Anti-CD137 antigen binding molecules for cancer treatment

Abstract

The purpose of the present invention is to provide: an anti-cancer agent which has an immune cell activating activity, cytotoxic activity, or anti-tumor activity, but has a low effect on non-tumor tissues such as normal tissues, and has few side effects; combination therapies using these anti-cancer agents and other anti-cancer agents; and pharmaceutical combinations for use in these combination therapies. Provided is an anti-cancer agent that is expected to be applied to various types of cancers by using, as an active ingredient, the anti-CD137 antigen-binding molecule of the present disclosure (whose binding activity to CD137 is changed according to various substances in a target tissue (e.g., a low molecular weight compound), said anti-cancer agent having an immune cell activating effect, a cytotoxic activity, or an anti-tumor activity, the effect on non-tumor tissues such as normal tissues is low, and the side effect is small. Combination therapies using these and other anti-cancer agents, as well as pharmaceutical compositions for use in these combination therapies, are also provided.

Description

[0001] This application is a divisional application of the international application PCT / JP2021 / 004871, international application date February 10, 2021, Chinese application number 202180024000.3, entitled "Anti-CD137 antigen-binding molecule for cancer treatment". Technical Field

[0002] This disclosure relates to anticancer agents comprising antiCD137 antigen-binding molecules, and treatment methods using such agents in combination with another anticancer agent. Background Technology

[0003] Cancer is a deadly disease that, with certain exceptions, is difficult to cure completely. Treatment outcomes with chemotherapy agents, the primary treatment, are not very good. It has been proposed that not only the heterogeneity of cancer cells themselves plays a significant role, but also the tumor microenvironment as a factor contributing to the difficulty of cancer treatment (Non-Patent Literature 1). Recently, it has been shown that unresectable malignant melanomas, among others, may be curable with anti-CTLA-4 antibodies that inhibit the immunosuppressive function of CTLA-4, thereby promoting T-cell activation (Non-Patent Literature 2). In 2011, the anti-human CTLA-4 monoclonal antibody (ipilimumab) was approved by the U.S. Food and Drug Administration (FDA) as the world's first immune-activating antibody drug. Furthermore, inhibitory antibodies targeting PD-1 and PD-L1 (other immune checkpoint molecules besides CTLA-4) have been reported to have therapeutic effects (Non-Patent Literature 3) and have received FDA approval.

[0004] It is understood that T cells, which play an important role in tumor immunity, are activated by two signals: 1) the binding of the T cell receptor (TCR) to antigen peptides presented by major histocompatibility complex (MHC) class I molecules, and the activation of the TCR; 2) the binding of co-stimulatory molecules on the T cell surface to their ligands on antigen-presenting cells, and the activation of these co-stimulatory molecules. Furthermore, the activation of co-stimulatory molecules belonging to the tumor necrosis factor receptor superfamily (TNFRSF), including CD137 (4-1BB), on the T cell surface has been described as important for T cell activation (Non-Patent Literature 4).

[0005] The tumor necrosis factor receptor superfamily includes molecules such as CD137, CD40, OX40, RANK, and GITR. CD137 has been reported to be expressed not only on the surface of T cells but also on the surface of other immune cells such as dendritic cells (DCs), B cells, NK cells, macrophages, and neutrophils (NPL 5).

[0006] CD137 agonist antibodies have been shown to exhibit antitumor activity in mouse models, primarily due to activation of CD8-positive T and NK cells in mouse model experiments (NPL 6). However, side effects caused by nonspecific hepatotoxicity of CD137 agonist antibodies have become clinical and non-clinical problems, hindering anticipated progress in drug development (Non-Patent Literature 7, Non-Patent Literature 8). It is suggested that these side effects are mainly caused by activation of immune cells in non-tumor, non-immune tissues (e.g., the liver), involving the binding of antibodies to Fcγ receptors via their constant regions (Non-Patent Literature 9). On the other hand, it has been reported that in order for agonist anti-TNF receptor superfamily members to exhibit agonistic activity in vivo, the antibody needs to be cross-linked with cells expressing Fcγ receptors (cells expressing FcγRII) (Non-Patent Literature 10). That is, the binding of CD137 agonist antibodies to Fcγ receptors is involved both in the efficacy of the antibody's antitumor activity and in its side effects such as hepatotoxicity. Therefore, increasing the binding between the antibody and the Fcγ receptor is expected to enhance efficacy, but may also increase hepatotoxicity. Conversely, decreasing the binding between the antibody and the Fcγ receptor may reduce side effects but also decrease efficacy. To date, there are no separate reports on the efficacy and side effects of CD137 agonist antibodies. Furthermore, the antitumor effects of CD137 agonist antibodies are not always effective clinically, and there is a desire to further improve efficacy while avoiding toxicity. Therefore, there is a desire to develop a new drug that can induce an antitumor immune response while reducing those side effects.

[0007] When therapeutic antibodies are administered in vivo, it is desirable that their target antigens be specifically expressed only at the lesion site. However, in many cases, the same antigens are also expressed in non-lesion sites, i.e., normal tissues, which may be the cause of undesirable side effects from a therapeutic point of view. For example, although antibodies against tumor antigens may exhibit cytotoxic activity against tumor cells through ADCC, they may also damage normal cells if the same antigens are expressed in normal cells. To address the above problem, focusing on the phenomenon that certain compounds are abundant in target tissues (e.g., tumor tissue), techniques have been developed to identify antigen-binding molecules whose antigen-binding activity changes depending on the concentration of such compounds (e.g., Patent Document 1).

[0008] In recent years, the efficacy of immunotherapies targeting immune checkpoint molecules such as CTLA-4, PD-1, and PD-L1 has been clinically proven. However, these drugs are not effective for all patients, and further improvements in efficacy are needed. Regarding the combined use of immunotherapies, it has been confirmed that the combination of nivolumab and ipilimumab improves efficacy in melanoma compared to ipilimumab alone (Non-Patent Literature 11).

[0009] Reference List

[0010] Patent documents

[0011] [PTL 1] WO2013 / 180200

[0012] Non-patent literature

[0013] [NPL 1] Hanahan, Cell, 2011, 144, 646-74

[0014] [NPL 2] Prieto, Clin Cancer Res. 2012, 18, 2039-47

[0015] [NPL 3] Hamid, Expert Opin. Biol. Ther., 2013, 6, 847-61

[0016] [NPL 4] Summers, Nat Rev Immunol, 2012, 12, 339-51

[0017] [NPL 5] Vinay, Cellular & Molecular Immunology, 2011, 8, 281-284

[0018] [NPL 6] Houot, Blood, 2009, 114, 3431-8

[0019] [NPL 7] Ascierto, Semin Oncol, 2010, 37, 508-16

[0020] [NPL 8] Dubrot, Cancer Immunol Immunother, 2010, 59, 1223-33

[0021] [NPL 9] Schabowsky, Vaccine, 2009, 28, 512-22

[0022] [NPL 10] Li, Proc Natl Acad Sci US A. 2013, 110(48), 19501-6

[0023] [NPL 11] N Eng J Med (2015) vol.373, p.23-34 Summary of the Invention

[0024] Technical issues

[0025] This disclosure relates to an anticancer agent comprising an anti-CD137 antigen-binding molecule, and a combination therapy with another anticancer agent.

[0026] Solution to the problem

[0027] To provide an anti-CD137 antigen-binding molecule with immune cell activating, cytotoxic, or antitumor activity, while exhibiting reduced activity and fewer side effects in non-tumor tissues such as normal tissues, and to provide a method of using the anti-CD137 antigen-binding molecule, this invention provides an anticancer agent comprising the anti-CD137 antigen-binding molecule as an active ingredient, characterized in that its binding activity to CD137 varies depending on various compounds (e.g., small molecule compounds) in the target tissue (e.g., tumor tissue). This disclosure also provides a combination therapy using such an anticancer agent comprising the anti-CD137 antigen-binding molecule and another agent.

[0028] Specifically, this disclosure provides anticancer agents comprising anti-CD137 antigen-binding molecules, methods of using them, combination therapies using such anticancer agents and another anticancer agent, kits, etc., as exemplarily described below. [1]

[0030] An anticancer agent comprising an anti-CD137 antigen-binding molecule as an active ingredient, said anti-CD137 antigen-binding molecule comprising any combination of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 selected from (a) to (m):

[0031] (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO:7; HVR-H2, which contains the amino acid sequence of SEQ ID NO:8; HVR-H3, which contains the amino acid sequence of SEQ ID NO:17; HVR-L1, which contains the amino acid sequence of SEQ ID NO:21; HVR-L2, which contains the amino acid sequence of SEQ ID NO:26; and HVR-L3, which contains the amino acid sequence of SEQ ID NO:27;

[0032] (b) HVR-H1, comprising the amino acid sequence of SEQ ID NO:7; HVR-H2, comprising the amino acid sequence of SEQ ID NO:9; HVR-H3, comprising the amino acid sequence of SEQ ID NO:17; HVR-L1, comprising the amino acid sequence of SEQ ID NO:22; HVR-L2, comprising the amino acid sequence of SEQ ID NO:26; and HVR-L3, comprising the amino acid sequence of SEQ ID NO:27;

[0033] (c) HVR-H1, comprising the amino acid sequence of SEQ ID NO:7; HVR-H2, comprising the amino acid sequence of SEQ ID NO:10; HVR-H3, comprising the amino acid sequence of SEQ ID NO:17; HVR-L1, comprising the amino acid sequence of SEQ ID NO:22; HVR-L2, comprising the amino acid sequence of SEQ ID NO:26; and HVR-L3, comprising the amino acid sequence of SEQ ID NO:27;

[0034] (d) HVR-H1, which contains the amino acid sequence of SEQ ID NO:7; HVR-H2, which contains the amino acid sequence of SEQ ID NO:11; HVR-H3, which contains the amino acid sequence of SEQ ID NO:18; HVR-L1, which contains the amino acid sequence of SEQ ID NO:21; HVR-L2, which contains the amino acid sequence of SEQ ID NO:26; and HVR-L3, which contains the amino acid sequence of SEQ ID NO:27;

[0035] (e) HVR-H1, comprising the amino acid sequence of SEQ ID NO:7; HVR-H2, comprising the amino acid sequence of SEQ ID NO:8; HVR-H3, comprising the amino acid sequence of SEQ ID NO:18; HVR-L1, comprising the amino acid sequence of SEQ ID NO:21; HVR-L2, comprising the amino acid sequence of SEQ ID NO:26; and HVR-L3, comprising the amino acid sequence of SEQ ID NO:27;

[0036] (f) HVR-H1, comprising the amino acid sequence of SEQ ID NO:7; HVR-H2, comprising the amino acid sequence of SEQ ID NO:12; HVR-H3, comprising the amino acid sequence of SEQ ID NO:18; HVR-L1, comprising the amino acid sequence of SEQ ID NO:21; HVR-L2, comprising the amino acid sequence of SEQ ID NO:26; and HVR-L3, comprising the amino acid sequence of SEQ ID NO:28;

[0037] (g) HVR-H1, comprising the amino acid sequence of SEQ ID NO:7; HVR-H2, comprising the amino acid sequence of SEQ ID NO:13; HVR-H3, comprising the amino acid sequence of SEQ ID NO:18; HVR-L1, comprising the amino acid sequence of SEQ ID NO:21; HVR-L2, comprising the amino acid sequence of SEQ ID NO:26; and HVR-L3, comprising the amino acid sequence of SEQ ID NO:29;

[0038] (h) HVR-H1, comprising the amino acid sequence of SEQ ID NO:7; HVR-H2, comprising the amino acid sequence of SEQ ID NO:14; HVR-H3, comprising the amino acid sequence of SEQ ID NO:19; HVR-L1, comprising the amino acid sequence of SEQ ID NO:23; HVR-L2, comprising the amino acid sequence of SEQ ID NO:26; and HVR-L3, comprising the amino acid sequence of SEQ ID NO:27;

[0039] (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO:7; HVR-H2, which contains the amino acid sequence of SEQ ID NO:15; HVR-H3, which contains the amino acid sequence of SEQ ID NO:20; HVR-L1, which contains the amino acid sequence of SEQ ID NO:24; HVR-L2, which contains the amino acid sequence of SEQ ID NO:26; and HVR-L3, which contains the amino acid sequence of SEQ ID NO:27;

[0040] (j) HVR-H1, which contains the amino acid sequence of SEQ ID NO:7; HVR-H2, which contains the amino acid sequence of SEQ ID NO:15; HVR-H3, which contains the amino acid sequence of SEQ ID NO:20; HVR-L1, which contains the amino acid sequence of SEQ ID NO:25; HVR-L2, which contains the amino acid sequence of SEQ ID NO:26; and HVR-L3, which contains the amino acid sequence of SEQ ID NO:27;

[0041] (k) HVR-H1, comprising the amino acid sequence of SEQ ID NO:7; HVR-H2, comprising the amino acid sequence of SEQ ID NO:16; HVR-H3, comprising the amino acid sequence of SEQ ID NO:20; HVR-L1, comprising the amino acid sequence of SEQ ID NO:25; HVR-L2, comprising the amino acid sequence of SEQ ID NO:26; and HVR-L3, comprising the amino acid sequence of SEQ ID NO:27;

[0042] (l) HVR-H1, comprising the amino acid sequence of SEQ ID NO:7; HVR-H2, comprising the amino acid sequence of SEQ ID NO:14; HVR-H3, comprising the amino acid sequence of SEQ ID NO:19; HVR-L1, comprising the amino acid sequence of SEQ ID NO:24; HVR-L2, comprising the amino acid sequence of SEQ ID NO:26; and HVR-L3, comprising the amino acid sequence of SEQ ID NO:27; and

[0043] (m) HVR-H1, which contains the amino acid sequence of SEQ ID NO:7; HVR-H2, which contains the amino acid sequence of SEQ ID NO:14; HVR-H3, which contains the amino acid sequence of SEQ ID NO:17; HVR-L1, which contains the amino acid sequence of SEQ ID NO:21; HVR-L2, which contains the amino acid sequence of SEQ ID NO:26; and HVR-L3, which contains the amino acid sequence of SEQ ID NO:27. [1.1]

[0045] An anticancer agent comprising an anti-CD137 antigen-binding molecule as an active ingredient, said anti-CD137 antigen-binding molecule comprising:

[0046] (a) VH, which has at least 95% sequence identity with any amino acid sequence of SEQ ID NO: 43 to 53; or

[0047] (b) VL, which has at least 95% sequence identity with any of the amino acid sequences of SEQ ID NO: 54 to 60. [1.2]

[0049] An anticancer agent comprising an antiCD137 antigen-binding molecule, said antiCD137 antigen-binding molecule comprising any combination of VH and VL selected from (a) to (m):

[0050] (a) VH, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 43; and VL, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 54;

[0051] (b) VH, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 44; and VL, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 55;

[0052] (c) VH, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 45; and VL, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 55;

[0053] (d) VH, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 46; and VL, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 54;

[0054] (e) VH, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 47; and VL, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 54;

[0055] (f) VH, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 48; and VL, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 56;

[0056] (g) VH, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 49; and VL, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 57;

[0057] (h) VH, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 50; and VL, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 58;

[0058] (i) VH, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 51; and VL, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 59;

[0059] (j) VH, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 51; and VL, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 60;

[0060] (k) VH, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 52; and VL, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 60;

[0061] (l) VH, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 50; and VL, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 59; and

[0062] (m) VH, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 53; and VL, which has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 54. [2]

[0064] An anticancer agent comprising an antiCD137 antigen-binding molecule, said antiCD137 antigen-binding molecule comprising any combination of VH and VL selected from (a) to (m):

[0065] (a) VH, which contains the amino acid sequence of SEQ ID NO: 43; and VL, which contains the amino acid sequence of SEQ ID NO: 54;

[0066] (b) VH, which contains the amino acid sequence of SEQ ID NO: 44; and VL, which contains the amino acid sequence of SEQ ID NO: 55;

[0067] (c) VH, which contains the amino acid sequence of SEQ ID NO: 45; and VL, which contains the amino acid sequence of SEQ ID NO: 55;

[0068] (d) VH, which contains the amino acid sequence of SEQ ID NO: 46; and VL, which contains the amino acid sequence of SEQ ID NO: 54;

[0069] (e) VH, which contains the amino acid sequence of SEQ ID NO: 47; and VL, which contains the amino acid sequence of SEQ ID NO: 54;

[0070] (f) VH, which contains the amino acid sequence of SEQ ID NO: 48; and VL, which contains the amino acid sequence of SEQ ID NO: 56;

[0071] (g) VH, which contains the amino acid sequence of SEQ ID NO: 49; and VL, which contains the amino acid sequence of SEQ ID NO: 57;

[0072] (h) VH, which contains the amino acid sequence of SEQ ID NO: 50; and VL, which contains the amino acid sequence of SEQ ID NO: 58;

[0073] (i) VH, which contains the amino acid sequence of SEQ ID NO: 51; and VL, which contains the amino acid sequence of SEQ ID NO: 59;

[0074] (j) VH, which contains the amino acid sequence of SEQ ID NO: 51; and VL, which contains the amino acid sequence of SEQ ID NO: 60;

[0075] (k) VH, which contains the amino acid sequence of SEQ ID NO: 52; and VL, which contains the amino acid sequence of SEQ ID NO: 60;

[0076] (l) VH, comprising the amino acid sequence of SEQ ID NO: 50; and VL, comprising the amino acid sequence of SEQ ID NO: 59; and

[0077] (m) VH, which contains the amino acid sequence of SEQ ID NO: 53; and VL, which contains the amino acid sequence of SEQ ID NO: 54. [2.1]

[0079] The anticancer agent described in any one of [1] to [2], wherein the antiCD137 antigen-binding molecule has CD137 binding activity dependent on small molecule compounds. [2.2]

[0081] An anticancer agent containing an antiCD137 antigen-binding molecule, said antiCD137 antigen-binding molecule having CD137 binding activity dependent on a small molecule compound, wherein said antiCD137 antigen-binding molecule competitively binds to CD137 in the presence of an antigen-binding molecule according to any one of [1] to [2.1] in the presence of a small molecule compound of 10 μM or more, 50 μM or more, 100 μM or more, 150 μM or more, 200 μM or more, or 250 μM or more. [2.3]

[0083] An anticancer agent containing an antiCD137 antigen-binding molecule having CD137 binding activity dependent on a small molecule compound, wherein, in the presence of a small molecule compound of 10 μM or more, 50 μM or more, 100 μM or more, 150 μM or more, 200 μM or more, or 250 μM or more, the antiCD137 antigen-binding molecule binds to the same CD137 epitope as the antigen-binding molecule bound by any one of [1] to [2.1]. [2.4]

[0085] The anticancer agent according to any one of [2.1] to [2.3], wherein the small molecule compound is an adenosine-containing compound. [2.5]

[0087] The anticancer agent according to any one of [2.1] to [2.4], wherein the small molecule compound is ATP. [2.6]

[0089] The anticancer agent described in any one of [1] to [2.5], wherein the antiCD137 antigen-binding molecule is a monoclonal antibody or its antigen-binding fragment. [3]

[0091] The anticancer agent described in any one of [1] to [2.6], wherein the antiCD137 antigen-binding molecule is a human antibody, a humanized antibody or a chimeric antibody, or an antigen-binding fragment of any of them. [3.1]

[0093] The anticancer agent described in any one of [1] to [3], wherein the antiCD137 antigen-binding molecule is a full-length IgG1 antibody. [3.2]

[0095] The anticancer agent according to any one of [1] to [3.1], wherein the antiCD137 antigen-binding molecule comprises an altered Fc region in which at least one amino acid is altered, wherein the altered Fc region has increased binding activity with FcγRIIb compared to the parental Fc region which does not contain the altered amino acid. [3.3]

[0097] [3.2] The anticancer agent wherein the at least one amino acid change is a substitution of at least one amino acid selected from the group consisting of G236N, H268D and A330K according to EU numbers. [3.4]

[0099] The anticancer agent described in [3.2] or [3.3], wherein the at least one amino acid alteration is a combination of amino acid substitutions G236N / H268D / A330K according to the EU number. [3.5]

[0101] The anticancer agent according to any one of [1] to [3.4], wherein the antiCD137 antigen-binding molecule comprises an altered Fc region in which at least one amino acid is altered, wherein the antiCD137 antigen-binding molecule has an increased isoelectric point (pI) compared to a parental antiCD137 antigen-binding molecule comprising a parental Fc region not comprising an amino acid altered region. [3.6]

[0103] [3.5] The anticancer agent wherein the at least one amino acid change is a substitution of at least one amino acid selected from the group consisting of Q311R, P343R and D413K according to EU numbers. [3.7]

[0105] The anticancer agent described in [3.5] or [3.6], wherein the at least one amino acid alteration is (i) an amino acid substitution of P343R, (ii) an amino acid substitution of a combination of Q311R / P343R, or (iii) an amino acid substitution of a combination of Q311R / D413K according to the EU designation. [4]

[0107] The anticancer agent according to any one of [1] to [3.7], wherein the antiCD137 antigen-binding molecule comprises a modified Fc region, and the modified Fc region comprises any combination of amino acid modifications selected from the following according to EU numbers:

[0108] L235W / G236N / H268D / Q295L / K326T / A330K / P343R / D413K;

[0109] K214R / L235W / G236N / H268D / Q295L / K326T / A330K / P343R / D413K;

[0110] L234Y / P238D / T250V / V264I / T307P / A330K / P343R / D413K;

[0111] L234Y / P238D / V264I / A330K / P343R / D413K;

[0112] L234Y / G237D / P238D / T250V / T307P / A330K / P343R / D413K;

[0113] L234Y / G237D / P238D / A330K / P343R / D413K;

[0114] L235W / G236N / H268D / Q295L / K326T / A330K / Q311R / P343R;

[0115] L234Y / P238D / T250V / V264I / T307P / A330K / Q311R / P343R;

[0116] L234Y / P238D / V264I / A330K / Q311R / P343R;

[0117] L234Y / G237D / P238D / T250V / T307P / A330K / Q311R / P343R;

[0118] L234Y / G237D / P238D / A330K / Q311R / P343R;

[0119] L235W / G236N / H268D / Q295L / K326T / A330K / P343R;

[0120] K214R / L235W / G236N / H268D / Q295L / K326T / A330K / P343R;

[0121] L235W / G236N / H268D / Q295L / K326T / A330K / D413K;

[0122] K214R / G236N / H268D / A330K / P343R;

[0123] K214R / L235W / G236N / H268D / A330K / P343R;

[0124] K214R / G236N / H268D / A330K / D413K;

[0125] K214R / G236N / H268D / A330K / P343R / D413K;

[0126] K214R / L235W / G236N / H268D / A330K / P343R / D413K;

[0127] K214R / G236N / H268D / A330K / Q311R;

[0128] K214R / L235W / G236N / H268D / A330K / Q311R;

[0129] K214R / G236N / H268D / A330K / Q311R / P343R;

[0130] K214R / L235W / G236N / H268D / A330K / Q311R / P343R;

[0131] K214R / G236N / H268D / A330K / Q311R / D413K;

[0132] K214R / L235W / G236N / H268D / A330K / Q311R / D413K; and

[0133] K214R / L235W / G236N / H268D / Q295L / K326T / A330K / Q311R. [4.1]

[0135] The anticancer agent described in any one of [3.2] to [4], wherein the altered Fc region is derived from the human IgG1 Fc region. [4.2]

[0137] The anticancer agent according to any one of [3.2] to [4.1], wherein the altered Fc region further comprises a deletion at positions 446 and 447 according to EU numbering. [4.3]

[0139] The anticancer agent according to any one of [1] to [4.2], wherein the antiCD137 antigen-binding molecule comprises a heavy chain constant region comprising any one of the amino acid sequences in SEQ ID NO: 64 to 85. [5]

[0141] An anticancer agent comprising an antiCD137 antigen-binding molecule, said antiCD137 antigen-binding molecule comprising any combination of VH, VL, CH and CL selected from (i) to (xxxviii):

[0142] (i) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 64; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0143] (ii) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 66; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0144] (iii) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 67; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0145] (iv) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 68; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0146] (v) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 69; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0147] (vi) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 70; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0148] (vii) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 71; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0149] (viii) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 73; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0150] (ix) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 75; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0151] (x) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 78; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0152] (xi) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 80; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0153] (xii) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 82; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0154] (xiii) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 84; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0155] (xiv) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 85; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0156] (xv) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 65; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0157] (xvi) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 72; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0158] (xvii) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 74; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0159] (xviii) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 75; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0160] (xix) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 77; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0161] (xx) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 78; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0162] (xxi) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 79; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0163] (xxii) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 80; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0164] (xxiii) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 81; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0165] (xxiv) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 82; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0166] (xxv) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 83; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0167] (xxvi) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 84; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0168] (xxvii) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 72; VL, which contains the amino acid sequence of SEQ ID NO: 60; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0169] (xxviii) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 74; VL, which contains the amino acid sequence of SEQ ID NO: 60; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0170] (xxix) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 75; VL, which contains the amino acid sequence of SEQ ID NO: 60; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0171] (xxx) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 77; VL, which contains the amino acid sequence of SEQ ID NO: 60; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0172] (xxxi) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 78; VL, which contains the amino acid sequence of SEQ ID NO: 60; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0173] (xxxii) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 79; VL, which contains the amino acid sequence of SEQ ID NO: 60; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0174] (xxxiii) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 80; VL, which contains the amino acid sequence of SEQ ID NO: 60; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0175] (xxxiv) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 81; VL, which contains the amino acid sequence of SEQ ID NO: 60; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0176] (xxxv) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 82; VL, which contains the amino acid sequence of SEQ ID NO: 60; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0177] (xxxvi) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 83; VL, which contains the amino acid sequence of SEQ ID NO: 60; and CL, which contains the amino acid sequence of SEQ ID NO: 63;

[0178] (xxxvii) VH, comprising the amino acid sequence of SEQ ID NO: 51; CH, comprising the amino acid sequence of SEQ ID NO: 84; VL, comprising the amino acid sequence of SEQ ID NO: 60; and CL, comprising the amino acid sequence of SEQ ID NO: 63; and

[0179] (xxxviii) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 85; VL, which contains the amino acid sequence of SEQ ID NO: 60; and CL, which contains the amino acid sequence of SEQ ID NO: 63. [6]

[0181] The anticancer agent described in any one of [1] to [5] is administered to a cancer patient with the following conditions: (i) selected from B cells, dendritic cells, natural killer cells, macrophages and CD8 cells. + Solid cancer with one or more cell types infiltrating T cells, and / or (ii) regulatory T (Treg) cells or CD4 cells. + Solid cancer with T-cell infiltration. [6.1]

[0183] The anticancer agent described in any one of [1] to [6], which is administered to patients with CD8 + Cancer patients with solid tumors infiltrated by T cells. [7]

[0185] The anticancer agent described in any one of [1] to [6.1] is administered to a patient with cancer that is refractory to immune checkpoint inhibitor therapy. [8]

[0187] The anticancer agent described in any one of [1] to [7] is administered to a cancer patient suffering from one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle tumor, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, Merkel cell carcinoma, bladder cancer, thyroid cancer, schwannoma, adrenal cancer, anal cancer, central nervous system tumors, neuroendocrine tissue tumors, penile cancer, pleural tumors, salivary gland tumors, vulvar cancer, thymoma, lymphoma, myeloid leukemia, and pediatric cancer. [8.1]

[0189] The anticancer agent described in any one of [1] to [8] is administered to a cancer patient suffering from one or more cancers selected from the group consisting of: gastric cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma, and myeloid leukemia. [9]

[0191] The anticancer agent described in any one of [1] to [5] is used in combination with at least one other anticancer agent. [9.1]

[0193] [9] The anticancer agent described herein, wherein the anticancer agent is characterized by being administered simultaneously with other anticancer agents. [9.2]

[0195] [9] The anticancer agent described herein, wherein the anticancer agent is characterized in that it is applied before or after the application of other anticancer agents.

[10]

[0197] The anticancer agent described in any one of [9] to [9.2], wherein the other anticancer agent is at least one selected from the group consisting of: chemotherapeutic agents, T-cell activating agonists, immune checkpoint inhibitors, T-cell redirecting antigen-binding molecules, antifibrotic agents, and angiogenesis inhibitors. [10.1]

[0199]

[10] The anticancer agents described herein, wherein the other anticancer agents are chemotherapeutic agents. [10.2]

[0201] The anticancer agent described in

[10] or [10.1], wherein the other anticancer agent is at least one chemotherapeutic agent selected from antimetabolites, plant alkaloids and platinum compounds. [10.3]

[0203]

[10] The anticancer agent, wherein the other anticancer agent is a T-cell activating agonist. [10.4]

[0205] The anticancer agents described in

[10] or [10.3], wherein the other anticancer agent is an agonist antibody against TNFRSF.

[11]

[0207]

[10] The anticancer agents described herein, wherein the other anticancer agents are immune checkpoint inhibitors.

[12]

[0209] The anticancer agent described in

[10] or

[11] , wherein the other anticancer agent is at least one immune checkpoint inhibitor selected from antiPD1 antibody, antiPDL1 antibody, antiTIGIT antibody, antiTIM3 antibody, and antiLAG3 antibody. [12.1]

[0211] The anticancer agent described in any one of

[10] ,

[11] and

[12] , wherein the other anticancer agent is an antiPDL1 antibody and / or an antiTIGIT antibody. [12.2]

[0213] The anticancer agent described in any one of [9] to [12.1] is administered to a patient with cancer that is refractory to immune checkpoint inhibitor therapy. [12.3]

[0215] [12.2] The anticancer agent, wherein the immune checkpoint inhibitor treats refractory cancers that have gene mutations in JAK1, JAK2 and / or B2M.

[13]

[0217]

[10] The anticancer agent, wherein the other anticancer agent is a T-cell redirected antigen-binding molecule. [13.1]

[0219] The anticancer agents described in

[10] or

[13] , wherein the other anticancer agents are multispecific antibodies that have binding activity with CD3 and cancer antigens. [13.2]

[0221] [13.1] The anticancer agent, wherein the multispecific antibody is a bispecific antibody.

[14]

[0223] The anticancer agent described in any one of [9] to [9.2], wherein the other anticancer agent is a substance that consumes and / or inactivates substances selected from regulatory T cells, CD4+, etc. + A reagent for one or more types of cells, including T cells, B cells, NK cells, and macrophages.

[15]

[0225] The anticancer agent described in any one of [9] to

[14] is administered to a cancer patient suffering from one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle tumor, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, Merkel cell carcinoma, bladder cancer, thyroid cancer, schwannoma, adrenal cancer, anal cancer, central nervous system tumors, neuroendocrine tissue tumors, penile cancer, pleural tumors, salivary gland tumors, vulvar cancer, thymoma, lymphoma, myeloid leukemia, and pediatric cancer. [15.1]

[0227] The anticancer agent described in any one of [9] to

[15] is administered to a cancer patient suffering from one or more cancers selected from the group consisting of: gastric cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma, and myeloid leukemia.

[16]

[0229] The anticancer agent described in any one of [1] to [5] is used in combination with at least one other anticancer agent to enhance the activation of T cells in an individual tumor tissue. [16.1]

[0231]

[16] The anticancer agent wherein when the expression level of at least one gene selected from CD8b1, Gzmb, Prf1 and Ifng in the tumor tissue is increased, it is determined that the activation of T cells in the tumor tissue has been enhanced.

[17]

[0233] The anticancer agent described in any one of [1] to [5], used in combination with at least one other anticancer agent to promote CD8 in individual tumor tissue.+ T cell proliferation. [17.1]

[0235] The anticancer agent described in any one of

[16] to

[17] is characterized in that it is administered simultaneously with other anticancer agents. [17.2]

[0237] The anticancer agent described in any one of

[16] to

[17] is characterized in that it is applied before or after the application of other anticancer agents. [17.3]

[0239] The anticancer agent described in any one of

[16] to [17.2], wherein the other anticancer agent is at least one selected from the group consisting of: chemotherapeutic agents, T-cell activating agonists, immune checkpoint inhibitors, T-cell redirecting antigen-binding molecules, antifibrotic agents, and angiogenesis inhibitors. [17.4]

[0241] The anticancer agent described in any one of

[16] to [17.2], wherein the other anticancer agent is a substance that consumes and / or inactivates substances selected from regulatory T cells, CD4+, etc. + A reagent for one or more types of cells, including T cells, B cells, NK cells, and macrophages.

[18]

[0243] A pharmaceutical composition for treating cancer comprising any one of [1] to [5] an antiCD137 antigen-binding molecule and at least one other anticancer agent. [18.1]

[0245]

[18] The pharmaceutical composition wherein the anti-CD137 antigen-binding molecule is used for simultaneous administration to the patient with other anticancer agents. [18.2]

[0247]

[18] The pharmaceutical composition wherein the anti-CD137 antigen-binding molecule is used to administer to a patient before or after administration of other anticancer agents.

[19]

[0249] The pharmaceutical composition of any one of

[18] to [18.2], wherein the other anticancer agent is at least one anticancer agent selected from the group consisting of: chemotherapeutic agents, T-cell activating agonists, immune checkpoint inhibitors, T-cell redirecting antigen-binding molecules, antifibrotic agents, and angiogenesis inhibitors. [19.1]

[0251]

[19] The pharmaceutical composition wherein the other anticancer agent is a chemotherapeutic agent. [19.2]

[0253] The pharmaceutical composition described in

[19] or [19.1], wherein the other anticancer agent is at least one chemotherapeutic agent selected from antimetabolites, plant alkaloids and platinum compounds. [19.3]

[0255]

[19] The pharmaceutical composition wherein the other anticancer agent is a T-cell activating agonist. [19.4]

[0257] The pharmaceutical composition described in

[19] or [19.3], wherein the other anticancer agent is an agonist antibody against TNFRSF.

[20]

[0259]

[19] The pharmaceutical composition wherein the other anticancer agent is an immune checkpoint inhibitor. [twenty one]

[0261] The pharmaceutical composition described in

[19] or

[20] , wherein the other anticancer agent is at least one immune checkpoint inhibitor selected from antiPD1 antibody, antiPDL1 antibody, antiTIGIT antibody, antiTIM3 antibody, and antiLAG3 antibody. [21.1]

[0263] The pharmaceutical composition of any one of

[19] ,

[20] and

[21] , wherein the other anticancer agent is an antiPDL1 antibody and / or an antiTIGIT antibody. [21.2]

[0265] The pharmaceutical composition described in any one of

[18] to [21.1] is used to treat cancers refractory to immune checkpoint inhibitor therapy. [21.3]

[0267] [21.2] The pharmaceutical composition wherein the immune checkpoint inhibitor treats refractory cancers that have gene mutations in JAK1, JAK2 and / or B2M. [twenty two]

[0269]

[19] The pharmaceutical composition wherein the other anticancer agent is a T-cell redirected antigen-binding molecule. [22.1]

[0271] The pharmaceutical composition described in

[19] or

[22] , wherein the other anticancer agent is a multispecific antibody that has binding activity with CD3 and cancer antigens. [22.2]

[0273] [22.1] The pharmaceutical composition thereof, wherein the multispecific antibody is a bispecific antibody. [twenty three]

[0275] The pharmaceutical composition of any one of

[18] to [18.2], wherein the other anticancer agent is consumed and / or inactivated by regulatory T cells, CD4+, etc. + A reagent for one or more of the following cell types: T cells, B cells, NK cells, and macrophages. [twenty four]

[0277] The pharmaceutical composition of any one of

[18] to

[23] , wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle tumor, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, Merkel cell carcinoma, bladder cancer, thyroid cancer, schwannoma, adrenal cancer, anal cancer, central nervous system tumors, neuroendocrine tissue tumors, penile cancer, pleural tumors, salivary gland tumors, vulvar cancer, thymoma, lymphoma, myeloid leukemia, and pediatric cancer. [24.1]

[0279] The pharmaceutical composition of any one of

[18] to

[24] , wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma, and myeloid leukemia.

[25]

[0281] The combination of any one of the antiCD137 antigen-binding molecules in [1] to [5] with at least one other anticancer agent is used to treat cancer. [25.1]

[0283]

[25] The combination described herein, wherein the anti-CD137 antigen-binding molecule is used to be administered to the patient simultaneously with other anticancer agents. [25.2]

[0285]

[25] The combination described herein, wherein the anti-CD137 antigen-binding molecule is used to administer to the patient before or after the administration of other anticancer agents.

[26]

[0287] The combination of any one of

[25] to [25.2], wherein the other anticancer agent is at least one anticancer agent selected from the group consisting of: chemotherapeutic agents, T-cell activating agonists, immune checkpoint inhibitors, T-cell redirecting antigen-binding molecules, antifibrotic agents, and angiogenesis inhibitors. [26.1]

[0289]

[26] The combination described herein, wherein the other anticancer agent is a chemotherapeutic agent. [26.2]

[0291] The combination described in

[26] or [26.1], wherein the other anticancer agent is at least one chemotherapeutic agent selected from antimetabolites, plant alkaloids and platinum compounds. [26.3]

[0293]

[26] The combination described herein, wherein the other anticancer agent is a T-cell activating agonist. [26.4]

[0295] The combination described in

[26] or [26.3], wherein the other anticancer agent is an agonist antibody against TNFRSF.

[27]

[0297]

[26] The combination described herein, wherein the other anticancer agent is an immune checkpoint inhibitor.

[28]

[0299] The combination described in

[26] or

[27] , wherein the other anticancer agent is at least one immune checkpoint inhibitor selected from antiPD1 antibody, antiPDL1 antibody, antiTIGIT antibody, antiTIM3 antibody, and antiLAG3 antibody. [28.1]

[0301] The combination of any one of

[26] ,

[27] and

[28] , wherein the other anticancer agent is an antiPDL1 antibody and / or an antiTIGIT antibody. [28.2]

[0303] The combination of any one of

[25] to [28.1] is used to treat cancers refractory to immune checkpoint inhibitor therapy. [28.3]

[0305] [28.2] The combination described herein, wherein the immune checkpoint inhibitor treats refractory cancers that have gene mutations in JAK1, JAK2 and / or B2M.

[29]

[0307]

[26] The combination described herein, wherein the other anticancer agent is a T-cell redirected antigen-binding molecule. [29.1]

[0309] The combination described in

[26] or

[29] , wherein the other anticancer agent is a multispecific antibody that has binding activity with CD3 and cancer antigens. [29.2]

[0311] [29.1] The combination described herein, wherein the multispecific antibody is a bispecific antibody.

[30]

[0313] The combination of any one of

[25] to [25.2], wherein the other anticancer agent is consumed and / or inactivated by regulatory T cells, CD4+, etc. + A reagent for one or more of the following cell types: T cells, B cells, NK cells, and macrophages.

[31]

[0315]

[25] to

[30] any combination thereof, wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle tumor, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, Merkel cell carcinoma, bladder cancer, thyroid cancer, schwannoma, adrenal cancer, anal cancer, central nervous system tumors, neuroendocrine tissue tumors, penile cancer, pleural tumors, salivary gland tumors, vulvar cancer, thymoma, lymphoma, myeloid leukemia and pediatric cancer. [31.1]

[0317] The combination of any one of

[25] to

[31] , wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma, and myeloid leukemia.

[32]

[0319] The use of any one of the antiCD137 antigen-binding molecules described in [1] to [5] in combination with at least one other anticancer agent as an anticancer agent. [32.1]

[0321]

[32] The use described herein, wherein the anti-CD137 antigen-binding molecule is used to be administered to a patient simultaneously with other anticancer agents. [32.2]

[0323]

[32] The use described herein, wherein the anti-CD137 antigen-binding molecule is used to administer to a patient before or after the administration of other anticancer agents.

[33]

[0325] The use of any one of

[32] to [32.2], wherein the other anticancer agent is at least one anticancer agent selected from the group consisting of: chemotherapeutic agents, T-cell activating agonists, immune checkpoint inhibitors, T-cell redirecting antigen-binding molecules, antifibrotic agents, and angiogenesis inhibitors. [33.1]

[0327]

[33] The use described herein, wherein the other anticancer agent is a chemotherapeutic agent. [33.2]

[0329] The use described in

[33] or [33.1], wherein the other anticancer agent is at least one chemotherapeutic agent selected from antimetabolites, plant alkaloids and platinum compounds. [33.3]

[0331]

[33] The other anticancer agent described herein is a T-cell activating agonist. [33.4]

[0333] The use described in

[33] or [33.3], wherein the other anticancer agent is an agonist antibody against TNFRSF.

[34]

[0335]

[33] The other anticancer agent described herein is an immune checkpoint inhibitor.

[35]

[0337] The use described in

[33] or

[34] , wherein the other anticancer agent is at least one immune checkpoint inhibitor selected from antiPD1 antibody, antiPDL1 antibody, antiTIGIT antibody, antiTIM3 antibody, and antiLAG3 antibody. [35.1]

[0339] The use of any one of

[33] ,

[34] and

[35] , wherein the other anticancer agent is an antiPDL1 antibody and / or an antiTIGIT antibody. [35.2]

[0341] The use of any one of

[32] to [35.1] is for the treatment of cancers refractory to immune checkpoint inhibitor therapy. [35.3]

[0343] [35.2] The use described herein, wherein the immune checkpoint inhibitor is used to treat refractory cancers that are cancers with gene mutations in JAK1, JAK2 and / or B2M.

[36]

[0345]

[33] The other anticancer agent described herein is a T-cell redirected antigen-binding molecule. [36.1]

[0347] The uses described in

[33] or

[36] , wherein the other anticancer agents are multispecific antibodies that have binding activity with CD3 and cancer antigens. [36.2]

[0349] [36.1] The use described herein, wherein the multispecific antibody is a bispecific antibody.

[37]

[0351] The use of any one of

[32] to [32.2], wherein the other anticancer agent is consumed and / or inactivated by regulatory T cells, CD4+, etc. + A reagent for one or more of the following cell types: T cells, B cells, NK cells, and macrophages.

[38]

[0353] The use of any one of

[32] to

[37] , wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle tumor, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, Merkel cell carcinoma, bladder cancer, thyroid cancer, schwannoma, adrenal cancer, anal cancer, central nervous system tumors, neuroendocrine tissue tumors, penile cancer, pleural tumors, salivary gland tumors, vulvar cancer, thymoma, lymphoma, myeloid leukemia and pediatric cancer. [38.1]

[0355] The use of any one of

[32] to

[38] , wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma and myeloid leukemia.

[39]

[0357] A kit for treating cancer, comprising:

[0358] (1) A pharmaceutical composition comprising, as an active ingredient, any one of [1] to [5]; and

[0359] (2) Packaging instructions or labels that indicate the administration of at least one other anticancer agent before, during or after the administration of the pharmaceutical composition. [39.1]

[0361]

[39] The kit described herein, wherein the pharmaceutical composition is filled in a container. [39.2]

[0363] The kit described in any one of

[39] to [39.1] uses a pharmaceutical composition containing an anti-CD137 antigen-binding molecule as an active ingredient for simultaneous administration to a patient with other anticancer agents. [39.3]

[0365] The kit described in any one of

[39] or [39.1] uses a pharmaceutical composition containing an anti-CD137 antigen-binding molecule as an active ingredient for administration to a patient before or after administration of other anticancer agents.

[40]

[0367] The kit described in any one of

[39] to [39.2], wherein the other anticancer agent is at least one anticancer agent selected from the group consisting of: chemotherapeutic agents, T-cell activating agonists, immune checkpoint inhibitors, T-cell redirecting antigen-binding molecules, antifibrotic agents, and angiogenesis inhibitors. [40.1]

[0369]

[40] The kit described herein, wherein the other anticancer agent is a chemotherapeutic agent. [40.2]

[0371] The kit described in

[40] or [40.1], wherein the other anticancer agent is at least one chemotherapeutic agent selected from antimetabolites, plant alkaloids and platinum compounds. [40.3]

[0373]

[40] The kit described herein, wherein the other anticancer agent is a T-cell activating agonist. [40.4]

[0375] The kit described in

[40] or [40.3], wherein the other anticancer agent is an agonist antibody against TNFRSF.

[41]

[0377]

[40] The kit described herein, wherein the anticancer agent is an immune checkpoint inhibitor.

[42]

[0379] The kit described in

[40] or

[41] , wherein the anticancer agent is at least one immune checkpoint inhibitor selected from antiPD1 antibody, antiPDL1 antibody, antiTIGIT antibody, antiTIM3 antibody, and antiLAG3 antibody. [42.1]

[0381] The kit described in any one of

[40] ,

[41] and

[42] , wherein the anticancer agent is an antiPDL1 antibody and / or an antiTIGIT antibody. [42.2]

[0383] The kit described in any one of

[39] to [42.1] is used to treat cancers refractory to immune checkpoint inhibitor therapy. [42.3]

[0385] [42.2] The kit described herein, wherein the immune checkpoint inhibitor is used to treat refractory cancers that are cancers with gene mutations in JAK1, JAK2 and / or B2M.

[43]

[0387]

[40] The kit described herein, wherein the anticancer agent is a T-cell redirected antigen-binding molecule. [43.1]

[0389] The kit described in

[40] or

[43] , wherein the anticancer agent is a multispecific antibody that has binding activity with CD3 and cancer antigens. [43.2]

[0391] [43.1] The kit described herein, wherein the multispecific antibody is a bispecific antibody.

[44]

[0393] The kit described in any one of

[39] to [39.2], wherein the other anticancer agent is consumed and / or inactivated by regulatory T cells, CD4+, etc. + A reagent for one or more of the following cell types: T cells, B cells, NK cells, and macrophages.

[45]

[0395] The kit described in any one of

[39] to

[44] , wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle tumor, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, Merkel cell carcinoma, bladder cancer, thyroid cancer, schwannoma, adrenal cancer, anal cancer, central nervous system tumors, neuroendocrine tissue tumors, penile cancer, pleural tumors, salivary gland tumors, vulvar cancer, thymoma, lymphoma, myeloid leukemia, and pediatric cancer. [45.1]

[0397] The kit described in any one of

[39] to

[44] , wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma, and myeloid leukemia.

[46]

[0399] A method for treating cancer, comprising administering an anti-CD137 antigen-binding molecule, as described in any one of [1] to [5], to a patient with a disease in which: (i) cells are selected from B cells, dendritic cells, natural killer cells, macrophages, and CD8+ cells. + Solid cancer with one or more cell types infiltrating T cells, and / or (ii) regulatory T (Treg) cells or CD4 cells.+ Solid cancer with T-cell infiltration. [46.1]

[0401]

[46] The method wherein the patient has CD8 + Patients with T-cell infiltrated solid cancer.

[47]

[0403] The method described in

[46] or [46.1], wherein the patient has cancer that is refractory to immune checkpoint inhibitor therapy.

[48]

[0405] The method of any one of

[46] to

[47] , wherein the patient suffers from one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle tumor, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, Merkel cell carcinoma, bladder cancer, thyroid cancer, schwannoma, adrenal cancer, anal cancer, central nervous system tumors, neuroendocrine tissue tumors, penile cancer, pleural tumors, salivary gland tumors, vulvar cancer, thymoma, lymphoma, myeloid leukemia, and pediatric cancer. [48.1]

[0407] The method of any one of

[46] to

[48] , wherein the patient is a patient suffering from one or more cancers selected from the group consisting of: gastric cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma, and myeloid leukemia.

[49]

[0409] Methods for treating cancer include administering an antiCD137 antigen-binding molecule, as described in any one of [1] to [5], in combination with at least one other anticancer agent to a patient. [49.1]

[0411]

[49] The method wherein the anti-CD137 antigen-binding molecule is administered to the patient simultaneously with other anticancer agents. [49.2]

[0413]

[49] The method wherein the anti-CD137 antigen-binding molecule is administered to the patient before or after administration of other anticancer agents.

[50]

[0415] The method of any one of

[49] to [49.2], wherein the other anticancer agent is at least one anticancer agent selected from the group consisting of: chemotherapeutic agents, T-cell activating agonists, immune checkpoint inhibitors, T-cell redirecting antigen-binding molecules, antifibrotic agents, and angiogenesis inhibitors. [50.1]

[0417]

[50] The method wherein the other anticancer agent is a chemotherapeutic agent. [50.2]

[0419] The method described in

[50] or [50.1], wherein the other anticancer agent is at least one chemotherapeutic agent selected from antimetabolites, plant alkaloids and platinum compounds. [50.3]

[0421]

[50] The method wherein the other anticancer agent is a T-cell activating agonist. [50.4]

[0423] The method described in

[50] or [50.3], wherein the other anticancer agent is an agonist antibody against TNFRSF.

[51]

[0425]

[50] The method wherein the other anticancer agent is an immune checkpoint inhibitor.

[52]

[0427] The method described in

[50] or

[51] , wherein the other anticancer agent is at least one immune checkpoint inhibitor selected from antiPD1 antibody, antiPDL1 antibody, antiTIGIT antibody, antiTIM3 antibody, and antiLAG3 antibody. [52.1]

[0429] The method of any one of

[50] ,

[51] and

[52] , wherein the other anticancer agent is an antiPDL1 antibody and / or an antiTIGIT antibody. [52.2]

[0431] The method described in any one of

[49] to [52.1] is used to treat cancers refractory to immune checkpoint inhibitor therapy. [52.3]

[0433] [52.2] The method wherein the immune checkpoint inhibitor treats refractory cancers that are cancers with gene mutations in JAK1, JAK2 and / or B2M.

[53]

[0435]

[50] The method wherein the other anticancer agent is a T-cell redirected antigen-binding molecule. [53.1]

[0437] The method described in

[50] or

[53] , wherein the other anticancer agent is a multispecific antibody that has binding activity with CD3 and cancer antigens. [53.2]

[0439] [53.1] The method wherein the multispecific antibody is a bispecific antibody.

[54]

[0441] The method of any one of

[49] to [49.2], wherein the other anticancer agent is consumed and / or inactivated by regulatory T cells, CD4+, etc. + A reagent for one or more of the following cell types: T cells, B cells, NK cells, and macrophages.

[55]

[0443] The method of any one of

[49] to

[54] , wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle tumor, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, Merkel cell carcinoma, bladder cancer, thyroid cancer, schwannoma, adrenal cancer, anal cancer, central nervous system tumors, neuroendocrine tissue tumors, penile cancer, pleural tumors, salivary gland tumors, vulvar cancer, thymoma, lymphoma, myeloid leukemia, and pediatric cancer. [55.1]

[0445] The method of any one of

[49] to

[55] , wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma, and myeloid leukemia.

[56]

[0447] The anti-CD137 antigen-binding molecule described in any one of [1] to [5] is used for the treatment of:

[0448] (i) Selected from B cells, dendritic cells, natural killer cells, macrophages, and CD8+ cells. + Solid cancers with one or more cell types infiltrating T cells, and / or

[0449] (ii) Regulatory T (Treg) cells or CD4 + Solid cancer with T-cell infiltration. [56.1]

[0451]

[56] The anti-CD137 antigen-binding molecule, wherein the cancer is CD8 + Solid cancer with T-cell infiltration.

[57]

[0453] The anti-CD137 antigen-binding molecule described in

[56] or [56.1], wherein the cancer is a cancer refractory to immune checkpoint inhibitor therapy.

[58]

[0455]

[56] to

[57] any one of the anti-CD137 antigen-binding molecules, wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle tumor, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, Merkel cell carcinoma, bladder cancer, thyroid cancer, schwannoma, adrenal cancer, anal cancer, central nervous system tumors, neuroendocrine tissue tumors, penile cancer, pleural tumors, salivary gland tumors, vulvar cancer, thymoma, lymphoma, myeloid leukemia, and pediatric cancer. [58.1]

[0457] The anti-CD137 antigen-binding molecule described in any one of

[56] to

[58] , wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma, and myeloid leukemia.

[59]

[0459] The anti-CD137 antigen-binding molecule described in any one of [1] to [5] is used in combination with at least one other anticancer agent for the treatment of cancer. [59.1]

[0461]

[59] The anti-CD137 antigen-binding molecule, wherein the treatment is characterized by the simultaneous administration of the anti-CD137 antigen-binding molecule with other anticancer agents. [59.2]

[0463]

[59] The anti-CD137 antigen-binding molecule, wherein the treatment is characterized by the application of the anti-CD137 antigen-binding molecule before or after the administration of other anticancer agents.

[60]

[0465] The anti-CD137 antigen-binding molecule described in any one of

[59] to [59.2], wherein the other anticancer agent is at least one selected from the group consisting of: chemotherapeutic agents, T-cell activating agonists, immune checkpoint inhibitors, T-cell redirecting antigen-binding molecules, antifibrotic agents, and angiogenesis inhibitors. [60.1]

[0467]

[60] The anti-CD137 antigen-binding molecule, wherein the other anticancer agent is a chemotherapeutic agent. [60.2]

[0469] The anti-CD137 antigen-binding molecule described in

[60] or [60.1], wherein the other anticancer agent is at least one chemotherapeutic agent selected from antimetabolites, plant alkaloids and platinum compounds. [60.3]

[0471]

[60] The anti-CD137 antigen-binding molecule, wherein the other anticancer agent is a T-cell activating agonist. [60.4]

[0473] The anti-CD137 antigen-binding molecule described in

[60] or [60.3], wherein the other anticancer agent is an agonist antibody against TNFRSF.

[61]

[0475]

[60] The anti-CD137 antigen-binding molecule, wherein the other anticancer agent is an immune checkpoint inhibitor.

[62]

[0477]

[60] or

[61] the anti-CD137 antigen-binding molecule, wherein the other anticancer agent is at least one immune checkpoint inhibitor selected from anti-PD1 antibody, anti-PDL1 antibody, anti-TIGIT antibody, anti-TIM3 antibody, and anti-LAG3 antibody. [62.1]

[0479] The anti-CD137 antigen-binding molecule described in any one of

[60] ,

[61] and

[62] , wherein the other anticancer agent is an anti-PDL1 antibody and / or an anti-TIGIT antibody. [62.2]

[0481] The anti-CD137 antigen-binding molecule described in any one of

[59] to [62.1] is used to treat cancers refractory to immune checkpoint inhibitor therapy. [62.3]

[0483] [62.2] The anti-CD137 antigen-binding molecule, wherein the immune checkpoint inhibitor treats refractory cancers that are cancers with gene mutations in JAK1, JAK2 and / or B2M.

[63]

[0485]

[60] The anti-CD137 antigen-binding molecule, wherein the other anticancer agent is a T-cell redirected antigen-binding molecule. [63.1]

[0487] The anti-CD137 antigen-binding molecule described in

[60] or

[63] , wherein the other anticancer agent is a multispecific antibody with binding activity to CD3 and cancer antigens. [63.2]

[0489] [63.1] The anti-CD137 antigen-binding molecule, wherein the multispecific antibody is a bispecific antibody.

[64]

[0491]

[59] to [59.2], wherein the other anticancer agent is a substance that consumes and / or inactivates substances selected from regulatory T cells, CD4+, and other antigen-binding molecules. + A reagent for one or more of the following cell types: T cells, B cells, NK cells, and macrophages.

[65]

[0493]

[59] to

[64] any one of the anti-CD137 antigen-binding molecules, wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle tumor, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, Merkel cell carcinoma, bladder cancer, thyroid cancer, schwannoma, adrenal cancer, anal cancer, central nervous system tumors, neuroendocrine tissue tumors, penile cancer, pleural tumors, salivary gland tumors, vulvar cancer, thymoma, lymphoma, myeloid leukemia, and pediatric cancer. [65.1]

[0495]

[59] to

[65] any one of the anti-CD137 antigen-binding molecules, wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma and myeloid leukemia.

[66]

[0497] Use of any one of [1] to [5] of the antiCD137 antigen-binding molecule in the preparation of a medicament for treating cancer, wherein the cancer is

[0498] (i) Selected from B cells, dendritic cells, natural killer cells, macrophages, and CD8+ cells. + Solid cancers with one or more cell types infiltrating T cells, and / or

[0499] (ii) Regulatory T (Treg) cells or CD4 + Solid cancer with T-cell infiltration. [66.1]

[0501]

[66] The use described herein, wherein the cancer is CD8 + Solid cancer with T-cell infiltration.

[67]

[0503] The use described in

[66] or [66.1], wherein the cancer is a cancer refractory to immune checkpoint inhibitor therapy.

[68]

[0505] For any of the uses described in

[66] to

[67] , wherein said cancer is one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle tumor, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, Merkel cell carcinoma, bladder cancer, thyroid cancer, schwannoma, adrenal cancer, anal cancer, central nervous system tumors, neuroendocrine tissue tumors, penile cancer, pleural tumors, salivary gland tumors, vulvar cancer, thymoma, lymphoma, myeloid leukemia, and pediatric cancers. [68.1]

[0507] The use of any one of

[66] to

[68] , wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma and myeloid leukemia.

[69]

[0509] Use of any one of [1] to [5] in the preparation of a medicament for treating cancer, wherein the medicament is characterized in that it is used in combination with at least one other anticancer agent. [69.1]

[0511]

[69] The use described herein, wherein the drug is characterized by being administered simultaneously with other anticancer agents. [69.2]

[0513]

[69] The use described herein, wherein the drug is characterized by being administered before or after the administration of other anticancer agents.

[70]

[0515] The use of any one of

[69] to [69.2], wherein the other anticancer agent is at least one selected from the group consisting of: chemotherapeutic agents, T-cell activating agonists, immune checkpoint inhibitors, T-cell redirecting antigen-binding molecules, antifibrotic agents, and angiogenesis inhibitors. [70.1]

[0517]

[70] The use described herein, wherein the other anticancer agent is a chemotherapeutic agent. [70.2]

[0519] The use described in

[70] or [70.1], wherein the other anticancer agent is at least one chemotherapeutic agent selected from antimetabolites, plant alkaloids and platinum compounds. [70.3]

[0521]

[70] The other anticancer agent described herein is a T-cell activating agonist. [70.4]

[0523] The use described in

[70] or [70.3], wherein the other anticancer agent is an agonist antibody against TNFRSF.

[71]

[0525]

[70] The other anticancer agent described herein is an immune checkpoint inhibitor.

[72]

[0527] The use described in

[70] or

[71] , wherein the other anticancer agent is at least one immune checkpoint inhibitor selected from antiPD1 antibody, antiPDL1 antibody, antiTIGIT antibody, antiTIM3 antibody, and antiLAG3 antibody. [72.1]

[0529] The use of any one of

[70] ,

[71] and

[72] , wherein the other anticancer agent is an antiPDL1 antibody and / or an antiTIGIT antibody. [72.2]

[0531] The use of any one of

[69] to [72.1] for the treatment of cancers refractory to immune checkpoint inhibitor therapy. [72.3]

[0533] [72.2] The use described herein, wherein the immune checkpoint inhibitor is used to treat refractory cancers that are cancers with gene mutations in JAK1, JAK2 and / or B2M.

[73]

[0535]

[70] The other anticancer agent described herein is a T-cell redirected antigen-binding molecule. [73.1]

[0537] The uses described in

[70] or

[73] , wherein the other anticancer agents are multispecific antibodies that have binding activity with CD3 and cancer antigens. [73.2]

[0539] [73.1] The use described herein, wherein the multispecific antibody is a bispecific antibody.

[74]

[0541] The use of any one of

[69] to [69.2], wherein the other anticancer agent is consumed and / or inactivated by regulatory T cells, CD4+, etc. +A reagent for one or more of the following cell types: T cells, B cells, NK cells, and macrophages.

[75]

[0543] The use of any one of

[69] to

[74] , wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle tumor, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, Merkel cell carcinoma, bladder cancer, thyroid cancer, schwannoma, adrenal cancer, anal cancer, central nervous system tumors, neuroendocrine tissue tumors, penile cancer, pleural tumors, salivary gland tumors, vulvar cancer, thymoma, lymphoma, myeloid leukemia and pediatric cancer. [75.1]

[0545] The use of any one of

[69] to

[75] , wherein the cancer is one or more cancers selected from the group consisting of: gastric cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma and myeloid leukemia. Attached Figure Description

[0546] Figure 1 The antitumor effect of A551-MB110 / B379-ml0r in a mouse model transplanted with LLC1 / OVA / GPC3 clone C5 cells was demonstrated.

[0547] Each point shows the average of a set (n=7) of tumor volumes.

[0548] Figure 2 The gene expression levels in tumor samples treated with A551-MB110 / B379-ml0r were shown in a mouse model transplanted with LLC1 / OVA / GPC3 clone C5 cells.

[0549] Subplot (A) shows the expression level of CD8b1, subplot (B) shows the expression level of Gzmb, subplot (C) shows the expression level of Prf1, and subplot (D) shows the expression level of Ifng.

[0550] Figure 3 This demonstrates the CD8+ levels in tumor tissues treated with A551-MB110 / B379-ml0r in a mouse model transplanted with LLC1 / OVA / GPC3 clone C5 cells. + The degree of T cell activation.

[0551] Subgraph (A) shows the OVA tetramer + T cells in CD8 + The proportion of T cells. Subfigure (B) shows granzyme B.+ T cells in CD8 + The proportion of T cells. Subfigure (C) shows PD-1. + T cells in CD8 + The proportion of T cells. Subplot (D) shows KLRG-1. + T cells in CD8 + The proportion of T cells. Subplot (E) shows ICOS + T cells in CD8 + The proportion of T cells.

[0552] Figure 4 The antitumor effect of A551-MB110 / B379-ml0r in a mouse model transplanted with E.G7-OVA cells was demonstrated.

[0553] Subplot (A) shows the tumor volume changes in each mouse in the excipient administration group. Subplot (B) shows the tumor volume changes in each mouse in the A551-MB110 / B379-ml0r (2.5 mg / kg) administration group.

[0554] Figure 5 The antitumor effect of A375-mIgG1 / B167-ml0r in a mouse model transplanted with E.G7-OVA cells was demonstrated.

[0555] Subplot (A) shows the tumor volume changes in each mouse in the excipient administration group. Subplot (B) shows the tumor volume changes in each mouse in the A375-mIgG1 / B167-ml0r (2.5 mg / kg) administration group.

[0556] Figure 6 The antitumor effect of A551-MB110 / B379-ml0r in a mouse model transplanted with C1498 cells was demonstrated.

[0557] Subplot (A) shows the tumor volume changes in each mouse in the excipient administration group. Subplot (B) shows the tumor volume changes in each mouse in the A551-MB110 / B379-ml0r (2.5 mg / kg) administration group.

[0558] Figure 7 The antitumor effect of A551-MB110 / B379-ml0r in a mouse model transplanted with Hepa1-6 / hGPC3 cells was demonstrated.

[0559] Subplot (A) shows the tumor volume changes in each mouse in the excipient administration group. Subplot (B) shows the tumor volume changes in each mouse in the A551-MB110 / B379-ml0r (2.5 mg / kg) administration group.

[0560] Figure 8 The antitumor effect of A375-mIgG1 / B167-ml0r in a mouse model transplanted with Hepa1-6 / hGPC3 cells was demonstrated.

[0561] Subplot (A) shows the tumor volume changes in each mouse in the excipient administration group. Subplot (B) shows the tumor volume changes in each mouse in the A375-mIgG1 / B167-ml0r (7.5 mg / kg) administration group.

[0562] Figure 9 Various immune cells (CD4) were shown. + T cells, CD8 + The effect of depletion (removal) of T cells, B cells, NK cells, granulocytes, and macrophages on the antitumor effect of A551-MB110 / B379-ml0r in a mouse model transplanted with LLC1 / OVA / GPC3 clone C5 cells.

[0563] Figure 10 The antitumor effects of A551-MB110 / B379-ml0r, anti-mouse PD-L1 antibody and their combination were demonstrated in a mouse model transplanted with MC38 cells.

[0564] Each point shows the average of a set (n=6) of tumor volumes.

[0565] Figure 11 The study demonstrated the antitumor effects (weight change) of A551-MB110 / B379-ml0r, anti-mouse PD-L1 antibody, or combinations thereof in a mouse model transplanted with MC38 cells.

[0566] Subplot (A) shows the weight change of each mouse in the excipient administration group. Subplot (B) shows the weight change of each mouse in the A551-MB110 / B379-ml0r administration group. Subplot (C) shows the weight change of each mouse in the anti-mouse PD-L1 antibody administration group. Subplot (D) shows the weight change of each mouse in the A551-MB110 / B379-ml0r and anti-mouse PD-L1 antibody combination administration group.

[0567] Figure 12 This study shows changes in the expression of various immune-related genes before and after administration of A551-MB110 / B379-ml0r, anti-mouse PD-L1 antibody, or combinations thereof to a mouse model transplanted with MC38 cells.

[0568] Figure 13The extent of CD8 expression in tumor tissues of mouse models transplanted with MC38 cells was shown by administration of A551-MB110 / B379-ml0r, anti-mouse PD-L1 antibody, or a combination thereof.

[0569] Subfigure (A) shows the expression level of CD8 in tumor tissues of the excipient-treated group, subfigure (B) shows the expression level of CD8 in tumor tissues of the A551-MB110 / B379-ml0r-treated group, subfigure (C) shows the expression level of CD8 in tumor tissues of the anti-mouse PD-L1 antibody-treated group, and subfigure (D) shows the expression level of CD8 in tumor tissues of the combined A551-MB110 / B379-ml0r and anti-mouse PD-L1 antibody-treated group.

[0570] Figure 14 The study demonstrated the expression level of PD-L1 in tumor tissues by administering A551-MB110 / B379-ml0r, anti-mouse PD-L1 antibody, or a combination thereof to a mouse model transplanted with MC38 cells.

[0571] Subfigure (A) shows the expression level of PD-L1 in tumor tissues of the excipient-treated group, subfigure (B) shows the expression level of PD-L1 in tumor tissues of the A551-MB110 / B379-ml0r-treated group, subfigure (C) shows the expression level of PD-L1 in tumor tissues of the anti-mouse PD-L1 antibody-treated group, and subfigure (D) shows the expression level of PD-L1 in tumor tissues of the combined A551-MB110 / B379-ml0r and anti-mouse PD-L1 antibody-treated group.

[0572] Figure 15 The extent of liver function markers detectable by blood tests was shown by administration of A551-MB110 / B379-ml0r, anti-mouse PD-L1 antibody, or a combination thereof to a mouse model transplanted with MC38 cells.

[0573] The top subplot shows ALT(U / L), and the bottom subplot shows AST(U / L).

[0574] Figure 16 This demonstrates the effect of administration of A551-MB110 / B379-ml0r, anti-mouse PD-L1 antibody, or a combination thereof to tumor tissues transplanted with MC38 cells in a mouse model, resulting in increased CD8 levels. + The degree of increase in the number of T cells.

[0575] Figure 17The antitumor effects of A551-MB110 / B379-ml0r, anti-mouse PD-L1 antibody, or combinations thereof were demonstrated in a mouse model transplanted with LLC1 / OVA / GPC3 clone C5 cells.

[0576] Each point shows the average of a set (n=5) of tumor volumes.

[0577] Figure 18 The antitumor effects of A375-mIgG1 / B167-ml0r, anti-mouse PD-L1 antibody, or combinations thereof were demonstrated in a mouse model transplanted with C1498 cells.

[0578] Each point shows the average of a set (n=5) of tumor volumes.

[0579] Figure 19 The antitumor effects of A375-mIgG1 / B167-ml0r, anti-mouse PD-L1 antibody, or combinations thereof were demonstrated in a mouse model transplanted with AE17 cells.

[0580] Each point shows the average of a set (n=5) of tumor volumes.

[0581] Figure 20 The antitumor effects of A551-MB110 / B379-ml0r, anti-hGPC3-mCD3 antibody, or combinations thereof were demonstrated in a mouse model transplanted with LLC1 / hGPC3.

[0582] Each point shows the average of a set (n=5) of tumor volumes.

[0583] Figure 21 This study shows gene expression levels in tumor samples in a mouse model transplanted with LLC1 / hGPC3, resulting from administration of A551-MB110 / B379-ml0r, anti-hGPC3-mCD3 antibody, or a combination thereof.

[0584] Specifically, subplot (A) shows the expression level of CD3e, subplot (B) shows the expression level of CD8b1, subplot (C) shows the expression level of Gzmb, subplot (D) shows the expression level of Prf1, and subplot (E) shows the expression level of Ifng.

[0585] Figure 22 This is a graph showing the agonist activity of various anti-CD137 antibodies tested using Jurkat cells in the presence or absence of ATP.

[0586] The X-axis shows the antibody concentration (μg / mL), and the Y-axis shows the relative light units.

[0587] Figure 23This is a graph showing the agonist activity of various anti-CD137 antibodies tested using Jurkat cells in the presence or absence of ADP.

[0588] The X-axis shows the antibody concentration (μg / mL), and the Y-axis shows the relative light units.

[0589] Figure 24 This is a graph showing the agonist activity of various anti-CD137 antibodies tested using human T cells in the presence or absence of ADPβS.

[0590] Figure 25 This is a graph showing the agonist activity of dBBAT119-P253 / dBBAT119L-LamLib (small molecule converted anti-CD137 antibody) or NS1-P253 (non-converted anti-CD137 antibody) as tested with human T cells in the presence or absence of ADPβS.

[0591] The X-axis shows the antibody concentration (μg / mL), and the Y-axis shows the IFN-γ production (ng / mL).

[0592] Figure 26 This is a graph showing the ATP-dependent antigen-binding activity of various anti-CD137 antibodies (converted anti-CD137 antibodies with enhanced binding activity) as tested by phage ELISA.

[0593] The Y-axis shows the S / N ratio of absorbance in the presence / absence of ATP, and the X-axis shows the S / N ratio of absorbance in the presence / absence of antigen.

[0594] Figure 27 This is a diagram showing the binding activity of various variants of the anti-CD137 antibody (dBBAT119H-P253 / dBBAT119L-LamLib) to human CD137 in the presence or absence of ATP.

[0595] The top row shows the binding activity with human CD137 in the absence of ATP, and the bottom row shows the binding activity with human CD137 in the presence of ATP.

[0596] Figure 28 This is a graph showing the agonist activity of dBBAT119H-P253 / dBBAT119L-LamLib, dBBATk119H024-P253 / dBBATk119L020-LamLib, IC17HdK-hIgG1 / IC17L-k0 (control), or NS1-P253 (non-converted anti-CD137 antibody) as tested with human T cells in the presence or absence of ADPβS.

[0597] Subplot (A) shows the test results in the absence of ADPβS, and subplot (B) shows the test results in the presence of ADPβS.

[0598] The X-axis shows the antibody concentration (μg / mL), and the Y-axis shows the IFN-γ production (ng / mL).

[0599] Figure 29 This is a graph showing the agonist activity of various converted anti-CD137 antibodies as tested using the 4-1BB Jurkat reporter assay in the presence or absence of ATP.

[0600] Subplot (A) shows the test results without ATP, and subplot (B) shows the test results with ATP.

[0601] Figure 30 This is a graph showing the enhanced agonist activity of various anti-CD137 antibodies in the presence of ATP, as tested using human peripheral blood mononuclear cells, due to increased binding activity of the heavy chain constant region to the Fcγ receptor.

[0602] Subplot (A) shows the agonist activity measured using IL-2 production as an indicator, and subplot (B) shows the agonist activity measured using IFN-γ production as an indicator.

[0603] Figure 31 This is a graph showing the enhanced agonist activity of various anti-CD137 antibodies in the presence of ATP, as tested using human peripheral blood mononuclear cells, due to increased binding activity of the heavy chain constant region to the Fcγ receptor or increased pI of the heavy chain constant region.

[0604] Subplot (A) shows the agonist activity measured using IL-2 production as an indicator, and subplot (B) shows the agonist activity measured using IFN-γ production as an indicator.

[0605] Figure 32 This is a graph showing the enhanced agonist activity of various anti-CD137 antibodies, as tested using human peripheral blood mononuclear cells, in the presence or absence of ATP, due to increased binding activity of the heavy chain constant region to the Fcγ receptor.

[0606] Subplot (A) shows the agonist activity measured using IL-2 production as an indicator, and subplot (B) shows the agonist activity measured using IFN-γ production as an indicator.

[0607] Figure 33 This is a graph showing the enhanced agonist activity of various anti-CD137 antibodies, as tested using human peripheral blood mononuclear cells, in the presence or absence of ATP, due to increased binding activity of the heavy chain constant region to the Fcγ receptor.

[0608] Subplot (A) shows the agonist activity measured using IL-2 production as an indicator, and subplot (B) shows the agonist activity measured using IFN-γ production as an indicator.

[0609] Figure 34 This is a graph showing the enhanced agonist activity of various anti-CD137 antibodies, as tested using human peripheral blood mononuclear cells, in the presence or absence of ATP, due to increased binding activity of the heavy chain constant region to the Fcγ receptor.

[0610] Subplot (A) shows the agonist activity measured using IL-2 production as an indicator, and subplot (B) shows the agonist activity measured using IFN-γ production as an indicator.

[0611] Figure 35 This is a graph showing the enhanced agonist activity of various anti-CD137 antibodies, as tested using human peripheral blood mononuclear cells, in the presence or absence of ATP, due to increased binding activity of the heavy chain constant region to the Fcγ receptor.

[0612] Subplot (A) shows the agonist activity measured using IL-2 production as an indicator, and subplot (B) shows the agonist activity measured using IFN-γ production as an indicator.

[0613] Figure 36 This is a graph showing the enhanced agonist activity of various anti-CD137 antibodies, as tested using human peripheral blood mononuclear cells, in the presence or absence of ATP, due to increased binding activity of the heavy chain constant region to the Fcγ receptor.

[0614] Subplot (A) shows the agonist activity measured using IL-2 production as an indicator, and subplot (B) shows the agonist activity measured using IFN-γ production as an indicator.

[0615] Figure 37 This is a graph showing the enhanced agonist activity of various anti-CD137 antibodies in the presence or absence of ATP, as tested using human peripheral blood mononuclear cells, due to increased pI in the heavy chain constant region.

[0616] Subplot (A) shows the agonist activity measured using IL-2 production as an indicator, and subplot (B) shows the agonist activity measured using IFN-γ production as an indicator.

[0617] Figure 38 This is a graph showing the enhanced agonist activity of various anti-CD137 antibodies in the presence or absence of ATP, as tested using human peripheral blood mononuclear cells, due to increased pI in the heavy chain constant region.

[0618] Subplot (A) shows the agonist activity measured using IL-2 production as an indicator, and subplot (B) shows the agonist activity measured using IFN-γ production as an indicator.

[0619] Figure 39 This is a graph showing the enhanced agonist activity of various anti-CD137 antibodies in the presence or absence of ATP, as tested using human peripheral blood mononuclear cells, due to increased pI in the heavy chain constant region.

[0620] Subplot (A) shows the agonist activity measured using IL-2 production as an indicator, and subplot (B) shows the agonist activity measured using IFN-γ production as an indicator.

[0621] Figure 40 This is a graph showing the enhanced agonist activity of various anti-CD137 antibodies in the presence or absence of ATP, as tested using human peripheral blood mononuclear cells, due to increased pI in the heavy chain constant region.

[0622] Subplot (A) shows the agonist activity measured using IL-2 production as an indicator, and subplot (B) shows the agonist activity measured using IFN-γ production as an indicator.

[0623] Figure 41 This is a graph showing the enhanced agonist activity of various anti-CD137 antibodies in the presence or absence of ATP, as tested using human peripheral blood mononuclear cells, due to increased pI in the heavy chain constant region.

[0624] Subplot (A) shows the agonist activity measured using IL-2 production as an indicator, and subplot (B) shows the agonist activity measured using IFN-γ production as an indicator.

[0625] Figure 42 This is a graph showing the enhanced agonist activity of various anti-CD137 antibodies in the presence or absence of ATP, as tested using human peripheral blood mononuclear cells, due to increased pI in the heavy chain constant region.

[0626] Subplot (A) shows the agonist activity measured using IL-2 production as an indicator, and subplot (B) shows the agonist activity measured using IFN-γ production as an indicator.

[0627] Figure 43 This is a graph showing the enhanced agonist activity of various anti-CD137 antibodies in the presence or absence of ATP, as tested using human peripheral blood mononuclear cells, due to increased pI in the heavy chain constant region.

[0628] Subplot (A) shows the agonist activity measured using IL-2 production as an indicator, and subplot (B) shows the agonist activity measured using IFN-γ production as an indicator.

[0629] Figure 44 This is a graph showing the enhanced agonist activity of various anti-CD137 antibodies, as tested using human peripheral blood mononuclear cells, in the presence or absence of ATP, due to increased binding activity of the heavy chain constant region to the Fcγ receptor.

[0630] Subplot (A) shows the agonist activity measured using IL-2 production as an indicator, and subplot (B) shows the agonist activity measured using IFN-γ production as an indicator.

[0631] Figure 45 This is a graph showing the plasma concentrations of various converted and non-converted anti-CD137 antibodies tested using human CD137 knock-in mice.

[0632] All Fc cells are mIgG1.

[0633] Figure 46 This is a graph showing the plasma concentrations of various converted and non-converted anti-CD137 antibodies tested using human CD137 knock-in mice.

[0634] Fc is MB110.

[0635] Figure 47 This is a graph showing the plasma concentrations of various converted and non-converted anti-CD137 antibodies tested using human CD137 knock-in mice.

[0636] Fc is MB492.

[0637] Figure 48 This is a graph illustrating the antitumor effect of A375-mIgG1 / B167-ml0r in a mouse model transplanted with MC38 cells.

[0638] Each point represents the average of a set (n = 5) of tumor volumes.

[0639] Figure 49 This is a graph showing the weight of organs in a mouse model transplanted with MC38 cells after administration of antibodies (NO1-mIgG1 or A375-mIgG1 / B167-ml0r).

[0640] Subplot (A) shows the weight of the lymph nodes, and subplot (B) shows the weight of the spleen.

[0641] Figure 50 This is a graph showing the degree of T cell activation in lymph nodes of a mouse model transplanted with MC38 cells after administration of NO1-mIgG1 or A375-mIgG1 / B167-ml0r.

[0642] Subplot (A) shows the percentage of PD-1 positive T cells in CD8 positive T cells, subplot (B) shows the percentage of ICOS positive T cells in CD8 positive T cells, and subplot (C) shows the percentage of granzyme B positive T cells in CD8 positive T cells.

[0643] Figure 51 This is a graph showing the level of T cell activation in the spleen of a mouse model transplanted with MC38 cell lines after administration of NO1-mIgG1 or A375-mIgG1 / B167-ml0r.

[0644] Subplot (A) shows the percentage of PD-1 positive T cells in CD8 positive T cells, subplot (B) shows the percentage of ICOS positive T cells in CD8 positive T cells, and subplot (C) shows the percentage of granzyme B positive T cells in CD8 positive T cells.

[0645] Figure 52 This is a graph showing the level of T cell activation in the liver of mouse models transplanted with MC38 cell lines after administration of NO1-mIgG1 or A375-mIgG1 / B167-ml0r.

[0646] Subplot (A) shows the percentage of PD-1 positive T cells in CD8 positive T cells, and subplot (B) shows the percentage of granzyme B positive T cells in CD8 positive T cells.

[0647] Figure 53 This is a graph illustrating the antitumor effect of A356-MB110 / B040-ml0r in a mouse model transplanted with the MC38 cell line.

[0648] Each point represents the average of a set (n = 5) of tumor volumes.

[0649] Figure 54 This is a graph showing the weight of organs in mouse models transplanted with MC38 cell lines after administration of NS2-MB110 or A356-MB110 / B040-ml0r.

[0650] Subplot (A) shows the weight of the lymph nodes, and subplot (B) shows the weight of the spleen.

[0651] Figure 55 This is a graph showing the level of T cell activation in the livers of mouse models transplanted with MC38 cell lines after administration of NS2-MB110 or A356-MB110 / B040-ml0r.

[0652] Subplot (A) shows the percentage of PD-1 positive T cells in CD8 positive T cells, and subplot (B) shows the percentage of ICOS positive T cells in CD8 positive T cells.

[0653] Figure 56 The figure shows the antitumor effect of A372-mIgG1 / B040-ml0r in a mouse model transplanted with the MC38 cell line.

[0654] Each point represents the average of a set (n = 5) of tumor volumes.

[0655] Figure 57 The number of lymph node cells (subfigure (A)) and spleen weight (subfigure (B)) in a mouse model transplanted with the MC38 cell line are shown after administration of A372-mIgG1 / B040-ml0r.

[0656] Figure 58 This is a graph showing the degree of T cell activation in the liver of a mouse model transplanted with the MC38 cell line after administration of A372-mIgG1 / B040-ml0r (percentage of CD8-positive T cells and granzyme B-positive T cells).

[0657] Figure 59 The figure shows the antitumor effect of A372-MB110 / B040-ml0r in a mouse model transplanted with the MC38 cell line.

[0658] Each point represents the average of a set (n = 5) of tumor volumes.

[0659] Figure 60 This is a graph showing the weight of organs in mouse models transplanted with the MC38 cell line after administration of NS2-MB110 or A372-MB110 / B040-ml0r.

[0660] Subplot (A) shows the weight of the lymph nodes, and subplot (B) shows the weight of the spleen.

[0661] Figure 61 This is a graph showing the degree of T cell activation in the liver of a mouse model transplanted with MC38 cell lines after administration of NS2-MB110 or A372-MB110 / B040-ml0r (percentage of PD-1 positive T cells among CD8 positive T cells).

[0662] Figure 62 The figure shows the antitumor effect of A372-MB492 / B040-ml0r in a mouse model transplanted with the MC38 cell line.

[0663] Each point represents the average of a set (n = 5) of tumor volumes.

[0664] Figure 63The figure shows the number of lymph node cells and spleen weight in mouse models transplanted with MC38 cell lines after administration of NS1-MB492 or A372-MB492 / B040-ml0r.

[0665] Subplot (A) shows the number of cells in the lymph nodes, and subplot (B) shows the organ weight of the spleen.

[0666] Figure 64 This is a graph showing the degree of T cell activation in the livers of mouse models transplanted with MC38 cell lines after administration of NS1-MB492 or A372-MB492 / B040-ml0r (percentage of CD8-positive T cells and granzyme B-positive T cells).

[0667] Figure 65 The figure shows the antitumor effects of A486-MB492 / B167-ml0r or A488-MB492 / B226-ml0r in a mouse model transplanted with the MC38 cell line.

[0668] Each point represents the average of a set (n = 5) of tumor volumes.

[0669] Figure 66 The figure shows the number of cells in each lymph node and the weight of the spleen in a mouse model transplanted with the MC38 cell line after administration of NS1-MB492, A486-MB492 / B167-ml0r, or A488-MB492 / B226-ml0r.

[0670] Subplot (A) shows the number of cells in each lymph node, and subplot (B) shows the weight of the spleen.

[0671] Figure 67 This is a graph showing the level of effector cell infiltration in the liver of mouse models transplanted with MC38 cell lines after administration of NS1-MB492, A486-MB492 / B167-ml0r, or A488-MB492 / B226-ml0r (percentage of CD3-positive and CD8-positive T cells in CD45-positive T cells).

[0672] Figure 68 The figure shows the antitumor effect of A489-MB492 / B223-ml0r in a mouse model transplanted with the MC38 cell line.

[0673] Each point represents the average of a set (n = 5) of tumor volumes.

[0674] Figure 69This is a graph showing the number of lymph node cells and the number of cells in the splenic lymphocyte fraction in a mouse model transplanted with the MC38 cell line after administration of NS1-MB492 or A489-MB492 / B223-ml0r.

[0675] Subgraph (A) shows the number of cells in the lymph nodes, and subgraph (B) shows the number of cells in the lymphocyte fraction of the spleen.

[0676] Figure 70 This is a graph showing the degree of T cell activation in the liver of a mouse model transplanted with the MC38 cell line after administration of NS1-MB492 or A489-MB492 / B223-ml0r (percentage of CD45-positive T cells and CD8-positive T cells).

[0677] Figure 71 The figure shows the antitumor effects of A548-mIgG1 / B256-ml0r and A551-mIgG1 / B256-ml0r in a mouse model transplanted with the MC38 cell line.

[0678] Subfigure (A) shows the antitumor activity of A548-mIgG1 / B256-ml0r, and subfigure (B) shows the antitumor activity of A551-mIgG1 / B256-ml0r.

[0679] Figure 72 This is a graph showing the weight of organs in mouse models transplanted with MC38 cell lines after administration of NS1-mIgG1, A548-mIgG1 / B256-ml0r, or A551-mIgG1 / B256-ml0r.

[0680] Subplot (A) shows the weight of the lymph nodes, and subplot (B) shows the weight of the spleen.

[0681] Figure 73 This is a graph showing the degree of T cell activation in the livers of mouse models transplanted with MC38 cell lines after administration of NS1-mIgG1, A548-mIgG1 / B256-ml0r, or A551-mIgG1 / B256-ml0r.

[0682] Subplot (A) shows the percentage of PD-1 positive T cells in CD8 positive T cells, and subplot (B) shows the percentage of granzyme B positive T cells in CD8 positive T cells.

[0683] Figure 74 The figure shows the antitumor effect of A551-MB110 / B379-ml0r in a mouse model transplanted with the MC38 cell line.

[0684] Figure 75 This is a graph showing the weight of organs in mouse models transplanted with the MC38 cell line after administration of NS1-mIgG1 or A551-MB110 / B379-ml0r.

[0685] Subplot (A) shows the weight of the lymph nodes, and subplot (B) shows the weight of the spleen.

[0686] Figure 76 This is a graph showing the degree of T cell activation in the spleen of a mouse model transplanted with MC38 cell lines after administration of NS1-mIgG1 or A551-MB110 / B379-ml0r.

[0687] Subplot (A) shows the percentage of PD-1 positive T cells in CD8 positive T cells, subplot (B) shows the percentage of ICOS positive T cells in CD8 positive T cells, and subplot (C) shows the percentage of granzyme B positive T cells in CD8 positive T cells.

[0688] Figure 77 This is a graph showing the degree of T cell activation in the livers of mouse models transplanted with MC38 cell lines after administration of NS1-mIgG1 or A551-MB110 / B379-ml0r.

[0689] Subplot (A) shows the percentage of PD-1 positive T cells in CD8 positive T cells, subplot (C) shows the percentage of ICOS positive T cells in CD8 positive T cells, and subplot (B) shows the percentage of granzyme B positive T cells in CD8 positive T cells.

[0690] The X-axis shows the antibody concentration (μg / mL), and the Y-axis shows the relative light units.

[0691] Figure 78 This is a graph showing the agonist activity of various anti-CD137 antibodies tested using 4-1BB Jurkat cells in the presence or absence of small molecule compounds (ATP or ADP).

[0692] The X-axis shows the antibody concentration (μg / mL), and the Y-axis shows the relative light units.

[0693] Figure 79 This is a graph showing the agonist activity of various converted anti-CD137 antibodies as tested using the 4-1BB Jurkat reporter assay in the presence of ATP.

[0694] Figure 80This is a graph comparing the plasma kinetics of each anti-CD137 conversion antibody, A375-SCF041aPh / B167-Lamlib and A375-MY201aPh / B167-Lamlib. The vertical axis of the graph shows the plasma concentration of each antibody.

[0695] Figure 81 This figure illustrates the antitumor effects of A375 / B167-SCF041aPh and A375 / B167-MY201aPh in a mouse model established by transplanting LLC1 / OVA / GPC3 cell lines into hCD137KI / mFcγR2bKO / hFcγR2bTg#90 mice.

[0696] Each point represents the average of a set (n = 5) of tumor volumes.

[0697] Figure 82 The study demonstrated that administration of A551-MB110 / B379-ml0r, anti-mouse PD-L1 antibody, or a combination thereof to a mouse model transplanted with MC38 cells resulted in increased CD8 levels in tumor tissue. + The number of cells.

[0698] Figure 83 This demonstrates that administration of A551-MB110 / B379-ml0r, anti-mouse PD-L1 antibody, or a combination thereof to a mouse model transplanted with MC38 cells resulted in increased PD-L1 levels in tumor tissue. + The number of cells.

[0699] Figure 84 The study showed the number of cells with or high expression of PD-L1 in tumor tissue after administration of A551-MB110 / B379-ml0r, anti-mouse PD-L1 antibody, or a combination thereof to a mouse model transplanted with MC38 cells.

[0700] Figure 85 The study demonstrated the tumor growth inhibition effect in a mouse model transplanted with MC38 cells by administration of A551-MB110 / B379-ml0r (“Sta-MB”), UreH-MB110 / UreL-mk1 (“Ure-MB”) or anti-mouse PD-L1 antibody (“PD-L1-Ab”) alone, or by administration of A551-MB110 / B379-ml0r (“Sta-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”) in combination, or by administration of UreH-MB110 / UreL-mk1 (“Ure-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”) in combination.

[0701] Figure 86The study shows the degree of fluctuation in blood parameters (leukocyte concentration, platelet concentration, and lymphocyte concentration) in mouse models transplanted with MC38 cells due to administration of A551-MB110 / B379-ml0r (“Sta-MB”), UreH-MB110 / UreL-mk1 (“Ure-MB”), or anti-mouse PD-L1 antibody (“PD-L1-Ab”), or a combination thereof, in the case of A551-MB110 / B379-ml0r (“Sta-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”), or in the case of UreH-MB110 / UreL-mk1 (“Ure-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”).

[0702] Figure 87 The organ weights of the spleen and tumor draining lymph nodes (DLNs) in a mouse model transplanted with MC38 cells are shown after administration of A551-MB110 / B379-ml0r (“Sta-MB”), UreH-MB110 / UreL-mk1 (“Ure-MB”) or anti-mouse PD-L1 antibody (“PD-L1-Ab”) alone, or in combination with A551-MB110 / B379-ml0r (“Sta-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”), or in combination with UreH-MB110 / UreL-mk1 (“Ure-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”).

[0703] Figure 88 The study showed that, following administration of A551-MB110 / B379-ml0r (“Sta-MB”), UreH-MB110 / UreL-mk1 (“Ure-MB”), or anti-mouse PD-L1 antibody (“PD-L1-Ab”), or a combination of A551-MB110 / B379-ml0r (“Sta-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”), or a combination of UreH-MB110 / UreL-mk1 (“Ure-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”), KLRG-1, ICOS, PD-1, and LAG-3 levels in the spleen of a mouse model transplanted with MC38 cells were significantly increased in CD8+. + The expression rate of Foxp3 in T cells and Foxp3 + Regulatory T cells in CD4 + The ratio in T cells.

[0704] Figure 89The study showed the CD45 concentration in the spleen of a mouse model transplanted with MC38 cells after administration of A551-MB110 / B379-ml0r (“Sta-MB”), UreH-MB110 / UreL-mk1 (“Ure-MB”), or anti-mouse PD-L1 antibody (“PD-L1-Ab”), or a combination thereof, of A551-MB110 / B379-ml0r (“Sta-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”), or a combination thereof, of UreH-MB110 / UreL-mk1 (“Ure-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”). + The percentage of positive CD8+ T cells and CD45+ activation markers in white blood cells. + Foxp3 in white blood cells + The ratio of regulatory T cells.

[0705] Figure 90 The study demonstrated that, following administration of A551-MB110 / B379-ml0r (“Sta-MB”), UreH-MB110 / UreL-mk1 (“Ure-MB”), or anti-mouse PD-L1 antibody (“PD-L1-Ab”), or a combination thereof, A551-MB110 / B379-ml0r (“Sta-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”), or a combination thereof, UreH-MB110 / UreL-mk1 (“Ure-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”), KLRG-1, ICOS, PD-1, and LAG-3 in tumor draining lymph nodes (DLN) of a mouse model transplanted with MC38 cells, showed increased activity of KLRG-1, ICOS, PD-1, and LAG-3 on CD8+ in the MD8+ region. + The expression rate of Foxp3 in T cells and Foxp3 + Regulatory T cells in CD4 + The ratio in T cells.

[0706] Figure 91 The study demonstrated the positive CD8 activation markers in tumor draining lymph nodes (DLNs) of a mouse model transplanted with MC38 cells after administration of A551-MB110 / B379-ml0r (“Sta-MB”), UreH-MB110 / UreL-mk1 (“Ure-MB”), or anti-mouse PD-L1 antibody (“PD-L1-Ab”), or in combination with A551-MB110 / B379-ml0r (“Sta-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”), or in combination with UreH-MB110 / UreL-mk1 (“Ure-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”).+ Absolute number of T cells and CD4 + Foxp3 in T cells + The absolute number of regulatory T cells.

[0707] Figure 92 The study demonstrated that, following administration of A551-MB110 / B379-ml0r (“Sta-MB”), UreH-MB110 / UreL-mk1 (“Ure-MB”), or anti-mouse PD-L1 antibody (“PD-L1-Ab”), or a combination thereof, A551-MB110 / B379-ml0r (“Sta-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”), or a combination thereof, UreH-MB110 / UreL-mk1 (“Ure-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”), KLRG-1, ICOS, PD-1, and LAG-3 levels in the liver of a mouse model transplanted with MC38 cells were significantly reduced in CD8+. + The expression rate of Foxp3 in T cells and Foxp3 + Regulatory T cells in CD4 + The ratio in T cells.

[0708] Figure 93 The study showed the CD45 levels in the livers of mouse models transplanted with MC38 cells after administration of A551-MB110 / B379-ml0r (“Sta-MB”), UreH-MB110 / UreL-mk1 (“Ure-MB”), or anti-mouse PD-L1 antibody (“PD-L1-Ab”), or a combination thereof, of A551-MB110 / B379-ml0r (“Sta-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”), or a combination thereof, of UreH-MB110 / UreL-mk1 (“Ure-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”). + Positive CD8 activation markers in white blood cells + T cell ratio and CD45 + Foxp3 in white blood cells + The ratio of regulatory T cells.

[0709] Figure 94The binding extent of Alexa488-labeled A551-MB110 / B379-ml0r, Alexa488-labeled IC17HdK-MB110 / IC17L-mk (negative control), and non-converted anti-CD137 antibody Alexa488-labeled UreH-MB110 / UreL-mk1 (positive control) to lymphocyte fractions in tumors, spleen, and liver in a mouse model transplanted with MC38 cells, as analyzed by flow cytometry (FCM).

[0710] Figure 95 A heatmap of the genome is shown, illustrating changes in expression associated with extracellular ATP in various normal organ tissues and various tumor tissues.

[0711] Figure 96 The expression levels of CD73 in various cancer cell lines were shown, as analyzed by flow cytometry (FCM).

[0712] Figure 97 The image shows tumor cells and CD4 from mice transplanted with the LLC1 / OVA / GPC3 clone C5 cell line. + T cells, CD8 + The expression rates of CD39 and CD73 in T cells and non-T cells, as analyzed by flow cytometry (FCM).

[0713] Regarding the expression of CD39 and CD73, tumor cells, CD4 + T cells, CD8 + T cells and non-T cells were tested five times using different samples, and the average value was calculated.

[0714] Figure 98 This is a representative figure showing tumor cells and CD4+ from mice transplanted with the LLC1 / OVA / GPC3 clone C5 cell line. + T cells, CD8 + The expression rates of CD39 and CD73 in T cells and non-T cells, as analyzed by flow cytometry (FCM).

[0715] Figure 99 The image shows tumor cells and CD4 from mice transplanted with the MC38 cell line. + T cells, CD8 + The expression rates of CD39 and CD73 in T cells and non-T cells, as analyzed by flow cytometry (FCM).

[0716] Regarding the expression of CD39 and CD73, tumor cells, CD4 + T cells, CD8+ T cells and non-T cells were tested five times using different samples, and the average value was calculated.

[0717] Figure 100 This is a representative figure showing tumor cells and CD4+ from mice transplanted with the MC38 cell line. + T cells, CD8 + The expression rates of CD39 and CD73 in T cells and non-T cells, as analyzed by flow cytometry (FCM).

[0718] Figure 101 Antitumor effects were demonstrated in a mouse model transplanted with LLC1 / OVA cells by administration of A551-MB110 / B379-ml0r (“Sta-MB”), UreH-MB110 / UreL-mk1 (“Ure-MB”) or anti-mouse PD-L1 antibody (“PD-L1-Ab”) alone, or by administration of A551-MB110 / B379-ml0r (“Sta-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”) in combination, or by administration of UreH-MB110 / UreL-mk1 (“Ure-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”) in combination.

[0719] Figure 102 The study demonstrated the presence of CD8+ in tumor-draining lymph nodes (DLNs) of a mouse model transplanted with LLC1 / OVA cells after administration of A551-MB110 / B379-ml0r (“Sta-MB”), UreH-MB110 / UreL-mk1 (“Ure-MB”), or anti-mouse PD-L1 antibody (“PD-L1-Ab”), or a combination thereof, of A551-MB110 / B379-ml0r (“Sta-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”), or a combination thereof, of UreH-MB110 / UreL-mk1 (“Ure-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”). + Expression rates of KLRG-1, ICOS, and PD-1 in T cells.

[0720] Figure 103The results show positive CD8 activation markers in tumor draining lymph nodes (DLNs) of a mouse model transplanted with LLC1 / OVA cells after administration of A551-MB110 / B379-ml0r (“Sta-MB”), UreH-MB110 / UreL-mk1 (“Ure-MB”), or anti-mouse PD-L1 antibody (“PD-L1-Ab”), or in combination with A551-MB110 / B379-ml0r (“Sta-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”), or in combination with UreH-MB110 / UreL-mk1 (“Ure-MB”) and anti-mouse PD-L1 antibody (“PD-L1-Ab”). + The absolute number of T cells.

[0721] Figure 104 The study demonstrated the tumor growth inhibition effect of A551-MB110 / B379-ml0r (“Sta-MB”) alone in a mouse model transplanted with LLC1 / OVA cells.

[0722] Figure 105 This study demonstrated the tumor growth inhibition effect of converting anti-CD137 antibody A551-MB110 / B379-ml0r or anti-TIGIT antibody alone, or the combination of converting anti-CD137 antibody A551-MB110 / B379-ml0r and anti-TIGIT antibody in a mouse model transplanted with the mouse AMLC1498 cell line.

[0723] Figure 106 The antitumor effect of the anti-CD137 antibody A551-MB110 / B379-ml0r was demonstrated in a mouse model transplanted with the MC38-hGPC3#G64B2M KO clone 5 cell line. Detailed Implementation

[0724] I. Definition

[0725] The term "binding activity" refers to the strength of the sum of non-covalent interactions between one or more binding sites of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). In this document, "binding activity" is not strictly limited to a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). For example, when members of a binding pair reflect a monovalent 1:1 interaction, binding activity is specifically referred to as intrinsic binding affinity (affinity). When members of a binding pair can bind both monovalently and multivalently, binding activity is the sum of the strengths of each binding. The binding activity of molecule X to its partner Y is typically expressed as a dissociation constant (KD) or "amount of analyte bound per unit amount of ligand" (hereinafter referred to as "binding amount"). Those skilled in the art will understand that, generally, a lower dissociation constant (KD) indicates higher binding activity, and a higher value for "amount of analyte bound per unit amount of ligand" or "binding amount" indicates higher binding activity. Binding activity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding activity are described below.

[0726] "Mature binding activity" antigen-binding molecules or antibodies, or "enhanced binding activity" antigen-binding molecules or antibodies, refer to antibodies with one or more altered hypervariable regions (HVRs) compared to parental antigen-binding molecules or antibodies without alterations, resulting in increased binding activity of the antigen-binding molecule or antibody to the antigen.

[0727] The terms "anti-CD137 antigen-binding molecule" or "anti-CD137 antibody" and "antigen-binding molecule that binds to CD137" or "antibody that binds to CD137" refer to antigen-binding molecules or antibodies capable of binding CD137 with sufficient binding activity, such that the antigen-binding molecule or antibody can be used as a diagnostic and / or therapeutic agent targeting CD137. In some embodiments, anti-CD137 antibodies bind to conserved CD137 epitopes among CD137 in different species.

[0728] The term "anti-CD137 antigen-binding molecule or anti-CD137 antibody with small molecule compound-dependent CD137 binding activity" refers to an antigen-binding molecule or antibody that exhibits higher binding activity to CD137 in the presence of a small molecule compound compared to its binding activity to CD137 in the absence of the small molecule compound. In one embodiment, "presence of a small molecule compound" means that the small molecule compound is present at a concentration of 10 μmol or higher, 50 μmol or higher, 100 μmol or higher, 150 μmol or higher, 200 μmol or higher, or 250 μmol or higher. In one embodiment, in the presence of the small molecule compound, the degree of binding activity of the anti-CD137 antigen-binding molecule or antibody to unrelated non-CD137 proteins is less than about 10% of the binding activity of the antigen-binding molecule or antibody to CD137, for example, as measured by radioimmunoassay (RIA) or surface plasmon resonance (SPR). In some embodiments, in the presence of low molecular weight compounds, the dissociation constant (KD) of the anti-CD137 antigen-binding molecule or antibody is less than 1 μM, less than 100 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, less than 0.01 nM, or less than 0.001 nM (e.g., 10 μM). -6 M and below, 10 -7 M and below, 10 -8 M and below, 10 -9 M and below, 10 -10 Below M, for example, 10 -6 M to 10 -10 M, 10 -7 M to 10 -9 M, for example, 10 -7 M to 10 -8 M).

[0729] In this document, the term "antigen-binding molecule" is used in its broadest sense to refer to a molecule that specifically binds to an antigenic determinant. In one embodiment, the antigen-binding molecule is an antibody, an antibody fragment, or an antibody derivative.

[0730] As used herein, an “agonistic antigen-binding molecule” or “agonistic antibody” is an antigen-binding molecule or antibody that significantly induces or enhances the biological activity of the antigens it binds to (e.g., CD137 and CD3).

[0731] Therefore, if the antigen is, for example, CD137, then such an antigen-binding molecule or antibody with agonistic activity is referred to as a "CD137 agonist antigen-binding molecule" or a "CD137 agonist antibody," respectively. Similarly, if the antigen is, for example, CD3, then such an antigen-binding molecule or antibody with agonistic activity is referred to as a "CD3 agonist antigen-binding molecule" or a "CD3 agonist antibody," respectively.

[0732] The term “antibody” is used in the broadest sense in this document and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0733] "Antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen bound by the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0734] "Antigen-binding molecule that binds to the same epitope" or "antibody that binds to the same epitope" refers to an antibody or antigen-binding molecule that, in a competitive assay, blocks the binding of the reference antibody or reference antigen-binding molecule to its antigen by 50% or more, and conversely, in a competitive assay, the reference antibody blocks the binding of the antibody to its antigen by 50% or more. Exemplary competitive assays are provided herein. In one embodiment, a competitive assay is performed in the presence of a low molecular weight compound when the reference antigen-binding molecule or reference antibody exhibits antigen-binding activity in a manner dependent on the low molecular weight compound.

[0735] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a specific source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0736] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. Antibodies are mainly classified into five classes: IgA, IgD, IgE, IgG, and IgM. Some of these can be further divided into subclasses (subtypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ.

[0737] "Effective functions" refer to those biological activities attributed to the Fc region of an antibody, which vary across antibody isotypes. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0738] "Cytotoxicity" refers to activities that inhibit or prevent cell function and / or cause cell death or destruction. Cytotoxicity can be, for example, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and T cell cytotoxicity; it can also be induced by cytotoxic agents (e.g., radioisotopes and chemotherapeutic agents) (e.g., immunoconjugates).

[0739] The term "Fc region" as used herein is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes both native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) of the Fc region may or may not be present. Unless otherwise stated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system (also known as the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0740] The term "variant Fc region" as used herein refers to an amino acid sequence that differs from the native Fc region due to at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution compared to the native Fc region or the Fc region of the parent polypeptide; for example, about one to about ten amino acid substitutions, preferably about one to about five amino acid substitutions, in the native Fc region or the Fc region of the parent polypeptide. The variant Fc region herein preferably shares at least about 80% homology with the native Fc region and / or with the Fc region of the parent polypeptide, most preferably at least about 90% homology, and more preferably at least about 95% homology.

[0741] In this paper, amino acid changes or substitutions within the Fc region or constant region can be represented by a combination of the EU numbering system and the amino acid. For example, S424N represents a substitution from serine (Ser) to asparagine (Asn) at position 424 of the EU number. EU424N represents a substitution from amino acid (of any type) to asparagine (Asn) at position 424 of the EU number.

[0742] The term "antibody containing an Fc region" as used herein refers to an antibody that contains an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) or C-terminal glycine-lysine (residues 446-447) of the Fc region can be removed, for example, during antibody purification or by recombinantly engineering the nucleic acid encoding the antibody. Therefore, compositions containing antibodies having an Fc region according to this disclosure can contain antibodies having G446-K447, antibodies having G446 and not having K447, antibodies with all G446-K447 removed, or mixtures of the above three types of antibodies.

[0743] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably in this document and refer to an antibody that has a structure substantially similar to that of a natural antibody or that has a heavy chain containing an Fc region or a variant Fc region as defined herein.

[0744] "Human antibody" refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody generated by a human or human cell or derived from a non-human source using a human antibody library or other human antibody encoding sequences. This definition of human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues.

[0745] "Frame" or "FR" refers to the variable domain residues excluding the hypervariable region (HVR) residues. A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences usually appear in the VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0746] For the purposes of this document, a “recipient human frame” is a frame comprising the amino acid sequence of a light chain variable domain (VL) frame or a heavy chain variable domain (VH) frame derived from the human immunoglobulin frame or the human common frame, as defined below. A recipient human frame “derived” from the human immunoglobulin frame or the human common frame may contain the same amino acid sequence or may contain variations in the amino acid sequence. In some embodiments, the number of amino acid variations is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer. In some embodiments, the VL recipient human frame sequence is identical to the VL human immunoglobulin frame sequence or the human common frame sequence.

[0747] "Human common framework" is a framework representing the most common amino acid residues in the selection of the VL or VH framework sequence of human immunoglobulins. Typically, the selection of the VL or VH sequence of human immunoglobulins is derived from a subgroup of variable domain sequences. Typically, the sequence subgroup is as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one embodiment, for VL, the subgroup is the κI subgroup as described above by Kabat et al. In one embodiment, for VH, the subgroup is subgroup III as described above by Kabat et al.

[0748] A "humanized" antibody is a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of the non-human antibody, and all or substantially all of the FRs correspond to those of the human antibody. The humanized antibody may optionally comprise at least a portion of the antibody constant region derived from the human antibody. The "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization.

[0749] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies binding to specific antigens can be isolated from antigen-binding antibodies using either the VH or VL domain to screen libraries of complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0750] As used herein, the term "hypervariant region" or "HVR" refers to each region in an antibody variable domain that is sequence-highly variable ("complementarity-determining region" or "CDR") and / or forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact"). Typically, an antibody contains six HVRs: three in the VH domain (H1, H2, H3) and three in the VL domain (L1, L2, L3). Exemplary HVRs in this document include:

[0751] (a) Hypervariable rings 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. (Molecular Biology Journal) 196: 901-917 (1987));

[0752] (b) CDRs 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., Immunologically Significant Protein Sequences, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MA, (1991)).

[0753] (c) Antigen contacts 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

[0754] (d) Combinations of (a), (b) and / or (c) including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

[0755] Unless otherwise stated, HVR residues and other residues (e.g., FR residues) within the variable domain are numbered herein according to the Kabat numbering system as described above. In this document, HVR residues or other residues (e.g., FR residues) within the variable domain, as well as amino acid alterations or substitutions at these residues, can be represented by combinations of the Kabat numbering system and amino acids. For example, N99 represents asparagine (Asn) at position 99 in the Kabat numbering system, while N99A represents a substitution from asparagine (Asn) to alanine (Ala) at position 99 in the Kabat numbering system.

[0756] "Immune conjugates" are antibodies conjugated to one or more foreign molecules, including but not limited to cytotoxic agents.

[0757] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 Radioactive isotopes of Pb and Lu; chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinblastine alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, donomycin or other intercalating agents); growth inhibitors; enzymes and fragments thereof, such as lysozymes; antibiotics; toxins, such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and various antitumor or anticancer agents disclosed below.

[0758] "Isolated" antibodies are antibodies that have been separated from components of their natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, which is determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0759] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules that are normally contained in cells, but which are located outside chromosomes or at chromosomal locations other than their natural chromosomal locations.

[0760] As used herein, the term "vector" refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures, as well as vectors integrated into the host cell genome to which they have been introduced. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0761] "Encoding nucleic acid for anti-CD137 antigen-binding molecule" refers to one or more nucleic acid molecules that encode a polypeptide that constitutes the antigen-binding molecule. "Isolated nucleic acid for anti-CD137 antibody" refers to one or more nucleic acid molecules that encode the antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or in a separate vector, as well as such nucleic acid molecules present at one or more locations in the host cell.

[0762] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably to refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformations” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of the number of passages. The nucleic acid content of progeny cells may not be exactly the same as that of the parent cells, but may contain mutations. Mutant progeny cells that have the same function or biological activity as those screened or selected in the original transformed cells are included herein.

[0763] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitopes, except for possible variant antibodies, such as variant antibodies containing naturally occurring mutations or variant antibodies that occur during the production of a monoclonal antibody formulation, which are typically present in small amounts. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies used according to this disclosure can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0764] "Naked antibody" refers to an antibody that is not conjugated to a heterologous portion (e.g., a cytotoxic portion) or a radiolabel. Naked antibodies can be present in pharmaceutical formulations.

[0765] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. Each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, from the N-terminus to the C-terminus, followed by three constant domains (CH1, CH2, and CH3). Similarly, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, from the N-terminus to the C-terminus, followed by a constant light chain structure (CL) domain. The light chains of an antibody can be assigned to one of two types based on the amino acid sequence of its constant domains, called kappa (κ) and lambda (λ).

[0766] The "percentage of amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after the sequences have been aligned and vacancies have been introduced (if necessary) to achieve maximum percentage sequence identity and any conserved substitutions have not been considered part of the sequence identity. Alignment can be performed in various ways within the scope of the art to determine the percentage of amino acid sequence identity, for example, using public computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENTYX (registered trademark) (Genetyx Ltd.). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared.

[0767] The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc. The source code, along with user documentation, has been filed with the U.S. Copyright Office in Washington, D.C. 20559 and registered under U.S. Copyright Registry No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems (including Digital UNIX V4.0D). All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged. When using ALIGN-2 for amino acid sequence comparison, the percentage of amino acid sequence identity (which can be expressed in the phrase "a given amino acid sequence A has or contains a certain percentage of amino acid sequence identity with, and or for a given amino acid sequence B") is calculated as follows:

[0768] 100 multiplied by the fraction X / Y

[0769] Where X is the number of amino acid residues that the sequence alignment program ALIGN-2 rated as identical matches in the alignment of A and B, and Y is the total number of amino acid residues in B. It should be understood that if the length of amino acid sequence A differs from the length of amino acid sequence B, the amino acid sequence identity % between A and B will not be equal to the amino acid sequence identity % between B and A. Unless otherwise specified, all amino acid sequence identity % values ​​used herein were obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0770] The term "pharmaceutical formulation" refers to a formulation in a form that allows the biological activity of the active ingredient contained therein to be effective and does not contain any other components that would have unacceptable toxicity to the subject to whom the formulation is administered.

[0771] "Pharmaceutically acceptable carriers" refer to components in a pharmaceutical preparation that are non-toxic to the subject, excluding the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0772] The "effective amount" of a reagent, such as a pharmaceutical preparation, refers to the amount that effectively achieves the desired therapeutic or preventative outcome within the required dosage and time period.

[0773] "Individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0774] Unless otherwise stated, the term "CD137" as used herein refers to any naturally occurring CD137 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term covers "full-length" untreated CD137 as well as any form of CD137 produced through cellular processing. The term also covers naturally occurring variants of CD137, such as splice variants or allelic variants.

[0775] The amino acid sequence of an exemplary human full-length CD137 is shown in SEQ ID NO: 1 (NCBI reference sequence: NP_001552.2), and the amino acid sequence of the extracellular region of an exemplary human CD137 is shown in SEQ ID NO: 2. The amino acid sequence of an exemplary mouse full-length CD137 is shown in SEQ ID NO: 3 (NCBI reference sequence: NP_035742.1), and the amino acid sequence of the extracellular region of an exemplary mouse CD137 is shown in SEQ ID NO: 4. The amino acid sequence of an exemplary monkey full-length CD137 is shown in SEQ ID NO: 5 (NCBI reference sequence: ABY47575.1), and the amino acid sequence of the extracellular region of an exemplary monkey CD137 is shown in SEQ ID NO: 6.

[0776] CD137 is a member of the tumor necrosis factor (TNF) receptor family. Its alternative names are tumor necrosis factor receptor superfamily member 9 (TNFRSF9), 4-1BB, and ILA. Besides being activated on CD4... + T cells and CD8 + In addition to being expressed on T cells, CD137 can also be expressed on B cells, dendritic cells, natural killer (NK) and NK-T cells, macrophages, monocytes, neutrophils, and CD4+ cells. + CD25 +It is expressed in regulatory T cells and vascular endothelial cells. Expression in cancer cells has also been reported (Labiano et al., Oncoimmunology, Vol. 24: e1062967 (2015)). The natural CD137 ligand CD137L is presented by antigen-presenting cells such as B cells, monocytes / macrophages, and dendritic cells (Watts et al., Annu. Rev. Immunol., Vol. 23: 23–68 (2005)). Through interaction with the ligand, CD137 leads to increased TCR-induced T cell proliferation, cytokine production, functional maturation, inhibition of apoptosis, and CD8+. + Long-term survival of T cells (Nam et al., Recent advances in cancer drug target research (Curr. Cancer Drug Targets, Vol. 5: pp. 357-363 (2005); Watts et al., Annual review of immunology (Annu. Rev. Immunol., Vol. 23: pp. 23-68 (2005)).

[0777] The term "regulatory T cell" or "Treg cell" refers to T cells responsible for the suppressive regulation (immune tolerance) of the immune response. In one implementation, the regulatory T cell is a CD4+ T cell. + and / or CD25 + T cells.

[0778] The terms “cancer,” “cancer,” and “cancerous” refer to or describe a physiological condition in mammals that is typically characterized by uncontrolled cell growth / proliferation.

[0779] The term "tumor" refers to all proliferative cell growth and proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. The terms "cancer," "cancer," "cancerous," "proliferative disorder," "proliferative disease," and "tumor" are not mutually exclusive in this document.

[0780] The terms "cell proliferation disorder" and "proliferative disease" refer to diseases associated with some degree of abnormal cell proliferation. In one implementation, a cell proliferation disorder is cancer.

[0781] As used herein, “treatment” (and its grammatical variations, such as “treat” or “treating”) refers to a clinical intervention that attempts to alter the natural course of disease in an individual being treated, and can be used for prevention or during the clinicopathological process. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, alleviating or reducing the state of the disease, and alleviating or improving prognosis. In some embodiments, the antibodies of this disclosure are used to delay the onset of disease or slow its progression.

[0782] II. Composition and Method (Anti-CD137 Agonist Antigen-Binding Molecule)

[0783] On one hand, this invention is based in part on anti-CD137 agonist antigen-binding molecules and their uses. In some embodiments, antibodies binding to CD137 are provided. The antibodies of this disclosure may exhibit activation of immune cells, cytotoxicity, or antitumor activity, and therefore may be used, for example, for the diagnosis or treatment of cancer.

[0784] A. Exemplary anti-CD137 antigen-binding molecules or antibodies

[0785] On the one hand, this disclosure provides isolated antigen-binding molecules or antibodies that bind to CD137. In some embodiments, anti-CD137 antigen-binding molecules or antibodies...

[0786] - It has CD137 binding activity that depends on small molecule compounds;

[0787] - Binds to the extracellular region of CD137;

[0788] - Forms a ternary complex with low molecular weight compounds and CD137;

[0789] - Combining human-derived CD137 and monkey-derived CD137;

[0790] - It has an agonistic effect on CD137 activity;

[0791] - It exhibits agonistic activity against CD137 in the presence of low molecular weight compounds;

[0792] - Exhibits low agonistic activity toward CD137 in the absence of low molecular weight compounds; and / or

[0793] - In the absence of low molecular weight compounds, it essentially does not exhibit agonistic activity against CD137.

[0794] [The binding activity of antigen-binding molecules or antibodies]

[0795] In some embodiments, in the presence of low molecular weight compounds, the dissociation constant (KD) of the binding activity of the antigen-binding molecules or antibodies provided herein is less than 1 μM, less than 100 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, less than 0.01 nM, or less than 0.001 nM (e.g., 10 μM). -6 Below M, 10 -7 Below M, 10 -8 Below M, 10 -9 Below M, 10 -10 Below M, for example, 10 -6 M to 10 -10 M, 10 -7 M to 10 -9 M, for example, 10 -7 M to 10 -8 M).

[0796] In one embodiment, the binding activity of the antigen-binding molecule or antibody is measured by a radiolabeled antigen binding assay (RIA) and expressed by KD. In one embodiment, the radiolabeled antigen binding assay is performed using the Fab form of the antibody of interest and its antigen. For example, by using the minimum concentration in the presence of a series of unlabeled antigen titrants. 125 I) Label the antigen to balance Fab, then capture the bound antigen with a plate coated with anti-Fab antibody to measure the solution-binding affinity of Fab to the antigen (see, e.g., Chen et al., Journal of Molecular Biology, 293: 865-881 (1999)). To establish the assay conditions, MICROTITER (trademark) multiwell plates (ThermoScientific) were coated overnight with 5 μg / mL capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6). Subsequently, they were blocked with 2% (w / v) bovine serum albumin in PBS at room temperature (approximately 23°C) for 2 to 5 hours. In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125I] The antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12, Presta et al., Cancer Res., 57:4593-4599 (1997). The Fab of interest is then incubated overnight. However, incubation may be prolonged (e.g., about 65 hours) to ensure equilibration. After this, the mixture is transferred to a capture plate and incubated at room temperature (e.g., one hour). The solution is then removed and the plate is washed eight times with 0.1% polysorbate 20 (Tween 20 (registered trademark)) in PBS. Once the plate is dry, 150 μL / well of scintillation agent (MICROSCINT-20) is added. TM Packard), and the board will be placed in the topcouncil. TM Count for several tens of minutes using a gamma counter (Packard). Select a concentration for each Fab that produces less than or equal to 20% of the maximum binding for the competitive binding assay.

[0797] In one embodiment, to measure antibody binding activity, a ligand capture method is used, for example, using BIACORE (registered trademark) T200 or BIACORE (registered trademark) 4000 (GE Healthcare, Uppsala, Sweden), which relies on surface plasmon resonance analysis as the measurement principle. BIACORE (registered trademark) control software is used for device operation. In one embodiment, an amine conjugation kit (GE Healthcare, Uppsala, Sweden) is used according to the manufacturer's instructions to immobilize molecules such as anti-tag antibodies, anti-IgG antibodies, protein A, etc., onto a sensor chip (GE Healthcare, Uppsala, Sweden) coated with carboxymethyl dextran. The ligand-capturing molecules are diluted with 10 mM sodium acetate solution at an appropriate pH and injected at an appropriate flow rate and injection time. A buffer containing 0.05% polysorbate 20 (i.e., Tween-20) is used as the measurement buffer, and binding activity is measured at a flow rate of 10-30 μL / min and at a preferred measurement temperature of 25°C or 37°C. For measurements using antibodies captured by ligand-capturing molecules as ligands, the antibody is injected to capture the target amount, followed by a series of dilutions of the antigen and / or Fc receptor (analyte) prepared with measurement buffer.

[0798] In one implementation, BIACORE (registered trademark) evaluation software is used to analyze the measurement results. Kinetic parameters are calculated by simultaneously fitting the sensor maps of association and dissociation using a 1:1 binding model, including the association rate (kon or ka), dissociation rate (koff or kd), and equilibrium dissociation constant (KD). For cases of weak binding activity, particularly those with rapid dissociation and difficult-to-calculate kinetic parameters, a steady-state model can be used to calculate the equilibrium dissociation constant (KD). As an additional parameter regarding binding activity, the "analyte binding per unit ligand amount" can be calculated by dividing the amount of analyte bound (resonance unit: RU) at a specific concentration by the amount of ligand captured.

[0799] [Small molecule-dependent binding activity]

[0800] On one hand, the anti-CD137 antigen-binding molecule or antibody exhibits small molecule-dependent CD137 binding activity. In one non-limiting embodiment, the anti-CD137 antigen-binding molecule or antibody shows higher binding activity to CD137 in the presence of a small molecule compound compared to the binding activity in the absence of the small molecule compound. In different embodiments, the anti-CD137 antigen-binding molecule or antibody shows higher binding activity to CD137 in the presence of a high concentration of a small molecule compound compared to the CD137 binding activity in the presence of a low concentration of the small molecule compound. In a preferred embodiment, the binding activity of the anti-CD137 antigen-binding molecule or antibody to CD137 in the presence of the small molecule compound is more than 2, 3, 5, 10, 15, 20, 25, 30, 50, 100, 200, 300, 500, or 1 x 10⁻⁶ times greater than the binding activity in the absence of the small molecule compound. 3 More than twice, 2 x 10 3 More than twice, 3 x 10 3 More than twice, 5 x 10 3 More than twice, 1 x 10 4 More than twice, 2 x 10 4 More than twice, 3 x 10 4 More than twice, 5 x 10 4 More than 1 times or 1 x 10 5More than twice as high. In different preferred embodiments, the binding activity of the anti-CD137 antigen-binding molecule or antibody to CD137 in the presence of the small molecule compound is 2 times, 3 times, 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 50 times, 100 times, 200 times, 300 times, 500 times, or 1 x 10^137 times higher than that in the absence of the small molecule compound. 3 Double the height, 2 x 10 3 Double the height, 3 x 10 3 Double the height, 5 x 10 3 Double the height, 1 x 10 4 Double the height, 2×10 4 Double the height, 3×10 4 Double the height, 5×10 4 Double the height or 1×10 5 Double high.

[0801] The concentration of the small molecule compound can be any concentration, as long as a difference in binding activity of the anti-CD137 antigen-binding molecule or antibody is detected. In one embodiment, the concentration of the small molecule compound "in the presence of the small molecule compound" and / or "in the presence of a high concentration of the small molecule compound" is, for example, 100 nM or more, 500 nM or more, 1 μM or more, 3 μM or more, 5 μM or more, 10 μM or more, 50 μM or more, 100 μM or more, 150 μM or more, 200 μM or more, 250 μM or more, 300 μM or more, 400 μM or more, 500 μM or more, or 1 mM or more. Alternatively, the concentration can be defined as an amount sufficient to cause the anti-CD137 antigen-binding molecule or antibody to exhibit maximum binding activity. Furthermore, in one embodiment, the concentration of the small molecule compound "in the presence of a low concentration" can be, for example, below 500 μM, below 250 μM, below 200 μM, below 150 μM, below 100 μM, below 50 μM, below 10 μM, below 1 μM, below 500 nM, below 100 nM, below 50 nM, or below 10 nM or 1 nM. A low concentration embodiment can also be selected where the concentration of the small molecule compound is zero or essentially zero.

[0802] Here, the term "basic concentration of zero" means, for example, that even if small molecule compounds are present, their concentration is so small that they cannot be detected by current technology.

[0803] In one embodiment, in the presence of a small molecule compound at concentrations of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM, the binding activity against CD137 is more than 2, 5, 10, 15, 16, 17, 18, 19, or 20 times greater than the binding activity against CD137 in the absence of the small molecule compound. In another embodiment, in the presence of a small molecule compound at a concentration of 10 μM or higher, the binding activity against CD137 of the anti-CD137 antigen-binding molecule or antibody is more than 2, 5, 10, 15, 16, 17, 18, 19, or 20 times greater than the binding activity against CD137 in the absence of the small molecule compound. In one embodiment, when the small molecule compound is present at a concentration of 100 μM or higher, the binding activity of the anti-CD137 antigen-binding molecule or antibody to CD137 is more than 2, 5, 10, 15, 16, 17, 18, 19, or 20 times that of the binding activity to CD137 in the absence of the small molecule compound.

[0804] In one embodiment, when the small molecule compound is present at a concentration of 10 μM or higher, the binding activity (KD) of the anti-CD137 antigen-binding molecule or antibody to CD137 is 9 x 10⁻⁶. -7 Below M, 8 x 10 -7 Below M, 7 x 10 -7 Below M, 6x 10 -7 Below M, 5 x 10 -7 Below M, or 4 x 10 -7 The dissociation constant (KD) is less than M, or preferably 5 x 10⁻⁶. -7 The dissociation constant (KD) is below M. In another embodiment, in the absence of small molecule compounds, the binding activity (KD) of the anti-CD137 antigen-binding molecule or antibody to CD137 is too large to be calculated by Biacore (weak binding activity), or the dissociation constant (KD) is 1 x 10⁻⁶. -7 M and above, 5 x 10 -7 M and above, 7 x 10 -7 M and above, 8 x 10 -7 M and above, 9 x 10 -7 M and above, 1×10 -6 M and above, 2×10 -6 M and above, 3×10 -6 M or above, or 4×10 -6M or higher, or preferably, the dissociation constant (KD) is 1 x 10⁻⁶. -6 M or higher. In another embodiment, when the small molecule compound is present at a concentration of 100 μM or higher, the binding activity (KD) of the anti-CD137 antigen-binding molecule or antibody to CD137 is 9 x 10⁻⁶. -7 Below M, 8 x 10 -7 Below M, 7 x 10 -7 Below M, 6 x 10 -7 Below M, 5 x 10 -7 Below M, 4 x 10 -7 Below M, 3 x 10 -7 Below M, 2×10 -7 Below M, or 1×10 -7 Below M, or preferably, the dissociation constant (KD) is 2 × 10⁻⁶. -7 Below M. In another embodiment, in the absence of small molecule compounds, the binding activity (KD) of the anti-CD137 antigen-binding molecule or antibody to CD137 is too large to be calculated by Biacore (weak binding activity), or the dissociation constant (KD) is 1 x 10⁻⁶. -7 M and above, 5 x 10 -7 M and above, 7 x 10 -7 M and above, 8 x 10 -7 M and above, 9 x 10 -7 M and above, 1 x 10 -6 M and above, 2 x 10 -6 M and above, 3 x 10 -6 M or larger, or 4 x 10 -6 M or higher, or preferably, the dissociation constant (KD) is 1 x 10⁻⁶. -6 M and above.

[0805] In one embodiment, when the small molecule compound is present at a concentration of 10 μM or higher, the binding activity (KD) of the anti-CD137 antigen-binding molecule or antibody to CD137 is 8 x 10⁻⁶. -8 The dissociation constant (KD) is below M, and the binding activity (KD) to CD137 in the absence of the compound is too large to be calculated by Biacore (weak binding activity). In another embodiment, the binding activity (KD) of the anti-CD137 antigen-binding molecule or antibody to CD137 in the presence of a small molecule compound at 100 μM is 2 x 10⁻⁶. -8The dissociation constant (KD) below M is too large for binding activity to CD137 in the absence of small molecule compounds, making calculations using Biacore impossible (weak binding activity).

[0806] On one hand, this disclosure provides an anti-CD137 antigen-binding molecule or antibody, wherein the value of [binding activity (binding amount) of CD137 in the presence of a low molecular weight compound at 10 μM or higher] / [binding activity (binding amount) of CD137 in the absence of a small molecular weight compound] is equal to or greater than the value of a reference anti-CD137 antigen-binding molecule. On another aspect, this disclosure provides an anti-CD137 antigen-binding molecule or antibody, wherein the value of [binding activity (binding amount) of CD137 in the presence of a low molecular weight compound at 100 μM or higher] / [binding activity (binding amount) of CD137 in the absence of a small molecular weight compound] is equal to or greater than the value of a reference anti-CD137 antigen-binding molecule. In any of the foregoing aspects, the reference anti-CD137 antigen-binding molecule may be selected from anti-CD137 antibodies containing HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, wherein HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 have the same properties as A375 / B167, A372 / B040, and A356 described in Table 37. The amino acid sequences that are identical to HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contained in / B040, A486 / B167, A487 / B167, A488 / B226, A489 / B223, A548 / B376, A551 / B256, A551 / B379, A555 / B379, A548 / B256, or A549 / B167 are all present.

[0807] In one embodiment, the reference anti-CD137 antigen-binding molecule is an antibody comprising the amino acid sequence of A375 / B167, A372 / B040, A356 / B040, A486 / B167, A487 / B167, A488 / B226, A489 / B223, A548 / B376, A551 / B256, A551 / B379, A555 / B379, A548 / B256, or A549 / B167 as a combination of heavy chain variable region / light chain variable region. In various preferred embodiments, the reference antigen-binding molecule is an anti-CD137 antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, wherein HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 have the same amino acid sequence as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contained in A375 / B167. In another embodiment, the reference anti-CD137 antigen-binding molecule is an anti-CD137 antibody comprising A375 / B167 as a combination of heavy chain variable region / light chain variable region. In various preferred embodiments, the reference antigen-binding molecule is an anti-CD137 antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, wherein HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 have the same amino acid sequence as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contained in A551 / B379. In another embodiment, the reference anti-CD137 antigen-binding molecule is an anti-CD137 antibody comprising A551 / B379 as a combination of heavy chain variable region / light chain variable region. In a preferred embodiment, the reference antigen binding molecule comprises human heavy and light chain constant regions (e.g., G1T3 (SEQ ID NO: 138) as the heavy chain constant region and human λ chain Lamlib (SEQ ID NO: 63) as the light chain constant region).

[0808] On one hand, this disclosure provides an anti-CD137 antigen-binding molecule or antibody, wherein, in the absence of a small molecule compound, its binding activity (binding amount) to CD137 is the same as or lower than that of a reference anti-CD137 antigen-binding molecule, and its binding activity (binding amount) to CD137 in the presence of a small molecule compound of 10 μM or higher is equal to or higher than that of the reference anti-CD137 antigen-binding molecule under the same conditions. On another aspect, this disclosure provides an anti-CD137 antigen-binding molecule or antibody, wherein, in the absence of a small molecule compound, its binding activity to CD137 is the same as or lower than that of a reference anti-CD137 antigen-binding molecule, and its binding activity (binding amount) to CD137 in the presence of a small molecule compound of 10 μM or higher is equal to or higher than that of the reference anti-CD137 antigen-binding molecule under the same conditions. In any of the foregoing aspects, the reference anti-CD137 antigen-binding molecule may be selected from anti-CD137 antibodies comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, wherein said HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 have the same properties as A375 / B167, A372 / B040, and A356 described in Table 37. The same amino acid sequences of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contained in / B040, A486 / B167, A487 / B167, A488 / B226, A489 / B223, A548 / B376, A551 / B256, A551 / B379, A555 / B379, A548 / B256, or A549 / B167.

[0809] In one embodiment, the reference anti-CD137 antigen-binding molecule is an anti-CD137 antibody comprising the amino acid sequences of A375 / B167, A372 / B040, A356 / B040, A486 / B167, A487 / B167, A488 / B226, A489 / B223, A548 / B376, A551 / B256, A551 / B379, A555 / B379, A548 / B256, or A549 / B167 as heavy chain variable region / light chain variable region combinations as described in Table 37. In a preferred embodiment, the reference anti-CD137 antigen-binding molecule is an anti-CD137 antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, wherein HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 have the same amino acid sequence as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contained in A375 / B167. In another embodiment, the reference anti-CD137 antigen-binding molecule is an anti-CD137 antibody comprising A375 / B167 as a combination of heavy chain variable region / light chain variable region. In various preferred embodiments, the reference anti-CD137 antigen-binding molecule is an anti-CD137 antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, wherein HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 have the same amino acid sequence as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contained in A551 / B379. In another embodiment, the reference anti-CD137 antigen-binding molecule is an anti-CD137 antibody comprising A551 / B379 as a combination of heavy chain variable region / light chain variable region. In a preferred embodiment, the reference antigen binding molecule comprises a human heavy chain constant region and a light chain constant region (e.g., G1T3 (SEQ ID NO: 138) as the heavy chain constant region and human λ chain Lamlib (SEQ ID NO: 63) as the light chain constant region).

[0810] In one aspect, this disclosure provides an anti-CD137 antigen-binding molecule or antibody, wherein the value of [binding activity (KD) against CD137 in the presence of a low molecular weight compound of 1 μM] / [binding activity (KD) against CD137 in the presence of a low molecular weight compound of 10 μM or more] is equal to or greater than the value of a reference antigen-binding molecule. In another aspect, this disclosure provides an anti-CD137 antigen-binding molecule or antibody, wherein the value of [binding activity (KD) against CD137 in the presence of a low molecular weight compound of 1 μM] / [binding activity (KD) against CD137 in the presence of a low molecular weight compound of 100 μM or more] is equal to or greater than the value of a reference antigen-binding molecule. In any of the foregoing aspects, the reference antigen binding molecule may be selected from antibodies comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, wherein said HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 have the same properties as those listed in Table 37, namely A375 / B167, A372 / B040, A356 / B040, and A. The same amino acid sequence of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contained in 486 / B167, A487 / B167, A488 / B226, A489 / B223, A548 / B376, A551 / B256, A551 / B379, A555 / B379, A548 / B256, or A549 / B167.

[0811] In one embodiment, the reference antigen-binding molecule is an antibody comprising the amino acid sequence of A375 / B167, A372 / B040, A356 / B040, A486 / B167, A487 / B167, A488 / B226, A489 / B223, A548 / B376, A551 / B256, A551 / B379, A555 / B379, A548 / B256, or A549 / B167 as a combination of heavy chain variable region / light chain variable region. In a preferred embodiment, the reference antigen-binding molecule is an antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, wherein the amino acid sequences of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 are identical to the amino acid sequences of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contained in A375 / B167. In another embodiment, the reference antigen-binding molecule is an antibody comprising A375 / B167 as a combination of heavy chain variable region / light chain variable region. In various embodiments, the reference antigen-binding molecule is an antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, wherein the amino acid sequences of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 are identical to the amino acid sequences of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contained in A551 / B379. In another embodiment, the reference antigen-binding molecule is an antibody comprising A551 / B379 as a combination of heavy chain variable region / light chain variable region. In a preferred embodiment, the reference antigen-binding molecule comprises human-derived heavy chain constant region and light chain constant region (e.g., G1T3 (SEQ ID NO: 138) as the heavy chain constant region and human λ chain Lamlib (SEQ ID NO: 63) as the light chain constant region).

[0812] In one embodiment, the binding activity of the anti-CD137 antibody to CD137 in the presence of a small molecule compound, in the absence of a small molecule compound, or at high and / or low concentrations of a small molecule compound is measured, for example, by a ligand capture method using a BIACORE (registered trademark) T200 with surface plasmon resonance spectroscopy as the measurement principle.

[0813] The following describes details of an exemplary method for measuring the binding activity of an anti-CD137 antibody to CD137. In one embodiment, the binding activity of the anti-CD137 antibody to CD137 is assessed using a BIACORE (registered trademark) T200. In a preferred embodiment, the assay uses 20 mM ACES (pH 7.4), 150 mM NaCl, 2 mM MgCl2, and 0.05% Tween 20 as a run buffer and is performed at 37°C. In one embodiment, the measurement is performed after the antibody has been captured as a ligand onto a ligand-capturing molecule. Specifically, an appropriate amount (e.g., about 100 RU, 200 RU, 300 RU, 400 RU, or 500 RU) of antibody is captured by interacting an antibody solution prepared using the run buffer with a chip first prepared by immobilizing Sure Protein A (GE Healthcare) onto an S-Series sensor chip CM3 (GE Healthcare).

[0814] In a preferred embodiment, an antibody of approximately 100 to 500 RU, preferably approximately 250 to 400 RU, was captured. Next, the binding activity to CD137 in the presence and absence of the small molecule compound was evaluated by interacting with a CD137 solution prepared using a run buffer to which the small molecule compound was added to a target concentration (e.g., 1 μM, 10 μM, 50 µM, or 100 µM), or by interacting with a CD137 solution prepared using a run buffer without the small molecule compound. Although the concentration of CD137 in the CD137 solution can be suitably determined, for example, when hCD137-HisBAP (see Reference Examples 1-1) is used as the antigen, measurements were performed using antigen concentrations of 0 nM, 15.625 nM, 62.5 nM, 250 nM, and 1000 nM, respectively. In one embodiment, the dissociation constant (KD) of the anti-CD137 antibody with human CD137 was calculated using Biacore T200 evaluation software 2.0. Specifically, the association rate constant ka (L / mol / s) and the dissociation rate constant kd (1 / s) were calculated by globally fitting the sensor map obtained through measurement using a 1:1 Langmuir combination model. The dissociation constant KD (mol / L) was then calculated from these values.

[0815] Other exemplary assays for measuring the binding activity of anti-CD137 antibodies to CD137 are described in detail below. The binding of anti-CD137 antibodies to human CD137 was assessed using a Biacore T200. Binding to human CD137 was measured using a run buffer of 20 mM ACES (pH 7.4), 150 mM NaCl, 2 mM MgCl2, and 0.05% Tween 20, and was performed at 37°C. First, approximately 250 to 400 RU of antibody was captured by interacting an antibody solution prepared using the run buffer with a chip in which Sure Protein A (GE Healthcare) was immobilized on an S-Series sensor chip CM3 (GE Healthcare). Next, human CD137 solutions prepared using run buffer supplemented with a target concentration (e.g., 1 μM, 10 μM, 50 μM, or 100 μM) of ATP, or human CD137 solutions prepared using run buffer without ATP, were interacted with to assess the binding activity to CD137 in the presence and absence of ATP. hCD137-HisBAP, prepared according to the method in Reference Example (1-1), was used as the human CD137 antigen, and measurements were performed at antigen concentrations of 0 nM, 15.625 nM, 62.5 nM, 250 nM, and 1000 nM. The chip was regenerated using 25 mM NaOH and 10 mM glycine-HCl (pH 1.5), and measurements were performed by repeatedly capturing the antibody. The dissociation constant for each antibody against human CD137 was calculated using Biacore T200 evaluation software 2.0. Specifically, the association rate constant ka (L / mol / s) and the dissociation rate constant kd (1 / s) were calculated by globally fitting the sensor map obtained through measurements using a 1:1 Langmuir binding model. The dissociation constant KD (mol / L) was calculated from these values.

[0816] In one embodiment, the binding activity of the anti-CD137 antibody to CD137 (preferably human CD137) can also be rewritten as "CD137 binding amount per unit volume of antibody". Specifically, the "CD137 binding amount per unit volume of antibody" is calculated by dividing the amount of CD137 bound to the antibody (RU) by the amount of antibody captured using the sensor map obtained by the assay method described above using the BIACORE (registered trademark) T200. In one embodiment, the binding activity of the anti-CD137 antibody to CD137 (preferably human CD137) can also be measured by the method described in Examples 5-3 or 6-2.

[0817] The terms "small molecule" and "small molecule compound" refer to naturally occurring chemical substances other than "biopolymers" present in living organisms, or non-naturally occurring chemical substances. Preferably, they are tissue-specific compounds or non-naturally occurring compounds, but are not limited thereto. In one embodiment, the "small molecule compound" in this disclosure is a "cancer tissue-specific compound" or a "cancer tissue-specific metabolite." The term "cancer tissue-specific compound (cancer tissue-specific compound)" in this disclosure refers to a compound that is present differently in tumor tissue compared to non-tumor tissue. As used herein, the term "cancer" is generally used to describe malignant tumors and can be metastatic or non-metastatic. The term "metabolism" refers to chemical changes that occur within the tissues of an organism, including "assimilation" and "catabolism." Assimilation refers to the biosynthesis or accumulation of molecules, while catabolism refers to the degradation of molecules. A "metabolite" is an intermediate or product produced by the metabolism of a substance.

[0818] The term "target tissue" refers to any living tissue to which the antigen-binding molecules of this disclosure are to be delivered. Target tissue can be histologically distinguishable tissue, such as various organs, or pathologically distinguishable tissue, such as normal tissue and diseased tissue. In some embodiments, the target tissue is tumor tissue. Conversely, "non-target tissue" refers to living tissue other than the target tissue.

[0819] The term "tumor tissue" refers to tissue containing at least one tumor cell. Typically, tumor tissue consists of a mass of tumor cells (parenchyma) that forms the main body of the tumor and connective tissue and blood vessels (stromal) that exist between the tumor cells and support the tumor. In some cases, these are clearly distinguishable, but in others, they are mixed together. In some cases, there are cells such as immune cells that have infiltrated the tumor tissue. Conversely, "non-tumor tissue" refers to tissue in the living body other than tumor tissue. Healthy, disease-free tissue / normal tissue is representative of this type of non-tumor tissue.

[0820] As a non-limiting embodiment of the cancer tissue-specific compound or cancer tissue-specific metabolite used in this disclosure, examples may be suitably selected from at least one of the compounds detailed below. "At least one compound" means, in addition to cases where the binding activity of the same antigen-binding domain to the antigen depends on one type of cancer tissue-specific compound or cancer tissue-specific metabolite, as described below, also includes cases where the binding activity depends on several types of cancer tissue-specific compounds or cancer tissue-specific metabolites.

[0821] As used herein, the term "target tissue-specific compound" refers to a compound that is present in a target tissue differently from non-target tissue. In several embodiments, the target tissue-specific compound may be a compound defined by qualitative target tissue specificity, such as being present in the target tissue but not in the non-target tissue, or being present in the non-target tissue but not in the target tissue. In different embodiments, the target tissue-specific compound may be a compound defined by quantitative target tissue specificity, such as being present in the target tissue at a different concentration than in the non-target tissue (e.g., a higher concentration or a lower concentration). In specific implementation schemes, the concentration of the target tissue-specific compound in the target tissue is, for example, 1.05 times, 1.1 times, 1.15 times, 1.2 times, 1.25 times, 1.3 times, 1.35 times, 1.4 times, 1.45 times, 1.5 times, 1.55 times, 1.6 times, 1.65 times, 1.7 times, 1.75 times, 1.8 times, 1.85 times, 1.9 times, 1.95 times, 2 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 3 times, 5 times, 10 times, 50 times, 100 times, or more than 10 times higher than in non-target tissues. 3 More than 10 times 4 More than 10 times 5 More than 10 times 6 More than 1.05 times higher. In another embodiment, the concentration of the target tissue-specific compound in the target tissue is, for example, more than 1.05 times, more than 1.1 times, more than 1.15 times, more than 1.2 times, more than 1.25 times, more than 1.3 times, more than 1.35 times, more than 1.4 times, more than 1.45 times, more than 1.5 times, more than 1.55 times, more than 1.6 times, more than 1.65 times, more than 1.7 times, more than 1.75 times, more than 1.8 times, more than 1.85 times, more than 1.9 times, more than 1.95 times, more than 2 times, more than 2.1 times, more than 2.2 times, more than 2.3 times, more than 2.4 times, more than 2.5 times, more than 3 times, more than 5 times, more than 10 times, more than 50 times, more than 100 times, more than 10 3 More than 10 times 4 More than 10 times 5 More than 10 times 6 The concentration of the target tissue-specific compound in the target tissue is statistically significantly higher or lower than that in the non-target tissue (i.e., p-value less than 0.05 and / or q-value less than 0.10, as determined by Welch's t-test or Wilcoxon's rank-sum test). In the specific implementation, the target tissue-specific compound is a tumor tissue-specific compound.

[0822] In a specific embodiment, the tumor tissue-specific compound is a metabolite produced by specific metabolism of tumor cells. The metabolite can be a product of metabolism essential for life (major metabolites) or a product of metabolism not essential for life (secondary metabolites). Examples of major metabolites include sugars, proteins, lipids, nucleic acids, etc. Examples of secondary metabolites include antibiotics and dyes. The metabolite can be a biopolymer or a small molecule. In a specific embodiment, a biopolymer is a molecule with a molecular weight of about 5000 or higher, composed of one or more types of repeating units, including, for example, polysaccharides, polypeptides, and polynucleotides. In a specific embodiment, a small molecule is a molecule with a molecular weight of less than about 500 and is a chemical substance present in living organisms. In a further embodiment, the tumor tissue-specific compound is a small molecule metabolite specifically produced in tumor cells (Eva Gottfried, Katrin Peter, and Marina P. Kreutz, Molecular and Modular Cancer Therapy (2010) 3(2), 111-132). In a further embodiment, the tumor tissue-specific compound is a metabolite specifically produced by cells infiltrating the tumor tissue (e.g., immune cells) or by stromal cells present in the tumor tissue (e.g., cancer-associated fibroblasts (CAF)). Examples of immune cells infiltrating the tumor tissue include dendritic cells, suppressor dendritic cells, regulatory T cells, depleted T cells, myeloma-derived suppressor cells (MDSCs), etc. In a further embodiment, metabolites produced by cells present in the tumor tissue (e.g., tumor cells, immune cells, stromal cells, etc.) and released extracellularly when the cells die due to apoptosis or necrosis, etc., may also be included in the tumor tissue-specific compounds of this disclosure.

[0823] To identify tumor tissue-specific compounds, appropriate analysis can be used at the transcriptomic level (e.g., Dhanasekaran et al. (Nature (2001) 41 2, 822-826), Lapointe et al. (Proc. Natl. Acad. Sci. USA (2004) 101, 811-816) or Perou et al. (Nature, (2000) 406, 747-752)), at the proteomic level (e.g., Ahram et al. (Mol. Carcinog. (2002) 33, 9-15) and Hood et al. (Molecular Cell Proteomics (Mol. Cell. Proteomics (2005) 4, 1741-1753)), and metabolomics analysis centered on metabolomics analysis (metabolicomics). That is, in order to identify metabolites in test samples, high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) (Brindle et al. (J. Mol. Recognit. (1997) 10, 182-187), mass spectrometry (GC / MS and LC / MS) (Gates and Sweeley (Clin. Chem. (1978) 24, 1663-1673)) and metabolospectral analysis using ELISA can be used alone and / or in combination.

[0824] In a specific embodiment, the tumor tissue-specific compound is at least one compound selected from the group consisting of: nucleosides having a purine ring structure, amino acids and their metabolites, lipids and their metabolites, major metabolites of carbohydrate metabolism, and nicotinamide and its metabolites. In a further embodiment, the tumor tissue-specific compound is at least one compound selected from the group consisting of (1) to (6):

[0825] (1) Nucleosides with purine structures, such as adenosine (ADO), adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and inosine;

[0826] (2) Amino acids, such as alanine, glutamic acid and aspartic acid;

[0827] (3) Metabolites of amino acids, such as kynurenine, anthranilic acid, 3-hydroxykynurenine and kynurenic acid.

[0828] (4) Metabolites of arachidonic acid, such as prostaglandin E2;

[0829] (5) Major metabolites of the glycolytic pathway or Krebs cycle, such as lactic acid, succinic acid, and citric acid; and,

[0830] (6) Metabolites of nicotinamide, such as 1-methylnicotinamide.

[0831] (1) Nucleosides with purine structures, such as adenosine (ADO), adenosine triphosphate (ATP), and adenosine diphosphate (ADP). adenosine monophosphate (AMP) and inosine

[0832] It is well known that when tumor cells die, a large amount of intracellular ATP leaks out. Therefore, the ATP concentration in tumor tissue is significantly higher than that in normal tissue (PLoS One. (2008) 3, e2599). AMP is metabolized by enzymes on the cell surface, such as extracellular 5'-nucleotidase (eco-5'-nucleotidase) (CD73) (Resta and Thompson (Immunol. Rev., (1998) 161, 95-109) and Sadej et al. (Melanoma Research, 2006) 16, 21 3-222). Adenosine is a purine nucleoside that is constitutively present in low concentrations in the extracellular environment, but significantly increased extracellular adenosine concentrations have been reported in hypoxic tissues found in solid tumors (Blay and Hoskin (Cancer Research, (1997) 57, 260 2-2605)). CD73 is expressed on the surface of tumor and immune cells (Kobie et al., Journal of Immunology, 2006, 17(7), 6780-6786), and has been found in breast cancer (Canbolat et al., Breast Cancer Res. Treat., 1996, 37, 189-193), gastric cancer (Durak et al., Cancer Letters, 1994, 84, 199-202), pancreatic cancer (Flocke and Mannherz, Biochim. Biophys. Acta, 1991, 1076, 273-281) and glioblastoma (Bardot et al., Br. J. Immunol., 2006, 17(7), 6780-6786)). Increased activity of adenosine in tumor tissues (Cancer (1994) 70, 212-218) has been observed. It has been proposed that the accumulation of adenosine in tumor tissues is a result of increased dephosphorylation of AMP by cytoplasmic 5'-nucleotidases (Headrick and Willis (Biochem.J., (1989) 261, 541-550)). Furthermore, regulatory T cells infiltrating tumor tissues also express ATPases and produce adenosine (Proc. Natl. Acad. Sci. USA (2006) 103(35), 13132-13137; Curr. Med. Chem. (2011) 18: 5217-5223. It is believed that the produced adenosine maintains tumor tissue in an immunosuppressive environment via adenosine receptors such as A2A receptors (Curr. Med. Chem. (2011) 18, 5217-5223). Therefore, ATP, ADP, AMP, adenosine, etc., which are believed to accumulate in high concentrations in tumor tissue through the metabolism of purine nucleotides, are examples of tumor tissue-specific compounds used in this disclosure. Furthermore, as adenosine is degraded to inosine by adenosine deaminase, inosine accumulates in high concentrations.

[0833] In specific embodiments, nucleosides having a purine ring structure include compounds containing adenosine. In particular embodiments, compounds containing adenosine include, for example, adenosine (ADO), adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), cyclic adenosine monophosphate (cAMP), deoxyadenosine (dADO), deoxyadenosine triphosphate (dATP), deoxyadenosine diphosphate (dADP), deoxyadenosine monophosphate (dAMP), and [γ-thio]adenosine triphosphate (ATPγS). In another embodiment, nucleosides having a purine ring structure include inosine, which is a metabolite of adenosine.

[0834] Furthermore, in specific implementations, the nucleosides having a purine ring structure include commercially available nucleosides having a purine ring structure, such as ADPbetaS (Sigma).

[0835] (2) Amino acids, such as alanine, glutamic acid, aspartic acid, etc.

[0836] In tumor cells, the uptake rate of glutamine, a nitrogen carrier in vivo, is increased, and the incorporation of glutamine and its subsequent conversion into glutamate and lactate (glutamine degradation) is considered a characteristic of tumor cells (Mazurek and Eigenbrodt, *Anticancer Res.*, (2003) 23, 1149-1154; and Mazurek et al., *Journal of Cell Physiology*, (1999) 181, 136-146). Plasma glutamine levels are decreased in cancer patients, while glutamate concentrations are increased (Droge et al., *Immunobiology*, (1987) 174, 473-479), and glutamine levels are also increased in lung cancer tissues. 13 Studies on C-labeled glucose metabolism 13 C-labeled succinic acid, 13 C-labeled alanine, 13 C-labeled glutamic acid and 13 There is a correlation between the concentrations of C-labeled citrates. For these reasons, alanine, glutamic acid, aspartic acid, etc., which are considered to accumulate in high concentrations in tumor tissues, for example due to glutamine degradation, are examples of tumor tissue-specific compounds used in this disclosure.

[0837] (3) Metabolites of amino acids, such as kynurenine, anthranilic acid, 3-hydroxykynurenine and kynuric acid.

[0838] Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-metabolizing enzyme that is highly expressed in many cancers such as melanoma, colon cancer, and kidney cancer (Uyttenhove et al. (Nat. Med. (2003) 9, 1269-1274). IDO catalyzes the conversion of tryptophan to kynurenine. In gliomas that do not express IDO, kynurenine is produced from tryptophan by tryptophan 2,3-dioxygenase (TDO) in the liver (Opitz et al. (Nature (2011) 478 (7368), 197-203)). IDO is also expressed on dendritic cells infiltrating tumor tissue, and these dendritic cells also produce kynurenine (J. Immunol., (2008) 181, 5396-5404). Furthermore, IDO is also expressed in myeloid-derived suppressor cells (MDSCs) of tumor tissue, and these MDSCs also produce kynurenine (Yu et al., J. Immunol., (2013) 190, 3783-3797). Kynurenase converts kynurenine to anthranilic acid, and kynurenine 3-hydroxylase converts kynurenine to 3-hydroxykynurenine. Both anthranilic acid and 3-hydroxykynurenine are converted to 3-hydroxyanthranilic acid, a precursor of NAD. Kynurenine transaminase converts kynurenine to kynurenic acid. For these reasons, kynurenine and its metabolites, namely anthranilic acid, 3-hydroxykynurenine, and kynurenic acid, are examples of tumor tissue-specific compounds, particularly tumor cell-specific metabolites, used in this disclosure.

[0839] (4) Metabolites of arachidonic acid, such as prostaglandin E2

[0840] Prostaglandin E2 (PGE2) promotes the growth of colon cancer cells and inhibits their apoptosis (Sheng et al., Cancer Res., 1998, 58, 362-366). Among PGE2 synthases, COX-1 is found to be constitutively expressed in almost all tissues, while COX-2 is induced by certain inflammatory cytokines and oncogenes in tumors (Warner and Mitchell, FASEB J., 2004, 18, 790-804). Overexpression of COX-2 has been reported to be associated with poor prognosis in breast cancer (Denkert et al., Clin. Breast Cancer, 2004, 4, 428-433) and rapid disease progression in ovarian cancer (Denker et al., Mod. Pathol., 2006, 19, 1261-1269). In addition, regulatory T cells infiltrating tumor tissue also produce PGE2. 2 (Curr. Med. Chem. (2011) 18, 5217-5223). For these reasons, arachidonic acid metabolites such as PGE2 are examples of tumor tissue-specific compounds, particularly tumor cell-specific metabolites or immune cell-specific metabolites infiltrating tumor tissues. In addition to PGE2, the production of thromboxane A2 (TXA2) is enhanced in tumor tissues such as colon cancer (J. Lab. Clin. Med. (1993) 122, 518-523).

[0841] (5) Major metabolites of the glycolytic pathway or Krebs cycle, such as lactic acid, succinic acid and citric acid.

[0842] A glycolytic phenotype characterized by the upregulation of glycolytic enzymes (Embden-Meyerhof pathway) such as pyruvate kinase, hexokinase, and lactate dehydrogenase (LDH) is commonly referred to as the Warburg effect, a characteristic feature of solid tumors. Lactate, the end product of glycolysis, as well as succinate and citrate produced by the Krebs cycle, are known to accumulate in tumor tissues (Teresa et al. (Molecular Cancer (2009) 8, 41-59)). For these reasons, the major metabolites produced by glycolysis, such as lactate, succinate, and citrate, are examples of tumor tissue-specific compounds, particularly tumor cell-specific metabolites, used in this disclosure. Furthermore, it is known that succinate, present in high concentrations in cells, leaks out of cells due to cell death (Nature Immunology (2008) 9, 1261-1269). This is thought to be the reason for the increased succinate concentration in tumor tissues where cell death occurs frequently.

[0843] (6) Metabolites of nicotinamide, such as 1-methylnicotinamide

[0844] Nicotinamide N-methyltransferase is known to be highly expressed in tumor tissues of many individuals. It is also known that 1-methylnicotinamide (a stable metabolite of nicotinamide produced by this enzyme) is secreted outside tumor cells (Yamada et al. (J. Nutr. Sci. Vitaminol. (2010) 56, 83-86)). For this reason, 1-methylnicotinamide and the like, which are thought to accumulate in high concentrations in tumor tissues due to the metabolism of nicotinamide, are examples of tumor tissue-specific compounds used in this disclosure.

[0845] The "antigen-binding molecule" disclosed herein comprises an "antigen-binding domain". As the "antigen-binding domain", any structural domain can be used, as long as it binds to the target antigen. In one embodiment, the antigen-binding domain of this disclosure includes, for example, a variable region of an antibody heavy chain and / or light chain, an Avimer (International Publications WO2004 / 044011 and WO2005 / 040229) comprising a module (A domain) of about 35 amino acids contained in various cell membrane proteins of organisms, an Adnectin (International Publication WO2002 / 032925) comprising the 10Fn3 domain of fibronectin, a glycoprotein expressed on the cell membrane, using protein A. The disclosure includes Affibodies (WO1995 / 001937) with a 58-amino acid IgG-binding domain as a scaffold, DARPins (designed ankyrin repeat proteins) based on ankyrin repeat sequences (AR) with a 33-amino acid repeat sequence (International Publication WO2002 / 020565), Anticalin (International Publication WO2003 / 029462) based on lipid carrier proteins (e.g., neutrophil gelatinase-associated lipid carrier protein (NGAL)), and variable lymphocyte receptors (VLRs), which are proteins that function in the adaptive immune systems of jawless vertebrates such as Japanese lampreys and hagfish, and modules (LRRs) containing leucine-rich repeat sequences (International Publication WO2008 / 016854), etc. In specific embodiments, the antigen-binding domain of this disclosure comprises variable regions of the heavy and light chains of the antibody. In further embodiments, the antigen-binding domain of this disclosure includes, for example, scFv (single-chain Fv), single-chain antibody, Fv, scFv2 (single-chain Fv2), Fab, or F(ab')2.

[0846] [HVR and Variable Area]

[0847] On the one hand, this disclosure provides an anti-CD137 antigen-binding molecule or antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, comprising any one amino acid sequence selected from SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, and 16; and (c) HVR-H3, comprising an amino acid sequence selected from any one of SEQ ID NO: 17, 18, 19, or 20. In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises: (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, which comprises the amino acid sequence selected from any one of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15 and 16; and (c) HVR-H3, which comprises the amino acid sequence selected from any one of SEQ ID NO: 17, 18, 19 or 20.

[0848] In one embodiment, the anti-CD137 antigen-binding molecule is an antibody comprising the amino acid sequence of A375 / B167, A372 / B040, A356 / B040, A486 / B167, A487 / B167, A488 / B226, A489 / B223, A548 / B376, A551 / B256, A551 / B379, A555 / B379, A548 / B256, or A549 / B167 as a combination of heavy chain variable region / light chain variable region. In a preferred embodiment, the antigen-binding molecule is an anti-CD137 antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, wherein HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 have the same amino acid sequence as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 included in A375 / B167. In a further embodiment, the anti-CD137 antigen-binding molecule is an anti-CD137 antibody comprising A375 / B167 as a combination of heavy chain variable region / light chain variable region. In various preferred embodiments, the anti-CD137 antigen-binding molecule is an anti-CD137 antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, wherein HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 have the same amino acid sequence as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 included in A551 / B379. In a further embodiment, the anti-CD137 antigen-binding molecule is an anti-CD137 antibody comprising A551 / B379 as a combination of heavy chain variable region / light chain variable region.

[0849] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises: (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO: 8; and (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 17.

[0850] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises: (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO: 9; and (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 17.

[0851] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises: (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO: 10; and (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 17.

[0852] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises: (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO: 11; and (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 18.

[0853] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises: (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO: 8; and (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 18.

[0854] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises: (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO: 12; and (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 18.

[0855] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises: (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO: 13; and (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 18.

[0856] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises: (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO: 14; and (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 19.

[0857] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises: (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO: 15; and (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 20.

[0858] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises: (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO: 16; and (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 20.

[0859] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises: (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO: 14; and (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 17.

[0860] In various aspects, this disclosure provides an anti-CD137 antigen-binding molecule or antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1, comprising an amino acid sequence selected from any one of SEQ ID NO: 21, 22, 23, 24, and 25; (b) HVR-L2, comprising an amino acid sequence selected from SEQ ID NO: 26; and (c) HVR-L3, comprising an amino acid sequence selected from any one of SEQ ID NO: 27, 28, and 29. In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises (a) HVR-L1, comprising an amino acid sequence selected from any one of SEQ ID NO: 21, 22, 23, 24, and 25; (b) HVR-L2, comprising an amino acid sequence selected from SEQ ID NO: 26; and (c) HVR-L3, comprising an amino acid sequence selected from any one of SEQ ID NO: 27, 28, and 29.

[0861] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises: (a) HVR-L1, which comprises the amino acid sequence of SEQ ID NO: 21; (b) HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3, which comprises the amino acid sequence of SEQ ID NO: 27.

[0862] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises: (a) HVR-L1, which comprises the amino acid sequence of SEQ ID NO: 22; (b) HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3, which comprises the amino acid sequence of SEQ ID NO: 27.

[0863] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises: (a) HVR-L1, which comprises the amino acid sequence of SEQ ID NO: 21; (b) HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3, which comprises the amino acid sequence of SEQ ID NO: 28.

[0864] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises: (a) HVR-L1, which comprises the amino acid sequence of SEQ ID NO: 21; (b) HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3, which comprises the amino acid sequence of SEQ ID NO: 29.

[0865] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises: (a) HVR-L1, which comprises the amino acid sequence of SEQ ID NO: 23; (b) HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3, which comprises the amino acid sequence of SEQ ID NO: 27.

[0866] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises: (a) HVR-L1, which comprises the amino acid sequence of SEQ ID NO: 24; (b) HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3, which comprises the amino acid sequence of SEQ ID NO: 27.

[0867] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises: (a) HVR-L1, which comprises the amino acid sequence of SEQ ID NO: 25; (b) HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3, which comprises the amino acid sequence of SEQ ID NO: 27.

[0868] On the other hand, the anti-CD137 antigen-binding molecule or antibody of this disclosure comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i), (ii), and (iii) below: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (ii) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, and 16; and (iii) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NO: 17, 18, 19, or 20; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i), (ii), and (iii) below: (i) HVR-L1 comprising the amino acid sequence of any one of SEQ ID NO: 21, 22, 23, 24, and 25; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 7; (iii) HVR-H1 comprising the amino acid sequence of any one of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, and 16; and (iii) HVR-H3 comprising the amino acid sequence of any one of SEQ ID NO: 7; and (iv) HVR-H2 comprising the amino acid sequence of any one of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, and 16; and (iii) HVR-H3 ... (iii) HVR-L3, which contains an amino acid sequence selected from any one of SEQ ID NO: 27, 28 and 29.

[0869] In another aspect, this disclosure provides an anti-CD137 antigen-binding molecule or antibody comprising: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 8; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 17; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 21; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 27.

[0870] In another aspect, this disclosure provides an anti-CD137 antigen-binding molecule or antibody comprising: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 9; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 17; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 22; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 27.

[0871] In another aspect, this disclosure provides an anti-CD137 antigen-binding molecule or antibody comprising: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 10; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 17; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 22; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 27.

[0872] In another aspect, this disclosure provides an anti-CD137 antigen-binding molecule or antibody comprising: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 11; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 18; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 21; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 27.

[0873] In another aspect, this disclosure provides an anti-CD137 antigen-binding molecule or antibody comprising: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 8; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 18; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 21; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 27.

[0874] In another aspect, this disclosure provides an anti-CD137 antigen-binding molecule or antibody comprising: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 12; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 18; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 21; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 28.

[0875] In another aspect, this disclosure provides an anti-CD137 antigen-binding molecule or antibody comprising: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 13; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 18; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 21; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 29.

[0876] In another aspect, this disclosure provides an anti-CD137 antigen-binding molecule or antibody comprising: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 14; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 19; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 27.

[0877] In another aspect, this disclosure provides an anti-CD137 antigen-binding molecule or antibody comprising: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 15; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 20; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 24; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 27.

[0878] In another aspect, this disclosure provides an anti-CD137 antigen-binding molecule or antibody comprising: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 15; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 20; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 25; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 27.

[0879] In another aspect, this disclosure provides an anti-CD137 antigen-binding molecule or antibody comprising: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 16; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 20; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 25; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 27.

[0880] In another aspect, this disclosure provides an anti-CD137 antigen-binding molecule or antibody comprising: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 14; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 19; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 24; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 27.

[0881] In another aspect, this disclosure provides an anti-CD137 antigen-binding molecule or antibody comprising: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 14; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 17; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 21; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 27.

[0882] In the specific implementation scheme, any one or more amino acids of the above-mentioned anti-CD137 antibody are substituted at the following HVR positions:

[0883] In HVR-H2 (SEQ ID NO: 30): positions 5, 6, 7, 10, 13, 14 and / or 17;

[0884] In HVR-H3 (SEQ ID NO: 31): positions 3 and / or 6;

[0885] In HVR-L1 (SEQ ID NO: 32): positions 4, 5, 9 and / or 11;

[0886] In HVR-L3 (SEQ ID NO: 33): positions 6, 7 and / or 8.

[0887] In specific implementation schemes, the substitutions provided in this specification are conservative substitutions. In specific implementation schemes, any one or more of the following substitutions can be combined arbitrarily:

[0888] In HVR-H2 (SEQ ID NO: 8): K5H or S; S6G; T7S; E10Y; D13E; S14Q; V17G or L;

[0889] In HVR-H3 (SEQ ID NO: 17): A3P, K, or I; F6E;

[0890] In HVR-L1 (SEQ ID NO: 21): R4S; Y5T; Y9F; E11N;

[0891] In HVR-L3 (SEQ ID NO: 27): E6P; H7A; Q8I

[0892] For HVR-H2, HVR-H3, HVR-L1, and HVR-L3, all possible combinations of the above substitutions are contained in the common sequences of SEQ ID NO: 30, 31, 32, and 33, respectively.

[0893] In any of the above embodiments, the anti-CD137 antigen-binding molecule or antibody is humanized. In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises any HVR as described in the above embodiments and further comprises a recipient human framework, such as a human immunoglobulin framework or a human common framework. In another embodiment, the anti-CD137 antigen-binding molecule or antibody comprises any HVR as described in the above embodiments and further comprises a heavy chain variable region (VH) or a light chain variable region (VL) containing a framework (FR) sequence. In one embodiment, FR1 in the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 35, FR2 in the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 36, FR3 in the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 37, and FR4 in the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 38. In one embodiment, FR1 of the light chain variable region contains the amino acid sequence of SEQ ID NO: 39, FR2 of the light chain variable region contains the amino acid sequence of SEQ ID NO: 40, FR3 of the light chain variable region contains the amino acid sequence of SEQ ID NO: 41, and FR4 of the light chain variable region contains the amino acid sequence of SEQ ID NO: 42.

[0894] On the other hand, the anti-CD137 antigen-binding molecule or antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53. In some embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence; however, the anti-CD137 antigen-binding molecule or antibody comprising this sequence retains its ability to bind to CD137. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53. In some embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the anti-CD137 antibody comprises the VH sequence in SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53, including post-translational modifications of that sequence. In a particular embodiment, VH comprises one, two, or three HVRs selected from the following: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2, comprising the amino acid sequence of any one of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, and 16; and (c) HVR-H3, comprising the amino acid sequence of any one of SEQ ID NO: 17, 18, 19, or 20. Post-translational modifications include, but are not limited to, modifying the N-terminus of the heavy or light chain with pyroglutamic acid via pyroglutamylation.

[0895] On the other hand, an anti-CD137 antigen-binding molecule or antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 54, 55, 56, 57, 58, 59, or 60. In some embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the anti-CD137 antigen-binding molecule or antibody comprising this sequence retains its ability to bind to CD137. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 54, 55, 56, 57, 58, 59, or 60. In some embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the anti-CD137 antigen-binding molecule or antibody comprises the VL sequence of SEQ ID NO: 54, 55, 56, 57, 58, 59, or 60, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1, which comprises any one amino acid sequence selected from SEQ ID NO: 21, 22, 23, 24, and 25; (b) HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3, which comprises any one amino acid sequence selected from SEQ ID NO: 27, 28, and 29. Post-translational modifications include, but are not limited to, modifying the N-terminus of the heavy or light chain glutamine or glutamic acid to pyroglutamic acid via pyroglutamylation.

[0896] On the other hand, an anti-CD137 antigen-binding molecule or antibody is provided, wherein the antigen-binding molecule or antibody comprises VH as in any of the embodiments provided above and VL as in any of the embodiments provided above.

[0897] - In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises the VH and VL sequences in SEQ ID NO: 43 and SEQ ID NO: 54, respectively, including post-translational modifications of those sequences.

[0898] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises the VH and VL sequences in SEQ ID NO: 44 and SEQ ID NO: 55, respectively, including post-translational modifications of those sequences.

[0899] - In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises the VH and VL sequences in SEQ ID NO: 45 and SEQ ID NO: 55, respectively, including post-translational modifications of those sequences.

[0900] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises the VH and VL sequences in SEQ ID NO: 46 and SEQ ID NO: 54, respectively, including post-translational modifications of those sequences.

[0901] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises the VH and VL sequences in SEQ ID NO: 47 and SEQ ID NO: 54, respectively, including post-translational modifications of those sequences.

[0902] - In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises the VH and VL sequences in SEQ ID NO: 48 and SEQ ID NO: 56, respectively, including post-translational modifications of those sequences.

[0903] - In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises the VH and VL sequences in SEQ ID NO: 49 and SEQ ID NO: 57, respectively, including post-translational modifications of those sequences.

[0904] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises the VH and VL sequences in SEQ ID NO: 50 and SEQ ID NO: 58, respectively, including post-translational modifications of these sequences.

[0905] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises the VH and VL sequences in SEQ ID NO: 51 and SEQ ID NO: 59, respectively, including post-translational modifications of those sequences.

[0906] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises the VH and VL sequences in SEQ ID NO: 51 and SEQ ID NO: 60, respectively, including post-translational modifications of those sequences.

[0907] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises the VH and VL sequences in SEQ ID NO: 52 and SEQ ID NO: 60, respectively, including post-translational modifications of those sequences.

[0908] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises the VH and VL sequences in SEQ ID NO: 50 and SEQ ID NO: 59, respectively, including post-translational modifications of those sequences.

[0909] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises the VH and VL sequences in SEQ ID NO: 53 and SEQ ID NO: 54, respectively, including post-translational modifications of those sequences.

[0910] The above-mentioned post-translational modifications include, but are not limited to, modifying the N-terminus of the heavy or light chain glutamine or glutamic acid to pyroglutamic acid by pyroglutamylation.

[0911] For each anti-CD137 antigen-binding molecule or antibody of this disclosure, the amino acid sequences corresponding to the preferred heavy chain variable region and light chain variable region, and their HVR1, HVR2 and HVR3, are shown in the table below.

[0912] [Table 1]

[0913]

[0914] When the anti-CD137 antigen-binding molecule or antibody provided herein has glutamine as the N-terminal amino acid of the heavy or light chain, this amino acid can be replaced by glutamate. When the anti-CD137 antibody provided herein has glutamate as the N-terminal amino acid of the heavy or light chain, this amino acid can be replaced by glutamine.

[0915] In a preferred embodiment, the anti-CD137 antigen-binding molecule or antibody containing the above-mentioned HVR, heavy chain variable region and / or light chain variable region has the low molecular weight compound-dependent binding activity to CD137 as described above.

[0916] [Constant Region]

[0917] On the other hand, the anti-CD137 antigen-binding molecule or antibody includes a constant region. The constant region can be a heavy chain constant region (including the Fc region), a light chain constant region, or both. On the other hand, the anti-CD137 antigen-binding molecule or antibody includes an Fc region. In some embodiments, the constant region is a constant region having a native sequence. Examples of heavy chain constant regions derived from natural antibodies include, for example, the heavy chain constant regions of human IgG1 (SEQ ID NO: 61, 62), human IgG2, human IgG3, human IgG4, etc. Examples of light chain constant regions derived from natural antibodies include, for example, the human κ chain, the human λ chain (e.g., SEQ ID NO: 63), etc.

[0918] As used herein, the term "parental constant region" or "parental Fc region" refers to the constant region or Fc region prior to the introduction of the amino acid alterations described herein. A "parental antigen-binding molecule" is an antigen-binding molecule containing the parental constant region or parental Fc region. In some embodiments, the parental Fc region is an Fc region having a natural sequence (or the Fc region of a natural antibody). Antibodies include, for example, IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc. Antibodies can be derived from humans or monkeys (e.g., cynomolgus monkeys, rhesus monkeys, marmosets, chimpanzees, or baboons). Natural antibodies can also include naturally occurring mutations. NIH Publication No. 91-3242, "Protein Sequences of Immunological Significance," describes several IgG allotype sequences arising from genetic polymorphism, and any of these can be used in this disclosure. In one embodiment, the parental Fc region is an Fc region derived from the heavy chain constant region of human IgG1, as shown in SEQ ID NO: 61, 62 or 182.

[0919] On the one hand, anti-CD137 antigen-binding molecules or antibodies have an increased isoelectric point (pI) compared to anti-CD137 antigen-binding molecules or antibodies containing a native or parental Fc region. In some embodiments, the variant Fc region contains at least one amino acid alteration. In a further embodiment, the amino acid alteration results in an increased isoelectric point (pI) of the variant Fc region compared to the parental Fc region. Without being bound by any particular theory, it is believed that the pH of biological fluids (e.g., plasma) is within a neutral pH range. In biological fluids, the net positive charge of antigen-binding molecules or antibodies with increased pI increases due to the increased pI, resulting in a more robust physicochemical coulombic interaction that attracts the antigen-binding molecules or antibodies to the surface of endothelial cells, where the endothelial cell surface has a net negative charge compared to antigen-binding molecules or antibodies without increased pI. Thus, agonistic antigen-binding molecules (or antibodies) or agonistic antigen-binding molecules (or antibodies) that bind antigens can approach the cell surface expressing Fc-γ receptors more closely, leading to increased binding of antigen-binding molecules or antibodies to Fc-γ receptor-expressing cells. For anti-CD137 agonist antigen-binding molecules or antibodies that exhibit CD137 agonist activity based on their contribution to Fc-γ receptor binding activity, those that increase binding to Fc-γ receptor-expressing cells due to amino acid changes that increase pI can exhibit stronger CD137 agonist activity compared to those that do not have amino acid changes that increase pI.

[0920] In this disclosure, pI can be a theoretically or experimentally determined pI. The value of pI can be determined, for example, by isoelectric focusing known to those skilled in the art. The theoretical pI value can be calculated using, for example, gene and amino acid sequence analysis software (Genetyx, etc.). In the calculation, the properties of the antibody can be reflected in the calculation formula. For example, (i) generally, conserved Cys in the antibody form disulfide bonds and do not carry side chain charges; therefore, such Cys can be excluded from the calculation, and only free forms of Cys that do not form disulfide bonds can be included in the calculation. Alternatively, (ii) the charge state or isoelectric point of the antibody may change due to post-translational modifications; therefore, taking into account such post-translational modifications, the calculation formula can be modified as follows: (a) when the N-terminus of the heavy chain is Q (glutamine), pyroglutamylation is assumed, and the N-terminal amino group is not included in the calculation; (b) when the C-terminus of the heavy chain is K (lysine), truncation is assumed, and K (only one residue) is excluded from the calculation; and (c) assuming all these Cs form disulfide bonds intramolecularly, side chains of all Cs (cysteine) present in normally conserved positions are excluded from the calculation. In a preferred embodiment, both (i) and (ii) above can be reflected in the calculation formula.

[0921] In one implementation, the pI value can be increased by, for example, at least 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, or 0.5, at least 0.6, 0.7, 0.8, or 0.9, at least 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5, or at least 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, or 3.0, compared to the original value.

[0922] In one embodiment, the amino acid alterations associated with pI increase and the methods for increasing the pI of an antigen-binding molecule or antibody are described in detail in Section III. Compositions and Methods (Comprising Agonistant Antigen-Binding Molecules with Variant Fc Regions Having Increased Isoelectric Points (pI)). Those skilled in the art will understand that any amino acid alterations and methods for increasing pI described in Section III. Compositions and Methods (Comprising Agonistant Antigen-Binding Molecules with Variant Fc Regions Having Increased Isoelectric Points (pI)) can be applied to anti-CD137 antigen-binding molecules or antibodies.

[0923] In one embodiment, the anti-CD137 antigen-binding molecule or antibody has a variant Fc region with increased pI, and the variant Fc region contains at least one amino acid change at at least one position selected from the group consisting of: positions 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422, and 431 according to EU numbers. In a further embodiment, the variant Fc region with increased pI contains Arg or Lys at each selected position.

[0924] In a further embodiment, the anti-CD137 antigen-binding molecule or antibody has a variant Fc region with increased pI, and the variant Fc region contains at least one amino acid change at at least one position selected from the group consisting of positions 311, 343, and 413 according to EU numbers. In a further embodiment, the variant Fc region with increased pI includes an amino acid change at position 311, 343, or 413 according to EU numbers. In a further embodiment, the variant Fc region with increased pI contains Arg or Lys at each selected position.

[0925] On the other hand, this disclosure provides an anti-CD137 antigen-binding molecule or antibody comprising a variant Fc region having an increased pI, said variant Fc region comprising an amino acid change of any one of the following (1) to (3): (1) at positions 311 and 343 according to EU designations; (2) at positions 311 and 413; and (3) at positions 343 and 413. In a further embodiment, the variant Fc region having an increased pI comprises Arg or Lys at each selected position.

[0926] In one embodiment, the anti-CD137 antigen-binding molecule or antibody of this disclosure comprises a variant Fc region containing amino acid alterations identified in Table 2 below.

[0927] Amino acid alterations used to increase pI in the Fc region

[0928] [Table 2]

[0929]

[0930] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises a variant Fc region prepared by altering the amino acid composition of an Fc region having a native sequence. In one embodiment, the variant Fc region exhibits increased binding activity to at least one Fc-γ receptor selected from the group consisting of Fc-γRIa, Fc-γRIIa, Fc-γRIIb, Fc-γRIIIa, and Fc-γRIIIb, compared to an Fc region having a native sequence or a parental Fc region. Preferably, the variant Fc region exhibits increased binding activity to Fc-γRIIb compared to an Fc region having a native sequence or a parental Fc region. It has been reported that anti-CD137 antibodies comprising a variant Fc region exhibiting increased binding activity to Fc-γRIIb have increased agonistic activity compared to anti-CD137 antibodies comprising an Fc region having a native sequence. In one embodiment, as an amino acid change to increase the binding activity to Fc-γRIIb, for example, the amino acid changes taught in WO2012 / 115241, WO2014 / 030728, WO2014 / 163101 and / or WO2017 / 104783 can be used. In a preferred embodiment, the change to increase the binding activity to Fc-γRIIb is an amino acid change at at least one position selected from the group consisting of: positions 234, 235, 236, 237, 238, 264, 268, 295, 326 and 330 according to EU numbers.

[0931] "Fcγ receptor" (referred to as Fcγ receptor, FcγR or FcgR in this document) refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3 and IgG4 monoclonal antibodies, and in particular any member of the protein family encoded by the Fcγ receptor gene. In humans, this family includes FcγRI (CD64), which includes allotypes FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes allotypes FcγRIIa (including allotypes H131 (H type) and R131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) and FcγRIIc; and FcγRIII (CD16), which includes allotypes FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any FcγR, and any undiscovered FcγR allotypes or allotypes, but not limited to these. FcγRIIb1 and FcγRIIb2 are reported splice variants of human FcγRIIb. Additionally, a splice variant named FcγRIIb3 has been reported (J Exp Med, 1989, 170: 1369-1385). Besides these splice variants, human FcγRIIb includes AAI46679.1 registered in the NCBI and all splice variants registered in the NCBI, namely NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1, and NP_003992.3. In addition, human FcγRIIb includes each previously reported genetic polymorphism, as well as FcγRIIb (Arthritis Rheum. 48:3242-3252 (2003); Kono et al., Hum.Mol.Genet. 14:2881-2892 (2005); and Kyogoju et al., Arthritis Rheum. 46:1242-1254 (2002)), and each genetic polymorphism to be reported in the future.

[0932] In FcγRIIa, there are two allotypes: one with histidine at position 131 (H type), and the other with arginine replaced at position 131 (R type) (Warrmerdam, J. Exp. Med. 172: 19-25 (1990)).

[0933] FcγRs include, but are not limited to, human, mouse, rat, rabbit, and monkey-derived FcγRs, and can be derived from any organism. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any mouse FcγR or FcγR isotype.

[0934] On the other hand, this disclosure provides an anti-CD137 antigen-binding molecule or antibody comprising a variant Fc region having increased binding activity to Fc-γRIIb, said variant Fc region comprising any of the following amino acid changes (1) to (8): (1) at positions 234, 238, 264 and 330 according to EU numbers; (2) at positions 234, 238 and 330; (3) at positions 234, 237, 238 and 330; (4) at positions 236, 268 and 330; (5) at positions 235, 236, 268, 295, 326 and 330.

[0935] In one embodiment, the anti-CD137 antigen-binding molecule or antibody of the present invention comprises a variant Fc region comprising the amino acid alterations identified in Table 3 below. In another embodiment, the anti-CD137 antigen-binding molecule or antibody of the present disclosure comprises a variant Fc region comprising, in addition to the amino acid alterations described in Table 2 (amino acid alterations involved in increasing the pI of the Fc region), any combination of amino acid alterations identified in Table 3 below.

[0936] Amino acid alterations used to increase Fc-γRIIb binding activity in the Fc region

[0937] [Table 3]

[0938]

[0939] In one embodiment, this disclosure provides variant Fc regions comprising those having at least one amino acid alteration and having binding activity to Fc-γRIIb equal to or greater than that of a reference Fc region. In one embodiment, the reference Fc region is an Fc region comprising any combination of the amino acid alterations identified in Table 3 above. In a preferred embodiment, the reference Fc region is an Fc region contained in the heavy chain constant region TT14 (SEQ ID NO: 149), TT16 (SEQ ID NO: 150), MY201 (SEQ ID NO: 153), or MY518 (SEQ ID NO: 154). In a preferred embodiment, the reference Fc region is an Fc region contained in the heavy chain constant region MY201 (SEQ ID NO: 153) or MY518 (SEQ ID NO: 154).

[0940] On the other hand, this disclosure provides isolated agonist antigen-binding molecules or antibodies comprising a variant Fc region having increased binding activity to the Fc-γ receptor (preferably Fc-γRIIb) and an increased pI. In one embodiment, the variant Fc region described herein comprises at least two amino acid alterations in the parental Fc region. As stated above, antigen-binding molecules or antibodies with increased pI are more strongly attracted to the surface of endothelial cells with a net negative charge by physicochemical coulombic interactions compared to antigen-binding molecules or antibodies without increased pI. Therefore, for agonist antigen-binding molecules or antibodies that exhibit agonist activity based on their contribution to the binding activity to the Fc-γ receptor (preferably Fc-γRIIb), the agonist activity of the antigen-binding molecule or antibody can be enhanced by combining amino acid alterations that increase the Fc-γ receptor (preferably Fc-γRIIb) or amino acid alterations that increase the pI.

[0941] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises a variant Fc region containing both an amino acid alteration that increases binding activity to the Fc-γ receptor (e.g., Fc-γRIIb) and an amino acid alteration that increases the isoelectric point (pI), as described above. As mentioned above, antigen-binding molecules or antibodies with increased pI are more strongly attracted to the surface of endothelial cells with a net negative charge via physicochemical coulombic interactions compared to antigen-binding molecules or antibodies without increased pI. Therefore, for anti-CD137 agonist antigen-binding molecules or antibodies that exhibit CD137 agonist activity based on their contribution to binding activity to the Fc-γ receptor (preferably Fc-γRIIb), the agonist activity of the anti-CD137 antigen-binding molecule or antibody can be increased by combining the amino acid alteration that increases the Fc-γ receptor (preferably Fc-γRIIb) and the amino acid alteration that increases the pI.

[0942] On one hand, this disclosure provides a polypeptide comprising a variant Fc region having increased binding activity to Fc-γRIIb and having an increased pI, the variant Fc region comprising at least three amino acid alterations, the amino acid alterations comprising (a) at least one amino acid alteration at at least one position selected from the group consisting of positions 234, 235, 236, 237, 238, 264, 268, 295, 326 and 330 according to EU numbers, and (b) at least two amino acid alterations at at least two positions selected from the group consisting of positions 311, 343 and 413 according to EU numbers.

[0943] In another aspect, this disclosure provides a polypeptide comprising a variant Fc region having increased binding activity to Fc-γRIIb and increased pI, comprising any of the following amino acid alterations (1) to (26): according to EU number

[0944] (1) Positions 235, 236, 268, 295, 326, 330, 343 and 413;

[0945] (2) Positions 214, 235, 236, 268, 295, 326, 330, 343 and 413;

[0946] (3) Positions 234, 238, 250, 264, 307, 330, 343 and 413;

[0947] (4) Positions 234, 238, 264, 330, 343 and 413;

[0948] (5) Positions 234, 237, 238, 250, 307, 330, 343 and 413;

[0949] (6) Positions 234, 237, 238, 330, 343 and 413;

[0950] (7) Positions 235, 236, 268, 295, 326, 330, 311 and 343;

[0951] (8) Positions 234, 238, 250, 264, 307, 330, 311 and 343;

[0952] (9) Positions 234, 238, 264, 330, 311 and 343;

[0953] (10) Positions 234, 237, 238, 250, 307, 330, 311 and 343;

[0954] (11) Positions 234, 237, 238, 330, 311 and 343;

[0955] (12) Positions 235, 236, 268, 295, 326, 330 and 343;

[0956] (13) Positions 214, 235, 236, 268, 295, 326, 330 and 343;

[0957] (14) Positions 235, 236, 268, 295, 326, 330 and 413;

[0958] (15) Positions 214, 236, 268, 330 and 343;

[0959] (16) Positions 214, 235, 236, 268, 330 and 343;

[0960] (17) Positions 214, 236, 268, 330 and 413;

[0961] (18) Positions 214, 236, 268, 330, 343 and 413;

[0962] (19) Positions 214, 235, 236, 268, 330, 343 and 413;

[0963] (20) Positions 214, 236, 268, 330 and 311;

[0964] (21) Positions 214, 235, 236, 268, 330 and 311;

[0965] (22) Positions 214, 236, 268, 330, 311 and 343;

[0966] (23) Positions 214, 235, 236, 268, 330, 311 and 343;

[0967] (24) Positions 214, 236, 268, 330, 311 and 413;

[0968] (25) Positions 214, 235, 236, 268, 330, 311 and 413;

[0969] (26) Positions 214, 235, 236, 268, 295, 326, 330 and 311.

[0970] In one embodiment, the variant Fc region of this disclosure comprises any combination of amino acid alterations identified in Table 4 below.

[0971] [Table 4]

[0972]

[0973] In one embodiment, the variant containing any combination of amino acid changes described in Table 4 above lacks the amino acid at position 447 according to the EU number in its Fc region. In a preferred embodiment, the variant containing any combination of amino acid changes described in Table 4 above lacks the amino acids at positions 446 and 447 according to the EU number in its Fc region.

[0974] Those skilled in the art will understand that, in addition to the modifications exemplified above, at least one amino acid modification that increases the binding activity to Fc-γR (including Fc-γRIIb) compared to the parental Fc region, as described or implied in, for example, WO2013 / 047752, WO2013 / 125667, WO2014 / 030728, WO2014 / 163101 or WO2017104783, and at least one amino acid modification that increases pI compared to the parental Fc region, as described or implied in, for example, WO2017 / 104783, WO2017 / 046994, and any combination of these amino acid modifications, may be used.

[0975] In addition, amino acid alterations made for other purposes can be incorporated into the variant Fc region described herein. For example, the following amino acid substitutions can be added: amino acid substitutions that increase FcRn binding activity (Hinton et al., J. Immunol. 176(1): 346-356 (2006); Dall'Acqua et al., J. Biol. Chem. 281(33): 23514-23524 (2006); Petkova et al., Intl. Immunol. 18(12): 1759-1769 (2006); Zalevsky et al., Nat. Biotechnol. 28(2): 157-159 (2010); WO 2006 / 019447; WO 2006 / 053301; and WO 2009 / 086320), and amino acid substitutions to improve antibody heterogeneity or stability (WO 2009 / 041613). Alternatively, peptides with properties that promote antigen clearance, as described in WO 2011 / 122011, WO 2012 / 132067, WO 2013 / 046704, or WO 2013 / 180201; peptides with properties that bind specifically to target tissues, as described in WO 2013 / 180200; and peptides with properties that repeatedly bind multiple antigen molecules, as described in WO 2009 / 125825, WO 2012 / 073992, or WO 2013 / 047752, may be combined with the variant Fc region described herein. Alternatively, to confer binding activity with other antigens, amino acid changes disclosed in EP1752471 and EP1772465 may be combined in the CH3 region of the variant Fc region described herein.

[0976] In one embodiment, the anti-CD137 antigen-binding molecule or antibody of this disclosure comprises a heavy chain constant region, said heavy chain constant region comprising any amino acid sequence selected from SEQ ID NO: 64-85. Preferably, the anti-CD137 antigen-binding molecule or antibody of this disclosure comprises a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 75 or 82.

[0977] In a preferred embodiment, the anti-CD137 antigen-binding molecule or antibody containing the above-described variant Fc region has the above-described small molecule-dependent CD137 binding activity.

[0978] In one embodiment, the anti-CD137 antigen-binding molecule or antibody of this disclosure comprises the following variable and constant regions: a variable region comprising the HVR, heavy chain variable region and / or light chain variable region described above; and the variant Fc region described above. In a preferred embodiment, the anti-CD137 antigen-binding molecule or antibody of this disclosure may be any anti-CD137 antibody selected from the antibodies described in Table 72.

[0979] In another aspect, this disclosure provides antigen-binding molecules or antibodies that bind to the same epitope on CD137 as the anti-CD137 antigen-binding molecules or antibodies provided herein, in the presence of low molecular weight compounds (e.g., in the presence of said low molecular weight compounds of 10 μM or more, 50 μM or more, 100 μM or more, 150 μM or more, 200 μM or more, or 250 μM or more). For example, in one embodiment, an antigen-binding molecule or antibody is provided that binds to the same antigenic epitope as an anti-CD137 antigen-binding molecule or antibody comprising A375 / B167, A372 / B040, A356 / B040, A486 / B167, A487 / B167, A488 / B226, A489 / B223, A548 / B376, A551 / B256, A551 / B379, A555 / B379, A548 / B256 and / or A549 / B167 as a combination of heavy chain variable region / light chain variable region. In one embodiment, the anti-CD137 antigen-binding molecule or antibody of this disclosure having CD137 binding activity that depends on the small molecule compound recognizes an epitope formed by a complex of an antigen (e.g., CD137) and a low molecular weight compound (e.g., ATP).

[0980] In another aspect, this disclosure provides an antigen-binding molecule or antibody that competes with the anti-CD137 antigen-binding molecule or antibody provided herein for binding to CD137 in the presence of a low molecular weight compound (e.g., in the presence of more than 10 μM, more than 50 μM, more than 100 μM, more than 150 μM, more than 200 μM, or more than 250 μM). For example, in one embodiment, these anti-CD137 antigen-binding molecules or antibodies compete with anti-CD137 antigen-binding molecules or antibodies comprising A375 / B167, A372 / B040, A356 / B040, A486 / B167, A487 / B167, A488 / B226, A489 / B223, A548 / B376, A551 / B256, A551 / B379, A555 / B379, A548 / B256 and / or A549 / B167 as combinations of heavy chain variable regions / light chain variants for binding sites with CD137.

[0981] In another aspect of this disclosure, the anti-CD137 antigen-binding molecule or antibody according to any of the above embodiments is a monoclonal antibody, including chimeric antibodies, humanized antibodies, or human antibodies. In one embodiment, the anti-CD137 antibody is an antibody fragment, such as Fv, Fab, Fab', scFv, a bisomatic antibody, or an F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as a complete IgG1 antibody or other antibody types or isotypes as defined herein.

[0982] On the other hand, the anti-CD137 antigen-binding molecule or antibody according to any of the above embodiments may, alone or in combination, contain any of the features described in sections 1-7 below:

[0983] 1. Agonistaltic activity of anti-CD137 antigen-binding molecules or antibodies

[0984] In specific embodiments, the anti-CD137 antigen-binding molecules or antibodies of this disclosure possess CD137 agonist activity. CD137 signaling not only stimulates IFN-γ secretion and proliferation of NK cells (Buechele et al., 2012; Lin et al., 2008; Melero et al., 1998), but also enhances their survival and DC activation as indicated by upregulated co-stimulatory molecules and cytokine secretion (Choi et al., 2009; Futagawa et al., 2002; Wilcox et al., 2002). However, CD137 is best characterized in T cells on CD4+. + and CD8 + Subgroups of co-stimulatory molecules that regulate TCR-induced activation. In combination with TCR activation, anti-CD137 agonist antibodies enhance T cell proliferation, stimulate lymphokine secretion, and reduce the sensitivity of T lymphocytes to activation-induced cell death (Snelet et al., 2011 review). Among these phenomena, the physiological phenomena observed on T cells after CD137 signaling are mediated by downstream signals activated by CD137 signaling, such as TRAF2, TRAF1, especially NF-κB, JNK, Erk, Akt, survivin, Bcl-XL, and / or Bcl-2 (Ward-Kavanagh et al., Immunity, 44:1005 (2016)).

[0985] In one embodiment, an "anti-CD137 agonist antigen-binding molecule" or "anti-CD137 agonist antibody" is an antigen-binding molecule or antibody that transduces CD137 signaling by binding to CD137 and significantly induces or enhances IFN-γ secretion, proliferation, and increased survival of NK cells; DC activation indicated by cytokine secretion and upregulation of co-stimulatory molecules; TCR induction; T cell proliferation; and / or lymphokine secretion. In different embodiments, an "anti-CD137 agonist antigen-binding molecule" or "anti-CD137 agonist antibody" is an antigen-binding molecule or antibody that transduces CD137 signaling by binding to CD137 on T cells and significantly induces activation of NF-κB on T cells. Furthermore, the antigen-binding molecule or antibody "exhibiting CD137 agonist activity" means that any of the above-mentioned physiological phenomena are observed when the antigen-binding molecule or antibody binds to CD137. Methods for measuring CD137 agonist activity are described in detail in "C. Assay" below.

[0986] In specific embodiments, the anti-CD137 antigen-binding molecule or antibody of this disclosure has small molecule-dependent CD137 agonistic activity. In one non-limiting embodiment, the anti-CD137 antigen-binding molecule or antibody exhibits higher CD137 agonistic activity in the presence of the small molecule compound than in the absence of the small molecule compound. In different embodiments, the anti-CD137 antigen-binding molecule or antibody exhibits higher CD137 agonistic activity in the presence of the small molecule compound compared to CD137 agonistic activity in the presence of low concentrations of the small molecule compound. In further embodiments, the CD137 agonistic activity of the anti-CD137 antigen-binding molecule or antibody in the presence of the small molecule compound is more than 2, 3, 5, 10, 20, 30, 50, 100, 200, 300, 500, or 1 x 10^137 times higher than the CD137 agonistic activity in the absence of the small molecule compound. 3 More than twice, 2 x 10 3 More than twice, 3 x 10 3 More than twice, 5 x 10 3 More than twice, 1 x 10 4 More than twice, 2 x 10 4 More than twice, 3 x 10 4 More than twice, 5 x 10 4 More than twice, or 1 x 10 5 More than twice.

[0987] Any suitable concentration of the small molecule compound can be chosen as the concentration, as long as a difference in the binding activity of the anti-CD137 antigen-binding molecule or antibody is detected. In one embodiment, the anti-CD137 antigen-binding molecule or antibody transduces CD137 signaling by binding to CD137 on the cell surface. Therefore, those skilled in the art will understand that an anti-CD137 antigen-binding molecule or antibody having small molecule-dependent CD137 binding activity has small molecule-dependent CD137 agonist activity. However, on the other hand, since the methods for measuring binding activity and agonist activity are different, those skilled in the art will understand that the concentration of the small molecule compound that detects a difference in binding activity may be different from the concentration of the small molecule compound that detects a difference in agonist activity (e.g., for an anti-CD137 antigen-binding molecule or antibody whose CD137 binding activity in the presence of 10 μM of the small molecule compound is more than twice that in the absence of the small molecule compound, the CD137 agonist activity (measured value) in the presence of 10 μM of the small molecule compound may be less than twice that in the absence of the small molecule compound). Furthermore, those skilled in the art will understand that the determination of agonistic activity can vary based on the determination of CD137 agonistic activity (see “C. Determination”).

[0988] In one embodiment, the anti-CD137 antigen-binding molecule or antibody (i) exhibits agonistic activity against CD137 in the presence of a small molecule compound of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM, and (ii) exhibits substantially no agonistic activity against CD137 in the absence of the small molecule compound, or exhibits low agonistic activity against CD137 in the absence of the small molecule compound (compared to the presence of the small molecule compound).

[0989] In one embodiment, when the agonistic activity of the anti-CD137 antigen-binding molecule or antibody is assessed by the "a) agonistic activity assay (PBMC)" described in detail in "C. Assays", the anti-CD137 antigen-binding molecule or antibody (i) exhibits agonistic activity against CD137 in the presence of a 250 μM small molecule compound, and (ii) exhibits low agonistic activity against CD137 in the absence of the small molecule compound (compared to the presence of the small molecule compound). In a further embodiment, the anti-CD137 antigen-binding molecule or antibody (i) exhibits agonistic activity against CD137 in the presence of a 250 μM small molecule compound, and (ii) exhibits substantially no agonistic activity against CD137 in the absence of the small molecule compound.

[0990] In one embodiment, when the agonistic activity of the anti-CD137 antigen-binding molecule or antibody is evaluated by the agonistic activity assay (reporter gene assay) described in detail in “C. Assay”, the anti-CD137 antigen-binding molecule or antibody (i) exhibits agonistic activity against CD137 in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM small molecule compound, and (ii) has substantially no agonistic activity against CD137 or low agonistic activity (compared to the presence of small molecule compound) in the absence of small molecule compound. The antibody concentration in the reporter gene assay can be arbitrarily selected, for example, a final antibody concentration of 0, 0.001, 0.01, 0.1, 1, or 10 μg / mL. In a preferred embodiment, the final antibody concentration is 0.1 μg / mL or 1 μg / mL.

[0991] In one implementation, when the final concentration of the antibody is 0.1 μg / mL, in “b. Agonistaltic Activity Assay (Reporter Gene Assay)” described in detail in “C. Assay”, (i) in the presence of 10 μM small molecule compound, the CD137 agonist activity (relative optical units) of the anti-CD137 antigen-binding molecule or antibody is more than 2, 3, 5, 10, 20, 30, 50, 60, 70, 80, or 90 times higher than the CD137 agonist activity (relative optical units) in the absence of small molecule compound. In one implementation, when the final concentration of the antibody is 0.1 μg / mL in the “b. agonist activity assay (reporter gene assay)” described in detail in “C. Assay”, (i) the CD137 agonist activity (relative optical units) of the anti-CD137 antigen-binding molecule or antibody in the presence of 100 μM small molecule compound is more than 2, 3, 5, 10, 20, 30, 50, 60, 70, 80, or 90 times higher than the CD137 agonist activity (relative optical units) in the absence of small molecule compound. In one embodiment, when the final antibody concentration is 0.1 μg / mL in the "b. Agonistant Activity Assay (Reporter Gene Assay)" described in detail in "C. Assay", (i) the CD137 agonist activity (relative optical units) of the anti-CD137 antigen-binding molecule or antibody in the presence of 250 μM small molecule compound is 2, 3, 5, 10, 20, 30, 50, 60, 70, 80, or 90 times higher than the CD137 agonist activity (relative optical units) in the absence of small molecule compound. In any of the above embodiments, furthermore, in the absence of small molecule compound, 0.1 μg / mL of the anti-CD137 antigen-binding molecule or antibody substantially does not exhibit CD137 agonist activity.

[0992] In one implementation, when the final concentration of the antibody is “1 μg / mL”, in the “b. agonist activity assay (reporter gene assay)” described in detail in “C. Assay”, (i) in the presence of 10 μM small molecule compound, the CD137 agonist activity (relative optical units) of the anti-CD137 antigen-binding molecule or antibody is more than 2, 3, 5, 10, 20, 30, 50, 60, 70, 80, or 90 times higher than the CD137 agonist activity (relative optical units) in the absence of small molecule compound. In one implementation, when the final concentration of the antibody is 0.1 μg / mL, in “b. agonist activity assay (reporter gene assay)” described in detail in “C. Assay”, (i) the CD137 agonist activity (relative optical units) of the anti-CD137 antigen-binding molecule or antibody in the presence of 100 μM small molecule compound is more than 2, 3, 5, 10, 20, 30, 50, 60, 70, 80, or 90 times higher than the CD137 agonist activity (relative optical units) in the absence of small molecule compound. In one embodiment, when the final antibody concentration is 0.1 μg / mL, in “b. Agonistaltic Activity Assay (Reporter Gene Assay)” described in detail in “C. Assay”, (i) in the presence of 250 μM small molecule compound, the CD137 agonist activity (relative optical units) of the anti-CD137 antigen-binding molecule or antibody is 2, 3, 5, 10, 20, 30, 50, 60, 70, 80, or 90 times higher than the CD137 agonist activity (relative optical units) in the absence of small molecule compound. In any of the above embodiments, furthermore, in the absence of small molecule compound, 1 μg / mL of the anti-CD137 antigen-binding molecule or antibody substantially does not show CD137 agonist activity.

[0993] 2. Antibody fragments

[0994] In some embodiments, the antibodies described herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson et al., Nat. Med., 9: 129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds. (Springer-Verlag, New York), pp. 269-315 (1994); also see WO 93 / 16185; U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments containing rescue receptor-binding epitope residues and having an increased in vivo half-life, see U.S. Patent No. 5,869,046.

[0995] Bisomic antibodies are antibody fragments with two antigen-binding sites and can be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med., 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993). Trisomic and tetrasomic antibodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0996] A single-domain antibody is an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain. In some embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6,248,516 B1).

[0997] Antibody fragments can be prepared using a variety of techniques, including but not limited to the proteolytic digestion of intact antibodies and the production of them from recombinant host cells (such as Escherichia coli or bacteriophages), as described herein.

[0998] 3. Chimeric antibodies and humanized antibodies

[0999] In some embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,56; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one instance, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In another instance, a chimeric antibody is a “class-switching” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include their antigen-binding fragments.

[1000] In some embodiments, the chimeric antibody is a humanized antibody. Typically, a nonhuman antibody is humanized to reduce its immunogenicity to humans while retaining the specificity and affinity of the parent nonhuman antibody. Typically, a humanized antibody contains one or more variable domains, wherein the HVR, such as the CDR (or a portion thereof), is derived from the nonhuman antibody, and the FR (or a portion thereof) is derived from the human antibody sequence. Optionally, the humanized antibody will also contain at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are replaced by corresponding residues from the nonhuman antibody (e.g., the antibody from which the HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[1001] Humanized antibodies and their preparation methods have been reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US patent numbers 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity-determining region (SDR) transplantation); Padlan, Mol. Immunol. 28:489-498 (1991). (Description of “re-tiling”); Dall'Acqua et al., Methods 36:43-60 (2005) (Description of “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (Description of “guided selection” method for FR shuffling).

[1002] Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using a “best fit” method (see, for example, Sims et al., J. Immunol. 151: 2296 (1993)); and frame regions derived from the common sequence of human antibodies from specific subgroups of light chain variable regions or heavy chain variable regions (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); and Presta et al., J. Immunol. 151: 2 623 (1993)); human mature (somatic mutant) frame regions or human germline frame regions (see, e.g., Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); frame regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[1003] 4. Human antibodies

[1004] In some embodiments, the antibodies provided herein are human antibodies. Human antibodies can be generated using a variety of techniques known in the art. Human antibodies are commonly described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[1005] Human antibodies can be prepared by administering an immunogen to transgenic animals that have been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci, which replace endogenous immunoglobulin loci, or are located extrachromosomally or randomly integrated into the animal's chromosome. In such transgenic mice, endogenous immunoglobulin loci are typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, a description of XENOMOUSE. TMThe technologies described are referenced in U.S. Patent Nos. 6,075,181 and 6,150,584; U.S. Patent No. 5,770,429 describing the HuMab (registered trademark) technology; U.S. Patent No. 7,041,870 describing the KM MOUSE (registered trademark) technology; and U.S. Patent Application Publication No. US2007 / 0061900 describing the VelociMouse (registered trademark) technology. The human variable region of the intact antibody produced from such animals can be further modified, for example, by binding to different human constant regions.

[1006] Human antibodies can also be prepared using hybridoma-based methods. Human myeloma and mouse-human heterologous myeloma cell lines used to produce human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Technology and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner Li et al., J. Immunol., 147:86 (1991)). Human antibodies produced via human B-cell hybridoma technology are described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Other methods include, for example, those described in U.S. Patent No. 7,189,826 (description of production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (description of human-human hybridoma). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[1007] Human antibodies can also be generated by isolating variable domain sequences of Fv clones selected from human phage display libraries. These variable domain sequences can then be bound to desired human constant domains. The technique for selecting human antibodies from an antibody library is described below.

[1008] 5. Antibodies derived from a library

[1009] The antibodies disclosed herein can be isolated by screening a combinatorial library for antibodies possessing the desired activity or multiple activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding properties. Such methods are reviewed, for example, in Hoogenboom et al., Molecular Biology Methods, 178:1-37 (edited by O'Brien et al., Human Press, Totoa, NJ, 2001), and further described below: e.g., McCafferty et al., Nature, 348:552-554; Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology, 248:161-175 (Lo, ed., Human Press). Press), Totoa, NJ, 2003; Sidhu et al., J. Mol. Biol., 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004) and Lee et al., J. Immunol. Methods, 284(1-2): 119-132 (2004).

[1010] In some phage display methods, VH and VL gene libraries are cloned separately by polymerase chain reaction (PCR) and randomly recombined in the phage library. Antigen-binding phages can then be screened as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments, either single-chain Fv (scFv) fragments or Fab fragments. Libraries derived from immunogenic sources provide high-affinity antibodies against immunogens without the need for hybridoma construction. Alternatively, juvenile libraries (e.g., from humans) can be cloned, as described in Griffiths et al., EMBO J., 12:725-734 (1993), to provide a single source of antibodies against a wide range of non-self and self antigens without any immunization. Finally, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992), naive libraries can also be synthesized by cloning an unrearranged V gene fragment from stem cells, encoding a highly variable CDR3 region using PCR primers containing random sequences, and performing rearrangement in vitro. Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Publications Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[1011] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments in this paper.

[1012] The antigen-binding molecules or antibodies of this disclosure that exhibit antigen-binding activity dependent on low molecular weight compounds can be selected by screening a library of antigen-binding molecules. As such a library, the aforementioned combined libraries can be used. The library of antigen-binding molecules can be a library of unbiased antigen-binding molecules (naive libraries), or it can be a library of biased antigen-binding molecules. Examples of the latter type of library include libraries of antigen-binding molecules pre-conferred with binding activity against a specific compound. In one embodiment, the antigen-binding molecule library is a library to which an amino acid-modified antigen-binding molecule conferred binding activity against a specific compound has been pre-introduced. Examples of this type of library include, for example, the libraries described in International Publication WO 2015 / 083764.

[1013] 6. Multispecific antibodies

[1014] In some embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. A multispecific antibody is a monoclonal antibody that has binding specificity to at least two different sites. In some embodiments, one binding specificity is against CD137, while the other is against any other antigen. In some embodiments, a bispecific antibody can bind to two different epitopes of CD137. Bispecific antibodies can also be used to target cytotoxic agents to cells expressing CD137. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[1015] In one embodiment, the anti-CD137 antigen-binding molecules or antibodies disclosed herein are bispecific antibodies, one arm of which has CD137-binding activity dependent on a small molecule compound, while the other arm binds to an antigen different from CD137. The "antigen" different from CD137 is not particularly structurally limited. In other words, the antigen can be inorganic or organic. Exemplary antigens are disclosed below. In one embodiment, the antigen is preferably an antigen expressed in cancerous or inflammatory tissues, such as cancer cells, immune cells, or stromal cells.

[1016] In this document, the structure of "antigen" is not particularly limited, as long as it contains the epitope to which the antigen-binding molecule of the present invention binds. Antigens can be inorganic or organic substances. In some implementations, examples of antigens include: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressins, aFGF, ALCAM, ALK, ALK-1, ALK-7, α-1-antitrypsin, α-V / β-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic peptide, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulating factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 bone morphogenetic protein, BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3). BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, β-NGF, BOK, bufotalin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H.Cathepsin L, Cathepsin O, Cathepsin S, Cathepsin V, Cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7 CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19,CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55,CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138,CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Clostridium perfringens toxin, CKb8-1, Claudin-6, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, PD1, PDL1, LAG3, TIM3, galectin-9, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, complement regulator (decay accelerator), des (1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DLL3, DNAM-1, DNA enzyme, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eosinophil activation chemokine 1, EpCAM, liver glycoside B2 / EphB4, EPO, ERCC, E-selectin. ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX, Fibroblast Activating Protein (FAP), Fas, FcR1, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF-3, FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Follicle-Stimulating Hormone, CXXXC Chemotactic Molecule (fractalkine), FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myosin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-α1, GFR-α2, GFR-α3, GITR, glucagon, Glut4, glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, growth hormone-releasing hormone, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparinase,Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), Herpes simplex virus (HSV) gB glycoprotein, HSVgD glycoprotein, HGFA, High molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, Human cardiac myosin, Human cytomegalovirus (HCMV), Human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-21, IL-23, IL-27, Interferon (INF)-α, INF-β, INF-γ, Inhibin, iNOS, Insulin A chain, Insulin B chain, Insulin-like growth factor 1, Integrin α2, Integrin α3, Integrin α4, Integrin α4 / β1, Integrin α4 / β7, Integrin α5 (αV), Integrin α5 / β1, Integrin α5 / β3, Integrin α6, Integrin β1, Integrin β2, Interferon γ, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1 bp1, LBP, LDGF, LECT2, lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing hormone.Lymphotoxin β receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-α, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, Mucin (Muc1), MUC18, Müllerian tubule inhibitor, Mug, MuSK, NAIP, NAP, NCAD, NC adherin, NCA 90, NCAM, NCAM Neprilysin, neurotrophic factor-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-β, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA. Prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat,STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptors (e.g., T cell receptor α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-β panspecific, TGF-βRI (ALK-5), TGF-βRII, TGF-βRIIb, TGF-βRIII, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-α, TNF-αβ, 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 (OPGOCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17(BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60), TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1,CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRSF23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-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-a Conectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TLR1 (Toll-like receptor 1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expression Lewis-Y related carbohydrates, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherinVE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aβ, CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp90 IL-17 / IL-17R, IL-20 / IL-20R, Oxidized LDL, PCSK9, Prokaryotic release enzyme, RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B, tau, VAP1, High molecular weight kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, Factor B, Factor D, Factor H, Complementin Sclerostin, fibrinogen, fibrin, prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, polyligand-1, polyligand-2, polyligand-3, polyligand-4, LPAAnd S1P. In some embodiments, examples of antigens include hormone receptors and growth factors. In some embodiments, antigens are those expressed or secreted in cells present in tumor tissue (e.g., tumor cells, immune cells, stromal cells, etc.).

[1017] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of heavy-light chain pairs of two immunoglobulins with different specificities (see Milstein and Cuello, Nature, 305:537 (1983), WO 93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), and "knob-in-hole" engineering (see, for example, U.S. Patent No. 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic redirection effects to prepare antibody Fc-heterodimer molecules (WO). 2009 / 089004A1); crosslinking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); generating bispecific antibodies using leucine zippers (see, for example, Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)); preparing bispecific antibody fragments using “bibody antibody” technology (see, for example, Hollinger et al., Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 90: 6444-6448 (1993)); and the preparation of trispecific antibodies using single-chain Fv (scFv) dimers (see, for example, Gruber et al., J. Immunol., 152: 5368 (1994)); and the preparation of trispecific antibodies as described, for example, Tutt et al., Journal of Immunology (J. Immunol.), 147: 60 (1991).

[1018] This article also includes engineered antibodies with three or more functional antigen-binding sites, including “octopus antibodies” (see, for example, US 2006 / 0025576A1).

[1019] The antibodies or fragments described herein also include “dual-action Fab” or “DAF”, which contain antigen-binding sites that bind to CD137 as well as another different antigen (see, for example, US 2008 / 0069820).

[1020] 7. Antibody variants

[1021] In some embodiments, amino acid sequence variants of the antibodies provided herein are considered. For example, it may be necessary to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the antibody amino acid sequence. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct possesses the desired properties, such as antigen binding.

[1022] a) Substitution, insertion, and deletion variants

[1023] In some embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVR and FR. Conserved substitutions are shown under the heading “Preferred Substitutions” in Table 5. More substantial variations are provided under the heading “Exemplary Substitutions” in Table 5 and are further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the antibody of interest, and products with desired activities can be screened, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[1024] [Table 5]

[1025]

[1026] Amino acids can be grouped according to common side chain characteristics:

[1027] (1) Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile;

[1028] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[1029] (3) Acidic: Asp, Glu;

[1030] (4) Alkaline: His, Lys, Arg;

[1031] (5) Residues that affect chain orientation: Gly, Pro;

[1032] (6) Aromatics: Trp, Tyr, Phe.

[1033] A non-conservative permutation would result in swapping members of one of these categories with members of another category.

[1034] One type of substitution variant involves replacing one or more hypervariable residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variant selected for further research will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody, and / or will substantially retain some of the biological properties of the parent antibody. Exemplary substitution variants are affinity-matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques, such as those described herein. In short, one or more HVR residues are mutated, and the variant antibody is displayed on a phage and screened for specific biological activities (e.g., binding affinity).

[1035] Modifications (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such modifications can be made in HVR “hotspots,” which are residues encoded by codons that mutate frequently during somatic maturation (see, e.g., Chowdhury, Methods in Molecular Biology, 207:179-196 (2008)), and / or residues in contact with the antigen, testing the binding affinity of the resulting variant VH or VL. Affinity maturation by constructing and reselecting from secondary libraries is described, for example, in Hoogenboom et al., Methods in Molecular Biology, 178:1-37 (O'Brien et al., Human Press, Totoa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variant gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves HVR-directed methods, where several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scan mutagenesis or modeling. CDR-H3 and CDR-L3 are particularly commonly targeted.

[1036] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, provided that such changes do not substantially reduce the antibody's ability to bind to the antigen. For example, conserved changes that do not substantially reduce binding affinity (e.g., the conserved substitutions provided herein) may be made in the HVRs. Such changes may, for example, be outside the antigen-contacting residues in the HVRs. In some embodiments of the variant VH and VL sequences provided above, each HVR either remains unchanged or contains no more than one, two, or three amino acid substitutions.

[1037] As described by Cunningham and Wells (1989), Science, 244:1081-1085, a useful method for identifying residues or regions of antibodies that can be targeted for mutagenesis is called "alanine scan mutagenesis." In this method, the target residues or groups (e.g., charged residues, such as arg, asp, his, lys, and glu) are identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and the antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex can be analyzed to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

[1038] Amino acid sequence insertions include amino and / or carboxyl terminus fusions ranging in length from one residue to peptides containing hundreds or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminus insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of enzymes (e.g., for ADEPT) or peptides that increase the plasma half-life of the antibody with the N-terminus or C-terminus of the antibody.

[1039] b) Glycosylation variants

[1040] In some implementations, the antibodies provided herein are modified to increase or decrease the degree of glycosylation. The addition or deletion of glycosylation sites in the antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[1041] When an antibody contains an Fc region, the carbohydrates associated with it can be altered. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, which are usually linked to Asn297 of the CH2 domain of the Fc region via N-bonds. See, for example, Wright et al., TIBTECH, 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose of GlcNAc attached to the “stem” of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of this disclosure can be modified to produce antibody variants with certain improved properties.

[1042] In one embodiment, an antibody variant is provided having a carbohydrate structure lacking (directly or indirectly) fucose linked to the Fc region. For example, the fucose content in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose at Asn297 in the sugar chain relative to the sum of all sugar structures (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue (EU number of Fc region residues) located at approximately position 297 in the Fc region. However, due to minor sequence variations in the antibody, Asn297 can also be located approximately + / - 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants can possess improved ADCC function. See, for example, U.S. Patent Publication Nos. 2003 / 0157108 (Presta, L.); and 2004 / 0093621 (Kyowa Hakko Kogyo, Inc.). Examples of publications relating to “defucosylated” or “fucose-deficient” antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotechnology and Bioengineering (Biotech. Bioeng.), 87:614 (2004).Examples of cell lines capable of producing defucosylation antibodies include Lec13 CHO cells with protein fucosylation defects (Ripka et al., Arch. Biochem. Biophys. 249: 533-545 (1986); US Patent Application No. US 2003 / 0157108 A1, Presta, L.; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and knockout cell lines, such as α-1,6-fucosylation gene FUT8, knockout CHO cells (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng., 87: 614 (2004); Kanda, Y. et al., Biotech. Bioeng., 94(4): 680-688 (2006); and WO2003 / 085107).

[1043] Antibody variants further provide branched oligosaccharides, for example, wherein the biantennary oligosaccharide attached to the Fc region of the antibody is branched by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, WO1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[1044] c) Fc region variants

[1045] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein, thereby creating an Fc region variant (also referred to as a "modified Fc region"). The Fc region variant may contain a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) with amino acid alterations (e.g., substitutions) at one or more amino acid positions.

[1046] In some embodiments, this disclosure considers antibody variants possessing some, but not all, effector functions, making them desirable candidates for applications where the antibody's in vivo half-life is important and certain effector functions (e.g., complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding activity. The primary cells mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991), page 464, Table 3 summarizes FcR expression on hematopoietic cells. Non-limiting examples of in vitro assays for assessing ADCC activity of target molecules are described in U.S. Patent Nos. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (e.g., see ACT1 for flow cytometry). TMNon-radioactive cytotoxicity assays (Cell Technology, Mountain View, California; and CytoTox 96 (registered trademark) non-radioactive cytotoxicity assay (Promega, Madison, Michigan). Useful effector cells for this assay include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, ADCC activity of the target molecule can be assessed in vivo, for example in animal models disclosed in the Proceedings of the National Academy of Sciences of the United States of America (Proc. Nat'l Acad. Sci. USA) 95:652-656 (1998) by Clynes et al. C1q binding assays can also be performed to confirm that the antibody cannot bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., Journal of Immunomethods (J. Immunol.). Methods), 202:163 (1996); Cragg, MS et al., Blood, 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood, 103:2738-2743 (2004). FcRn binding and in vivo clearance / half-life can also be determined using methods known in the art (see, for example, Petkova, SB et al., International Immunology, 18(12):1759-1769 (2006)).

[1047] Antibodies with reduced effector function include those with one or more substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant, in which residues 265 and 297 are substituted with alanine (US Patent No. 7,332,581).

[1048] Certain antibody variants are described as having increased or decreased binding to FcR (see, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

[1049] In some embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbers of the residues).

[1050] In some implementations, such as those described in U.S. Patent Nos. 6,194,551, WO 99 / 51642, and Idusogie et al., Journal of Immunology, 164: 4178-4184 (2000), alterations are made in the Fc region resulting in altered (i.e., increased or decreased) C1q binding and / or complement-dependent cytotoxicity (CDC).

[1051] Antibodies with prolonged half-lives and increased binding to the neonatal Fc receptor (FcRn), responsible for transferring maternal IgG to the fetus (Guyer et al., Journal of Immunology, 117:587 (1976) and Kim et al., Journal of Immunology, 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). These antibodies contain an Fc region with one or more substitutions that increase the binding of the Fc region to the FcRn. Such Fc variants include Fc variants in which one or more of the following Fc region residues are substituted: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, such as the substitution of Fc region residue 434 (US Patent No. 7,371,826).

[1052] See also Duncan & Winter, Nature, 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 concerning other examples of Fc region variants.

[1053] In one implementation, the binding activity of the antibody Fc region (including variant Fc regions, hereinafter also applicable) to each person's Fc-γ receptor (Fc-γR) can be measured, for example, using a ligand capture method with BIACORE (registered trademark) T200, which relies on surface plasmon resonance analysis as the measurement principle.

[1054] The following describes details of an exemplary method for measuring the binding activity of the antibody Fc region to various human Fc-γ receptors (Fc-γRs). In one embodiment, the binding activity of the antibody Fc region to Fc-γRs is assessed using BIACORE (registered trademark) T200. In a preferred embodiment, the measurement is performed at 25°C using a measurement buffer of 50 mM phosphate, 150 mM NaCl, 0.05 w / v %-P20, pH 7.4. Specifically, approximately 1000 RU of antibody containing the variant Fc region is first captured onto a sensor chip, and CaptureSelect (trademark) human Fab-λ kinetic biotin conjugate (Thermo Fisher Scientific) is immobilized as the molecule of the capture ligand. Human Fc-γRs are diluted with the measurement buffer to a concentration of 8 nM for Fc-γRIa and 1000 nM for other Fc-γRs, and then bound to the captured antibody. The binding activity of each antibody to each Fc-γR was evaluated by calculating the amount of Fc-γR bound per unit amount of antibody (RU) using BiacoreT200 evaluation software 2.0. In one embodiment, the binding activity of the antibody Fc region to various human Fc-γ receptors (Fc-γRs) can be measured by referring to the methods described in Examples 7-4.

[1055] In a preferred embodiment, the Fc-γR used in the above measurement method can be the extracellular domain of Fc-γR prepared by the following method. First, the gene for the extracellular domain of Fc-γR is synthesized using methods known to those skilled in the art. For this synthesis, the sequence of each Fc-γR is prepared based on information registered in NCBI. More specifically, the sequence of Fc-γRI is prepared based on the sequence of NCBI accession number #NM_000566.3, the sequence of Fc-γRIIa is prepared based on the sequence of NCBI accession number #NM_001136219.1, the sequence of Fc-γRIIb is prepared based on the sequence of NCBI accession number NM_004001.3, and the sequence of Fc-γRIIIa is prepared based on the sequence of NCBI accession number NM_001127593.1, and a His tag is added to the C-terminus. The polymorphic sites of Fc-γRIIa were prepared according to J. Exp. Med., 1990, 172, 19-25, and those of Fc-γRIIIa according to J. Clin. Invest., 1997, 100, 1059-1070. The obtained gene fragments were inserted into expression vectors in animal cells to prepare expression vectors. The prepared expression vectors were transiently introduced into FreeStyle293 cells (Invitrogen) derived from human embryonic renal cell carcinoma cells, and the protein of interest was expressed. The culture supernatant was collected and filtered through a 0.22 μm filter, and then purified essentially through the following four steps: First, cation exchange column chromatography (SP Sepharose FF); second, His-tag affinity column chromatography (HisTrap HP); third, gel filtration column chromatography (Superdex 200); fourth, sterile filtration. Note that for Fc-γRI, the first step was anion exchange column chromatography using QSepharose FF. The concentration of purified protein is calculated based on the absorption coefficient, which is calculated by measuring the absorbance at 280 nm using a spectrophotometer and PACE or similar methods for measurement (Protein Science, 1995, 4, 2411-2423).

[1056] In one implementation, the binding activity of the antibody Fc region to human FcRn can be measured by using a ligand capture method of the BIACORE (registered trademark) T200, for example, which relies on surface plasmon resonance analysis as the measurement principle.

[1057] Details of an exemplary method for measuring the binding activity of the antibody Fc region to human FcRn are described below. In one embodiment, the binding activity of the antibody Fc region to human FcRn is assessed using a BIACORE (registered trademark) T200. In a preferred embodiment, the measurement is performed at 25°C using a measurement buffer of 50 mM phosphate, 150 mM NaCl, 0.05 w / v %-P20, pH 6.0. Specifically, approximately 400 RU of antibody containing the Fc region is first captured onto a sensor chip, on which a CaptureSelect (trademark) human Fab-λ kinetic biotin conjugate (Thermo Fisher Scientific) is immobilized as a capture ligand molecule, and then human FcRn diluted with the measurement buffer is bound to it. The binding activity of each antibody to FcRn is assessed by calculating KD(M) using a steady-state model in the Biacore T200 evaluation software 2.0. In a preferred embodiment, the human FcRn protein for this measurement is prepared according to the method described in Reference Example 2 of WO2010107110. In one implementation, the binding activ...

Claims

1. A pharmaceutical composition for treating cancer, comprising a combination of an anti-CD137 antigen-binding molecule and at least one other anticancer agent, wherein the anti-CD137 antigen-binding molecule comprises any combination of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 selected from (a) to (m): (a) HVR-H1, which contains the amino acid sequence of SEQ ID NO:7; HVR-H2, which contains the amino acid sequence of SEQ ID NO:8; HVR-H3, which contains the amino acid sequence of SEQ ID NO:17; HVR-L1, which contains the amino acid sequence of SEQ ID NO:21; HVR-L2, which contains the amino acid sequence of SEQ ID NO:26; and HVR-L3, which contains the amino acid sequence of SEQ ID NO:27; (b) HVR-H1, comprising the amino acid sequence of SEQ ID NO:7; HVR-H2, comprising the amino acid sequence of SEQ ID NO:9; HVR-H3, comprising the amino acid sequence of SEQ ID NO:17; HVR-L1, comprising the amino acid sequence of SEQ ID NO:22; HVR-L2, comprising the amino acid sequence of SEQ ID NO:26; and HVR-L3, comprising the amino acid sequence of SEQ ID NO:27; (c) HVR-H1, comprising the amino acid sequence of SEQ ID NO:7; HVR-H2, comprising the amino acid sequence of SEQ ID NO:10; HVR-H3, comprising the amino acid sequence of SEQ ID NO:17; HVR-L1, comprising the amino acid sequence of SEQ ID NO:22; HVR-L2, comprising the amino acid sequence of SEQ ID NO:26; and HVR-L3, comprising the amino acid sequence of SEQ ID NO:27; (d) HVR-H1, which contains the amino acid sequence of SEQ ID NO:7; HVR-H2, which contains the amino acid sequence of SEQ ID NO:11; HVR-H3, which contains the amino acid sequence of SEQ ID NO:18; HVR-L1, which contains the amino acid sequence of SEQ ID NO:21; HVR-L2, which contains the amino acid sequence of SEQ ID NO:26; and HVR-L3, which contains the amino acid sequence of SEQ ID NO:27; (e) HVR-H1, comprising the amino acid sequence of SEQ ID NO:7; HVR-H2, comprising the amino acid sequence of SEQ ID NO:8; HVR-H3, comprising the amino acid sequence of SEQ ID NO:18; HVR-L1, comprising the amino acid sequence of SEQ ID NO:21; HVR-L2, comprising the amino acid sequence of SEQ ID NO:26; and HVR-L3, comprising the amino acid sequence of SEQ ID NO:27; (f) HVR-H1, comprising the amino acid sequence of SEQ ID NO:7; HVR-H2, comprising the amino acid sequence of SEQ ID NO:12; HVR-H3, comprising the amino acid sequence of SEQ ID NO:18; HVR-L1, comprising the amino acid sequence of SEQ ID NO:21; HVR-L2, comprising the amino acid sequence of SEQ ID NO:26; and HVR-L3, comprising the amino acid sequence of SEQ ID NO:28; (g) HVR-H1, comprising the amino acid sequence of SEQ ID NO:7; HVR-H2, comprising the amino acid sequence of SEQ ID NO:13; HVR-H3, comprising the amino acid sequence of SEQ ID NO:18; HVR-L1, comprising the amino acid sequence of SEQ ID NO:21; HVR-L2, comprising the amino acid sequence of SEQ ID NO:26; and HVR-L3, comprising the amino acid sequence of SEQ ID NO:29; (h) HVR-H1, comprising the amino acid sequence of SEQ ID NO:7; HVR-H2, comprising the amino acid sequence of SEQ ID NO:14; HVR-H3, comprising the amino acid sequence of SEQ ID NO:19; HVR-L1, comprising the amino acid sequence of SEQ ID NO:23; HVR-L2, comprising the amino acid sequence of SEQ ID NO:26; and HVR-L3, comprising the amino acid sequence of SEQ ID NO:27; (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO:7; HVR-H2, which contains the amino acid sequence of SEQ ID NO:15; HVR-H3, which contains the amino acid sequence of SEQ ID NO:20; HVR-L1, which contains the amino acid sequence of SEQ ID NO:24; HVR-L2, which contains the amino acid sequence of SEQ ID NO:26; and HVR-L3, which contains the amino acid sequence of SEQ ID NO:27; (j) HVR-H1, which contains the amino acid sequence of SEQ ID NO:7; HVR-H2, which contains the amino acid sequence of SEQ ID NO:15; HVR-H3, which contains the amino acid sequence of SEQ ID NO:20; HVR-L1, which contains the amino acid sequence of SEQ ID NO:25; HVR-L2, which contains the amino acid sequence of SEQ ID NO:26; and HVR-L3, which contains the amino acid sequence of SEQ ID NO:27; (k) HVR-H1, comprising the amino acid sequence of SEQ ID NO:7; HVR-H2, comprising the amino acid sequence of SEQ ID NO:16; HVR-H3, comprising the amino acid sequence of SEQ ID NO:20; HVR-L1, comprising the amino acid sequence of SEQ ID NO:25; HVR-L2, comprising the amino acid sequence of SEQ ID NO:26; and HVR-L3, comprising the amino acid sequence of SEQ ID NO:27; (l) HVR-H1, comprising the amino acid sequence of SEQ ID NO:7; HVR-H2, comprising the amino acid sequence of SEQ ID NO:14; HVR-H3, comprising the amino acid sequence of SEQ ID NO:19; HVR-L1, comprising the amino acid sequence of SEQ ID NO:24; HVR-L2, comprising the amino acid sequence of SEQ ID NO:26; and HVR-L3, comprising the amino acid sequence of SEQ ID NO:27; and (m) HVR-H1, which contains the amino acid sequence of SEQ ID NO:7; HVR-H2, which contains the amino acid sequence of SEQ ID NO:14; HVR-H3, which contains the amino acid sequence of SEQ ID NO:17; HVR-L1, which contains the amino acid sequence of SEQ ID NO:21; HVR-L2, which contains the amino acid sequence of SEQ ID NO:26; and HVR-L3, which contains the amino acid sequence of SEQ ID NO:

27.

2. A pharmaceutical composition for treating cancer, comprising a combination of an anti-CD137 antigen-binding molecule and at least one other anticancer agent, wherein the anti-CD137 antigen-binding molecule comprises any combination of VH and VL selected from (a) to (m): (a) VH, which contains the amino acid sequence of SEQ ID NO: 43; and VL, which contains the amino acid sequence of SEQ ID NO: 54; (b) VH, which contains the amino acid sequence of SEQ ID NO: 44; and VL, which contains the amino acid sequence of SEQ ID NO: 55; (c) VH, which contains the amino acid sequence of SEQ ID NO: 45; and VL, which contains the amino acid sequence of SEQ ID NO: 55; (d) VH, which contains the amino acid sequence of SEQ ID NO: 46; and VL, which contains the amino acid sequence of SEQ ID NO: 54; (e) VH, which contains the amino acid sequence of SEQ ID NO: 47; and VL, which contains the amino acid sequence of SEQ ID NO: 54; (f) VH, which contains the amino acid sequence of SEQ ID NO: 48; and VL, which contains the amino acid sequence of SEQ ID NO: 56; (g) VH, which contains the amino acid sequence of SEQ ID NO: 49; and VL, which contains the amino acid sequence of SEQ ID NO: 57; (h) VH, which contains the amino acid sequence of SEQ ID NO: 50; and VL, which contains the amino acid sequence of SEQ ID NO: 58; (i) VH, which contains the amino acid sequence of SEQ ID NO: 51; and VL, which contains the amino acid sequence of SEQ ID NO: 59; (j) VH, which contains the amino acid sequence of SEQ ID NO: 51; and VL, which contains the amino acid sequence of SEQ ID NO: 60; (k) VH, which contains the amino acid sequence of SEQ ID NO: 52; and VL, which contains the amino acid sequence of SEQ ID NO: 60; (l) VH, comprising the amino acid sequence of SEQ ID NO: 50; and VL, comprising the amino acid sequence of SEQ ID NO: 59; and (m) VH, which contains the amino acid sequence of SEQ ID NO: 53; and VL, which contains the amino acid sequence of SEQ ID NO:

54.

3. The pharmaceutical composition according to claim 1 or 2, wherein, The anti-CD137 antigen-binding molecule is a human antibody, a humanized antibody, or a chimeric antibody, or an antigen-binding fragment of any of these.

4. The pharmaceutical composition according to claim 1 or 2, wherein, The anti-CD137 antigen-binding molecule comprises a modified Fc region, and the modified Fc region comprises any combination of amino acid modifications selected according to EU numbers: L235W / G236N / H268D / Q295L / K326T / A330K / P343R / D413K; K214R / L235W / G236N / H268D / Q295L / K326T / A330K / P343R / D413K; L234Y / P238D / T250V / V264I / T307P / A330K / P343R / D413K; L234Y / P238D / V264I / A330K / P343R / D413K; L234Y / G237D / P238D / T250V / T307P / A330K / P343R / D413K; L234Y / G237D / P238D / A330K / P343R / D413K; L235W / G236N / H268D / Q295L / K326T / A330K / Q311R / P343R; L234Y / P238D / T250V / V264I / T307P / A330K / Q311R / P343R; L234Y / P238D / V264I / A330K / Q311R / P343R; L234Y / G237D / P238D / T250V / T307P / A330K / Q311R / P343R; L234Y / G237D / P238D / A330K / Q311R / P343R; L235W / G236N / H268D / Q295L / K326T / A330K / P343R; K214R / L235W / G236N / H268D / Q295L / K326T / A330K / P343R; L235W / G236N / H268D / Q295L / K326T / A330K / D413K; K214R / G236N / H268D / A330K / P343R; K214R / L235W / G236N / H268D / A330K / P343R; K214R / G236N / H268D / A330K / D413K; K214R / G236N / H268D / A330K / P343R / D413K; K214R / L235W / G236N / H268D / A330K / P343R / D413K; K214R / G236N / H268D / A330K / Q311R; K214R / L235W / G236N / H268D / A330K / Q311R; K214R / G236N / H268D / A330K / Q311R / P343R; K214R / L235W / G236N / H268D / A330K / Q311R / P343R; K214R / G236N / H268D / A330K / Q311R / D413K; K214R / L235W / G236N / H268D / A330K / Q311R / D413K; and K214R / L235W / G236N / H268D / Q295L / K326T / A330K / Q311R.

5. A pharmaceutical composition for treating cancer, comprising a combination of an anti-CD137 antigen-binding molecule and at least one other anticancer agent, wherein the anti-CD137 antigen-binding molecule comprises any combination of VH, VL, CH and CL selected from (i) to (xxxviii): (i) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 64; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (ii) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 66; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (iii) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 67; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (iv) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 68; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (v) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 69; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (vi) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 70; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (vii) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 71; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (viii) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 73; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (ix) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 75; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (x) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 78; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xi) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 80; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xii) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 82; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xiii) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 84; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xiv) VH, which contains the amino acid sequence of SEQ ID NO: 43; CH, which contains the amino acid sequence of SEQ ID NO: 85; VL, which contains the amino acid sequence of SEQ ID NO: 54; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xv) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 65; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xvi) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 72; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xvii) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 74; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xviii) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 75; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xix) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 77; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xx) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 78; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xxi) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 79; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xxii) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 80; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xxiii) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 81; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xxiv) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 82; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xxv) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 83; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xxvi) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 84; VL, which contains the amino acid sequence of SEQ ID NO: 59; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xxvii) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 72; VL, which contains the amino acid sequence of SEQ ID NO: 60; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xxviii) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 74; VL, which contains the amino acid sequence of SEQ ID NO: 60; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xxix) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 75; VL, which contains the amino acid sequence of SEQ ID NO: 60; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xxx) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 77; VL, which contains the amino acid sequence of SEQ ID NO: 60; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xxxi) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 78; VL, which contains the amino acid sequence of SEQ ID NO: 60; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xxxii) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 79; VL, which contains the amino acid sequence of SEQ ID NO: 60; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xxxiii) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 80; VL, which contains the amino acid sequence of SEQ ID NO: 60; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xxxiv) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 81; VL, which contains the amino acid sequence of SEQ ID NO: 60; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xxxv) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 82; VL, which contains the amino acid sequence of SEQ ID NO: 60; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xxxvi) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 83; VL, which contains the amino acid sequence of SEQ ID NO: 60; and CL, which contains the amino acid sequence of SEQ ID NO: 63; (xxxvii) VH, comprising the amino acid sequence of SEQ ID NO: 51; CH, comprising the amino acid sequence of SEQ ID NO: 84; VL, comprising the amino acid sequence of SEQ ID NO: 60; and CL, comprising the amino acid sequence of SEQ ID NO: 63; and (xxxviii) VH, which contains the amino acid sequence of SEQ ID NO: 51; CH, which contains the amino acid sequence of SEQ ID NO: 85; VL, which contains the amino acid sequence of SEQ ID NO: 60; and CL, which contains the amino acid sequence of SEQ ID NO: 63.

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