necessarily limited to these values. The dosage and method of administration will vary depending on the patient's weight, age, symptoms, etc., but a person skilled in the art can set an appropriate dosage and method of administration considering these conditions. 【0316】 The polynucleotides in this invention are typically loaded (inserted) into a suitable vector and introduced into host cells. The vector is not particularly limited as long as it stably retains the inserted nucleic acid. For example, if E. coli is used as the host, the pBluescript vector (manufactured by Stratagene) is preferred as a cloning vector, but various commercially available vectors can be used. When a vector is used for the purpose of producing polypeptides (e.g., chimeric receptors, IgG antibodies, bispecific antibodies, antigen-binding molecules, etc.) used in the implementation of this invention, an expression vector is particularly useful. The expression vector is not particularly limited as long as it is a vector that expresses polypeptides in vitro, in E. coli, in cultured cells, or in living organisms. However, for example, the pBEST vector (Promega) is preferred for in vitro expression, the pET vector (Invitrogen) is preferred for E. coli, the pME18S-FL3 vector (GenBank Accession No. AB009864) is preferred for cultured cells, and the pME18S vector (Mol Cell Biol. 8:466-472 (1988)) is preferred for living organisms. Insertion of the DNA of the present invention into the vector can be carried out by conventional methods, for example, by a ligase reaction using restriction enzyme sites (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.4-11.11). 【0317】 There are no particular restrictions on the host cells used, and various host cells can be used depending on the purpose. Examples of cells used to express polypeptides include bacterial cells (e.g., Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, Bacillus subtilis), fungal cells (e.g., yeast, Aspergillus), insect cells (e.g., Drosophila S2, Spodoptera SF9), animal cells (e.g., CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells), and plant cells. Vector introduction into host cells can be performed by known methods such as calcium phosphate precipitation, electroporation (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), lipofectamine method (GIBCO-BRL), and microinjection. 【0318】 To cause polypeptides expressed in host cells to be secreted into the lumen of the endoplasmic reticulum, the pericellular lumen, or the extracellular environment, appropriate secretory signals can be incorporated into the target polypeptide. These signals may be endogenous or heterologous to the target polypeptide. 【0319】 In the above manufacturing method, if the polypeptide of the present invention is secreted into the culture medium, the culture medium is recovered. If the polypeptide of the present invention is produced inside cells, the cells are first lysed, and then the polypeptide is recovered. 【0320】 To recover and purify the polypeptide of the present invention from recombinant cell cultures, known methods can be used, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyl apatite chromatography, and lectin chromatography. 【0321】 Furthermore, monodomain antibodies are used as antigen-binding domains in several embodiments of the present invention, in which the antigen-binding activity of the monodomain antibody is suppressed by association with a specific VL, or by association with a specific VH, or by association with a specific VHH. The present invention also relates to a method for screening such monodomain antibodies. 【0322】 To suppress the antigen-binding activity of single-domain antibodies, VL / VH / VHH sequences with known sequences, such as those registered in the IMGT or Kabat database, can be used. Additionally, sequences newly identified as VL / VH / VHH from human antibody libraries can also be used. By combining these sequences to prepare proteins and measuring their binding activity using the aforementioned method, VL / VH / VHH sequences that suppress the binding activity of single-domain antibodies can be selected. 【0323】 One embodiment of the present invention, the following steps: (a) A step to obtain a monodomain antibody having target antigen-binding activity; (b) A step of associating the single-domain antibody obtained in step (a) with a specific VL; (c) A step to confirm that the binding activity of the monodomain antibody associated with a specific VL in step (b) to the antigen has been weakened or lost; The present invention provides a method for screening single-domain antibodies whose antigen-binding activity is suppressed upon association with a specific VL, including [specific VL]. In this invention, "weakened binding activity" means that the binding activity to the target antigen is reduced compared to before association, regardless of the degree of reduction. 【0324】 One embodiment of the present invention, the following steps: (a) A step to obtain a monodomain antibody having target antigen-binding activity; (b) A step of associating the single-domain antibody obtained in step (a) with a specific VH; (c) A step to confirm that the binding activity of the monodomain antibody associated with a specific VH in step (b) to the antigen has been weakened or lost; The present invention provides a method for screening single-domain antibodies whose antigen-binding activity is suppressed upon association with a specific VH, including [specific VH]. In this invention, "weakened binding activity" means that the binding activity to the target antigen is reduced compared to before association, regardless of the degree of reduction. 【0325】 One embodiment of the present invention, the following steps: (a) A step to obtain a monodomain antibody having target antigen-binding activity; (b) A step of associating the single-domain antibody obtained in step (a) with a specific VHH; (c) A step to confirm that the binding activity of the monodomain antibody associated with a specific VHH in step (b) to the antigen has been weakened or lost; The present invention provides a method for screening single-domain antibodies whose antigen-binding activity is suppressed by association with a specific VHH, including [specific VHH]. In this invention, "weakened binding activity" means that the binding activity to the target antigen is reduced compared to before association, regardless of the degree of reduction. 【0326】 An example of a method for associating a single-domain antibody with a specific VL / VH / VHH sequence is to design a molecule that uses the sequence of a single-domain antibody instead of one of the VH or VL sequences in an antibody or antibody fragment containing both VH and VL, such as a complete antibody, Fab, Fab', or (Fab)2, and then express a polypeptide having that sequence. 【0327】 Furthermore, the present invention relates not only to screening for monodomain antibodies whose antigen-binding activity is suppressed by association with a specific VL, or by association with a specific VH, or by association with a specific VHH, but also to a method for producing monodomain antibodies whose antigen-binding activity is suppressed by promoting association with a specific VL / VH / VHH, or by promoting association with a specific VL, or by promoting association with a specific VH, or by promoting association with a specific VHH. 【0328】 One embodiment of the present invention, the following steps: The present invention provides a method for producing a single-domain antibody whose antigen-binding activity is suppressed by association with a specific VL, comprising the step of (a) producing a modified single-domain antibody in which an amino acid residue involved in association with an antibody VL is substituted in the single-domain antibody, thereby maintaining the binding activity of the single-domain antibody to a target antigen; 【0329】 In a particular embodiment, the following steps are further taken: (b) A step of associating the modified single-domain antibody prepared in step (a) with a specific VL; (c) A step to confirm that the antigen-binding activity of the modified single-domain antibody associated with the VL is weakened or lost; The present invention provides a method for producing a single-domain antibody whose antigen-binding activity is suppressed by association with a specific VL, including [specific VL]. In this invention, "weakened binding activity" means that the binding activity to the target antigen is reduced compared to before association, regardless of the degree of reduction. 【0330】 One embodiment of the present invention, the following steps: (a) A step of producing a modified single-domain antibody in which an amino acid residue involved in association with antibody VH is substituted in the single-domain antibody, thereby maintaining the binding activity of the single-domain antibody against the target antigen; This invention provides a method for producing a single-domain antibody whose antigen-binding activity is suppressed by association with a specific VH, including [specific VH]. 【0331】 In a particular embodiment, the following steps are further taken: (b) A step of associating the modified single-domain antibody prepared in step (a) with a specific VH; (c) A step to confirm that the antigen-binding activity of the modified single-domain antibody associated with the VH has been weakened or lost; The present invention provides a method for producing a single-domain antibody whose antigen-binding activity is suppressed by association with a specific VH, including [specific VH]. In this invention, "weakened binding activity" means that the binding activity to the target antigen is reduced compared to before association, regardless of the degree of reduction. 【0332】 One embodiment of the present invention, the following steps: (a) A step of producing a modified single-domain antibody in which an amino acid residue involved in association with VHH is substituted in the single-domain antibody, thereby maintaining the binding activity of the single-domain antibody to the target antigen; This invention provides a method for producing a single-domain antibody whose antigen-binding activity is suppressed by association with a specific VHH, including [specific VHH]. 【0333】 In a particular embodiment, the following steps are further taken: (b) A step of associating the modified single-domain antibody prepared in step (a) with a specific VHH; (c) A step to confirm that the antigen-binding activity of the modified single-domain antibody associated with the VHH is weakened or lost; The present invention provides a method for producing a single-domain antibody whose antigen-binding activity is suppressed by association with a specific VHH, including [specific VHH]. In this invention, "weakened binding activity" means that the binding activity to the target antigen is reduced compared to before association, regardless of the degree of reduction. 【0334】 The process of associating a single-domain antibody with a specific VL / VH / VHH involves designing an antibody or antibody fragment containing both VH and VL, such as a complete antibody, Fab, Fab', (Fab)2, etc., which uses the sequence of a single-domain antibody instead of one of the VH or VL sequences, and then expressing a polypeptide having that sequence. 【0335】 According to one embodiment of the present invention, a monodomain antibody whose antigen-binding activity is suppressed or lost upon association with a specific VL / VH / VHH of the present invention can be obtained from a library containing multiple fusion polypeptides in which a monodomain antibody is linked to a first association-supporting domain. 【0336】 As an embodiment of the "library" in this specification, it is possible to provide a library that can efficiently obtain single-domain antibodies whose antigen-binding activity is suppressed or lost upon association with a specific VL / VH / VHH. 【0337】 In this specification, "library" means a set of fusion polypeptides having different sequences, or a set of nucleic acids or polynucleotides encoding these fusion polypeptides. The fusion polypeptides contained in the library are not a single sequence, but rather fusion polypeptides with sequences that differ from each other. 【0338】 In this specification, the term "differently sequenced" in the description of multiple fusion polypeptides with different sequences means that the sequences of the individual fusion polypeptides in the library are different from each other. More preferably, it means that the sequences of the single-domain antibody portions in the individual fusion polypeptides in the library are different. That is, the number of different sequences in the library reflects the number of independent clones with different sequences in the library and is sometimes referred to as the "library size." In a typical phage display library, 10 6 from 10 12Therefore, by applying known techniques such as ribosome display, it is possible to expand the library size up to 10¹⁴. However, the actual number of phage particles used during panning selection of a phage library is usually 10 to 10,000 times larger than the library size. This excess multiple is also called the "library equivalent number," and it indicates that there may be 10 to 10,000 individual clones having the same amino acid sequence. Thus, the term "different sequences from each other" in this invention means that the sequences of individual polypeptides in the library, excluding the library equivalent number, are different from each other, or more specifically, that there may be 10 different sequences of polypeptides. 6 from 10 14 Molecules, preferably 10 7 from 10 12 It means that molecules exist. 【0339】 Furthermore, the term "multiple" in the description of the present invention as a library mainly consisting of multiple fusion polypeptides means that, for example, the polypeptides, polynucleotide molecules, vectors, or viruses of the present invention usually refer to a collection of two or more types of the substance. For example, if two or more substances differ from each other with respect to a particular trait, it means that there are two or more types of that substance. An example is a mutant amino acid observed at a particular amino acid position in the amino acid sequence. For example, if there are two or more polypeptides of the present invention that are substantially the same, preferably identical in sequence, except for a particular mutant amino acid at a very diverse range of amino acid positions exposed on the surface, then there are multiple polypeptides of the present invention. In another example, if there are two or more polynucleotide molecules of the present invention that are substantially the same, preferably identical in sequence, except for the bases encoding a particular mutant amino acid at a very diverse range of amino acid positions exposed on the surface, then there are multiple polynucleotide molecules of the present invention. 【0340】 As a screening method for fusion polypeptides using binding activity as an indicator, a panning method using phage vectors is also suitably employed. A fusion polypeptide can be formed by linking a gene encoding a single-domain antibody with a gene encoding the IgG antibody CH1 domain or the light chain constant region in an appropriate embodiment. By inserting the gene encoding the fusion polypeptide into a phage vector, a phage expressing the fusion polypeptide on its surface can be obtained. After contact between this phage and the desired antigen, the phage bound to the antigen can be recovered, thereby recovering the DNA encoding the fusion polypeptide having the desired binding activity. By repeating this operation as needed, fusion polypeptides with the desired binding activity can be enriched. 【0341】 In addition to phage display, other techniques for obtaining fusion polypeptides by panning using libraries include techniques using cell-free translation systems, techniques for presenting fusion polypeptides on the surface of cells or viruses, and techniques using emulsions. For example, techniques using cell-free translation systems include ribosome display, which forms a complex of mRNA and translated protein via ribosomes by removing stop codons, cDNA display, mRNA display, which covalently bonds gene sequences and translated proteins using compounds such as puromycin, and CIS display, which forms a complex of genes and translated proteins using nucleic acid-binding proteins. Furthermore, techniques for presenting fusion polypeptides on the surface of cells or viruses, in addition to phage display, may include E. coli display, Gram-positive bacterium display, yeast display, mammalian cell display, and virus display. Techniques using emulsions may include in vitro virus display, which encapsulates genes and translation-related molecules in an emulsion. These methods are already publicly known (Nat Biotechnol. 2000 Dec;18(12):1287-92, Nucleic Acids Res. 2006;34(19):e127, Proc Natl Acad Sci US A. 2004 Mar 2;101(9):2806-10, Proc Natl Acad Sci US A. 2004 Jun 22;101(25):9193-8, Protein Eng Des Sel. 2008 Apr;21(4):247-55, Proc Natl Acad Sci US A. 2000 Sep 26;97(20):10701-5, MAbs. 2010 Sep-Oct;2(5):508-18, Methods Mol Biol. 2012;911:183-98). 【0342】 In another embodiment of the present invention, a library is provided comprising a plurality of fusion polypeptides in which a single-domain antibody and the constant region of an IgG antibody light chain are linked, wherein the single-domain antibody contains a single-domain antibody whose antigen-binding activity is suppressed or lost upon association with a specific VL / VH / VHH, and a method is provided for screening single-domain antibodies from the library whose antigen-binding activity is suppressed or lost upon association with a specific VL / VH / VHH. 【0343】 "Antigen binding activity below a certain value" can refer to antigen binding activity that falls below a certain standard when measured by the method illustrated in this specification, for example. "Antigen binding activity above a certain value" can similarly refer to antigen binding activity that exceeds a certain standard when measured by the method illustrated in this specification, for example. A fusion polypeptide with antigen binding activity above a certain value binds to the antigen more strongly than a fusion polypeptide with antigen binding activity below a certain value. 【0344】 The following describes several embodiments in which the IgG antibody CH1 domain is used as the first association support domain and the IgG antibody CL is used as the second association support domain. 【0345】 A library containing multiple fusion polypeptides, each linked to a single-domain antibody and the CH1 domain of an IgG antibody, can be used to screen for fusion polypeptides containing the desired single-domain antibody. 【0346】 In some embodiments of the present invention, a library is provided comprising a plurality of fusion polypeptides in which a single-domain antibody and an IgG antibody CH1 domain are linked, wherein the single-domain antibody contains a single-domain antibody whose antigen-binding activity is suppressed or lost upon association with a specific VL / VH / VHH, and a method is provided for screening fusion polypeptides from the library containing a single-domain antibody whose antigen-binding activity is suppressed or lost upon association with a specific VL / VH / VHH. 【0347】 In a particular embodiment, a method is provided for screening a library containing multiple fusion polypeptides in which a single-domain antibody is linked to the CH1 domain of an IgG antibody, for fusion polypeptides containing a single-domain antibody whose antigen-binding activity is suppressed or lost upon association with a specific VL. Specifically, the following steps are taken: (a) A step of in vitro displaying the fused polypeptide of the library according to the present invention; (b) A step of preparing an association partner by fusing a specific VL with the constant region of an IgG antibody light chain; (c) A step of associating the fusion polypeptide displayed in step (a) with the association partner prepared in step (b), and selecting a fusion polypeptide that does not bind to the antigen when the single-domain antibody and the VL are associated, or whose antigen-binding activity is below a certain value; (d) A step of selecting a fusion polypeptide in which the single-domain antibody contained in the fusion polypeptide selected in step (c) binds to the antigen without the VL being associated, or in which the antigen-binding activity is above a certain value; A screening method for single-domain antibodies, including those containing such antibodies, is provided. 【0348】 The association partner prepared in step (b) further includes a protease cleavage sequence, and in step (d), the association between the monodomain antibody and the VL is dissolved by protease treatment, making it possible to confirm the antigen-binding activity of the monodomain antibody in a state where the monodomain antibody and VL are not associated. The position of the protease cleavage sequence in the association partner is not limited as long as the association between the monodomain antibody and VL is dissolved during cleavage. As an example of the position of the protease cleavage sequence, it can be located near the boundary between the VL of the association partner and the constant region of the IgG antibody light chain, preferably between amino acid 96 (Kabat numbering) of the VL and amino acid 130 (EU numbering) (Kabat numbering 130) of the antibody light chain constant region, and more preferably between amino acid 104 (Kabat numbering) of the VL and amino acid 113 (EU numbering) (Kabat numbering 113) of the antibody light chain constant region. 【0349】 Alternatively, instead of using an association partner containing a protease cleavage sequence, it is possible to introduce a protease cleavage sequence into the fusion polypeptide in the library, thereby dissociating the monodomain antibody from the VL by cleavage of the fusion polypeptide by the protease. The position of the protease cleavage sequence in the fusion polypeptide is not limited, as long as the association between the monodomain antibody and the VL is dissociated upon cleavage, and the antigen-binding activity of the monodomain antibody is maintained after cleavage. For example, the protease cleavage sequence can be located near the boundary between the monodomain antibody and the IgG antibody CH1 domain in the fusion polypeptide. 【0350】 Furthermore, in step (d), it is also possible to display the full-length or single-domain antibody portion of the fusion polypeptide selected in step (c) again, and confirm the antigen-binding activity of the single-domain antibody without association between the single-domain antibody and VL. 【0351】 In a particular embodiment, a method is provided for screening a library containing multiple fusion polypeptides, each consisting of a single-domain antibody linked to the constant region of an IgG antibody light chain, for fusion polypeptides containing a single-domain antibody whose antigen-binding activity is suppressed or lost upon association with a specific VH. Specifically, the following steps are taken: (a) A step of in vitro displaying the fused polypeptide of the library according to the present invention; (b) A step of preparing an association partner in which a specific VH and the IgG antibody CH1 domain are fused; (c) A step of associating the fusion polypeptide displayed in step (a) with the association partner prepared in step (b), and selecting a fusion polypeptide that does not bind to the antigen when the single-domain antibody and the VH are associated, or whose antigen-binding activity is below a certain value; (d) A step of selecting a fusion polypeptide in which the single-domain antibody contained in the fusion polypeptide selected in step (c) binds to the antigen without associating with the VH, or in which the antigen-binding activity is above a certain value; A method for screening fusion polypeptides containing a single-domain antibody is provided. 【0352】 The association partner prepared in step (b) further includes a protease cleavage sequence, and in step (d), the association between the monodomain antibody and the VH is dissolved by protease treatment, making it possible to confirm the antigen-binding activity of the monodomain antibody in a state where the monodomain antibody and VH are not associated. The position of the protease cleavage sequence in the association partner is not limited as long as the association between the monodomain antibody and VH is dissolved during cleavage. As an example of the position of the protease cleavage sequence, it can be located near the boundary between the VH of the association partner and the CH1 domain of the IgG antibody, preferably between amino acid 101 (Kabat numbering) of VH and amino acid 140 (EU numbering) of the antibody heavy chain constant region, and more preferably between amino acid 109 (Kabat numbering) of VH and amino acid 122 (EU numbering) of the antibody heavy chain constant region. 【0353】 Alternatively, instead of using an association partner containing a protease cleavage sequence, it is possible to introduce a protease cleavage sequence into the fusion polypeptide in the library, thereby dissociating the monodomain antibody from VH when the fusion polypeptide is cleaved by the protease. The location of the protease cleavage sequence in the fusion polypeptide is not limited, as long as the association between the monodomain antibody and VH is dissociated upon cleavage, and the antigen-binding activity of the monodomain antibody is maintained after cleavage. For example, the protease cleavage sequence can be located near the boundary between the monodomain antibody and the constant region of the IgG antibody light chain in the fusion polypeptide. 【0354】 Furthermore, in step (d), it is also possible to display the full-length or monodomain antibody portion of the fusion polypeptide selected in step (c) again, and confirm the antigen-binding activity of the monodomain antibody without association between the monodomain antibody and VH. 【0355】 The amino acids included in the amino acid sequence described in this invention may undergo post-translational modifications (for example, modification to pyroglutamic acid by pyroglutamylation of the N-terminal glutamine is a modification well known to those skilled in the art), but even when amino acids are modified post-translation in this way, they are naturally still included in the amino acid sequence described in this invention. 【0356】 Methods for producing antibodies with desired binding activity are known to those skilled in the art. In the present invention, antigen-binding molecules can be used in which a molecule expressed on the surface of a target cell (lesion cell) is the antigen (target antigen). When the target cell is a tumor cell or cancer cell, the antigen is exemplified herein as a tumor antigen. The following is an example of a method for producing an antibody that binds to a tumor antigen. 【0357】 Antibodies that bind to tumor antigens can be obtained as polyclonal or monoclonal antibodies using known methods. Mammalian-derived monoclonal antibodies are preferably produced as such antibodies. Mammalian-derived monoclonal antibodies include those produced by hybridomas and those produced by host cells transformed with expression vectors containing antibody genes using genetic engineering techniques. 【0358】 Monoclonal antibody-producing hybridomas can be produced using known techniques, for example, as follows: Mammals are immunized according to a standard immunization method using a tumor antigen protein as the sensitizing antigen. The resulting immune cells are fused with known parent cells by a standard cell fusion method. Next, hybridomas that produce anti-tumor antigen antibodies can be selected by screening monoclonal antibody-producing cells using a standard screening method. 【0359】 Specifically, the production of monoclonal antibodies is carried out as follows: First, a tumor antigen protein can be obtained by expressing a tumor antigen gene, which can then be used as a sensitizing antigen for antibody acquisition. That is, a suitable host cell is transformed by inserting the gene sequence encoding the tumor antigen into a known expression vector. The desired human tumor antigen protein is purified from the host cell or culture supernatant by a known method. To obtain a soluble tumor antigen from the culture supernatant, for example, a protein in which the hydrophobic region of the tumor antigen polypeptide sequence has been deleted can be used. Similarly, purified natural GPC3 protein can also be used as a sensitizing antigen. 【0360】 The purified tumor antigen protein can be used as a sensitizing antigen for immunization against mammals. Partial peptides of the tumor antigen can also be used as sensitizing antigens. In this case, the partial peptide can be obtained by chemical synthesis from the amino acid sequence of the human tumor antigen. It can also be obtained by incorporating a part of the tumor antigen gene into an expression vector and expressing it. Furthermore, it can be obtained by degrading the tumor antigen protein using a protease, but the region and size of the tumor antigen peptide used as a partial peptide are not particularly limited to any special form. Preferably, the number of amino acids constituting the peptide to be used as a sensitizing antigen is at least 5, for example, 6 or more, or 7 or more. More specifically, peptides with 8 to 50, preferably 10 to 30 residues, can be used as sensitizing antigens. 【0361】 Furthermore, fusion proteins obtained by fusing a desired partial polypeptide or peptide of a tumor antigen protein with a different polypeptide can be used as sensitizing antigens. For example, antibody Fc fragments or peptide tags can be suitably used to produce fusion proteins used as sensitizing antigens. A vector expressing a fusion protein can be produced by fusing genes encoding two or more desired polypeptide fragments in-frame, and then inserting the fusion gene into an expression vector as described above. Methods for producing fusion proteins are described in Molecular Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58 (1989) Cold Spring Harbor Lab. press). As an example, methods for obtaining GPC3 used as a sensitizing antigen and immunization methods using it are specifically described in WO2003 / 000883, WO2004 / 022754, WO2006 / 006693, etc. 【0362】 While the mammals immunized with the sensitizing antigen are not limited to specific animals, it is preferable to select them considering their compatibility with the parent cells used for cell fusion. Generally, rodents such as mice, rats, hamsters, rabbits, and monkeys are preferred. 【0363】 The animals described above are immunized with the sensitizing antigen according to known methods. For example, a common method is to administer the sensitizing antigen to mammals by injection intraperitoneal or subcutaneous injection. Specifically, the sensitizing antigen, diluted to an appropriate dilution ratio with PBS (Phosphate-Buffered Saline) or physiological saline, is mixed with a conventional adjuvant, such as Freund's complete adjuvant, if desired, and emulsified. After emulsification, the sensitizing antigen is administered to mammals several times every 4 to 21 days. A suitable carrier may also be used during immunization with the sensitizing antigen. In particular, when a partial peptide with a small molecular weight is used as the sensitizing antigen, it may be desirable to immunize with the sensitizing antigen peptide bound to a carrier protein such as albumin or keyhole limpet hemocyanin. 【0364】 Furthermore, hybridomas that produce the desired antibody can also be produced using DNA immunization as follows. DNA immunization is an immunization method in which a vector DNA constructed in such a manner that a gene encoding an antigen protein can be expressed in the immunized animal is administered, and the sensitized antigen is expressed in the immunized animal, thereby providing immune stimulation. Compared to general immunization methods in which protein antigens are administered to immunized animals, DNA immunization is expected to have the following advantages. - The structure of membrane proteins can be maintained, allowing for immune stimulation. - There is no need to purify immune antigens. 【0365】 To obtain the monoclonal antibody of the present invention by DNA immunization, first, DNA expressing a tumor antigen protein is administered to an immunized animal. The DNA encoding the tumor antigen can be synthesized by known methods such as PCR. The obtained DNA is inserted into a suitable expression vector and administered to an immunized animal. Commercial expression vectors such as pcDNA3.1 can be suitably used as the expression vector. Commonly used methods can be used to administer the vector into a living organism. For example, DNA immunization is performed by introducing gold particles to which the expression vector is adsorbed into the cells of an immunized animal using a gene gun. Furthermore, antibodies that recognize tumor antigens can also be produced using the method described in International Publication WO2003 / 104453. 【0366】 After the mammal has been immunized in this manner and an increase in antibody titers binding to tumor antigens in the serum has been confirmed, immune cells are collected from the mammal and used for cell fusion. Splenocytes, in particular, may be used as preferred immune cells. 【0367】 Mammalian myeloma cells are used as the cells fused with the aforementioned immune cells. It is preferable that the myeloma cells possess appropriate selection markers for screening. A selection marker refers to a trait that allows (or prevents) survival 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). Cells lacking HGPRT or TK are hypoxanthine-aminopterin-thymidine sensitive (hereinafter abbreviated as HAT sensitive). HAT-sensitive cells cannot synthesize DNA in HAT-selective medium and die, but when fused with normal cells, they can continue DNA synthesis using the normal cell's salvage pathway and thus proliferate even in HAT-selective medium. 【0368】 HGPRT-deficient and TK-deficient cells 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 their DNA will die. On the other hand, cells lacking these enzymes and unable to incorporate these pyrimidine analogs can survive in the selective medium. Another selection marker, known as G418 resistance, confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) via the neomycin resistance gene. Various myeloma cells suitable for cell fusion are known. 【0369】 Examples of such myeloma cells include P3 (P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (C. Eur. J. Immunol. (1976) 6 (7), 511-519), MPC-11 (Cell (1976) 8 (3), 405-415), SP2 / 0 (Nature (1978) 276 (5685), 269-270), FO (J. Immunol. Methods (1980) 35 (1-2), 1-21), S194 / 5.XX0.BU.1 (J. Exp. Med.(1978)148 (1), 313-323), R210 (Nature(1979)277 (5692), 131-133), etc., can be suitably used. 【0370】 Cell fusion between the immune cells and myeloma cells is basically performed according to known methods, such as the method of Köhler and Myrstein et al. (Methods Enzymol. (1981) 73, 3-46). 【0371】 More specifically, the cell fusion can be carried out, for example, in a normal nutrient culture medium in the presence of a cell fusion promoter. Examples of fusion promoters include polyethylene glycol (PEG) and Sendai virus (HVJ), and additional adjuvants such as dimethyl sulfoxide may be added as desired to further enhance fusion efficiency. 【0372】 The ratio of immune cells to myeloma cells can be set arbitrarily. For example, it is preferable to use 1 to 10 times more immune cells than myeloma cells. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium, MEM culture medium, or other common culture mediums used for this type of cell culture can be used, and serum supplements such as fetal bovine serum (FCS) may be suitably added. 【0373】 Cell fusion is performed by thoroughly mixing predetermined amounts of the immune cells and myeloma cells in the culture medium, and then adding a PEG solution (for example, with an average molecular weight of about 1000 to 6000) that has been preheated to about 37°C, usually at a concentration of 30 to 60% (w / v). The desired fused cells (hybridomas) are formed by the gradual mixing of the mixture. Subsequently, the appropriate culture medium mentioned above is added sequentially, and the process of centrifugation and removal of the supernatant is repeated, thereby removing cell fusion agents and other substances unfavorable to hybridoma growth. 【0374】 The hybridomas obtained in this manner can be selected by culturing them in a standard selective culture medium, such as HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Culturing with the HAT culture medium can be continued for a sufficient time (usually several days to several weeks) to kill cells other than the desired hybridoma (non-fusion cells). Subsequently, screening and single cloning of hybridomas that produce the desired antibody is performed using a standard limiting dilution method. 【0375】 The hybridomas obtained in this way can be selected by using a selective culture medium corresponding to the selection markers present in 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 successfully fuse with normal cells can be selectively proliferated in the HAT culture medium. Culturing with the HAT culture medium is continued for a sufficient amount of time for cells other than the desired hybridoma (non-fused cells) to die. Specifically, generally, the desired hybridoma can be selected by culturing for several days to several weeks. Subsequently, screening and single cloning of hybridomas that produce the desired antibody can be performed using the usual limiting dilution method. 【0376】 Screening and single cloning of desired antibodies can be suitably carried out by known antigen-antibody reaction-based screening methods. For example, a monoclonal antibody that binds to GPC3 can bind to GPC3 expressed on the cell surface. Such monoclonal antibodies can be screened, for example, by FACS (fluorescence activated cell sorting). FACS is a system that allows for the measurement of antibody binding to the cell surface by analyzing cells contacted with a fluorescent antibody using laser light and measuring the fluorescence emitted by individual cells. 【0377】 To screen hybridomas that produce the monoclonal antibody of the present invention by FACS, cells expressing GPC3 are first prepared. Preferred cells for screening are mammalian cells that forcibly express the tumor antigen used. By using untransformed mammalian cells as the host cells as a control, the antibody binding activity to the tumor antigen on the cell surface can be selectively detected. That is, by selecting hybridomas that produce antibodies that do not bind to host cells but bind to GPC3-forcibly expressing cells, hybridomas that produce tumor antigen monoclonal antibodies can be obtained. 【0378】 Alternatively, the binding activity of antibodies against immobilized tumor antigen-expressing cells can be evaluated based on the principles of ELISA. For example, GPC3-expressing cells are immobilized in the wells of an ELISA plate. The culture supernatant of hybridomas is brought into contact with the immobilized cells in the wells, and antibodies that bind to the immobilized cells are detected. If the monoclonal antibody is derived from a mouse, the antibody bound to the cells can be detected by an anti-mouse immunoglobulin antibody. Hybridomas that produce the desired antibody with antigen-binding ability, selected through these screenings, can be cloned by methods such as limiting dilution. 【0379】 The hybridomas producing monoclonal antibodies thus created can be subcultured in a normal culture medium. Furthermore, these hybridomas can be stored for extended periods in liquid nitrogen. 【0380】 The hybridoma can be cultured according to conventional methods, and the desired monoclonal antibody can be obtained from the culture supernatant. Alternatively, the hybridoma can be administered to a compatible mammal to proliferate, and the monoclonal antibody can be obtained from its ascites fluid. The former method is suitable for obtaining high-purity antibodies. 【0381】 Antibodies encoded by antibody genes cloned from antibody-producing cells such as hybridomas can also be suitably utilized. By incorporating the cloned antibody gene into a suitable vector and introducing it into a host, the antibody encoded by the gene is expressed. Methods for isolating antibody genes, introducing them into vectors, and transforming host cells have already been established, for example, by Vandamme et al. (Eur.J. Biochem.(1990)192 (3), 767-775). Methods for producing recombinant antibodies are also known, as described below. 【0382】 For example, cDNA encoding the variable region (V region) of an antibody can be obtained from hybridoma cells that produce antibodies that bind to tumor antigens. To do this, total RNA is usually extracted from the hybridoma first. Methods for extracting mRNA from cells include, for example, the following: - Guanidine ultracentrifugation (Biochemistry (1979) 18 (24), 5294-5299) - AGPC method (Anal. Biochem. (1987) 162 (1), 156-159). 【0383】 The extracted mRNA can be purified using an mRNA Purification Kit (GE Healthcare Biosciences), etc. Alternatively, kits for directly extracting total mRNA from cells are commercially available, such as the QuickPrep mRNA Purification Kit (GE Healthcare Biosciences). mRNA can be obtained from hybridomas using such kits. From the obtained mRNA, cDNA encoding the antibody V region can be synthesized using reverse transcriptase. cDNA can be synthesized using an AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation), etc. Furthermore, for cDNA synthesis and amplification, the SMART RACE cDNA amplification kit (Clontech) and the 5'-RACE method using PCR (Proc. Natl. Acad. Sci. USA (1988) 85 (23), 8998-9002, Nucleic Acids Res. (1989) 17 (8), 2919-2932) may be used as appropriate. Furthermore, during the process of synthesizing cDNA, appropriate restriction enzyme sites, as described later, can be introduced at both ends of the cDNA. 【0384】 The target cDNA fragment is purified from the obtained PCR product and then ligated to vector DNA. A recombinant vector is thus prepared, introduced into E. coli or other organisms, and after colony selection, the desired recombinant vector can be prepared from the E. coli that formed the colonies. Then, whether or not the recombinant vector possesses the target cDNA base sequence is confirmed by known methods, such as dideoxynucleotide chain intermination. 【0385】 To obtain genes encoding variable regions, the 5'-RACE method using primers for variable region gene amplification is a convenient approach. First, cDNA is synthesized using RNA extracted from hybridoma cells as a template, yielding a 5'-RACE cDNA library. Commercially available kits, such as the SMART RACE cDNA amplification kit, can be used as appropriate for synthesizing the 5'-RACE cDNA library. 【0386】 The obtained 5'-RACE cDNA library is used as a template to amplify the antibody gene by PCR. Primers for mouse antibody gene amplification can be designed based on known antibody gene sequences. 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). 【0387】 Specifically, for example, when the goal is to obtain genes encoding mouse IgG, primers capable of amplifying genes encoding γ1, γ2a, γ2b, and γ3 as heavy chains, and κ and λ chains as light chains, can be used. To amplify the variable region genes of IgG, a primer that anneals to the constant region close to the variable region is generally used for the 3' side. On the other hand, for the 5' side primer, the primers included with the 5' RACE cDNA library preparation kit are used. 【0388】 Using the PCR product thus amplified, an immunoglobulin consisting of a combination of heavy and light chains can be reconstituted. The binding activity of the reconstituted immunoglobulin to the antigen can be used as an indicator to screen for the desired antibody. For example, when the goal is to obtain an antibody against GPC3, it is even more preferable that the antibody binds to GPC3 specifically. Antibodies used in this invention can be screened, for example, as follows: (1) A step of contacting antigen-expressing cells with an antibody containing a V region encoded by cDNA obtained from a hybridoma, (2) A step of detecting the binding of antigen-expressing cells to antibodies, (3) A step of selecting an antibody that binds to antigen-expressing cells. 【0389】 Methods for detecting the binding of antibodies to tumor antigen-expressing cells are well known. Specifically, the binding of antibodies to tumor antigen-expressing cells can be detected using techniques such as FACS, as mentioned earlier. Fixed specimens of tumor antigen-expressing cells may be used as appropriate to evaluate the binding activity of antibodies. 【0390】 As a screening method for antibodies using binding activity as an indicator, the panning method using phage vectors is also suitably employed. When antibody genes are obtained from a polyclonal antibody-expressing cell population as a library of heavy chain and light chain subclasses, the screening method using phage vectors is advantageous. Genes encoding the variable regions of the heavy chain and light chain can be linked with a suitable linker sequence to form a single-chain Fv (scFv). By inserting the gene encoding scFv into a phage vector, a phage expressing scFv on its surface can be obtained. After contact between this phage and the desired antigen, the phage bound to the antigen can be recovered, thereby recovering the DNA encoding scFv with the desired binding activity. By repeating this operation as needed, scFv with the desired binding activity can be enriched. 【0391】 After obtaining cDNA encoding the V region of an antibody that binds to the target tumor antigen, the cDNA is digested by restriction enzymes that recognize restriction enzyme sites inserted at both ends of the cDNA. Preferred restriction enzymes recognize and digest base sequences that appear infrequently in the base sequence constituting the antibody gene. Furthermore, to insert one copy of the digested fragment into the vector in the correct orientation, insertion of a restriction enzyme that provides an adhesive end is preferable. 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 constant region and the variable region originate from different sources. Therefore, in addition to heterologous chimeric antibodies such as mouse-human, human-human allologous chimeric antibodies are also included in the chimeric antibodies of this invention. A chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector that already has a constant region. Specifically, for example, a restriction enzyme recognition sequence for a restriction enzyme that digests the V region gene can be appropriately placed on the 5' end of an expression vector containing DNA encoding the desired antibody constant region (C region). A chimeric antibody expression vector is constructed by in-frame fusion of the two, which have been digested with the same combination of restriction enzymes. 【0392】 To produce monoclonal antibodies, the antibody gene is incorporated into an expression vector so that it is expressed under the control of an expression regulatory region. This expression regulatory region includes, for example, enhancers and promoters. Furthermore, an appropriate signal sequence may 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 is used as the signal sequence, but other suitable signal sequences can also be added. The expressed polypeptide is cleaved at the carboxyl terminus of the above sequence, and the cleaved polypeptide can be secreted extracellularly as a mature polypeptide. Subsequently, by transforming a suitable host cell with this expression vector, recombinant cells expressing DNA encoding an antibody that binds to a target tumor antigen can be obtained. 【0393】 For antibody gene expression, the DNA encoding the antibody heavy chain (H chain) and light chain (L chain) is incorporated into separate expression vectors. The vectors containing both the H and L chains can simultaneously transform (co-transfect) the same host cells, thereby expressing antibody molecules with both H and L chains. Alternatively, the host cells can be transformed by incorporating the DNA encoding both the H and L chains into a single expression vector (see International Publication WO 94 / 11523). 【0394】 Many combinations of host cells and expression vectors are known for producing antibodies by introducing isolated antibody genes into suitable hosts. These expression systems can all be applied to isolate the domain containing the antibody variable region of the present invention. When eukaryotic cells are used as host cells, animal cells, plant cells, or fungal cells may be used as appropriate. Specifically, the following are examples of animal cells: (1) Mammalian cells: CHO, COS, myeloma, BHK (baby hamster kidney), Hela, Vero, etc. (2) Amphibian cells: African clawed frog oocytes, etc. (3) Insect cells: sf9, sf21, Tn5, etc. 【0395】 Alternatively, as for plant cells, antibody gene expression systems using cells from the Nicotiana genus, such as Nicotiana tabacum, are known. Callus-cultured cells can be used as appropriate for plant cell transformation. 【0396】 Furthermore, the following types of fungal cells can be used: - Yeast: Saccharomyces species such as Saccharomyces serevisiae, and Pichia species such as methanol-utilizing yeast Pichia pastoris. - Filamentous fungi: Aspergillus species such as Aspergillus niger. 【0397】 Furthermore, antibody gene expression systems using prokaryotic cells are also known. For example, when using bacterial cells, bacterial cells such as Escherichia coli (E. coli) and Bacillus subtilis can be used as appropriate. 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. 【0398】 In addition to the host cells mentioned above, transgenic animals can also be used to produce recombinant antibodies. That is, antibodies can be obtained from animals into which the 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 a gene encoding a protein that is specifically produced in milk. As the protein secreted in milk, for example, goat β-casein can be used. The 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. From the milk produced by the transgenic goat (or its offspring) born from the goat that received the embryo, the desired antibody can be obtained as a fusion protein with the milk protein. Furthermore, 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). 【0399】 When the antigen-binding molecules described herein are administered to humans, the domain containing the antibody variable region in the antigen-binding molecule may appropriately be a domain derived from a recombinant antibody that has been artificially modified for purposes such as reducing heterologous antigenicity to humans. Recombinant antibodies include, for example, humanized antibodies. These modified antibodies can be appropriately manufactured using known methods. 【0400】 The variable region of an antibody used to create a domain containing the antibody variable region in the antigen-binding molecule described herein typically consists of three complementarity-determining regions (CDRs) flanked by four framework regions (FRs). The CDRs are essentially the regions that determine the antibody's binding specificity. The amino acid sequences of CDRs are highly diverse. On the other hand, the amino acid sequences constituting the FRs often exhibit high identity even among antibodies with different binding specificities. Therefore, it is generally believed that the binding specificity of one antibody can be transferred to another antibody by transplanting the CDRs. 【0401】 Humanized antibodies are also called reshaped human antibodies. Specifically, known examples include humanized antibodies obtained by transplanting the CDR of an antibody from a non-human animal, such as a mouse antibody, into a human antibody. General genetic recombination methods for obtaining humanized antibodies are also known. Specifically, Overlap Extension PCR is a known method for transplanting the CDR of a mouse antibody into the FR of a human antibody. In Overlap Extension PCR, the nucleotide sequence encoding the mouse antibody CDR to be transplanted is added to the primer for synthesizing the human antibody FR. Primers are prepared for each of the four FRs. Generally, when transplanting mouse CDRs into human FRs, it is considered advantageous to select human FRs with high identity to the mouse FRs in order to maintain the function of the CDRs. That is, it is generally preferable to use human FRs with amino acid sequences that have high identity to the amino acid sequences of the FRs adjacent to the mouse CDR to be transplanted. 【0402】 Furthermore, the nucleotide sequences to be linked are designed to connect in-frame. Human FRs are synthesized individually using each primer. As a result, products are obtained in which DNA encoding mouse CDRs is attached to each FR. The nucleotide sequences encoding mouse CDRs in each product are designed to overlap with each other. Subsequently, a complementary chain synthesis reaction takes place by annealing the overlapping CDR regions of products synthesized using the human antibody gene as a template. Through this reaction, human FRs are linked via the mouse CDR sequences. 【0403】 The V-region gene, which ultimately consists of three CDRs and four FRs linked together, is amplified to its full length by primers that anneal to its 5' and 3' ends and have appropriate restriction enzyme recognition sequences added. A vector for human antibody expression can be created by inserting the DNA obtained as described above into an expression vector so as to fuse it in-frame with the DNA encoding the human antibody C-region. After introducing this integration vector into a host to establish recombinant cells, the recombinant cells are cultured and the DNA encoding the humanized antibody is expressed, thereby producing the humanized antibody in the cultured cell culture (see European Patent Publication EP 239400, International Publication WO1996 / 002576). 【0404】 By qualitatively or quantitatively measuring and evaluating the antigen-binding activity of the humanized antibody prepared as described above, a human antibody FR that forms a good antigen-binding site for the CDR when linked via the CDR can be suitably selected. If necessary, amino acid residues of the FR can be substituted so that the reconstituted human antibody CDR forms an appropriate antigen-binding site. For example, amino acid sequence mutations can be introduced into the FR by applying the PCR method used for transplanting mouse CDRs into human FRs. Specifically, partial nucleotide sequence mutations can be introduced into primers that anneal to the FR. The FR synthesized by such primers will have nucleotide sequence mutations introduced. By measuring and evaluating the antigen-binding activity of the mutant antibody with substituted amino acids using the method described above, a mutant FR sequence with the desired properties can be selected (Sato, K. et al., Cancer Res, 1993, 53, 851-856). 【0405】 Furthermore, transgenic animals possessing the entire repertoire of human antibody genes (see International Publications WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, WO1996 / 033735) can be used as immunized animals, and desired human antibodies can be obtained by DNA immunization. 【0406】 Furthermore, a technique for obtaining human antibodies by panning using a human antibody library is also known. For example, the V region of a human antibody is expressed as a single-chain antibody (scFv) on the surface of a phage using phage display. A phage expressing an scFv that binds to an antigen can be selected. By analyzing the genes of the selected phage, the DNA sequence encoding the V region of the human antibody that binds to the antigen can be determined. After determining the DNA sequence of the scFv that binds to the antigen, an expression vector can be created by fusing the V region sequence in-frame with the sequence of the desired human antibody C region and then inserting it into a suitable expression vector. The human antibody can be obtained by introducing this expression vector into suitable expression cells as described above and expressing the gene encoding the human antibody. These methods are already publicly known (see International Publications WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, WO1995 / 015388). 【0407】 Domain containing an antibody variable region with T cell receptor complex binding activity. In this specification, "domain containing an antibody variable region having T cell receptor complex binding activity" refers to a portion of a T cell receptor complex antibody that includes a region that specifically binds to and is complementary to part or all of the T cell receptor complex. The T cell receptor complex may be the T cell receptor itself, or an adapter molecule that, together with the T cell receptor, constitutes the T cell receptor complex. CD3 is a suitable adapter. 【0408】 Domain containing an antibody variable region with T cell receptor binding activity In this specification, "domain containing an antibody variable region having T cell receptor binding activity" refers to a portion of a T cell receptor antibody that includes a region that specifically binds to and is complementary to a part or all of the T cell receptor. The portion of the T cell receptor to which the domain of the present invention binds may be a variable region or a constant region, but preferably an epitope located in the constant region. Examples of constant region sequences include the T cell receptor alpha chain of RefSeq registry number CAA26636.1, the T cell receptor beta chain of RefSeq registry number C25777, the T cell receptor gamma 1 chain of RefSeq registry number A26659, the T cell receptor gamma 2 chain of RefSeq registry number AAB63312.1, and the T cell receptor delta chain of RefSeq registry number AAA61033.1. 【0409】 Domain containing an antibody variable region having CD3 binding activity. In this specification, "domain containing an antibody variable region having CD3 binding activity" means a portion of a CD3 antibody comprising a region that specifically binds to and is complementary to a part or all of CD3. Preferably, the domain includes the light chain variable region (VL) and the heavy chain variable region (VH) of the anti-CD3 antibody. 【0410】 The domain containing the antibody variable region having CD3 binding activity according to the present invention may bind to any epitope present in the gamma, delta, or epsilon chain sequence constituting human CD3. In the present invention, a domain containing the light chain variable region (VL) and heavy chain variable region (VH) of an anti-CD3 antibody that binds to an epitope present in the extracellular region of the epsilon chain of the human CD3 complex is preferably used. In addition to the light chain variable region (VL) and heavy chain variable region (VH) of an anti-CD3 antibody described in the examples, CD3-binding domains containing the light chain variable region (VL) and heavy chain variable region (VH) of an OKT3 antibody (Proc. Natl. Acad. Sci. USA (1980) 77, 4914-4917) or various known CD3 antibodies are preferably used. Furthermore, a domain containing an antibody variable region originating from an anti-CD3 antibody having desired properties, obtained by immunizing a desired animal with the gamma, delta, or epsilon chain constituting human CD3 using the method described above, may be used as appropriate. As described above, a humanized antibody or a human antibody may be used as the anti-CD3 antibody that originates from the domain containing the antibody variable region having CD3 binding activity. The structure of the gamma, delta, or epsilon chain constituting CD3 is described in RefSeq registry numbers NM_000073.2, NM_000732.4, and NM_000733.3 for its polynucleotide sequence, and in RefSeq registry numbers NP_000064.1, NP_000723.1, and NP_000724.1 for its polypeptide sequence. 【0411】 specific Specificity refers to a state in which one molecule of a specifically binding molecule does not show any significant binding to any molecules other than the one or more molecules it binds to. It is also used when a domain containing an antibody variable region is specific to a particular epitope among several epitopes contained in a given antigen. Furthermore, if the epitopes to which the domain containing the antibody variable region binds are contained in multiple different antigens, the antigen-binding molecule having the domain containing the antibody variable region can bind to various antigens containing those epitopes. 【0412】 Epitope An epitope, meaning an antigenic determinant present in an antigen, refers to a site on the antigen to which a domain containing an antibody variable region in an antigen-binding molecule disclosed herein binds. Therefore, for example, an epitope can be defined by its structure. Alternatively, an epitope can be defined by the binding activity of the antigen-binding molecule that recognizes it. If the antigen is a peptide or polypeptide, the epitope can also be identified by the amino acid residues that constitute it. Furthermore, if the epitope is a glycan, it can also be identified by a specific glycan structure. 【0413】 A linear epitope is an epitope that contains an epitope whose primary amino acid sequence has been recognized. A linear epitope typically contains at least three, and most commonly at least five, for example, about 8 to 10, or 6 to 20 amino acids in its unique sequence. 【0414】 A structural epitope, in contrast to a linear epitope, is an epitope in which the primary sequence of amino acids containing the epitope is not a single defining component of the recognized epitope (for example, an epitope whose primary sequence of amino acids is not necessarily recognized by the antibody defining the epitope). A structural epitope may contain a larger number of amino acids than a linear epitope. In relation to the recognition of structural epitopes, antibodies recognize the three-dimensional structure of the peptide or protein. For example, if a protein molecule folds to form a three-dimensional structure, certain amino acids and / or polypeptide backbone that form the structural epitope are parallel, allowing the antibody to recognize the epitope. Methods for determining the three-dimensional structure of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-specific spin labeling and electromagnetic paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.). 【0415】 The following is an example of how to confirm the binding of an antigen-binding molecule to an epitope to a tumor antigen. 【0416】 For example, the recognition of a linear epitope present in the tumor antigen molecule by an antigen-binding molecule can be confirmed, for instance, as follows: A linear peptide consisting of the amino acid sequence constituting the extracellular domain of the tumor antigen is synthesized for the above purpose. This peptide can be synthesized chemically, or obtained by genetic engineering using the region of the tumor antigen's cDNA that codes for the amino acid sequence corresponding to the extracellular domain. Next, the binding activity of the linear peptide consisting of the amino acid sequence constituting the extracellular domain with a test antigen-binding molecule having a domain containing an antibody variable region that has binding activity to the tumor antigen is evaluated. For example, the binding activity of the antigen-binding molecule to the immobilized linear peptide can be evaluated by ELISA using the immobilized linear peptide as the antigen. Alternatively, the binding activity to the linear peptide can be determined based on the level of inhibition by the linear peptide in the binding of the antigen-binding molecule to tumor antigen-expressing cells. Through these tests, the binding activity of the antigen-binding molecule to the linear peptide can be determined. 【0417】 Furthermore, the recognition of a structural epitope by a test antigen-binding molecule having a domain containing an antibody variable region with binding activity to a tumor antigen can be confirmed as follows. For the above purpose, cells expressing a tumor antigen are prepared. When a test antigen-binding molecule having a domain containing an antibody variable region with binding activity to a tumor antigen comes into contact with a tumor antigen-expressing cell, it binds strongly to the cell, while the antigen-binding molecule does not substantially bind to a linear peptide consisting of amino acid sequences constituting the extracellular domain of the immobilized tumor antigen. Here, substantially non-binding means a binding activity of 80% or less, usually 50% or less, preferably 30% or less, and particularly preferably 15% or less of the binding activity to human tumor antigen-expressing cells. 【0418】 Methods for measuring the binding activity of a test antigen-binding molecule containing an antigen-binding domain to tumor antigens on tumor antigen-expressing cells include, for example, the method described in Antibodies A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). Specifically, it can be evaluated using the principles of ELISA or FACS (fluorescence-activated cell sorting) with GPC3-expressing cells as the antigen. 【0419】 In the ELISA format, the binding activity of a test antigen-binding molecule containing an antigen-binding domain for a target tumor antigen to tumor antigen-expressing cells is quantitatively evaluated by comparing the signal levels generated by the enzymatic reaction. Specifically, the test antigen-binding molecule is added to an ELISA plate immobilized with tumor antigen-expressing cells, and the antigen-binding molecule bound to the cells is detected using an enzyme-labeled antibody that recognizes the test antigen-binding molecule. Alternatively, in FACS, the binding activity of the test antigen-binding molecule to tumor antigen-expressing cells can be compared by creating a dilution series of the test antigen-binding molecule and determining the antibody binding titer against tumor antigen-expressing cells. 【0420】 The binding of a target antigen-binding molecule to an antigen expressed on the cell surface suspended in a buffer solution can be detected by a flow cytometer. Examples of known flow cytometers include the following: FACSCanto TM II FACSAria™ FACSArray™ FACSVantage TM SE FACSCalibur™ (both 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) 【0421】 For example, the following method is a suitable method for measuring the binding activity of a test antigen-binding molecule to an antigen. First, cells expressing the target tumor antigen are stained with a FITC-labeled secondary antibody that recognizes the test antigen-binding molecule. The test antigen-binding molecule is diluted with a suitable buffer to prepare the aggregate to the desired concentration for use. For example, it can be used at a concentration between 10 μg / ml and 10 ng / ml. Next, the fluorescence intensity and cell number are measured using FACSCalibur (BD). The amount of antibody bound to the cells is reflected in the fluorescence intensity, i.e., the Geometric Mean value, obtained by analysis using CELL QUEST Software (BD). That is, by obtaining the Geometric Mean value, the binding activity of the test antigen-binding molecule, expressed by the amount of the test antigen-binding molecule bound, can be measured. 【0422】 The sharing of an epitope between a test antigen-binding molecule and another antigen-binding molecule can be confirmed by competition between the two molecules for the same epitope. Competition between antigen-binding molecules can be detected by cross-blocking assays, for example. A competitive ELISA assay is a preferred cross-blocking assay. 【0423】 Specifically, in a cross-blocking assay, tumor antigen proteins coated on wells of a microtiter plate are pre-incubated in the presence or absence of candidate competing antigen-binding molecules, after which the test antigen-binding molecule is added. The amount of the test antigen-binding molecule bound to the tumor antigen protein in the well is indirectly correlated with the binding ability of the candidate competing antigen-binding molecules that compete for binding to the same epitope. In other words, the greater the affinity of the competing antigen-binding molecule for the same epitope, the lower the binding activity of the test antigen-binding molecule to the well coated with tumor antigen protein. 【0424】 The amount of antigen-binding molecules bound to the wells via tumor antigen proteins can be easily measured by pre-labeling the antigen-binding molecules. For example, biotin-labeled antigen-binding molecules can be measured using an avidin peroxidase conjugate and an appropriate substrate. Cross-blocking assays utilizing enzymatic labeling such as peroxidase are specifically called competitive ELISA assays. Antigen-binding molecules can be labeled with other detectable or measurable labeling substances. Specifically, radiolabeling and fluorescent labeling are well known. 【0425】 If, compared to the binding activity obtained in a control test performed in the absence of the candidate competing antigen-binding molecule, the competing antigen-binding molecule can block the binding of the test antigen-binding molecule containing the antigen-binding domain to the tumor antigen by at least 20%, preferably at least 20-50%, and more preferably at least 50%, then the test antigen-binding molecule is either binding to substantially the same epitope as the competing antigen-binding molecule, or is an antigen-binding molecule that competes for binding to the same epitope. 【0426】 If the structure of the epitope to which the test antigen-binding molecule, which contains an antigen-binding domain for the target tumor antigen, binds has been identified, the sharing of the epitope between the test antigen-binding molecule and the control antigen-binding molecule can be evaluated by comparing the binding activity of both antigen-binding molecules to a peptide into which an amino acid mutation has been introduced in the peptide constituting the epitope. 【0427】 One method for measuring such binding activity is to compare the binding activity of a test antigen-binding molecule and a control antigen-binding molecule to a linear peptide into which a mutation has been introduced in the aforementioned ELISA format. Alternatively, the binding activity to the mutated peptide bound to a column can be measured by quantitatively determining the antigen-binding molecule eluted into the eluate after the test antigen-binding molecule and the control antigen-binding molecule have been passed through the column. Methods for adsorbing the mutated peptide onto a column as a fusion peptide with, for example, GST, are well known. 【0428】 Furthermore, if the identified epitope is a stereoepitope, the sharing of the epitope between the test antigen-binding molecule and the control antigen-binding molecule can be evaluated by the following method. First, cells expressing the tumor antigen and cells expressing the tumor antigen with a mutation introduced into the epitope are prepared. The test antigen-binding molecule and the control antigen-binding molecule are added to the cell suspension, in which these cells are suspended in a suitable buffer such as PBS. Next, FITC-labeled antibodies capable of recognizing the test antigen-binding molecule and the control antigen-binding molecule are added to the cell suspension, which has been washed with a buffer as appropriate. The fluorescence intensity and cell count of the cells stained with the labeled antibody are measured using FACSCalibur (BD). The concentrations of the test antigen-binding molecule and the control antigen-binding molecule are adjusted to the desired concentration by appropriately diluting them with a suitable buffer. For example, concentrations between 10 μg / ml and 10 ng / ml are used. The amount of labeled antibody bound to the cells is reflected in the fluorescence intensity, i.e., the Geometric Mean value, obtained by analysis using CELL QUEST Software (BD). In other words, by obtaining the Geometric Mean value, it is possible to measure the binding activity of the test antigen-binding molecule and the control antigen-binding molecule, which is represented by the amount of labeled antibody bound. 【0429】 In this method, it can be determined that the substance does not bind to tumor antigen-expressing cells containing the mutation by the following method. First, the test antigen-binding molecule and the control antigen-binding molecule bound to cells expressing the mutational tumor antigen are stained with a labeled antibody. Next, 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. By calculating this comparison value (ΔGeo-Mean) from the Geometric Mean values ​​in the presence and absence of the antigen-binding molecule based on the following formula, the percentage increase in fluorescence intensity due to the binding of the antigen-binding molecule can be determined. 【0430】 ΔGeo-Mean=Geo-Mean (in the presence of antigen-binding molecules) / Geo-Mean (in the absence of antigen-binding molecules) 【0431】 Fv (variable fragment) In this specification, the term "Fv (variable fragment)" refers to the smallest unit of an antibody-derived antigen-binding domain, consisting of a pair of the antibody's light chain variable region (VL) and heavy chain variable region (VH). In 1988, Skerra and Pluckthun found that Fv could be prepared from the periplasmic fraction of E. coli in a homogeneous and active state by inserting the antibody gene downstream of a bacterial signal sequence and inducing the expression of the gene in E. coli (Science (1988) 240 (4855), 1038-1041). The Fv prepared from the periplasmic fraction had VH and VL associated in a manner that allowed for binding to the antigen. 【0432】 In this specification, Fv refers to, for example, the following antigen-binding molecules; The antigen-binding molecule includes (1) a divalent antigen-binding domain in which a monovalent scFv is linked to one polypeptide constituting an Fc region via a heavy chain Fv fragment constituting a CD3-binding domain, and the other monovalent scFv is linked to another polypeptide constituting an Fc region via a light chain Fv fragment constituting a CD3-binding domain, and the other monovalent scFv is linked to another polypeptide constituting an Fc region via a light chain Fv fragment constituting a CD3-binding domain, and the divalent antigen-binding domain is a divalent scFv, (2) a domain containing an Fc region among the amino acids constituting the Fc region of IgG1, IgG2a, IgG3, or IgG4 that does not have binding activity to the Fc gamma receptor, and (3) at least a monovalent CD3-binding domain, in which a pair of Fv fragments, including a light chain Fv fragment and a heavy chain Fv fragment, associate in a manner that has binding to the antigen CD3 and constitutes a CD3-binding domain. 【0433】 scFv, single-chain antibody, or sc(Fv)2 In this specification, the terms “scFv,” “monochain antibody,” or “sc(Fv)2” refer to an antibody fragment that contains variable regions derived from both the heavy and light chains within a single polypeptide chain, but lacks a constant region. Generally, monochain antibodies further include a polypeptide linker between the VH and VL domains, which enables the formation of a desired structure that is expected to allow antigen binding. Monochain antibodies are discussed in detail by Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg, and Moore (ed.), Springer-Verlag, New York, 269–315 (1994). See also International Patent Application Publication WO1988 / 001649 and U.S. Patents 4,946,778 and 5,260,203. In certain embodiments, monochain antibodies may also be bispecific and / or humanized. 【0434】 scFv is an antigen-binding domain in which the VH and VL components of Fv are linked by a peptide linker (Proc. Natl. Acad. Sci. USA (1988) 85 (16), 5879-5883). This peptide linker allows VH and VL to be kept in close proximity. 【0435】 sc(Fv)2 is a single-chain antibody in which four variable regions, 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), 177-189). These two VHs and VLs may originate from different monoclonal antibodies. For example, bispecific sc(Fv)2, which recognizes two different epitopes present in the same antigen, is also preferred, as disclosed in Journal of Immunology (1994) 152 (11), 5368-5374. sc(Fv)2 can be prepared by methods known to those skilled in the art. For example, it can be prepared by linking scFv with a linker such as a peptide linker. 【0436】 In this specification, the antigen-binding domain configuration of sc(Fv)2 is characterized by two VHs and two VLs arranged in the order VH, VL, VH, VL ([VH]linker[VL]linker[VH]linker[VL]) starting from the N-terminus of the single-chain polypeptide. However, the order of the two VHs and two VLs is not limited to the above configuration and may be arranged in any order. For example, the following configuration can also be given. 【0437】 [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] 【0438】 The molecular morphology of sc(Fv)2 is also described in detail in WO2006 / 132352, and those skilled in the art can use these descriptions to appropriately prepare the desired sc(Fv)2 for the preparation of the antigen-binding molecules disclosed herein. 【0439】 Furthermore, the antigen-binding molecule of the present invention may be conjugated with carrier polymers such as PEG or organic compounds such as anticancer agents. Additionally, a glycosylation sequence may be inserted, and the glycosylation may be suitably added to achieve a desired effect. 【0440】 As the linker for binding the variable region of the antibody, any peptide linker that can be introduced by genetic engineering, or a synthetic compound linker (see, for example, Protein Engineering, 9 (3), 299-305, 1996) can be used, but 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 depending on the purpose, but a 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. If sc(Fv)2 contains three peptide linkers, peptide linkers of the same length may be used, or peptide linkers of different lengths may be used. 【0441】 For example, in the case of a peptide linker: Ser Gly·Ser Gly·Gly·Ser Ser·Gly·Gly Gly·Gly·Gly·Ser Ser·Gly·Gly·Gly Gly·Gly·Gly·Gly·Ser Ser·Gly·Gly·Gly·Gly Gly·Gly·Gly·Gly·Gly·Ser Ser·Gly·Gly·Gly·Gly·Gly Gly·Gly·Gly·Gly·Gly·Gly·Ser Ser·Gly·Gly·Gly·Gly·Gly·Gly (Gly·Gly·Gly·Gly·Ser) n (Ser·Gly·Gly·Gly·Gly) n Examples include [n is an integer greater than or equal to 1]. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose. 【0442】 Synthetic chemical linkers (chemical crosslinking agents) are crosslinking agents commonly used for crosslinking peptides, such as N-hydroxysuccinimide (NHS), disuccinimidylsverate (DSS), bis(sulfosuccinimidyl)sverate (BS3), dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethylene glycol bis(succinimidylsuccinate) (EGS), ethylene glycol bis(sulfosuccinimidylsuccinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimideoxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimideoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES), and these crosslinking agents are commercially available. 【0443】 When binding four antibody variable regions, typically three linkers are required, but the same linker may be used for all of them, or different linkers may be used. 【0444】 "Fab" consists of a light chain and a heavy chain with a CH1 region and a variable region. The heavy chain of the Fab molecule cannot form disulfide bonds with other heavy chain molecules. 【0445】 "F(ab')2" and "Fab'" refer to antibody fragments produced by treating immunoglobulins (monoclonal antibodies) with proteolytic enzymes such as pepsin or papain, and digesting them before and after the disulfide bond between the two H chains in the hinge region. For example, by treating IgG with papain, the disulfide bond between the two H chains in the hinge region is cleaved upstream, producing two homologous antibody fragments: an L chain consisting of VL (variable L chain region) and CL (constant L chain region), and an H chain fragment consisting of VH (variable H chain region) and CH gamma 1 (gamma 1 region in the constant H chain region), which are linked by a disulfide bond at the C-terminal region. These two homologous antibody fragments are each called Fab'. 【0446】 "F(ab')2" comprises two light chains and two heavy chains containing constant regions of the CH1 domain and a portion of the CH2 domain such that interchain disulfide bonds are formed between the two heavy chains. The antigen-binding molecules F(ab')2 disclosed herein can be suitably obtained by partially digesting a full-length monoclonal antibody having a desired antigen-binding domain with a protease such as pepsin, and then removing the Fc fragment by adsorption onto a protein A column. The protease is not particularly limited as long as it can digest a full-length antibody to produce F(ab')2 restrictively by appropriately setting the reaction conditions of the enzyme, such as pH, for example, pepsin and ficin can be cited. 【0447】 The Fc region constituting the antigen-binding molecule disclosed herein can be suitably obtained by partially digesting an antibody, such as a monoclonal antibody, with a proteolytic enzyme such as pepsin, adsorbing the fragment onto a protein A column or a protein G column, and then eluting it with an appropriate elution buffer. The proteolytic enzyme is not particularly limited as long as it can digest an antibody, such as a monoclonal antibody, by appropriately setting the reaction conditions of the enzyme, such as pH. Examples include pepsin and ficin. 【0448】 The antigen-binding molecules described herein include Fc regions of IgG1, IgG2, IgG3, or IgG4 in which the binding activity to the Fc gamma receptor is reduced. 【0449】 The isotype of an antibody is determined by the structure of its constant region. The constant regions of the IgG1, IgG2, IgG3, and IgG4 isotypes are called C-gamma 1, C-gamma 2, C-gamma 3, and C-gamma 4, respectively. 【0450】 The Fc region refers to the region excluding F(ab')2, which includes two light chains and two heavy chains that include a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. The Fc region constituting the antigen-binding molecule disclosed herein can be suitably obtained by partially digesting IgG1, IgG2, IgG3, IgG4 monoclonal antibodies, etc., with a proteolytic enzyme such as pepsin, and then re-eluting the fraction adsorbed onto a protein A column. Such a proteolytic enzyme is not particularly limited as long as it can digest the full-length antibody in a restrictive manner to produce F(ab')2 by appropriately setting the reaction conditions of the enzyme, such as pH, for example, pepsin and ficin can be given as examples. 【0451】 An Fcγ receptor is a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, and essentially refers to any member of the family of proteins encoded by the Fcγ receptor gene. In humans, this family includes, but is not limited to, FcγRI(CD64), which includes isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII(CD32), which includes isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII(CD16), which includes isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any undiscovered human FcγR species or FcγR isoforms or allotypes. FcγR may be derived from any organism, including, but is not limited to, humans, mice, rats, rabbits, and monkeys. Mouse FcγR receptors include, but are not limited to, FcγRI(CD64), FcγRII(CD32), FcγRIII(CD16), and FcγRIII-2(CD16-2), as well as any undiscovered mouse FcγR receptors, FcγR isoforms, or allotypes. Preferred examples of such Fcγ receptors include human FcγRI(CD64), FcγRIIA(CD32), FcγRIIB(CD32), FcγRIIIA(CD16), and / or FcγRIIIB(CD16).The polynucleotide and amino acid sequences of FcγRI are listed under RefSeq registry numbers NM_000566.3 and NP_000557.1, respectively; the polynucleotide and amino acid sequences of FcγRIIA are listed under RefSeq registry numbers BC020823.1 and 30AAH20823.1, respectively; the polynucleotide and amino acid sequences of FcγRIIB are listed under RefSeq registry numbers BC146678.1 and AAI46679.1, respectively; the polynucleotide and amino acid sequences of FcγRIIIA are listed under RefSeq registry numbers BC033678.1 and AAH33678.1, respectively; and the polynucleotide and amino acid sequences of FcγRIIIB are listed under RefSeq registry numbers BC128562.1 and AAI28563.1, respectively. Whether or not the Fcγ receptor has binding activity to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies can be confirmed by the FACS and ELISA formats described above, as well as by ALPHA screening (Amplified Luminescent Proximity Homogeneous Assay) and the BIACORE method utilizing surface plasmon resonance (SPR) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010). 【0452】 Furthermore, "Fc ligand" or "effector ligand" means 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. Binding of an Fc ligand to Fc preferably induces one or more effector functions. Fc ligands include, but are not limited to, Fc receptors, FcγR, FcαR, FcεR, FcRn, C1q, C3, mannan-binding lectins, mannose receptors, 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), a family of Fc receptors homologous to FcγR. Fc ligands may also include undiscovered molecules that bind to Fc. 【0453】 The reduced binding activity of the Fc region to any of the Fcγ receptors FcγI, FcγIIA, FcγIIB, FcγIIIA, and / or FcγIIIB can be confirmed by the FACS and ELISA formats described above, as well as by ALPHA screening (Amplified Luminescent Proximity Homogeneous Assay) and the BIACORE method utilizing surface plasmon resonance (SPR) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010). 【0454】 The ALPHA screen is performed using ALPHA technology, which employs two beads, a donor and an acceptor, based on the following principle: Molecules bound to the donor bead biologically interact with molecules bound to the acceptor bead, and an emission signal is detected only when the two beads are in close proximity. A photosensitiver within the donor bead, excited by a laser, converts surrounding oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and, upon reaching the nearby acceptor bead, triggers a chemiluminescent reaction within the bead, ultimately emitting light. When the molecules bound to the donor bead and the molecules bound to the acceptor bead do not interact, the singlet oxygen produced by the donor bead does not reach the acceptor bead, and therefore no chemiluminescent reaction occurs. 【0455】 For example, a biotin-labeled antigen-binding molecule is bound to a donor bead, and a glutathione S-transferase (GST)-tagged Fcγ receptor is bound to an acceptor bead. In the absence of competing antigen-binding molecules with mutant Fc regions, antigen-binding molecules with wild-type Fc regions interact with the Fcγ receptor, producing a signal in the 520-620 nm range. Antigen-binding molecules with untagged mutant Fc regions compete with the interaction between antigen-binding molecules with wild-type Fc regions and the Fcγ receptor. The relative binding affinity can be determined by quantifying the decrease in fluorescence resulting from this competition. Biotinylation of antigen-binding molecules such as antibodies using sulfo-NHS-biotin is well known. As a method for tagging the Fcγ receptor with GST, a fusion gene obtained by in-frame fusion of a polynucleotide encoding the Fcγ receptor and a polynucleotide encoding GST is expressed in cells containing such a fusion gene in an expressionable vector, and then purified using a glutathione column can be appropriately employed. The obtained signals can be suitably analyzed by fitting them to a one-site competition model that utilizes nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego). 【0456】 When one of the substances whose interaction is to be observed (ligand) is fixed onto a gold thin film on a sensor chip, and light is shone from the back of the sensor chip so as to cause total internal reflection at the interface between the gold thin film and glass, a region of reduced reflection intensity (SPR signal) is formed in a part of the reflected light. When the other substance whose interaction is to be observed (analyte) is flowed onto the surface of the sensor chip and the ligand and analyte bind, the mass of the immobilized ligand molecule increases, and the refractive index of the solvent on the surface of the sensor chip changes. This change in refractive index causes the position of the SPR signal to shift (conversely, when the bond dissociates, the signal position returns to its original position). The Biacore system plots the amount of the above shift, i.e., the change in mass on the sensor chip surface, on the vertical axis and displays the change in mass over time as measurement data (sensorgram). From the sensorgram curve, kinetics: the binding rate constant (ka) and the dissociation rate constant (kd) can be determined, and affinity (KD) can be determined from the ratio of these constants. Inhibition measurement methods are also suitably used in the BIACORE method. Examples of inhibitory assay methods are described in Proc.Natl.Acad.Sci.USA (2006) 103 (11), 4005-4010. 【0457】 Several therapeutic antibodies exhibiting antitumor effects exert their antitumor effects against cancer cells by inhibiting signals necessary for cancer cell proliferation, inducing cell death signals, or through ADCC (Antibody-Dependent Cell-mediated Cytotoxicity) and CDC (Complement-dependent Cytotoxicity). ADCC is cytotoxicity exerted by effector cells such as NK cells and macrophages when the Fc region of an antibody binds to Fc receptors present on these effector cells, against the target cancer cells to which the antibody has bound. CDC is cytotoxicity caused by the binding of the complement complex to the complement binding site present in the antibody structure. When the complement components present in the complex form pores on the cell membrane of the cell to which the antibody has bound, the influx of water and ions into the cell is promoted, leading to cell destruction. Among Fc receptors, Fcγ receptors are those that can bind to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies. When the binding activity to Fcγ receptors is low, the receptors expressed on T cells and NK cells or macrophages, etc., are not cross-linked independently of cancer antigens. Therefore, cancer antigen-independent cytokine induction does not occur. Antibodies in which the Fc region has reduced binding activity to any of the Fcγ receptors FcγI, FcγIIA, FcγIIB, FcγIII...

Claims

[Claim 1] A pharmaceutical composition comprising cells expressing a chimeric receptor, for use in combination with the administration of an antigen-binding molecule that recognizes a molecule expressed on the surface of a target cell as an antigen, Cells that express chimeric receptors are T cells or NK cells. The antigen-binding molecule comprises a variable region and a constant region of the antibody, and a linker that is cleaved by a protease, and after cleavage of the linker, it has the ability to bind to the target antigen. The antigen-binding molecule is an IgG antibody or a heavy chain antibody. Due to the linker being cut, Fab, (Fab) ２ VL, VH, or VHH, or their antigen-binding fragments are obtained. A pharmaceutical composition comprising a chimeric receptor comprising an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular binding domain has the ability to bind to an antigen-binding molecule after linker cleavage, and can bind to the target cell via binding to the antigen-binding molecule after linker cleavage, and the target cell is a cancer cell or an inflammatory cell. [Claim 2] A pharmaceutical composition containing an antigen-binding molecule for use in combination with the administration of cells expressing a chimeric receptor that recognizes a molecule expressed on the surface of a target cell as an antigen, Cells that express chimeric receptors are T cells or NK cells. The antigen-binding molecule comprises a variable region and a constant region of the antibody, and a linker that is cleaved by a protease, and after cleavage of the linker, it has the ability to bind to the target antigen. The antigen-binding molecule is an IgG antibody or a heavy chain antibody. Due to the linker being cut, Fab, (Fab) ２ VL, VH, or VHH, or their antigen-binding fragments are obtained. Chimeric receptors include an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain. The extracellular binding domain has the ability to bind to antigen-binding molecules after linker cleavage. A pharmaceutical composition that can bind to the target cells via binding to an antigen-binding molecule after antigen cleavage, wherein the target cells are cancer cells or inflammatory cells. [Claim 3] K for antigen of antigen-binding molecule before linker cleavage Ｄ K for the antigen of the antigen after linker cleavage relative to the value Ｄ Ratio of values ​​(K Ｄ (After cutting) / K Ｄ The pharmaceutical composition according to claim 1 or 2, wherein (before cutting) is 0.1 or 0.01 or less. [Claim 4] The pharmaceutical composition according to any one of claims 1 to 3, wherein the antigen-binding molecule is an IgG antibody. [Claim 5] The pharmaceutical composition according to any one of claims 1 to 4, wherein the antigen-binding molecule whose linker is cleaved by a protease comprises an antigen-binding domain and a portion of the cleaved linker. [Claim 6] The pharmaceutical composition according to any one of claims 1 to 5, wherein the linker cleaved by the protease of the antigen-binding molecule is located near the boundary between the variable region and the constant region, or near the boundary between CH1 and CH2 within the constant region. [Claim 7] The pharmaceutical composition according to any one of claims 1 to 6, wherein the antigen-binding molecule after linker cleavage is the VL, VH, VHH of the antibody or its antigen-binding fragment. [Claim 8] The pharmaceutical composition according to any one of claims 1 to 7, wherein the antigen-binding molecule is a single-domain antibody containing a linker that is cleaved by a protease, and the antigen-binding molecule after linker cleavage includes the antigen-binding domain and a part of the linker of the single-domain antibody. [Claim 9] The pharmaceutical composition according to any one of claims 1 to 8, wherein the linker cleaved by the protease comprises a protease cleavage sequence. [Claim 10] The pharmaceutical composition according to any one of claims 1 to 8, wherein the linker cleaved by the protease comprises a peptide having any of the protease cleavage sequences of SEQ ID NOs: 1 to 725. [Claim 11] The pharmaceutical composition according to any one of claims 1 to 10, wherein the target cell is a cancer cell. [Claim 12] A composition comprising nucleic acid for use in the production of a pharmaceutical composition according to any one of claims 1 to 11, wherein the nucleic acid is an isolated nucleic acid encoding an antigen-binding molecule or a chimeric receptor contained in the pharmaceutical composition as defined in any one of claims 1 to 11. [Claim 13] A composition comprising a vector containing an isolated nucleic acid as described in claim 12, for use in the production of a pharmaceutical composition as described in any one of claims 1 to 11. [Claim 14] The composition according to claim 13, wherein the vector is operably bound to at least one regulatory element for the expression of an antigen-binding molecule or a chimeric receptor. [Claim 15] A composition comprising cells transfected or transduced with the isolated nucleic acid described in claim 12 or the vector described in claim 13 or 14, for use in the production of the pharmaceutical composition described in any one of claims 1 to 11.

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