CD3-targeting antigen-binding molecule with improved stability

Abstract

The present disclosure provides a novel CD3-binding domain with superior stability compared to CD3-binding domains used in conventional T cell redirecting antibodies, and an antigen-binding molecule comprising the CD3-binding domain. A bispecific antigen-binding molecule prepared by combining the CD3-binding domain of the present disclosure with an antigen-binding domain binding to an antigen expressed on the surface of a target cell such as a cancer cell can induce T-cell-dependent cytotoxicity to the target cell and can be used for the treatment or prevention of various cancers.

Description

CD3-targeted antigen-binding molecules with improved stability

[0001] The present disclosure relates to antigen-binding molecules comprising a CD3-binding domain with improved stability, pharmaceutical compositions comprising the antigen-binding molecules, and uses thereof.

[0002] Antibodies have attracted attention as pharmaceuticals due to their high stability in plasma and minimal side effects (Non-Patent Documents 1 and 2). Antibodies are known to exert antitumor effects against cancer cells by inducing effector cell-mediated cytotoxicity (ADCC), antibody-dependent cytotoxicity (ADCP), complement-dependent cytotoxicity (CDC), and other cytotoxicity functions (also known as effector functions) through antigen binding, agonistic activity, and antagonistic activity (Non-Patent Document 3). ADCC is the cytotoxicity exerted by effector cells, such as NK cells and macrophages, against antibody-bound target cancer cells when the Fc region of the antibody binds to Fc receptors present on these effector cells.

[0003] Natural immunoglobulins bind to antigens through their variable regions and to receptors such as FcγR, FcRn, FcαR, and FcεR, as well as complement, through their constant regions. Conventional natural IgG antibodies recognize and bind to a single epitope through their variable region (Fab), and can therefore only bind to a single antigen. However, it is known that multiple proteins are involved in cancer and inflammation, and crosstalk between these proteins can occur.

[0004] Bispecific antibodies, which bind to two or more antigens, have been studied as molecules that inhibit multiple targets. By modifying natural IgG antibodies, it is possible to confer binding activity to two different antigens (first and second antigens) (Non-Patent Document 4). Therefore, it is possible to neutralize two or more antigens with a single molecule or enhance antitumor activity by crosslinking cytotoxic cells with cancer cells.

[0005] T cell-redirecting antibodies, which are one type of bispecific antibody and whose antitumor effect is mediated by cytotoxicity, recruiting T cells as effector cells, have been known since the 1980s (Non-Patent Documents 5, 6, 7). Unlike antibodies that use ADCC, which recruits NK cells and macrophages as effector cells, T cell-redirecting antibodies are bispecific antibodies that contain an antibody against one of the constituent subunits of the T cell receptor (TCR) complex on T cells, specifically an antibody that binds to the CD3ε chain, and an antibody that binds to an antigen on the target cancer cell. The simultaneous binding of T cell-redirecting antibodies to the CD3ε chain and the cancer antigen allows T cells to approach the cancer cells. It is believed that the cytotoxicity of T cells results in the antitumor effect against cancer cells.

[0006] Catumaxomab, a known T cell-redirecting antibody, uses two Fab fragments that bind to a cancer antigen (EpCAM) and the CD3ε chain expressed on T cells. Catumaxomab is a trifunctional antibody that simultaneously binds to both a cancer antigen and CD3ε, thereby inducing cytotoxic activity by T cells, and simultaneously binds to both a cancer antigen and FcγR, thereby inducing cytotoxic activity by antigen-presenting cells such as NK cells and macrophages. However, because trifunctional antibodies simultaneously bind to CD3ε and FcγR even in the absence of cancer antigens, CD3ε-expressing T cells and FcγR-expressing cells are cross-linked, resulting in the massive production of various cytokines, even in the absence of cancer cells. Due to this cancer antigen-independent induction of cytokine production, trifunctional antibody administration is currently limited to intraperitoneal administration (Non-Patent Document 8), and systemic administration is extremely difficult due to the severe cytokine storm-like side effects.

[0007] On the other hand, unlike catumaxomab, BiTE (bispecific T-cell engager) does not have a binding site for Fcγ receptors, and therefore does not cross-link T cells with receptors expressed on NK cells, macrophages, etc. in a cancer antigen-independent manner. Therefore, it has been shown that the cancer antigen-independent cytokine induction observed when catumaxomab is administered does not occur. However, because BiTE is a low-molecular-weight engineered antibody molecule lacking the Fc region, there is a problem in that the blood half-life of BiTE administered to patients is significantly shorter than that of IgG antibodies commonly used as therapeutic antibodies.

[0008] Recently, new polypeptide complexes have been provided that utilize an Fc region with reduced FcγR-binding activity to bring T cells into close proximity with target cancer cells, exhibiting excellent safety characteristics by not inducing cytokine storms or other events in a cancer antigen-independent manner, and have a long blood half-life (Patent Document 1). New T cell-redirecting antibodies with such advantageous effects have been reported to be useful not only for targeting cancer cells, but also for targeting regulatory T cells and exhausted T cells, which are present in the tumor microenvironment and have the function of suppressing immune responses (Patent Documents 2 and 3).

[0009] WO2012 / 073985WO2016 / 047722WO2015 / 174439

[0010] Nat. Biotechnol. (2005) 23, 1073-1078Eur J Pharm Biopharm. (2005) 59 (3), 389-396Drug Des Devel Ther (2009) 3, 7-16MAbs. (2012) Mar 1, 4(2)Nature (1985) 314 (6012), 628-31Int J Cancer (1988) 41 (4), 609-15.Proc Natl Acad Sci USA (1986) 83 (5), 1453-7Cancer Immunol Immunother. (2007) 56(9), 1397-406

[0011] The present invention was made in light of the above circumstances, and focuses particularly on the CD3-binding domain used in T cell redirecting antibodies. It is an object of the present invention to provide a novel CD3-binding domain that has superior stability to the CD3-binding domains used in conventional T cell redirecting antibodies, an antigen-binding molecule comprising the CD3-binding domain, a method for producing the antigen-binding molecule, and a pharmaceutical composition comprising the antigen-binding molecule as an active ingredient, as well as uses thereof.

[0012] The present inventors introduced various modifications into the CDRs of the CD3-binding Fab contained in the bispecific antibody described in WO2015174439 and succeeded in obtaining modified antibodies that showed a smaller decrease in CD3-binding activity when stored at room temperature or above than the parent Fab. The present inventors further combined these modified CD3-binding domains with other antigen-binding domains, such as a cancer-specific antigen-binding domain, to prepare bispecific antigen-binding molecules and found that the degree of decrease in T cell-dependent cytotoxicity when stored at room temperature or above was also small. Based on these findings, the present inventors demonstrated that the multispecific antigen-binding molecules of the present invention can damage tissues containing target cells expressing a target antigen, such as a cancer-specific antigen.

[0013] The present disclosure is based on these findings and specifically includes the following exemplary embodiments: [1] An antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises an antibody heavy chain variable region and light chain variable region having binding activity to CD3, wherein the heavy chain variable region comprises: an heavy chain CDR1 comprising the amino acid sequence NAWMH (SEQ ID NO: 1); an heavy chain CDR2 comprising the amino acid sequence QIX1DKSQNYATX2VAESVKG (SEQ ID NO: 2), wherein X1 is K or R and X2 is Y or F; and an heavy chain CDR3 comprising the amino acid sequence VHYX3AGYGVDX4 (SEQ ID NO: 3), wherein X3 is A or P and X4 is I, M, or L, wherein the light chain variable region comprises: An antigen-binding molecule comprising: an L-chain CDR1 comprising the amino acid sequence RSX5X6X7VVHENRX8TYLH (SEQ ID NO: 4), wherein X5 is S or T, X6 is Q or M, X7 is S or T, and X8 is Q or N; an L-chain CDR2 comprising the amino acid sequence KVSNRFS (SEQ ID NO: 5); and an L-chain CDR3 comprising the amino acid sequence GQGTQVPYT (SEQ ID NO: 6). [2] The antigen-binding molecule of [1], wherein the antibody H chain variable region and L chain variable region having binding activity to CD3 comprise any combination of H chain CDR1, CDR2, and CDR3, and L chain CDR1, CDR2, and CDR3 selected from the following (a1) to (a8): (a1) a combination of H chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, H chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, H chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10, L chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, L chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and L chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (a2)(a3) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (a4) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (a5) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (a6) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 13, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (a7)(a8) A combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 18, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (a9) A combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 19, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6. [3] The antigen-binding molecule of [1] or [2], wherein the antibody H-chain variable region and L-chain variable region having binding activity to CD3 comprise any combination of H-chain variable region and L-chain variable region selected from the following (a1) to (a8): (a1) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 20 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25; (a2) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 21 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25; (a3) ​​a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 21 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 26; (a4) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25; (a5) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 23 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25; (a6) (a7) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 27; (a8) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 28;A combination of an H chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L chain variable region comprising the amino acid sequence shown in SEQ ID NO: 29. [4-1] The antigen-binding molecule of any of [1] to [3], further comprising an Fc region. [4-2] The antigen-binding molecule of any of [1] to [3], further comprising an Fc region with reduced binding activity to an Fcγ receptor. [4-3] The antigen-binding molecule according to [4-1] or [4-2], wherein the antigen-binding molecule comprises any of the combinations of heavy chains and light chains selected from the following (a1) to (a8): (a1) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 30 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35, (a2) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 31 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35, (a3) ​​a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 31 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 36, (a4) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 32 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35, (a5) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 33 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35, (a6) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 32 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 37, (a7) (a8) a combination of a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 32 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 38. [5-1] The antigen-binding molecule of any of [1] to [4], which is a monospecific antigen-binding molecule. [5-2] The antigen-binding molecule of any of [1] to [4], wherein the first antigen-binding domain and the second antigen-binding domain comprise the same amino acid sequence. [5-3] The antigen-binding molecule of any of [1] to [4], wherein the first antigen-binding domain and the second antigen-binding domain comprise different amino acid sequences. [6-1] The antigen-binding molecule of any of [1] to [4], which is a multispecific antigen-binding molecule. [6-2] The antigen-binding molecule of any of [1] to [4], which is a bispecific antigen-binding molecule.[6-3] The antigen-binding molecule of any of [1] to [4], which is an antigen-binding molecule with triple or higher specificity. [7-1] The antigen-binding molecule of [6], wherein the second antigen-binding domain comprises an antibody variable region having binding activity for a cancer antigen. [7-2] The antigen-binding molecule of [6], wherein the second antigen-binding domain comprises an antibody variable region having binding activity for a molecule expressed on the surface of a cell that has the function of suppressing an immune response. [8] The antigen-binding molecule of any of [1] to [7], which is an antibody. [9] A nucleic acid encoding the antigen-binding molecule of any of [1] to [8].

[10] A vector into which the nucleic acid of [9] has been introduced.

[11] A cell comprising the nucleic acid of [9] or the vector of

[10] .

[12] A method for producing the antigen-binding molecule of any of [1] to [8], which comprises a step of culturing the cell of

[11] .

[13] An antigen-binding molecule produced by the method of

[12] .

[14] A pharmaceutical composition comprising the antigen-binding molecule of any of [1] to [8] and a pharmaceutically acceptable carrier. [15-1] The pharmaceutical composition of

[14] for inducing cytotoxicity. [15-2] The pharmaceutical composition of

[14] for inducing T cell-dependent cytotoxicity. [15-3] The pharmaceutical composition of

[14] for use in the treatment or prevention of cancer. [16-1] A method for inducing cytotoxicity, comprising the step of administering the antigen-binding molecule of any of [1] to [8] or the pharmaceutical composition of

[14] . [16-2] A method for inducing T cell-dependent cytotoxicity, comprising the step of administering the antigen-binding molecule of any of [1] to [8] or the pharmaceutical composition of

[14] . [16-3] A method for treating or preventing cancer, comprising the step of administering the antigen-binding molecule of any of [1] to [8] or the pharmaceutical composition of

[14] . [17-1] A kit for inducing cytotoxicity, comprising the antigen-binding molecule of any of [1] to [8] or the pharmaceutical composition of

[14] , and instructions for use. [17-2] A kit for inducing T cell-dependent cytotoxicity, comprising the antigen-binding molecule of any of [1] to [8] or the pharmaceutical composition of

[14] , and instructions for use. [17-3]A kit for treating or preventing cancer, comprising the antigen-binding molecule of any of [1] to [8] or the pharmaceutical composition of

[14] , and instructions for use. [18-1] The antigen-binding molecule of any of [1] to [8], for use in inducing cytotoxicity. [18-2] The antigen-binding molecule of any of [1] to [8], for use in inducing T cell-dependent cytotoxicity. [18-3] The antigen-binding molecule of any of [1] to [8], for use in treating or preventing cancer. [19-1] Use of the antigen-binding molecule of any of [1] to [8] in the manufacture of a cytotoxicity inducer. [19-2] Use of the antigen-binding molecule of any of [1] to [8] in the manufacture of a T cell-dependent cytotoxicity inducer. [19-3] Use of the antigen-binding molecule of any of [1] to [8] in the manufacture of a cancer therapeutic or preventive agent. [A1] A monovalent antigen-binding molecule comprising a single antigen-binding domain, wherein the single antigen-binding domain comprises an antibody heavy chain variable region and light chain variable region having binding activity to CD3, wherein the heavy chain variable region comprises: an heavy chain CDR1 comprising the amino acid sequence NAWMH (SEQ ID NO: 1); an heavy chain CDR2 comprising the amino acid sequence QIX1DKSQNYATX2VAESVKG (SEQ ID NO: 2), in which X1 is K or R and X2 is Y or F; and an heavy chain CDR3 comprising the amino acid sequence VHYX3AGYGVDX4 (SEQ ID NO: 3), in which X3 is A or P and X4 is I, M, or L, and wherein the light chain variable region comprises: [A2] An antigen-binding molecule comprising: an L chain CDR1 comprising the amino acid sequence RSX5X6X7VVHENRX8TYLH (SEQ ID NO: 4), wherein X5 is S or T, X6 is Q or M, X7 is S or T, and X8 is Q or N; an L chain CDR2 comprising the amino acid sequence KVSNRFS (SEQ ID NO: 5); and an L chain CDR3 comprising the amino acid sequence GQGTQVPYT (SEQ ID NO: 6).The antigen-binding molecule of [A1], wherein the antibody H chain variable region and L chain variable region having binding activity to CD3 comprise any combination of H chain CDR1, CDR2, and CDR3, and L chain CDR1, CDR2, and CDR3 selected from the following (a1) to (a8): (a1) a combination of H chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, H chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, H chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10, L chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, L chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and L chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (a2) (a3) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (a4) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (a5)(a6) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 13, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (a7) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (a8) A combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 18, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (a9) A combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 19, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6. [A3] The antigen-binding molecule of [A1] or [A2], wherein the antibody H chain variable region and L chain variable region having binding activity to CD3 comprise any combination of H chain variable region and L chain variable region selected from the following (a1) to (a8): (a1) a combination of an H chain variable region comprising the amino acid sequence shown in SEQ ID NO: 20 and an L chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25; (a2) a combination of an H chain variable region comprising the amino acid sequence shown in SEQ ID NO: 21 and an L chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25; (a3)(a4) a combination of an H chain variable region comprising the amino acid sequence shown in SEQ ID NO: 21 and an L chain variable region comprising the amino acid sequence shown in SEQ ID NO: 26, (a4) a combination of an H chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25, (a5) a combination of an H chain variable region comprising the amino acid sequence shown in SEQ ID NO: 23 and an L chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25, (a6) a combination of an H chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L chain variable region comprising the amino acid sequence shown in SEQ ID NO: 27, (a7) a combination of an H chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L chain variable region comprising the amino acid sequence shown in SEQ ID NO: 28, (a8) a combination of an H chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L chain variable region comprising the amino acid sequence shown in SEQ ID NO: 29. [A4-1] The antigen-binding molecule of any of [A1] to [A3], further comprising an Fc region. [A4-2] The antigen-binding molecule of any of [A1] to [A3], further comprising an Fc region with reduced binding activity to an Fcγ receptor. [A4-3] The antigen-binding molecule according to [A4-1] or [A4-2], wherein the antigen-binding molecule comprises any of the combinations of heavy chains and light chains selected from the following (a1) to (a8): (a1) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 30 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35, (a2) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 31 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35, (a3) ​​a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 31 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 36, (a4) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 32 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35, (a5) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 33 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35, (a6) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 32 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 37, (a7)(a8) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 32 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 38. [A5] A nucleic acid encoding the antigen-binding molecule according to any of [A1] to [A4]. [A6] A vector into which the nucleic acid according to [A5] has been introduced. [A7] A cell comprising the nucleic acid according to [A5] or the vector according to [A6]. [A8] A method for producing the antigen-binding molecule according to any of [A1] to [A4], comprising the step of culturing the cell according to [A7]. [A9] An antigen-binding molecule produced by the method according to [A8].

[0014] I. Definitions The following definitions are provided to facilitate understanding of the invention described herein.

[0015] The term "antigen-binding molecule" refers to a molecule capable of binding to its antigen with sufficient affinity. In one embodiment, the degree of binding of an antigen-binding molecule to proteins unrelated to the antigen is less than about 10% of the binding of the antigen-binding molecule to the antigen, as measured (e.g., by radioimmunoassay (RIA)). In certain embodiments, the antigen-binding molecule has an affinity for the antigen of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., ≦10 -8 M or less, e.g. 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 In certain embodiments, the antigen-binding molecule binds to an epitope of the antigen that is conserved among the antigens from different species. In some embodiments, the antigen-binding molecule is an antibody.

[0016] As used herein, the term "antibody" refers to a natural or partially or fully synthetically produced immunoglobulin. Antibodies can be isolated from natural sources such as plasma or serum where they occur, or from the culture supernatant of antibody-producing hybridoma cells, or can be partially or fully synthesized using techniques such as genetic recombination. Preferred examples of antibodies include immunoglobulin isotypes and their isotype subclasses. Nine known classes (isotypes) of human immunoglobulins are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. Of these isotypes, antibodies of the present invention may include IgG1, IgG2, IgG3, and IgG4.

[0017] 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 major 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 the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0018] "Native antibodies" refer to immunoglobulin molecules with various naturally occurring structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain contains a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain contains a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be assigned to one of two types, called kappa (κ) or lambda (λ).

[0019] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies typically have similar structures, each containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by screening a complementary library of VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to that antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0020] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence (the "complementarity determining region" or "CDR"), and / or forms structurally defined loops (the "hypervariable loops"), and / or contains antigen-contacting residues (the "antigen contacts"). Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs 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) antigenic contacts 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) combinations 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).Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0021] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0022] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain, with the exception that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) residue at the Fc region may be present or absent. In one embodiment, the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) residue at the Fc region may be removed by degradation. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0023] A "native-sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. Native-sequence human Fc regions include native-sequence human IgG1 Fc regions (non-A and A allotypes), native-sequence human IgG2 Fc regions, native-sequence human IgG3 Fc regions, and native-sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0024] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution, e.g., about one to about ten amino acid substitutions, preferably about one to about five amino acid substitutions, in the native-sequence Fc region or in the Fc region of the parent polypeptide compared to the native-sequence Fc region or the Fc region of the parent polypeptide. The variant Fc region herein preferably has at least about 80% sequence identity with the native-sequence Fc region and / or the Fc region of the parent polypeptide, more preferably at least about 90% sequence identity thereto, and most preferably at least about 95% sequence identity thereto.

[0025] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR binds to IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997).) FcRs are reviewed, e.g., in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med 126:330-41 (1995). Other FcRs, including those identified in the future, are also encompassed by the term "FcR" herein.

[0026] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for regulating maternal IgG transfer to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO2004 / 92219 (Hinton et al.)).

[0027] In vivo binding to human FcRn and plasma half-life of human FcRn high-affinity binding polypeptides can be measured, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which polypeptides with mutant Fc regions are administered. Instead of plasma half-life, blood half-life or serum half-life can be measured. WO2000 / 42072 (Presta) describes antibody variants with increased or decreased binding to FcR. See, for example, Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

[0028] The term "Fc region-containing antibody" refers to an antibody that contains an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) or the C-terminal glycine-lysine (residues 446-447) of the Fc region can be removed, for example, during antibody purification or by recombinant manipulation of a nucleic acid encoding the antibody. Thus, a composition containing an antibody with an Fc region of the present invention can include an antibody with G446-K447, an antibody with G446 but no K447, an antibody from which G446-K447 have been completely removed (in the case of IgG, an antibody whose C-terminus is P445 or an antibody with an amide group (NH2) added to P445 to form proline amide (Pro-NH2)), or a mixture of the above three types of antibodies.

[0029] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0030] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcR) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), thereby enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill them with cytotoxins. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Patent Nos. 5,500,362 or 5,821,337 or U.S. Patent No. 6,737,056 (Presta), may be performed. Useful effector cells for such assays include PBMCs and NK cells. Alternatively, or additionally, the ADCC activity of a molecule of interest may be assessed in vivo in an animal model, such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0031] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical and / or bind the same epitope (determinant), except for possible variants (e.g., variants containing naturally occurring mutations or variants that arise during the production of a monoclonal antibody preparation; such variants are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different epitopes, each monoclonal antibody in a monoclonal antibody preparation is directed against a single epitope on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention may be produced by a variety of techniques, including, but not limited to, hybridoma technology, recombinant DNA technology, phage display technology, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.

[0032] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0033] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0034] A "human antibody" is an antibody with an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a human antibody repertoire or other non-human source that uses human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0035] The terms "full length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0036] "Antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antigen-binding molecules formed from antibody fragments.

[0037] As used herein, the term "Fv (variable fragment)" refers to the minimum unit of an antibody-derived antigen-binding domain consisting of a pair of an antibody light chain variable region (VL (light chain variable region)) and an antibody heavy chain variable region (VH (heavy chain variable region)). In 1988, Skerra and Pluckthun discovered that homogeneous and active Fv could be prepared from the periplasmic fraction of E. coli by inserting an antibody gene downstream of a bacterial signal sequence and inducing expression of the gene in E. coli (Science (1988) 240 (4855), 1038-1041). The Fv prepared from the periplasmic fraction contained an association of VH and VL in a manner that allowed it to bind to an antigen.

[0038] As used herein, the terms "scFv," "single-chain antibody," or "sc(Fv)2" refer to an antibody fragment that contains, in a single polypeptide chain, variable regions from both the heavy and light chains but lacks constant regions. Generally, single-chain antibodies further comprise a polypeptide linker between the VH and VL domains that enables them to form the desired structure that may enable antigen binding. Single-chain antibodies are discussed in detail by Plückthun in *The Pharmacology of Monoclonal Antibodies*, Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York, pp. 269-315 (1994). See also International Patent Application Publication No. WO 1988 / 001649 and U.S. Pat. Nos. 4,946,778 and 5,260,203. In certain embodiments, single-chain antibodies may also be bispecific and / or humanized.

[0039] An scFv is an antigen-binding domain in which the VH and VL constituting the Fv are linked by a peptide linker (Proc. Natl. Acad. Sci. USA (1988) 85(16), 5879-5883). The VH and VL can be held in close proximity by the peptide linker.

[0040] sc(Fv)2 is a single-chain antibody in which four variable regions, two VL and two VH, are linked by a linker such as a peptide linker to form a single chain (J Immunol. Methods (1999) 231 (1-2), 177-189). The two VH and VL may be derived from different monoclonal antibodies. Suitable examples include bispecific sc(Fv)2s that recognize two different epitopes present in the same antigen, as disclosed in Journal of Immunology (1994) 152 (11), 5368-5374. sc(Fv)2s can be produced by methods known to those skilled in the art. For example, they can be produced by linking scFvs with a linker such as a peptide linker.

[0041] As used herein, the configuration of the antigen-binding domain constituting sc(Fv)2 includes antigen-binding molecules characterized in that two VHs and two VLs are arranged in the order of VH, VL, VH, VL ([VH] linker [VL] linker [VH] linker [VL]) starting from the N-terminus of the single-chain polypeptide. However, the order of the two VHs and two VLs is not particularly limited to the above configuration, and they may be arranged in any order. For example, the following order configurations are also possible: [VL] linker [VH] linker [VH] linker [VL] [VH] linker [VL] linker [VL] linker [VH] [VH] linker [VH] linker [VL] linker [VH] [VL] linker [VH] linker [VL] linker [VH] [VL] linker [VH] linker [VL] linker [VH]

[0042] The molecular form of sc(Fv)2 is also described in detail in WO2006 / 132352, and based on these descriptions, a person skilled in the art would be able to appropriately prepare a desired sc(Fv)2 for producing the antigen-binding molecules disclosed herein.

[0043] The antigen-binding molecules of the present invention may be conjugated with a carrier polymer such as PEG or an organic compound such as an anticancer agent. Furthermore, a glycosylation sequence can be inserted into the amino acid sequence of the antigen-binding molecules of the present invention to suitably add a glycosylation chain to the antigen-binding molecules in order to obtain the desired effect of the glycosylation.

[0044] The linker linking the antibody variable regions can be any peptide linker that can be introduced by genetic engineering or a synthetic compound linker (e.g., the linkers disclosed in Protein Engineering, 9 (3), 299-305, 1996). However, in the present invention, a peptide linker is preferred. The length of the peptide linker is not particularly limited and can be selected appropriately by those skilled in the art depending on the purpose. A preferred length is 5 amino acids or more (the upper limit is not particularly limited, but is usually 30 amino acids or less, preferably 20 amino acids or less), with 15 amino acids being particularly preferred. When sc(Fv)2 contains three peptide linkers, all peptide linkers may be the same length, or peptide linkers of different lengths may be used. A widely known example of a peptide linker is a flexible polypeptide linker composed of glycine and serine. The length and sequence of the peptide linker can be selected appropriately by those skilled in the art depending on the purpose.

[0045] Synthetic chemical linkers (chemical cross-linkers) are cross-linkers commonly used for cross-linking peptides, such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES), and these cross-linkers are commercially available.

[0046] When four antibody variable regions are linked, three linkers are usually required, and the same linkers may be used for all of them, or different linkers may be used.

[0047] "Fab" is composed of one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0048] "F(ab')2" and "Fab'" refer to antibody fragments produced by treating immunoglobulins (monoclonal antibodies) with protease enzymes such as pepsin or papain, resulting in digestion across the disulfide bond between the two heavy chains in the hinge region. For example, treating IgG with papain cleaves the antibody upstream of the disulfide bond between the two heavy chains in the hinge region, producing two homologous antibody fragments in which an light chain consisting of a VL (light chain variable region) and a CL (light chain constant region) and an heavy chain fragment consisting of a VH (heavy chain variable region) and a CHγ1 (the γ1 region of the heavy chain constant region) are linked by a disulfide bond at their C-terminal regions. These two homologous antibody fragments are each referred to as Fab'.

[0049] "F(ab')2" comprises two light chains and two heavy chains comprising constant regions of the CH1 domain and a portion of the CH2 domain such that an interchain disulfide bond is formed between the two heavy chains. The F(ab')2 constituting the antigen-binding molecule disclosed herein can be suitably obtained by partially digesting a full-length monoclonal antibody or the like having the desired antigen-binding domain with a protease such as pepsin, followed by removal of the Fc fragment by adsorption onto a protein A column. Such a protease is not particularly limited, as long as it can digest a full-length antibody so as to produce F(ab')2 in a limited manner by appropriately setting the enzyme reaction conditions, such as pH, and examples thereof include pepsin and ficin.

[0050] "Affinity" refers to the strength of the total non-covalent interactions between one binding site of a molecule (e.g., an antibody) and the molecule's binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity," as used herein, refers to the intrinsic binding affinity, reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0051] The term "specific binding" refers to the state in which one of two specifically binding molecules does not exhibit any significant binding to any molecules other than the one or more molecules to which it binds. The term is also used when a domain containing an antibody variable region is specific to a particular epitope among multiple epitopes contained in an antigen. When the epitopes to which the domain containing an antibody variable region binds are contained in multiple different antigens, an antigen-binding molecule having the domain containing the antibody variable region can bind to various antigens containing the epitope.

[0052] The term "epitope" refers to a site on an antigen to which an antigen-binding molecule (e.g., an antibody), whether proteinaceous or non-proteinaceous, binds. For example, an epitope can be defined by its structure. Alternatively, an epitope can be defined by the binding activity of an antigen-binding molecule (e.g., an antibody) that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope can also be identified by the amino acid residues that constitute the epitope. When the epitope is a glycan, the epitope can also be identified by a specific glycan structure.

[0053] A linear epitope is one that comprises a recognized primary amino acid structure. A linear epitope typically includes at least three, and most usually at least five, e.g., about 8 to about 10, or 6 to 20, amino acids in a unique sequence.

[0054] In contrast to linear epitopes, conformational epitopes are typically epitopes in which the amino acids constituting the epitope are not contiguous as a primary structure but are composed of discontinuous amino acid residues (sometimes referred to as discontinuous epitopes). Conformational epitopes may include an increased number of amino acids compared to linear epitopes. In recognizing conformational epitopes, antigen-binding molecules (e.g., antibodies) recognize the three-dimensional structure of a peptide or protein. For example, when a protein molecule folds to form a three-dimensional structure, certain amino acids and / or polypeptide backbones that form a conformational epitope are juxtaposed, allowing antigen-binding molecules (e.g., antibodies) to recognize the epitope. Methods for determining the conformational structure of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-directed spin labeling and electromagnetic paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

[0055] The structure of an antigen-binding molecule (e.g., an antibody) that binds to an epitope is called a "paratope," "antigen-binding site," "antigen-binding region," or "antigen-binding domain." The epitope and paratope are stably bound by hydrogen bonds, electrostatic forces, van der Waals forces, hydrophobic bonds, and other forces acting between them. The binding strength between the epitope and paratope is called affinity. The sum of the binding strengths when multiple antigen-binding molecules (e.g., antibodies) bind to multiple antigens is called avidity. For example, when an antigen-binding molecule containing multiple antigen-binding domains (i.e., a multivalent antigen-binding molecule) binds to multiple epitopes, the affinities act synergistically, resulting in a higher avidity than affinity.

[0056] An "antigen-binding molecule that competes with a reference antigen-binding molecule" refers to an antigen-binding molecule that blocks the binding of the reference antigen-binding molecule to its own antigen by 50% or more in a competition assay. Conversely, the reference antigen-binding molecule blocks the binding of the antigen-binding molecule to its own antigen by 50% or more in a competition assay. Competing antigen-binding molecules include antigen-binding molecules that bind to the same or overlapping epitope as the reference antigen-binding molecule, and antigen-binding molecules that bind to an adjacent epitope that is sufficiently close to the epitope bound by the reference antigen-binding molecule and cause steric hindrance. Exemplary competition assays are provided herein.

[0057] An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as measured by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0058] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its original environment. Isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0059] An "isolated nucleic acid encoding an antigen-binding molecule" refers to one or more nucleic acid molecules encoding an antigen-binding molecule, preferably comprising an antibody heavy chain variable region and a light chain variable region, and includes nucleic acid molecules carried on a single vector or separate vectors, and nucleic acid molecules present at one or more locations in a host cell.

[0060] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which they are introduced. Some vectors are capable of conferring expression of a nucleic acid to which they are operatively linked. Such vectors are also referred to herein as "expression vectors." Vectors can be introduced into host cells by viral or electroporation methods, but vector introduction is not limited to ex vivo introduction; vectors can also be introduced directly into a living body.

[0061] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as that for which the original transformed cell was screened or selected are also included herein.

[0062] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0063] A "pharmaceutically acceptable carrier" refers to an ingredient, other than an active ingredient, in a pharmaceutical formulation or composition that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0064] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is human.

[0065] An "effective amount" of an agent (eg, a pharmaceutical formulation) refers to an amount, at dosages and for periods of time necessary, effective to achieve a desired therapeutic or prophylactic result.

[0066] The term "package insert" is used to refer to instructions typically included in commercial packaging of therapeutic products that contain information about the indications, usage, dosage, administration, concomitant therapy, contraindications, and / or warnings regarding the use of such therapeutic product.

[0067] As used herein, "treatment" (and its grammatical derivatives, such as "treat," "treating," etc.) refers to a clinical intervention intended to alter the natural course of the individual being treated and can be performed prophylactically or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or slow the progression of disease.

[0068] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0069] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[0070] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive as used herein.

[0071] "Inhibiting cell growth or proliferation" means reducing cell growth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducing cell death.

[0072] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, and excluding any conservative substitutions from the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetyx Corporation). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0073] The ALIGN-2 sequence comparison computer program is the copyright of Genentech, Inc., and its source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, one can say that a given amino acid sequence A has or contains a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored by the sequence alignment program ALIGN-2 as identical matches in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0074] The term "and / or" is used herein to refer to each of the objects listed before and after "and / or" or any combination thereof. For example, "A, B and / or C" includes each of the objects "A," "B," and "C," as well as the combinations "A and B," "A and C," "B and C," and "A and B and C."

[0075] II. Detailed Description of the Invention The invention of this disclosure generally relates to antigen-binding molecules comprising a CD3-binding domain with excellent stability, pharmaceutical compositions comprising them, and uses thereof. Specifically, the antigen-binding molecules of the present invention comprise a CD3-binding domain with improved stability compared to the CD3-binding domain contained in conventional T cell-redirecting antibodies (e.g., the bispecific antibodies described in WO2015174439). The structure of the antigen-binding molecules of the present invention is not particularly limited, as long as they comprise the CD3-binding domain of the present invention with improved stability. The antigen-binding molecules may be molecules having a natural antibody structure or polypeptides having other artificially designed structures.

[0076] In some embodiments, the antigen-binding molecule of the present invention is an antibody. In some embodiments, the antigen-binding molecule of the present invention is a monoclonal antibody, including a chimeric, humanized, or human antibody. In one embodiment, the antigen-binding molecule of the present invention is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, single-domain antibody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as a complete IgG1 antibody, a complete IgG2 antibody, a complete IgG3 antibody, or a complete IgG4 antibody.

[0077] Antigen-binding molecules comprising a CD3-binding domain with excellent stability. In one aspect, the present invention provides antigen-binding molecules comprising a first antigen-binding domain (CD3-binding domain) having binding activity to CD3 and a second antigen-binding domain. In another aspect, the present invention provides monovalent antigen-binding molecules comprising a single antigen-binding domain (CD3-binding domain) having binding activity to CD3. The CD3-binding domain contained in the antigen-binding molecules of the present invention is characterized by excellent stability and a small decrease in CD3-binding activity after storage at room temperature or higher. In one embodiment, the CD3-binding activity of the CD3-binding domain contained in the antigen-binding molecules of the present invention after storage at room temperature or higher (e.g., 50°C) for a predetermined period of time (e.g., 3 days) maintains 65% or more, 70% or more, or 75% or more of the CD3-binding activity after storage at a low temperature (e.g., 4°C). In some embodiments, the CD3-binding domain contained in the antigen-binding molecules of the present invention has binding activity to the CD3 epsilon chain. In some embodiments, the CD3 is mammalian CD3. In a specific embodiment, the CD3 is human CD3. In some embodiments, the antigen-binding domain comprised in the antigen-binding molecule of the present invention is Fv, Fab, Fab', or scFv.

[0078] CD3-binding domain In one embodiment, the CD3-binding domain comprised in the antigen-binding molecule of the present invention comprises an antibody H-chain variable region and L-chain variable region having binding activity to CD3, wherein the H-chain variable region comprises: an H-chain CDR1 comprising the amino acid sequence NAWMH (SEQ ID NO: 1); an H-chain CDR2 comprising the amino acid sequence QIX1DKSQNYATX2VAESVKG (SEQ ID NO: 2), in which X1 is K or R, and X2 is Y or F; and an H-chain CDR3 comprising the amino acid sequence VHYX3AGYGVDX4 (SEQ ID NO: 3), in which X3 is A or P, and X4 is I, M, or L, and the L-chain variable region comprises: an L-chain CDR1 comprising the amino acid sequence RSX5X6X7VVHENRX8TYLH (SEQ ID NO: 4), in which X5 is S or T, X6 is Q or M, X7 is S or T, and X8 is Q or N; and an L-chain CDR3 comprising the amino acid sequence GQGTQVPYT (SEQ ID NO: 6). In this specification, the terms H chain and heavy chain are used interchangeably, and the terms L chain and light chain are used interchangeably.

[0079] In a specific embodiment, the antibody H chain variable region and L chain variable region having binding activity to CD3 comprise any combination of H chain CDR1, CDR2, and CDR3, and L chain CDR1, CDR2, and CDR3 selected from the following (a1) to (a8): (a1) a combination of H chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, H chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, H chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10, L chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, L chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and L chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (a2) (a3) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (a4) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (a5)(a6) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 13, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (a7) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (a8) A combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 18, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (a9) A combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 19, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.

[0080] In one embodiment, the antibody heavy chain variable region and light chain variable region having binding activity to CD3 include any combination of heavy chain variable region and light chain variable region selected from the following (a1) to (a8): (a1) a combination of an heavy chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 20 and an light chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 25; (a2) a combination of an heavy chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 21 and an light chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 25; (a3) ​​a combination of an heavy chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 21 and an light chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 26; (a4) (a5) a combination of an H-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 25; (a6) a combination of an H-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 27; (a7) (a8) a combination of a heavy chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22 and a light chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 28;A combination of an H chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 22 and an L chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 29.

[0081] In one embodiment, the antibody H-chain variable region and L-chain variable region having binding activity to CD3 comprise any of the combinations of H-chain variable regions and L-chain variable regions selected from the following (a1) to (a8): (a1) a combination of an H-chain variable region that has at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 20, and comprises an H-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an H-chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an H-chain CDR3 comprising the amino acid sequence of SEQ ID NO: 10, and an L-chain variable region that has at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 25, and comprises an L-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an L-chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an L-chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (a2) (a3) a combination of an H-chain variable region which has at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 21 and which comprises an H-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an H-chain CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and an H-chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11, and an L-chain variable region which has at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 25 and which comprises an L-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an L-chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an L-chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6;(a4) a combination of an H-chain variable region which has at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 21 and which comprises an H-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an H-chain CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and an H-chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11, and an L-chain variable region which has at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 26 and which comprises an L-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 16, an L-chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an L-chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (a5) a combination of an H-chain variable region which has at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 22 and which comprises an H-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an H-chain CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and an H-chain CDR3 comprising the amino acid sequence of SEQ ID NO: 12, and an L-chain variable region which has at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 25 and which comprises an L-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an L-chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an L-chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (a6) a combination of an H-chain variable region which has at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 23 and which comprises an H-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an H-chain CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and an H-chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and an L-chain variable region which has at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 25 and which comprises an L-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an L-chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an L-chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6;(a7) a combination of an H-chain variable region which has at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 22 and which comprises an H-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an H-chain CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and an H-chain CDR3 comprising the amino acid sequence of SEQ ID NO: 12, and an L-chain variable region which has at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 27 and which comprises an L-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 17, an L-chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an L-chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (a8) a combination of an H-chain variable region which has at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 22 and which comprises an H-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an H-chain CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and an H-chain CDR3 comprising the amino acid sequence of SEQ ID NO: 12, and an L-chain variable region which has at least 90% or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 28 and which comprises an L-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an L-chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an L-chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; A combination of an H-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 22 and comprising an H-chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, an H-chain CDR2 having the amino acid sequence shown in SEQ ID NO: 9, and an H-chain CDR3 having the amino acid sequence shown in SEQ ID NO: 12, and an L-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 29 and comprising an L-chain CDR1 having the amino acid sequence shown in SEQ ID NO: 19, an L-chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and an L-chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6.

[0082] In a specific embodiment, the antibody heavy chain variable region and light chain variable region having binding activity to CD3 comprise any of the combinations of heavy chain variable region and light chain variable region selected from the following (a1) to (a8): (a1) a combination of an heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 20 and an light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 25; (a2) a combination of an heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 21 and an light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 25; (a3) ​​a combination of an heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 21 and an light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 26; (a4) a combination of an heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 22 and an light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 25; (a5) a combination of an heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 23 and an light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 25; (a6) (a7) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 27; (a8) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 29.

[0083] In one embodiment, the antigen-binding molecule of the present invention comprises, as heavy and light chains having binding activity to CD3, any of the following combinations of heavy and light chains selected from (a1) to (a8): (a1) a combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 30 and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 35; (a2) a combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 31 and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 35; (a3) ​​a combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 31 and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 36; (a4) (a5) a combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 32 and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 35; (a6) a combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 32 and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 37; (a7) a combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 32 and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 38; (a8)A combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 32 and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 39.

[0084] In one embodiment, the antigen-binding molecule of the present invention comprises, as heavy and light chains having binding activity to CD3, any of the combinations of heavy and light chains selected from the following (a1) to (a8): (a1) a combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 30, and comprising an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 10, and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 35, and comprising an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (a2) (a3) a combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 31 and comprising an H chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 having the amino acid sequence shown in SEQ ID NO: 8, and an H chain CDR3 having the amino acid sequence shown in SEQ ID NO: 11, and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 35 and comprising an L chain CDR1 having the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6; (a4) A combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 31 and comprising an H chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 having the amino acid sequence shown in SEQ ID NO: 8, and an H chain CDR3 having the amino acid sequence shown in SEQ ID NO: 11, and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 36 and comprising an L chain CDR1 having the amino acid sequence shown in SEQ ID NO: 16, an L chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6.(a5) A combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 32 and comprising an H chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 having the amino acid sequence shown in SEQ ID NO: 9, and an H chain CDR3 having the amino acid sequence shown in SEQ ID NO: 12, and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 35 and comprising an L chain CDR1 having the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6. (a6) A combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 33 and comprising an H chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 having the amino acid sequence shown in SEQ ID NO: 9, and an H chain CDR3 having the amino acid sequence shown in SEQ ID NO: 13, and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 35 and comprising an L chain CDR1 having the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6. (a7) A combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 32 and comprising an H chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 having the amino acid sequence shown in SEQ ID NO: 9, and an H chain CDR3 having the amino acid sequence shown in SEQ ID NO: 12, and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 37 and comprising an L chain CDR1 having the amino acid sequence shown in SEQ ID NO: 17, an L chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6.(a8) A combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 32 and comprising an H chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 having the amino acid sequence shown in SEQ ID NO: 9, and an H chain CDR3 having the amino acid sequence shown in SEQ ID NO: 12, and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 38 and comprising an L chain CDR1 having the amino acid sequence shown in SEQ ID NO: 18, an L chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6. A combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 32 and comprising an H chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 having the amino acid sequence shown in SEQ ID NO: 9, and an H chain CDR3 having the amino acid sequence shown in SEQ ID NO: 12, and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 39 and comprising an L chain CDR1 having the amino acid sequence shown in SEQ ID NO: 19, an L chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6.

[0085] In a specific embodiment, the antigen-binding molecule of the present invention comprises a heavy chain and a light chain having binding activity to CD3, and the heavy chain and the light chain are any of the combinations of heavy chain and light chain selected from the following (a1) to (a8): (a1) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 30 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35, (a2) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 31 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35, (a3) ​​a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 31 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 36, (a4) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 32 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35, (a5) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 33 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35, (a6) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 32 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 37, (a7) (a8) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 32 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 38; (a9) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 32 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 39.

[0086] Second antigen-binding domain In the antigen-binding molecules of the present invention, the structure of the second antigen-binding domain is not particularly limited, as long as it has binding activity to a target antigen. In some embodiments, the second antigen-binding domain comprises an antibody heavy chain variable region and light chain variable region.

[0087] Monospecific antigen-binding molecules In some embodiments, the antigen-binding molecules of the present invention are monospecific antigen-binding molecules, and both the first and second antigen-binding domains have binding activity to CD3 (e.g., human CD3). In one embodiment, the first and second antigen-binding domains have binding activity to the same epitope on CD3. In a specific embodiment, both the first and second antigen-binding domains have binding activity to the CD3ε chain (e.g., human CD3ε chain). In a specific embodiment, the first and second antigen-binding domains comprise the same amino acid sequence. In another specific embodiment, the first and second antigen-binding domains comprise different amino acid sequences.

[0088] Multispecific antigen-binding molecules In some embodiments, the antigen-binding molecules of the present invention are multispecific antigen-binding molecules having binding specificities at at least two different sites. In certain embodiments, the multispecific antigen-binding molecules of the present invention are bispecific antigen-binding molecules. In another specific embodiment, the multispecific antigen-binding molecules of the present invention are antigen-binding molecules with three or more specificities, such as trispecific, tetraspecific, pentaspecific, or hexaspecific antigen-binding molecules.

[0089] In one embodiment of the multispecific antigen-binding molecules of the present invention, the first antigen-binding domain has binding activity to CD3, and the second antigen-binding domain has binding activity to an antigen other than CD3. In this embodiment, the "antigen other than CD3" recognized and bound by the second antigen-binding domain is preferably a molecule expressed on the surface of cells targeted by the cytotoxic effect of T cells (T cell-dependent cytotoxicity), and examples thereof include molecules specifically expressed on the surface of cancer cells (also referred to herein as cancer antigens or cancer-specific antigens) and molecules expressed on the surface of cells that have the function of suppressing immune responses (also referred to herein as immunosuppressive cell surface antigens).

[0090] In one embodiment, the second antigen-binding domain has binding activity to a cancer antigen. In such an embodiment, the multispecific antigen-binding molecule of the present invention binds to CD3 via the first antigen-binding domain and to a cancer antigen via the second antigen-binding domain, thereby bringing T cells into proximity with target cancer cells and enabling cancer treatment or prevention through cytotoxic activity of T cells against tissues containing target cancer cells. In another embodiment, the second antigen-binding domain has binding activity to an immunosuppressive cell surface antigen. In such an embodiment, the multispecific antigen-binding molecule of the present invention binds to CD3 via the first antigen-binding domain and to an immunosuppressive cell surface antigen via the second antigen-binding domain, thereby bringing T cells into proximity with cells that function to suppress immune responses (also referred to herein as immunosuppressive cells), enabling cancer treatment or prevention through cytotoxic activity of T cells against immune response-suppressive cells. In the multispecific antigen-binding molecules of the present invention, the second antigen-binding domain can be any known antigen-binding domain or other antigen-binding domain to be identified in the future, which has binding activity to a cancer antigen or an immunosuppressive cell surface antigen.

[0091] In a preferred embodiment, the multispecific antigen-binding molecule of the present invention has a cytotoxic activity that is equivalent to or greater than that of a control multispecific antigen-binding molecule that comprises, as a first antigen-binding domain having CD3-binding activity, an antibody heavy chain variable region comprising an heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 10 (e.g., an heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20), and an antibody light chain variable region comprising an light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14, an light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6 (e.g., an light chain variable region comprising the amino acid sequence of SEQ ID NO: 24). Examples of such multispecific antigen-binding molecules include multispecific antigen-binding molecules (e.g., bispecific antigen-binding molecules, e.g., bispecific antibodies) that comprise, as a first antigen-binding domain, the heavy and light chain CDRs, heavy and light chain variable regions, or heavy and light chains of antibody Nos. 2, 14, 25, 29, 30 to 32, and 34 listed in Table 5 below.

[0092] Domain Comprising an Antibody Variable Region with CD3-Binding Activity As used herein, the terms "antibody variable region with CD3-binding activity" and "domain comprising an antibody variable region with CD3-binding activity" are used interchangeably and refer to a portion of an anti-CD3 antibody comprising a region that specifically binds to and is complementary to part or all of CD3, an adaptor molecule that, together with the T cell receptor, forms a T cell receptor complex. Preferably, the domain comprises the light chain variable region (VL) and heavy chain variable region (VH) of an anti-CD3 antibody. Suitable examples of such domains include "scFv (single chain Fv)," "single chain antibody," "Fv," "scFv2 (single chain Fv 2)," "Fab," and "F(ab')2."

[0093] The CD3-binding domain of the present invention (a domain comprising an antibody variable region having binding activity to CD3) may bind to any epitope present in the γ chain, δ chain, or ε chain sequence constituting CD3. In the present invention, a domain comprising a light chain variable region (VL) and a heavy chain variable region (VH) of an anti-CD3 antibody that binds to an epitope present in the extracellular domain of the ε chain of the human CD3 complex is preferably used. The light chain variable region (VL) and heavy chain variable region (VH) of an anti-CD3 antibody described in the Examples are preferably used as such a domain. As described above, an appropriate humanized antibody or human antibody is used as the anti-CD3 antibody that serves as the source of the domain comprising an antibody variable region having CD3-binding activity. The polynucleotide sequences of the gamma, delta, and epsilon chains that make up CD3 are registered under RefSeq accession numbers NM_000073.2, NM_000732.4, and NM_000733.3, respectively, and their polypeptide sequences are registered under RefSeq accession numbers NP_000064.1, NP_000723.1, and NP_000724.1, respectively.

[0094] Domains Comprising Antibody Variable Regions with Binding Activity to Antigens Other Than CD3 As used herein, the terms "antibody variable region with binding activity to an antigen other than CD3" and "domains comprising antibody variable regions with binding activity to an antigen other than CD3" are used interchangeably and refer to a portion of an antibody comprising a region that specifically binds to and is complementary to part or all of the antigen (e.g., a cancer antigen; also referred to herein as a cancer-specific antigen). A domain comprising an antibody variable region can be provided by one or more antibody variable domains. Preferably, a domain comprising an antibody variable region comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Suitable examples of such domains comprising antibody variable regions include "scFv (single chain Fv)," "single chain antibody," "Fv," "scFv2 (single chain Fv 2)," "Fab," and "F(ab')2."

[0095] Antigen As used herein, the term "antigen" is limited to only including the epitope to which the antigen-binding domain binds. Suitable examples of antigens include, but are not limited to, peptides, polypeptides, and proteins derived from animals or humans. Suitable examples of antigens used to treat diseases caused by target tissues include, but are not limited to, molecules expressed on the surface of target cells (e.g., cancer cells and inflammatory cells), molecules expressed on the surface of other cells in tissues containing target cells, molecules expressed on the surface of cells that play an immunological role in target cells and tissues containing target cells, and large molecules present in the interstitium of tissues containing target cells.

[0096] In one embodiment, the antigen may be derived from any animal species (e.g., human; or non-human animal, e.g., mouse, rat, hamster, guinea pig, rabbit, monkey, cynomolgus monkey, rhesus monkey, hamadryas baboon, chimpanzee, goat, sheep, dog, horse, pig, cow, or camel), or any bird; the antigen is preferably derived from human, rabbit, monkey, rat, or mouse.

[0097] The "antigen" (e.g., CD3) recognized and bound by the antigen-binding molecules of the present invention refers to any native "antigen" (e.g., CD3) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. This term encompasses "full-length" unprocessed "antigens" (e.g., CD3) as well as any form of "antigen" (e.g., CD3) resulting from processing in cells. These terms also encompass naturally occurring variants of "antigens" (e.g., CD3), such as splice variants and allelic variants.

[0098] In some embodiments, antigens include 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, addressin, 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 stimulatory 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-6, Vgr-1, BMP-7 (OP-1), BMP-8 (BMP8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C) 3), 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, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (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, Welch bacillus toxin, CKb8-1, Claudin 6, CLC, CMV, CMV UL, 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, Cytokeratin tumor-related antigen, DAN, DCC, DcR3, DC-SIGN, Complement control factor (Decay accelerating factor), des(1-3)-IGF-I (Brain IGF-1), Dhh, Digoxin, DLL3, 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 / EphB4, EPO, ERCC, E-selectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF-3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, 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), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, 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, HMFG PEM, 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 growth 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-1 bp1, 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, metalloproteinase, M GDF 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 inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, NC adherin,NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, 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, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, 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, Thymic 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(WHO 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 RII, CD120b, 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). Apo-2 Liquid TL2) TNFSF11(TRANCE / RANK Liquid ODF OPG Liquid) TNFSF12(TWEAK Apo-3リガンド, DR3リンド, TNFSF13(APRIL TALL2), TNFSF13B(BAFF LEAD, TALL1, THANK, TNFSF20), TNFSF14(LIGHT HVEM LTG) TNFSF15(TL1A / VEGI) TNFSF18(GITR TL AITR 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-like receptor) receptor)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, WNT 5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL 1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aβ, CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp9 0, IL-17 / IL-17R, IL-20 / IL-20R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1, chromogranin A, chromogranin B, tau, VAP1, high molecular weight kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6Nav1.7, 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 VII Antigens include factor Ia, factor IX, factor IXa, factor X, factor 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, and S1P. In some embodiments, the antigen includes receptors for hormones and growth factors. In certain embodiments, the antigen is an antigenic molecule expressed or secreted by cells contained in tumor tissue (e.g., tumor cells, immune cells, stromal cells, etc.), particularly an antigen specifically expressed by cancer cells (cancer-specific antigens). In certain embodiments, the antigen is an antigenic molecule expressed on the surface of cells that function to suppress immune responses.

[0099] Cancer-specific antigens: Non-limiting examples of antigens on target cells include "cancer-specific antigens" that are expressed by cancer cells and enable the differentiation of cancer cells from healthy cells. Examples include antigens that are expressed in association with the malignant transformation of cells, and abnormal glycans that appear on the cell surface or protein molecules when cells become cancerous. Specific examples include ALK, pleiotrophin (PTN), EpCAM, CA125, prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), TYRP1, HMW-MAA, prostate-specific membrane antigen (PSMA), CEA, MUC1, HMFG1, TAG-72, GICA (CA19-9), NY-ESO-1, LEA, CD15, CD17, CD19, CD20, CD22, CD30, CD33, CD38, CD77, CD79b, CD147, CD228, GD2, GD3, GM2, GM3, TSTA, and virus-induced tumors. Antigens (e.g., envelope antigens of DNA tumor viruses and RNA tumor viruses), alpha-fetoprotein (AFP), 5T4, differentiation antigens (e.g., L6 and L20 antigens), CD165, EGFR, ANKRD17, ErbB2, APO-1, SSEA-1, SCP-1, LeY, oligosaccharide antigens, SSEA-3, SSEA-4, CTAGE1, MART-1, sialyl Tn (STn), NY-CO-45, NY-LU-12, ART1, MA2, NOVA2, TSPAN8, MAGE-C1, MAGE-B1, MAGE-B2, MAGE-4A, Examples of polypeptides include MAGE-X2, YKL-40, EREG, CA15-3, CLEC12A, Nectin4, Trop2, BCMA, Tissue factor, FRα (FOLR1), ErbB3, Claudin18 (Claudin18.2), B7-H3 (CD276), MET, PSCA, PTK7, MSLN (Mesothelin), CCR4, CDH6, IL13RA2, GPC3, XPR1, NOX1, MARVELD3 isoform 1, MARVELD3 isoform 2, SPINT2, MANSC1, SLC12A2, CDCP1, SEZ6L2, FLVCR1, SLC7A5, STEAP1, MMP14, TNFRSF21, TMPRSS4, claudin 6, and DLL3, as well as any fragments of these polypeptides and modified structures thereof.

[0100] Antigens expressed on the surface of cells that have the function of suppressing an immune response "Cells that have the function of suppressing an immune response" are not particularly limited as long as they have the function of suppressing an immune response, and examples include regulatory T cells (Treg), exhausted T cells, myeloma-derived stromal cells (MDSC), tumor-associated macrophages (TAM), induced regulatory T cells (Tr1), tumor-associated dendritic cells (TADC), tumor-associated neutrophils (TAN), cancer-associated fibroblasts (CAF), and regulatory B cells (Breg). Regulatory T cells and exhausted T cells are particularly preferred as target cells of the present invention. Specific examples of molecules expressed on the surface of cells that have the function of suppressing these immune responses include CTLA4, PD1, TIM3, LAG-3, CD244 (2B4), CD160, GARP, OX40, CD137 (4-1BB), CD25, VISTA, VISATA, BTLA, TNFR25, CD57, KLRG1, CCR2, CCR5, CCR6, CD39, CD73, CD4, CD18, CD49b, CD1d, CD5, CD21, TIM1, CD19, CD20, CD23, CD24, CD38, CD93, IgM, B220 (CD45R), CD317, PD-L1, CD11b, Ly6G, ICAM-1, FAP, PDGFR, podoplanin, and TIGIT. Among these molecules, preferred molecules that are targets of the binding domain of the present invention include, for example, cell fractions (CD4 + , CD25 high , CD45RA -Examples of such molecules include CTLA4, TIM3, LAG3, CD137 (4-1BB), CD25, CCR5, CCR6, CD38, and TIGIT, which are cell surface molecules specifically expressed on the IL-16 receptor tyrosine kinase (IL-1) receptor. Preferred target molecules for the binding domains of the present invention include CTLA4, LAG3, and OX40.

[0101] Antigen-binding activity

[0113] Methods for confirming epitope binding by test antigen-binding molecules having domains containing antibody variable regions with binding activity to CD3 are exemplified below, but methods for confirming epitope binding by test antigen-binding molecules having domains containing antibody variable regions with binding activity to other antigens can also be appropriately performed in accordance with the examples below.

[0102] For example, whether a test antigen-binding molecule containing a domain comprising an antibody variable region with binding activity to CD3 recognizes a linear epitope present in the CD3 molecule can be confirmed, for example, as follows. For this purpose, a linear peptide consisting of the amino acid sequence constituting the extracellular domain of CD3 is synthesized. The peptide can be chemically synthesized. Alternatively, it can be obtained by genetic engineering techniques using a region in CD3 cDNA that encodes 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 to a test antigen-binding molecule containing a domain comprising an antibody variable region with binding activity to CD3 is assessed. For example, the binding activity of the antigen-binding molecule to the peptide can be assessed by ELISA using an immobilized linear peptide as the antigen. Alternatively, the binding activity of the antigen-binding molecule to the linear peptide can be determined based on the level of inhibition by the linear peptide of binding of the antigen-binding molecule to CD3-expressing cells. These tests can determine the binding activity of the antigen-binding molecule to the linear peptide.

[0103] Furthermore, whether a test antigen-binding molecule having a domain containing an antibody variable region with CD3-binding activity recognizes a conformational epitope can be confirmed as follows. For this purpose, CD3-expressing cells are prepared. Examples of such cases include when a test antigen-binding molecule having a domain containing an antibody variable region with CD3-binding activity strongly binds to CD3-expressing cells upon contact with the cells, but does not substantially bind to a linear peptide consisting of the amino acid sequence constituting the extracellular domain of immobilized CD3. Here, "not substantially binding" refers to a binding activity that is 80% or less, typically 50% or less, preferably 30% or less, and particularly preferably 15% or less of the binding activity toward human CD3-expressing cells.

[0104] Methods for measuring the binding activity of test antigen-binding molecules containing a CD3 antigen-binding domain toward CD3-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, the binding activity can be assessed by ELISA or fluorescence activated cell sorting (FACS) using CD3-expressing cells as antigens.

[0105] In the ELISA format, the binding activity of a test antigen-binding molecule containing a CD3 antigen-binding domain for CD3-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 on which CD3-expressing cells have been immobilized, and the test antigen-binding molecule bound to the cells is detected using an enzyme-labeled antibody that recognizes the test antigen-binding molecule. Alternatively, in FACS, a dilution series of the test antigen-binding molecule is prepared, and the antibody binding titer for CD3-expressing cells is determined, allowing the binding activity of the test antigen-binding molecule for CD3-expressing cells to be compared.

[0106] The binding of a test antigen-binding molecule to an antigen expressed on the surface of cells suspended in a buffer solution or the like can be detected using a flow cytometer. Known flow cytometers include, for example, the FACSCanto TM II FACSAria TM FACSArray TM FACSVantage TM SE FACSCalibur TM (All are trade names of BD Biosciences) EPICS ALTRA HyPerSort Cytomics FC 500 EPICS XL-MCL ADC EPICS XL ADC Cell Lab Quanta / Cell Lab Quanta SC (All are trade names of Beckman Coulter)

[0107] For example, the following method is a suitable example of a method for measuring the antigen-binding activity of a test antigen-binding molecule containing a CD3 antigen-binding domain. First, the test antigen-binding molecule is reacted with cells expressing CD3 and stained with an FITC-labeled secondary antibody that recognizes the test antigen-binding molecule. The test antigen-binding molecule is diluted with an appropriate buffer solution to prepare the complex at the desired concentration. For example, it can be used at any concentration between 10 μg / ml and 10 ng / ml. Next, fluorescence intensity and cell number are measured using a FACSCalibur (BD). The amount of antibody binding 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 molecule, represented by the amount of binding of the test antigen-binding molecule, can be measured.

[0108] Whether a test antigen-binding molecule containing a CD3 antigen-binding domain shares an epitope with another antigen-binding molecule can be confirmed by competition between the two for the same epitope. Competition between antigen-binding molecules can be detected by cross-blocking assays, for example. For example, competitive ELISA assays are preferred cross-blocking assays.

[0109] Specifically, in a cross-blocking assay, CD3 protein coated on the wells of a microtiter plate is preincubated in the presence or absence of a candidate competing antigen-binding molecule, and then a test antigen-binding molecule is added. The amount of test antigen-binding molecule bound to CD3 protein in the well is indirectly correlated with the binding ability of the candidate competing antigen-binding molecule that competes 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 CD3 protein-coated wells.

[0110] The amount of test antigen-binding molecules bound to the wells via CD3 protein can be easily measured by labeling the antigen-binding molecules in advance. For example, biotin-labeled antigen-binding molecules can be measured using an avidin-peroxidase conjugate and an appropriate substrate. Cross-blocking assays using enzyme labels such as peroxidase are particularly known as competitive ELISA assays. Antigen-binding molecules can also be labeled with other detectable or measurable labeling substances. Specific examples include radiolabels and fluorescent labels.

[0111] If a competitor antigen-binding molecule can block the binding of a test antigen-binding molecule comprising an antigen-binding domain to CD3 by at least 20%, preferably at least 20-50%, and more preferably at least 50%, compared to the binding activity obtained in a control test performed in the absence of the candidate competitor antigen-binding molecule, the test antigen-binding molecule binds to substantially the same epitope as the competitor antigen-binding molecule, or is an antigen-binding molecule that competes for binding to the same epitope.

[0112] When the structure of the epitope to which a test antigen-binding molecule containing a CD3 antigen-binding domain binds has been identified, whether the test antigen-binding molecule and a control antigen-binding molecule share a common epitope can be assessed by comparing the binding activity of both antigen-binding molecules toward peptides in which amino acid mutations have been introduced into the peptide constituting the epitope.

[0113] For example, such binding activity can be measured by comparing the binding activity of test and control antigen-binding molecules to a mutated linear peptide in the ELISA format described above. Alternatively, binding activity to the mutant peptide bound to a column can be measured by flowing the test and control antigen-binding molecules down the column and then quantifying the antigen-binding molecules eluted in the eluate. Methods for adsorbing mutant peptides to a column, for example, as fusion peptides with GST, are known.

[0114] Furthermore, if the identified epitope is a conformational epitope, whether the test and control antigen-binding molecules share the same epitope can be assessed by the following method. First, CD3-expressing cells and CD3-expressing cells with a mutated epitope are prepared. These cells are suspended in an appropriate buffer, such as PBS, and the test and control antigen-binding molecules are added to the cell suspension. Next, an FITC-labeled antibody capable of recognizing the test and control antigen-binding molecules is added to the cell suspension after washing with an appropriate buffer. The fluorescence intensity and cell count of cells stained with the labeled antibody are measured using a FACSCalibur (BD). The test and control antigen-binding molecules are diluted with a suitable buffer to the desired concentration and used. For example, they are used at a concentration between 10 μg / ml and 10 ng / ml. 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 and control antigen-binding molecules, represented by the amount of bound labeled antibody, can be measured.

[0115] In this method, for example, "substantially no binding to mutant CD3-expressing cells" can be determined by the following method. First, test and control antigen-binding molecules bound to mutant CD3-expressing cells are stained with a labeled antibody. The fluorescence intensity of the cells is then detected. When a FACSCalibur is used for flow cytometry to detect fluorescence, the obtained fluorescence intensity can be analyzed using CELL QUEST Software. The percentage increase in fluorescence intensity due to antigen-binding molecule binding can be determined by calculating the comparative value (ΔGeo-Mean) from the Geometric Mean values ​​in the presence and absence of the antigen-binding molecule using the following formula:

[0116] ΔGeo-Mean = Geo-Mean (in the presence of antigen-binding molecules) / Geo-Mean (in the absence of antigen-binding molecules)

[0117] The Geometric Mean comparison value (mutant CD3 molecule ΔGeo-Mean value) obtained by analysis, which reflects the binding amount of the test antigen-binding molecule to mutant CD3-expressing cells, is compared with a ΔGeo-Mean comparison value, which reflects the binding amount of the test antigen-binding molecule to CD3-expressing cells. In this case, it is particularly preferred that the concentrations of the test antigen-binding molecule used to determine the ΔGeo-Mean comparison values ​​for mutant CD3-expressing cells and CD3-expressing cells are adjusted to be identical or substantially identical to each other. An antigen-binding molecule previously confirmed to recognize an epitope in CD3 is used as a control antigen-binding molecule.

[0118] A test antigen-binding molecule is deemed to "not substantially bind to mutant CD3-expressing cells" if the ΔGeo-Mean comparison value for the test antigen-binding molecule against mutant CD3-expressing cells is at least 80%, preferably 50%, more preferably 30%, and particularly preferably 15% of the ΔGeo-Mean comparison value for the test antigen-binding molecule against CD3-expressing cells. The formula for calculating the Geo-Mean value (Geometric Mean) is described in the CELL QUEST Software User's Guide (BD biosciences). When the comparison values ​​are substantially equivalent, the epitopes of the test and control antigen-binding molecules can be determined to be identical.

[0119] Antigen-binding molecules containing an FcRn-binding domain contained in the Fc region A preferred example of the antigen-binding molecule of the present invention is an antigen-binding molecule containing an FcRn-binding domain contained in the Fc region of an antibody. A well-known method for extending the blood half-life of a protein administered to the body involves adding an antibody FcRn-binding domain to the protein of interest and utilizing the FcRn-mediated recycling function.

[0120] In the present invention, the term "FcRn-binding domain" is not particularly limited as long as it has binding activity to FcRn, and may be a domain that directly binds to FcRn or a domain that indirectly binds to FcRn. Examples of domains that directly bind to FcRn include the variable region of an antibody whose antigen is FcRn, Fab, the Fc region of an antibody, fragments thereof, albumin, albumin domain 3, human serum albumin (HSA), and transferrin. Furthermore, examples of domains that indirectly bind to FcRn include domains that have binding activity to the above-mentioned domains that directly bind to FcRn. In the context of the present invention, one embodiment of the FcRn-binding domain is the Fc region of an antibody, or a fragment of the Fc region that contains the FcRn-binding region. Here, examples of the "Fc region" include Fc regions of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM types, and an Fc region derived from a native IgG can be used. Native IgG refers to a polypeptide belonging to the class of antibodies that includes the same amino acid sequence as that of naturally occurring IgG and is substantially encoded by immunoglobulin gamma genes. For example, native human IgG refers to native human IgG1, native human IgG2, native human IgG3, native human IgG4, etc. Native IgG also includes naturally occurring mutants thereof. For the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, multiple allotype sequences due to genetic polymorphisms are described in "Sequences of proteins of immunological interest," NIH Publication No. 91-3242, and any of these sequences may be used in the present invention. In particular, for the human IgG1 sequence, the amino acid sequence at positions 356 to 358 (EU numbering) may be either DEL or EEM.

[0121] In some embodiments, the antigen-binding molecules of the present invention comprise an antibody Fc region. When the antigen-binding molecules of the present invention comprise a single antigen-binding domain, the antigen-binding domain may be linked to one of the two polypeptides constituting the dimeric Fc region. When the antigen-binding molecules of the present invention comprise a first antigen-binding domain and a second antigen-binding domain, the first antigen-binding domain may be linked to one of the two polypeptides constituting the dimeric Fc region, and the second antigen-binding domain may be linked to the other, but this is not a limitation. In certain embodiments, the antigen-binding molecules of the present invention comprise an antibody Fc region with reduced Fcγ receptor-binding activity. When the multispecific antigen-binding molecules of the present invention comprise an antibody Fc region, it is preferable to use an antibody Fc region with reduced Fcγ receptor-binding activity to avoid the target antigen-independent induction of various cytokine production, as seen in trifunctional antibodies such as catumaxomab. Various amino acid mutations (modifications) in the Fc region of an antibody that result in reduced binding activity to Fcγ receptors are known, and Fc regions bearing such known amino acid mutations (modifications) or other mutations (modifications) that will be identified in the future can be used in the antigen-binding molecules of the present invention. Furthermore, the strength of the effector function acting through the binding of the antibody Fc region to the Fcγ receptor varies depending on the IgG subclass, and in humans, it is known to be high in IgG1 and IgG3 and low in IgG2 and IgG4. Therefore, native Fc regions of human IgG2 or IgG4 can also be used as the Fc region of an antibody with reduced binding activity to Fcγ receptors that is included in the antigen-binding molecules of the present invention.

[0122] Fcγ Receptor An Fcγ receptor refers to a receptor that can bind to the Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody, and refers to any member of a family of proteins substantially encoded by the Fcγ receptor gene. In humans, this family includes, but is not limited to, FcγRI (CD64), which includes the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any unidentified human FcγRs or FcγR isoforms or allotypes. FcγRs may be derived from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any unidentified mouse FcγRs or 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 registered under RefSeq accession numbers NM_000566.3 and NP_000557.1, respectively; the polynucleotide and amino acid sequences of FcγRIIA are registered under RefSeq accession numbers BC020823.1 and AAH20823.1, respectively; the polynucleotide and amino acid sequences of FcγRIIB are registered under RefSeq accession numbers BC146678.1 and AAI46679.1, respectively; the polynucleotide and amino acid sequences of FcγRIIIA are registered under RefSeq accession numbers BC033678.1 and AAH33678.1, respectively; and the polynucleotide and amino acid sequences of FcγRIIIB are registered under RefSeq accession numbers BC128562.1 and AAI28563.1, respectively. Whether an Fcγ receptor has binding activity to the Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be confirmed by the FACS or ELISA formats described above, as well as by ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) and the BIACORE method using surface plasmon resonance (SPR) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0123] Fcγ Receptor-Binding Activity Whether an Fc region has reduced binding activity 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 ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) and the BIACORE method using surface plasmon resonance (SPR) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0124] The ALPHA screen is performed using ALPHA technology, which uses two beads, donor and acceptor, based on the following principle: A luminescent signal is detected only when a molecule bound to the donor bead biologically interacts with a molecule bound to the acceptor bead and the two beads are in close proximity. A photosensitizer inside the donor bead, excited by a laser, converts surrounding oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and, when it reaches a nearby acceptor bead, triggers a chemiluminescent reaction within the bead, ultimately emitting light. If 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 no chemiluminescent reaction occurs.

[0125] For example, biotin-labeled antigen-binding molecules are bound to donor beads, and glutathione S-transferase (GST)-tagged Fcγ receptors are bound to acceptor beads. In the absence of competing antigen-binding molecules with mutant Fc regions, antigen-binding molecules with wild-type Fc regions interact with Fcγ receptors, generating a signal at 520-620 nm. Antigen-binding molecules with untagged mutant Fc regions compete with the interaction between antigen-binding molecules with wild-type Fc regions and Fcγ receptors. Relative binding affinity can be determined by quantifying the decrease in fluorescence that occurs as a result of competition. Biotinylation of antigen-binding molecules such as antibodies using sulfo-NHS-biotin or similar is known. Methods for tagging Fcγ receptors with GST include expressing a fusion gene in which a polynucleotide encoding the Fcγ receptor and a polynucleotide encoding GST are fused in frame in cells harboring such a fusion gene in an expression vector, and purifying the gene using a glutathione column. The resulting signals are suitably analyzed by fitting to a one-site competition model using non-linear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0126] One of the substances to be observed for interaction (ligand) is immobilized on a thin gold film on a sensor chip. When light is shone from the back of the sensor chip so that it is totally reflected at the interface between the gold film and the glass, a portion of the reflected light exhibits a reduced reflection intensity (SPR signal). When the other substance to be observed for interaction (analyte) is poured over the surface of the sensor chip, binding occurs between the ligand and the analyte, increasing the mass of the immobilized ligand molecule and changing the refractive index of the solvent on the sensor chip surface. This change in refractive index shifts the position of the SPR signal (conversely, dissociation returns the signal position). The Biacore system plots the amount of 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). The kinetics (association rate constant (ka) and dissociation rate constant (kd)) can be calculated from the sensorgram curve, and the affinity (KD) can be calculated from the ratio of these constants. Inhibition assays are also suitable for use with the BIACORE method. An example of an inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0127] As used herein, "decreased Fcγ receptor-binding activity" refers to, for example, when the competitive activity of a test antigen-binding molecule is 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, or 15% or less, and particularly preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, compared to the binding activity of a control antigen-binding molecule having an Fc region, based on the above-mentioned analytical methods.

[0128] Antigen-binding molecules having the Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be used as a control. These Fc regions may have structures in which an A is added to the N-terminus of the amino acid sequences registered under RefSeq accession numbers AAC82527.1, AAB59393.1, CAA27268.1, and AAB59394.1, respectively. In one embodiment, when an antigen-binding molecule having a mutant Fc region of an antibody of a certain isotype is used as a test substance, the effect of the mutation in the mutant on Fcγ receptor-binding activity can be verified by using an antigen-binding molecule having an Fc region of the antibody of the certain isotype as a control. As described above, antigen-binding molecules having Fc region mutants verified to have reduced Fcγ receptor-binding activity can be appropriately prepared. In another embodiment, the Fc region of an antibody of a certain isotype is compared with the Fc region of an antibody of another certain isotype in terms of Fcγ receptor-binding activity, and the Fc region with lower Fcγ receptor-binding activity can be used in the antigen-binding molecules of the present invention. Furthermore, an amino acid mutation can be introduced into such an Fc region with lower Fcγ receptor-binding activity to obtain an Fc region with even lower Fcγ receptor-binding activity.

[0129] Known examples of such mutations in the Fc region include a deletion of amino acids 231A-238S, as specified by EU numbering (WO 2009 / 011941), C226S, C229S, P238S, (C220S) (J. Rheumatol (2007) 34, 11), C226S, C229S (Hum. Antibod. Hybridomas (1990) 1(1), 47-54), and C226S, C229S, E233P, L234V, and L235A (Blood (2007) 109, 1185-1192).

[0130] Preferred examples of such antigen-binding molecules include those having an Fc region in which any of the following amino acids, as specified by EU numbering, have been substituted among the amino acids constituting the Fc region of an antibody of a particular isotype: 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331, or 332. The antibody isotype from which the Fc region originates is not particularly limited, and Fc regions derived from IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies can be used as appropriate.

[0131] For example, any of the following substitutions, specified according to EU numbering, among the amino acids constituting the Fc region of an IgG1 antibody (numbers indicate the amino acid residue positions specified according to EU numbering, the single-letter amino acid code preceding the number indicates the amino acid residue before substitution, and the single-letter amino acid code following the number indicates the amino acid residue before substitution): (a) L234F, L235E, P331S, (b) C226S, C229S, P238S, (c) C226S, C229S, (d) C226S, C229S, E233P, L234V, L235A, (e) L234A, L235A or L235R, N297A, (f) L235A or L235R, S239K, N297A. Alternatively, antigen-binding molecules having an Fc region in which the amino acid sequence at positions 231 to 238 has been deleted may also be used as appropriate.

[0132] Furthermore, antigen-binding molecules having an Fc region in which any of the following substitutions specified according to EU numbering have been made in the amino acids constituting the Fc region of an IgG2 antibody (numbers indicate the amino acid residue position specified according to EU numbering, the single-letter amino acid code preceding the number indicates the amino acid residue before substitution, and the single-letter amino acid code following the number indicates the amino acid residue before substitution): (g) H268Q, V309L, A330S, P331S (h) V234A (i) G237A (j) V234A, G237A (k) A235E, G237A (l) V234A, A235E, G237A may also be used as appropriate.

[0133] Furthermore, antigen-binding molecules having an Fc region in which any of the following substitutions specified according to EU numbering have been made in the amino acids constituting the Fc region of an IgG3 antibody (numbers indicate amino acid residue positions specified according to EU numbering, the single-letter amino acid code preceding the number indicates the amino acid residue before substitution, and the single-letter amino acid code following the number indicates the amino acid residue before substitution): (m) F241A, (n) D265A, and (o) V264A may also be used as appropriate.

[0134] Furthermore, antigen-binding molecules having an Fc region in which any of the following substitutions specified according to EU numbering have been made in the amino acids constituting the Fc region of an IgG4 antibody (numbers indicate amino acid residue positions specified according to EU numbering, the single-letter amino acid code preceding the number indicates the amino acid residue before substitution, and the single-letter amino acid code following the number indicates the amino acid residue before substitution): (p) L235A, G237A, E318A; (q) L235E; or (r) F234A, L235A may also be used as appropriate.

[0135] Other preferred examples include antigen-binding molecules having an Fc region in which any of the amino acids at positions 233, 234, 235, 236, 237, 327, 330, and 331 (EU numbering) constituting the Fc region of an IgG1 antibody is substituted with an amino acid corresponding to the corresponding EU numbering in the corresponding IgG2 or IgG4.

[0136] Other preferred examples include antigen-binding molecules having an Fc region in which one or more of the following amino acids, identified according to EU numbering, among the amino acids constituting the Fc region of an IgG1 antibody, have been substituted with other amino acids: 234, 235, and 297. The type of amino acid present after substitution is not particularly limited, but particularly preferred are antigen-binding molecules having an Fc region in which one or more of the amino acids at positions 234, 235, and 297 have been substituted with alanine.

[0137] Other preferred examples include antigen-binding molecules having an Fc region in which the amino acid at position 265 (EU numbering) of the amino acids constituting the Fc region of an IgG1 antibody has been substituted with another amino acid. The type of amino acid present after substitution is not particularly limited, but particularly preferred are antigen-binding molecules having an Fc region in which the amino acid at position 265 has been substituted with alanine.

[0138] In certain embodiments, the antigen-binding molecules of the present invention comprise an Fc region with reduced Fcγ receptor binding activity. Examples of such embodiments include antibodies with reduced effector functions attributable to the Fc region, which are desirable when certain effector functions (such as complement and ADCC) are unnecessary or harmful. Examples of antibodies with reduced effector function include those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with two or more substitutions at amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant (U.S. Patent No. 7,332,581), which involves substitutions of residues 265 and 297 with alanine.

[0139] For other examples of Fc region variants, see also US Pat. No. 5,648,260; US Pat. No. 5,624,821; and WO94 / 29351.

[0140] Method for Producing Antibodies One preferred embodiment of the antigen-binding molecule of the present invention is an antibody comprising the variable region of the antibody of the present invention.

[0141] Methods for producing antibodies with the desired binding activity are known to those skilled in the art, and antibodies can be obtained as polyclonal or monoclonal antibodies. The antibodies of the present invention are preferably produced as monoclonal antibodies derived from mammals. Mammalian monoclonal antibodies include those produced by hybridomas and those produced by host cells transformed by genetic engineering techniques with an expression vector containing an antibody gene.

[0142] The mammal to be immunized to obtain antibodies is not limited to a specific animal, but is preferably selected in consideration of compatibility with the parent cells used in cell fusion to produce hybridomas. Generally, rodents such as mice, rats, hamsters, rabbits, and monkeys are preferably used.

[0143] The above-mentioned animals are immunized with the sensitizing antigen according to known methods. For example, immunization is generally carried out by intraperitoneal or subcutaneous injection of the sensitizing antigen into a mammal. Specifically, the sensitizing antigen is diluted at an appropriate dilution ratio with PBS (phosphate-buffered saline) or physiological saline, and optionally mixed with a conventional adjuvant, such as Freund's complete adjuvant, and emulsified. The sensitizing antigen is then administered to the mammal several times every 4 to 21 days. Furthermore, a suitable carrier can 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.

[0144] Hybridomas that produce the desired antibodies can also be prepared 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 to the immunized animal, and a sensitizing antigen is expressed in the body of the immunized animal, thereby providing immune stimulation. Compared to general immunization methods in which a protein antigen is administered to an immunized animal, DNA immunization is expected to have the following advantages: - Immunostimulation can be provided while maintaining the structure of membrane proteins - There is no need to purify the immunizing antigen

[0145] To obtain the monoclonal antibody of the present invention by DNA immunization, first, DNA expressing an antigen protein is administered to an animal to be immunized. DNA encoding the antigen protein can be synthesized by known methods such as PCR. The obtained DNA is inserted into an appropriate expression vector and administered to the animal to be immunized. Commercially available expression vectors such as pcDNA3.1 can be suitably used as the expression vector. Commonly used methods can be used to administer the vector to a living body. For example, DNA immunization is performed by introducing gold particles to which the expression vector is adsorbed into the cells of an individual animal to be immunized using a gene gun.

[0146] After a mammal is immunized in this manner and an increase in the antibody titer binding to the antigen in the serum is confirmed, immune cells are collected from the mammal and subjected to cell fusion. Splenocytes are particularly preferred as immune cells.

[0147] Mammalian myeloma cells are used as the cells to be fused with the immune cells. The myeloma cells preferably contain an appropriate selection marker 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) and thymidine kinase deficiency (hereinafter abbreviated as TK deficiency). Cells deficient in 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; however, when fused with normal cells, they can continue DNA synthesis by utilizing the salvage pathway of normal cells, allowing them to proliferate even in HAT selective medium.

[0148] HGPRT-deficient or TK-deficient cells can be selected on media containing 6-thioguanine, 8-azaguanine (hereafter abbreviated as 8AG), or 5'-bromodeoxyuridine, respectively. Normal cells that incorporate these pyrimidine analogs into their DNA die. On the other hand, cells lacking these enzymes and unable to incorporate these pyrimidine analogs can survive in selective media. Another selectable marker, called G418 resistance, confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) via the neomycin resistance gene. Various myeloma cell lines suitable for cell fusion are known.

[0149] 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), and 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.

[0150] Basically, cell fusion between the immune cells and myeloma cells is carried out according to known methods, such as the method of Kohler and Milstein et al. (Methods Enzymol. (1981) 73, 3-46).

[0151] More specifically, the cell fusion can be carried out in a conventional nutrient medium in the presence of a cell fusion promoter, such as polyethylene glycol (PEG) or Sendai virus (HVJ), with the addition of an adjuvant such as dimethyl sulfoxide, if desired, to further enhance the fusion efficiency.

[0152] The ratio of immune cells to myeloma cells can be set arbitrarily. For example, the ratio of immune cells to myeloma cells is preferably 1 to 10. The culture medium used for the cell fusion may be, for example, RPMI1640 culture medium, MEM culture medium, or other conventional culture medium suitable for growing the myeloma cell line, and may further contain a serum supplement such as fetal calf serum (FCS).

[0153] For cell fusion, a predetermined amount of the immune cells and myeloma cells are thoroughly mixed in the culture medium, and a PEG solution (e.g., an average molecular weight of approximately 1,000 to 6,000) preheated to approximately 37°C is added, usually at a concentration of 30 to 60% (w / v). The mixture is gently mixed to form the desired fused cells (hybridomas). Next, an appropriate culture medium such as those listed above is successively added, and the mixture is centrifuged and the supernatant is removed. This procedure can be repeated to remove cell fusion agents and other substances that are undesirable for hybridoma growth.

[0154] The hybridomas thus obtained can be selected by culturing them in a conventional selective culture medium, such as HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Culture can be continued in the HAT culture medium for a sufficient period of time (usually several days to several weeks) for cells other than the desired hybridoma (unfused cells) to die. Hybridomas producing the desired antibody are then screened and single-cloned by the conventional limiting dilution method.

[0155] The hybridomas thus obtained can be selected using a selective medium corresponding to the selection marker possessed by the myeloma used in cell fusion. For example, cells lacking HGPRT or TK can be selected by culturing them in HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that have successfully fused with normal cells can selectively grow in HAT medium. Culture in the above HAT medium is continued for a period of time sufficient for cells other than the desired hybridoma (non-fused cells) to die. Specifically, the desired hybridoma can generally be selected by culturing for several days to several weeks. Hybridomas producing the desired antibody can then be screened and single-cell cloned by conventional limiting dilution.

[0156] Screening and single cloning of the desired antibody can be preferably performed by a known screening method based on antigen-antibody reactions. The desired antibody can be screened, for example, by fluorescence activated cell sorting (FACS). FACS is a system that analyzes cells contacted with a fluorescent antibody using laser light and measures the fluorescence emitted by individual cells, thereby enabling measurement of antibody binding to the cell surface.

[0157] To screen hybridomas producing the monoclonal antibody of the present invention by FACS, cells expressing the antigen to which the produced antibody binds are first prepared. Preferred cells for screening are mammalian cells overexpressing the antigen. By using non-transformed mammalian cells as a control, the binding activity of the antibody to the cell surface antigen can be selectively detected. That is, hybridomas producing the desired monoclonal antibody can be obtained by selecting hybridomas producing antibodies that do not bind to host cells but bind to cells overexpressing the antigen.

[0158] Alternatively, cells expressing a target antigen can be immobilized, and the binding activity of an antibody to the antigen-expressing cells can be evaluated based on the principles of ELISA. For example, antigen-expressing cells are immobilized in the wells of an ELISA plate. Hybridoma culture supernatant is contacted with the immobilized cells in the wells, and antibodies binding to the immobilized cells are detected. If the monoclonal antibody is derived from a mouse, the antibody bound to the cells can be detected with an anti-mouse immunoglobulin antibody. Hybridomas producing the desired antibody capable of binding to the antigen selected by these screening methods can be cloned by limiting dilution or other methods. Hybridomas producing the monoclonal antibodies thus prepared can be passaged in a conventional culture medium. Furthermore, the hybridomas can be stored for long periods in liquid nitrogen.

[0159] The hybridomas are cultured according to conventional methods, and the desired monoclonal antibodies can be isolated from the culture supernatant. Alternatively, the hybridomas can be administered to a compatible mammal to grow, and the monoclonal antibodies can be isolated from the ascites. The former method is suitable for obtaining highly purified antibodies.

[0160] Antibodies encoded by antibody genes cloned from antibody-producing cells such as hybridomas can also be suitably used. The cloned antibody genes are incorporated into an appropriate vector and introduced into a host, whereby the antibodies encoded by the genes are 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.

[0161] To obtain cDNA encoding the variable region (V region) of an antibody, total RNA is usually extracted from hybridomas. The following methods can be used to extract mRNA from cells: - Guanidine ultracentrifugation (Biochemistry (1979) 18 (24), 5294-5299) - AGPC method (Anal. Biochem. (1987) 162 (1), 156-159)

[0162] The extracted mRNA can be purified using, for example, an mRNA Purification Kit (GE Healthcare Biosciences). Alternatively, kits for extracting total mRNA directly from cells, such as the QuickPrep mRNA Purification Kit (GE Healthcare Biosciences), are commercially available. Using such kits, mRNA can be obtained from hybridomas. cDNA encoding antibody V regions can be synthesized from the obtained mRNA using reverse transcriptase. cDNA can be synthesized using an AMV Reverse Transcriptase First-Strand cDNA Synthesis Kit (Seikagaku Corporation) or similar. Alternatively, 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) can be used appropriately for cDNA synthesis and amplification. Furthermore, during the process of synthesizing such cDNA, appropriate restriction enzyme sites, which will be described later, can be introduced at both ends of the cDNA.

[0163] The desired cDNA fragment is purified from the resulting PCR product and then ligated to vector DNA. The recombinant vector thus constructed is introduced into E. coli or other bacteria, and colonies are selected. The desired recombinant vector can then be prepared from the E. coli that formed the colonies. Whether the recombinant vector contains the nucleotide sequence of the desired cDNA is then confirmed by known methods, such as the dideoxynucleotide chain termination method.

[0164] A convenient way to obtain genes encoding variable regions is to use the 5'-RACE method, which uses primers specifically designed for amplifying variable region genes. First, cDNA is synthesized using RNA extracted from hybridoma cells as a template, and a 5'-RACE cDNA library is obtained. A commercially available kit, such as the SMART RACE cDNA Amplification Kit, can be used to synthesize the 5'-RACE cDNA library.

[0165] The resulting 5'-RACE cDNA library is used as a template to amplify antibody genes by PCR. Primers for amplifying mouse antibody genes can be designed based on known antibody gene sequences. These primers have different base sequences for each immunoglobulin subclass. Therefore, it is desirable to determine the subclass in advance using a commercially available kit such as the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche Diagnostics).

[0166] Specifically, for example, when the goal is to obtain a gene encoding mouse IgG, primers capable of amplifying genes encoding γ1, γ2a, γ2b, and γ3 heavy chains and κ and λ light chains can be used. To amplify IgG variable region genes, the 3' primer generally anneals to a region corresponding to the constant region close to the variable region. Meanwhile, the 5' primer used is a primer included in the 5' RACE cDNA library construction kit.

[0167] The PCR products thus amplified can be used to reconstitute immunoglobulins consisting of a combination of heavy and light chains. The antigen-binding activity of the reconstituted immunoglobulins can be used as an index to screen for desired antibodies. Screening can be performed, for example, as follows: (1) contacting a desired antigen-expressing cell with an antibody containing a V region encoded by a cDNA obtained from a hybridoma, (2) detecting binding of the antibody to the antigen-expressing cell, and (3) selecting an antibody that binds to the antigen-expressing cell.

[0168] Methods for detecting the binding between an antibody and the antigen-expressing cells are known. Specifically, the binding between an antibody and the antigen-expressing cells can be detected by techniques such as the above-mentioned FACS. Fixed specimens of the antigen-expressing cells can be used as appropriate to evaluate the binding activity of the antibody.

[0169] Panning methods using phage vectors are also suitable for screening antibodies based on binding activity. When antibody genes are obtained as a library of heavy and light chain subclasses from a polyclonal antibody-expressing cell population, screening methods using phage vectors are advantageous. Genes encoding the heavy and light chain variable regions can be linked with an appropriate linker sequence to form single-chain Fvs (scFvs). Phages expressing scFvs on their surface can be obtained by inserting a gene encoding an scFv into a phage vector. DNA encoding scFvs with the desired binding activity can be recovered by contacting the phage with the desired antigen and then recovering the phage bound to the antigen. Repeating this procedure as necessary allows the enrichment of scFvs with the desired binding activity.

[0170] After obtaining cDNA encoding the V region of the desired antibody, the cDNA is digested with restriction enzymes that recognize restriction enzyme sites inserted at both ends of the cDNA. Preferred restriction enzymes recognize and digest nucleotide sequences that appear rarely in the nucleotide sequence constituting the antibody gene. Furthermore, to insert one copy of the digested fragment into a vector in the correct orientation, insertion of a restriction enzyme that generates a cohesive end is preferred. An antibody expression vector can be obtained by inserting the cDNA encoding the V region of the antibody digested as described above into an appropriate expression vector. If a gene encoding the antibody constant region (C region) and a gene encoding the V region are fused in frame, a chimeric antibody can be obtained. Here, a chimeric antibody refers to an antibody in which the constant region and variable region are derived from different sources. Therefore, in addition to heterogeneous chimeric antibodies such as mouse-human, human-human allogeneic chimeric antibodies are also included in the chimeric antibodies of the present invention. A chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector that already contains 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 at the 5' end of an expression vector carrying DNA encoding the desired antibody constant region (C region). The two genes are digested with the same combination of restriction enzymes and fused in frame to construct a chimeric antibody expression vector.

[0171] To produce a monoclonal antibody, an antibody gene is incorporated into an expression vector so that its expression is controlled by an expression control region. Expression control regions for antibody expression include, for example, enhancers and promoters. An appropriate signal sequence can also be added to the amino terminus so that the expressed antibody is secreted extracellularly. The signal sequence is cleaved from the carboxyl terminal of the expressed polypeptide, allowing the antibody to be secreted extracellularly. Next, a suitable host cell can be transformed with this expression vector to obtain a recombinant cell that expresses the antibody-encoding DNA.

[0172] For antibody gene expression, DNA encoding the antibody heavy chain (H chain) and light chain (L chain) are incorporated into separate expression vectors. By co-transfecting the same host cells with vectors incorporating the H chain and L chain, antibody molecules comprising both H and L chains can be expressed. Alternatively, host cells can be transformed by incorporating DNA encoding the H chain and L chain into a single expression vector (see International Publication WO 94 / 11523).

[0173] Many combinations of host cells and expression vectors are known for producing antibodies by introducing isolated antibody genes into suitable hosts. Any of these expression systems can be applied to isolating antigen-binding domains contained in the antigen-binding molecules of the present invention.

[0174] When eukaryotic cells are used as host cells, animal cells, plant cells, or fungal cells can be used as appropriate. Specific examples of animal cells include the following: (1) mammalian cells: CHO, COS, myeloma, BHK (baby hamster kidney), Hela, Vero, etc. (2) amphibian cells: Xenopus oocytes, etc. (3) insect cells: sf9, sf21, Tn5, etc.

[0175] Alternatively, an antibody gene expression system using plant cells derived from the genus Nicotiana, such as Nicotiana tabacum, is known. Callus cultured cells can be appropriately used for transformation of plant cells.

[0176] Furthermore, the following fungal cells can be used: - Yeast: Saccharomyces genus such as Saccharomyces cerevisiae, Pichia genus such as Pichia pastoris - Filamentous fungi: Aspergillus genus such as Aspergillus niger

[0177] Expression systems for antibody genes using prokaryotic cells are also known. For example, when bacterial cells are used, bacterial cells such as Escherichia coli (E. coli) and Bacillus subtilis can be used as appropriate. An expression vector containing the antibody gene of interest is introduced into these cells by transformation. The transformed cells are cultured in vitro, and the desired antibody can be obtained from the culture of the transformed cells.

[0178] In addition to the host cells described above, transgenic animals can also be used to produce recombinant antibodies. That is, the antibody can be obtained from an animal into which a gene encoding the desired antibody has been introduced. For example, an antibody gene can be constructed as a fusion gene by inserting it in-frame into a gene encoding a protein specifically produced in milk. Examples of proteins secreted into milk include goat beta-casein. A DNA fragment containing the fusion gene with the antibody gene inserted is injected into a goat embryo, and the injected embryo is then introduced into a female goat. The transgenic goat (or its offspring) born to the embryo recipient produces milk from which the desired antibody can be isolated as a fusion protein with a milk protein. Furthermore, hormones can be administered to transgenic goats to increase the amount of milk containing the desired antibody produced by the transgenic goat (Bio / Technology (1994), 12 (7), 699-702).

[0179] When the antigen-binding molecules described herein are administered to humans, for example, when domains containing antibody variable regions are used as various binding domains in the molecules, antigen-binding domains derived from recombinant antibodies that have been artificially modified for purposes such as reducing heterologous antigenicity to humans can be appropriately used. Recombinant antibodies include, for example, humanized antibodies. These modified antibodies are appropriately produced using known methods.

[0180] The antibody variable regions used to generate the various binding domains of the antigen-binding molecules described herein typically consist of three complementarity-determining regions (CDRs) sandwiched between four framework regions (FRs). CDRs essentially determine the binding specificity of an antibody. The amino acid sequences of CDRs are highly diverse. However, the amino acid sequences constituting FRs often show high identity even among antibodies with different binding specificities. Therefore, it is generally believed that CDR grafting can transfer the binding specificity of one antibody to another.

[0181] Humanized antibodies are also called reshaped human antibodies. Specifically, humanized antibodies are known in which CDRs from non-human animals, such as mouse antibodies, are grafted onto human antibodies. Common genetic recombination techniques for obtaining humanized antibodies are also known. Specifically, overlap extension PCR is a well-known method for grafting mouse antibody CDRs onto human FRs. In overlap extension PCR, the nucleotide sequence encoding the mouse antibody CDR to be grafted is added to a primer used to synthesize the human antibody FR. Primers are prepared for each of the four FRs. In general, when grafting mouse CDRs onto human FRs, selecting human FRs with high identity to the mouse FRs is considered advantageous in terms of maintaining CDR function. In other words, it is generally preferable to use human FRs whose amino acid sequences are highly identical to the amino acid sequences of the FRs adjacent to the mouse CDR to be grafted.

[0182] The nucleotide sequences to be linked are designed to be connected in frame with each other. Human FRs are synthesized individually using each primer. As a result, products are obtained in which DNA encoding mouse CDRs is added to each FR. The nucleotide sequences encoding the mouse CDRs of each product are designed to overlap with each other. Next, the overlapping CDR portions of the products synthesized using the human antibody gene as a template are annealed to each other to perform complementary strand synthesis. This reaction links the human FRs via the mouse CDR sequences.

[0183] The V region gene, which finally contains three CDRs and four FRs, is amplified in its entirety using primers that anneal to its 5' and 3' ends and have appropriate restriction enzyme recognition sequences added. A humanized antibody expression vector can be constructed by inserting the DNA obtained as described above and DNA encoding a human antibody C region into an expression vector so that they are fused in frame. After introducing the integration vector into a host to establish recombinant cells, the recombinant cells are cultured to express the DNA encoding the humanized antibody, resulting in the production of the humanized antibody in the cultured cell culture (see European Patent Publication EP 239400 and International Publication WO 1996 / 002576).

[0184] By qualitatively or quantitatively measuring and evaluating the antigen-binding activity of the humanized antibody prepared as described above, it is possible to suitably select FRs of a human antibody that form a good antigen-binding site when linked via the CDRs. If necessary, amino acid residues in the FRs can be substituted so that the CDRs of a reshaped human antibody form a suitable antigen-binding site. For example, amino acid sequence mutations can be introduced into the FRs by applying the PCR method used to graft mouse CDRs onto human FRs. Specifically, partial nucleotide sequence mutations can be introduced into primers annealing to the FRs. Nucleotide sequence mutations are introduced into the FRs synthesized using such primers. Mutant FR sequences with desired properties can be selected by measuring and evaluating the antigen-binding activity of mutant antibodies with amino acid substitutions using the above method (Sato, K. et al., Cancer Res, 1993, 53, 851-856).

[0185] Alternatively, transgenic animals carrying the entire repertoire of human antibody genes (see International Publications WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, and WO1996 / 033735) can be used as immunized animals to obtain desired human antibodies by DNA immunization.

[0186] Furthermore, techniques for obtaining human antibodies by panning using a human antibody library are also known. For example, the V region of a human antibody is expressed on the surface of a phage as a single-chain antibody (scFv) by phage display. Phages expressing scFvs that bind to an antigen can be selected. By analyzing the genes of the selected phage, the DNA sequence encoding the V region of a human antibody that binds to the antigen can be determined. After determining the DNA sequence of the scFv that binds to the antigen, the V region sequence can be fused in frame with the sequence of the C region of a desired human antibody and then inserted into an appropriate expression vector to prepare an expression vector. The expression vector is introduced into a suitable expression cell such as those listed above, and the gene encoding the human antibody is expressed to obtain the human antibody. These methods are already known (see International Publications WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, and WO1995 / 015388).

[0187] In addition to phage display, other known techniques for obtaining human antibodies by panning using a human antibody library include techniques using cell-free translation systems, techniques for displaying antigen-binding molecules 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 between mRNA and the translated protein via ribosomes by removing a stop codon, cDNA display, which covalently binds a gene sequence to a translated protein using a compound such as puromycin, and mRNA display, and CIS display, which forms a complex between a gene and the translated protein using a nucleic acid-binding protein. Furthermore, in addition to phage display, techniques for displaying antigen-binding molecules on the surface of cells or viruses include E. coli display, Gram-positive bacteria display, yeast display, mammalian cell display, and viral display. Techniques using emulsions include in vitro viral display, which involves encapsulating genes and translation-related molecules in an emulsion. These methods are already 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).

[0188] Method for Producing Multispecific Antibodies One preferred embodiment of the antigen-binding molecules of the present invention is a multispecific antibody. When an Fc region with reduced Fcγ receptor-binding activity is used as the Fc region of a multispecific antibody of the present invention, an Fc region derived from a known multispecific antibody can also be used as appropriate. Bispecific antibodies are particularly preferred as multispecific antibodies of the present invention.

[0189] For the aggregation of multispecific antibodies, a technique can be applied that suppresses undesired association of H chains by introducing electric charge repulsion at the interface of the second constant region (CH2) or third constant region (CH3) of the antibody H chain (WO2006 / 106905).

[0190] In techniques that suppress unintended association between H chains by introducing electric charge repulsion at the CH2 or CH3 interface, examples of amino acid residues that contact the interface of other H chain constant regions include the regions corresponding to residues 356 (EU numbering), 439 (EU numbering), 357 (EU numbering), 370 (EU numbering), 399 (EU numbering), and 409 (EU numbering) in the CH3 region.

[0191] More specifically, for example, an antibody comprising two types of H chain CH3 regions can be an antibody in which one to three pairs of amino acid residues selected from the pairs of amino acid residues shown in (1) to (3) below in the first H chain CH3 region have the same charge: (1) amino acid residues contained in the H chain CH3 region at positions 356 and 439 (EU numbering), (2) amino acid residues contained in the H chain CH3 region at positions 357 and 370 (EU numbering), and (3) amino acid residues contained in the H chain CH3 region at positions 399 and 409 (EU numbering).

[0192] Furthermore, the antibody can be one in which a second H chain CH3 region different from the first H chain CH3 region has a set of amino acid residues selected from the sets of amino acid residues shown in (1) to (3) above, and one to three sets of amino acid residues corresponding to the sets of amino acid residues shown in (1) to (3) that have the same type of charge in the first H chain CH3 region have an opposite charge to the corresponding amino acid residues in the first H chain CH3 region.

[0193] The amino acid residues described in (1) to (3) above are close to each other when associated. Those skilled in the art can find the positions corresponding to the amino acid residues described in (1) to (3) above for the desired H chain CH3 region or H chain constant region by homology modeling using commercially available software, and can appropriately modify the amino acid residues at those positions.

[0194] In the above-mentioned antibody, the "charged amino acid residue" is preferably selected from amino acid residues included in either the following group (a) or (b): (a) glutamic acid (E), aspartic acid (D), (b) lysine (K), arginine (R), histidine (H).

[0195] In the above-mentioned antibodies, "having the same charge" means, for example, that two or more amino acid residues all have an amino acid residue included in one of the above groups (a) or (b). "Having opposite charges" means, for example, that when at least one amino acid residue among two or more amino acid residues has an amino acid residue included in one of the above groups (a) or (b), the remaining amino acid residues have an amino acid residue included in a different group.

[0196] In a preferred embodiment, the antibody may have the first H chain CH3 region and the second H chain CH3 region cross-linked by a disulfide bond.

[0197] The amino acid residues to be modified in the present invention are not limited to those in the antibody variable region or constant region described above. Those skilled in the art can identify amino acid residues that form an interface between polypeptide mutants or heteromultimers by homology modeling using commercially available software, and can modify the amino acid residues at those sites to control association.

[0198] Other known techniques can also be used to aggregate multispecific antibodies of the present invention. For example, an amino acid side chain in one of the antibody H chain variable regions can be replaced with a larger side chain (knob) and an amino acid side chain in the opposing H chain variable region can be replaced with a smaller side chain (hole) so that the knob can be positioned in the hole, thereby efficiently facilitating aggregation between polypeptides having different amino acids that have Fc domains (WO 1996 / 027011, Ridgway JB et al., Protein Engineering (1996) 9, 617-621, Merchant AM et al., Nature Biotechnology (1998) 16, 677-681, US20130336973).

[0199] In addition, other known techniques can also be used to form multispecific antibodies of the present invention. By using a strand-exchange engineered domain CH3, in which a portion of the CH3 of one antibody H chain is modified with a corresponding IgA-derived sequence and the complementary portion of the CH3 of the other H chain is modified with a corresponding IgA-derived sequence, association of polypeptides with different sequences can be efficiently induced by complementary association of CH3s (Protein Engineering Design & Selection, 23; 195-202, 2010). This known technique can also be used to efficiently form desired multispecific antibodies.

[0200] Other methods for forming multispecific antibodies include antibody production techniques utilizing the association of antibody CH1 and CL or VH and VL, as described in WO2011 / 028952, WO2014 / 018572, and Nat Biotechnol. 2014 Feb;32(2):191-8; techniques for producing bispecific antibodies using separately prepared monoclonal antibodies (Fab Arm Exchange), as described in WO2008 / 119353 and WO2011 / 131746; techniques for controlling the association between CH3s of antibody heavy chains, as described in WO2012 / 058768 and WO2013 / 063702; techniques for producing bispecific antibodies composed of two types of light chains and one type of heavy chain, as described in WO2012 / 023053; and techniques for producing bispecific antibodies composed of two types of light chains and one type of heavy chain, as described in Christoph et al. (Nature Biotechnology Vol. 31, pp. 753-758). Alternatively, a technique for producing a bispecific antibody using two bacterial cell lines each expressing one half of an antibody chain consisting of one H chain and one L chain, as described in (2013), can be used.

[0201] One embodiment of multispecific antibody formation, as described above, involves mixing two monoclonal antibodies in the presence of a reducing agent, cleaving the core-hinge disulfide bonds, and then reassociating them to obtain heterodimerized bispecific antibodies (FAE). However, by introducing electrostatic interactions (WO2006 / 106905) into the interaction interface of the CH3 region, heterodimerization can be induced more efficiently during reassociation (WO2015 / 046467). In FAE using native IgG, reassociation occurs randomly, so theoretically bispecific antibodies can only be obtained with a 50% efficiency, but this method allows for the production of bispecific antibodies with high yields.

[0202] Even if the desired multispecific antibody cannot be efficiently formed, it can still be obtained by isolating and purifying the desired multispecific antibody from the produced antibodies. For example, a method has been reported in which amino acid substitutions are introduced into the variable regions of two types of H chains to impart a difference in isoelectric point, thereby enabling the purification of two types of homoantibodies and the desired heteroantibody by ion exchange chromatography (WO2007114325). Another method for purifying heteroantibodies has been reported, in which a heterodimerized antibody consisting of a mouse IgG2a H chain that binds to Protein A and a rat IgG2b H chain that does not bind to Protein A is purified using Protein A (WO98050431, WO95033844). Furthermore, by using an H chain in which the amino acid residues at positions 435 and 436 (EU numbering), which are the binding sites between IgG and Protein A, are substituted with amino acids that have different binding strengths to Protein A, such as Tyr or His, the interaction between each H chain and Protein A can be changed, and by using a Protein A column, it is possible to efficiently purify only the heterodimerized antibody.

[0203] Alternatively, a common L chain capable of conferring binding ability to multiple different H chains may be obtained and used as the common L chain for a multispecific antibody. By expressing IgG by introducing genes for multiple H chains different from such a common L chain into cells, efficient expression of multispecific IgG becomes possible (Nature Biotechnology (1998) 16, 677-681). When selecting a common H chain, a method can also be used to select a common L chain that exhibits high binding ability in response to any different H chains (WO2004 / 065611).

[0204] Furthermore, Fc regions with reduced C-terminal heterogeneity can be used as appropriate as Fc regions of the present invention. More specifically, Fc regions are provided in which glycine at position 446 and lysine at position 447, as specified by EU numbering, in the amino acid sequences of two polypeptides that constitute an Fc region derived from IgG1, IgG2, IgG3, or IgG4 are deleted.

[0205] Multiple of these techniques, for example, two or more in combination, can also be used. These techniques can also be applied separately, as appropriate, to the two H chains to be associated. Furthermore, these techniques can also be used in combination with the above-mentioned Fc region with reduced binding activity to Fcγ receptors. The antigen-binding molecules of the present invention may also be antigen-binding molecules having the same amino acid sequence as those modified as above, which have been separately prepared.

[0206] The structure of the multispecific antigen-binding molecule of the present invention is not limited, as long as it comprises (1) a first antigen-binding domain having CD3-binding activity, comprising the antibody H-chain variable region and L-chain variable region; (2) a second antigen-binding domain comprising an amino acid sequence different from that of the first antigen-binding domain; and, optionally, (3) a domain comprising an Fc region with reduced Fcγ receptor-binding activity. In the present invention, the above domains can be directly linked via peptide bonds. For example, when F(ab')2 is used as the antigen-binding domains of (1) and (2), and these Fc regions are used as the domain comprising an Fc region with reduced Fcγ receptor-binding activity, linking the antigen-binding domains described in (1) and (2) to the domain comprising the Fc region of (3) via a peptide bond results in the linked polypeptide forming an antibody structure. To produce such an antibody, it can be purified from the culture medium of the hybridoma described above, or from the culture medium of a desired host cell in which a polynucleotide encoding the polypeptide constituting the antibody is stably maintained.

[0207] Preferred antibody variable regions of the present invention that have CD3-binding activity include antibody variable regions that have CD3ε-binding activity. Examples of such antibody variable regions include the heavy chain variable regions of antibody Nos. 2, 14, 25, 29, 30 to 32, and 34 listed in Table 5 below, or antibody H chain variable regions having the amino acid sequences of heavy chain CDR1, CDR2, and CDR3, and the light chain variable regions of antibody Nos. 2, 14, 25, 29, 30 to 32, and 34 listed in Table 5, or antibody L chain variable regions having the amino acid sequences of light chain CDR1, CDR2, and CDR3, as well as antibody H chain variable regions and L chain variable regions functionally equivalent to these variable regions.

[0208] Here, in the present invention, "functionally equivalent" means that the binding affinity to an antigen under specified conditions is equivalent, or that, when used as a multispecific antigen-binding molecule, the cytotoxic activity against cells expressing the target antigen or tissues containing said cells is equivalent. Binding affinity and cytotoxic activity can be measured based on the description herein. The cells for which cytotoxic activity is measured may be desired cells expressing the target antigen or desired tissues containing said cells, but for example, human cancer cell lines expressing the target antigen can be used. Furthermore, with respect to antibody constant regions, the reduction in binding activity to Fcγ receptors may also be equivalent.

[0209] For example, an antibody H-chain variable region functionally equivalent to the antibody H-chain variable region described herein (the original H-chain variable region) means that when combined with the antibody L-chain variable region described herein as the pair of the original H chain, it has equivalent binding affinity, or when used as a multispecific antigen-binding molecule, it has equivalent cytotoxic activity against cells expressing the target antigen or tissues containing said cells. Furthermore, an antibody L-chain variable region functionally equivalent to the antibody L-chain variable region described herein (the original L-chain variable region) means that when combined with the antibody H-chain variable region described herein as the pair of the original L chain, it has equivalent binding affinity, or when used as a multispecific antigen-binding molecule, it has equivalent cytotoxic activity against cells expressing the target antigen or tissues containing said cells.

[0210] Furthermore, "equivalent" does not necessarily mean that the activity is of the same level; the activity may be enhanced, and therefore can also be expressed as "equivalent or greater." Specifically, in the case of binding affinity to an antigen, "equivalent" can be when the value (KD value / parent KD value) compared to the binding affinity (parent KD value) of a control antibody variable region is 2.0 or less. The KD value / parent KD value is preferably 1.5 or less, more preferably 1.3 or less, 1.2 or less, 1.1 or less, 1.0 or less, 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, or 0.5 or less. There is no lower limit, but it can be, for example, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , or 10 -6 Specifically, in the present invention, the KD value / parent KD value may be 10 -6 Preferably up to 2.0, more preferably 10 -3 ~1.5, more preferably 10 -1The value of the cytotoxicity ratio is preferably 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, or 1 nM, and more preferably 0.05 nM or 0.1 nM. The value of the cytotoxicity ratio / parental cytotoxicity ratio is preferably 0.8 or higher, more preferably 0.9 or higher, 1.0 or higher, 1.2 or higher, 1.5 or higher, 2 or higher, 3 or higher, 5 or higher, 10 or higher, or 20 or higher. There is no upper limit, but the value can be, for example, 10 or 10 2 , 10 3 , 10 4 , 10 5 , or 10 6 It may be.

[0211] In the case of cytotoxic activity, examples of such activity include a value (concentration for 50% cell growth inhibition / concentration for 50% parent cell growth inhibition) of 1.5 or less, as compared with the concentration of the original multispecific antigen-binding molecule that inhibits 50% cell growth (concentration for 50% parent cell growth inhibition). The concentration of multispecific antigen-binding molecule required to halve the cell growth rate compared to when the multispecific antigen-binding molecule is not added. The value of "concentration for 50% cell growth inhibition / concentration for 50% parent cell growth inhibition" is preferably 1.3 or less, and more preferably 1.2 or less, 1.1 or less, 1.0 or less, 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, or 0.5 or less. There is no lower limit, but it can be, for example, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , or 10 -6 Specifically, 10 -6 ~1.5x10 -0 is preferred, and more preferably 10 -6 ~10 -1 , more preferably 10 -6 ~10 -2, more preferably 10 -6 ~10 -3 is.

[0212] Cytotoxic activity can be assessed by in vitro TDCC activity as described in Example 4. In this case, "equivalent" refers to a value of 0.7 or higher compared to the TDCC activity of a control multispecific antigen-binding molecule (TDCC activity / parent TDCC activity). The value of TDCC activity / parent TDCC activity is preferably 0.8 or higher, more preferably 0.9 or higher, 1.0 or higher, 1.2 or higher, 1.5 or higher, 2 or higher, 3 or higher, 5 or higher, 10 or higher, or 20 or higher.

[0213] Furthermore, the second antigen-binding domain having binding activity to the target antigen has a KD value of, for example, 5x10 -9 M or less, preferably 4x10 -9 M or less, e.g. 3x10 -9 M or less, 2x10 -9 M or less, 1x10 -9 M or less, 8x10 -10 M or less, 5x10 -10 M or less, 4x10 -10 M or less, 3x10 -10 M or less, 2x10 -10 M or less, 1x10 -10 M or less, 8x10 -11 M or less, 5x10 -11 M or less, 4x10 -11 M or less, 3x10 -11 M or less, 2x10 -11 M or less, 1x10 -11 M or less, 8x10 -12 M or less, 5x10 -12 M or less, 4x10 -12 M or less, 3x10 -12 M or less, 2x10 -12 M or less, 1x10 -12 M or less, 8x10 -13 M or less, 5x10 -13 M or less, 4x10 -13 M or less, 3x10 -13 M or less, 2x10 -13 M or smaller, or 1x10 -13It may be M or less.

[0214] Furthermore, for an antigen-binding domain containing an antibody variable region having binding activity to CD3, the KD value for CD3, for example, human CD3, more specifically, for example, human CD3ε chain, is, for example, 5x10 -7 M or less, preferably 2x10 -7 M or less, e.g. 1.5x10 -7 M or less, 1.4x10 -7 M or less, 1.3x10 -7 M or less, 1.2x10 -7 M or less, 1x10 -7 M or less, 3x10 -8 M or less, 2x10 -8 M or less, 1x10 -8 M or less, 8x10 -9 M or less, 5x10 -9 M or less, 4x10 -9 M or less, 3x10 -9 M or less, 2x10 -9 M or less, 1x10 -9 M or less, 8x10 -10 M or less, 5x10 -10 M or less, 4x10 -10 M or less, 3x10 -10 M or less, 2x10 -10 M or less, 1x10 -10 M or less, 8x10 -11 M or less, 5x10 -11 M or less, 4x10 -11 M or less, 3x10 -11 M or less, 2x10 -11 M or less, 1x10 -11 M or less, 8x10 -12 M or less, 5x10 -12 M or less, 4x10 -12 M or less, 3x10 -12 M or less, 2x10 -12 M or smaller, or 1x10 -12 It is below M.

[0215] The multispecific antigen-binding molecules of the present invention preferably have KD values ​​of 5x10 for the target antigen and human CD3 (e.g., human CD3ε chain), respectively. -9 M and below and 5x10 -7M or less, and more preferably 1 x 10 -9 M and below and 5x10 -8 It is below M.

[0216] In the present invention, "functionally equivalent" antibody variable regions are not particularly limited as long as they are antibody heavy chain variable regions and / or antibody light chain variable regions that satisfy the above-mentioned conditions. Such antibody variable regions may, for example, have one or more amino acids (e.g., 1, 2, 3, 4, 5, or 10 amino acids) substituted, deleted, added, and / or inserted in the amino acid sequence of the variable regions of antibodies Nos. 2, 14, 25, 29, 30 to 32, and 34 listed in Table 5. Methods for substituting, deleting, adding, and / or inserting one or more amino acids in an amino acid sequence that are well known to those skilled in the art include methods for introducing mutations into proteins.For example, those skilled in the art will be familiar with site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene 152, 271-275; Zoller, MJ, and Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 100, 468-500; Kramer, W, Drutsa, V, Jansen, HW, Kramer, B, Pflugfelder, M, and Fritz, HJ (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456; Kramer, W, and Fritz, HJ (1987) By appropriately introducing mutations into the amino acid sequence using techniques such as those described in "Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367" and "Kunkel, TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 82, 488-492," variable regions functionally equivalent to the antibody variable regions having the above-mentioned functions can be prepared.

[0217] When modifying an amino acid residue, it is desirable to mutate it into another amino acid that preserves the properties of the amino acid side chain. For example, the properties of the amino acid side chain can include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids with aliphatic side chains (G, A, V, L, I, P), amino acids with hydroxyl group-containing side chains (S, T, Y), amino acids with sulfur atom-containing side chains (C, M), amino acids with carboxylic acid- and amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, H), and amino acids with aromatic side chains (H, F, Y, W) (the characters in parentheses represent the single-letter symbols of the amino acids). Substitution of amino acids within each of these groups is referred to as a conservative substitution. It is already known that polypeptides having an amino acid sequence modified by deletion, addition, and / or substitution of one or more amino acid residues with other amino acids can retain their biological activity (Mark, D.F. et al., Proc. Natl. Acad. Sci. USA (1984) 81:5662-6; Zoller, M.J. and Smith, M., Nucleic Acids Res. (1982) 10:6487-500; Wang, A. et al., Science (1984) 224:1431-3; Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79:6409-13). The variable regions of the present invention containing such amino acid modifications have an amino acid sequence identity of at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% with the amino acid sequence of the CDR sequence, FR sequence, or entire variable region before modification. As used herein, sequence identity is defined as the percentage of residues identical to those in the original H-chain or L-chain variable region amino acid sequence, after aligning the sequences as necessary to maximize sequence identity and introducing gaps as appropriate. Amino acid sequence identity can be determined by the method described below.

[0218] Furthermore, "functionally equivalent antibody variable regions" can also be obtained from nucleic acids that hybridize under stringent conditions to nucleic acids consisting of nucleotide sequences encoding the amino acid sequences of the variable regions of antibodies Nos. 2, 14, 25, 29, 30 to 32, and 34 listed in Table 5, for example. Examples of stringent hybridization conditions for isolating nucleic acids that hybridize under stringent conditions to nucleic acids consisting of nucleotide sequences encoding the amino acid sequences of the variable regions include 6 M urea, 0.4% SDS, 0.5 x SSC, and 37°C, or hybridization conditions of equivalent stringency. Higher stringency conditions, such as 6 M urea, 0.4% SDS, 0.1 x SSC, and 42°C, are expected to result in the isolation of nucleic acids with higher homology. Washing conditions after hybridization include, for example, 0.5xSSC (1xSSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) and 0.1% SDS at 60°C, more preferably 0.2xSSC and 0.1% SDS at 60°C, more preferably 0.2xSSC and 0.1% SDS at 62°C, more preferably 0.2xSSC and 0.1% SDS at 65°C, and more preferably 0.1xSSC and 0.1% SDS at 65°C. The sequence of the isolated nucleic acid can be determined by known methods described below. The homology of the isolated nucleic acid is at least 50% or more, more preferably 70% or more, and even more preferably 90% or more (e.g., 95%, 96%, 97%, 98%, 99% or more) sequence identity across the entire base sequence. Instead of the above-mentioned method utilizing hybridization techniques, it is also possible to isolate nucleic acids that hybridize under stringent conditions with nucleic acids consisting of a nucleotide sequence encoding the amino acid sequence of the variable region by using gene amplification methods, such as polymerase chain reaction (PCR), which use primers synthesized based on the nucleotide sequence information encoding the amino acid sequence of the variable region.

[0219] The identity of nucleotide sequences and amino acid sequences can be determined by the BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA (1993) 90:5873-7). Based on this algorithm, programs called BLASTN and BLASTX have been developed (Altschul et al., J. Mol. Biol. (1990) 215:403-10). When analyzing nucleotide sequences using BLASTN based on BLAST, parameters are set, for example, as score = 100 and word length = 12. When analyzing amino acid sequences using BLASTX based on BLAST, parameters are set, for example, as score = 50 and word length = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific techniques for these analysis methods are known (see the BLAST (Basic Local Alignment Search Tool) website of the NCBI (National Center for Biotechnology Information); http: / / www.ncbi.nlm.nih.gov).

[0220] Nucleic Acids, Vectors, and Cells In one aspect, the present invention relates to nucleic acids (polynucleotides) encoding the antigen-binding molecules of the present invention. The antigen-binding molecules of the present invention can be incorporated into any expression vector. An appropriate host can be transformed with the expression vector to produce cells expressing the antigen-binding molecules. The antigen-binding molecules encoded by the nucleic acids (polynucleotides) can be obtained by culturing the cells expressing the antigen-binding molecules and recovering the expression product from the culture supernatant. Specifically, the present invention relates to vectors containing nucleic acids (polynucleotides) encoding the antigen-binding molecules of the present invention, cells harboring the vectors, and methods for producing antigen-binding molecules, which include culturing the cells and recovering the antigen-binding molecules from the culture supernatant. These can be obtained, for example, by techniques similar to those used for the recombinant antibodies described above.

[0221] Pharmaceutical Compositions In one aspect, the present invention provides pharmaceutical compositions comprising, as an active ingredient, an antigen-binding molecule of the present invention (particularly a multispecific antigen-binding molecule). In one embodiment, the present invention provides pharmaceutical compositions that induce cytotoxicity, comprising, as an active ingredient, the antigen-binding molecule. The pharmaceutical compositions of the present invention induce cytotoxicity, particularly T-cell-dependent cytotoxicity, and are preferably administered to subjects suffering from or at risk of recurrence of a disease for which such cytotoxicity is necessary for prevention or treatment. In one embodiment, the pharmaceutical compositions of the present invention are for use in the treatment or prevention of cancer.

[0222] Pharmaceutical compositions containing the antigen-binding molecules of the present invention (particularly multispecific antigen-binding molecules) as an active ingredient can also be described as cytotoxicity inducers and cytostatic agents containing the antigen-binding molecules as an active ingredient, methods for inducing cytotoxicity and methods for inhibiting cell proliferation comprising the step of administering the antigen-binding molecules to a subject, the antigen-binding molecules for use in inducing cytotoxicity and inhibiting cell proliferation, or use of the antigen-binding molecules in the manufacture of cytotoxicity inducers and cytostatic agents. Furthermore, pharmaceutical compositions containing the antigen-binding molecules of the present invention (particularly multispecific antigen-binding molecules) as an active ingredient can also be described as cancer therapeutic or preventive agents containing the antigen-binding molecules as an active ingredient, methods for treating or preventing cancer comprising the step of administering the antigen-binding molecules to a subject, the antigen-binding molecules for use in cancer therapeutic or preventive agents, or use of the antigen-binding molecules in the manufacture of cancer therapeutic or preventive agents.

[0223] In the present invention, the phrase "containing an antigen-binding molecule as an active ingredient" means containing the antigen-binding molecule as the main active ingredient, and does not limit the content of the antigen-binding molecule.

[0224] In one embodiment, the antigen-binding molecule of the present invention can be administered by administering or incorporating a nucleic acid encoding the antigen-binding molecule of the present invention into a living body using a vector or the like, and directly expressing the antigen-binding molecule of the present invention in the living body; however, the antigen-binding molecule may also be administered without using a vector. Examples of vectors include viral vectors and plasmid vectors, and further examples include adenovirus vectors and adeno-associated virus vectors. The nucleic acid encoding the antigen-binding molecule of the present invention may be administered directly to a living body, or cells into which a nucleic acid encoding the antigen-binding molecule of the present invention has been introduced may be administered to a living body. For example, the antigen-binding molecule of the present invention can be administered by chemically modifying mRNA encoding the antigen-binding molecule of the present invention to increase mRNA stability in the living body, and then directly administering the mRNA to a human to express the antigen-binding molecule of the present invention in the living body (see EP2101823B, WO2013 / 120629). Alternatively, B cells into which a nucleic acid encoding the antigen-binding molecule of the present invention has been introduced may be administered (Sci Immunol. (2019) 4(35), eaax0644). Alternatively, bacteria into which nucleic acids encoding the antigen-binding molecules of the present invention have been introduced may be administered (Nature Reviews Cancer (2018) 18, 727-743).

[0225] Furthermore, if necessary, the antigen-binding molecules of the present invention can be encapsulated in microcapsules (microcapsules made of hydroxymethylcellulose, gelatin, poly(methyl methacrylate), etc.) to form colloid drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules, etc.) ("Remington's Pharmaceutical Science 16 th edition", Oslo Ed. (1980), etc. Furthermore, methods for making drugs sustained-release drugs are also known, and these methods can be applied to the antigen-binding molecules of the present invention (J. Biomed. Mater. Res. (1981) 15, 267-277; Chemtech. (1982) 12, 98-105; U.S. Pat. No. 3,773,719; European Patent Publications EP 58481 and EP 133988; Biopolymers (1983) 22, 547-556).

[0226] The pharmaceutical composition, cytotoxicity inducer, cytostatic agent, or cancer therapeutic or preventive agent (hereinafter collectively referred to as the pharmaceutical composition of the present invention) of the present invention can be administered to a patient either orally or parenterally. Parenteral administration is preferred. Specific administration methods include injection, nasal administration, pulmonary administration, and transdermal administration. Injection methods include intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection. For example, the pharmaceutical composition of the present invention can be administered systemically or locally by injection. Furthermore, an appropriate administration method can be selected depending on the patient's age and symptoms. The dosage can be selected, for example, from 0.0001 mg to 1,000 mg per kg of body weight per administration. Alternatively, the dosage can be selected, for example, from 0.001 mg / body weight to 100,000 mg / body weight per patient. However, the pharmaceutical composition of the present invention is not limited to these dosages.

[0227] The pharmaceutical compositions of the present invention can be formulated according to conventional methods (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA) and may contain pharmaceutically acceptable carriers and additives. Examples include surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, and flavoring agents. Furthermore, other commonly used carriers can be used as appropriate, without being limited to these. Specific examples of carriers include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglycerides, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, and inorganic salts.

[0228] A non-limiting example of a technique that can be combined with the antigen-binding molecules of the present invention is the creation of T cells that secrete T cell-redirecting antibodies using a CD3-binding domain (Trends Immunol. (2019) 40(3) 243-257). One non-limiting example of a creation method involves introducing nucleic acids encoding a bispecific antibody comprising the CD3-binding domain described herein and a cancer antigen-binding domain into effector cells such as T cells using gene modification techniques.

[0229] Therapeutic Methods, etc. In one aspect, the present invention provides a method for inducing (inducing) damage to a target cell or inhibiting its proliferation, comprising the step of contacting the target cell with a multispecific antigen-binding molecule of the present invention that binds to an antigen specifically expressed on the cell surface. In one embodiment, the present invention provides a method for inducing (inducing) damage to a cell expressing a target cancer-specific antigen or a tumor tissue containing the antigen-expressing cell, or a method for inhibiting the proliferation of the cell or tumor tissue, comprising the step of contacting the cell expressing the target cancer-specific antigen with a multispecific antigen-binding molecule of the present invention that binds to the antigen. In another embodiment, the present invention provides a method for inducing (inducing) damage to a cell or inhibiting the proliferation of the cell, comprising the step of contacting a cell having the function of suppressing an immune response with a multispecific antigen-binding molecule of the present invention that binds to an antigen expressed on the cell surface. The multispecific antigen-binding molecule that binds to the antigen is as described above for the antigen-binding molecule of the present invention contained in the cytotoxicity-inducing agent and cell proliferation inhibitor of the present invention. The cells to which the multispecific antigen-binding molecule of the present invention that binds to the antigen are not particularly limited, as long as the antigen is expressed.

[0230] In the present invention, "contact" is carried out, for example, by adding a multispecific antigen-binding molecule of the present invention that binds to the antigen to a culture medium of target antigen-expressing cells cultured in a test tube. In this case, the antigen-binding molecule to be added may be in the form of a solution or a solid obtained by lyophilization or the like, as appropriate. When added as an aqueous solution, it may be an aqueous solution containing only the multispecific antigen-binding molecule of the present invention, or it may be a solution containing, for example, the above-mentioned surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, flavoring agents, etc. The concentration to be added is not particularly limited, but a final concentration in the culture medium of preferably 1 pg / ml to 1 g / ml, more preferably 1 ng / ml to 1 mg / ml, and even more preferably 1 μg / ml to 1 mg / ml, is suitable.

[0231] In another embodiment, "contact" in the present invention can also be achieved by administering to a non-human animal into which cells expressing the target antigen have been transplanted or to an animal that has cells endogenously expressing the antigen. Administration can be carried out either orally or parenterally. Parenteral administration is particularly preferred, and specific examples of such administration methods include injection, intranasal administration, pulmonary administration, and transdermal administration. Examples of injection include intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection. For example, the pharmaceutical composition of the present invention, or the cytotoxicity inducer and cell proliferation inhibitor can be administered systemically or locally by injection. An appropriate administration method can be selected depending on the age and symptoms of the subject animal. When administered as an aqueous solution, the solution may contain only the multispecific antigen-binding molecule of the present invention, or may contain, for example, the above-mentioned surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, and flavoring agents. The dosage can be selected, for example, from the range of 0.0001 mg to 1,000 mg per kg of body weight per administration. Alternatively, the dosage can be selected, for example, from the range of 0.001 to 100,000 mg per patient. However, the dosage of the multispecific antigen-binding molecules of the present invention is not limited to these dosages.

[0232] The following methods are suitable for assessing or measuring cytotoxicity induced in target cells (particularly cells expressing a target antigen other than CD3, to which the second antigen-binding domain of the multispecific antigen-binding molecule of the present invention binds) by contact with the multispecific antigen-binding molecule of the present invention. Methods for assessing or measuring cytotoxic activity in vitro include methods for measuring cytotoxic T cell activity. Whether or not the multispecific antigen-binding molecule of the present invention has T cell cytotoxic activity can be measured by known methods (e.g., Current protocols in Immunology, Chapter 7. Immunologic studies in humans, Editor, John E., Coligan et al., John Wiley & Sons, Inc., (1993)). When measuring activity, an antigen-binding molecule that binds to an antigen different from the target antigen and not expressed by the cells used in the test is used as a control, similar to the multispecific antigen-binding molecule of the present invention, and the activity can be determined by determining whether the multispecific antigen-binding molecule of the present invention exhibits stronger cytotoxic activity than the antigen-binding molecule used as a control.

[0233] Furthermore, to evaluate or measure cytotoxic activity in vivo, cells expressing the target antigen are transplanted intradermally or subcutaneously into a non-human test animal, and the test antigen-binding molecule is then administered intravenously or intraperitoneally daily or at intervals of several days from the same day or the following day. By measuring tumor size over time, the difference in the change in tumor size can be defined as cytotoxic activity. Similar to in vitro evaluation, a control antigen-binding molecule is administered, and cytotoxic activity can be determined if the tumor size in the group administered with the antigen-binding molecule of the present invention is significantly smaller than that in the group administered with the control antigen-binding molecule.

[0234] Suitable methods for assessing or measuring the inhibitory effect on the proliferation of cells expressing a target antigen include measuring the cellular uptake of isotope-labeled thymidine and the MTT assay. Furthermore, suitable methods for assessing or measuring the cell proliferation inhibitory activity in vivo include the same methods as those for assessing or measuring the cytotoxic activity in vivo described above.

[0235] Kits The present invention also provides kits for use in the methods of the present invention, comprising the antigen-binding molecules of the present invention or antigen-binding molecules produced by the production methods of the present invention. The kits can also be packaged with other components, such as pharmaceutically acceptable carriers, vehicles, and instructions describing how to use the kit. The present invention also relates to the antigen-binding molecules of the present invention or antigen-binding molecules produced by the production methods of the present invention, for use in the methods of the present invention.

[0236] While the foregoing invention has been described in detail by way of illustration and illustration for purposes of clarity of understanding, the descriptions and illustrations herein should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein by reference in their entireties.

[0237] The present invention will be described in more detail below with reference to examples, but these examples should not be construed as limiting the scope of the present invention.

[0238] [Example 1] Preparation of CDR-modified antibodies with improved binding stability to CD3 antigen. Bispecific antibodies that damage target cells by recognizing the T cell marker CD3 and an antigen on the target cell have been reported, but the reported CD3-binding antibody variable regions still had room for improvement in binding stability. Improving this profile was expected to result in sustained in vivo efficacy and easier in vitro evaluation. Specifically, the antibody variable region described in WO2015174439 lost its binding ability over time in buffer, and further modification to improve binding stability was considered necessary. First, an antibody heavy chain gene TR01H113-F760mnP17 (SEQ ID NO: 30) was prepared, which contained the heavy chain variable region of an antibody against human CD3 described in WO2015174439 and the constant region sequence of human IgG1. Another antibody light chain gene L0011-KT0 (SEQ ID NO: 34) was prepared, which contained the light chain variable region of an antibody against human CD3 and the constant region sequence of human κ chain. Plasmids encoding CDR-modified antibodies were obtained by introducing modifications into the bases encoding the CDR amino acid sequence of this antibody gene using methods known to those skilled in the art. Plasmids encoding the heavy and light chains were mixed and transfected into human embryonic kidney cell-derived HEK293 cells. After 4 days of culture, the supernatant was purified using methods known to those skilled in the art to obtain antibodies. The absorbance of the purified antibody solution at 280 nm was measured using a spectrophotometer. The concentration of the purified antibody was calculated from the measured value using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4, 2411-2423).

[0239]

[0240] Example 2: Evaluation of CD3 antigen binding of antibodies with modified CDRs. Polynucleotides encoding antigens consisting of human CD3ε and CD3γ and a FLAG tag were transfected into HEK293 cells, and the culture supernatant was purified by ion exchange chromatography, FLAG tag affinity chromatography, and gel filtration chromatography to obtain the antigen (hereafter referred to as CD3eg-linker). Analysis of the interaction between the prepared antibodies and CD3eg-linker was performed using an Octet HTX as follows. Phosphate-buffered saline (pH 7.4) containing 0.05% TWEEN 20 was used as the buffer. A Protein A chip (Sartorius) was used as the sensor chip. The antibodies listed in Table 1 were each prepared in buffer at 10 μg / mL, and human CD3eg-linker was serially diluted to 900, 300, 100, and 33 nM. Antibody was loaded onto the sensor chip for 60 seconds after a 30-second baseline, followed by a 30-second baseline step and subsequent 180-second antigen binding and dissociation steps. The binding value was defined as the magnitude of the response at 295 seconds into the measurement. The equilibrium dissociation constant (KD) was determined by global fitting of the response curves at multiple concentrations (Octet BLI Analysis Version: 12.2.2.4). This global fitting used a 1:1 binding model for the entire region of the association and dissociation phases.

[0241]

[0242] [Example 3] Evaluation of binding stability of antibodies with modified CDRs to CD3eg-linker The prepared antibodies were diluted to 0.1 mg / mL and stored at 4°C for 3 days or at 50°C for 3 days, and antigen binding was evaluated using the same method as in Example 2.

[0243]

[0244] The "binding H / I ratio" in the table refers to the ratio of the binding measurement values ​​for each antibody after storage at 4°C to those after storage at 50°C, and indicates the extent to which each antibody maintains its binding activity at 50°C. Antibodies Nos. 2, 14, 25, 29, 30-32, and 34 created in the present invention had binding values ​​at 50°C that were equal to or greater than 0.078, which was the value of the previously reported control antibody 1. Furthermore, the binding H / I values ​​for these antibodies were 75% or greater, exceeding the 61% value of the previously reported control antibody 1. Therefore, it was confirmed that these variants had significantly improved binding activity at 50°C in buffer.

[0245] Example 4: Evaluation of antibodies with modified CDRs by TDCC Reporter Bioassay Bispecific antibodies were prepared using each antibody prepared in the present invention and an anti-MarvelD3 antibody using methods known to those skilled in the art. The prepared antibodies were diluted to 0.044 mg / mL and stored at 4°C or 50°C for 3 days, as in Example 3. The in vitro TDCC activity of these antibodies was measured, and differences in TDCC activity due to storage temperature were evaluated. In vitro TDCC activity was measured using TCR / CD3 Effector Cells (NFAT) and the Bio-Glo luciferase assay system (Promega), and TDCC activity was quantified by luminescence from the reporter gene luciferase. 3 x 10 cells were added to each well of a 384-well plate using medium. 6 NFAT-RE-luc2 Jurkat cells were prepared at a concentration of 5 × 10 / mL. 5The reaction was initiated by adding 10 μL of 10 nM or 100 nM antibody solution to LS 174T cells (ATCC) prepared at 1 mL. LS 174T is a cell line expressing the MarvelD3 antigen. The reaction was carried out overnight at 37°C. After incubation, luciferase substrate was added to each well, and reporter gene expression was quantified by measuring luminescence using a plate reader. The luciferase response (counts per second: CPS) represents the average of the values ​​measured for the 10 nM and 100 nM antibody solutions.

[0246] NA indicates that the measurement was not performed.

[0247] The "activated H / I ratio" in the table is the ratio of the luciferase response values ​​for each antibody after storage at 4°C to those after storage at 50°C, and indicates the extent to which each antibody lost binding activity at 50°C. Antibodies Nos. 2, 14, 25, 29, 30-32, and 34 selected in Example 3 exhibited luciferase responses of 4.3.E+05 or higher when stored at 4°C, which corresponds to the response of the previously reported control antibody 1 when stored at 50°C. Furthermore, antibodies Nos. 14, 25, 29, 30-32, and 34 exhibited activated H / I TDCC activity ratios of 67% or higher when stored at 50°C. This value was superior to that of the previously reported control antibody 1, indicating that these antibodies were modified antibodies with improved activated H / I ratios while maintaining TDCC activity. The amino acid sequences of antibody numbers 1, 2, 14, 25, 29, 30-32, and 34 are shown in Table 5.

[0248]

[0249] The present invention provides a novel CD3-binding domain that has superior stability compared to CD3-binding domains used in conventional T cell-redirecting antibodies, as well as antigen-binding molecules containing the CD3-binding domain. Bispecific antigen-binding molecules prepared by combining the CD3-binding domain of the present invention with an antigen-binding domain that binds to an antigen expressed on the surface of target cells such as cancer cells can induce T cell-dependent cytotoxicity against the target cells and can be used to treat or prevent various cancers. Antigen-binding molecules containing the CD3-binding domain of the present invention show only a small decrease in CD3-binding activity at room temperature or higher, and are expected to maintain their T cell-dependent cytotoxicity for a long period of time when administered to patients.

Claims

1. An antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises an antibody heavy chain variable region and light chain variable region having binding activity to CD3, wherein the heavy chain variable region comprises: an heavy chain CDR1 comprising the amino acid sequence NAWMH (SEQ ID NO: 1); an heavy chain CDR2 comprising the amino acid sequence QIX1DKSQNYATX2VAESVKG (SEQ ID NO: 2), wherein X1 is K or R and X2 is Y or F; and an heavy chain CDR3 comprising the amino acid sequence VHYX3AGYGVDX4 (SEQ ID NO: 3), wherein X3 is A or P and X4 is I, M, or L, and wherein the light chain variable region comprises: An antigen-binding molecule comprising: an L-chain CDR1 comprising the amino acid sequence RSX5X6X7VVHENRX8TYLH (SEQ ID NO: 4), wherein X5 is S or T, X6 is Q or M, X7 is S or T, and X8 is Q or N; an L-chain CDR2 comprising the amino acid sequence KVSNRFS (SEQ ID NO: 5); and an L-chain CDR3 comprising the amino acid sequence GQGTQVPYT (SEQ ID NO: 6).

2. The antigen-binding molecule of claim 1, wherein the antibody H chain variable region and L chain variable region having binding activity to CD3 comprise any combination of H chain CDR1, CDR2, and CDR3, and L chain CDR1, CDR2, and CDR3 selected from the following (a1) to (a8): (a1) a combination of H chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, H chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, H chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10, L chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, L chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and L chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (a2) (a3) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (a4) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (a5)(a6) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 13, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (a7) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (a8) A combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 18, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (a9) A combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 19, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:

6.

3. The antigen-binding molecule of claim 1 or 2, wherein the antibody H-chain variable region and L-chain variable region having binding activity to CD3 comprise any combination of H-chain variable region and L-chain variable region selected from the following (a1) to (a8): (a1) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 20 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25; (a2) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 21 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25; (a3) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 21 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 26; (a4) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25; (a5) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 23 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25; (a6) (a7) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 27; (a8) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO:

29.

4. An antigen-binding molecule according to any one of claims 1 to 3, further comprising an Fc region.

5. The antigen-binding molecule of any one of claims 1 to 4, which is a monospecific antigen-binding molecule.

6. The antigen-binding molecule of any one of claims 1 to 4, which is a bispecific antigen-binding molecule.

7. The antigen-binding molecule of claim 6, wherein the second antigen-binding domain comprises an antibody variable region having binding activity against a cancer antigen.

8. The antigen-binding molecule of any one of claims 1 to 7, which is an antibody.

9. A nucleic acid encoding the antigen-binding molecule of any one of claims 1 to 8.

10. A vector into which the nucleic acid according to claim 9 has been introduced.

11. A cell comprising the vector of claim 10.

12. A method for producing the antigen-binding molecule of any one of claims 1 to 8, comprising the step of culturing the cell of claim 11.

13. An antigen-binding molecule produced by the method of claim 12.

14. A pharmaceutical composition comprising the antigen-binding molecule of any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition according to claim 14 for use in the treatment or prevention of cancer.

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