Antigen binding molecules capable of binding to CD3 and CD137 but not at the same time

Abstract

To provide antigen-binding molecules that bind to CD3 and CD137 (4-1BB); compositions comprising the antigen-binding molecules; and methods of using the same.SOLUTION: The present invention provides antigen-binding molecules comprising a variable region capable of binding to CD3 and CD137 (4-1BB), but not to CD3 and CD137 at the same time, and a variable region binding to a third antigen different from CD3 and CD137. Such antigen binding molecules show enhanced T-cell dependent cytotoxic activity induced by these antigen binding molecules through binding to three different antigens.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to antigen-binding molecules that bind to CD3 and CD137 (4-1BB), and methods of using the same. [Background technology]

[0002] Antibodies are attracting attention as pharmaceuticals because they are highly stable in plasma and rarely cause adverse reactions (Nat. Biotechnol. (2005) 23, 1073-1078 (Non-Patent Document 1) and Eur J Pharm Biopharm. (2005) 59 (3), 389-396 (Non-Patent Document 2)). Antibodies not only bind to antigens and have agonistic or antagonistic effects, but also induce effector cell-mediated cytotoxicity (also known as effector function), such as ADCC (antibody-dependent cellular cytotoxicity), ADCP (antibody-dependent cellular phagocytosis), or CDC (complement-dependent cytotoxicity). In particular, antibodies of the IgG1 subclass exhibit effector function against cancer cells, and therefore many antibody drugs have been developed in the field of oncology.

[0003] For an antibody to exert ADCC, ADCP, or CDC, its Fc region must bind to antibody receptors (FcγR) and various complement components present on effector cells (e.g., NK cells or macrophages). In humans, the FcγR protein family has been reported to include FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb isoforms, and their respective allotypes have also been reported (Immunol. Lett. (2002) 82, 57-65 (Non-Patent Document 3)). Of these isoforms, FcγRIa, FcγRIIa, and FcγRIIIa contain a domain called ITAM (immunoreceptor tyrosine-based activation motif) in their intracellular domain, which transduces activation signals. In contrast, only FcγRIIb contains a domain called ITIM (immunoreceptor tyrosine-based inhibitory motif) in its intracellular domain, which transduces inhibitory signals. All of these isoforms of FcγR are known to transmit signals by cross-linking with immune complexes, etc. (Nat. Rev. Immunol. (2008) 8, 34-47 (Non-Patent Document 4)). In fact, when antibodies exert their effector function against cancer cells, FcγR molecules on the effector cell membrane are clustered by the Fc regions of multiple antibodies bound to the cancer cell membrane, thereby transmitting an activation signal through the effector cells. As a result, a cytocidal effect is exerted. In this regard, FcγR cross-linking is limited to effector cells located near cancer cells, indicating that immune activation is localized to cancer cells (Ann. Rev. Immunol. (1988). 6, 251-81 (Non-Patent Document 5)).

[0004] Native immunoglobulins bind to antigens via their variable regions and to receptors such as FcγR, FcRn, FcαR, and FcεR or complement via their constant regions. Each FcRn (a binding molecule that interacts with the Fc region of IgG) binds to each heavy chain of the antibody, one molecule at a time. Therefore, it has been reported that two FcRn molecules bind to one IgG antibody molecule. Unlike FcRn, FcγR interacts with the antibody via the hinge region and CH2 domain, and only one FcγR molecule binds to one IgG antibody molecule (J. Bio. Chem., (20001) 276, 16469-16477). It has been found that several amino acid residues in the hinge region and CH2 domain of an antibody, as well as the sugar chain attached to Asn 297 (EU numbering) in the CH2 domain, are important for the binding between FcγR and the Fc region of an antibody (Chem. Immunol. (1997), 65, 88-110 (Non-Patent Document 6), Eur. J. Immunol. (1993) 23, 1098-1104 (Non-Patent Document 7), and Immunol. (1995) 86, 319-324 (Non-Patent Document 8)). Focusing on this binding site, Fc region variants with various FcγR-binding properties have been studied, and Fc region variants with higher binding activity to activating FcγRs have been obtained (WO2000 / 042072 (Patent Document 1) and WO2006 / 019447 (Patent Document 2)). For example, Lazar et al. succeeded in increasing the binding activity of human IgG1 to human FcγRIIIa (V158) by approximately 370-fold by substituting Ser 239, Ala 330, and Ile 332 (EU numbering) of human IgG1 with Asn, Leu, and Glu, respectively (Proc. Natl. Acad. Sci. USA (2006) 103, 4005-4010 (Non-Patent Document 9) and WO2006 / 019447 (Patent Document 2)). This modified form has approximately 9-fold higher binding activity than the wild-type form in terms of the ratio of FcγRIIIa to FcγIIb (A / I ratio).Alternatively, Shinkawa et al. succeeded in increasing the FcγRIIIa-binding activity by approximately 100-fold by deleting the fucose in the sugar chain attached to Asn 297 (EU numbering) (J. Biol. Chem. (2003) 278, 3466-3473 (Non-Patent Document 10)). These methods can significantly improve the ADCC activity of human IgG1 compared to native human IgG1.

[0005] Natural IgG antibodies typically recognize and bind to a single epitope via their variable region (Fab), and therefore can only bind to a single antigen. However, cancer and inflammation are known to involve multiple proteins, and these proteins may crosstalk with each other. For example, several inflammatory cytokines (TNF, IL1, and IL6) are known to be involved in immune diseases (Nat. Biotech., (2011) 28, 502-10 (Non-Patent Document 11)). Furthermore, activation of other receptors is known to be one of the mechanisms underlying the acquisition of drug resistance in cancer (Endocr Relat Cancer (2006) 13, 45-51 (Non-Patent Document 12)). In such cases, conventional antibodies that recognize a single epitope cannot inhibit multiple proteins.

[0006] Antibodies that bind to two or more antigens with a single molecule (these antibodies are called bispecific antibodies) are being studied as molecules that inhibit multiple targets. By modifying natural IgG antibodies, it is possible to confer binding activity to two different antigens (first antigen and second antigen) (mAbs. (2012) Mar 1, 4(2)). Therefore, such antibodies not only neutralize these two or more antigens with a single molecule, but also enhance antitumor activity by crosslinking cytotoxic cells to cancer cells. Previously reported molecular forms of bispecific antibodies include molecules in which antigen-binding sites are added to the N- or C-terminus of an antibody (DVD-Ig, TCB, and scFv-IgG), molecules in which the two Fab regions of an antibody have different sequences (common L-chain bispecific antibodies and hybrid hybridomas), molecules in which one Fab region recognizes two antigens (two-in-one IgG and DutaMab), and molecules in which the CH3 domain loop serves as a separate antigen-binding site (Fcab) (Nat. Rev. (2010), 10, 301-316 (Non-Patent Document 13) and Peds (2010), 23(4), 289-297 (Non-Patent Document 14)). Since all of these bispecific antibodies interact with FcγR via their Fc regions, the effector functions of the antibody are preserved.

[0007] If all antigens recognized by a bispecific antibody are specifically expressed in cancer, a bispecific antibody that binds to one of the antigens will exhibit cytotoxic activity against cancer cells, and thus is expected to have a more effective anticancer effect than conventional antibody drugs that recognize a single antigen. However, if one of the antigens recognized by the bispecific antibody is expressed in normal tissues or immune cells, cross-linking with FcγR will cause damage to normal tissues or release of cytokines (J. Immunol. (1999) Aug. 1, 163(3), 1246-52 (Non-Patent Document 15)). As a result, strong adverse reactions will be induced.

[0008] For example, catumaxomab is known as a bispecific antibody that recognizes a protein expressed on T cells and a protein expressed on cancer cells (cancer antigens). Catumaxomab binds to a cancer antigen (EpCAM) and the CD3ε chain expressed on T cells, respectively, via its two Fab fragments. Catumaxomab induces T cell-mediated cytotoxicity by simultaneously binding to a cancer antigen and CD3ε, and induces NK cell- or antigen-presenting cell (e.g., macrophage)-mediated cytotoxicity by simultaneously binding to a cancer antigen and FcγR. By utilizing these two cytotoxic activities, catumaxomab has demonstrated a high therapeutic effect against malignant ascites when administered intraperitoneally, and has therefore been approved in Europe (Cancer Treat Rev. (2010) Oct 36(6), 458-67 (Non-Patent Document 16)). Furthermore, cases have been reported in which administration of catumaxomab resulted in the appearance of antibodies that react with cancer cells, demonstrating the induction of adaptive immunity (Future Oncol. (2012) Jan 8(1), 73-85 (Non-Patent Document 17)). Based on these results, such antibodies that possess both T cell-mediated cytotoxic activity and the effect mediated by cells such as NK cells or macrophages via FcγR (these antibodies are particularly referred to as trifunctional antibodies) are attracting attention because they are expected to have strong antitumor effects and induce adaptive immunity.

[0009] However, trifunctional antibodies simultaneously bind to CD3ε and FcγR even in the absence of cancer antigens. Therefore, even in the absence of cancer cells, they crosslink CD3ε-expressing T cells to FcγR-expressing cells, resulting in the production of large amounts of various cytokines. Due to this induction of cytokine production independent of cancer antigens, trifunctional antibodies are currently administered only intraperitoneally (Cancer Treat Rev. 2010 Oct 36(6), 458-67 (Non-Patent Document 16)). Systemic administration of trifunctional antibodies is extremely difficult due to the severe cytokine storm-like adverse reactions (Cancer Immunol Immunother. 2007 Sep; 56(9): 1397-406 (Non-Patent Document 18)). Bispecific antibodies of the prior art can bind to both antigens, i.e., a cancer antigen (EpCAM) as a first antigen and CD3ε as a second antigen, simultaneously while binding to FcγR. Therefore, due to their molecular structure, such adverse reactions caused by simultaneous binding to FcγR and CD3ε as a second antigen cannot be avoided. In recent years, improved antibodies have been provided that induce T cell-mediated cytotoxicity while avoiding adverse reactions by using an Fc region with reduced binding activity to FcγR ( WO2012 / 073985 ). However, even such antibodies cannot bind to cancer antigens while acting on two immune receptors, namely, CD3ε and FcγR, given their molecular structure. No antibody is known that exerts both T cell-mediated cytotoxicity and non-T cell-mediated cytotoxicity in a cancer antigen-specific manner while avoiding adverse reactions.

[0010] T cells play an important role in tumor immunity and are known to be activated by two signals: 1) T cell receptor (TCR) binding to antigen peptides presented by major histocompatibility complex (MHC) class I molecules and TCR activation; and 2) costimulatory molecules on the surface of T cells binding to ligands on antigen-presenting cells and activation of costimulatory molecules. Furthermore, activation of molecules belonging to the tumor necrosis factor (TNF) superfamily and the TNF receptor superfamily, such as CD137 (4-1BB) on the surface of T cells, has been described as important for T cell activation (Vinay, 2011, Cellular & Molecular Immunology, 8, 281-284 (Non-Patent Document 19)).

[0011] CD137 agonist antibodies have been experimentally demonstrated to exhibit antitumor effects, primarily through the activation of CD8-positive T cells and NK cells (Houot, 2009, Blood, 114, 3431-8). T cells engineered to carry chimeric antigen receptor molecules consisting of a tumor antigen-binding domain as the extracellular domain and CD3 and CD137 signaling domains as the intracellular domain (CAR-T cells) can enhance the durability of efficacy (Porter, N ENGL J MED, 2011, 365;725-733). However, the side effects of such CD137 agonist antibodies due to their nonspecific hepatotoxicity are a clinical and nonclinical problem, preventing progress in drug development (Dubrot, Cancer Immunol. Immunother., 2010, 28, 512-22). It has been suggested that the main cause of side effects is antibody binding to Fcγ receptors via the antibody constant region (Schabowsky, Vaccine, 2009, 28, 512-22 (Non-Patent Document 23)). Furthermore, it has been reported that antibody cross-linking by Fcγ receptor-expressing cells (FcγRII-expressing cells) is required for agonist antibodies targeting receptors belonging to the TNF receptor superfamily to exert their agonist activity in vivo (Li, Proc Natl Acad Sci USA. 2013, 110(48), 19501-6 (Non-Patent Document 24)). WO2015 / 156268 (Patent Document 3) describes that a bispecific antibody having a binding domain with CD137 agonist activity and a binding domain for a tumor-specific antigen can exert CD137 agonist activity and activate immune cells only in the presence of cells expressing the tumor-specific antigen, thereby avoiding the hepatotoxic adverse event of a CD137 agonist antibody while maintaining the antitumor activity of the antibody.WO2015 / 156268 further describes that the antitumor activity can be further enhanced and these adverse events can be avoided by combining this bispecific antibody with another bispecific antibody having a binding domain with CD3 agonist activity and a binding domain for a tumor-specific antigen. A trispecific antibody having three binding domains for CD137, CD3, and a tumor-specific antigen (EGFR) has also been reported (WO2014 / 116846 (Patent Document 4)). However, no antibody has been known that exerts both T cell-mediated cytotoxicity and CD137-mediated activation of T cells and other immune cells in a cancer antigen-specific manner while avoiding adverse reactions. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] WO2000 / 042072 [Patent Document 2] WO2006 / 019447 [Patent Document 3] WO2015 / 156268 [Patent Document 4] WO2014 / 116846 [Non-patent literature]

[0013] [Non-Patent Document 1] Nat. Biotechnol. (2005) 23, 1073-1078 [Non-patent document 2] Eur J Pharm Biopharm. (2005) 59 (3), 389-396 [Non-patent document 3] Immunol. Lett. (2002) 82, 57-65 [Non-patent document 4] Nat. Rev. Immunol. (2008) 8, 34-47 [Non-Patent Document 5] Ann. Rev. Immunol. (1988). 6. 251-81

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[0014] A trispecific antibody comprising a tumor-specific antigen (EGFR)-binding domain, a CD137-binding domain, and a CD3-binding domain has already been reported (WO2014116846). However, because an antibody having such a molecular format can simultaneously bind to three different antigens, the present inventors speculated that the trispecific antibody might simultaneously bind to CD3 and CD137, thereby cross-linking CD3ε-expressing T cells and CD137-expressing cells (e.g., T cells, B cells, NK cells, DCs, etc.). Furthermore, it has been reported that bispecific antibodies against CD8 and CD3ε cross-link the two and thereby induce mutual cytotoxic activity between CD8-positive T cells (Wong, Clin. Immunol. Immunopathol. 1991, 58(2), 236-250). Therefore, the present inventors speculated that bispecific antibodies against a molecule expressed on T cells and CD3ε would also cross-link cells expressing the molecule with cells expressing CD3ε, thereby inducing mutual cytotoxic activity between T cells. [Means for solving the problem]

[0015] The present invention provides antigen-binding domains that bind to CD3 and CD137, and methods for using the same. The present invention also provides methods for more efficiently obtaining antigen-binding molecules that induce T cell-dependent cytotoxicity.

[0016] The present inventors have successfully prepared antigen-binding molecules comprising an antibody variable region capable of binding to CD3 and CD137 (4-1BB) but not simultaneously binding to CD3 and CD137, and a variable region that binds to a third antigen different from CD3 and CD137, preferably a molecule specifically expressed in cancer tissue, more preferably glypican-3 (GPC3). By improving the binding activity to CD3 and / or CD137, the present inventors have successfully prepared antigen-binding molecules that exhibit enhanced T cell-dependent cytotoxicity induced by these antigen-binding molecules through binding to three different antigens. Such antigen-binding molecules can be used in immunotherapy, avoiding the cross-linking between different cells that occurs when conventional multispecific antigen-binding molecules bind to antigens expressed on different cells, which is thought to cause adverse reactions when multispecific antigen-binding molecules are used as pharmaceuticals.

[0017] More specifically, the present invention relates to the following: [1] An antigen-binding molecule comprising an antibody variable region capable of binding to CD3 and CD137 but not simultaneously binding to CD3 and CD137, preferably determined by SPR under the following conditions: 37°C, pH 7.4, 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3; antigen-binding molecules are immobilized on a CM4 sensor chip, and antigen is the analyte. When measured at 5 x 10 -6 Less than M, 5 x 10 -7 Less than M, 5 x 10 -8 Less than M or 3 x 10 -8 The antigen-binding molecule binds to CD137 with an equilibrium dissociation constant (KD) of less than M. [1A] Preferably, SPR is performed under the following conditions: 37°C, pH 7.4, 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3; antigen-binding molecules are immobilized on a CM4 sensor chip, and antigen is the analyte. When measured at 5 x 10 -6 M~3×10 -8 An antigen-binding molecule [1] that binds to CD137 with an equilibrium dissociation constant (KD) of M. [1B] Preferably, SPR is performed under the following conditions: 25°C, pH 7.4, 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3; antigen-binding molecules are immobilized on a CM4 sensor chip, and antigen is the analyte. When measured at 2 x 10 -6 M~1×10 -8 An antigen-binding molecule [1]-[1A] that binds to CD3 with an equilibrium dissociation constant (KD) of M. [2] (a) at least one, two, three, or more amino acid residues of the extracellular domain of CD3ε (CD3 epsilon) comprising the amino acid sequence of SEQ ID NO: 159; and / or (b) at least one, two, three, or more amino acid residues of the N-terminal region of human CD137, including the amino acid sequence LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAEC (SEQ ID NO: 152), preferably LQDPCSN, NNRNQI, and / or GQRTCDI. An antigen-binding molecule of [1] to [1B] that binds to [3] The antigen-binding molecule of [1] or [2], wherein the antibody variable region has 1 to 25 amino acid alterations, and the altered amino acids are selected from amino acids in a loop, amino acids in the FR3 region, or amino acids selected from positions 31 to 35, 50 to 65, 71 to 74, and 95 to 102, according to the Kabat numbering, in the antibody heavy-chain variable domain, and positions 24 to 34, 50 to 56, and 89 to 97, according to the Kabat numbering, in the antibody light-chain variable domain. [3A] The antigen-binding molecule of any of [1] to [3], wherein the heavy chain variable domain (VH) and / or light chain variable domain (VL) comprises one or more amino acid substitutions selected from Tables 1.3(a) to 1.3(d), and the one or more amino acid substitutions exhibit an increase in binding affinity to CD3 and / or CD137 of at least 0.2, 0.3, 0.5, 0.8, 1, 1.5, or 2-fold as shown in Tables 1.3(a) to 1.3(d). In some embodiments, the antibody variable region comprises: (a) a heavy chain variable domain amino acid sequence comprising, at each of the following positions (all according to Kabat numbering), one or more of the following amino acid residues as indicated for that position: A, D, E, I, G, K, L, M, N, R, T, W, or Y at amino acid position 26; D, F, G, I, M, or L at amino acid position 27; D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 28; F or W at amino acid position 29; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 30; F, I, N, R, S, T, or V at amino acid position 31; A, H, I, K, L, N, Q, R, S, T, or V at amino acid position 32; W at amino acid position 33; F, I, L, M, or V at amino acid position 34; F, H, S, T, V, or Y at amino acid position 35; E, F, H, I, K, L, M, N, Q, S, T, W, or Y at amino acid position 50; I, K, or V at amino acid position 51; K, M, R, or T at amino acid position 52; A, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y at amino acid position 52b; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 52c; A, E, F, H, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 53; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 54; E, F, G, H, L, M, N, Q, W, or Y at amino acid position 55; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 56; A, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, or V at amino acid position 57; A, F, H, K, N, P, R, or Y at amino acid position 58; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 59; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 60; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 61; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 62; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 63; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 64; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 65; H or R at amino acid position 93; F, G, H, L, M, S, T, V, or Y at amino acid position 94; I or V at amino acid position 95; F, H, I, K, L, M, T, V, W, or Y at amino acid position 96; F, Y, or W at amino acid position 97; A, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 98; A, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 99; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100a; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100b; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100c; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100d; A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y at amino acid position 100e; A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100f; Approximately 100g of amino acids A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; A, D, E, G, H, I, L, M, N, P, S, T, or V at amino acid position 100h; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100i; A, D, F, I, L, M, N, Q, S, T, or V at amino acid position 101; A, D, E, F, G, H, IK, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 102; and / or (b) a light chain variable domain amino acid sequence comprising, at each of the following positions (all according to Kabat numbering), one or more of the following amino acid residues as indicated for that position: A, D, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 24; A, G, N, P, S, T, or V at amino acid position 25; A, D, E, F, G, I, K, L, M, N, Q, R, S, T, or V at amino acid position 26; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 27; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 27a; A, I, L, M, P, T, or V at amino acid position 27b; A, E, F, H, I, K, L, M, N, P, Q, R, T, W, or Y at amino acid position 27c; A, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 27d; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 27e; G, N, S, or T at amino acid position 28; A, F, G, H, K, L, M, N, Q, R, S, T, W, or Y at amino acid position 29; A, F, G, H, I, K, L, M, N, Q, R, V, W, or Y at amino acid position 30; I, L, Q, S, T, or V at amino acid position 31; F, W, or Y at amino acid position 32; A, F, H, L, M, Q, or V at amino acid position 33; A, H, or S at amino acid position 34; I, K, L, M, or R at amino acid position 50; A, E, I, K, L, M, Q, R, S, T, or V at amino acid position 51; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 52; A, E, F, G, H, K, L, M, N, P, Q, R, S, V, W, or Y at amino acid position 53; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 54; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y at amino acid position 55; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 56; A, G, K, S, or Y at amino acid position 89; Q at amino acid position 90; G at amino acid position 91; A, D, H, K, N, Q, R, S, or T at amino acid position 92; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 93; A, D, H, I, M, N, P, Q, R, S, T, or V at amino acid position 94; P at amino acid position 95; F or Y at amino acid position 96; and A, D, E, G, H, I, K, L, M, N, Q, R, S, T, or V at amino acid position 97 It is preferred that the compound contains: [4] The antigen-binding molecule of any one of [1] to [3A], wherein the antibody variable region comprises any one of the following: (a1) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 16, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 30, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 44, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a2) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 17, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 31, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 45, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 64, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 69, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 74; (a3) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 18, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 32, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 46, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a4) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 19, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 33, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 47, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a5) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 19, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 33, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 47, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 65, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 70, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 75; (a6) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 20, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 34, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 48, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a7) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 22, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 36, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 50, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a8) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 23, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 37, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 51, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a9) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 23, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 37, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 51, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 66, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 71, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 76; (a10) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 24, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 38, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 52, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a11) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 25, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 39, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 53, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 66, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 71, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 76; (a12) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 26, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 40, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 54, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 66, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 71, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 76; (a13) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 26, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 40, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 54, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a14) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 27, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 41, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 55, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a15) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 28, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 42, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 56, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (b1) HCDR1 comprising the amino acid sequence of SEQ ID NO: 16, HCDR2 comprising the amino acid sequence of SEQ ID NO: 30, HCDR3 comprising the amino acid sequence of SEQ ID NO: 44, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b2) HCDR1 comprising the amino acid sequence of SEQ ID NO: 17, HCDR2 comprising the amino acid sequence of SEQ ID NO: 31, HCDR3 comprising the amino acid sequence of SEQ ID NO: 45, LCDR1 comprising the amino acid sequence of SEQ ID NO: 64, LCDR2 comprising the amino acid sequence of SEQ ID NO: 69, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 74; (b3) HCDR1 comprising the amino acid sequence of SEQ ID NO: 18, HCDR2 comprising the amino acid sequence of SEQ ID NO: 32, HCDR3 comprising the amino acid sequence of SEQ ID NO: 46, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b4) HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, HCDR2 comprising the amino acid sequence of SEQ ID NO: 33, HCDR3 comprising the amino acid sequence of SEQ ID NO: 47, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b5) HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, HCDR2 comprising the amino acid sequence of SEQ ID NO: 33, HCDR3 comprising the amino acid sequence of SEQ ID NO: 47, LCDR1 comprising the amino acid sequence of SEQ ID NO: 65, LCDR2 comprising the amino acid sequence of SEQ ID NO: 70, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 75; (b6) HCDR1 comprising the amino acid sequence of SEQ ID NO: 20, HCDR2 comprising the amino acid sequence of SEQ ID NO: 34, HCDR3 comprising the amino acid sequence of SEQ ID NO: 48, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b7) HCDR1 comprising the amino acid sequence of SEQ ID NO: 22, HCDR2 comprising the amino acid sequence of SEQ ID NO: 36, HCDR3 comprising the amino acid sequence of SEQ ID NO: 50, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b8) HCDR1 comprising the amino acid sequence of SEQ ID NO: 23, HCDR2 comprising the amino acid sequence of SEQ ID NO: 37, HCDR3 comprising the amino acid sequence of SEQ ID NO: 51, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b9) HCDR1 comprising the amino acid sequence of SEQ ID NO: 23, HCDR2 comprising the amino acid sequence of SEQ ID NO: 37, HCDR3 comprising the amino acid sequence of SEQ ID NO: 51, LCDR1 comprising the amino acid sequence of SEQ ID NO: 66, LCDR2 comprising the amino acid sequence of SEQ ID NO: 71, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 76; (b10) HCDR1 comprising the amino acid sequence of SEQ ID NO: 24, HCDR2 comprising the amino acid sequence of SEQ ID NO: 38, HCDR3 comprising the amino acid sequence of SEQ ID NO: 52, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b11) HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, HCDR2 comprising the amino acid sequence of SEQ ID NO: 39, HCDR3 comprising the amino acid sequence of SEQ ID NO: 53, LCDR1 comprising the amino acid sequence of SEQ ID NO: 66, LCDR2 comprising the amino acid sequence of SEQ ID NO: 71, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 76; (b12) HCDR1 comprising the amino acid sequence of SEQ ID NO: 26, HCDR2 comprising the amino acid sequence of SEQ ID NO: 40, HCDR3 comprising the amino acid sequence of SEQ ID NO: 54, LCDR1 comprising the amino acid sequence of SEQ ID NO: 66, LCDR2 comprising the amino acid sequence of SEQ ID NO: 71, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 76; (b13) HCDR1 comprising the amino acid sequence of SEQ ID NO: 26, HCDR2 comprising the amino acid sequence of SEQ ID NO: 40, HCDR3 comprising the amino acid sequence of SEQ ID NO: 54, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b14) HCDR1 comprising the amino acid sequence of SEQ ID NO: 27, HCDR2 comprising the amino acid sequence of SEQ ID NO: 41, HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b15) HCDR1 comprising the amino acid sequence of SEQ ID NO: 28, HCDR2 comprising the amino acid sequence of SEQ ID NO: 42, HCDR3 comprising the amino acid sequence of SEQ ID NO: 56, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (c1) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 2, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c2) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 3, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 59; (c3) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 4, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c4) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c5) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 60; (c6) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 6, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c7) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 8, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c8) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 9, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c9) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 9, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 61; (c10) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 10, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c11) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 11, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 61; (c12) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 12, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 61; (c13) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 12, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c14) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 13, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c15) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 14, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (d1) a heavy chain variable domain (VH) of SEQ ID NO: 2, and a light chain variable domain (VL) of SEQ ID NO: 58; (d2) a heavy chain variable domain (VH) of SEQ ID NO: 3, and a light chain variable domain (VL) of SEQ ID NO: 59; (d3) a heavy chain variable domain (VH) of SEQ ID NO: 4, and a light chain variable domain (VL) of SEQ ID NO: 58; (d4) a heavy chain variable domain (VH) of SEQ ID NO: 5, and a light chain variable domain (VL) of SEQ ID NO: 58; (d5) a heavy chain variable domain (VH) of SEQ ID NO: 5, and a light chain variable domain (VL) of SEQ ID NO: 60; (d6) a heavy chain variable domain (VH) of SEQ ID NO: 6, and a light chain variable domain (VL) of SEQ ID NO: 58; (d7) a heavy chain variable domain (VH) of SEQ ID NO: 8, and a light chain variable domain (VL) of SEQ ID NO: 58; (d8) a heavy chain variable domain (VH) of SEQ ID NO: 9, and a light chain variable domain (VL) of SEQ ID NO: 58; (d9) a heavy chain variable domain (VH) of SEQ ID NO: 9, and a light chain variable domain (VL) of SEQ ID NO: 61; (d10) a heavy chain variable domain (VH) of SEQ ID NO: 10, and a light chain variable domain (VL) of SEQ ID NO: 58; (d11) a heavy chain variable domain (VH) of SEQ ID NO: 11, and a light chain variable domain (VL) of SEQ ID NO: 61; (d12) a heavy chain variable domain (VH) of SEQ ID NO: 12, and a light chain variable domain (VL) of SEQ ID NO: 61; (d13) a heavy chain variable domain (VH) of SEQ ID NO: 12, and a light chain variable domain (VL) of SEQ ID NO: 58; (d14) a heavy chain variable domain (VH) of SEQ ID NO: 13, and a light chain variable domain (VL) of SEQ ID NO: 58; (d15) a heavy chain variable domain (VH) of SEQ ID NO: 14, and a light chain variable domain (VL) of SEQ ID NO: 58; (e) an antibody variable region that competes with any one of the antibody variable regions (a1) to (d15) for binding to CD3; (f) an antibody variable region that competes with any one of the antibody variable regions (a1) to (d15) for binding to CD137; (g) an antibody variable region that binds to the same epitope on CD3 as any one of the antibody variable regions of (a1) to (d15); (h) An antibody variable region that binds to the same epitope on CD137 as any one of the antibody variable regions of (a1) to (d15). [4A] An antigen-binding molecule according to [4][c1] to [c15], wherein the heavy chain variable domain (VH) and / or light chain variable domain (VL) comprises one or more amino acid substitutions selected from Tables 1.3(a) to 1.3(d), and the one or more amino acid substitutions exhibit an increase in binding affinity to CD3 and / or CD137 of at least 0.2, 0.3, 0.5, 0.8, 1, 1.5, or 2-fold as shown in Tables 1.3(a) to 1.3(d). [4B] The antibody variable region (a) a heavy chain variable domain amino acid sequence comprising, at each of the following positions (all according to Kabat numbering), one or more of the following amino acid residues as indicated for that position: A, D, E, I, G, K, L, M, N, R, T, W, or Y at amino acid position 26; D, F, G, I, M, or L at amino acid position 27; D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 28; F or W at amino acid position 29; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 30; F, I, N, R, S, T, or V at amino acid position 31; A, H, I, K, L, N, Q, R, S, T, or V at amino acid position 32; W at amino acid position 33; F, I, L, M, or V at amino acid position 34; F, H, S, T, V, or Y at amino acid position 35; E, F, H, I, K, L, M, N, Q, S, T, W, or Y at amino acid position 50; I, K, or V at amino acid position 51; K, M, R, or T at amino acid position 52; A, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y at amino acid position 52b; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 52c; A, E, F, H, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 53; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 54; E, F, G, H, L, M, N, Q, W, or Y at amino acid position 55; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 56; A, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, or V at amino acid position 57; A, F, H, K, N, P, R, or Y at amino acid position 58; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 59; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 60; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 61; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 62; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 63; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 64; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 65; H or R at amino acid position 93; F, G, H, L, M, S, T, V, or Y at amino acid position 94; I or V at amino acid position 95; F, H, I, K, L, M, T, V, W, or Y at amino acid position 96; F, Y, or W at amino acid position 97; A, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 98; A, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 99; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100a; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100b; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100c; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100d; A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y at amino acid position 100e; A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100f; Approximately 100g of amino acids A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; A, D, E, G, H, I, L, M, N, P, S, T, or V at amino acid position 100h; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100i; A, D, F, I, L, M, N, Q, S, T, or V at amino acid position 101; A, D, E, F, G, H, IK, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 102; and / or (b) a light chain variable domain amino acid sequence comprising, at each of the following positions (all according to Kabat numbering), one or more of the following amino acid residues as indicated for that position: A, D, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 24; A, G, N, P, S, T, or V at amino acid position 25; A, D, E, F, G, I, K, L, M, N, Q, R, S, T, or V at amino acid position 26; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 27; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 27a; A, I, L, M, P, T, or V at amino acid position 27b; A, E, F, H, I, K, L, M, N, P, Q, R, T, W, or Y at amino acid position 27c; A, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 27d; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 27e; G, N, S, or T at amino acid position 28; A, F, G, H, K, L, M, N, Q, R, S, T, W, or Y at amino acid position 29; A, F, G, H, I, K, L, M, N, Q, R, V, W, or Y at amino acid position 30; I, L, Q, S, T, or V at amino acid position 31; F, W, or Y at amino acid position 32; A, F, H, L, M, Q, or V at amino acid position 33; A, H, or S at amino acid position 34; I, K, L, M, or R at amino acid position 50; A, E, I, K, L, M, Q, R, S, T, or V at amino acid position 51; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 52; A, E, F, G, H, K, L, M, N, P, Q, R, S, V, W, or Y at amino acid position 53; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 54; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y at amino acid position 55; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 56; A, G, K, S, or Y at amino acid position 89; Q at amino acid position 90; G at amino acid position 91; A, D, H, K, N, Q, R, S, or T at amino acid position 92; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 93; A, D, H, I, M, N, P, Q, R, S, T, or V at amino acid position 94; P at amino acid position 95; F or Y at amino acid position 96; and A, D, E, G, H, I, K, L, M, N, Q, R, S, T, or V at amino acid position 97 The antigen-binding molecule of [4A], [5] An antigen-binding molecule according to any one of [1] to [4B] above, which has at least one characteristic selected from the group consisting of the following (1) to (3): (1) The antigen-binding molecule does not simultaneously bind to CD3 and CD137, each of which is expressed on different cells; (2) the antigen-binding molecule has agonistic activity against CD137; and (3) The antigen-binding molecule has a KD value for binding to human CD137 that is equivalent to or 10-fold, 20-fold, 50-fold, or 100-fold lower than a reference antibody comprising the VH sequence of SEQ ID NO: 1 and the VL sequence of SEQ ID NO: 57, wherein the KD value is preferably measured by SPR under the following conditions: 37°C, pH 7.4, 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3; antigen-binding molecules are immobilized on a CM4 sensor chip, and antigen is the analyte. It is measured in [6] The antigen-binding molecule of any one of [1] to [5], further comprising an antibody variable region capable of binding to a third antigen different from CD3 and CD137. [7] The antigen-binding molecule of [6], wherein the third antigen is a molecule that is specifically expressed in cancer tissue. [7A] The antigen-binding molecule of any one of [6] to [7], wherein the third antigen is glypican-3 (GPC3). [7B] The antigen-binding molecule of [7A], wherein the antibody variable region capable of binding to glypican-3 (GPC3) comprises a VH sequence having the amino acid sequence of SEQ ID NO: 206 and a VL sequence having the amino acid sequence of SEQ ID NO: 207. [7C] Any one of the antigen-binding molecules of [6] to [7B], which has at least one characteristic selected from the group consisting of the following (1) to (5): (1) the antigen-binding molecule induces CD3 activation of T cells against cells expressing a molecule of a third antigen, but does not induce CD3 activation of T cells against cells expressing CD137; (2) the antigen-binding molecule induces T cell cytotoxicity against cells expressing a molecule of a third antigen, but does not induce T cell cytotoxicity against cells expressing CD137; (3) the antigen-binding molecule does not induce cytokine release from PBMCs in the absence of cells expressing a molecule of the third antigen; (4) the antigen-binding molecule induces equivalent or 2-fold, 5-fold, 10-fold, 20-fold, or 100-fold higher CD137 activation and / or cytotoxic activity of T cells against cells expressing a molecule of a third antigen compared to a reference antibody comprising the VH sequence of SEQ ID NO: 1 and the VL sequence of SEQ ID NO: 57; and / or (5) The antigen-binding molecule induces 2-fold, 5-fold, 10-fold, 20-fold, or 100-fold higher T cell cytotoxicity against cells expressing a molecule of the third antigen compared to a reference bispecific antibody targeting the third antigen and CD3, while not inducing cytokine (IL-6) release from PBMCs. [8] The antigen-binding molecule of any one of [1] to [7C], further comprising an antibody Fc region. [9] The antigen-binding molecule of [8], wherein the Fc region has reduced FcγR-binding activity compared to the Fc region of a native human IgG1 antibody.

[10] A pharmaceutical composition comprising the antigen-binding molecule according to any one of [1] to [9] and a pharmaceutically acceptable carrier. [10A] The pharmaceutical composition of

[10] or the antigen-binding molecule of [1] to [9] for use in the treatment of cancer. [10B] Use of the pharmaceutical composition of

[10] or the antigen-binding molecule of [1] to [9] for the manufacture of a drug for use in the treatment of cancer. [10C] A method for preventing, treating, or suppressing cancer, comprising administering the pharmaceutical composition of

[10] or the antigen-binding molecule of [5] to [9] to a mammalian subject suffering from cancer. [10D] A method for inducing cytotoxic activity, preferably T cell-dependent cytotoxic activity, in a subject, comprising the step of administering to a mammalian subject suffering from cancer the pharmaceutical composition of

[10] or the antigen-binding molecule of [5] to [9]. [10E] A method for reducing or killing cancer cells in a subject, comprising administering to a mammalian subject suffering from cancer the pharmaceutical composition of

[10] or the antigen-binding molecule of [5] to [9]. [10F] A method for extending the lifespan or survival rate of a cancer patient, comprising administering the pharmaceutical composition of

[10] or the antigen-binding molecule of [5] to [9] to a mammalian subject suffering from cancer. [10G] The pharmaceutical composition for use or antigen-binding molecule, use, or method according to any one of [10A] to [10F], wherein the cancer is characterized by expression or upregulated expression of a third antigen, preferably glypican-3 (GPC3).

[11] An isolated polynucleotide comprising a nucleotide sequence encoding any one of the antigen-binding molecules of [1] to [9].

[12] An expression vector comprising the polynucleotide described in

[11] .

[13] A host cell transformed or transfected with the polynucleotide according to

[11] or the expression vector according to

[12] .

[14] A method for producing a multispecific antigen-binding molecule or a multispecific antibody, comprising the step of culturing the host cell of

[13] .

[15] A multispecific antigen-binding molecule or a multispecific antibody produced by the method of

[14] .

[16] A method for obtaining or screening for an antibody variable region that can bind to CD3 and CD137 but does not simultaneously bind to CD3 and CD137, comprising the steps of: (a) providing a library comprising a plurality of antibody variable regions; (b) contacting the library provided in step (a) with either CD3 or CD137 as a first antigen and collecting antibody variable regions that bind to the first antigen; (c) contacting the antibody variable regions collected in step (b) with a second antigen selected from CD3 and CD137, and collecting the antibody variable regions bound to the second antigen; and (d) an antibody variable region: (1) Preferably, by SPR under the following conditions: 37°C, pH 7.4, 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3; antigen-binding molecules are immobilized on a CM4 sensor chip, and antigen is the analyte. When measured at 1000 kJ / s, it is approximately 5 x 10 -6 Less than M or 5 x 10 -6 M~3×10 -8 an antibody variable region that binds to CD137 with an equilibrium dissociation constant (KD) of M; and / or (2) Preferably by SPR under the following conditions: 25°C, pH 7.4, 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3; antigen-binding molecules are immobilized on a CM4 sensor chip, and antigen is the analyte. When measured at 2 x 10 -6 M~1×10 -8 Antibody variable region that binds to CD3 with an equilibrium dissociation constant (KD) of M A process of selecting. [16A] The method of

[16] , further comprising the step of introducing one or more amino acid modifications into the antibody variable region collected in step (c) between steps (c) and (d).

[17] The method of either

[16] or [16A], wherein the antibody variable region in step (a) or steps (c) and (d) is an antibody variable region having 1 to 25 amino acid modifications, and the modified amino acids are amino acids in a loop, amino acids in the FR3 region, or amino acids selected from positions 31 to 35, 50 to 65, 71 to 74, and 95 to 102, according to the Kabat numbering, in the antibody heavy-chain variable domain, and positions 24 to 34, 50 to 56, and 89 to 97, according to the Kabat numbering, in the antibody light-chain variable domain.

[18] The method of

[17] , wherein the heavy chain variable domain (VH) and / or light chain variable domain (VL) comprise one or more amino acid substitutions selected from Tables 1.3(a)-1.3(d), and the one or more amino acid substitutions exhibit at least a 0.2, 0.3, 0.5, 0.8, 1, 1.5, or 2-fold increase in binding affinity to CD3 and / or CD137 as set forth in Tables 1.3(a)-1.3(d). In some embodiments, the antibody variable region comprises: (a) a heavy chain variable domain amino acid sequence comprising, at each of the following positions (all according to Kabat numbering), one or more of the following amino acid residues as indicated for that position: A, D, E, I, G, K, L, M, N, R, T, W, or Y at amino acid position 26; D, F, G, I, M, or L at amino acid position 27; D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 28; F or W at amino acid position 29; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 30; F, I, N, R, S, T, or V at amino acid position 31; A, H, I, K, L, N, Q, R, S, T, or V at amino acid position 32; W at amino acid position 33; F, I, L, M, or V at amino acid position 34; F, H, S, T, V, or Y at amino acid position 35; E, F, H, I, K, L, M, N, Q, S, T, W, or Y at amino acid position 50; I, K, or V at amino acid position 51; K, M, R, or T at amino acid position 52; A, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y at amino acid position 52b; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 52c; A, E, F, H, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 53; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 54; E, F, G, H, L, M, N, Q, W, or Y at amino acid position 55; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 56; A, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, or V at amino acid position 57; A, F, H, K, N, P, R, or Y at amino acid position 58; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 59; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 60; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 61; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 62; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 63; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 64; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 65; H or R at amino acid position 93; F, G, H, L, M, S, T, V, or Y at amino acid position 94; I or V at amino acid position 95; F, H, I, K, L, M, T, V, W, or Y at amino acid position 96; F, Y, or W at amino acid position 97; A, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 98; A, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 99; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100a; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100b; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100c; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100d; A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y at amino acid position 100e; A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100f; Approximately 100g of amino acids A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; A, D, E, G, H, I, L, M, N, P, S, T, or V at amino acid position 100h; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100i; A, D, F, I, L, M, N, Q, S, T, or V at amino acid position 101; A, D, E, F, G, H, IK, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 102; and / or (b) a light chain variable domain amino acid sequence comprising, at each of the following positions (all according to Kabat numbering), one or more of the following amino acid residues as indicated for that position: A, D, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 24; A, G, N, P, S, T, or V at amino acid position 25; A, D, E, F, G, I, K, L, M, N, Q, R, S, T, or V at amino acid position 26; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 27; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 27a; A, I, L, M, P, T, or V at amino acid position 27b; A, E, F, H, I, K, L, M, N, P, Q, R, T, W, or Y at amino acid position 27c; A, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 27d; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 27e; G, N, S, or T at amino acid position 28; A, F, G, H, K, L, M, N, Q, R, S, T, W, or Y at amino acid position 29; A, F, G, H, I, K, L, M, N, Q, R, V, W, or Y at amino acid position 30; I, L, Q, S, T, or V at amino acid position 31; F, W, or Y at amino acid position 32; A, F, H, L, M, Q, or V at amino acid position 33; A, H, or S at amino acid position 34; I, K, L, M, or R at amino acid position 50; A, E, I, K, L, M, Q, R, S, T, or V at amino acid position 51; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 52; A, E, F, G, H, K, L, M, N, P, Q, R, S, V, W, or Y at amino acid position 53; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 54; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y at amino acid position 55; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 56; A, G, K, S, or Y at amino acid position 89; Q at amino acid position 90; G at amino acid position 91; A, D, H, K, N, Q, R, S, or T at amino acid position 92; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 93; A, D, H, I, M, N, P, Q, R, S, T, or V at amino acid position 94; P at amino acid position 95; F or Y at amino acid position 96; and A, D, E, G, H, I, K, L, M, N, Q, R, S, T, or V at amino acid position 97 It is preferred that the compound contains: In another aspect, the present invention relates to an antigen-binding molecule, e.g., an antibody, that binds to at least one, two, three, or more amino acid residues in the N-terminal region of CD137, which comprises the amino acid sequence of LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAEC (SEQ ID NO: 152) of human CD137, preferably LQDPCSN, NNRNQI, and / or GQRTCDI.

[0018] In some embodiments, the antigen-binding molecules of the present invention can activate T cells through their agonistic activity against CD3, and can induce the cytotoxic activity of T cells against target cells and enhance T cell activation, survival, and differentiation into memory T cells through their costimulatory agonistic activity against CD137 and CD3. On the other hand, the antigen-binding molecules of the present invention do not simultaneously bind to CD3 and CD137, thereby avoiding adverse events caused by cross-linking of CD137 and CD3.

[0019] In some embodiments, the antigen-binding molecules of the present invention can also activate immune cells expressing CD137 through their agonistic activity against CD137, thereby enhancing the immune response to target cells. [Brief explanation of the drawings]

[0020] [Figure 1.1] Measurement of CD3 agonist activity of affinity-matured GPC3 / Dual-Ig variant trispecific antibodies. Mean luminescence units + / - standard deviation (sd) detected by SK-pca60 cell line co-cultured with NFAT-luc2 Jurkat reporter cells at an E:T ratio of 5 for 24 hours with selected antibodies split across plate 1 (top panel) and plate 2 (bottom panel). Antibodies were added at 0.02, 0.2, and 2 nM. [Figure 1.2] Measurement of CD137 agonist activity of affinity-matured GPC3 / Dual-Ig variant trispecific antibodies. Mean luminescence units + / - standard deviation (sd) detected by SK-pca60 cell line co-cultured with Jurkat NFκB reporter cells overexpressing CD137 for 5 hours at an E:T ratio of 5 with selected antibodies split across plate 1 (top panel) and plate 2 (bottom panel). Antibodies were added at 0.5, 2.5, and 5 nM. [Figure 1.3a] Cytotoxic activity against the GPC3-expressing SK-pca60 cell line upon co-culture with PBMCs in the presence of selected GPC3 / Dual-Ig trispecific molecules (Plate 1). Mean % cell growth inhibition values ​​+ / - sd obtained at approximately 120 hours are plotted. [Figure 1.3b] Cytotoxic activity against the GPC3-expressing SK-pca60 cell line upon co-culture with PBMCs in the presence of selected GPC3 / Dual-Ig trispecific molecules (Plate 2). Mean % cell growth inhibition values ​​+ / - sd obtained at approximately 120 hours are plotted. [Figure 1.3c]Cytokine (IFNγ) release measured in co-cultures of SK-pca60 cell lines expressing GPC3 with PBMCs in the presence of selected GPC3 / Dual-Ig trispecific molecules. Co-culture supernatants were analyzed at 48 hours. Graphs show mean IFNγ concentrations + / - sd. Antibodies were split between plate 1 (upper panel) and plate 2 (lower panel) for evaluation. [Figure 1.3d] Cytokine (IL-2) release measured in co-cultures of SK-pca60 cell lines expressing GPC3 with PBMCs in the presence of selected GPC3 / Dual-Ig trispecific molecules. Co-culture supernatants were analyzed at 48 hours. The graph shows the mean IL-2 concentration + / - sd. Antibodies were split into plate 1 (upper panel) and plate 2 (lower panel) for evaluation. [Figure 1.3e] Cytokine (IL-6) release measured in co-cultures of SK-pca60 cell lines expressing GPC3 with PBMCs in the presence of selected GPC3 / Dual-Ig trispecific molecules. Co-culture supernatants were analyzed at 48 hours. The graph shows the mean IL-6 concentration + / - sd. Antibodies were split into plate 1 (upper panel) and plate 2 (lower panel) for evaluation. [Figure 2.1] Design and construction of trispecific antibodies (mAb AB). [Figure 2.2] Naming rules for prepared trispecific antibodies. [Figure 2.3a] Antigen-independent Jurkat activation on GPC3-negative cells. Parental CHO cells were co-cultured with NFAT-luc2 Jurkat reporter cells for 24 hours at E:T 5. Graph illustrating the mean luminescence units + / - standard deviation (sd) of various antibody formats incubated at 0.5, 5, and 50 nM. [Figure 2.3b]Antigen-independent Jurkat activation on GPC3-negative cells. CD137-overexpressing CHO cells were co-cultured with NFAT-luc2 Jurkat reporter cells for 24 hours at E:T 5. Graph illustrating the mean luminescence units + / - standard deviation (sd) of various antibody formats incubated at 0.5, 5, and 50 nM. [Figure 2.4a] Antigen-independent cytokine (IFNγ) release in PBMC solutions. Supernatants of affinity-matured GPC3 / Dual-Ig variants or GPC3 / CD137xCD3 trispecific antibodies added to PBMC solutions at 3.2, 16, and 80 nM were analyzed at 48 hours. Graph shows mean IFNγ concentrations + / - sd. Antibodies were split between plate 1 (top panel) and plate 2 (bottom panel) for evaluation. [Figure 2.4b] Antigen-independent cytokine (TNFα) release in PBMC solutions. Supernatants of affinity-matured GPC3 / Dual-Ig variants or GPC3 / CD137xCD3 trispecific antibodies added to PBMC solutions at 3.2, 16, and 80 nM were analyzed at 48 hours. Graph shows mean TNFα concentrations + / - sd. Antibodies were split between plate 1 (top panel) and plate 2 (bottom panel) for evaluation. [Figure 2.4c] Antigen-independent cytokine (IL-6) release in PBMC solutions. Supernatants of affinity-matured GPC3 / Dual-Ig variants or GPC3 / CD137xCD3 trispecific antibodies added to PBMC solutions at 3.2, 16, and 80 nM were analyzed at 48 hours. Graph shows mean IL-6 concentrations + / - sd. Antibodies were split between plate 1 (top panel) and plate 2 (bottom panel) for evaluation. [Figure 3.1a] In vivo efficacy of antibodies against LLC1 / hGPC3 xenografts in a humanized CD3 / CD137 mouse model. Y-axis represents tumor volume (mm3), and X-axis represents days after tumor implantation. [Figure 3.1b]In vivo efficacy of antibodies against LLC1 / hGPC3 xenografts in a humanized CD3 / CD137 mouse model. Y-axis represents tumor volume (mm3), and X-axis represents days after tumor implantation. [Figure 3.1c] Plasma IL-6 concentrations. Blood was collected from mice 2 hours after antibody injection, and plasma IL-6 concentrations were measured using the Bio-Plex Pro Mouse Cytokine Th1 Panel. [Figure 3.2] In vivo efficacy of antibodies against sk-pca-13a xenografts in the huNOG mouse model. Y-axis represents tumor volume (mm3), and X-axis represents days after tumor implantation. [Figure 3.3a] Epitope of the H0868L0581 Fab contact region on CD137. Epitope mapping in the CD137 amino acid sequence (black: closer than 3.0 Å to H0868L0581; striped: closer than 4.5 Å). [Figure 3.3b] Epitope of the H0868L0581 Fab contact region on CD137. Epitope mapping in the crystal structure (dark grey spheres: closer than 3.0 Å to H0868L0581, light grey bars: closer than 4.5 Å). [Figure 4] 1 shows the design of C3NP1-27, a CD3ε peptide antigen, which is biotinylated with a disulfide bond linker. [Figure 5] Graph showing the results of phage ELISA of clones obtained by phage display against CD3 and CD137. The Y axis indicates the specificity for CD137-Fc, and the X axis indicates the specificity of each clone for CD3. [Figure 6] Graph showing the results of phage ELISA of clones obtained by phage display against CD3 and CD137. The Y axis indicates the specificity of each clone for CD137-Fc in bead ELISA, and the X axis indicates the specificity for CD3 in the same plate ELISA as in FIG. [Figure 7]1 shows comparative data between the amino acid sequence of human CD137 and that of cynomolgus monkey CD137. [Figure 8] Graph showing the results of ELISA of IgG obtained by phage display against CD3 and CD137. The Y axis indicates the specificity of each clone for cynomolgus monkey CD137-Fc, and the X axis indicates the specificity for human CD137. [Figure 9] Graph showing the results of ELISA of IgG obtained by phage display against CD3 and CD137. The Y-axis indicates specificity for CD3e. [Figure 10] Graph showing the results of competitive ELISA of IgG obtained by phage display against CD3 and CD137. The Y axis indicates the ELISA response to biotin-human CD137-Fc or biotin-human Fc. Excess human CD3 or human Fc was used as a competitor. [Figure 11A] 1 is a graph showing the results of phage ELISA of phage display panning output pools against CD3 and CD137. The Y axis indicates specificity for human CD137. The X axis indicates panning output pools, where "initial" indicates the pool before phage display panning, and R1 to R6 indicate the panning output pools after rounds 1 to 6 of phage display panning, respectively. [Figure 11B] 1 is a graph showing the results of phage ELISA of phage display panning output pools against CD3 and CD137. The Y axis indicates specificity for cynomolgus monkey CD137. The X axis indicates panning output pools, where "initial" indicates the pool before phage display panning, and R1 to R6 indicate the panning output pools after rounds 1 to 6 of phage display panning, respectively. [Figure 11C]1 is a graph showing the results of phage ELISA of phage display panning output pools for CD3 and CD137. The Y axis indicates specificity for CD3. The X axis indicates panning output pools, where "initial" indicates the pool before phage display panning, and R1 to R6 indicate the panning output pools after rounds 1 to 6 of phage display panning, respectively. [Figure 12.1] 1 is a series of graphs showing the results of ELISA of IgG obtained by phage display against CD3 and CD137. The Y axis indicates the specificity of each clone for human CD137-Fc, and the X axis indicates the specificity for cynomolgus monkey CD137 or CD3. [Figure 12.2] 1 is a series of graphs showing the results of ELISA of IgG obtained by phage display against CD3 and CD137. The Y axis indicates the specificity of each clone for human CD137-Fc, and the X axis indicates the specificity for cynomolgus monkey CD137 or CD3. [Figure 12.3] 1 is a series of graphs showing the results of ELISA of IgG obtained by phage display against CD3 and CD137. The Y axis indicates the specificity of each clone for human CD137-Fc, and the X axis indicates the specificity for cynomolgus monkey CD137 or CD3. [Figure 13] 1 is a series of graphs showing the results of ELISA of IgG obtained by phage display against CD3 and CD137. The Y axis indicates the specificity of each clone for human CD137-Fc, and the X axis indicates the specificity for cynomolgus monkey CD137 or CD3. [Figure 14] Graph showing the results of competitive ELISA of IgG obtained by phage display against CD3 and CD137. The Y axis represents the ELISA response to biotin-human CD137-Fc or biotin-human Fc. An excess amount of human CD3 was used as a competitor. [Figure 15]Graph showing the results of ELISA of IgG obtained by phage display against CD3 and CD137 to identify the epitope domain of each clone. The Y axis represents the ELISA response to each domain of human CD137. [Figure 16] 1 is a series of graphs showing the results of ELISA of IgG obtained by phage display affinity maturation against CD3 and CD137. The Y axis indicates the specificity of each clone for human CD137-Fc, and the X axis indicates the specificity for cynomolgus monkey CD137 or CD3. [Figure 17.1]

[0023] Figure 1 is a series of graphs showing the results of competitive ELISA of phage-displayed IgG against CD3 and CD137. The Y-axis represents the ELISA response to biotin-human CD137-Fc or biotin-human Fc. An excess of human CD3 was used as a competitor. [Figure 17.2]

[0023] Figure 1 is a series of graphs showing the results of competitive ELISA of phage-displayed IgG against CD3 and CD137. The Y-axis represents the ELISA response to biotin-human CD137-Fc or biotin-human Fc. An excess of human CD3 was used as a competitor. [Figure 17.3]

[0023] Figure 1 is a series of graphs showing the results of competitive ELISA of phage-displayed IgG against CD3 and CD137. The Y-axis represents the ELISA response to biotin-human CD137-Fc or biotin-human Fc. An excess of human CD3 was used as a competitor. [Figure 17.4]

[0023] Figure 1 is a series of graphs showing the results of competitive ELISA of phage-displayed IgG against CD3 and CD137. The Y-axis represents the ELISA response to biotin-human CD137-Fc or biotin-human Fc. An excess of human CD3 was used as a competitor. [Figure 17.5]

[0023] Figure 1 is a series of graphs showing the results of competitive ELISA of phage-displayed IgG against CD3 and CD137. The Y-axis represents the ELISA response to biotin-human CD137-Fc or biotin-human Fc. An excess of human CD3 was used as a competitor. [Figure 18A] FIG. 1 is a diagram showing the mechanism of IL-6 secretion from activated B cells mediated by anti-human GPC3 / Dual-Fab antibody. [Figure 18B] 1 is a graph showing the results of evaluating the CD137-mediated agonistic activity of various anti-human GPC3 / Dual-Fab antibodies based on the level of IL-6 production secreted by activated B cells. Ctrl indicates a negative control human IgG1 antibody. [Figure 19A] FIG. 1 is a diagram showing the mechanism of luciferase expression in activated Jurkat T cells mediated by anti-human GPC3 / Dual-Fab antibodies. [Figure 19B]

[0023] Figure 1 is a series of graphs showing the results of evaluating the CD3-mediated agonist activity of various anti-human GPC3 / Dual-Fab antibodies based on the level of luciferase production expressed in activated Jurkat T cells. Ctrl indicates a negative control human IgG1 antibody. [Figure 20]

[0023] Figure 1 is a series of graphs showing the results of assessing cytokine (IL-2, IFN-γ, and TNF-α) release from human PBMC-derived T cells in the presence of each immobilized antibody. The Y-axis represents the concentration of each secreted cytokine, and the X-axis represents the concentration of immobilized antibody. A control anti-CD137 antibody (B), a control anti-CD3 antibody (CE115), a negative control antibody (Ctrl), and one of the dual antibodies (H183L072) were used for the assay. [Figure 21]This is a series of graphs showing the results of assessing T cell-dependent cytotoxicity (TDCC) of each bispecific antibody against GPC3-positive target cells (SK-pca60 and SK-pca13a). The Y axis represents the percentage of cytostasis (CGI), and the X axis represents the concentration of each bispecific antibody. Anti-GPC3 / Dual bispecific antibody (GC33 / H183L072), negative control / Dual bispecific antibody (Ctrl / H183L072), anti-GPC3 / anti-CD137 bispecific antibody (GC33 / B), and negative control / anti-CD137 bispecific antibody (Ctrl / B) were used for this assay. Five times the amount of effector (E) cells was added to tumor (T) cells (ET5). [Figure 22] 1 is a graph showing the results of cell ELISA of CE115 against CD3e. [Figure 23] FIG. 1 shows the molecular form of EGFR_ERY22_CE115. [Figure 24] 1 is a graph showing the results of TDCC (SK-pca13a) of EGFR_ERY22_CE115. [Figure 25] 1 is an exemplary sensorgram of an antibody with a binding ratio of less than 0.8. [Figure 26]

[0023] Figure 1 is a series of graphs showing the results of Biacore analysis of simultaneous binding of GPC3 / CD137xCD3 trispecific antibody and anti-GPC3 / dual-Fab antibody. The Y-axis represents the binding response to each antigen. First, human CD3 (hCD3) was used as the analyte, followed by hCD3 (shown as a dashed line) or a mixture of human CD137 (hCD137) and hCD3 (shown as a solid line). [Figure 27]27A and 27B are a series of sensorgrams showing the results of FACS analysis of each antibody against CD137-positive CHO cells or Jurkat cells. Figures 27(a) and (c) show the results of binding to human CD137-positive CHO cells, and Figures 27(b) and (d) show the results for parental CHO cells. In Figures 27(a) and (b), the solid lines show the results for the anti-GPC3 / dual antibody (GC33 / H183L072, i.e., GPC33 / H183L072), and the solid areas show the results for the control antibody (Ctrl). In Figures 27(c) and (d), the solid lines, dark gray areas, and light gray areas show the results for the GPC3 / CD137xCtrl trispecific antibody, GPC3 / CD137xCD3 trispecific antibody, and Ctrl / CtrlxCD3 trispecific antibody, respectively. Figures 27(e) and (f) show the results of binding to Jurkat CD3-positive cells. In Figure 27(e), the solid line and the filled area indicate the results for the anti-GPC3 / dual antibody (GC33 / H183L072, i.e., GPC33 / H183L072) and the control antibody (Ctrl), respectively. In Figure 27(f), the solid line, the dark gray filled area, and the light gray filled area indicate the results for the GPC3 / CtrlxCD3 trispecific antibody, the GPC3 / CD137xCD3 trispecific antibody, and the Ctrl / CD137xCtrl trispecific antibody, respectively. [Figure 28] This graph shows the results of evaluating the CD3-mediated agonistic activity of various antibodies against GPC3-positive target cells SK-pca60 by measuring the level of luciferase production expressed in activated Jurkat T cells. Six types of trispecific antibodies, an anti-GPC3 / Dual-Fab antibody (GPC3 / H183L072), and a control / Dual-Fab antibody (Ctrl / H183L072) were used for this assay. The X-axis indicates the concentration of each antibody used. [Figure 29]This graph shows the results of evaluating the CD3-mediated agonistic activity of various antibodies against human CD137-positive CHO cells and parental CHO cells by measuring the level of luciferase production expressed in activated Jurkat T cells. Six types of trispecific antibodies, an anti-GPC3 / Dual-Fab antibody (GPC3 / H183L072), and a control / Dual-Fab antibody (Ctrl / H183L072) were used for this assay. The X-axis indicates the concentration of each antibody used. [Figure 30]

[0023] Figure 1 is a series of graphs showing the results of assessing cytokine (IL-2, IFN-γ, and TNF-α) release from human PBMCs in the presence of each soluble antibody. The Y-axis represents the concentration of each secreted cytokine, and the X-axis represents the concentration of the antibody used. Ctrl / CD137xCD3 trispecific antibody and control / Dual-Fab antibody (Ctrl / H183L072) were used for this assay. DETAILED DESCRIPTION OF THE INVENTION

[0021] Description of Aspects In the present invention, the term "antibody variable region" generally refers to a region containing a domain composed of four framework regions (FR) and three flanking complementarity-determining regions (CDRs), and also includes subsequences thereof, as long as the subsequence has the activity of binding to part or all of an antigen. Regions containing an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH) are particularly preferred. The antibody variable regions of the present invention may have any sequence and may be derived from any antibody, such as mouse, rat, rabbit, goat, or camel antibodies, or humanized antibodies obtained by humanizing any of these non-human antibodies, or human antibodies. "Humanized antibodies," also known as reshaped human antibodies, are obtained by grafting the complementarity-determining regions (CDRs) of an antibody derived from a non-human mammal, such as a mouse antibody, onto the CDRs of a human antibody. Methods for identifying CDRs are known in the art (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institutes of Health, Bethesda, Md.; and Chothia et al., Nature (1989) 342: 877). General recombinant techniques for this purpose are also known in the art (see European Patent Application Publication No. EP 125023 and WO 96 / 02576).

[0022] An "antibody variable region" of the present invention that "does not simultaneously bind to CD3 and CD137 (4-1BB)" means that the antibody variable region of the present invention cannot bind to CD137 when bound to CD3, and conversely, the variable region cannot bind to CD3 when bound to CD137. Here, the phrase "does not simultaneously bind to CD3 and CD137" also includes not cross-linking cells expressing CD3 with cells expressing CD137, or not simultaneously binding to CD3 and CD137 expressed on different cells. This phrase also includes cases where CD3 and CD137 are not expressed on the cell membrane as soluble proteins, or when both are present on the same cell, the variable region can simultaneously bind to both CD3 and CD137, but cannot simultaneously bind to CD3 and CD137 expressed on different cells. Such antibody variable regions are not particularly limited as long as they have these functions. Examples of such antibodies include a variable region derived from an IgG antibody variable region, in which some of the amino acids have been modified to bind to a desired antigen. The modified amino acids are selected from, for example, amino acids in the variable region of an antibody that binds to CD3 or CD137, such that the modification does not abolish antigen binding. Here, the phrase "expressed on different cells" simply means that the antigens are expressed on separate cells, and such cell pairs may be of the same type, such as a T cell and another T cell, or may be of different types, such as a T cell and an NK cell.

[0023] An amino acid modification may be used alone, or multiple amino acid modifications may be used in combination. When multiple amino acid modifications are used in combination, the number of modifications to be combined is not particularly limited and can be appropriately set within the scope that allows the object of the invention to be achieved. The number of modifications to be combined is, for example, 2 to 30, preferably 2 to 25, 2 to 22, 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 2 to 3. The multiple amino acid modifications to be combined may be made only to the heavy or light chain variable domain of the antibody, or may be distributed appropriately among both the heavy and light chain variable domains.

[0024] One or more amino acid residues in the variable region are permissible for modification as long as antigen-binding activity is maintained. When amino acids in the variable region are modified, the resulting variable region preferably maintains the binding activity of the corresponding unmodified antibody, and preferably has a binding activity that is at least 50%, more preferably at least 80%, and even more preferably at least 100% higher than that before modification, although the variable region of the present invention is not limited thereto. The binding activity may be increased by the amino acid modification, for example, by 2-fold, 5-fold, or 10-fold compared to the binding activity before modification.

[0025] Examples of preferred regions for amino acid modification include solvent-exposed regions and loops in the variable region. CDR1, CDR2, CDR3, FR3, and loops are particularly preferred. Specifically, positions 31-35, 50-65, 71-74, and 95-102 (Kabat numbering) in the H-chain variable domain, and positions 24-34, 50-56, and 89-97 (Kabat numbering) in the L-chain variable domain are preferred. Positions 31, 52a-61, 71-74, and 97-101 (Kabat numbering) in the H-chain variable domain, and positions 24-34, 51-56, and 89-96 (Kabat numbering) in the L-chain variable domain are more preferred. Furthermore, amino acids that enhance antigen-binding activity may be introduced during amino acid modification.

[0026] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain whose sequences ("complementarity determining regions" or "CDRs") are hypervariable and / or form structurally defined loops ("hypervariable loops") and / or contain residues that contact the antigen ("antigen contacts"). Generally, antibodies contain six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include the following: (a) hypervariable loops present 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 present at amino acid residues 24–34 (L1), 50–56 (L2), 89–97 (L3), 31–35b (H1), 50–65 (H2), and 95–102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigen contact sites present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) A combination of (a), (b), and / or (c) including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). 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.

[0027] In the present invention, a "loop" means a region containing residues that are not involved in maintaining the β-barrel structure of an immunoglobulin. In the present invention, amino acid modification means substitution, deletion, addition, insertion, or modification, or a combination thereof. In the present invention, amino acid modification is used interchangeably with amino acid mutation, and can be used with the same meaning.

[0028] Substitution of an amino acid residue is carried out, for example, by replacing it with another amino acid residue for the purpose of altering any of the following (a) to (c): (a) the polypeptide backbone structure of a region having a sheet or helix structure; (b) the charge or hydrophobicity of the target site; and (c) the size of the side chain. Amino acid residues are classified into the following groups based on common side chain properties: (1) hydrophobic residues: norleucine, Met, Ala, Val, Leu, and Ile; (2) neutral hydrophilic residues: Cys, Ser, Thr, Asn, and Gln; (3) acidic residues: Asp and Glu; (4) basic residues: His, Lys, and Arg; (5) residues that affect chain orientation: Gly and Pro; and (6) aromatic residues: Trp, Tyr, and Phe.

[0029] Substitution of amino acid residues within each of these groups is referred to as a conservative substitution, while substitution of an amino acid residue in one of these groups with an amino acid residue in another group is referred to as a non-conservative substitution. Substitutions according to the present invention may be conservative or non-conservative substitutions, or a combination of conservative and non-conservative substitutions.

[0030] Modifications of amino acid residues also include the selection of variable regions capable of binding to CD3 and CD137 but incapable of simultaneously binding to these antigens from those obtained by randomly modifying amino acids in the variable regions of antibodies that bind to CD3 or CD137, where such modifications do not abolish antigen-binding; and modifications in which a peptide previously known to have binding activity against a desired antigen is inserted into the above-mentioned regions.

[0031] In the antibody variable region of the present invention, the above-mentioned modifications may be combined with modifications known in the art.For example, the modification of the N-terminal glutamine of the variable region to pyroglutamic acid by pyroglutamylation is a modification well known to those skilled in the art.Therefore, the antibody of the present invention having glutamine at the N-terminus of its heavy chain may contain a variable region in which this N-terminal glutamine is modified to pyroglutamic acid.

[0032] Such antibody variable regions may further have amino acid modifications to improve, for example, antigen binding, pharmacokinetics, stability, or antigenicity. The antibody variable regions of the present invention may be modified so that they have pH-dependent antigen-binding activity and are thereby capable of repeatedly binding to the antigen (WO2009 / 125825).

[0033] Furthermore, for example, amino acid modifications that change the antigen-binding activity depending on the concentration of a target tissue-specific compound may be added to such an antibody variable region that binds to a third antigen (WO2013 / 180200).

[0034] The variable region may be further modified to, for example, enhance avidity, improve specificity, lower pI, confer pH-dependent antigen binding properties, improve thermostability of the bond, improve solubility, improve stability against chemical modification, reduce glycosylation-induced heterogeneity, avoid T cell epitopes identified by in silico prediction or by using in vitro T cell-based assays to reduce immunogenicity, or introduce T cell epitopes to activate regulatory T cells (mAbs 3:243-247, 2011).

[0035] Whether an antibody variable region of the present invention is "capable of binding to CD3 and CD137" can be determined by methods known in the art. This can be determined, for example, by electrochemiluminescence (ECL) (BMC Research Notes 2011, 4:281). Specifically, for example, a region of a biotin-labeled test antigen-binding molecule capable of binding to CD3 and CD137, such as a small antibody composed of the Fab region, or a monovalent antibody thereof (an antibody lacking one of the two Fab regions of a normal antibody), is mixed with CD3 or CD137 labeled with a sulfo-tag (Ru complex), and the mixture is added to a streptavidin-immobilized plate. During this procedure, the biotin-labeled test antigen-binding molecule binds to the streptavidin on the plate. Light is generated from the sulfo-tag, and the luminescence signal is detected using a Sector Imager 600 or 2400 (MSD KK), etc., thereby confirming the binding of the above-mentioned region of the test antigen-binding molecule to CD3 or CD137. Alternatively, the assay may be performed by ELISA, FACS (fluorescence activated cell sorting), ALPHAScreen (amplified luminescence proximity homogeneous assay screen), BIACORE method based on the surface plasmon resonance (SPR) phenomenon, etc. (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0036] Specifically, the assay can be performed using, for example, Biacore (GE Healthcare Japan Corp.), an interaction analysis instrument based on the surface plasmon resonance (SPR) phenomenon. Biacore analysis instruments include any model, such as the Biacore T100, T200, X100, A100, 4000, 3000, 2000, 1000, or C. Any Biacore sensor chip, such as a CM7, CM5, CM4, CM3, C1, SA, NTA, L1, HPA, or Au chip, can be used as the sensor chip. Proteins for capturing the antigen-binding molecules of the present invention, such as protein A, protein G, protein L, anti-human IgG antibody, anti-human IgG-Fab, anti-human L chain antibody, anti-human Fc antibody, antigen protein, or antigen peptide, are immobilized on the sensor chip by a coupling method such as amine coupling, disulfide coupling, or aldehyde coupling. CD3 or CD137 is injected onto the sensor chip as an analyte, and the interaction is measured to obtain a sensorgram. In this procedure, the concentration of CD3 or CD137 can be selected within the range of several μM to several pM according to the strength of the interaction (eg, KD) of the assay sample.

[0037] Alternatively, CD3 or CD137 may be immobilized on a sensor chip instead of an antigen-binding molecule, and then the antibody sample to be evaluated may be allowed to interact with it. Whether the antibody variable region of the antigen-binding molecule of the present invention has binding activity to CD3 or CD137 can be confirmed based on the dissociation constant (KD) value calculated from the sensorgram of the interaction, or based on the degree of increase in the sensorgram after the action of the antigen-binding molecule sample above the level before the action.

[0038] In some embodiments, the binding activity or affinity of an antibody variable region of the present invention for an antigen of interest (i.e., CD3 or CD137) is evaluated, for example, using a Biacore T200 instrument (GE Healthcare) or a Biacore 8K instrument (GE Healthcare) at 37°C (for CD137) or 25°C (for CD3). Anti-human Fc (e.g., GE Healthcare) is immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (e.g., GE Healthcare). The antigen-binding molecule or antibody variable region is captured on the anti-Fc sensor surface, and then the antigen (CD3 or CD137) is injected onto the flow cell. The capture level of the antigen-binding molecule or antibody variable region may aim for 200 resonance units (RU). Recombinant human CD3 or CD137 may be injected at 400 to 25 nM prepared by two-fold serial dilution, followed by dissociation. All antigen-binding molecules or antibody variable regions and analytes are prepared in ACES pH 7.4 containing 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, and 0.005% NaN3. The sensor surface is regenerated with 3 M MgCl2 every cycle. Binding affinity is determined by processing data and fitting to a 1:1 binding model, for example, using Biacore T200 Evaluation software, version 2.0 (GE Healthcare) or Biacore 8K Evaluation software (GE Healthcare). To evaluate the specific binding activity or affinity of the antigen-binding domain of the present invention, KD values ​​are calculated.

[0039] ALPHAScreen is implemented using ALPHA technology, which uses two types of beads (donor and acceptor), based on the following principle: luminescence signals are detected only when a biological interaction between a molecule bound to a donor bead and a molecule bound to an acceptor bead brings these two beads into close proximity. A photosensitizer in the donor bead, excited by a laser, converts ambient oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and reaches the nearby acceptor bead, thereby triggering a chemiluminescent reaction in the bead, ultimately resulting in the emission of light. If there is no interaction between the molecules bound to the donor bead and the molecules bound to the acceptor bead, the singlet oxygen produced by the donor bead will not reach the acceptor bead. Therefore, the chemiluminescent reaction will not occur.

[0040] One of the substances (ligands) whose interaction is to be observed is immobilized on a thin gold film on a sensor chip. Light is shone on the back of the sensor chip to induce total internal reflection at the interface between the gold film and the glass. As a result, a region of reduced reflection intensity (SPR signal) is formed in a portion of the reflected light. The other substance (analyte) whose interaction is to be observed is injected onto the surface of the sensor chip. When the analyte binds to the ligand, the mass of the immobilized ligand molecule increases, causing a change in the refractive index of the solvent on the sensor chip surface. This change in refractive index causes a shift in the position of the SPR signal (conversely, when the bound molecule dissociates, the signal returns to its original position). The Biacore system plots the amount of shift, i.e., the change in mass on the sensor chip surface, on the ordinate, and displays the time-dependent change in mass as assay data (sensorgram). The amount of analyte bound to the ligand captured on the sensor chip surface (the amount of change in response on the sensorgram before and after analyte interaction) can be determined from the sensorgram. However, because the amount of binding also depends on the amount of ligand, comparisons must be performed under conditions using substantially the same amount of ligand. Kinetics, i.e., the association rate constant (ka) and dissociation rate constant (kd), can be determined from the curve of the sensorgram, while affinity (KD) can be determined from the ratio of these constants. Inhibition assays are also suitable for use in the BIACORE method. An example of an inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0041] Whether an antigen-binding molecule of the present invention "does not simultaneously bind to CD3 and CD137" can be confirmed by confirming that the antigen-binding molecule has binding activity to both CD3 and CD137; then, pre-binding either CD3 or CD137 to an antigen-binding molecule containing a variable region having this binding activity; and then determining the presence or absence of its binding activity to the other by the above-mentioned method. Alternatively, this can also be confirmed by determining whether the binding of the antigen-binding molecule to either CD3 or CD137 immobilized on an ELISA plate or sensor chip is inhibited by the addition of the other to the solution. In some embodiments, the binding of the antigen-binding molecule of the present invention to either CD3 or CD137 is inhibited by the binding of the antigen-binding molecule to the other by at least 50%, preferably 60% or more, more preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, or even more preferably 95% or more.

[0042] In one aspect, while one antigen (e.g., CD3) is immobilized, inhibition of binding of the antigen-binding molecule to CD3 can be determined in the presence of another antigen (e.g., CD137) by a method known in the prior art (i.e., ELISA, BIACORE, etc.). In another aspect, while CD137 is immobilized, inhibition of binding of the antigen-binding molecule to CD137 can also be determined in the presence of CD3. When either one of the above two aspects is performed, if binding is inhibited by at least 50%, preferably 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, or even more preferably 95% or more, it is determined that the antigen-binding molecule of the present invention does not bind to CD3 and CD137 simultaneously. In some embodiments, the concentration of the antigen injected as the analyte is at least 1-fold, 2-fold, 5-fold, 10-fold, 30-fold, 50-fold, or 100-fold higher than the concentration of the other antigen that is immobilized. In a preferred mode, the concentration of the antigen injected as analyte is 100 times higher than the concentration of the other antigen to be immobilized, and binding is inhibited by at least 80%. In one embodiment, the ratio of the KD value for the CD3 (analyte)-binding activity of the antigen-binding molecule to the KD value for the CD137 (immobilized)-binding activity of the antigen-binding molecule (KD(CD3) / KD(CD137)) is calculated, and a CD3 (analyte) concentration that is 10-fold, 50-fold, 100-fold, or 200-fold higher than the CD137 (immobilized) concentration by the KD value ratio (KD(CD3) / KD(CD137)) can be used for the above-mentioned competitive measurement. (For example, if the KD value ratio is 0.1, a concentration 1-fold, 5-fold, 10-fold, or 20-fold higher can be selected. Furthermore, if the KD value ratio is 10, a concentration 100-fold, 500-fold, 1000-fold, or 2000-fold higher can be selected.)

[0043] In one aspect, while one antigen (e.g., CD3) is immobilized, the attenuation of the binding signal of the antigen-binding molecule to CD3 can be determined in the presence of another antigen (e.g., CD137) by a method known in the prior art (i.e., ELISA, ECL, etc.). In another aspect, while CD137 is immobilized, the attenuation of the binding signal of the antigen-binding molecule to CD137 can also be determined in the presence of CD3. When either one of the above two aspects is performed, if the binding signal is attenuated by at least 50%, preferably 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, or even more preferably 95% or more, it is determined that the antigen-binding molecule of the present invention does not simultaneously bind to CD3 and CD137. In some embodiments, the concentration of the antigen injected as the analyte is at least 1-fold, 2-fold, 5-fold, 10-fold, 30-fold, 50-fold, or 100-fold higher than the concentration of the other antigen that is immobilized. In a preferred mode, the concentration of the antigen injected as analyte is 100 times higher than the concentration of the other antigen to be immobilized, and binding is inhibited by at least 80%. In one embodiment, the ratio of the KD value for the CD3 (analyte)-binding activity of the antigen-binding molecule to the KD value for the CD137 (immobilized)-binding activity of the antigen-binding molecule (KD(CD3) / KD(CD137)) is calculated, and a CD3 (analyte) concentration that is 10-fold, 50-fold, 100-fold, or 200-fold higher than the CD137 (immobilized) concentration by this KD value ratio (KD(CD3) / KD(CD137)) can be used for the above measurement. (For example, if the KD value ratio is 0.1, a concentration 1-fold, 5-fold, 10-fold, or 20-fold higher can be selected. Furthermore, if the KD value ratio is 10, a concentration 100-fold, 500-fold, 1000-fold, or 2000-fold higher can be selected.)

[0044] Specifically, for example, when using the ECL method, a biotin-labeled test antigen-binding molecule, CD3 labeled with a sulfo-tag (Ru complex), and unlabeled CD137 are prepared. If the test antigen-binding molecule can bind to both CD3 and CD137 but does not simultaneously bind to both CD3 and CD137, the mixture of the test antigen-binding molecule and labeled CD3 is added to a streptavidin-immobilized plate, and the luminescence signal of the sulfo-tag is detected in the absence of unlabeled CD137 by subsequent light emission. In contrast, the luminescence signal decreases in the presence of unlabeled CD137. The decrease in the luminescence signal can be quantified to determine the relative binding activity. This analysis can be similarly performed using labeled CD137 and unlabeled CD3.

[0045] In ALPHAScreen, a test antigen-binding molecule interacts with CD3 in the absence of competing CD137, generating a signal at 520-620 nm. Untagged CD137 competes with CD3 for interaction with the test antigen-binding molecule. The resulting decrease in fluorescence is quantified, thereby determining relative binding activity. Biotinylation of polypeptides using sulfo-NHS-biotin or similar techniques is known in the art. For example, CD3 can be tagged with GST by any suitable method, including fusing a polynucleotide encoding CD3 in frame with a polynucleotide encoding GST; expressing the resulting fusion gene in cells carrying a vector capable of expressing it; and then purifying it using a glutathione column. The resulting signal is preferably analyzed using, for example, the software GRAPHPAD PRISM (GraphPad Software, Inc., San Diego), which is fitted with a one-site competition model based on nonlinear regression analysis. This analysis can be performed similarly using tagged CD137 and untagged CD3. Alternatively, a method using fluorescence resonance energy transfer (FRET) may be used. FRET is a phenomenon in which excitation energy is directly transferred between two closely spaced fluorescent molecules due to electronic resonance. When FRET occurs, the excitation energy of the donor (a fluorescent molecule in an excited state) is transferred to the acceptor (another fluorescent molecule located near the donor), causing the fluorescence emitted from the donor to be quenched (more precisely, the fluorescence lifetime is shortened), and instead, fluorescence is emitted from the acceptor. This phenomenon can be used to analyze whether an antibody simultaneously binds to CD3 and CD137. For example, when CD3 containing a fluorescent donor and CD137 containing a fluorescent acceptor simultaneously bind to a test antigen-binding molecule, the fluorescence of the donor is quenched, while fluorescence is emitted from the acceptor. Therefore, a change in fluorescence wavelength is observed. Such an antibody is confirmed to simultaneously bind to CD3 and CD137. On the other hand, if mixing CD3, CD137, and the test antigen-binding molecule does not change the fluorescence wavelength of the fluorescent donor bound to CD3, the test antigen-binding molecule can be considered to be an antigen-binding domain that can bind to CD3 and CD137 but does not bind to CD3 and CD137 simultaneously.

[0046] For example, a biotin-labeled test antigen-binding molecule is bound to streptavidin on donor beads, while glutathione S-transferase (GST)-tagged CD3 is bound to acceptor beads. The test antigen-binding molecule interacts with CD3 in the absence of a competing second antigen, generating a signal at 520-620 nm. The untagged second antigen competes with CD3 for interaction with the test antigen-binding molecule. The resulting decrease in fluorescence is quantified, thereby determining relative binding activity. Biotinylation of polypeptides using sulfo-NHS-biotin or similar is known in the art. For example, CD3 can be tagged with GST by any suitable method, including fusing a polynucleotide encoding CD3 in frame with a polynucleotide encoding GST; expressing the resulting fusion gene in cells carrying a vector capable of expressing it; and then purifying it using a glutathione column. The resulting signals are preferably analyzed using, for example, the software GRAPHPAD PRISM (GraphPad Software, Inc., San Diego) fitted to a one-site competition model based on nonlinear regression analysis.

[0047] Tagging is not limited to GST tagging, and may be performed with any tag, including, but not limited to, histidine tag, MBP, CBP, Flag tag, HA tag, V5 tag, c-myc tag, etc. Binding of the test antigen-binding molecule to the donor beads is not limited to binding using biotin-streptavidin reaction. In particular, when the test antigen-binding molecule contains Fc, possible methods include binding the test antigen-binding molecule via an Fc-recognizing protein such as protein A or protein G on the donor beads.

[0048] In addition, when CD3 and CD137 are not expressed on the cell membrane as soluble proteins, or when both are present on the same cell, the ability of the variable region to simultaneously bind to CD3 and CD137, but not to CD3 and CD137 expressed on different cells, can also be assayed by methods known in the art. Specifically, a test antigen-binding molecule that has been confirmed to be positive in ECL-ELISA for detecting simultaneous binding to CD3 and CD137 is also mixed with cells expressing CD3 and cells expressing CD137. Unless the antigen-binding molecule and these cells simultaneously bind to each other, the test antigen-binding molecule can be shown not to simultaneously bind to CD3 and CD137 expressed on different cells. This assay can be performed, for example, by cell-based ECL-ELISA. CD3-expressing cells are immobilized on a plate in advance. After the test antigen-binding molecule is bound to it, CD137-expressing cells are added to the plate. Different antigens expressed only on CD137-expressing cells are detected using sulfo-tagged antibodies against these antigens. If the antigen-binding molecule simultaneously binds to two antigens expressed on two cells, respectively, a signal is observed. If the antigen-binding molecule does not simultaneously bind to these antigens, no signal is observed. Alternatively, this assay can be carried out by the ALPHAScreen method. Test antigen-binding molecule is mixed with the cells expressing CD3 bound to donor beads and the cells expressing CD137 bound to acceptor beads. When the antigen-binding molecule simultaneously binds to the two antigens expressed on the two cells, respectively, a signal is observed. When the antigen-binding molecule does not simultaneously bind to these antigens, no signal is observed. Alternatively, this assay can be performed using Octet interaction analysis. First, cells expressing peptide-tagged CD3 are bound to a biosensor that recognizes the peptide tag. CD137-expressing cells and a test antigen-binding molecule are placed in a well and analyzed for interaction. If the antigen-binding molecule simultaneously binds to two antigens expressed on two cells, respectively, a large wavelength shift is observed due to the binding of the test antigen-binding molecule and the CD137-expressing cells to the biosensor. If the antigen-binding molecule does not simultaneously bind to these antigens, a small wavelength shift is observed due to the binding of only the test antigen-binding molecule to the biosensor.

[0049] Instead of these binding activity-based methods, biological activity-based assays can be performed. For example, CD3-expressing cells and CD137-expressing cells are mixed and cultured with a test antigen-binding molecule. When the antigen-binding molecule simultaneously binds to the two antigens, the two antigens expressed on the two cells are mutually activated via the test antigen-binding molecule. Therefore, changes in activation signals, such as increases in the phosphorylation levels downstream of each antigen, can be detected. Alternatively, cytokine production is induced as a result of activation. Therefore, the amount of cytokine produced can be measured, thereby confirming simultaneous binding to the two cells. Alternatively, cytotoxic activity against CD137-expressing cells can be induced as a result of activation. Alternatively, reporter gene expression can be induced by a promoter activated downstream of the CD137 or CD3 signaling pathway as a result of activation. Therefore, the cytotoxic activity or the amount of reporter protein produced can be measured, thereby confirming simultaneous binding to the two cells.

[0050] In one embodiment, the cellular cytotoxic activity is T-cell-dependent cytotoxicity (TDCC). In another embodiment, the cytotoxic activity is cellular cytotoxicity against cells expressing CD3 or CD137 on their surface. The (cellular) cytotoxic activity or TDCC of an antibody (or antigen-binding molecule) of the present invention can be assessed by any suitable method known in the art. For example, TDCC can be measured by a real-time cytostatic assay as described in Example 2.3.2. In this assay, target cells are incubated with T cells (e.g., PBMCs) or expanded T cells in the presence of a test antibody on a 96-well plate, and the proliferation of the target cells is monitored by a method known in the art, for example, by using a suitable analytical instrument (e.g., xCELLigence Real-Time Cell Analyzer). The cytostatic rate (CGI:%) is calculated as follows: CGI(%)=100-(CI Ab ×100 / CI NoAb It is determined from the cell index value according to the formula given as "CI Ab ” represents the cell index value of the well containing the antibody at a particular experimental time, and “CI NoAb " represents the average cell index value of wells containing no antibody. If the CGI rate of an antibody is high, i.e., if it has a significantly positive value, it can be said that the antibody has TDCC activity.

[0051] In a preferred aspect, T cell activation can be assayed by methods known in the art, such as using an engineered T cell line (e.g., Jurkat / NFAT-RE Reporter Cell Line (T Cell Activation Bioassay, Promega)) that expresses a reporter gene (e.g., luciferase) in response to its activation. In this method, target cells (e.g., cells expressing CD3 and cells expressing CD137) are cultured with T cells in the presence of a test antibody, and the level or activity of the reporter gene expression product is then measured by an appropriate method as an indicator of T cell activation. When the reporter gene is a luciferase gene, luminescence resulting from the reaction between luciferase and its substrate may be measured as an indicator of T cell activation. If the T cell activation measured as described above is higher, the test antibody is determined to have higher T cell activation activity. In one aspect, when recombinant T cells that express a reporter gene in response to CD3 signaling are co-cultured with cells expressing CD137 in the presence of the antigen-binding molecule, if the expression of the reporter gene or the activity of the reporter gene product is at most about 50%, 30%, 20%, 10%, 5%, or 1%, it is determined that the antigen-binding molecule does not induce T cell activation against cells expressing CD137, where 100% activation is the level of activation achieved by an antigen-binding molecule that simultaneously binds to CD3 and CD137. In one aspect, when recombinant T cells that express a reporter gene in response to CD3 signaling are co-cultured with cells expressing CD137 in the presence of the antigen-binding molecule, if the expression of the reporter gene or the activity of the reporter gene product is at most about 50%, 30%, 20%, 10%, 5%, or 1%, it is determined that the antigen-binding molecule does not induce T cell activation against cells expressing CD137, where 100% activation is the level of activation achieved by the same antigen-binding molecule against cells expressing a molecule of a third antigen.

[0052] In one embodiment, whether an antigen-binding molecule does not induce cytokine release can be determined, for example, by incubating PBMCs with the antigen-binding molecule and measuring cytokines such as IL-2, IFNγ, and TNFα released from PBMCs into the culture supernatant using methods known in the art. If significant levels of cytokines are not detected or significant cytokine expression is not induced in the culture supernatant of PBMCs incubated with the antigen-binding molecule, the antigen-binding molecule is determined not to induce cytokine release from PBMCs. In one aspect, "significant levels of cytokines are not detected" also refers to a cytokine concentration level that is at most about 50%, 30%, 20%, 10%, 5%, or 1%, where 100% is the cytokine concentration achieved by an antigen-binding molecule that simultaneously binds to CD3 and CD137. In one aspect, "no significant levels of cytokines are detected" also refers to a level of cytokine concentration that is at most about 50%, 30%, 20%, 10%, 5%, or 1%, where 100% is the cytokine concentration achieved in the presence of cells expressing molecules of a third antigen. In one aspect, "no significant induction of cytokine expression" also refers to a level of cytokine concentration increase that is at most 5-fold, 2-fold, or 1-fold the concentration of each cytokine before adding the antigen-binding molecule.

[0053] In the present invention, the term "Fc region" refers to a region of an antibody molecule comprising a hinge or a portion thereof, and a fragment consisting of the CH2 and CH3 domains. The Fc region of an IgG class refers, for example, but is not limited to, the region from cysteine ​​226 (EU numbering (also referred to herein as the EU index)) to the C-terminus, or from proline 230 (EU numbering) to the C-terminus. The Fc region can be obtained, for example, by partially digesting an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody with a protease such as pepsin, followed by re-elution of the fraction adsorbed to a protein A or protein G column. There is no particular limitation on the type of protease, so long as it can digest a full-length antibody to form Fab or F(ab')2 under appropriately selected enzyme reaction conditions (e.g., pH). Examples include pepsin and papain.

[0054] In some embodiments, the "antigen-binding molecule" is not particularly limited as long as it comprises an "antibody variable region" of the present invention. The antigen-binding molecule may further comprise a peptide or protein having a length of approximately 5 amino acids or more. The peptide or protein is not limited to peptides or proteins derived from living organisms and may be, for example, a polypeptide consisting of an artificially designed sequence. Natural polypeptides, synthetic polypeptides, recombinant polypeptides, etc. may also be used.

[0055] In some embodiments, the "antigen-binding molecule" of the present invention is not particularly limited to molecules comprising an "antibody variable region." In certain embodiments, antigen-binding molecules other than antibodies comprising a variable region and capable of binding to two different antigens, such as affibodies, may be obtained by methods commonly known to those skilled in the art (PLoS One. 2011;6(10):e25791; PLoS One. 2012;7(8):e42288; J Mol Biol. 2011 Aug 5;411(1):201-19; Proc Natl Acad Sci US A. 2011 Aug 23;108(34):14067-72).

[0056] Preferred examples of the antigen-binding molecules of the present invention may include antigen-binding molecules comprising an antibody Fc region.

[0057] In the present invention, for example, an Fc region derived from a native IgG can be used as the "Fc region" of the present invention. Here, native IgG refers to a polypeptide that contains the same amino acid sequence as an IgG found in nature and belongs to the class of antibodies substantially encoded by the immunoglobulin γ gene. Native human IgG refers to, for example, native human IgG1, native human IgG2, native human IgG3, or native human IgG4. Native IgG also includes naturally occurring variants thereof. Multiple allotype sequences based on genetic polymorphisms are described in "Sequences of proteins of immunological interest," NIH Publication No. 91-3242, as constant region sequences for human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, and any of these can be used in the present invention. In particular, the sequence of human IgG1 may have DEL or EEM as the amino acid sequence at positions 356 to 358 (EU numbering).

[0058] Antibody Fc regions are found, for example, as IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM-type Fc regions. For example, an Fc region derived from a natural human IgG antibody can be used as the antibody Fc region of the present invention. For example, an Fc region derived from a natural IgG constant region, specifically, a constant region derived from natural human IgG1 (SEQ ID NO: 208), a constant region derived from natural human IgG2 (SEQ ID NO: 209), a constant region derived from natural human IgG3 (SEQ ID NO: 210), or a constant region derived from natural human IgG4 (SEQ ID NO: 211) can be used as the Fc region of the present invention. The constant region of natural IgG also includes naturally occurring variants thereof.

[0059] The Fc region of the present invention is particularly preferably an Fc region with reduced binding activity to an Fcγ receptor. Here, Fcγ receptor (also referred to herein as FcγR) refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, or IgG4, and refers to any member of a protein family substantially encoded by Fcγ receptor genes. In humans, this family includes FcγRI (CD64), which includes the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 (H type) and R131 (R type)), 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); and any unidentified human FcγR or FcγR isoform or allotype, but is not limited thereto. FcγRs include those derived from humans, mice, rats, rabbits, and monkeys. FcγR is not limited to these molecules and may be derived from any organism. Mouse FcγR includes, but is not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any unidentified mouse FcγR or FcγR isoform or allotype. Preferred examples of such Fcγ receptors include human FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16), and / or FcγRIIIb (CD16).

[0060] FcγRs are found in the form of activating receptors with ITAMs (immunoreceptor tyrosine-based activation motifs) and inhibitory receptors with ITIMs (immunoreceptor tyrosine-based inhibitory motifs). FcγRs are classified into activating FcγRs (FcγRI, FcγRIIa R, FcγRIIa H, FcγRIIIa, and FcγRIIIb) and inhibitory FcγRs (FcγRIIb). The polynucleotide and amino acid sequences of FcγRI are set forth in NM_000566.3 and NP_000557.1, respectively; the polynucleotide and amino acid sequences of FcγRIIa are set forth in BC020823.1 and AAH20823.1, respectively; the polynucleotide and amino acid sequences of FcγRIIb are set forth in BC146678.1 and AAI46679.1, respectively; the polynucleotide and amino acid sequences of FcγRIIIa are set forth in BC033678.1 and AAH33678.1, respectively; and the polynucleotide and amino acid sequences of FcγRIIIb are set forth in BC128562.1 and AAI28563.1, respectively (RefSeq accession numbers). FcγRIIa has two genetic polymorphisms in which the 131st amino acid of FcγRIIa is substituted with histidine (H type) or arginine (R type) (J. Exp. Med., 172, 19-25, 1990). FcγRIIb has two genetic polymorphisms in which the 232nd amino acid of FcγRIIb is substituted with isoleucine (I type) or threonine (T type) (Arthritis. Rheum., 46: 1242-1254 (2002)). FcγRIIIa has two genetic polymorphisms in which the 158th amino acid of FcγRIIIa is substituted with valine (V type) or phenylalanine (F type) (J. Clin. Invest., 100(5): 1059-1070 (1997)). There are two types of genetic polymorphisms (NA1 type and NA2 type) in FcγRIIIb (J. Clin. Invest. 85: 1287-1295 (1990)).

[0061] The reduced binding activity to Fcγ receptors can be confirmed by well-known methods such as FACS, ELISA format, ALPHAScreen (amplified luminescence proximity homogeneous assay screen), or the BIACORE method based on the surface plasmon resonance (SPR) phenomenon (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010). The ALPHAScreen method is based on the ALPHA technology, which uses two types of beads (donor and acceptor), and is based on the following principle: a luminescent signal is detected only when a molecule bound to a donor bead and a molecule bound to an acceptor bead are brought into close proximity due to a biological interaction between these two beads. A photosensitizer in the donor bead, excited by a laser, converts ambient oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and reaches the nearby acceptor bead, thereby triggering a chemiluminescent reaction in the bead, ultimately resulting in the emission of light. If there is no interaction between the molecules bound to the donor bead and the molecules bound to the acceptor bead, the singlet oxygen produced by the donor bead will not reach the acceptor bead. Therefore, the chemiluminescent reaction will not occur.

[0062] For example, a biotin-labeled test antigen-binding molecule is bound to donor beads, while an Fcγ receptor tagged with glutathione S-transferase (GST) is bound to acceptor beads. In the absence of a competing antigen-binding molecule with a mutant Fc region, the antigen-binding molecule with a wild-type Fc region interacts with the Fcγ receptor and generates a signal at 520-620 nm. The antigen-binding molecule with an untagged mutant Fc region competes with the antigen-binding molecule with the wild-type Fc region for interaction with the Fcγ receptor. The resulting decrease in fluorescence is quantified, allowing relative binding affinity to be determined. Biotinylation of antigen-binding molecules (e.g., antibodies) using sulfo-NHS-biotin or similar is known in the art. For example, an Fcγ receptor can be tagged with GST by an appropriate method, including fusing a polynucleotide encoding an Fcγ receptor with a polynucleotide encoding GST in frame, expressing the resulting fusion gene using a cell carrying a vector capable of expressing it, and then purifying it using a glutathione column. The resulting signal is preferably analyzed using, for example, the software GRAPHPAD PRISM (GraphPad Software, Inc., San Diego) fitted to a one-site competition model based on nonlinear regression analysis.

[0063] One of the substances (ligands) whose interaction is to be observed is immobilized on a thin gold film on a sensor chip. Light is shone on the back of the sensor chip to cause total internal reflection at the interface between the gold film and the glass. As a result, a region of reduced reflection intensity (SPR signal) is formed in a portion of the reflected light. The other substance (analyte) whose interaction is to be observed is injected onto the surface of the sensor chip. When the analyte binds to the ligand, the mass of the immobilized ligand molecule increases, 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, when the bound molecule dissociates, the signal returns to its original position). The Biacore system plots the amount of shift, i.e., the change in mass on the sensor chip surface, on the ordinate, and displays the time-dependent change in mass as assay data (sensorgram). Kinetics, i.e., the association rate constant (ka) and dissociation rate constant (kd), can be determined from the curve of the sensorgram, while affinity (KD) can be determined from the ratio of these constants. Inhibition assays are also suitable for use in the BIACORE method. Examples of inhibition assays are described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0064] As used herein, "decreased Fcγ receptor-binding activity" means that a test antigen-binding molecule exhibits, based on the above-described analytical method, a binding activity that is, for example, 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 comprising an Fc region. An antigen-binding molecule having an Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be used as a control antigen-binding molecule, as appropriate. The structure of the Fc region is set forth in SEQ ID NO: 212 (RefSeq Accession No. AAC82527.1 with an A added to the N-terminus), SEQ ID NO: 213 (RefSeq Accession No. AAB59393.1 with an A added to the N-terminus), SEQ ID NO: 214 (RefSeq Accession No. CAA27268.1 with an A added to the N-terminus), or SEQ ID NO: 215 (RefSeq Accession No. AAB59394.1 with an A added to the N-terminus). When an antigen-binding molecule having a variant of an Fc region of an antibody of a certain isotype is used as a test substance, the effect of mutations in the variant on Fcγ receptor-binding activity is tested using this antigen-binding molecule having an Fc region of this antibody of a certain isotype as a control. Antigen-binding molecules having an Fc region variant confirmed to have reduced Fcγ receptor-binding activity are appropriately prepared.

[0065] For example, variants such as the 231A-238S deletion (WO 2009 / 011941), C226S, C229S, P238S, (C220S) (J. Rheumatol (2007) 34, 11), C226S, C229S (Hum. Antibod. Hybridomas (1990) 1(1), 47-54), C226S, C229S, E233P, L234V, or L235A (Blood (2007) 109, 1185-1192) (these amino acids are defined according to EU numbering) are known in the art as such variants. Preferred examples include antigen-binding molecules having an Fc region derived from the Fc region of an antibody of a certain isotype by substitution of any of the following amino acids: 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, and 332 (EU numbering). The antibody isotype from which the Fc region is derived is not particularly limited, and Fc regions derived from IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies may be used as appropriate. An Fc region derived from a natural human IgG1 antibody is preferably used. For example, the following substitution groups of constituent amino acids, as defined according to EU numbering (numbers represent the positions of amino acid residues as defined according to EU numbering; the single-letter amino acid code preceding the number represents the amino acid residue before substitution; and the single-letter amino acid code following the number represents the amino acid residue before substitution): (a) L234F, L235E, and P331S, (b) C226S, C229S, and P238S; (c) C226S and C229S, and (d) C226S, C229S, E233P, L234V, and L235A Antigen-binding molecules having an Fc region derived from the IgG1 antibody Fc region by either of the above or by deletion of the amino acid sequence at positions 231 to 238 can also be used appropriately.

[0066] The following substitution groups of constituent amino acids, defined according to EU numbering (numbers represent the positions of amino acid residues defined according to EU numbering; the single-letter amino acid code preceding the number represents the amino acid residue before substitution; and the single-letter amino acid code following the number represents the amino acid residue before substitution): (e) H268Q, V309L, A330S, and P331S; (f) V234A, (g) G237A, (h) V234A and G237A, (i) A235E and G237A, and (j) V234A, A235E, and G237A Antigen-binding molecules having an Fc region derived from the IgG2 antibody Fc region, such as those described above, can also be used appropriately.

[0067] The following substitution groups of constituent amino acids, defined according to EU numbering (numbers represent the positions of amino acid residues defined according to EU numbering; the single-letter amino acid code preceding the number represents the amino acid residue before substitution; and the single-letter amino acid code following the number represents the amino acid residue before substitution): (k)F241A, (l) D265A, and (m)V264A Antigen-binding molecules having an Fc region derived from an IgG3 antibody Fc region, such as those described above, can also be used appropriately.

[0068] The following substitution groups of constituent amino acids, defined according to EU numbering (numbers represent the positions of amino acid residues defined according to EU numbering; the single-letter amino acid code preceding the number represents the amino acid residue before substitution; and the single-letter amino acid code following the number represents the amino acid residue before substitution): (n) L235A, G237A, and E318A, (o) L235E, and (p)F234A and L235A Antigen-binding molecules having an Fc region derived from an IgG4 antibody Fc region, such as those described above, can also be used appropriately.

[0069] Other preferred examples include antigen-binding molecules having an Fc region derived from the Fc region of a native human IgG1 antibody by substitution of any of the following amino acids at positions 233, 234, 235, 236, 237, 327, 330, and 331, as defined according to EU numbering, with amino acids at the corresponding positions according to EU numbering in the Fc region of the counterpart IgG2 or IgG4.

[0070] Other preferred examples include antigen-binding molecules having an Fc region derived from the Fc region of a native human IgG1 antibody by substitution of one or more of the following amino acids: 234, 235, and 297, as defined according to EU numbering, with different amino acids. The type of amino acid present after substitution is not particularly limited. 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 are substituted with alanine.

[0071] Other preferred examples include antigen-binding molecules having an Fc region derived from the Fc region of an IgG1 antibody by substituting a different amino acid for the amino acid at position 265 (as defined by EU numbering). The type of amino acid present after substitution is not particularly limited. Particularly preferred are antigen-binding molecules having an Fc region in which the amino acid at position 265 is substituted with alanine.

[0072] One preferred form of the "antigen-binding molecule" of the present invention may be, for example, a multispecific antibody comprising the antibody variable region of the present invention.

[0073] For the association of multispecific antibodies, a technique can be applied that suppresses unintended association between heavy chains by introducing charge repulsion at the interface between the second constant domain (CH2) or the third constant domain (CH3) of the antibody heavy chains (WO2006 / 106905). In a technique for suppressing unintended association between H chains by introducing charge repulsion at the CH2 or CH3 interface, examples of amino acid residues that contact each other at the interface between H chain constant domains include residues at EU numbering positions 356, 439, 357, 370, 399, and 409 in one CH3 domain, and their partner residues in the other CH3 domain.

[0074] More specifically, for example, an antibody comprising two H-chain CH3 domains can be prepared as an antigen-binding molecule in which one to three pairs of amino acid residues selected from the following pairs of amino acid residues (1) to (3) in the first H-chain CH3 domain have the same electric charge: (1) amino acid residues at positions 356 and 439 (EU numbering) contained in the H-chain CH3 domain; (2) amino acid residues at positions 357 and 370 (EU numbering) contained in the H-chain CH3 domain; and (3) amino acid residues at positions 399 and 409 (EU numbering) contained in the H-chain CH3 domain.

[0075] The antibody can also be prepared as an antibody in which one to three pairs of amino acid residues are selected from pairs of amino acid residues (1) to (3) in a second H-chain CH3 domain that is different from the first H-chain CH3 domain, so that they correspond to pairs of amino acid residues (1) to (3) in the first H-chain CH3 domain that have the same charge but have the opposite charge to the corresponding amino acid residues in the first H-chain CH3 domain.

[0076] Each amino acid residue in pairs (1) to (3) is located near its partner in the associated H chain. Those skilled in the art can identify the positions corresponding to the amino acid residues in each of pairs (1) to (3) for a desired H chain CH3 domain or H chain constant domain by homology modeling using commercially available software, and can appropriately modify the amino acid residues at those positions.

[0077] In the above-described antibodies, each of the "charged amino acid residues" is preferably selected from, for example, amino acid residues included in either of the following groups (a) and (b): (a) glutamic acid (E) and aspartic acid (D); and (b) Lysine (K), arginine (R), and histidine (H).

[0078] In the above-described antibodies, the phrase "having the same charge" means, for example, that all of the two or more amino acid residues are amino acid residues included in either one of groups (a) and (b). The phrase "having opposite charges" means, for example, that at least one amino acid residue among the two or more amino acid residues may be an amino acid residue included in either one of groups (a) and (b), while the remaining amino acid residues are amino acid residues included in the other group.

[0079] In a preferred embodiment, the antibody may have a first H chain CH3 domain and a second H chain CH3 domain cross-linked by a disulfide bond. The amino acid residues to be modified according to 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 the amino acid residues that constitute the interface of a polypeptide variant or heteromultimer by homology modeling using commercially available software, and can modify the amino acid residues at those positions to control the association.

[0080] The assembly of the multispecific antibodies of the present invention can also be achieved by alternative techniques known in the art. An amino acid side chain present in the variable domain of one antibody heavy chain is replaced with a larger side chain (knob), and its partner amino acid side chain present in the variable domain of the other heavy chain is replaced with a smaller side chain (hole). The knob can be positioned in the hole to ensure efficient assembly of polypeptides of Fc domains with different amino acid sequences (WO1996 / 027011; Ridgway JB et al., Protein Engineering (1996) 9, 617-621; and Merchant AM et al. Nature Biotechnology (1998) 16, 677-681).

[0081] In addition to this technique, further alternative techniques known in the art can be used to form the multispecific antibodies of the present invention. A portion of the CH3 of one antibody's H chain is converted to its corresponding IgA-derived sequence, and the complementary portion of the CH3 of the other antibody's H chain is converted to its corresponding IgA-derived sequence. The resulting chain-exchange engineered domain CH3 can be used to efficiently associate polypeptides with different sequences through complementary CH3 association (Protein Engineering Design & Selection, 23; 195-202, 2010). This technique, known in the art, can also be used to efficiently form the desired multispecific antibodies.

[0082] Alternatively, multispecific antibodies can be formed by, for example, antibody preparation techniques using antibody CH1-CL association and VH-VL association as described in WO2011 / 028952, techniques for preparing bispecific antibodies using separately prepared monoclonal antibodies (Fab arm exchange) as described in WO2008 / 119353 and WO2011 / 131746, techniques for controlling the association between antibody heavy chain CH3 domains as described in WO2012 / 058768 and WO2013 / 063702, techniques for preparing bispecific antibodies composed of two types of light chains and one type of heavy chain as described in WO2012 / 023053, or techniques for preparing bispecific antibodies using two bacterial cell lines each expressing antibody half molecules consisting of one H chain and one L chain as described in Christoph et al. (Nature Biotechnology Vol. 31, p 753-758 (2013)). In addition to these assembly techniques, CrossMab technology (Scaefer et al., Proc. Natl. Acad. Sci. USA (2011) 108, 11187-11192), a known heterologous light chain assembly technique in which a light chain forming a variable region binding to a first epitope and a light chain forming a variable region binding to a second epitope are assembled with a heavy chain forming a variable region binding to a first epitope and a heavy chain forming a variable region binding to a second epitope, respectively, can also be used to prepare the multispecific or multiparatopic antigen-binding molecules provided by the present invention. An example of a technique for preparing bispecific antibodies using separately prepared monoclonal antibodies includes a method comprising subjecting a monoclonal antibody in which specific amino acids have been substituted in the heavy chain CH3 domain to reducing conditions to promote antibody heterodimerization and obtain the desired bispecific antibody. Examples of preferred amino acid substitution sites for this method include residues at EU numbering positions 392 and 397 in the CH3 domain.Furthermore, bispecific antibodies can also be prepared by using antibodies in which one to three pairs of amino acid residues selected from the following pairs of amino acid residues (1) to (3) in a first H-chain CH3 domain have the same electric charge: (1) the amino acid residues at positions 356 and 439 (EU numbering) in the H-chain CH3 domain; (2) the amino acid residues at positions 357 and 370 (EU numbering) in the H-chain CH3 domain; and (3) the amino acid residues at positions 399 and 409 (EU numbering) in the H-chain CH3 domain. Bispecific antibodies can also be prepared by using antibodies in which one to three pairs of amino acid residues are selected from pairs of amino acid residues (1) to (3) in a second H-chain CH3 domain that is different from the first H-chain CH3 domain so that they correspond to pairs of amino acid residues (1) to (3) in the first H-chain CH3 domain that have the same electric charge but have the opposite electric charge to the corresponding amino acid residues in the first H-chain CH3 domain.

[0083] Even if the desired multispecific antibody cannot be efficiently formed, the multispecific antibody of the present invention can be obtained by separating and purifying the desired multispecific antibody from the produced antibodies. For example, a previously reported method involves introducing amino acid substitutions into the variable domains of two types of H chains to impart a difference in isoelectric point, so that two types of homodimers and the desired heterodimerized antibody can be separately purified by ion exchange chromatography (WO2007114325). A method for purifying heterodimerized antibodies consisting of a mouse IgG2a H chain capable of binding to Protein A and a rat IgG2b H chain that cannot bind to Protein A has previously been reported (WO98050431 and WO95033844). Alternatively, the amino acid residues at positions 435 and 436 (EU numbering), which constitute the Protein A-binding site of IgG, can be substituted with amino acids such as Tyr and His, which provide different Protein A binding strengths, and the resulting H chains are used to alter the interaction of each H chain with Protein A. As a result, only the heterodimerized antibody can be efficiently purified by use of a Protein A column.

[0084] Multiple techniques, for example, two or more, may be used in combination. These techniques may also be applied separately to the two H chains to be associated. The antigen-binding molecules of the present invention may be prepared based on such modified forms, but may also be prepared as antigen-binding molecules having the same amino acid sequence.

[0085] Amino acid sequence alterations can be accomplished by a variety of methods known in the art. Examples of these methods that may be performed include 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 (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456). HJ (1987) 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), PCR mutagenesis, and cassette mutagenesis may be used, but are not limited to these methods.

[0086] The "antigen-binding molecule" of the present invention may be an antibody fragment that contains both the heavy and light chains constituting the "antibody variable region" of the present invention in a single polypeptide chain, but lacks the constant region. Such an antibody fragment may be, for example, a diabody (Db), a single-chain antibody, or sc(Fab')2.

[0087] Db is a dimer composed of two polypeptide chains (e.g., Holliger P et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); EP404,097; and WO93 / 11161). These polypeptide chains are connected via a linker that is so short, for example, about 5 residues, that the light chain variable domain (VL) and the heavy chain variable domain (VH) on the same polypeptide chain cannot pair with each other. Because of this short linker, the VL and VH encoded on the same polypeptide chain cannot form a single-chain Fv, but instead dimerize with the VH and VL, respectively, on separate polypeptide chains to form two antigen-binding sites.

[0088] Examples of single-chain antibodies include sc(Fv)2. sc(Fv)2 is a single-chain antibody having one chain composed of four variable domains, i.e., two VL and two VH, linked via a linker such as a peptide linker (J Immunol. Methods (1999) 231 (1-2), 177-189). These two VH and VL may be derived from different monoclonal antibodies. Preferred examples include bispecific sc(Fv)2, which recognizes two different epitopes present in the same antigen, as disclosed in Journal of Immunology (1994) 152 (11), 5368-5374. sc(Fv)2 can be prepared by methods commonly known to those skilled in the art. For example, sc(Fv)2 can be prepared by connecting two scFvs via a linker such as a peptide linker.

[0089] Examples of the configuration of the antigen-binding domain that constitutes the sc(Fv)2 described herein include antibodies in which two VHs and two VLs are arranged in the order of VH, VL, VH, and VL (i.e., [VH]-linker-[VL]-linker-[VH]-linker-[VL]) starting from the N-terminus of the single-chain polypeptide. The order of the two VHs and two VLs is not particularly limited to the above configuration and may be in any order. Examples also include the following configuration: [VL]-linker-[VH]-linker-[VH]-linker-[VL], [VH]-linker-[VL]-linker-[VL]-linker-[VH], [VH]-linker-[VH]-linker-[VL]-linker-[VL], [VL]-linker-[VL]-linker-[VH]-linker-[VH], and [VL]-linker-[VH]-linker-[VL]-linker-[VH].

[0090] The molecular form of sc(Fv)2 is also described in detail in WO2006 / 132352. Based on the description therein, a person skilled in the art can appropriately prepare a desired sc(Fv)2 in order to prepare the antigen-binding molecules disclosed herein.

[0091] The antigen-binding molecules of the present invention may be conjugated to a carrier polymer such as PEG or an organic compound such as an anticancer agent. Furthermore, a glycosylation sequence can be inserted to suitably add a sugar chain to the antigen-binding molecules of the present invention to produce a desired effect.

[0092] For example, any peptide linker that can be introduced by genetic engineering or a synthetic compound linker (e.g., a linker disclosed in Protein Engineering, 9 (3), 299-305, 1996) can be used as a linker for linking antibody variable domains. In the present invention, peptide linkers are preferred. The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art depending on the purpose. The length is preferably 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), and particularly preferably 15 amino acids. When sc(Fv)2 contains three peptide linkers, all of these peptide linkers used may be the same length or different lengths.

[0093] Examples of peptide linkers include: Ser, Gly-Ser, Gly-Gly-Ser, Ser-Gly-Gly, Gly-Gly-Gly-Ser (SEQ ID NO: 216), Ser-Gly-Gly-Gly (SEQ ID NO: 217), Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 218), Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 219), Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 220), Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 221), Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 222), Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 223), (Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 218))n, and (Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 219))n, (where n is an integer of 1 or greater). However, the length or sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0094] The synthetic compound linker (chemical crosslinker) is a crosslinker commonly used for crosslinking 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), or bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES). These crosslinkers are commercially available. Three linkers are usually required to link four antibody variable domains, and all of the linkers used may be the same or different linkers.

[0095] F(ab')2 comprises two light chains and two heavy chains containing constant regions (part of the CH1 domain and CH2 domain) such that interchain disulfide bonds are formed between the two heavy chains. The F(ab')2 that constitutes the polypeptide complexes disclosed herein can be preferably obtained, for example, by partially digesting a full-length monoclonal antibody containing the desired antigen-binding domain with a protease such as pepsin, followed by removal of the Fc fragment adsorbed to a Protein A column. There is no particular limitation on the protease used, as long as it is capable of digesting the full-length antibody to form F(ab')2 under appropriately selected enzyme reaction conditions (e.g., pH). Examples include pepsin and ficin.

[0096] In addition to the amino acid modifications described above, the antigen-binding molecules of the present invention may further contain additional modifications. The additional modifications may be selected from, for example, amino acid substitutions, deletions, and modifications, and combinations thereof. For example, the antigen-binding molecules of the present invention can be further modified arbitrarily without substantially changing the intended function of the molecule. Such mutations can be made, for example, by conservative substitution of amino acid residues. Alternatively, modifications that alter the intended function of the antigen-binding molecules of the present invention may also be made, as long as the altered function is within the scope of the present invention.

[0097] The amino acid sequence modification according to the present invention also includes post-translational modification. Specifically, post-translational modification can refer to the addition or deletion of a sugar chain. For example, an antigen-binding molecule of the present invention having an IgG1-type constant region can have a sugar chain-modified amino acid residue at EU numbering position 297. The sugar chain structure used in the modification is not limited. Generally, antibodies expressed by eukaryotic cells contain sugar chain modifications in the constant region. Therefore, antibodies expressed by the following cells are usually modified with several sugar chains: mammalian antibody-producing cells; and A eukaryotic cell transformed with an expression vector containing DNA encoding an antibody. Here, eukaryotic cells include yeast and animal cells. For example, CHO cells or HEK293H cells are typical animal cells for transformation with an expression vector containing antibody-encoding DNA. On the other hand, the antibodies of the present invention also include antibodies that are not glycosylated at that position. Antibodies with constant regions that are not glycosylated can be obtained by expressing genes encoding these antibodies in prokaryotic cells such as E. coli.

[0098] More specifically, the additional modification according to the present invention may be, for example, the addition of sialic acid to a sugar chain in the Fc region (mAbs. 2010 Sep-Oct;2(5):519-27).

[0099] When the antigen-binding molecule of the present invention has an Fc region, for example, amino acid substitutions may be made to improve binding activity to FcRn (J Immunol. 2006 Jan 1;176(1):346-56; J Biol Chem. 2006 Aug 18;281(33):23514-24; Int Immunol. 2006 Dec;18(12):1759-69; Nat Biotechnol. 2010 Feb;28(2):157-9; WO2006 / 019447; WO2006 / 053301; and WO2009 / 086320), or to improve antibody heterogeneity or stability ((WO2009 / 041613)).

[0100] In the present invention, the term "antibody" is used in the broadest sense and includes any antibody, such as a monoclonal antibody (including a full-length monoclonal antibody), a polyclonal antibody, an antibody variant, an antibody fragment, a multispecific antibody (e.g., a bispecific antibody), a chimeric antibody, and a humanized antibody, so long as it exhibits the desired biological activity.

[0101] The antibody of the present invention is not limited by the type of its antigen, its origin, etc., and may be any antibody. Examples of antibody origins include, but are not limited to, human antibodies, mouse antibodies, rat antibodies, and rabbit antibodies.

[0102] Antibodies can be prepared by methods well known to those skilled in the art. For example, monoclonal antibodies can be produced by hybridoma method (Kohler and Milstein, Nature 256:495 (1975)) or recombinant method (U.S. Patent No. 4,816,567). Alternatively, monoclonal antibodies can be isolated from phage display antibody libraries (Clackson et al., Nature 352:624-628 (1991); and Marks et al., J. Mol. Biol. 222:581-597 (1991)). Monoclonal antibodies can also be isolated from single B cell clones (N. Biotechnol. 28(5): 253-457 (2011)).

[0103] Humanized antibodies are also called reshaped human antibodies. Specifically, for example, humanized antibodies consisting of human antibodies grafted with CDRs from non-human animal (e.g., mouse) antibodies are known in the art. General genetic recombination techniques for obtaining humanized antibodies are also known. Specifically, for example, overlap extension PCR is known in the art as a method for grafting CDRs from mouse antibodies onto human FRs.

[0104] A vector for expressing a humanized antibody can be prepared by inserting DNA encoding an antibody variable domain, each containing three linked CDRs and four FRs, and DNA encoding a human antibody constant domain into an expression vector so that the variable domain DNA is fused in frame with the constant domain DNA. These vectors with inserts are introduced into a host to establish recombinant cells. The recombinant cells are then cultured to express the DNA encoding the humanized antibody, and the humanized antibody is produced in culture (see European Patent Publication No. EP 239400 and International Publication No. WO1996 / 002576).

[0105] If necessary, amino acid residues in the FR may be substituted so that the CDRs of the reshaped human antibody form an appropriate antigen-binding site. For example, the amino acid sequence of the FR can be mutated by applying the PCR method used in grafting mouse CDRs onto human FRs.

[0106] The desired human antibody can be obtained by DNA immunization using transgenic animals carrying a full repertoire of human antibody genes (see International Publication Nos. WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, and WO1996 / 033735) as immunized animals.

[0107] In addition, techniques for obtaining human antibodies by panning using a human antibody library are also known. For example, human antibody V regions are expressed as single-chain antibodies (scFvs) on the surface of phages using phage display methods. Phages expressing antigen-binding scFvs can be selected. The genes of the selected phages can be analyzed to determine the DNA sequence encoding the V region of the antigen-binding human antibody. After determining the DNA sequence of the antigen-binding scFv, the V region sequence can be fused in frame with the sequence of the C region of the desired human antibody and then inserted into an appropriate expression vector to prepare an expression vector. The expression vector is then transfected into the preferred expression cells listed above for the expression of genes encoding the human antibody to obtain a human antibody. These methods are known in the art (see International Publication Nos. WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, and WO1995 / 015388).

[0108] In addition to phage display technology, techniques using cell-free translation systems, techniques displaying antigen-binding molecules on the surface of cells or viruses, and emulsion-based techniques are known as techniques for obtaining human antibodies by panning using human antibody libraries. For example, ribosome display, which involves forming a complex between mRNA and the translated protein via ribosomes by removing a stop codon, cDNA or mRNA display, which involves covalently linking the translated protein to a gene sequence using a compound such as puromycin, or CIS display, which involves forming a complex between a gene and the translated protein using a nucleic acid-binding protein, can be used as a cell-free translation system-based technique. Phage display, as well as E. coli display, Gram-positive bacteria display, yeast display, mammalian cell display, and virus display can be used as techniques for displaying antigen-binding molecules on the surface of cells or viruses. For example, in vitro virus display, which uses genes and translation-related molecules encapsulated in an emulsion, can be used as an emulsion-based technique. These methods are known in the art (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; and Methods Mol Biol. 2012;911:183-98).

[0109] The variable region that binds to the third antigen of the present invention may be a variable region that recognizes any antigen, or may be a variable region that recognizes a molecule that is specifically expressed in cancer tissue.

[0110] As used herein, the term "third antigen" is not particularly limited and may be any antigen. Examples of antigens include 17-IA, 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, and activin RIB. ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, adiponectin, ADP-ribosyl cyclase-1, aFGF, AGE, ALCAM, ALK, ALK-1, ALK-7, allergen, α1-antichemotrypsin, α1-antitrypsin, α-synuclein, α-V / β-1 Antagonist, aminin, amylin, amyloid beta, amyloid immunoglobulin heavy chain variable region, amyloid immunoglobulin light chain variable region, androgen, ANG, angiotensinogen, angiopoietin ligand-2, anti-Id, antithrombin III, anthrax, APAF-1, APE, APJ, apoA1, apo-serum amyloid A, Apo-SAA, APP, APRIL, AR, ARC, ART, artemin, ASPARTIC, atrial natriuretic factor, atrial natriuretic peptide, atrial natriuretic peptide A, atrial natriuretic peptide B, atrial natriuretic peptide C, av / b3 integrin, Axl, B7-1, B7-2, B7-H, BACE, BACE-1, Bacillus anthracis anthracis) protective antigen, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, BcI, BCMA, BDNF, b-ECGF, β-2-microglobulin, β-lactamase, bFGF, BID, Bik, BIM, BLC, BL -CAM, BLK, B lymphocyte stimulating factor (BlyS), BMP, BMP-2 (BMP-2a), BMP-3 (osteogenin), BMP-4 (BMP-2b), BMP-5, BMP-6 (Vgr-1), BMP-7 (OP-1), BMP-8 (BMP-8a), BMPR,BMPR-IA (ALK-3), BMPR-IB (ALK-6), BMPR-II (BRK-3), BMPs, BOK, bombesin, bone-derived neurotrophic factor, bovine growth hormone, BPDE, BPDE-DNA, BRK-2, BTC, B-lymphocyte cell adhesion molecule, C10, C1 inhibitor, C1q, C3, C3a, C4, C5, C5a (complement 5a), CA125, CAD-8, cadherin-3, calcitonin, cAMP, carbonic anhydrase-IX, carcinoembryonic antigen (CEA), cancer-associated antigen, cardiotrophin-1, 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 / I-309, CCL11 / eotaxin, CCL12 / MCP-5, CCL13 / MCP-4, CCL14 / HCC-1, CCL15 / HCC-2, CCL16 / HCC-4, CCL17 / TARC, CCL18 / PARC, CCL19 / ELC, CCL2 / MCP-1, CCL20 / MIP-3-α, CCL21 / SLC, CCL22 / MDC, CCL23 / MPIF-1, CCL24 / eotaxin-2, CCL25 / TECK, CCL26 / eotaxin-3, CCL27 / CTACK, CCL28 / MEC, CCL3 / M1P-1-α, CCL3Ll / LD-78-β, CCL4 / MIP-l-β, CCL5 / RANTES, C CL6 / C10, CCL7 / MCP-3, CCL8 / MCP-2, CCL9 / 10 / MTP-1-γ, CCR, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD10, CD105, CD11a, CD1 1b, CD11c, CD123, CD13, CD137, CD138, CD14, CD140a, CD146, CD147, CD148, CD15, CD152, CD16, CD164, CD18, CD19, CD2, CD20, CD21, CD22, CD23, CD25, CD 26, CD27L, CD28, CD29, CD3, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD37, CD38, CD3E, CD4, CD40, CD40L, CD44, CD45, CD46, CD49a, CD49b, CD5, CD51,CD52, CD54, CD55, CD56, CD6, CD61, CD64, CD66e, CD7, CD70, CD74, CD8, CD80 (B7-1), CD89, CD95, CD105, CD158a, CEA, CEACAM5, CFTR, cGMP, CGRP receptor, CINC, CKb8-1, claudin 18, CLC, Clostridium botulinum toxin, Clostridium difficile toxin, Clostridium perfringens toxin, c-Met, CMV, CMV UL, CNTF, CNTN-1, complement factor 3 (C3), complement factor D, corticosteroid-binding globulin, colony-stimulating factor-1 receptor, COX, C-Ret, CRG-2, CRTH2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1 / fractalkine, CX3CR1, CXCL, CXCL1 / Gro-α, CXCL10, CXCL11 / I-TAC, CXCL12 / SDF -l-α / β, CXCL13 / BCA-1, CXCL14 / BRAK, CXCL15 / Lungkine, CXCL16, CXCL16, CXCL2 / Gro-β, CXCL3 / G ro-γ, CXCL3, CXCL4 / PF4, CXCL5 / ENA-78, CXCL6 / GCP-2, CXCL7 / NAP-2, CXCL8 / IL-8, CXCL9 / Mig, CXCLlO / IP- 10, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cystatin C, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, Delta-like protein ligand 4, des(1-3)-IGF-1 (brain IGF-1), Dhh, DHICA oxidase, Dickkopf-1, digoxin, dipeptidyl peptidase IV, DKl, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EGF-like domain-containing protein 7, elastase, elastin, EMA, EMMPRIN, ENA, ENA-78, endosialin, endothelin receptor, endotoxin, enkephalinase, eNOSEot, eotaxin, eotaxin-2, eotaxini, EpCAM, ephrin B2 / EphB4, Epha2 tyrosine kinase receptor, epidermal growth factor receptor (EGFR), ErbB2 receptor, ErbB3 tyrosine kinase receptor, ERCC, EREG, erythropoietin (EPO), erythropoietin receptor, E-selectin, ET-1, Exodus-2, RSV F protein, F10, F11, F12, F13, F5, F9, factor Ia, factor IX, factor Xa, factor VII, factor VIII, factor VIIIc, Fas, FcαR, FcεRI, FcγIIb, FcγRI, FcγR IIa, FcγRIIIa, FcγRIIIb, FcRn, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF-2 receptor, FGF-3, FGF-8, FGF-acidic, FGF-basic, fibrin, fibroblast activation protein (FAP), fibroblast growth factor, fibroblast growth factor-10, fibronectin, FL, FLIP, Flt-3, FLT3 ligand, folate receptor, follicle-stimulating hormone (FSH), fractalkine (CX3C), free heavy chain, free light chain, FZD1, FZD10, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, G250, Gas 6, GCP-2, GCSF, G-CSF, G-CSF receptor, GD2, GD3, GDF, GDF-1, GDF-15 (MIC-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, GDNF, gelsolin, GFAP, GF-CSF, GFR-α1, GFR-α2, GFR-α3, GF-β1, gH envelope glycoprotein, GITR, glucagon, glucagon receptor, glucagon-like peptide 1 receptor, Glut 4, glutamate carboxypeptidase II, glycoprotein hormone receptor, glycoprotein IIb / IIIa (GP) IIb / IIIa), glypican-3, GM-CSF, GM-CSF receptor, gp130, gp140, gp72, granulocyte-CSF (G-CSF), GRO / MGSA, growth hormone-releasing factor, GRO-β, GRO-γ, H. pylori,Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCC 1, HCMV gB envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, heparin cofactor II, hepatic growth factor, anthrax protective antigen, hepatitis C virus E2 glycoprotein, hepatitis E, hepcidin, Her1, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HGF, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV envelope proteins such as GP120, HIV MIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HMGB-1, HRG, Hrk, HSP47, Hsp90, HSV gD glycoprotein, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (hGH), human serum albumin, human tissue-type plasminogen activator (t-PA), huntingtin, HVEM, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFN-α, IFN-β, IFN-γ, IgA, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1, IGF-1 R, IGF-2, IGFBP, IGFR, IL, IL-1, IL-10, IL-10 receptor, IL-11, IL-11 receptor, IL-12, IL-12 receptor, IL-13, IL-13 receptor, IL-15, IL-15 receptor, IL-1 6, IL-16 receptor, IL-17, IL-17 receptor, IL-18(IGIF), IL-18 receptor, IL-1α, IL-1β, IL-1 receptor, IL-2, IL-2 receptor, IL-20, IL-20 receptor, IL-21, IL-21 receptor, IL-23, IL-23 receptor, IL-2 receptor, IL-3, IL-3 receptor, IL-31, IL-31 receptor, IL-3 receptor, IL-4, IL-4 receptor, IL-5, IL-5 receptor, IL-6, IL-6 receptor, IL-7 , IL-7 receptor, IL-8, IL-8 receptor, IL-9, IL-9 receptor, immunoglobulin immune complex, immunoglobulin, INF-α, INF-α receptor, INF-β, INF-β receptor, INF-γ, INF-γ receptor, type I IFN,Type I IFN receptor, influenza, inhibin, inhibin α, inhibin β, iNOS, insulin, insulin A chain, insulin B chain, insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor binding protein, integrin, integrin α2, integrin α3, integrin α4, integrin α4 / β1, integrin α-V / β-3, integrin α-V / β-6, integrin α4 / β7, integrin α5 / β1, integrin α5 / β3, integrin α5 / β6, integrin ασ (αV), integrin αθ, integrin β1, integrin β2, integrin β3 (GPIIb-IIIa), IP-10, I-TA C, JE, kallikrein, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, kallistatin, KC, KDR, keratinocyte growth factor (KGF), keratinocyte growth factor 2 (KGF-2), KGF, killer immunoglobulin-like receptor, kit ligand (KL), Kit tyrosine kinase, laminin 5, LAMP, LAPP (amylin, islet amyloid polypeptide), LAP (TGF-1), latency-associated peptide, latent TGF-1, latent TGF-1 bp1, LBP, LDGF, LDL, LDL receptor, LECT2, Lefty, leptin, luteinizing hormone (LH), Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, LFA-3 receptor, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotactin, lymphotoxin β receptor, lysosphingolipid receptor, Mac-1, macrophage-CSF (M-CSF), MAdCAM, MAG, MAP2, MARC, maspin, MCAM, MCK-2, MCP, MCP-1, MCP-2, MCP-3, MCP-4, MCP-I (MCAF), M-CSF, MDC, MDC (67 aa), MDC (69 aa), megsin, Mer,MET tyrosine kinase receptor family, metalloproteases, membrane glycoprotein OX2, mesothelin, MGDF receptor, MGMT, MHC (HLA-DR), microbial proteins, MIF, MIG, MIP, MIP-1α, MIP-1β, MIP-3α, MIP-3β, MIP-4, 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, monocyte-attracting protein, monocyte colony inhibitory factor, mouse gonadotropin-related peptide, MPIF, Mpo, MSK, MSP, MUC-16, MUC18, mucin (Mud), Müllerian inhibitory substance, Mug, MuSK, myelin-associated glycoprotein, myeloid progenitor inhibitory factor-1 (MPIF-I), NAIP, nanobody, NAP, NAP-2, NCA 90, NCAD, N-cadherin, NCAM, neprilysin, neural cell adhesion molecule, neuroserpin, nerve growth factor (NGF), neurotrophin-3, neurotrophin-4, neurotrophin-6, neuropilin 1, neurturin, NGF-β, NGFR, NKG20, N-methionyl human growth hormone, nNOS, NO, Nogo-A, Nogo receptor, hepatitis C virus nonstructural protein type 3 (NS3), NOS, Npn, NRG-3, NT, NT-3, NT-4, NTN, OB, OGG1, oncostatin M, OP-2, OPG, OPN, OSM, OSM receptor, osteoinductive factor, osteopontin, OX40L, OX40R, oxidized LDL p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PCSK9, PDGF, PDGF receptor, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-D, PDK-1, PECAM, PEDF, PEM, PF-4, PGE, PGF, PGI2, PGJ2, PIGF, PIN, PLA2, placental growth factor, placental alkaline phosphatase (PLAP), placental lactogen, plasminogen activator inhibitor-1, platelet growth factor, plgR, PLP, polyglycol chains of various sizes (e.g., PEG-20, PEG-30, PEG40), PP14, prekallikrein,Prion protein, procalcitrate, Nin, programmed cell death protein 1, proinsulin, prolactin, proprotein convertase PC9, prorelaxin, prostate-specific membrane antigen (PSMA), protein A, protein C, protein D, protein S, protein Z, PS, PSA, PSCA, PsmAr, PTEN, PTHrp, Ptk, PTN, P-selectin glycoprotein ligand-1, R51, RAGE, RANK, RANKL, RANTES, relaxin, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, Ret, reticulon 4, rheumatoid factor, RLI P76, RPA2, RPK-1, RSK, RSV Fgp, S100, RON-8, SCF / KL, SCGF, sclerostin, SDF-1, SDF1α, SDF1β, SERINE, serum amyloid P, serum albumin, sFRP-3, Shh, Shiga-like toxin II, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, sphingosine 1-phosphate receptor 1, staphylococcal lipoteichoic acid, Stat, STEAP, STEAP-II, stem cell factor (SCF), streptokinase, superoxide dismutase, syndecan-1, TACE, TACI, TAG-72 (tumor Tumor-associated glycoprotein-72), TARC, TB, TCA-3, T-cell receptor α / β, TdT, TECK, TEM1, TEM5, TEM7, TEM8, tenascin, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, Pan-specific TGF-β, TGF-βRII, TGF-βRIIb, TGF-βRIII, TGF-βRl (ALK-5), TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, TGF-I, thrombin, thrombopoietin (TPO), thymic stromal lymphoprotein (Thymic stromallymphoprotein) and activator Ck-1 activates thyroid hormone (TSH). Commercial Tie, TIMP, TIQ. Built-in bathrooms, bathrooms, snowflakes Liquids TMEFF2, Tmpo, TMPRSS2, TNF complex I, TNF complex II, TNF-α TNF-β2, 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)、TNFRSF12A、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(WHO ATAR / HveA / LIGHT R / TR2、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA)、TNFRSF18(GITR AITR)、TNFRSF19(TROY CROWN / TRADE)、TNFRSF19L(RELT)、TNFRSF1A(TNF Rl CD120a / p55-60, TNFRSF1B (TNF RII CD120b / p75-80), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRSF25 (DR3). Apo-3 / LARD / TR-3 / TRAMP / WSL-1); Apo-1 / APT1 / CD95), TNFRSF6B(DcR3 M68 / TR6), TNFRSF7(CD27), TNFRSF8 (CD30), TNFRSF9(4-1 BB CD137 / ILA), TNFRST23(DcTRAIL R1 TNFRH1), TNFSF10 (TRAILApo-2 ligand / TL2), TNFSF11 (TRANCE / RANK ligand ODF / OPG ligand), TNFSF12 (TWEAK Apo-3 ligand / DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS / TALL1 / THANK / TNFSF20), TNFSF14 (LIGHT HVEM ligand / LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand / TL6), TNFSF1A (TNF-α Connectin / DIF / TNFSF2), TNFSF1B (TNF-b LTa / TNFSF1), TNFSF3 (LTb TNFSF / 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 (4-1 BB ligand CD137 ligand), TNF-α, TNF-β, TNIL-I, toxic metabolites, TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, transforming growth factors (TGFs), such as TGF-α and TGF-β, transmembrane glycoprotein NMB, transthyretin, TRF, Trk, TROP-2, trophoblast glycoprotein, TSG, TSLP, tumor necrosis factor (TNF), tumor-associated antigen CA125, tumor-associated antigen expressing Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VAP-1, vascular endothelial growth factor (VEGF), vaspin, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEFGR-2, VEGF receptor (VEGFR), VEGFR-3 (flt-4), VEGI, VIM, viral antigen, VitB12 receptor, vitronectin receptor, VL These include A, VLA-1, VLA-4, VNR integrin, von Willebrand factor (vWF), WIF-1, WNT1, WNT10A, WNT10B, WNT11, WNT16, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, XCL1, XCL2 / SCM-l-β, XCLl / lymphotactin, XCR1, XEDAR, XIAP, and XPD.

[0111] Specific examples of molecules specifically expressed on T cells include CD3 and T cell receptors. CD3 is particularly preferred. For example, in the case of human CD3, the site in CD3 to which the antigen-binding molecule of the present invention binds may be any epitope present in the sequence of the γ chain, δ chain, or ε chain that constitutes human CD3. In particular, an epitope present in the extracellular region of the ε chain in the human CD3 complex is preferred. The polynucleotide sequences of the structures of the γ chain, δ chain, and ε chain that constitute CD3 are shown in SEQ ID NOs: 224 (NM_000073.2), 226 (NM_000732.4), and 228 (NM_000733.3), and their polypeptide sequences are shown in SEQ ID NOs: 225 (NP_000064.1), 227 (NP_000723.1), and 229 (NP_000724.1) (RefSeq accession numbers are shown in parentheses).

[0112] One of the two variable regions of the antibody contained in the antigen-binding molecule of the present invention binds to a "third antigen" different from the above-mentioned "CD3" and "CD137." In some embodiments, the third antigen is derived from human, mouse, rat, monkey, rabbit, or dog. In some embodiments, the third antigen is a molecule specifically expressed on cells or organs derived from human, mouse, rat, monkey, rabbit, or dog. The third antigen is preferably a molecule not systemically expressed on cells or organs. The third antigen is preferably, for example, a tumor cell-specific antigen, and also includes antigens expressed in association with the malignant transformation of cells and abnormal sugar chains that appear on cell surfaces or protein molecules during the malignant transformation of cells. Specific examples include ALK receptor (pleiotrophin receptor), pleiotrophin, KS 1 / 4 pancreatic cancer antigen, ovarian cancer antigen (CA125), prostatic acid phosphate, prostate-specific antigen (PSA), melanoma-associated antigen p97, melanoma antigen gp75, high-molecular-weight melanoma antigen (HMW-MAA), prostate-specific membrane antigen, carcinoembryonic antigen (CEA), polymorphic epithelial mucin antigen, human milk fat globule antigen, colorectal tumor-associated antigens (e.g., CEA, TAG-72, CO17-1A, GICA 19-9, CTA-1, and LEA), and Burkitt's lymphoma antigen 38.13, CD19, human B lymphoma antigen CD20, CD33, melanoma-specific antigens (e.g., ganglioside GD2, ganglioside GD3, ganglioside GM2, and ganglioside GM3), tumor-specific transplantation antigens (TSTA), T antigens, virus-induced tumor antigens (e.g., envelope antigens of DNA tumor viruses and RNA tumor viruses), colon CEA, oncofetal antigen α-fetoprotein (e.g., oncofetal trophoblast glycoprotein 5T4 and oncofetal bladder tumor antigen), differentiation antigens (e.g., human Lung cancer antigens L6 and L20), fibrosarcoma antigen, human T-cell leukemia-associated antigen Gp37, neoglycoproteins, sphingolipids, breast cancer antigens (e.g., EGFR (epidermal growth factor receptor)), NY-BR-16, NY-BR-16 and HER2 antigen (p185HER2), polymorphic epithelial mucin (PEM), malignant human lymphocyte antigen APO-1, differentiation antigens such as I antigen found in fetal erythrocytes, early endoderm I antigen found in adult erythrocytes, I (Ma) found in preimplantation embryos or gastric cancer, and I found in mammary epithelium. M18, M39, SSEA-1 found in bone marrow cells, VEP8, VEP9, Myl, VIM-D5, D156-22 found in colorectal cancer, TRA-1-85 (blood type H), SCP-1 found in testicular and ovarian cancer, C14 found in colon cancer, F3 found in lung cancer, AH6 found in gastric cancer, Y hapten, Ley found in embryonal carcinoma cells, TL5 (blood type A), EGF receptor found in A431 cells, E1 series found in pancreatic cancer ( Blood type B), FC10.2 found in embryonal carcinoma cells, gastric cancer antigen, CO-514 (blood type Lea) found in adenocarcinoma, NS-10 found in adenocarcinoma, CO-43 (blood type Leb), G49 found in the EGF receptor of A431 cells, MH2 (blood type ALeb / Ley) found in colon cancer, 19.9 found in colon cancer, gastric cancer mucin, T5A7 found in bone marrow cells, R24 found in melanoma, 4.2, GD3, D1 found in embryonal carcinoma cells.1, OFA-1, GM2, OFA-2, GD2, and M1:22:25:8, SSEA-3 and SSEA-4 found in 4-cell to 8-cell embryos, cutaneous T-cell lymphoma-associated antigen, MART-1 antigen, sialyl Tn (STn) antigen, colon cancer antigen NY-CO-45, lung cancer antigen NY-LU-12 variant A, adenocarcinoma antigen ART1, paraneoplastic-associated brain-testis cancer antigen (tumor neural antigen MA2 and paraneoplastic neural antigen), neuro-oncological abdominal antigen 2 (NOVA2), blood cell cancer antigen gene 520, tumor-associated antigen CO-029, tumor These include tumor-associated antigens MAGE-C1 (cancer / testis antigen CT7), MAGE-B1 (MAGE-XP antigen), MAGE-B2 (DAM6), MAGE-2, MAGE-4a, MAGE-4b, MAGE-X2, cancer-testis antigen (NY-EOS-1), YKL-40, and any fragments of these polypeptides, as well as modified structures thereof (such as the aforementioned modified phosphate groups and sugar chains), EpCAM, EREG, CA19-9, CA15-3, sialyl SSEA-1 (SLX), HER2, PSMA, CEA, and CLEC12A.

[0113] As used herein, the term "CD137," also known as 4-1BB, is a member of the tumor necrosis factor (TNF) receptor family. Examples of factors belonging to the TNF superfamily or TNF receptor superfamily include CD137, CD137L, CD40, CD40L, OX40, OX40L, CD27, CD70, HVEM, LIGHT, RANK, RANKL, CD30, CD153, GITR, and GITRL.

[0114] In one aspect, the antigen-binding molecule of the present invention has at least one characteristic selected from the group consisting of the following (1) to (4): (1) the variable region binds to the extracellular domain of CD3ε (epsilon) comprising the amino acid sequence of SEQ ID NO: 159; (2) the antigen-binding molecule has agonistic activity against CD137; (3) the antigen-binding molecule induces CD3 activation of T cells against cells expressing a molecule of a third antigen, but does not induce activation of T cells against cells expressing CD137; and (4) The antigen-binding molecule does not induce cytokine release from PBMCs in the absence of cells expressing a third antigen molecule.

[0115] In one aspect, the antigen-binding molecule of the present invention has at least one characteristic selected from the group consisting of the following (1) to (4): (1) the variable region binds to the extracellular domain of CD3ε (epsilon) comprising the amino acid sequence of SEQ ID NO: 159; (2) the antigen-binding molecule has agonistic activity against CD137; (3) the antigen-binding molecule induces T cell cytotoxicity against cells expressing a third antigen molecule, but does not induce T cell activation against cells expressing CD137; and (4) The antigen-binding molecule does not induce cytokine release from PBMCs in the absence of cells expressing a third antigen molecule. In some embodiments, the antigen-binding molecules of the present invention have at least one characteristic selected from the group consisting of the following (1) and (2): (1) the antigen-binding molecule does not compete with a CD137 ligand for binding to CD137; and (2) The antigen-binding molecule induces T cell cytotoxicity against cells expressing a third antigen molecule, but does not induce T cell cytotoxicity against cells expressing CD137.

[0116] In one aspect, a "CD137 agonist antibody" or an "antigen-binding molecule having agonist activity against CD137" of the present invention refers to an antibody or antigen-binding molecule that, when added to cells, tissues, or organisms expressing CD137, activates at least about 5%, specifically at least about 10%, or more specifically at least about 15% of CD137-expressing cells, where 0% activation is the background level (e.g., IL6 secretion) of non-activated cells expressing CD137. In various embodiments, CD137 agonist antibodies for use as pharmaceutical compositions of the present invention can activate cellular activity by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 750%, or 1000%. In one aspect, the "CD137 agonist antibody" or "antigen-binding molecule having agonist activity against CD137" of the present invention also refers to an antibody or antigen-binding molecule that, when added to cells, tissues, or organisms expressing CD137, activates at least about 5%, specifically at least about 10%, or more specifically at least about 15% of cells expressing CD137, where 100% activation is the level of activation achieved by an equimolar amount of binding partner under physiological conditions. In various embodiments, CD137 agonist antibodies for use as pharmaceutical compositions of the present invention can activate cellular activity by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 750%, or 1000%. In some embodiments, a "binding partner," as used herein, is a molecule known to bind to CD137 and induce activation of cells expressing CD137. In a further embodiment, examples of binding partners include urelumab (CAS Registry Number 934823-49-1) and variants thereof, as described in WO2005 / 035584A1, utomilumab (CAS Registry Number 1417318-27-4) and variants thereof, as described in WO2012 / 032433A1, and various known CD137 agonist antibodies. In certain embodiments, examples of binding partners include CD137 ligands. In a further embodiment, the activation of CD137-expressing cells by anti-CD137 agonist antibodies may be determined using an ELISA that characterizes IL6 secretion (see, for example, Reference Example 5-2 herein). The anti-CD137 antibody used as a binding partner and the antibody concentration for measurement can refer to Reference Example 5-2, where 100% activation is the level of activation achieved by the antibody.In a further embodiment, an antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 142 and the light chain amino acid sequence of SEQ ID NO: 144 can be used as a binding partner at 30 μg / mL for measurement (see, e.g., Reference Example 5-2 herein). In some embodiments, activation of CD137-expressing cells by an anti-CD137 agonist antibody can be determined, for example, by using recombinant T cells that express a reporter gene (e.g., luciferase) in response to CD137 signaling and detecting reporter gene expression or activity of the reporter gene product as an indicator of T cell activation. When recombinant T cells that express a reporter gene in response to CD137 signaling are co-cultured with an antigen-binding molecule, if the expression of the reporter gene or the activity of the reporter gene product is 10%, 20%, 30%, 40%, 50%, 90%, 100%, or more greater than that of a negative control, the antigen-binding molecule is determined to induce activation of T cells against cells expressing CD137 (see, for example, Example 2.2 herein).

[0117] In a non-limiting embodiment, the present invention provides a "CD137 agonist antibody" comprising an Fc region, wherein the Fc region has enhanced binding activity to inhibitory Fcγ receptors.

[0118] In a non-limiting embodiment, CD137 agonist activity can be confirmed using B cells known to express CD137 on their surface. In a non-limiting embodiment, the HDLM-2 B cell line can be used as the B cells. Because IL-6 expression is induced as a result of CD137 activation, CD137 agonist activity can be assessed by the amount of human interleukin-6 (IL-6) produced. In this assessment, the amount of IL-6 is used to assess the increase in IL-6 expression from non-activated B cells as a 0% background level, thereby determining the percentage of CD137 agonist activity of the molecule being evaluated.

[0119] In some embodiments, the antigen-binding molecules of the present invention induce CD3 activation of T cells against cells expressing a molecule of a third antigen, but do not induce CD3 activation of T cells against cells expressing CD137. Whether an antigen-binding molecule induces CD3 activation of T cells against cells expressing a molecule of a third antigen can be determined, for example, by co-culturing T cells with cells expressing the third antigen in the presence of the antigen-binding molecule and assaying CD3 activation of T cells. T cell activation can be assayed, for example, by using recombinant T cells that express a reporter gene (e.g., luciferase) in response to CD3 signaling and detecting reporter gene expression or activity of the reporter gene product as an indicator of T cell activation. When recombinant T cells that express a reporter gene in response to CD3 signaling are co-cultured with cells expressing a third antigen in the presence of the antigen-binding molecule, detection of reporter gene expression or reporter gene product activity in a dose-dependent manner of the antigen-binding molecule indicates that the antigen-binding molecule induces T cell activation against cells expressing the third antigen. Similarly, whether an antigen-binding molecule does not induce CD3 activation of T cells against cells expressing CD137 can be determined, for example, by co-culturing T cells with cells expressing CD137 in the presence of the antigen-binding molecule and assaying CD3 activation of T cells as described above. If reporter gene expression or reporter gene product activity is absent, below the detection limit, or below that of a negative control when recombinant T cells that express a reporter gene in response to CD3 signaling are co-cultured with cells expressing CD137 in the presence of the antigen-binding molecule, it is determined that the antigen-binding molecule does not induce T cell activation against cells expressing CD137.In one aspect, when recombinant T cells that express a reporter gene in response to CD3 signaling are co-cultured with cells expressing CD137 in the presence of the antigen-binding molecule, if the expression of the reporter gene or the activity of the reporter gene product is at most about 50%, 30%, 20%, 10%, 5%, or 1%, it is determined that the antigen-binding molecule does not induce T cell activation against cells expressing CD137, where 100% activation is the level of activation achieved by an antigen-binding molecule that simultaneously binds to CD3 and CD137. In one aspect, when recombinant T cells that express a reporter gene in response to CD3 signaling are co-cultured with cells expressing CD137 in the presence of the antigen-binding molecule, if the expression of the reporter gene or the activity of the reporter gene product is at most about 50%, 30%, 20%, 10%, 5%, or 1%, it is determined that the antigen-binding molecule does not induce T cell activation against cells expressing CD137, where 100% activation is the level of activation achieved by the same antigen-binding molecule against cells expressing a molecule of a third antigen.

[0120] In some embodiments, the antigen-binding molecules of the present invention do not induce cytokine release from PBMCs in the absence of cells expressing a third antigen. Whether an antigen-binding molecule does not induce cytokine release in the absence of cells expressing a third antigen can be determined, for example, by incubating PBMCs with the antigen-binding molecule in the absence of cells expressing a third antigen and measuring cytokines such as IL-2, IFNγ, and TNFα released from PBMCs into the culture supernatant using methods known in the art. If no significant level of cytokines is detected in the culture supernatant of PBMCs incubated with the antigen-binding molecule in the absence of cells expressing a third antigen, or if no significant cytokine expression is induced, it is determined that the antigen-binding molecule does not induce cytokine release from PBMCs in the absence of cells expressing a third antigen. In one aspect, "no significant levels of cytokines are detected" also refers to a cytokine concentration level that is at most about 50%, 30%, 20%, 10%, 5%, or 1%, where 100% is the cytokine concentration achieved by an antigen-binding molecule that simultaneously binds to CD3 and CD137. In one aspect, "no significant levels of cytokines are detected" also refers to a cytokine concentration level that is at most about 50%, 30%, 20%, 10%, 5%, or 1%, where 100% is the cytokine concentration achieved in the presence of cells expressing a molecule of a third antigen. In one aspect, "no significant induction of cytokine expression occurs" also refers to a cytokine concentration increase that is at most 5-fold, 2-fold, or 1-fold the concentration of each cytokine before the addition of the antigen-binding molecule.

[0121] In some embodiments, the antigen binding molecule of the present invention competes for binding to CD137 or binds to the same epitope on CD137 as an antibody selected from the group consisting of: (a1) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 16, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 30, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 44, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a2) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 17, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 31, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 45, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 64, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 69, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 74; (a3) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 18, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 32, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 46, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a4) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 19, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 33, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 47, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a5) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 19, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 33, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 47, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 65, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 70, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 75; (a6) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 20, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 34, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 48, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a7) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 22, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 36, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 50, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a8) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 23, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 37, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 51, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a9) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 23, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 37, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 51, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 66, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 71, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 76; (a10) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 24, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 38, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 52, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a11) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 25, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 39, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 53, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 66, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 71, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 76; (a12) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 26, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 40, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 54, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 66, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 71, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 76; (a13) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 26, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 40, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 54, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a14) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 27, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 41, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 55, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a15) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 28, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 42, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 56, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (b1) HCDR1 comprising the amino acid sequence of SEQ ID NO: 16, HCDR2 comprising the amino acid sequence of SEQ ID NO: 30, HCDR3 comprising the amino acid sequence of SEQ ID NO: 44, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b2) HCDR1 comprising the amino acid sequence of SEQ ID NO: 17, HCDR2 comprising the amino acid sequence of SEQ ID NO: 31, HCDR3 comprising the amino acid sequence of SEQ ID NO: 45, LCDR1 comprising the amino acid sequence of SEQ ID NO: 64, LCDR2 comprising the amino acid sequence of SEQ ID NO: 69, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 74; (b3) HCDR1 comprising the amino acid sequence of SEQ ID NO: 18, HCDR2 comprising the amino acid sequence of SEQ ID NO: 32, HCDR3 comprising the amino acid sequence of SEQ ID NO: 46, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b4) HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, HCDR2 comprising the amino acid sequence of SEQ ID NO: 33, HCDR3 comprising the amino acid sequence of SEQ ID NO: 47, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b5) HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, HCDR2 comprising the amino acid sequence of SEQ ID NO: 33, HCDR3 comprising the amino acid sequence of SEQ ID NO: 47, LCDR1 comprising the amino acid sequence of SEQ ID NO: 65, LCDR2 comprising the amino acid sequence of SEQ ID NO: 70, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 75; (b6) HCDR1 comprising the amino acid sequence of SEQ ID NO: 20, HCDR2 comprising the amino acid sequence of SEQ ID NO: 34, HCDR3 comprising the amino acid sequence of SEQ ID NO: 48, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b7) HCDR1 comprising the amino acid sequence of SEQ ID NO: 22, HCDR2 comprising the amino acid sequence of SEQ ID NO: 36, HCDR3 comprising the amino acid sequence of SEQ ID NO: 50, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b8) HCDR1 comprising the amino acid sequence of SEQ ID NO: 23, HCDR2 comprising the amino acid sequence of SEQ ID NO: 37, HCDR3 comprising the amino acid sequence of SEQ ID NO: 51, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b9) HCDR1 comprising the amino acid sequence of SEQ ID NO: 23, HCDR2 comprising the amino acid sequence of SEQ ID NO: 37, HCDR3 comprising the amino acid sequence of SEQ ID NO: 51, LCDR1 comprising the amino acid sequence of SEQ ID NO: 66, LCDR2 comprising the amino acid sequence of SEQ ID NO: 71, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 76; (b10) HCDR1 comprising the amino acid sequence of SEQ ID NO: 24, HCDR2 comprising the amino acid sequence of SEQ ID NO: 38, HCDR3 comprising the amino acid sequence of SEQ ID NO: 52, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b11) HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, HCDR2 comprising the amino acid sequence of SEQ ID NO: 39, HCDR3 comprising the amino acid sequence of SEQ ID NO: 53, LCDR1 comprising the amino acid sequence of SEQ ID NO: 66, LCDR2 comprising the amino acid sequence of SEQ ID NO: 71, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 76; (b12) HCDR1 comprising the amino acid sequence of SEQ ID NO: 26, HCDR2 comprising the amino acid sequence of SEQ ID NO: 40, HCDR3 comprising the amino acid sequence of SEQ ID NO: 54, LCDR1 comprising the amino acid sequence of SEQ ID NO: 66, LCDR2 comprising the amino acid sequence of SEQ ID NO: 71, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 76; (b13) HCDR1 comprising the amino acid sequence of SEQ ID NO: 26, HCDR2 comprising the amino acid sequence of SEQ ID NO: 40, HCDR3 comprising the amino acid sequence of SEQ ID NO: 54, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b14) HCDR1 comprising the amino acid sequence of SEQ ID NO: 27, HCDR2 comprising the amino acid sequence of SEQ ID NO: 41, HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b15) HCDR1 comprising the amino acid sequence of SEQ ID NO: 28, HCDR2 comprising the amino acid sequence of SEQ ID NO: 42, HCDR3 comprising the amino acid sequence of SEQ ID NO: 56, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (c1) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 2, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c2) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 3, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 59; (c3) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 4, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c4) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c5) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 60; (c6) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 6, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c7) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 8, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c8) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 9, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c9) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 9, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 61; (c10) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 10, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c11) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 11, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 61; (c12) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 12, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 61; (c13) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 12, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c14) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 13, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c15) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 14, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (d1) a heavy chain variable domain (VH) of SEQ ID NO: 2, and a light chain variable domain (VL) of SEQ ID NO: 58; (d2) a heavy chain variable domain (VH) of SEQ ID NO: 3, and a light chain variable domain (VL) of SEQ ID NO: 59; (d3) a heavy chain variable domain (VH) of SEQ ID NO: 4, and a light chain variable domain (VL) of SEQ ID NO: 58; (d4) a heavy chain variable domain (VH) of SEQ ID NO: 5, and a light chain variable domain (VL) of SEQ ID NO: 58; (d5) a heavy chain variable domain (VH) of SEQ ID NO: 5, and a light chain variable domain (VL) of SEQ ID NO: 60; (d6) a heavy chain variable domain (VH) of SEQ ID NO: 6, and a light chain variable domain (VL) of SEQ ID NO: 58; (d7) a heavy chain variable domain (VH) of SEQ ID NO: 8, and a light chain variable domain (VL) of SEQ ID NO: 58; (d8) a heavy chain variable domain (VH) of SEQ ID NO: 9, and a light chain variable domain (VL) of SEQ ID NO: 58; (d9) a heavy chain variable domain (VH) of SEQ ID NO: 9, and a light chain variable domain (VL) of SEQ ID NO: 61; (d10) a heavy chain variable domain (VH) of SEQ ID NO: 10, and a light chain variable domain (VL) of SEQ ID NO: 58; (d11) a heavy chain variable domain (VH) of SEQ ID NO: 11, and a light chain variable domain (VL) of SEQ ID NO: 61; (d12) a heavy chain variable domain (VH) of SEQ ID NO: 12, and a light chain variable domain (VL) of SEQ ID NO: 61; (d13) a heavy chain variable domain (VH) of SEQ ID NO: 12, and a light chain variable domain (VL) of SEQ ID NO: 58; (d14) a heavy chain variable domain (VH) of SEQ ID NO: 13, and a light chain variable domain (VL) of SEQ ID NO: 58; (d15) A heavy chain variable domain (VH) of SEQ ID NO: 14, and a light chain variable domain (VL) of SEQ ID NO: 58.

[0122] Whether a test antibody shares a common epitope with a particular antibody can be evaluated based on the competition between the two antibodies for the same epitope. Competition between antibodies can be detected by cross-blocking assays, etc. For example, competitive ELISA assays are preferred cross-blocking assays. Specifically, in cross-blocking assays, the CD137 protein used to coat the wells of a microtiter plate is pre-incubated in the presence or absence of a candidate competing antibody, and then the anti-CD137 antibody of the present invention is added thereto. The amount of the anti-CD137 antibody of the present invention bound to the CD137 protein in the well is indirectly correlated with the binding ability of the candidate competing antibody (test antibody) competing for binding to the same epitope. That is, the higher the affinity of the test antibody for the same epitope, the lower the amount of the anti-CD137 antibody of the present invention bound to the wells coated with CD137 protein, and the higher the amount of the test antibody bound to the wells coated with CD137 protein.

[0123] The amount of antibody bound to the well can be easily determined by pre-labeling the antibody. For example, biotin-labeled antibody can be measured using an avidin / peroxidase conjugate and an appropriate substrate. In particular, cross-blocking assays using enzyme labels such as peroxidase are called "competitive ELISA assays." Antibodies can be labeled with other labeling substances that allow detection or measurement. Specifically, radiolabels, fluorescent labels, etc. are known.

[0124] Furthermore, if the test antibody has a constant region derived from a species different from that of the anti-CD137 antibody of the present invention, the amount of antibody bound to the wells can be measured by using a labeled antibody that recognizes the constant region of that antibody. Alternatively, if the antibodies are derived from the same species but belong to different classes, the amount of antibody bound to the wells can be measured using antibodies that distinguish between the individual classes.

[0125] A candidate competing antibody is either an antibody that substantially binds to the same epitope as an anti-CD137 antibody of the present invention, or an antibody that competes for binding to the same epitope, if the candidate antibody is able to block binding of an anti-CD137 antibody by at least 20%, preferably at least 20% to 50%, and even more preferably at least 50%, compared to the binding activity obtained in a control experiment performed in the absence of the candidate competing antibody.

[0126] In another embodiment, the ability of a test antibody to competitively or cross-competitively bind to another antibody can be determined by one of skill in the art using standard binding assays such as BIAcore analysis or flow cytometry, as known in the art.

[0127] Methods for determining spatial conformation of epitopes include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance (see Epitope Mapping Protocols in Methods in Molecular Biology, G.E. Morris (ed.), Vol. 66 (1996)).

[0128] Whether a test antibody shares a common epitope with a CD137 ligand can also be evaluated based on the competition between the test antibody and the CD137 ligand for the same epitope. The competition between an antibody and a CD137 ligand can be detected by a cross-blocking assay as described above. In another embodiment, the ability of a test antibody to competitively or cross-competitively bind to a CD137 ligand can be appropriately determined by a person skilled in the art using a standard binding assay known in the art, such as BIAcore analysis or flow cytometry.

[0129] In some embodiments, convenient examples of the antigen-binding molecules of the present invention include antigen-binding molecules that bind to the same epitope of human CD137 as that bound by an antibody selected from the group consisting of: In human CD137 protein an antibody recognizing a region including the sequence SPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (SEQ ID NO: 154); An antibody recognizing a region including the sequence DCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (SEQ ID NO: 149); An antibody that recognizes a region containing the sequence LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAEC (SEQ ID NO: 152), and An antibody that recognizes a region containing the sequence LQDPCSNCPAGTFCDNNRNQIC (sequence number: 147).

[0130] Depending on the cancer antigen to be targeted, those skilled in the art can appropriately select heavy chain variable region sequences and light chain variable region sequences contained in the cancer-specific antigen-binding domain that bind to the cancer antigen. When the epitope to which the antigen-binding domain binds is contained in multiple different antigens, the antigen-binding molecule containing the antigen-binding domain can bind to various antigens having the epitope.

[0131] "Epitope" refers to an antigenic determinant in an antigen and refers to the antigenic site to which various binding domains in the antigen-binding molecules disclosed herein bind. Thus, for example, an epitope can be defined according to its structure. Alternatively, an epitope may be defined according to the antigen-binding activity of an antigen-binding molecule that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope can be specified by the amino acid residues that form the epitope. Alternatively, when the epitope is a glycan, the epitope can be specified by its specific glycan structure.

[0132] A linear epitope is one whose primary amino acid sequence contains the epitope being recognized. Such a linear epitope typically contains at least 3, and most commonly at least 5, e.g., about 8-10 or 6-20 amino acids in its specific sequence.

[0133] In contrast to linear epitopes, "conformational epitopes" are epitopes in which the primary amino acid sequence containing the epitope is not the sole determinant of the recognized epitope (e.g., the primary amino acid sequence of a conformational epitope is not necessarily recognized by the antibody that defines the epitope). Conformational epitopes may contain a greater number of amino acids than linear epitopes. Antibodies that recognize conformational epitopes recognize the three-dimensional structure of a peptide or protein. For example, when a protein molecule folds to form a three-dimensional structure, the amino acids and / or polypeptide backbone that form the conformational epitope align, making the epitope recognizable by the antibody. Methods for determining the conformational structure of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, site-directed spin labeling, and electron paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

[0134] An example of a method for evaluating the binding of an epitope in a cancer-specific antigen by a test antigen-binding molecule is shown below. Methods for evaluating the binding of an epitope in a target antigen by another binding domain can also be performed as appropriate according to the following examples.

[0135] For example, whether a test antigen-binding molecule containing an antigen-binding domain against a cancer-specific antigen recognizes a linear epitope in an antigen molecule can be confirmed, for example, as described below. For example, a linear peptide containing the amino acid sequence forming the extracellular domain of a cancer-specific antigen is synthesized for the above purpose. The peptide can be chemically synthesized or obtained by genetic engineering techniques using a region in the cDNA of the cancer-specific antigen that encodes the amino acid sequence corresponding to the extracellular domain. The test antigen-binding molecule containing the antigen-binding domain against a cancer-specific antigen is then evaluated for its binding activity to the linear peptide containing the amino acid sequence forming the extracellular domain. For example, the binding activity of the antigen-binding molecule to the peptide can be evaluated by ELISA using an immobilized linear peptide as an antigen. Alternatively, the binding activity to the linear peptide can be evaluated based on the level at which the linear peptide inhibits the binding of the antigen-binding molecule to cancer-specific antigen-expressing cells. The binding activity of the antigen-binding molecule to the linear peptide can be demonstrated by these tests.

[0136] Whether a test antigen molecule containing an antigen-binding domain for the above-mentioned antigen recognizes a conformational epitope can be confirmed as follows. For example, an antigen-binding molecule containing an antigen-binding domain for a cancer-specific antigen binds strongly to cancer-specific antigen-expressing cells upon contact, but does not substantially bind to an immobilized linear peptide containing an amino acid sequence that forms the extracellular domain of the cancer-specific antigen. As used herein, "does not substantially bind" means that the binding activity to antigen-expressing cells is 80% or less, generally 50% or less, preferably 30% or less, and particularly preferably 15% or less, compared to the binding activity to antigen-expressing cells in ELISA or fluorescence-activated cell sorting (FACS) using the antigen-expressing cells as the antigen.

[0137] In the ELISA format, the binding activity of a test antigen-binding molecule containing an antigen-binding domain to antigen-expressing cells can be 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 antigen-expressing cells are immobilized. The cell-bound test antigen-binding molecule is then detected using an enzyme-labeled antibody that recognizes the test antigen-binding molecule. Alternatively, when using FACS, a dilution series of the test antigen-binding molecule can be prepared, and the antigen-binding titer to the antigen-expressing cells can be determined, and the binding activity of the test antigen-binding molecule to the antigen-expressing cells can be compared.

[0138] 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 following devices: FACSCanto(TM) II FACSAria(trademark) FACSArray™ FACSVantage(TM) SE FACSCalibur™ (all 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 trade names of Beckman Coulter).

[0139] Suitable methods for assaying the binding activity of a test antigen-binding molecule containing an antigen-binding domain for the above-mentioned antigen include, for example, the following method. First, antigen-expressing cells are reacted with the test antigen-binding molecule, which are then stained with an FITC-labeled secondary antibody and analyzed using a FACSCalibur (BD). The fluorescence intensity obtained by analysis using CELL QUEST Software (BD), i.e., the geometric mean value, reflects the amount of antibody bound to the cells. In other words, the binding activity of the test antigen-binding molecule, represented by the amount of bound test antigen-binding molecule, can be measured by determining the geometric mean value.

[0140] Whether a test antigen-binding molecule comprising an antigen-binding domain of the present invention shares a common epitope with another antigen-binding molecule can be evaluated based on the competition between the two molecules for the same epitope.Competition between antigen-binding molecules can be detected by cross-blocking assays, etc.For example, competitive ELISA assays are preferred cross-blocking assays.

[0141] Specifically, in cross-blocking assay, the antigen coating the well of a microtiter plate is pre-incubated in the presence or absence of a candidate competing antigen-binding molecule, and then a test antigen-binding molecule is added thereto. The amount of test antigen-binding molecule bound to the antigen 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. That is, the higher the affinity of the competing antigen-binding molecule for the same epitope, the lower the binding activity of the test antigen-binding molecule to the well coated with the antigen.

[0142] The amount of test antigen-binding molecules bound to the wells via antigen can be easily determined by pre-labeling the antigen-binding molecules. For example, biotin-labeled antigen-binding molecules can be measured using avidin / peroxidase conjugates and appropriate substrates. In particular, cross-blocking assays using enzyme labels such as peroxidase are called "competitive ELISA assays." Antigen-binding molecules can also be labeled with other labeling substances that allow detection or measurement. Specifically, radiolabels, fluorescent labels, etc. are known. When a candidate competitor antigen-binding molecule is able to block binding of a test antigen-binding molecule comprising an antigen-binding domain by at least 20%, preferably at least 20-50%, and more preferably at least 50%, compared to the binding activity in a control experiment performed in the absence of the competitor antigen-binding molecule, the test antigen-binding molecule is determined to substantially bind to the same epitope as the competitor antigen-binding molecule, or to compete for binding to the same epitope.

[0143] When the structure of the epitope bound by a test antigen-binding molecule comprising an antigen-binding domain of the present invention has already been identified, whether the test antigen-binding molecule and the control antigen-binding molecule share a common epitope can be evaluated by comparing the binding activities of the two antigen-binding molecules toward a peptide prepared by introducing amino acid mutations into the peptide that forms the epitope.

[0144] For example, the binding activity of a test antigen-binding molecule and a control antigen-binding molecule to a linear peptide containing a mutation can be measured by comparison in the ELISA format described above. In addition to ELISA, the binding activity of the test antigen-binding molecule and the control antigen-binding molecule can be determined by passing the test antigen-binding molecule and the control antigen-binding molecule through a column and then quantifying the eluted antigen-binding molecules in the eluate. For example, methods for adsorbing a mutant peptide to a column in the form of a GST-fusion peptide are known.

[0145] Alternatively, if the identified epitope is a conformational epitope, whether the test and control antigen-binding molecules share a common epitope can be assessed using the following method. First, cells expressing the antigen targeted by the antigen-binding domain and cells expressing the antigen bearing the mutated epitope are prepared. These cells are suspended in an appropriate buffer, such as PBS, to prepare a cell suspension, to which the test and control antigen-binding molecules are added. The cell suspension is then washed appropriately with buffer, and an FITC-labeled antibody capable of recognizing the test and control antigen-binding molecules is added. The fluorescence intensity and number of cells stained with the labeled antibody are determined using a FACSCalibur (BD). The test and control antigen-binding molecules are appropriately diluted with an appropriate buffer and used at the desired concentration. For example, they may be used at a concentration ranging from 10 μg / ml to 10 ng / ml. The fluorescence intensity, i.e., the geometric mean value, determined by analysis using CELL QUEST Software (BD) reflects the amount of labeled antibody bound to the cells. That is, the binding activity of the test and control antigen-binding molecules, represented by the amount of bound labeled antibody, can be measured by determining the geometric mean value.

[0146] In some embodiments, the antigen-binding molecule of the present invention comprises: (a) a heavy chain variable domain amino acid sequence comprising, at each of the following positions (all according to Kabat numbering), one or more of the following amino acid residues as indicated for that position: A, D, E, I, G, K, L, M, N, R, T, W, or Y at amino acid position 26; D, F, G, I, M, or L at amino acid position 27; D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 28; F or W at amino acid position 29; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 30; F, I, N, R, S, T, or V at amino acid position 31; A, H, I, K, L, N, Q, R, S, T, or V at amino acid position 32; W at amino acid position 33; F, I, L, M, or V at amino acid position 34; F, H, S, T, V, or Y at amino acid position 35; E, F, H, I, K, L, M, N, Q, S, T, W, or Y at amino acid position 50; I, K, or V at amino acid position 51; K, M, R, or T at amino acid position 52; A, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y at amino acid position 52b; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 52c; A, E, F, H, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 53; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 54; E, F, G, H, L, M, N, Q, W, or Y at amino acid position 55; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 56; A, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, or V at amino acid position 57; A, F, H, K, N, P, R, or Y at amino acid position 58; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 59; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 60; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 61; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 62; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 63; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 64; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 65; H or R at amino acid position 93; F, G, H, L, M, S, T, V, or Y at amino acid position 94; I or V at amino acid position 95; F, H, I, K, L, M, T, V, W, or Y at amino acid position 96; F, Y, or W at amino acid position 97; A, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 98; A, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 99; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100a; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100b; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100c; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100d; A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y at amino acid position 100e; A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100f; Approximately 100g of amino acids A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; A, D, E, G, H, I, L, M, N, P, S, T, or V at amino acid position 100h; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100i; A, D, F, I, L, M, N, Q, S, T, or V at amino acid position 101; A, D, E, F, G, H, IK, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 102; and / or (b) a light chain variable domain amino acid sequence comprising, at each of the following positions (all according to Kabat numbering), one or more of the following amino acid residues as indicated for that position: A, D, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 24; A, G, N, P, S, T, or V at amino acid position 25; A, D, E, F, G, I, K, L, M, N, Q, R, S, T, or V at amino acid position 26; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 27; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 27a; A, I, L, M, P, T, or V at amino acid position 27b; A, E, F, H, I, K, L, M, N, P, Q, R, T, W, or Y at amino acid position 27c; A, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 27d; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 27e; G, N, S, or T at amino acid position 28; A, F, G, H, K, L, M, N, Q, R, S, T, W, or Y at amino acid position 29; A, F, G, H, I, K, L, M, N, Q, R, V, W, or Y at amino acid position 30; I, L, Q, S, T, or V at amino acid position 31; F, W, or Y at amino acid position 32; A, F, H, L, M, Q, or V at amino acid position 33; A, H, or S at amino acid position 34; I, K, L, M, or R at amino acid position 50; A, E, I, K, L, M, Q, R, S, T, or V at amino acid position 51; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 52; A, E, F, G, H, K, L, M, N, P, Q, R, S, V, W, or Y at amino acid position 53; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 54; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y at amino acid position 55; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 56; A, G, K, S, or Y at amino acid position 89; Q at amino acid position 90; G at amino acid position 91; A, D, H, K, N, Q, R, S, or T at amino acid position 92; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 93; A, D, H, I, M, N, P, Q, R, S, T, or V at amino acid position 94; P at amino acid position 95; F or Y at amino acid position 96; and A, D, E, G, H, I, K, L, M, N, Q, R, S, T, or V at amino acid position 97 Includes:

[0147] The antigen-binding molecules of the present invention can be produced by methods commonly known to those skilled in the art. For example, antibodies can be prepared by the methods shown below, but methods for preparing the antibodies of the present invention are not limited thereto. Many combinations of host cells and expression vectors are known in the art for preparing antibodies by transferring an isolated gene encoding the polypeptide into an appropriate host. All of these expression systems can be applied to the isolation of the antigen-binding molecules of the present invention. When eukaryotic cells are used as host cells, animal cells, plant cells, or fungal cells can be used as appropriate. Specifically, examples of animal cells include the following cells: (1) Mammalian cells, such as CHO (Chinese hamster ovary cell line), COS (monkey kidney cell line), myeloma cells (Sp2 / O, NS0, etc.), BHK (baby hamster kidney cell line), HEK293 (human embryonic kidney cell line with sheared adenovirus (Ad) 5 DNA), PER.C6 cells (human embryonic retinal cell line transformed with adenovirus type 5 (Ad5) E1A and E1B genes), Hela, and Vero (Current Protocols in Protein Science (May 2001, Unit 5.9, Table 5.9.1)); (2) amphibian cells, such as Xenopus oocytes; and (3) Insect cells, such as sf9, sf21, and Tn5. Antibodies can also be prepared using E. coli (mAbs 2012 Mar-Apr; 4(2): 217-225) or yeast (WO2000023579). Antibodies prepared using E. coli are non-glycosylated, whereas antibodies prepared using yeast are glycosylated.

[0148] DNA encoding an antibody heavy chain, which encodes a heavy chain in which one or more amino acid residues in the variable domain are substituted with a different amino acid of interest, and DNA encoding an antibody light chain are expressed. DNA encoding a heavy chain or light chain in which one or more amino acid residues in the variable domain are substituted with a different amino acid of interest can be obtained, for example, by obtaining DNA encoding an antibody variable domain prepared by a method known in the art against a specific antigen, and then introducing appropriate substitutions so that codons encoding specific amino acids in the domain encode the different amino acid of interest.

[0149] Alternatively, DNA encoding a protein in which one or more amino acid residues in an antibody variable domain prepared by methods known in the art against a specific antigen have been substituted with a different amino acid of interest may be designed in advance and chemically synthesized to obtain DNA encoding a heavy chain in which one or more amino acid residues in the variable domain have been substituted with a different amino acid of interest. The amino acid substitution site and type are not particularly limited. Examples of preferred regions for amino acid modification include solvent-exposed regions and loops in the variable domain. CDR1, CDR2, CDR3, FR3, and loops are particularly preferred. Specifically, positions 31-35, 50-65, 71-74, and 95-102 (Kabat numbering) in the H-chain variable domain and positions 24-34, 50-56, and 89-97 (Kabat numbering) in the L-chain variable domain are preferred. More preferred are positions 31, 52a to 61, 71 to 74, and 97 to 101, according to the Kabat numbering, in the H-chain variable domain, and positions 24 to 34, 51 to 56, and 89 to 96, according to the Kabat numbering, in the L-chain variable domain. Amino acid alterations are not limited to substitutions, but may also be deletions, additions, insertions, or modifications, or combinations thereof.

[0150] The DNA encoding the heavy chain, in which one or more amino acid residues in the variable domain are replaced with different amino acids of interest, can also be prepared as separate partial DNAs. Examples of partial DNA combinations include, but are not limited to, DNA encoding the variable domain and DNA encoding the constant domain; and DNA encoding the Fab domain and DNA encoding the Fc domain. Similarly, the DNA encoding the light chain can also be prepared as separate partial DNAs.

[0151] These DNAs can be expressed by the following methods: for example, a heavy chain expression vector is constructed by incorporating a DNA encoding a heavy chain variable domain into an expression vector together with a DNA encoding a heavy chain constant domain. Similarly, a light chain expression vector is constructed by incorporating a DNA encoding a light chain variable domain into an expression vector together with a DNA encoding a light chain constant domain. The heavy and light chain genes may also be incorporated into a single vector.

[0152] The DNA encoding the antibody of interest is incorporated into an expression vector so that it is expressed under the control of an expression control region, such as an enhancer and a promoter.The resulting expression vector is then transformed into a host cell to express the antibody.In this case, a suitable host and expression vector can be used in combination.

[0153] Examples of vectors include M13-based vectors, pUC-based vectors, pBR322, pBluescript, and pCR-Script. In addition to these vectors, for example, pGEM-T, pDIRECT, or pT7 can also be used for the purpose of subcloning and excising cDNA.

[0154] In particular, expression vectors are useful for producing the antibody of the present invention.For example, when the host is E. coli such as JM109, DH5α, HB101 or XL1-Blue, it is essential that the expression vector has a promoter that allows efficient expression in E. coli, such as lacZ promoter (Ward et al., Nature (1989) 341, 544-546; and FASEB J. (1992) 6, 2422-2427, which are incorporated herein by reference in their entirety), araB promoter (Better et al., Science (1988) 240, 1041-1043, which are incorporated herein by reference in their entirety) or T7 promoter. Examples of such vectors include the vectors mentioned above, as well as pGEX-5X-1 (Pharmacia), the "QIAexpress system" (Qiagen NV), pEGFP, and pET (in this case, the host is preferably BL21 expressing T7 RNA polymerase).

[0155] The vector may contain a signal sequence for polypeptide secretion. In the case of production in the periplasm of E. coli, the pelB signal sequence (Lei, SP et al., J. Bacteriol. (1987) 169, 4397, the entire contents of which are incorporated herein by reference) can be used as the signal sequence for polypeptide secretion. The vector can be introduced into host cells, for example, by using the lipofectin method, the calcium phosphate method, or the DEAE-dextran method.

[0156] In addition to expression vectors for E. coli, examples of vectors for producing the polypeptides of the present invention include mammalian expression vectors (e.g., pcDNA3 (Invitrogen Corp.), pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p5322, the entire contents of which are incorporated herein by reference), pEF, and pCDM8), insect cell-derived expression vectors (e.g., "Bac-to-BAC Baculovirus Expression System" (GIBCO BRL), and pBacPAK8), plant-derived expression vectors (e.g., pMH1 and pMH2), animal virus-derived expression vectors (e.g., pHSV, pMV, and pAdexLcw), retrovirus-derived expression vectors (e.g., pZIPneo), yeast-derived expression vectors (e.g., "Pichia Expression Kit" (Invitrogen Corp.), pNV11, and SP-Q01), and Bacillus subtilis (Bacillus Examples of suitable expression vectors include those derived from L. subtilis (e.g., pPL608 and pKTH50).

[0157] For expression in animal cells such as CHO cells, COS cells, NIH3T3 cells, or HEK293 cells, the vector must have a promoter necessary for intracellular expression, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108, the entire contents of which are incorporated herein by reference), the MMTV-LTR promoter, the EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322, the entire contents of which are incorporated herein by reference), the CAG promoter (Gene. (1991) 108, 193, the entire contents of which are incorporated herein by reference), or the CMV promoter. More preferably, the vector has a gene for screening transformed cells (e.g., a drug resistance gene that can serve as a marker for drugs (e.g., neomycin, G418, etc.)). Examples of vectors with such properties include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13. Additionally, the EBNA1 protein may be coexpressed to increase gene copy number using a vector carrying the replication origin OriP (Biotechnol Bioeng. 2001 Oct 20;75(2):197-203; and Biotechnol Bioeng. 2005 Sep 20;91(6):670-7).

[0158] An exemplary method for stably expressing a gene and increasing its copy number in cells includes transforming CHO cells deficient in a nucleic acid synthesis pathway with a vector (e.g., pCHOI) carrying a complementing DHFR gene and using methotrexate (MTX) for gene amplification. An exemplary method for transiently expressing a gene includes transforming COS cells carrying the SV40 T antigen gene on their chromosome with a vector (e.g., pcD) carrying an SV40 origin of replication. Origins of replication derived from polyoma virus, adenovirus, bovine papilloma virus (BPV), and the like can also be used. To increase gene copy number in a host cell system, the expression vector can contain a selection marker such as the aminoglycoside phosphotransferase (APH) gene, the thymidine kinase (TK) gene, the Escherichia coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, or the dihydrofolate reductase (dhfr) gene.

[0159] Antibodies can be recovered, for example, by culturing the transformed cells and then isolating the antibody from the transformed cells or the culture medium. Antibodies can be isolated and purified by an appropriate combination of methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, C1q, FcRn, protein A, and protein G columns, affinity chromatography, ion exchange chromatography, and gel filtration chromatography.

[0160] The above-mentioned techniques, such as knob-into-hole technology (WO1996 / 027011; Ridgway JB et al., Protein Engineering (1996) 9, 617-621; and Merchant AM et al., Nature Biotechnology (1998) 16, 677-681) or the technique of suppressing unintended association between H chains by introducing charge repulsion (WO2006 / 106905), can be applied to methods for efficiently preparing multispecific antibodies.

[0161] The present invention further provides methods for producing the antigen-binding molecules of the present invention. Specifically, the present invention provides methods for producing antigen-binding molecules comprising an antibody variable region capable of binding to two different antigens (a first antigen and a second antigen) but not simultaneously binding to CD3 and CD137 (this variable region is referred to as a first variable region), and a variable region that binds to a third antigen different from CD3 and CD137 (this variable region is referred to as a second variable region), the method comprising the step of preparing an antigen-binding molecule library containing diverse amino acid sequences of the first variable region.

[0162] Examples may include a method of making comprising the steps of: (i) preparing a library of antigen-binding molecules each of which has at least one amino acid altered in its antibody variable region that binds to CD3 or CD137, wherein the at least one amino acid in the altered variable region differs from each other; (ii) selecting, from the prepared library, antigen-binding molecules comprising variable regions that have binding activity to CD3 and CD137 but do not simultaneously bind to CD3 and CD137; (iii) culturing host cells comprising a nucleic acid encoding the variable region of the antigen-binding molecule selected in step (ii) and a nucleic acid encoding the variable region of an antigen-binding molecule that binds to a third antigen to express an antigen-binding molecule comprising an antibody variable region that can bind to CD3 and CD137 but does not simultaneously bind to CD3 and CD137 and a variable region that binds to the third antigen; and (iv) recovering the antigen-binding molecule from the host cell culture.

[0163] In this method of preparation, step (ii) may be an optional step of: (v) selecting, from the prepared library, antigen-binding molecules comprising variable regions that have binding activity to CD3 and CD137 but do not simultaneously bind to CD3 and CD137, each of which is expressed on different cells.

[0164] The antigen-binding molecules used in step (i) are not particularly limited, as long as they each contain an antibody variable region. The antigen-binding molecules may be antibody fragments such as Fv, Fab, or Fab', or antibodies containing an Fc region.

[0165] The amino acids to be modified are selected, for example, from amino acids in the variable region of an antibody that binds to CD3 or CD137, the modification of which does not abolish binding to the antigen.

[0166] In the present invention, one amino acid modification may be used alone, or multiple amino acid modifications may be used in combination. When multiple amino acid modifications are used in combination, the number of modifications to be combined is not particularly limited, and may be, for example, 2 to 30, preferably 2 to 25, 2 to 22, 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 2 to 3. The combined amino acid modifications may be made only to the heavy or light chain variable domain of the antibody, or may be distributed appropriately among both the heavy and light chain variable domains.

[0167] Examples of preferred regions for amino acid modification include solvent-exposed regions and loops in the variable region. CDR1, CDR2, CDR3, FR3, and loops are particularly preferred. Specifically, positions 31-35, 50-65, 71-74, and 95-102 (Kabat numbering) in the H-chain variable domain, and positions 24-34, 50-56, and 89-97 (Kabat numbering) in the L-chain variable domain are preferred. Positions 31, 52a-61, 71-74, and 97-101 (Kabat numbering) in the H-chain variable domain, and positions 24-34, 51-56, and 89-96 (Kabat numbering) in the L-chain variable domain are more preferred.

[0168] Modification of amino acid residues also includes random modification of amino acids in the above-mentioned regions of the antibody variable region that binds to CD3 or CD137, and insertion of peptides previously known to have binding activity to CD3 or CD137 into the above-mentioned regions. The antigen-binding molecules of the present invention can be obtained by selecting variable regions that can bind to CD3 and CD137 but cannot simultaneously bind to these antigens from among antigen-binding molecules modified in this way.

[0169] Whether the variable region is capable of binding to CD3 and CD137 but is unable to simultaneously bind to these antigens, and further whether the variable region is capable of simultaneously binding to both CD3 and CD137 when either CD3 or CD137 is present on a cell and the other antigen is present alone, when both antigens are present alone, or when both antigens are present on the same cell, but is unable to simultaneously bind to these antigens each expressed on different cells, can also be confirmed using the methods described above.

[0170] The present invention further provides nucleic acids encoding the antigen-binding molecules of the present invention. The nucleic acids of the present invention may be in any form, such as DNA or RNA.

[0171] The present invention further provides a vector containing the nucleic acid of the present invention. The type of vector can be appropriately selected by those skilled in the art depending on the host cell to receive the vector. For example, any of the vectors described above can be used.

[0172] The present invention further relates to a host cell transformed with the vector of the present invention. The host cell can be appropriately selected by those skilled in the art. For example, any of the host cells described above can be used.

[0173] The present invention also provides pharmaceutical compositions comprising the antigen-binding molecules of the present invention and a pharmaceutically acceptable carrier. Pharmaceutical compositions of the present invention can be formulated according to methods known in the art by adding a pharmaceutically acceptable carrier to the antigen-binding molecules of the present invention. For example, the pharmaceutical compositions can be used in the form of parenteral injection of a sterile solution or suspension with water or any other pharmaceutically acceptable solution. For example, pharmaceutical compositions can be formulated by mixing the antigen-binding molecules with an appropriate combination of pharmacologically acceptable carriers or vehicles, specifically, sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, etc., in a unit dosage form required for commonly accepted pharmaceutical practice. 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, saccharides, carboxymethyl cellulose, corn starch, and inorganic salts. The amount of the active ingredient in such preparations is determined so that an appropriate dosage within the indicated range can be achieved.

[0174] Sterile compositions for injection can be formulated according to conventional pharmaceutical practice, using vehicles such as distilled water for injection.Examples of aqueous solutions for injection include isotonic solutions containing physiological saline, glucose and other auxiliary agents (for example, D-sorbitol, D-mannose, D-mannitol and sodium chloride).These solutions can be used in combination with suitable solubilizers, such as alcohol (particularly ethanol) or polyalcohols (for example, propylene glycol and polyethylene glycol), or nonionic surfactants, such as polysorbate 80 (trademark) or HCO-50.

[0175] Examples of oily solutions include sesame oil and soybean oil. These solutions may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizer. The solution may further be mixed with a buffer (e.g., phosphate buffer and sodium acetate buffer), a soothing agent (e.g., procaine hydrochloride), a stabilizer (e.g., benzyl alcohol and phenol), and an antioxidant. The injection solution prepared in this manner is usually filled into an appropriate ampule. The pharmaceutical composition of the present invention is preferably administered parenterally. Specific examples of dosage forms include injections, intranasal administration, pulmonary administration, and transdermal administration. Examples of injections include intravenous injections, intramuscular injections, intraperitoneal injections, and subcutaneous injections, through which the pharmaceutical composition can be administered systemically or locally.

[0176] The administration method can be selected appropriately depending on the patient's age and symptoms. The dose of a pharmaceutical composition containing a polypeptide or a polynucleotide encoding the polypeptide can be selected, for example, within the range of 0.0001 to 1,000 mg per kg of body weight per dose. Alternatively, the dose can be selected, for example, within the range of 0.001 to 100,000 mg per patient, but is not necessarily limited to these values. The dose and administration method vary depending on the patient's body weight, age, symptoms, etc., but those skilled in the art can select the dose and method appropriately.

[0177] The present invention also provides methods for treating cancer, which comprise a step of administering the antigen-binding molecule of the present invention; the antigen-binding molecule of the present invention for use in treating cancer; use of the antigen-binding molecule of the present invention in producing a cancer therapeutic agent; and a process for producing a cancer therapeutic agent, which comprises a step of using the antigen-binding molecule of the present invention.

[0178] As used herein, the three-letter amino acid codes and corresponding one-letter codes are defined as follows: alanine: Ala and A, arginine: Arg and R, asparagine: Asn and N, aspartic acid: Asp and D, cysteine: Cys and C, glutamine: Gln and Q, glutamic acid: Glu and E, glycine: Gly and G, histidine: His and H, isoleucine: Ile and I, leucine: Leu and L, lysine: Lys and K, methionine: Met and M, phenylalanine: Phe and F, proline: Pro and P, serine: Ser and S, threonine: Thr and T, tryptophan: Trp and W, tyrosine: Tyr and Y, and valine: Val and V.

[0179] It should be understood by those skilled in the art that any one or any combination of two or more of the aspects described herein is also included in the present invention, unless a technical contradiction arises based on the common general knowledge of those skilled in the art.

[0180] All references cited herein are incorporated by reference in their entirety.

[0181] The present invention is further illustrated with reference to the following examples, which, however, are not intended to be limiting. [Example]

[0182] Example 1 Screening of affinity-matured variants derived from the parent Dual-Fab H183L072 for improved in vitro cytotoxic activity against tumor cells 1.1. Sequences of affinity matured variants To increase the binding affinity of the parent Dual-Fab H183L072 (heavy chain: SEQ ID NO: 1; light chain: SEQ ID NO: 57), more than 1,000 Dual-Fab variants were generated by introducing single or multiple mutations into the variable regions using H183L072 as a template. Antibodies were expressed in Expi293 (Invitrogen) and purified by Protein A followed by gel filtration when necessary. The sequences of the 15 indicated variants with multiple mutations are listed in Tables 1.1 and 1.2, and the binding kinetics are evaluated at 25°C and / or 37°C using a Biacore T200 instrument (GE Healthcare) as described below in Example 1.2.2. The fold change in affinity for human CD137 and human CD3 due to single mutations in the variable regions is listed in Table 1.3.

[0183] [Table 1.1a]

[0184] [Table 1.1b]

[0185] [Table 1.2a]

[0186] [Table 1.2b] TIFF2026021604000005.tif227170TIFF2026021604000006.tif165170TIFF2026021604000007.tif65170

[0187] [Table 1.3a]

[0188] [Table 1.3b]

[0189] [Table 1.3c] [Table 1.3d]

[0190] In Tables 1.3a-1.3d, the mutated positions and the original amino acid at each position according to Kabat numbering are shown in the top two columns. Values ​​represent the fold change in affinity when each of the mutations shown in the leftmost column was introduced into each position.

[0191] 1.2. Binding kinetics of affinity matured variants 1.2.1. Expression and Purification of Human CD3 and CD137 The γ and ε subunits of the human CD3 complex (human CD3eg linker) were linked by a 29-mer linker, and a Flag tag was fused to the C-terminus of the γ subunit (SEQ ID NO: 84, Tables 1.1a and 1.2a). This construct was transiently expressed using the FreeStyle293F cell line (Thermo Fisher). Conditioned medium expressing the human CD3eg linker was concentrated using a column packed with Q HP resin (GE Healthcare) and then applied to FLAG-tag affinity chromatography. Fractions containing the human CD3eg linker were collected and subsequently applied to a Superdex 200 gel filtration column (GE Healthcare) equilibrated with 1x D-PBS. Fractions containing the human CD3eg linker were then pooled and stored at -80°C.

[0192] Human CD137 extracellular domain (ECD) (SEQ ID NO: 201, Tables 1.1a and 1.2a) bearing a hexahistidine (His-tag) and biotin acceptor peptide (BAP) at its C-terminus was transiently expressed using the FreeStyle293F cell line (Thermo Fisher). Conditioned medium expressing human CD137 ECD was applied to a HisTrap HP column (GE Healthcare) and eluted with a buffer containing imidazole (Nacalai). Fractions containing human CD137 ECD were collected and subsequently loaded onto a Superdex 200 gel filtration column (GE Healthcare) equilibrated with 1x D-PBS. Fractions containing human CD137 ECD were then pooled and stored at -80°C.

[0193] 1.2.2. Affinity Measurement for Human CD3 and CD137 The binding affinity of Dual-Fab antibodies (Dual-Ig) to human CD3 was evaluated at 25°C using a Biacore T200 instrument (GE Healthcare). Anti-human Fc (GE Healthcare) was immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). The antibody was captured on the anti-Fc sensor surface, and then recombinant human CD3 or CD137 was injected onto the flow cell. All antibodies and analytes were prepared in ACES pH 7.4 containing 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, and 0.005% NaN. The sensor surface was regenerated with 3 M MgCl after each cycle. Binding affinity was determined by processing the data and fitting to a 1:1 binding model using Biacore T200 Evaluation software, version 2.0 (GE Healthcare). The CD137 binding affinity assay was performed under the same conditions, except that the assay temperature was set to 37°C. The binding affinities of the Dual-Fab antibodies to recombinant human CD3 and CD137 are shown in Table 1.4.

[0194] [Table 1.4]

[0195] 1.3. Preparation of Bispecific and Trispecific Antibodies To evaluate the efficacy of Dual-Ig variants, bispecific or trispecific antibodies were generated, each with one arm recognizing a tumor antigen and another arm recognizing effector cells, primarily T cells. Anti-GPC3 (heavy chain: SEQ ID NO: 206; light chain: SEQ ID NO: 207), which targets the tumor antigen glypican-3 (GPC3), or a negative control keyhole limpet hemocyanin (KLH) (herein referred to as Ctrl) antibody was used as the anti-target binding arm. The antibodies described in Examples 1.1 and 1.2 were generated using Fab-arm exchange (FAE) according to the method described in (Proc Natl Acad Sci USA. 2013 Mar 26; 110(13): 5145-5150). The molecular format of the bispecific or trispecific antibody is the same as that of conventional IgG. For example, GPC3 / H1643L581 is a trispecific antibody capable of binding to GPC3, CD3, and CD137. To identify which Dual-Ig trispecific variants described in Example 1.1 contribute to the improved cytotoxic activity due to CD137 activity, a GPC3 / CD3ε bispecific antibody (Table 1.1) capable of binding to GPC3 and CD3 was included as a control. All generated antibodies contain a silent Fc with reduced affinity for Fcγ receptors.

[0196] [Example 2] Evaluation of in vitro cytotoxic activity of affinity-matured variants derived from the parent Dual-Fab H183L072 against tumor cells 2.1. Evaluation of CD3 Agonist Activity of Affinity-Matured Variants in Vitro To assess CD3 agonist activity as a result of affinity maturation, an NFAT-luc2 Jurkat luciferase assay is performed. Briefly, 4 x 10 cells expressing human GPC3 on the cell membrane are cultured. 3SK-pca60 cells (Reference Example 13) were used as target cells, and 2.0 × 10 cells / well were incubated in the presence of 0.02, 0.2, and 2 nM of the trispecific antibody for 24 hours. 4 The variants were co-cultured with NFAT-luc2 Jurkat cells (E:T ratio 5) at 1000 cells / well. The variants were divided into plate 1 in the upper panel of Figure 1.1 and plate 2 in the lower panel of Figure 1.1. After 24 hours, luciferase activity was detected using the Bio-Glo Luciferase Assay System (Promega, G7940) according to the manufacturer's instructions. Luminescence (units) was detected using the GloMax® Explorer System (Promega #GM3500), and capture values ​​were plotted using Graphpad Prism 7. The parent trispecific antibody GPC3 / H183L072 and bispecific antibody GPC3 / CD3ε were included at a concentration of 2 nM. Figure 1.1 shows that most variants have similar CD3 agonist activity. Especially at 2 nM, the variants have activity similar to that of the parent H183L072. The top panel of Figure 1.1 showed that all variants in plate 1 had similar CD3 agonist activity. The bottom panel of Figure 1.1 showed that among the variants in plate 2, H1610L939 had slightly weaker CD3 agonist activity, while H2591L581 had the strongest CD3 agonist activity.

[0197] 2.2. Evaluation of CD137 Agonist Activity of Affinity-Matured Variants In Vitro To assess which antibody variants could result in potent CD137 agonist activity as a result of affinity maturation, the GloResponse™ NF-κB-Luc2 / CD137 Jurkat cell line (Promega #CS196004) was used as effector cells, and the SK-pca60 cell line (Reference Example 13) was used as target cells, as described above. 3 cells / well of SK-pca60 cells (target cells) and 2.0 × 10 4Both NF-κB-Luc2 / CD137 Jurkat (effector cells) cells / well were added to each well of a white-bottom 96-well assay plate (Costar, 3917) at an E:T ratio of 5. Antibodies were added to each well at concentrations of 0.5 nM, 2.5 nM, and 5 nM and incubated at 37°C, 5% CO for 5 hours. Expressed luciferase was detected using the Bio-Glo Luciferase Assay System (Promega, G7940) according to the manufacturer's instructions. Luminescence (units) was detected using the GloMax® Explorer System (Promega #GM3500), and capture values ​​were plotted using Graphpad Prism 7.

[0198] In Figure 1.2, the antibody variants are divided into plate 1 (upper panel of Figure 1.2) and plate 2 (lower panel of Figure 1.2). All variants in both plates have detectable CD137 agonist activity compared to GPC3 / CD3ε, which was used as a negative control. The parent antibody GPC3 / H183L072 before affinity maturation was also used as a control in both plates. In Figure 1.2, all variants showed stronger CD137 agonist activity than the parent antibody GPC3 / H183L072 after affinity maturation for CD137 binding. Thus, GPC3 / H1643L581 and GPC3 / H868L581 in plate 1 (top panel of Figure 1.2) and GPC3 / H2594L581 and GPC3 / H2591L581 in plate 2 (bottom panel of Figure 1.2) were the top variants that conferred stronger CD137 agonist activity. In contrast, variants such as GPC3 / H1550L918 in plate 1 and GPC3 / H1610L581 and GPC3 / H1610L939 in plate 2 showed weaker CD137 activity.

[0199] Taken together, Figures 1.1 and 1.2 show that of the variants, GPC3 / H1643L581, GPC3 / H868L581 in plate 1 and GPC3 / H2591L581 in plate 2 appear to have similarly strong activity in Jurkat cells, whereas GPC3 / H1610L939 has weaker activity.

[0200] 2.3. Evaluation of in vitro cytotoxic activity of affinity matured variants To extend the findings regarding CD3 and CD137 activation to in vitro cytotoxic activity, the affinity-matured variants described above were subjected to evaluation of T cell-dependent cytotoxicity (TDCC) activity against SK-pca60 cells using human peripheral blood mononuclear cells.

[0201] 2.3.1. Preparation of frozen human PBMCs A cryovial containing commercially purchased PBMCs (STEMCELL Technologies.) was placed in a 37°C water bath to thaw the cells. The cells were then dispensed into a 15 mL Falcon tube containing 9 mL of medium (the medium used to culture target cells). The cell suspension was then centrifuged at 1,200 rpm for 5 minutes at room temperature. The supernatant was gently aspirated, and fresh warmed medium was added for resuspension, which was used as a human PBMC solution.

[0202] 2.3.2. Measurement of TDCC activity using anti-GPC3 affinity-matured dual-Fab trispecific antibody Cytotoxic activity was evaluated by observing the tumor cell growth inhibition rate in the presence of PBMC using the xCELLigence Real-Time Cell Analyzer (Roche Diagnostics). Figure 1.3 shows the TDCC activity of the anti-GPC3 affinity-matured dual-Fab trispecific antibody. SK-pca60 cell line was used as the target cell. Target cells were detached from the dish and 3.5 × 10 cells were cultured. 3Cells were plated onto E-plate 96 (Roche Diagnostics) in 100 μL / well aliquots by adjusting the cells / well, and cell proliferation measurements were initiated using an xCELLigence Real-Time Cell Analyzer. After 24 hours, the plate was removed, and 50 μL of each antibody prepared at each concentration (3-fold serial dilutions starting from 5 nM, i.e., 0.19, 0.56, 1.67, and 5 nM) was added to the plate. After 15 minutes of reaction at room temperature, 50 μL of fresh human PBMC solution prepared in (Example 2.3.1) was added to the plate at an effector:target ratio of 0.5 (i.e., 1.75 × 10 3 PBMCs were added at 100 μg / well (1000 cells / well), and cell proliferation measurements were resumed using an xCELLigence Real-Time Cell Analyzer. Reactions were carried out at 37°C under 5% carbon dioxide gas. Because CD137 signaling enhances T cell survival and prevents activation-induced cell death, the TDCC assay was performed at a low E:T ratio. To observe the superior cytotoxic activity attributed to CD137 activation, an extended period may be required. Therefore, approximately 120 hours after the addition of PBMCs, the cytostatic growth inhibition (CGI) rate (%) was determined using the following formula: The cell index value obtained from the xCELLigence Real-Time Cell Analyzer used in the calculation was a normalized value, with the cell index value immediately before antibody addition defined as 1. Cell proliferation inhibition rate (%) = (AB) × 100 / (A-1) A represents the mean Cell Index value in wells without added antibody (containing only target cells and human PBMCs), and B represents the mean Cell Index value in target wells. Tests were performed in triplicate.

[0203] As in the previous examples, the affinity matured variants were split onto two plates, with GPC3 / H1643L581 serving as an internal plate control for reference in Figure 1.3. While most variants exhibit similar TDCC activity, it can be observed that among the variants, H1643L581 exhibited relatively stronger TDCC activity at lower concentrations of 0.56 nM and 1.67 nM on both plates. At the 0.56 nM concentration, Figure 1.3a shows that GPC3 / H2591L581 was relatively weaker, while Figure 1.3b shows that GPC3 / H1610L939 was relatively weaker.

[0204] 2.3.3. Measurement of cytokine release using anti-GPC3 affinity-matured dual-Fab trispecific antibody To further confirm the in vitro potency of the antibodies, they were also evaluated for cytokine release. Supernatants at 48 hours from the TDCC assay, similarly performed in Example 2.3.2, were collected and evaluated for the presence of cytokines. Because most antibodies exhibit CD3 agonist activity similar to that of GPC3 / CD3ε in Figure 1.1, GPC3 / CD3ε was added to this assay to evaluate cytokine release as a result of synergistic activity with CD137. Similarly, GPC3 / H1643L581 was used as an internal plate control. Total cytokine release was evaluated using the cytometric bead array (CBA) Human Th1 / T2 Cytokine kit II (BD Biosciences #551809). IFNγ (Figure 1.3c), IL-2 (Figure 1.3d), and IL-6 (Figure 1.3e) were evaluated.

[0205] As shown in Figures 1.3c and 1.3d, GPC3 / H2591L581 and GPC3 / H1643L581 were the top two variants that produced high IFNγ and IL-2 at 5 nM and 1.67 nM in Plate 1. In Plate 2, GPC3 / H1610L939, GPC3 / H2594L581, and GPC3 / H1643L581 showed relatively strong cytokine release at 5 nM. However, only GPC3 / H1643L581 showed a stronger cytokine release at 1.67 nM. Regarding IL-6 levels, as shown in Figure 1.3e, all variants showed similar levels to GPC3 / CD3ε in Plate 1, except for GPC3 / H2591L581, which showed lower IL-6 levels at 0.56 nM and 0.19 nM. Similarly, all variants show similar cytokine release levels as GPC3 / H1643L581 in plate 2. In summary, the Dual Fab variants can show improved IFNγ and IL-2 release compared to GPC3 / CD3ε without significantly increasing IL-6 levels.

[0206] In summary, the affinity-matured variants exhibited stronger CD137 agonist activity, which was able to elicit TDCC activity corresponding to cytokine release. In particular, the variants showed improved levels of IFNγ and IL-2 compared to GPC3 / CD3ε.

[0207] [Example 3] Evaluation of off-target cytotoxic activity of GPC3 / CD3 / human CD137 (2 + 1) trispecific antibody and anti-GPC3 / Dual (1 + 1) trispecific antibody 3.1. Preparation of anti-GPC3 / CD137xCD3 (2+1) trispecific antibody To investigate target-independent cytotoxicity and cytokine release, trispecific antibodies were generated using CrossMab and FAE technology (Figures 2.1 and 2.2). Antibody A (mAb A), a tetravalent IgG-like molecule with two binding domains in each arm, resulting in four binding domains in one molecule, was generated using CrossMab as described above. Antibody B (mAb B), a bivalent IgG, has the same format as a conventional IgG. The Fc regions of both mAb A and mAb B are FcγR-silent, deglycosylated, and amenable to FAE. Six trispecific antibodies were constructed. The target antigens of each Fv region in the six trispecific antibodies are listed in Table 2.1. The naming conventions for the binding domains of mAb A, mAb B, and mAb AB are shown in Figure 2.2. The pairs of mAb A and mAb B used to generate each trispecific antibody, mAb AB, and their sequence numbers are shown in Tables 2.2 and 2.2. The antibody CD3D(2)_i121 (abbreviated as AN121), described in WO2005 / 035584A1, was used as the anti-CD3 antibody. The trispecific antibodies listed in Table 2 were expressed and purified by the methods described above.

[0208] [Table 2.1]

[0209] [Table 2.2]

[0210] [Table 2.3]

[0211] 3.2. Evaluation of GPC3 / CD137xCD3 Trispecific Antibody Binding The binding affinities of the trispecific antibodies to human CD3 and CD137 were evaluated at 37°C using a Biacore T200 instrument (GE Healthcare). Anti-human Fc antibodies (GE Healthcare) were immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). The antibodies were captured on the anti-Fc sensor surface, and then recombinant human CD3 or CD137 was injected onto the flow cell. All antibodies and analytes were prepared in ACES pH 7.4 containing 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, and 0.005% NaN. The sensor surface was regenerated with 3 M MgCl after each cycle. Binding affinities were determined by processing the data and fitting to a 1:1 binding model using Biacore T200 Evaluation software, version 2.0 (GE Healthcare).

[0212] The binding affinities of the trispecific antibodies to recombinant human CD3 and CD137 are shown in Table 2.4.

[0213] [Table 2.4]

[0214] 3.3.Evaluation of off-target cytotoxicity of GPC3 / CD137xCD3 trispecific antibody and anti-GPC3 / Dual-Fab trispecific antibody against human CD137-expressing cells The 2+1 format trispecific antibodies GPC3 / CD137xCD3, GPC3 / CtrlxCD3, or the 1+1 format GPC3 / H183L072 derived from the parent Dual-Fab H183L072 resulted in dose-dependent activation of Jurkat cells in the presence of GPC3-expressing target cells, SK-pca60 (Reference Example 15-5; Figure 28). It was also shown that only the 2+1 format trispecific format, but not the 1+1 format trispecific format using GPC3 / H183L072, resulted in Jurkat cell activation in the presence of hCD137-expressing CHO cells (Reference Example 15-6; Figure 29). This suggested that the 2+1 format could potentially result in tumor antigen-independent activation of T cells.

[0215] To investigate whether affinity maturation of H183L072 could result in potential off-target cytotoxic activity, hCD3-expressing Jurkat cells were co-cultured with hCD137-expressing CHO cells and the affinity-matured variants were subjected to the same evaluation compared against the trispecific 2+1 antibody format. 3 Cells / well of hCD137-expressing CHO (Figure 2.3b) or parental CHO (Figure 2.3a) were cultured at 2.5 x 10 in the presence of 0.5, 5, and 50 nM of the trispecific antibody for 24 hours. 4 The trispecific antibodies were co-cultured with NFAT-luc2 Jurkat cells. Figure 2.3a shows that all trispecific antibodies did not nonspecifically activate Jurkat cells when co-cultured with parental CHO cells. However, it was observed that both GPC3 / CD137xCD3 and Ctrl / CD137xCD3 could activate Jurkat cells in the presence of hCD137-expressing CHO cells. The affinity-matured variants in the 1+1 format did not activate Jurkat cells when co-cultured with hCD137-expressing CHO cells. Taken together, this suggests that the trispecific format GPC3 / CD137xCD3, unlike the GPC3 / Dual(1+1) format, can activate Jurkat cells independently of target or tumor antigen binding, even after affinity maturation for CD137 binding, and may result in off-target cytotoxicity.

[0216] 3.4. Evaluation of off-target cytokine release from PBMCs following GPC3 / CD137xCD3 trispecific antibody and GPC3 / Dual-Fab trispecific antibody A comparison of the trispecific format for off-target toxicity was also assessed using human PBMC solutions. Briefly, 2.0 x 10 cells prepared as described in Example 2.3.1 were used. 5 PBMCs were incubated with 80, 16, and 3.2 nM of the trispecific antibodies for 48 hours in the absence of target cells. Because IL-2 was not detected by either antibody, the levels of IL-6, IFNγ, and TNFα in the supernatants are shown in Figures 2.4a-2.4c. Cytokine release measurements were performed as described in Example 2.3.3. As in Example 2, affinity-matured variants were split into two plates. As shown in Figure 2.4, GPC3 / CD137xCD3, but not anti-GPC3 / Dual-Fab, resulted in the release of IFNγ (Figure 2.4a), TNFα (Figure 2.4b), and IL-6 (Figure 2.4c) from PBMCs. These results suggest that the GPC3 / CD137xCD3 trispecific format resulted in nonspecific activation of PBMCs in the absence of target cells. Finally, the data demonstrated that the Dual-Fab trispecific 1+1 format can confer target-specific effector cell activation without off-target toxicity.

[0217] [Example 4] Evaluation of the in vivo efficacy of GPC3 / CD3ε bispecific antibody and anti-GPC3 / Dual-Fab(1+1) trispecific antibody 4.1. Preparation of anti-GPC3 / Dual-Fab, GPC3 / CD3ε, and GPC3 / CD137 bispecific antibodies Antibodies for in vivo efficacy studies were generated as described in Example 1.3. In addition to the anti-GPC3 / Dual-Fab and GPC3 / CD3ε used in Example 1, anti-CD137 antibodies were generated in bivalent form to obtain GPC3 / CD137, as with the antibodies generated in Example 1.1 (Table 1.1) before FAE was performed in Example 1.3. For the humanized huNOG mouse study, the antibody contains a human Fc with reduced affinity for Fcγ receptors. In contrast, for the CD137 / CD3 double-humanized mouse study, the antibody contains a mouse Fc with reduced affinity for Fcγ receptors.

[0218] Generation of CD137 / CD3 double-humanized mice Human CD137 knock-in (KI) mouse strains were generated by replacing the endogenous CD137 genomic region with the human CD137 genomic sequence using mouse embryonic stem cells. Human CD3 EDG-replaced mice were established as strains in which all three components of the CD3 complex, CD3e, CD3d, and CD3g, were replaced with their human counterparts, CD3E, CD3D, and CD3G (Scientific Rep. 2018;8:46960). CD137 / CD3 double-humanized mouse strains were established by crossing human CD137 KI mice with human CD3 EDG-replaced mice.

[0219] 4.3. Preparation of LLC1 / hGPC3 cell line The mouse cancer cell line LL / 2 (LLC1) (ATCC) was transfected with pCXND3-hGPC3 and single-cell clones were isolated using 500 μg / ml G418. The expression of hGPC3 in the selected clone (LLC1 / hGPC3) was confirmed.

[0220] 4.4. Evaluation of the in vivo efficacy of anti-GPC3 / Dual-Fab trispecific antibodies in hCD3 / hCD137 mice The antibodies prepared in Example 4.1 were evaluated for their in vivo efficacy using a tumor-bearing model. For in vivo efficacy evaluation, the CD3 / CD137 double humanized mice established in Example 4.2, hereafter referred to as "hCD3 / hCD137 mice", were used. LLC1 / hGPC3 cells stably expressing human GPC3 were transplanted into hCD3 / hCD137 mice, and hCD3 / hCD137 mice in which tumor formation was confirmed were treated with GPC3 / H1643L0581, GPC3 / CD137, or GPC3 / CD3ε antibodies.

[0221] More specifically, the following tests were performed in a drug efficacy test of GPC3 / H1643L0581 using the LLC1 / hGPC3 model. LLC1 / hGPC3 (1 × 10 6 Cells) were implanted into the subcutaneous inguinal region of hCD3 / hCD137 mice. The day of implantation was defined as day 0. On day 9 after implantation, mice were randomized into groups according to their body weight and tumor size. On the day of randomization, GPC3 / H1643L0581, GPC3 / CD137, or GPC3 / CD3ε antibody was administered intravenously via the tail vein at 6 mg / kg. The combination therapy group was treated with 6 mg / kg of GPC3 / CD3ε and 6 mg / kg of GPC3 / CD137 antibody. The antibodies were administered only once. Tumor volume and body weight were measured every 3 to 4 days using an antitumor testing system (ANTES version 7.0.0.0).

[0222] As a result, the antitumor activity was more obviously observed in the GPC3 / H1643L0581 group than in the GPC3 / CD3ε and GPC3 / CD137 groups (Figure 3.1a). In another in vivo efficacy evaluation, LLC / hGPC3 cells were implanted into the right flank of hCD3 / hCD137 mice. On day 9, the mice were randomized into groups based on their tumor volume and body weight and intravenously injected with vehicle or the antibody prepared in Example 4.1. Tumor volume was measured twice weekly. Mice were bled 2 hours after treatment for IL-6 analysis. Plasma samples were analyzed using Bio-Plex Pro Mouse Cytokine Th1 Panel according to the manufacturer's protocol. As shown in Figures 3.1b and 3.1c, the GPC3 / Dual group exhibited stronger antitumor activity and less IL-6 production than the GPC3 / CD3ε group.

[0223] 4.5. Evaluation of the in vivo efficacy of anti-GPC3 / Dual-Fab trispecific antibodies in HuNOG mice The anti-tumor activity of the anti-GPC3 / Dual-Fab antibody, GPC3 / CD3ε bispecific antibody, and GPC3 / CD137 bispecific antibody prepared in Example 4.1 was tested in the sk-pca-13a human liver cancer model. The GPC3 / CD3ε bispecific antibody was also tested in combination with the GPC3 / CD137 bispecific antibody. sk-pca-13a cells were subcutaneously implanted into NOG humanized mice. To obtain the sk-pca-13a cell line, the human GPC3 gene was integrated into the chromosome of the human liver adenocarcinoma cell line SK-HEP-1 (ATCC No. HTB-52) by methods well known to those skilled in the art.

[0224] NOG female mice were purchased from In-Vivo Science. For humanization, the mice were sublethally irradiated and then injected with 100,000 human umbilical cord blood cells (ALLCELLS) one day later. Sixteen weeks later, sk-pca-13a cells (1 × 10 7The cells were mixed with Matrigel™ Basement Membrane Matrix (Corning) and implanted into the right flank of humanized NOG mice. The day of implantation was defined as day 0. On day 19, mice were randomized based on tumor volume and body weight and intravenously injected with either vehicle (PBS containing 0.05% Tween), 5 mg / kg GPC3 / CD3ε, 5 mg / kg GPC3 / H1643L0581, or a combination of 5 mg / kg GPC3 / CD3ε and 5 mg / kg GPC3 / CD137.

[0225] As a result, anti-GPC3 / Dual-Fab (GPC3 / H1643L0581) showed stronger antitumor activity than GPC3 / CD3ε (Figure 3.2).

[0226] [Example 5] X-ray crystal structure analysis of H0868L0581 / hCD137 complex 5.1. Preparation of antibodies for cocrystallography H0868L581 was selected for co-crystallography with hCD137 protein. The bivalent antibody was transiently transfected and expressed using the Expi293 Expression system (Thermo Fisher Scientific). The culture supernatant was collected, and the antibody was purified from the supernatant using MabSelect SuRe affinity chromatography (GE Healthcare) followed by gel filtration chromatography on a Superdex200 (GE Healthcare).

[0227] 5.2. Expression and purification of the extracellular domain (24-186) of human CD137 The extracellular domain of human CD137 fused to Fc via a factor Xa-cleavable linker (CD137-FFc, SEQ ID NO: 81) was expressed in HEK293 cells in the presence of kifunensine. CD137-FFc from the culture medium was purified by affinity chromatography (HiTrap MabSelect SuRe column, GE Healthcare) and size-exclusion chromatography (HiLoad 16 / 600 Superdex 200 pg column, GE Healthcare). The Fc was cleaved with factor Xa, and the resulting CD137 extracellular domain was further purified using a tandem gel filtration column (HiLoad 16 / 600 Superdex 200 pg, GE Healthcare) and a Protein A column (HiTrap MabSelect SuRe 1 ml, GE Healthcare), followed by purification using benzamidine Sepharose resin (GE Healthcare). Fractions containing the CD137 extracellular domain were pooled and stored at -80°C.

[0228] 5.3. Preparation of Fab Fragments of H0868L0581 and Anti-CD137 Control Antibody Antibodies for crystal structure analysis were transiently transfected and expressed using the Expi293 Expression system (Thermo Fisher Scientific). Culture supernatants were collected, and antibodies were purified from the supernatants using MabSelect SuRe affinity chromatography (GE Healthcare) followed by gel filtration chromatography using Superdex200 (GE Healthcare). Fab fragments of H0868L0581 and a known anti-CD137 control antibody (hereafter referred to as 137Ctrl; heavy chain SEQ ID NO: 82, light chain SEQ ID NO: 83) were prepared by conventional methods using restriction digestion with Lys-C (Roche), followed by loading onto a Protein A column (MabSelect SuRe, GE Healthcare) to remove the Fc fragment, a cation exchange column (HiTrap SP HP, GE Healthcare), and a gel filtration column (Superdex200 16 / 60, GE Healthcare). Fractions containing the Fab fragments were pooled and stored at -80°C.

[0229] 5.4. Preparation of H0868L0581 Fab, 137Ctrl, and Human CD137 Complex Purified CD137 was mixed with GST-tagged endoglycosidase F1 (in-house developed) for deglycosylation, followed by purification of CD137 using a gel filtration column (HiLoad 16 / 600 Superdex 200 pg, GE Healthcare) and a Protein A column (HiTrap MabSelect SuRe 1 ml, GE Healthcare). Purified CD137 was mixed with H0868L0581 Fab. The complex was purified using a gel filtration column (Superdex 200 Increase 10 / 300 GL, GE Healthcare). The purified H0868L0581 Fab and CD137 complex was then mixed with 137Ctrl. The ternary complex was purified by gel filtration chromatography (Superdex200 10 / 300 Increase, GE Healthcare) using a column equilibrated with 25 mM HEPES pH 7.3 and 100 mM NaCl.

[0230] 5.5.Crystallization The purified conjugate was concentrated to approximately 10 mg / mL and crystallized by sitting drop vapor diffusion at 21° C. The reservoir solution consisted of 0.1 M Tris-HCl pH 8.5, 25.0% v / v polyethylene glycol monomethyl ether 550.

[0231] 5.6. Data collection and structure determination X-ray diffraction data were collected using an X06SA SLS. The crystal was kept frozen in a nitrogen stream at -178°C throughout the measurements. A total of 1,440 X-ray diffraction images were collected using an Eiger X16M (DECTRIS) beamline, rotating the crystal 0.25° at a time. Cell parameter determination, diffraction spot indexing, and diffraction data processing from the diffraction images were performed using the autoPROC program (Acta. Cryst. 2011, D67: 293-302), XDS Package (Acta. Cryst. 2010, D66: 125-132), and AIMLESS (Acta. Cryst. 2013, D69: 1204-1214). Finally, diffraction intensity data with a maximum resolution of 3.705 Å were obtained. Crystallographic data statistics are...

Claims

1. An antigen-binding molecule comprising an antibody variable region capable of binding to CD3 and CD137 but not simultaneously binding to CD3 and CD137, Preferably by SPR under the following conditions: 37°C, pH 7.4, 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3; antigen-binding molecules are immobilized on a CM4 sensor chip, and antigen is the analyte. When measured at 5 x 10 -6 The antigen-binding molecule binds to CD137 with an equilibrium dissociation constant (KD) of less than M.

2. (a) at least one, two, three, or more amino acid residues of the extracellular domain of CD3ε (CD3 epsilon) comprising the amino acid sequence of SEQ ID NO: 159; and / or (b) at least one, two, three, or more amino acid residues in the N-terminal region of CD137 comprising the amino acid sequence of LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAEC (SEQ ID NO: 152) of human CD137, preferably LQDPCSN, NNRNQI, and / or GQRTCDI. The antigen-binding molecule of claim 1 .

3. The antigen-binding molecule of claim 1 or 2, wherein the antibody variable region comprises any one of the following: (a1) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 16, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 30, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 44, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a2) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 17, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 31, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 45, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 64, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 69, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 74; (a3) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 18, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 32, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 46, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a4) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 19, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 33, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 47, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a5) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 19, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 33, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 47, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 65, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 70, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 75; (a6) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 20, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 34, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 48, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a7) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 22, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 36, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 50, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a8) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 23, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 37, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 51, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a9) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 23, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 37, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 51, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 66, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 71, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 76; (a10) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 24, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 38, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 52, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a11) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 25, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 39, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 53, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 66, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 71, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 76; (a12) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 26, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 40, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 54, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 66, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 71, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 76; (a13) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 26, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 40, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 54, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a14) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 27, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 41, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 55, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (a15) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 28, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 42, a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 56, a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 63, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 68, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 73; (b1) HCDR1 comprising the amino acid sequence of SEQ ID NO: 16, HCDR2 comprising the amino acid sequence of SEQ ID NO: 30, HCDR3 comprising the amino acid sequence of SEQ ID NO: 44, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b2) HCDR1 comprising the amino acid sequence of SEQ ID NO: 17, HCDR2 comprising the amino acid sequence of SEQ ID NO: 31, HCDR3 comprising the amino acid sequence of SEQ ID NO: 45, LCDR1 comprising the amino acid sequence of SEQ ID NO: 64, LCDR2 comprising the amino acid sequence of SEQ ID NO: 69, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 74; (b3) HCDR1 comprising the amino acid sequence of SEQ ID NO: 18, HCDR2 comprising the amino acid sequence of SEQ ID NO: 32, HCDR3 comprising the amino acid sequence of SEQ ID NO: 46, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b4) HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, HCDR2 comprising the amino acid sequence of SEQ ID NO: 33, HCDR3 comprising the amino acid sequence of SEQ ID NO: 47, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b5) HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, HCDR2 comprising the amino acid sequence of SEQ ID NO: 33, HCDR3 comprising the amino acid sequence of SEQ ID NO: 47, LCDR1 comprising the amino acid sequence of SEQ ID NO: 65, LCDR2 comprising the amino acid sequence of SEQ ID NO: 70, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 75; (b6) HCDR1 comprising the amino acid sequence of SEQ ID NO: 20, HCDR2 comprising the amino acid sequence of SEQ ID NO: 34, HCDR3 comprising the amino acid sequence of SEQ ID NO: 48, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b7) HCDR1 comprising the amino acid sequence of SEQ ID NO: 22, HCDR2 comprising the amino acid sequence of SEQ ID NO: 36, HCDR3 comprising the amino acid sequence of SEQ ID NO: 50, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b8) HCDR1 comprising the amino acid sequence of SEQ ID NO: 23, HCDR2 comprising the amino acid sequence of SEQ ID NO: 37, HCDR3 comprising the amino acid sequence of SEQ ID NO: 51, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b9) HCDR1 comprising the amino acid sequence of SEQ ID NO: 23, HCDR2 comprising the amino acid sequence of SEQ ID NO: 37, HCDR3 comprising the amino acid sequence of SEQ ID NO: 51, LCDR1 comprising the amino acid sequence of SEQ ID NO: 66, LCDR2 comprising the amino acid sequence of SEQ ID NO: 71, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 76; (b10) HCDR1 comprising the amino acid sequence of SEQ ID NO: 24, HCDR2 comprising the amino acid sequence of SEQ ID NO: 38, HCDR3 comprising the amino acid sequence of SEQ ID NO: 52, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b11) HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, HCDR2 comprising the amino acid sequence of SEQ ID NO: 39, HCDR3 comprising the amino acid sequence of SEQ ID NO: 53, LCDR1 comprising the amino acid sequence of SEQ ID NO: 66, LCDR2 comprising the amino acid sequence of SEQ ID NO: 71, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 76; (b12) HCDR1 comprising the amino acid sequence of SEQ ID NO: 26, HCDR2 comprising the amino acid sequence of SEQ ID NO: 40, HCDR3 comprising the amino acid sequence of SEQ ID NO: 54, LCDR1 comprising the amino acid sequence of SEQ ID NO: 66, LCDR2 comprising the amino acid sequence of SEQ ID NO: 71, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 76; (b13) HCDR1 comprising the amino acid sequence of SEQ ID NO: 26, HCDR2 comprising the amino acid sequence of SEQ ID NO: 40, HCDR3 comprising the amino acid sequence of SEQ ID NO: 54, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b14) HCDR1 comprising the amino acid sequence of SEQ ID NO: 27, HCDR2 comprising the amino acid sequence of SEQ ID NO: 41, HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (b15) HCDR1 comprising the amino acid sequence of SEQ ID NO: 28, HCDR2 comprising the amino acid sequence of SEQ ID NO: 42, HCDR3 comprising the amino acid sequence of SEQ ID NO: 56, LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 73; (c1) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 2, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c2) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 3, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 59; (c3) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 4, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c4) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c5) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 5, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 60; (c6) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 6, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c7) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 8, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c8) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 9, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c9) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 9, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 61; (c10) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 10, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c11) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 11, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 61; (c12) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 12, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 61; (c13) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 12, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c14) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 13, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (c15) a heavy chain variable domain (VH) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 14, and a light chain variable domain (VL) comprising an amino acid sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 58; (d1) a heavy chain variable domain (VH) of SEQ ID NO: 2, and a light chain variable domain (VL) of SEQ ID NO: 58; (d2) a heavy chain variable domain (VH) of SEQ ID NO: 3, and a light chain variable domain (VL) of SEQ ID NO: 59; (d3) a heavy chain variable domain (VH) of SEQ ID NO: 4, and a light chain variable domain (VL) of SEQ ID NO: 58; (d4) a heavy chain variable domain (VH) of SEQ ID NO: 5, and a light chain variable domain (VL) of SEQ ID NO: 58; (d5) a heavy chain variable domain (VH) of SEQ ID NO: 5, and a light chain variable domain (VL) of SEQ ID NO: 60; (d6) a heavy chain variable domain (VH) of SEQ ID NO: 6, and a light chain variable domain (VL) of SEQ ID NO: 58; (d7) a heavy chain variable domain (VH) of SEQ ID NO: 8, and a light chain variable domain (VL) of SEQ ID NO: 58; (d8) a heavy chain variable domain (VH) of SEQ ID NO: 9, and a light chain variable domain (VL) of SEQ ID NO: 58; (d9) a heavy chain variable domain (VH) of SEQ ID NO: 9, and a light chain variable domain (VL) of SEQ ID NO: 61; (d10) a heavy chain variable domain (VH) of SEQ ID NO: 10, and a light chain variable domain (VL) of SEQ ID NO: 58; (d11) a heavy chain variable domain (VH) of SEQ ID NO: 11, and a light chain variable domain (VL) of SEQ ID NO: 61; (d12) a heavy chain variable domain (VH) of SEQ ID NO: 12, and a light chain variable domain (VL) of SEQ ID NO: 61; (d13) a heavy chain variable domain (VH) of SEQ ID NO: 12, and a light chain variable domain (VL) of SEQ ID NO: 58; (d14) a heavy chain variable domain (VH) of SEQ ID NO: 13, and a light chain variable domain (VL) of SEQ ID NO: 58; (d15) a heavy chain variable domain (VH) of SEQ ID NO: 14, and a light chain variable domain (VL) of SEQ ID NO: 58; (e) an antibody variable region that competes with any one of the antibody variable regions of (a1) to (d15) for binding to CD3; (f) an antibody variable region that competes with any one of the antibody variable regions (a1) to (d15) for binding to CD137; (g) an antibody variable region that binds to the same epitope on CD3 as any one of the antibody variable regions of (a1) to (d15); (h) An antibody variable region that binds to the same epitope on CD137 as any one of the antibody variable regions (a1) to (d15).

4. (a) a heavy chain variable domain amino acid sequence comprising, at each of the following positions (all according to Kabat numbering), one or more of the following amino acid residues as indicated for that position: A, D, E, I, G, K, L, M, N, R, T, W, or Y at amino acid position 26; D, F, G, I, M, or L at amino acid position 27; D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 28; F or W at amino acid position 29; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 30; F, I, N, R, S, T, or V at amino acid position 31; A, H, I, K, L, N, Q, R, S, T, or V at amino acid position 32; W at amino acid position 33; F, I, L, M, or V at amino acid position 34; F, H, S, T, V, or Y at amino acid position 35; E, F, H, I, K, L, M, N, Q, S, T, W, or Y at amino acid position 50; I, K, or V at amino acid position 51; K, M, R, or T at amino acid position 52; A, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y at amino acid position 52b; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 52c; A, E, F, H, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 53; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 54; E, F, G, H, L, M, N, Q, W, or Y at amino acid position 55; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 56; A, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, or V at amino acid position 57; A, F, H, K, N, P, R, or Y at amino acid position 58; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 59; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 60; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 61; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 62; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 63; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 64; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 65; H or R at amino acid position 93; F, G, H, L, M, S, T, V, or Y at amino acid position 94; I or V at amino acid position 95; F, H, I, K, L, M, T, V, W, or Y at amino acid position 96; F, Y, or W at amino acid position 97; A, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 98; A, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 99; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100a; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100b; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100c; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100d; A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y at amino acid position 100e; A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100f; Approximately 100g of amino acids A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; A, D, E, G, H, I, L, M, N, P, S, T, or V at amino acid position 100h; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 100i; A, D, F, I, L, M, N, Q, S, T, or V at amino acid position 101; A, D, E, F, G, H, IK, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 102; and / or (b) a light chain variable domain amino acid sequence comprising, at each of the following positions (all according to Kabat numbering), one or more of the following amino acid residues as indicated for that position: A, D, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 24; A, G, N, P, S, T, or V at amino acid position 25; A, D, E, F, G, I, K, L, M, N, Q, R, S, T, or V at amino acid position 26; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 27; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 27a; A, I, L, M, P, T, or V at amino acid position 27b; A, E, F, H, I, K, L, M, N, P, Q, R, T, W, or Y at amino acid position 27c; A, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 27d; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 27e; G, N, S, or T at amino acid position 28; A, F, G, H, K, L, M, N, Q, R, S, T, W, or Y at amino acid position 29; A, F, G, H, I, K, L, M, N, Q, R, V, W, or Y at amino acid position 30; I, L, Q, S, T, or V at amino acid position 31; F, W, or Y at amino acid position 32; A, F, H, L, M, Q, or V at amino acid position 33; A, H, or S at amino acid position 34; I, K, L, M, or R at amino acid position 50; A, E, I, K, L, M, Q, R, S, T, or V at amino acid position 51; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 52; A, E, F, G, H, K, L, M, N, P, Q, R, S, V, W, or Y at amino acid position 53; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 54; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y at amino acid position 55; A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y at amino acid position 56; A, G, K, S, or Y at amino acid position 89; Q at amino acid position 90; G at amino acid position 91; A, D, H, K, N, Q, R, S, or T at amino acid position 92; A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y at amino acid position 93; A, D, H, I, M, N, P, Q, R, S, T, or V at amino acid position 94; P at amino acid position 95; F or Y at amino acid position 96; and A, D, E, G, H, I, K, L, M, N, Q, R, S, T, or V at amino acid position 97 The antigen-binding molecule of any one of (a1) to (a15) or (c1) to (c15) of claim 3, comprising:

5. The antigen-binding molecule of any one of claims 1 to 4, having at least one characteristic selected from the group consisting of the following (1) to (3): (1) The antigen-binding molecule does not simultaneously bind to CD3 and CD137, each of which is expressed on different cells; (2) The antigen-binding molecule has agonist activity against CD137; and (3) The antigen-binding molecule has a KD value for binding to human CD137 that is equivalent to or 10-fold, 20-fold, 50-fold, or 100-fold lower than a reference antibody comprising the VH sequence of SEQ ID NO: 1 and the VL sequence of SEQ ID NO: 57, wherein the KD value is preferably measured by SPR under the following conditions: 37°C, pH 7.4, 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3; antigen-binding molecules are immobilized on a CM4 sensor chip, and antigen is the analyte. It is measured in

6. The antigen-binding molecule of any one of claims 1 to 5, further comprising an antibody variable region capable of binding to a third antigen different from CD3 and CD137.

7. The antigen-binding molecule of claim 6 , wherein the third antigen is a molecule that is specifically expressed in cancer tissue.

8. The antigen-binding molecule of any one of claims 1 to 7, further comprising an antibody Fc region.

9. The antigen-binding molecule of claim 8 , wherein the Fc region has reduced FcγR-binding activity compared to the Fc region of a native human IgG1 antibody.

10. A pharmaceutical composition comprising the antigen-binding molecule of any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

11. An isolated polynucleotide comprising a nucleotide sequence encoding the antigen-binding molecule of any one of claims 1 to 9.

12. An expression vector comprising the polynucleotide of claim 11.

13. A host cell transformed or transfected with a polynucleotide according to claim 11 or an expression vector according to claim 12.

14. A method for producing a multispecific antigen-binding molecule or a multispecific antibody, comprising culturing the host cell of claim 13.

15. A method for obtaining or screening for an antibody variable region that can bind to CD3 and CD137, but does not simultaneously bind to CD3 and CD137, comprising the steps of: (a) providing a library comprising a plurality of antibody variable regions; (b) contacting the library provided in step (a) with either CD3 or CD137 as a first antigen and collecting antibody variable regions that bind to the first antigen; (c) contacting the antibody variable regions collected in step (b) with a second antigen selected from CD3 and CD137, and collecting the antibody variable regions bound to the second antigen; and (d) an antibody variable region: (1) Preferably by SPR under the following conditions: 37°C, pH 7.4, 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3; antigen-binding molecules are immobilized on a CM4 sensor chip, and antigen is the analyte. When measured at 1000 kJ / s, it is approximately 5 x 10 -6 Less than M or 5 x 10 -6 M to 3 x 10 -8 an antibody variable region that binds to CD137 with an equilibrium dissociation constant (KD) of M; and / or (2) Preferably by SPR under the following conditions: 25°C, pH 7.4, 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% NaN3; antigen-binding molecules are immobilized on a CM4 sensor chip, and antigen is the analyte. When measured at 2 x 10 -6 M to 1 x 10 -8 An antibody variable region that binds to CD3 with an equilibrium dissociation constant (KD) of M A process of selecting.

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