Anti-CD137 antigen binding molecules and uses thereof

Abstract

To provide anti-CD137 antigen binding molecules with an immune cell-activating activity, cytotoxic activity or antitumor activity while with low effects on non-tumorous tissues such as normal tissues and little side effects, and use methods thereof.SOLUTION: The invention relates to an anti-CD137 antigen binding molecule having a CD137 binding activity depending on a low molecular compound, an isolated nucleic acid encoding the antigen binding molecule, a host cell comprising the nucleic acid, a method for producing the antigen binding molecule, and an immunoconjugate comprising the antigen binding molecule and a cytotoxic agent.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to anti-CD137 antigen binding molecules and methods of use thereof. [Background technology]

[0002] Cancer is a fatal disease that is difficult to cure, with some exceptions. Therapeutic outcomes using chemotherapy, the primary treatment, are not particularly favorable. It has been suggested that the heterogeneity of cancer cells as well as the tumor microenvironment play a major role in the difficulty of cancer treatment (Non-Patent Document 1). In recent years, anti-CTLA-4 antibodies, which inhibit the function of CTLA-4, which suppresses immune responses, and promote T cell activation, have demonstrated the potential for curing inoperable malignant melanoma and other cancers (Non-Patent Document 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 immunostimulatory antibody drug. Furthermore, the therapeutic effects of inhibitors of immune checkpoint molecules other than CTLA-4, such as PD-1 and PD-L1, have also been reported (Non-Patent Document 3), and these antibodies have been approved by the FDA. Activation of T cells, which play an important role in tumor immunity, is understood to be mediated by two signals: 1) binding and activation of the T cell receptor (TCR) to antigen peptides presented by major histocompatibility complex (MHC) class I molecules; and 2) binding and activation of costimulatory molecules on the T cell surface to their ligands on antigen-presenting cells. Furthermore, it has been reported that activation of costimulatory molecules belonging to the tumor necrosis factor receptor superfamily (TNFRSF), including CD137 (4-1BB) on the T cell surface, is important for T cell activation (Non-Patent Document 4).

[0003] TNFRSF includes molecules such as CD137, CD40, OX40, RANK, and GITR. It has been reported that CD137 is 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 (Non-Patent Document 5). The antitumor effects of CD137 agonist antibodies have already been demonstrated in mouse models, and experimental studies in mouse models have shown that this effect is primarily due to the activation of CD8+ T cells and NK cells (Non-Patent Document 6). However, the nonspecific hepatotoxicity of CD137 agonist antibodies has been a problem in both clinical and non-clinical settings, and drug development has not progressed as expected (Non-Patent Documents 7 and 8). The main cause of these side effects has been suggested to be the activation of immune cells in non-immune tissues other than tumors, such as the liver, which is mediated by binding to Fcγ receptors via the antibody constant region (Non-Patent Document 9). On the other hand, it has been reported that agonist antibodies of receptors belonging to the TNF receptor superfamily require cross-linking of the antibody by Fcγ receptor-expressing cells (FcγRII-expressing cells) in order to exhibit agonistic activity in vivo (Non-Patent Document 10). In other words, since both the antitumor efficacy of CD137 agonist antibodies and side effects such as hepatotoxicity are related to antibody binding to Fcγ receptors, increasing antibody Fcγ receptor binding is expected to improve efficacy but also increase hepatotoxicity, while reducing antibody Fcγ receptor binding is thought to reduce side effects but also reduce efficacy. To date, no CD137 agonist antibodies have been reported that separate efficacy from side effects. Furthermore, the antitumor effect of CD137 agonist antibodies in clinical settings is not particularly strong, and further enhancement of efficacy while avoiding toxicity is desired. Therefore, the development of new drugs that can induce antitumor immune responses while suppressing these side effects is desired.

[0004] When a therapeutic antibody is administered in vivo, it is desirable that the target antigen be specifically expressed only at the lesion site. However, in many cases, the same antigen is also expressed in non-lesioned normal tissues, which can cause undesirable side effects from a therapeutic perspective. For example, while an antibody against a tumor antigen can exhibit cytotoxic activity against tumor cells through ADCC or other mechanisms, if the same antigen is also expressed in normal tissues, it may also cytotoxicize normal cells. To solve the above problems, a technology has been developed that focuses on the phenomenon in which specific compounds are present in large amounts in target tissues (e.g., tumor tissues) and searches for antigen-binding molecules whose antigen-binding activity changes depending on the concentration of such compounds (e.g., Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. WO2013 / 180200 [Non-patent literature]

[0006] [Non-Patent Document 1] Hanahan, Cell, 2011, 144, 646-74 [Non-patent document 2] Prieto, Clin Cancer Res. 2012, 18, 2039-47 [Non-patent document 3] Hamid, Expert Opin. Biol. Ther., 2013, 6, 847-61 [Non-patent document 4] Summers, Nat Rev Immunol, 2012, 12, 339-51 [Non-patent document 5] Vinay, Cellular & Molecular Immunology, 2011, 8, 281-284 [Non-patent document 6] Houot, Blood, 2009, 114, 3431-8 [Non-Patent Document 7] Ascierto, Semin Oncol, 2010, 37, 508-16 [Non-patent document 8] Dubrot, Cancer Immunol Immunother, 2010, 59, 1223-33 [Non-Patent Document 9] Schabowsky, Vaccine, 2009, 28, 512-22 [Non-Patent Document 10] Li, Proc Natl Acad Sci US A. 2013, 110(48), 19501-6 Summary of the Invention [Problem to be solved by the invention]

[0007] The present disclosure relates to anti-CD137 antigen binding molecules and methods of use thereof. [Means for solving the problem]

[0008] The present disclosure provides anti-CD137 antigen-binding molecules that have immune cell activation, cytotoxic activity, or anti-tumor activity but have low activity against non-tumor tissues such as normal tissues and few side effects, as well as methods for using them. The present disclosure also provides anti-CD137 antigen-binding molecules whose CD137-binding activity varies depending on various substances (e.g., small molecular weight compounds) in target tissues (e.g., tumor tissues), as well as methods for using the same, pharmaceutical formulations, etc. In one aspect, the anti-CD137 antigen-binding molecules of the present disclosure have few side effects, so the dosage can be increased without concern for side effects, and as a result, stronger pharmaceutical efficacy (cytotoxic activity or anti-tumor activity) can be exhibited. Specifically, the present disclosure provides anti-CD137 antigen-binding molecules, methods for using the same, pharmaceutical formulations, and the like, which are exemplified below. [1] An anti-CD137 antigen-binding molecule that has CD137-binding activity dependent on the small molecule compound. [2] The anti-CD137 antigen-binding molecule of [1], whose binding activity to CD137 in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM of a small molecule compound is at least two-fold higher than its binding activity to CD137 in the absence of the small molecule compound. [2.1] The anti-CD137 antigen-binding molecule of [1] or [2], whose binding activity to CD137 in the presence of 10 μM or more of a small molecule compound is at least two-fold higher than its binding activity to CD137 in the absence of the small molecule compound. [2.2] KD value for CD137 in the presence of 10 μM or more of small molecule compounds is 5x10 -7 The anti-CD137 antigen-binding molecule of any of [1] to [2.1], which has a molecular weight of M or less. [2.3] KD value for CD137 in the absence of small molecule compounds is 1x10 -6 The anti-CD137 antigen-binding molecule of any of [1] to [2.2], having a molecular weight of M or more. [2.4] The KD value for CD137 in a solution prepared so that the concentration of the small molecule compound is 10 μM or higher is 5x10 -7 M or less, and the KD value for CD137 in a solution without the addition of small molecule compounds is 1x10 -6 The anti-CD137 antigen-binding molecule of [1], wherein the antibody has a nucleotide sequence of M or more. [2.5] The anti-CD137 antigen-binding molecule of [1], wherein the KD value for CD137 in a solution prepared so that the concentration of the small molecule compound is 10 μM or higher, and the KD value for CD137 in a solution to which no small molecule compound has been added, are each measured by Biacore assay within 24 hours after contacting CD137 with the anti-CD137 antigen-binding molecule in the solution. [2.6] The anti-CD137 antigen-binding molecule of any of [1] to [2.5], which forms a ternary complex together with a low molecular weight compound and CD137. [2.7] The anti-CD137 antigen-binding molecule of any of [1] to [2.6], which binds to human and monkey-derived CD137. [2.8] The anti-CD137 antigen-binding molecule of any of [1] to [2.7], wherein the low molecular weight compound is an adenosine-containing compound. [2.9] The anti-CD137 antigen-binding molecule of any of [1] to [2.8], wherein the low molecular weight compound is ATP. [3] The anti-CD137 antigen-binding molecule of any of [1] to [2.9], comprising any combination of HVR-H1, HVR-H2, and HVR-H3 selected from the following (a) to (k): (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17; (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, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17; (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, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17; (d) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (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, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (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, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (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, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (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, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19; (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20; (j) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 16, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20; and (k) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17. [3.1] The anti-CD137 antigen-binding molecule of any of [1] to [3], comprising any combination of HVR-L1, HVR-L2, and HVR-L3 selected from the following (a) to (g): (a) 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; (b) 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-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; (d) 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; (e) 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; (f) 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 (g) 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. [4] An anti-CD137 antigen binding molecule comprising any combination of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 selected from the following (a) to (m): (a) 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: 17, 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; (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 comprising the amino acid sequence of SEQ ID NO: 7, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 11, 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; (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 comprising the amino acid sequence of SEQ ID NO: 7, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20, 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; (j) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, 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; (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 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: 17, 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. [5] (a) a VH having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 43 to 53; or (b) An anti-CD137 antigen-binding molecule comprising a VL having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 54 to 60. [5.1] An anti-CD137 antigen-binding molecule comprising any combination of VH and VL selected from the following (a) to (m): (a) a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 43, and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 54; (b) a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 44, and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 55; (c) a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 45, and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 55; (d) a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 46, and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 54; (e) a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 47, and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 54; (f) a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 48, and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 56; (g) a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 49, and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 57; (h) a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 50, and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 58; (i) a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 51, and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 59; (j) a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 51, and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 60: (k) a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 52, and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 60; (l) a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 50, and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 59; and (m) a VH having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 53, and a VL having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 54. [5.2] An anti-CD137 antigen-binding molecule comprising any combination of VH and VL selected from the following (a) to (m): (a) a VH comprising the amino acid sequence of SEQ ID NO: 43, and a VL comprising the amino acid sequence of SEQ ID NO: 54; (b) a VH comprising the amino acid sequence of SEQ ID NO: 44, and a VL comprising the amino acid sequence of SEQ ID NO: 55; (c) a VH comprising the amino acid sequence of SEQ ID NO: 45, and a VL comprising the amino acid sequence of SEQ ID NO: 55; (d) a VH comprising the amino acid sequence of SEQ ID NO: 46, and a VL comprising the amino acid sequence of SEQ ID NO: 54; (e) a VH comprising the amino acid sequence of SEQ ID NO: 47, and a VL comprising the amino acid sequence of SEQ ID NO: 54; (f) a VH comprising the amino acid sequence of SEQ ID NO: 48, and a VL comprising the amino acid sequence of SEQ ID NO: 56; (g) a VH comprising the amino acid sequence of SEQ ID NO: 49, and a VL comprising the amino acid sequence of SEQ ID NO: 57; (h) a VH comprising the amino acid sequence of SEQ ID NO: 50, and a VL comprising the amino acid sequence of SEQ ID NO: 58; (i) a VH comprising the amino acid sequence of SEQ ID NO: 51, and a VL comprising the amino acid sequence of SEQ ID NO: 59; (j) a VH comprising the amino acid sequence of SEQ ID NO: 51, and a VL comprising the amino acid sequence of SEQ ID NO: 60: (k) a VH comprising the amino acid sequence of SEQ ID NO: 52, and a VL comprising the amino acid sequence of SEQ ID NO: 60; (l) a VH comprising the amino acid sequence of SEQ ID NO: 50, and a VL comprising the amino acid sequence of SEQ ID NO: 59; and (m) VH comprising the amino acid sequence of SEQ ID NO: 53, and VL comprising the amino acid sequence of SEQ ID NO: 54. [5.3] An anti-CD137 antigen-binding molecule in which the value of [CD137-binding activity (amount of binding) in the presence of 10 μM or more of a small molecule compound] / [CD137-binding activity (amount of binding) in the absence of the small molecule compound] is the same as or greater than that of a reference antigen-binding molecule, wherein the reference antigen-binding molecule is an anti-CD137 antigen-binding molecule comprising a combination of 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: 17, 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. [5.4] The anti-CD137 antigen-binding molecule of [5.3], wherein the reference antigen-binding molecule is an anti-CD137 antigen-binding molecule comprising a combination of a VH comprising the amino acid sequence of SEQ ID NO: 43 and a VL comprising the amino acid sequence of SEQ ID NO: 54. [5.5] an anti-CD137 antigen-binding molecule having a value of [binding activity (KD) to CD137 in the presence of 1 μM of a small molecule compound] / [binding activity (KD) to CD137 in the presence of 10 μM or more of the small molecule compound] that is the same as or greater than that of a reference antigen-binding molecule, wherein the reference antigen-binding molecule is an anti-CD137 antigen-binding molecule comprising a combination of: 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: 17; 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. [5.6] The anti-CD137 antigen-binding molecule of [5.5], wherein the reference antigen-binding molecule is an anti-CD137 antigen-binding molecule comprising a combination of a VH comprising the amino acid sequence of SEQ ID NO: 43 and a VL comprising the amino acid sequence of SEQ ID NO: 54. [5.7] An anti-CD137 antigen-binding molecule that competes with any of the antigen-binding molecules of [3] to [5.2] for binding to CD137 in the presence of a small molecular weight compound at 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 of the small molecular weight compound, and has CD137-binding activity dependent on the small molecular weight compound. [5.8] An anti-CD137 antigen-binding molecule that binds to the same CD137 epitope as that bound by any of the antigen-binding molecules of [3] to [5.2] in the presence of a small molecule compound at 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 of the small molecule compound, and has CD137-binding activity dependent on the small molecule compound. [5.8A] The anti-CD137 antigen-binding molecule of any of [5.3] to [5.8], wherein the low molecular weight compound is an adenosine-containing compound. [5.8B] The anti-CD137 antigen-binding molecule of any of [5.3] to [5.8A], wherein the low molecular weight compound is ATP. [5.9] The anti-CD137 antigen-binding molecule of any of [1] to [5.8B], which is a monoclonal antibody or an antigen-binding fragment thereof. [5.10] The anti-CD137 antigen-binding molecule of any of [1] to [5.9], which is a human antibody, a humanized antibody, or a chimeric antibody, or an antigen-binding fragment thereof. [5.11] The anti-CD137 antigen-binding molecule of any of [1] to [5.10], which is a full-length IgG1 antibody. [5.12] The anti-CD137 antigen-binding molecule of any of [1] to [5.11], which comprises an altered Fc region in which at least one amino acid has been altered, and the altered Fc region has increased FcγRIIb-binding activity compared to a parent Fc region that does not contain the amino acid alteration. [5.13] The anti-CD137 antigen-binding molecule of [5.12], wherein the FcγRIIb-binding activity of the modified Fc is the same as or higher than that of a reference Fc region, wherein the reference Fc is a human IgG1 Fc region containing a combination of amino acid substitutions of G236N / H268D / A330K according to EU numbering. [5.14] An anti-CD137 antigen-binding molecule according to [5.12] or [5.13], wherein the reference Fc region comprises the amino acid sequence of SEQ ID NO: 153. [5.15] The anti-CD137 antigen-binding molecule of [5.12], wherein the at least one amino acid modification is at least one amino acid substitution selected from the group consisting of G236N, H268D, and A330K (EU numbering). [5.16] The anti-CD137 antigen-binding molecule of [5.12] or [5.15], wherein the at least one amino acid modification is a combination of amino acid substitutions of G236N / H268D / A330K based on EU numbering. [5.17] The anti-CD137 antigen-binding molecule of any of [5.12] to [5.16], wherein the parent Fc region is derived from a human IgG1 Fc region. [5.18] The anti-CD137 antigen-binding molecule of any of [1] to [5.17], which comprises a modified Fc region in which at least one amino acid has been modified, and which has an increased isoelectric point (pI) compared to a parent anti-CD137 antigen-binding molecule comprising a parent Fc region that does not contain the amino acid modification. [5.19] The anti-CD137 antigen-binding molecule according to [5.18], wherein the at least one amino acid modification is a modification of an amino acid residue that can be exposed on the surface of the parent Fc region. [5.20] The at least one amino acid modification is (i) a modification in which at least one amino acid residue having a negative charge in its side chain in the parent Fc region is replaced with an amino acid residue having no charge in its side chain; (ii) a modification in which at least one uncharged amino acid residue in the parent Fc region is replaced with an amino acid residue having a positive charge in the side chain; and / or (iii) a modification in which at least one negatively charged amino acid residue in the parent Fc region is replaced with an amino acid residue having a positively charged side chain; The anti-CD137 antigen-binding molecule of [5.18] or [5.19], [5.21] The anti-CD137 antigen-binding molecule of any of [5.18] to [5.20], wherein the at least one amino acid modification is a combination of multiple amino acid substitutions, and the multiple amino acid substitutions are located at positions that are structurally close to each other. [5.22] The anti-CD137 antigen-binding molecule of any of [5.18] to [5.21], wherein the binding activity of the modified Fc region to Fcγ receptors (FcγR) is not substantially reduced compared to the parent Fc region. [5.23] The anti-CD137 antigen-binding molecule of [5.22], wherein the Fcγ receptor (FcγR) is FcγRIIb. [5.24] The anti-CD137 antigen-binding molecule of any of [5.18] to [5.23], wherein the at least one amino acid modification is at least one amino acid substitution selected from the group consisting of Q311R, P343R, and D413K, based on EU numbering. [5.25] The anti-CD137 antigen-binding molecule of any of [5.18] to [5.24], wherein the at least one amino acid modification is a combination of (i) a P343R amino acid substitution, (ii) a Q311R / P343R amino acid substitution, or (iii) a Q311R / D413K amino acid substitution, based on EU numbering. [6] The anti-CD137 antigen-binding molecule of any one of [1] to [5.25], comprising a modified Fc region, wherein the modified Fc region comprises any one combination of amino acid modifications selected from the following, based on EU numbering: 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. [6.1] The anti-CD137 antigen-binding molecule of any of [1] to [6], wherein the modified Fc region is derived from a human IgG1 Fc region. [6.2] The anti-CD137 antigen-binding molecule of any of [1] to [6.1], wherein the modified Fc region further comprises deletions at positions 446 and 447 according to EU numbering. [7] The anti-CD137 antigen-binding molecule of any of [1] to [6.2], comprising a heavy chain constant region comprising any one of the amino acid sequences of SEQ ID NOs: 64 to 85. [7.1] An anti-CD137 antigen-binding molecule comprising any combination of VH, VL, CH and CL selected from the following (i) to (xxxviii): (i) a VH comprising the amino acid sequence of SEQ ID NO: 43, a CH comprising the amino acid sequence of SEQ ID NO: 64, a VL comprising the amino acid sequence of SEQ ID NO: 54, and a CL comprising the amino acid sequence of SEQ ID NO: 63; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 43, a CH comprising the amino acid sequence of SEQ ID NO: 66, a VL comprising the amino acid sequence of SEQ ID NO: 54, and a CL comprising the amino acid sequence of SEQ ID NO: 63; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 43, a CH comprising the amino acid sequence of SEQ ID NO: 67, a VL comprising the amino acid sequence of SEQ ID NO: 54, and a CL comprising the amino acid sequence of SEQ ID NO: 63; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 43, a CH comprising the amino acid sequence of SEQ ID NO: 68, a VL comprising the amino acid sequence of SEQ ID NO: 54, and a CL comprising the amino acid sequence of SEQ ID NO: 63; (v) a VH comprising the amino acid sequence of SEQ ID NO: 43, a CH comprising the amino acid sequence of SEQ ID NO: 69, a VL comprising the amino acid sequence of SEQ ID NO: 54, and a CL comprising the amino acid sequence of SEQ ID NO: 63; (vi) a VH comprising the amino acid sequence of SEQ ID NO: 43, a CH comprising the amino acid sequence of SEQ ID NO: 70, a VL comprising the amino acid sequence of SEQ ID NO: 54, and a CL comprising the amino acid sequence of SEQ ID NO: 63; (vii) a VH comprising the amino acid sequence of SEQ ID NO: 43, a CH comprising the amino acid sequence of SEQ ID NO: 71, a VL comprising the amino acid sequence of SEQ ID NO: 54, and a CL comprising the amino acid sequence of SEQ ID NO: 63; (viii) a VH comprising the amino acid sequence of SEQ ID NO: 43, a CH comprising the amino acid sequence of SEQ ID NO: 73, a VL comprising the amino acid sequence of SEQ ID NO: 54, and a CL comprising the amino acid sequence of SEQ ID NO: 63; (ix) a VH comprising the amino acid sequence of SEQ ID NO: 43, a CH comprising the amino acid sequence of SEQ ID NO: 75, a VL comprising the amino acid sequence of SEQ ID NO: 54, and a CL comprising the amino acid sequence of SEQ ID NO: 63; (x) a VH comprising the amino acid sequence of SEQ ID NO: 43, a CH comprising the amino acid sequence of SEQ ID NO: 78, a VL comprising the amino acid sequence of SEQ ID NO: 54, and a CL comprising the amino acid sequence of SEQ ID NO: 63; (xi) a VH comprising the amino acid sequence of SEQ ID NO: 43, a CH comprising the amino acid sequence of SEQ ID NO: 80, a VL comprising the amino acid sequence of SEQ ID NO: 54, and a CL comprising the amino acid sequence of SEQ ID NO: 63; (xii) a VH comprising the amino acid sequence of SEQ ID NO: 43, a CH comprising the amino acid sequence of SEQ ID NO: 82, a VL comprising the amino acid sequence of SEQ ID NO: 54, and a CL comprising the amino acid sequence of SEQ ID NO: 63; (xiii) a VH comprising the amino acid sequence of SEQ ID NO: 43, a CH comprising the amino acid sequence of SEQ ID NO: 84, a VL comprising the amino acid sequence of SEQ ID NO: 54, and a CL comprising the amino acid sequence of SEQ ID NO: 63; (xiv) a VH comprising the amino acid sequence of SEQ ID NO: 43, a CH comprising the amino acid sequence of SEQ ID NO: 85, a VL comprising the amino acid sequence of SEQ ID NO: 54, and a CL comprising the amino acid sequence of SEQ ID NO: 63; (xv) a VH comprising the amino acid sequence of SEQ ID NO: 51, a CH comprising the amino acid sequence of SEQ ID NO: 65, a VL comprising the amino acid sequence of SEQ ID NO: 59, and a CL comprising the amino acid sequence of SEQ ID NO: 63; (xvi) a VH comprising the amino acid sequence of SEQ ID NO: 51, a CH comprising the amino acid sequence of SEQ ID NO: 72, a VL comprising the amino acid sequence of SEQ ID NO: 59, and a CL comprising the amino acid sequence of SEQ ID NO: 63; (xvii) a VH comprising the amino acid sequence of SEQ ID NO: 51, a CH comprising the amino acid sequence of SEQ ID NO: 74, a VL comprising the amino acid sequence of SEQ ID NO: 59, and a CL comprising the amino acid sequence of SEQ ID NO: 63; (xviii) VH comprising the amino acid sequence of SEQ ID NO: 51, CH comprising the amino acid sequence of SEQ ID NO: 75, VL comprising the amino acid sequence of SEQ ID NO: 59, and CL comprising the amino acid sequence of SEQ ID NO: 63; (xix) a VH comprising the amino acid sequence of SEQ ID NO: 51, a CH comprising the amino acid sequence of SEQ ID NO: 77, a VL comprising the amino acid sequence of SEQ ID NO: 59, and a CL comprising the amino acid sequence of SEQ ID NO: 63; (xx) VH comprising the amino acid sequence of SEQ ID NO: 51, CH comprising the amino acid sequence of SEQ ID NO: 78, VL comprising the amino acid sequence of SEQ ID NO: 59, and CL comprising the amino acid sequence of SEQ ID NO: 63; (xxi) a VH comprising the amino acid sequence of SEQ ID NO: 51, a CH comprising the amino acid sequence of SEQ ID NO: 79, a VL comprising the amino acid sequence of SEQ ID NO: 59, and a CL comprising the amino acid sequence of SEQ ID NO: 63; (xxii) VH comprising the amino acid sequence of SEQ ID NO: 51, CH comprising the amino acid sequence of SEQ ID NO: 80, VL comprising the amino acid sequence of SEQ ID NO: 59, and CL comprising the amino acid sequence of SEQ ID NO: 63; (xxiii) VH comprising the amino acid sequence of SEQ ID NO: 51, CH comprising the amino acid sequence of SEQ ID NO: 81, VL comprising the amino acid sequence of SEQ ID NO: 59, and CL comprising the amino acid sequence of SEQ ID NO: 63; (xxiv) VH comprising the amino acid sequence of SEQ ID NO: 51, CH comprising the amino acid sequence of SEQ ID NO: 82, VL comprising the amino acid sequence of SEQ ID NO: 59, and CL comprising the amino acid sequence of SEQ ID NO: 63; (xxv) VH comprising the amino acid sequence of SEQ ID NO: 51, CH comprising the amino acid sequence of SEQ ID NO: 83, VL comprising the amino acid sequence of SEQ ID NO: 59, and CL comprising the amino acid sequence of SEQ ID NO: 63; (xxvi) a VH comprising the amino acid sequence of SEQ ID NO: 51, a CH comprising the amino acid sequence of SEQ ID NO: 84, a VL comprising the amino acid sequence of SEQ ID NO: 59, and a CL comprising the amino acid sequence of SEQ ID NO: 63; (xxvii) VH comprising the amino acid sequence of SEQ ID NO: 51, CH comprising the amino acid sequence of SEQ ID NO: 72, VL comprising the amino acid sequence of SEQ ID NO: 60, and CL comprising the amino acid sequence of SEQ ID NO: 63; (xxviii) VH comprising the amino acid sequence of SEQ ID NO: 51, CH comprising the amino acid sequence of SEQ ID NO: 74, VL comprising the amino acid sequence of SEQ ID NO: 60, and CL comprising the amino acid sequence of SEQ ID NO: 63; (xxix) a VH comprising the amino acid sequence of SEQ ID NO: 51, a CH comprising the amino acid sequence of SEQ ID NO: 75, a VL comprising the amino acid sequence of SEQ ID NO: 60, and a CL comprising the amino acid sequence of SEQ ID NO: 63; (xxx) VH comprising the amino acid sequence of SEQ ID NO: 51, CH comprising the amino acid sequence of SEQ ID NO: 77, VL comprising the amino acid sequence of SEQ ID NO: 60, and CL comprising the amino acid sequence of SEQ ID NO: 63; (xxxi) VH comprising the amino acid sequence of SEQ ID NO: 51, CH comprising the amino acid sequence of SEQ ID NO: 78, VL comprising the amino acid sequence of SEQ ID NO: 60, and CL comprising the amino acid sequence of SEQ ID NO: 63; (xxxii) VH comprising the amino acid sequence of SEQ ID NO: 51, CH comprising the amino acid sequence of SEQ ID NO: 79, VL comprising the amino acid sequence of SEQ ID NO: 60, and CL comprising the amino acid sequence of SEQ ID NO: 63; (xxxiii) VH comprising the amino acid sequence of SEQ ID NO: 51, CH comprising the amino acid sequence of SEQ ID NO: 80, VL comprising the amino acid sequence of SEQ ID NO: 60, and CL comprising the amino acid sequence of SEQ ID NO: 63; (xxxiv) VH comprising the amino acid sequence of SEQ ID NO: 51, CH comprising the amino acid sequence of SEQ ID NO: 81, VL comprising the amino acid sequence of SEQ ID NO: 60, and CL comprising the amino acid sequence of SEQ ID NO: 63; (xxxv) VH comprising the amino acid sequence of SEQ ID NO: 51, CH comprising the 82 amino acid sequence of SEQ ID NO: , VL comprising the amino acid sequence of SEQ ID NO: 60, and CL comprising the amino acid sequence of SEQ ID NO: 63; (xxxvi) VH comprising the amino acid sequence of SEQ ID NO: 51, CH comprising the 83 amino acid sequence of SEQ ID NO: , VL comprising the amino acid sequence of SEQ ID NO: 60, and CL comprising the amino acid sequence of SEQ ID NO: 63; (xxxvii) a VH comprising the amino acid sequence of SEQ ID NO: 51, a CH comprising the amino acid sequence of SEQ ID NO: 84, a VL comprising the amino acid sequence of SEQ ID NO: 60, and a CL comprising the amino acid sequence of SEQ ID NO: 63; and (xxxviii) VH comprising the amino acid sequence of SEQ ID NO: 51, CH comprising the amino acid sequence of SEQ ID NO: 85, VL comprising the amino acid sequence of SEQ ID NO: 60, and CL comprising the amino acid sequence of SEQ ID NO: 63. [8] An isolated nucleic acid encoding the anti-CD137 antigen-binding molecule of any one of [1] to [7.1]. [9] A vector comprising the nucleic acid according to [8].

[10] A host cell comprising the nucleic acid of [8] or the vector of [9].

[11] A method for producing an anti-CD137 antigen-binding molecule, comprising culturing the host cell of

[10] so that the anti-CD137 antigen-binding molecule is produced.

[12] An immunoconjugate comprising the anti-CD137 antigen-binding molecule of any one of [1] to [7.1] and a cytotoxic agent.

[13] A pharmaceutical formulation comprising the anti-CD137 antigen-binding molecule of any of [1] to [7.1] or the immunoconjugate of

[12] ; and a pharmaceutically acceptable carrier.

[14] An anti-CD137 antigen-binding molecule according to any one of [1] to [7.1] or an immunoconjugate according to

[12] for use as a pharmaceutical. 14.1 An anti-CD137 antigen-binding molecule according to any one of [1] to [7.1], an immunoconjugate according to

[12] , or a pharmaceutical formulation according to

[13] , for use in treating a tumor. 14.2 The anti-CD137 antigen-binding molecule, immunoconjugate, or pharmaceutical preparation according to [14.1], wherein the tumor is a solid tumor infiltrated with B cells, dendritic cells, natural killer cells, macrophages, and / or CD8-positive T cells. 14.3 The anti-CD137 antigen-binding molecule, immunoconjugate, or pharmaceutical preparation according to [14.1], wherein the tumor is a solid tumor infiltrated with regulatory T (Treg) cells.

[15] An anti-CD137 antigen-binding molecule according to any one of [1] to [7.1], an immunoconjugate according to

[12] , or a pharmaceutical formulation according to

[13] , for use in activating immune cells. 15.1 The anti-CD137 antigen-binding molecule, immunoconjugate, or pharmaceutical preparation of

[15] , wherein the immune cells are B cells, dendritic cells, natural killer cells, macrophages, and / or T cells. 15.2 The anti-CD137 antigen-binding molecule of any one of [1] to [7.1] or the pharmaceutical preparation of

[13] for activating immune cells in tumor tissue. 15.3 The anti-CD137 antigen binding molecule or pharmaceutical preparation according to [15.2], wherein the immune cells are B cells, dendritic cells, natural killer cells, macrophages, and / or T cells. 15.4 An anti-CD137 antigen-binding molecule according to any one of [1] to [7.1], an immunoconjugate according to

[12] , or a pharmaceutical formulation according to

[13] , for use in cell damage.

[16] The anti-CD137 antigen-binding molecule of any of [1] to [7.1], the immunoconjugate of

[12] , or the pharmaceutical formulation of

[13] , which exhibits a lower level of immune activation in non-tumor tissues compared to an anti-CD137 antigen-binding molecule that does not have CD137-binding activity dependent on a small molecule compound. 16.1 The anti-CD137 antigen-binding molecule, immunoconjugate, or pharmaceutical preparation of

[16] , wherein the non-tumor tissue is a lymph node, spleen, and / or liver. 16.2 The anti-CD137 antigen-binding molecule of any of [1] to [7.1] or the immunoconjugate of

[12] , which does not substantially bind to CD137 expressed in non-tumor tissues. 16.3 The anti-CD137 antigen-binding molecule of any of [1] to [7.1] or the immunoconjugate of

[12] , which has a prolonged blood half-life compared to an anti-CD137 antigen-binding molecule that does not have CD137-binding activity dependent on a low molecular weight compound.

[17] The anti-CD137 antigen-binding molecule of any of [1] to [7.1], the immunoconjugate of

[12] , or the pharmaceutical formulation of

[13] , which has a reduced level of side effects compared to an anti-CD137 antigen-binding molecule that does not have CD137-binding activity dependent on a small molecule compound. 17.1 The anti-CD137 antigen-binding molecule, immunoconjugate, or pharmaceutical preparation of

[17] , wherein the adverse effects are increased AST, increased ALT, fever, nausea, acute hepatitis, liver damage, splenomegaly, enterocolitis, suppurative inflammation of the skin, neutropenia, lymphopenia, thrombocytopenia, transaminase expression, and / or hyperbilirubinemia.

[18] An anti-CD137 antigen-binding molecule that exhibits CD137 agonist activity dependent on the small molecule compound. 18.1 The anti-CD137 antigen-binding molecule of

[18] , whose agonistic activity against CD137 in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM of a small molecule compound is at least two-fold higher than the agonistic activity against CD137 in the absence of the small molecule compound. 18.2 The anti-CD137 antigen-binding molecule of

[18] or [18.1], wherein the agonist activity against CD137 in the presence of 10 μM or more of a small molecule compound is at least two-fold higher than the agonist activity against CD137 in the absence of the small molecule compound. 18.3 The anti-CD137 antigen-binding molecule of

[18] or [18.1], wherein the agonist activity against CD137 in the presence of 50 μM or more of a small molecule compound is at least two-fold higher than the agonist activity against CD137 in the absence of the small molecule compound. 18.4 The anti-CD137 antigen-binding molecule of

[18] or [18.1], wherein the agonist activity against CD137 in the presence of 250 μM or more of a small molecule compound is at least two-fold higher than the agonist activity against CD137 in the absence of the small molecule compound. 18.5 The anti-CD137 antigen-binding molecule of any of

[18] to [18.4], wherein the agonistic activity against CD137 is assessed by the amount of IL-2 and / or IFN-γ produced by CD137-expressing cells. 18.6 The anti-CD137 antigen-binding molecule according to [18.5], wherein the CD137-expressing cells are isolated human peripheral blood mononuclear cells (PBMCs) or human peripheral blood mononuclear cell (PBMC)-derived T cells. 18.7 The anti-CD137 antigen-binding molecule of any of

[18] to [18.4], wherein the agonist activity against CD137 is assessed by a reporter gene assay. 18.8 The anti-CD137 antigen-binding molecule of

[18] , which exhibits agonistic activity against CD137 in a solution prepared so that the final concentration of the low molecular weight compound is 50 μM or more, and which exhibits substantially no agonistic activity against CD137 in a solution to which the low molecular weight compound is not added. 18.9 The anti-CD137 antigen-binding molecule according to [18.8], wherein the agonistic activity against CD137 in a solution prepared so that the final concentration of the small molecule compound is 50 μM or more, and the agonistic activity against CD137 in a solution to which the small molecule compound is not added, are each assessed by the amount of IL-2, IFN-γ, and / or IL-6 produced, measured within 72 hours after contacting CD137-expressing cells with the anti-CD137 antigen-binding molecule in the solution. [18.10] The anti-CD137 antigen-binding molecule according to [18.8], wherein the agonistic activity against CD137 in a solution prepared so that the final concentration of the small molecule compound is 50 μM or more, and the agonistic activity against CD137 in a solution to which the small molecule compound is not added, are assessed by luciferase luminescence signals measured within 6 hours after contacting the anti-CD137 antigen-binding molecule with an NF-kappaB-luciferase reporter construct and T cells expressing CD137. [18.11] The anti-CD137 antigen-binding molecule of any of

[18] to [18.10], wherein the low molecular weight compound is an adenosine-containing compound. [18.12] The anti-CD137 antigen-binding molecule of any of

[18] to [18.11], wherein the low molecular weight compound is ATP.

[19] The anti-CD137 antigen-binding molecule of any of [1] to [7.1], which has CD137 agonist activity dependent on the small molecule compound. 19.1 The anti-CD137 antigen-binding molecule of

[19] , whose agonistic activity against CD137 in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM of a small molecule compound is at least two-fold higher than the agonistic activity against CD137 in the absence of the small molecule compound. 19.2 The anti-CD137 antigen-binding molecule of

[19] or [19.1], wherein the agonist activity against CD137 in the presence of 10 μM or more of a small molecule compound is at least two-fold higher than the agonist activity against CD137 in the absence of the small molecule compound. 19.3 The anti-CD137 antigen-binding molecule of

[19] or [19.1], wherein the agonist activity against CD137 in the presence of 50 μM or more of a small molecule compound is at least two-fold higher than the agonist activity against CD137 in the absence of the small molecule compound. 19.4 The anti-CD137 antigen-binding molecule of

[19] or [19.1], wherein the agonist activity against CD137 in the presence of 250 μM or more of a small molecule compound is at least two-fold higher than the agonist activity against CD137 in the absence of the small molecule compound. 19.5 The anti-CD137 antigen-binding molecule of any of

[19] to [19.4], wherein the agonistic activity against CD137 is assessed by the amount of IL-2 and / or IFN-γ produced by CD137-expressing cells. 19.6 The anti-CD137 antigen-binding molecule according to [19.5], wherein the CD137-expressing cells are isolated human peripheral blood mononuclear cells (PBMCs) or human peripheral blood mononuclear cell (PBMC)-derived T cells. 19.7 The anti-CD137 antigen-binding molecule of any of

[19] to [19.4], wherein the agonist activity against CD137 is assessed by a reporter gene assay. 19.8 The anti-CD137 antigen-binding molecule of

[19] , which exhibits agonistic activity against CD137 in a solution prepared so that the final concentration of the low molecular weight compound is 50 μM or more, and which exhibits substantially no agonistic activity against CD137 in a solution to which the low molecular weight compound is not added. 19.9 The anti-CD137 antigen-binding molecule according to [19.8], wherein the agonistic activity against CD137 in a solution prepared so that the final concentration of the small molecule compound is 50 μM or more, and the agonistic activity against CD137 in a solution to which the small molecule compound is not added, are each assessed by the amount of IL-2, IFN-γ, and / or IL-6 produced, measured within 72 hours after contacting CD137-expressing cells with the anti-CD137 antigen-binding molecule in the solution. [19.10] The anti-CD137 antigen-binding molecule according to [19.8], wherein the agonistic activity against CD137 in a solution prepared so that the final concentration of the small molecule compound is 50 μM or more, and the agonistic activity against CD137 in a solution to which the small molecule compound is not added, are assessed by luciferase luminescence signals measured within 6 hours after contacting the anti-CD137 antigen-binding molecule with an NF-kappaB-luciferase reporter construct and T cells expressing CD137. [19.11] The anti-CD137 antigen-binding molecule of any of

[19] to [19.10], wherein the low molecular weight compound is an adenosine-containing compound. [19.12] The anti-CD137 antigen-binding molecule of any of

[19] to [19.11], wherein the low molecular weight compound is ATP.

[20] An agonist antigen-binding molecule comprising an altered Fc region, wherein the altered Fc region contains at least one amino acid modification that results in an increase in the isoelectric point (pI) compared to a parent agonist antigen-binding molecule comprising the parent Fc region, and wherein the altered Fc region has increased agonist activity compared to the parent agonist antigen-binding molecule. 20.1 The agonist antigen-binding molecule of

[20] , wherein the at least one amino acid modification is a modification of an amino acid residue that can be exposed on the surface of the parent Fc region. 20.2 The at least one amino acid modification is (i) a modification in which at least one amino acid residue having a negative charge in its side chain in the parent Fc region is replaced with an amino acid residue having no charge in its side chain; (ii) a modification in which at least one uncharged amino acid residue in the parent Fc region is replaced with an amino acid residue having a positive charge in the side chain; and / or (iii) a modification in which at least one negatively charged amino acid residue in the parent Fc region is replaced with an amino acid residue having a positively charged side chain; The agonist antigen-binding molecule of

[20] or [20.1], 20.3 The agonist antigen-binding molecule of any of

[20] to [20.2], wherein the at least one amino acid modification is a combination of multiple amino acid substitutions, and the multiple amino acid substitutions are located at positions that are structurally close to each other. 20.4 The agonist antigen-binding molecule of any of

[20] to [20.3], wherein the binding activity of the modified Fc region to an Fcγ receptor is not substantially reduced compared to the parent Fc region. 20.5 The agonist antigen-binding molecule of [20.4], wherein the Fcγ receptor is FcγRIIb. 20.6 The agonist antigen-binding molecule of any of

[20] to [20.4], wherein the at least one amino acid modification is at least one amino acid substitution selected from the group consisting of Q311R, P343R, and D413K based on EU numbering. 20.7 The agonist antigen-binding molecule of any of

[20] to [20.6], wherein the at least one amino acid alteration is (i) P343R / D413K, (ii) Q311R / P343R, (iii) P343R, (iv) D413K, (v) Q311R, or (vi) Q311R / D413K, or a combination thereof, based on EU numbering. 20.8 The agonist antigen-binding molecule of any of

[20] to [20.7], which is an anti-CD137 antigen-binding molecule. 20.9 The agonist antigen-binding molecule of any of

[20] to [20.8], which is an anti-CD137 antibody. 〔twenty one〕 A method for producing an agonist antigen-binding molecule comprising a modified Fc region, comprising: introducing at least one amino acid modification into the parent Fc region that results in an increase in the isoelectric point (pI) compared to a parent agonist antigen-binding molecule comprising the parent Fc region; The method, wherein the agonist activity of the agonist antigen-binding molecule comprising the modified Fc region is increased compared to the parent agonist antigen-binding molecule. 21.1 The method of

[21] , wherein the agonist activity of the agonist antigen-binding molecule against an antigen in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM of a small molecule compound is at least two-fold higher than the agonist activity against the antigen in the absence of the small molecule compound. 21.2 The method of

[21] or [21.1], wherein the agonist activity of the agonist antigen-binding molecule against the antigen in the presence of 10 μM or more of a small molecule compound is at least two-fold higher than the agonist activity against the antigen in the absence of the small molecule compound. 21.3 The method of

[21] or [21.1], wherein the agonist activity of the agonist antigen-binding molecule against the antigen in the presence of 50 μM or more of a small molecule compound is at least two-fold higher than the agonist activity against the antigen in the absence of the small molecule compound. 21.4 The method of

[21] or [21.1], wherein the agonist activity of the agonist antigen-binding molecule against the antigen in the presence of 250 μM or more of a small molecule compound is at least two-fold higher than the agonist activity against the antigen in the absence of the small molecule compound. 21.5 The method according to any one of

[21] to [21.4], wherein the agonist activity against the antigen is evaluated based on the amount of IL-2 and / or IFN-γ produced by the antigen-expressing cells. 21.6 The method described in [21.5], wherein the antigen-expressing cells are isolated human peripheral blood mononuclear cells (PBMCs) or human peripheral blood mononuclear cell (PBMC)-derived T cells. 21.7 The method according to any one of

[21] to [21.4], wherein the agonist activity against the antigen is evaluated by a reporter gene assay. 21.8 moreover, (i) obtaining an expression vector comprising a suitable promoter operably linked to a gene encoding the agonist antigen-binding molecule prepared by the method of any one of

[21] to [21.7]; (ii) introducing the vector into a host cell and culturing the host cell to produce the agonist antigen-binding molecule; (iii) recovering the agonist antigen-binding molecule from the host cell culture; The method according to any one of

[21] to [21.7], comprising: 21.9 The method of any of

[21] to [21.8], wherein the agonist antigen-binding molecule is an anti-CD137 antigen-binding molecule. [21.10] The method of any of

[21] to [21.9], wherein the agonist antigen-binding molecule is an anti-CD137 antibody. [21.11] The method according to any one of [21.1] to [21.10], wherein the low molecular weight compound is an adenosine-containing compound. [21.12] The method according to any one of [21.1] to [21.11], wherein the low molecular weight compound is ATP. 〔twenty two〕 A method for increasing the agonist activity of an agonist antigen-binding molecule comprising an Fc region, comprising introducing into the Fc region at least one amino acid modification that results in an increase in the isoelectric point (pI) compared to a parent agonist antigen-binding molecule comprising the parent Fc region. 22.1 The method of

[22] , wherein the agonist activity of the agonist antigen-binding molecule against an antigen in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM of a small molecule compound is at least two-fold higher than the agonist activity against the antigen in the absence of the small molecule compound. 22.2 The method of

[22] or [22.1], wherein the agonist activity of the agonist antigen-binding molecule against the antigen in the presence of 10 μM or more of a small molecule compound is at least two-fold higher than the agonist activity against the antigen in the absence of the small molecule compound. 22.3 The method of

[22] or [22.1], wherein the agonist activity of the agonist antigen-binding molecule against the antigen in the presence of 50 μM or more of a small molecule compound is at least two-fold higher than the agonist activity against the antigen in the absence of the small molecule compound. 22.4 The method of

[22] or [22.1], wherein the agonist activity of the agonist antigen-binding molecule against the antigen in the presence of 250 μM or more of a small molecule compound is at least two-fold higher than the agonist activity against the antigen in the absence of the small molecule compound. [22.5] The method according to any one of

[22] to [22.4], wherein the agonist activity against the antigen is evaluated based on the amount of IL-2 and / or IFN-γ produced by the antigen-expressing cells. 22.6 The method described in [22.5], wherein the antigen-expressing cells are isolated human peripheral blood mononuclear cells (PBMCs) or human peripheral blood mononuclear cell (PBMC)-derived T cells. 22.7 The method according to any one of

[22] to [22.4], wherein the agonist activity against the antigen is evaluated by a reporter gene assay. 22.8 The method of any of

[22] to [22.7], wherein the agonist antigen-binding molecule is an anti-CD137 antigen-binding molecule. 22.9 The method of any of

[22] to [22.8], wherein the agonist antigen-binding molecule is an anti-CD137 antibody. [22.10] The method according to any one of [22.1] to [22.9], wherein the low molecular weight compound is an adenosine-containing compound. [22.11] The method according to any one of [22.1] to [22.10], wherein the low molecular weight compound is ATP. 〔twenty three〕 A method for increasing the agonist activity of an agonist antigen-binding molecule comprising an Fc region, comprising using at least one amino acid modification that results in an increase in the isoelectric point (pI) compared to a parent agonist antigen-binding molecule comprising the parent Fc region. 23.1 The method of

[23] , wherein the agonist activity of the agonist antigen-binding molecule against an antigen in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM of a small molecule compound is at least two-fold higher than the agonist activity against the antigen in the absence of the small molecule compound. 23.2 The method of

[23] or [23.1], wherein the agonist activity of the agonist antigen-binding molecule against the antigen in the presence of 10 μM or more of a small molecule compound is at least two-fold higher than the agonist activity against the antigen in the absence of the small molecule compound. 23.3 The method of

[23] or [23.1], wherein the agonist activity of the agonist antigen-binding molecule against the antigen in the presence of 50 μM or more of a small molecule compound is at least two-fold higher than the agonist activity against the antigen in the absence of the small molecule compound. 23.4 The method of

[23] or [23.1], wherein the agonist activity of the agonist antigen-binding molecule against the antigen in the presence of 250 μM or more of a small molecule compound is at least two-fold higher than the agonist activity against the antigen in the absence of the small molecule compound. [23.5] The method according to any one of

[23] to [23.4], wherein the agonist activity against the antigen is evaluated based on the amount of IL-2 and / or IFN-γ produced by the antigen-expressing cells. 23.6 The method described in [23.5], wherein the antigen-expressing cells are isolated human peripheral blood mononuclear cells (PBMCs) or human peripheral blood mononuclear cell (PBMC)-derived T cells. 23.7 The method according to any one of

[23] to [23.4], wherein the agonist activity against the antigen is evaluated by a reporter gene assay. 23.8 The method of any of

[23] to [23.7], wherein the agonist antigen-binding molecule is an anti-CD137 antigen-binding molecule. 23.9 The method of any of

[23] to [23.8], wherein the agonist antigen-binding molecule is an anti-CD137 antibody. [23.10] The method according to any one of [23.1] to [23.9], wherein the low molecular weight compound is an adenosine-containing compound. [23.11] The method according to any one of [23.1] to [23.10], wherein the low molecular weight compound is ATP. 〔twenty four〕 A method for screening for an antigen-binding domain or antigen-binding molecule having antigen-binding activity dependent on a small molecule compound, comprising: (a) contacting an antigen-binding domain or antigen-binding molecule, or a library thereof, with a fusion molecule in which two or more units of an antigen are fused per unit of a fusion partner molecule in the presence of a low molecular weight compound; (b) placing the antigen-binding domain or antigen-binding molecule that has bound to the antigen in the fusion molecule in step (a) in the absence or presence of a low concentration of the small molecule compound; and (c) isolating the antigen-binding domain or antigen-binding molecule dissociated in step (b); A screening method comprising: 24.1 The method of

[24] , wherein the fusion partner molecule is a dimeric Fc region. 24.2 The method described in [24.1], wherein the Fc region comprises a first Fc subunit and a second Fc subunit, and the antigen is fused to each of the first and second Fc subunits. 24.3 The method described in [24.1] or [24.2], wherein the antigen is fused to the N-terminus of each of the first and second Fc subunits. 24.4 The method of any of

[24] to [24.3], wherein the library of antigen-binding domains or antigen-binding molecules is a phage library. [24.5] The method of any of

[24] to [24.4], wherein the phages contained in the phage library display two or more antigen-binding domains or antigen-binding molecules on their surface. 24.6 The method according to any one of

[24] to [24.5], wherein the phages contained in the phage library are phages having a deletion in the pIII gene derived from a helper phage. 〔twenty five〕 A method for screening for antigen-binding domains or antigen-binding molecules having antigen-binding activity dependent on two or more different low molecular weight compounds, comprising: (a) contacting an antigen with an antigen-binding domain or antigen-binding molecule, or a library thereof, in the presence of a first small molecule compound; (b) placing the antigen-binding domain or antigen-binding molecule that bound to the antigen in step (a) in the absence or presence of a low concentration of the first small molecule compound; (c) isolating the antigen-binding domain or antigen-binding molecule dissociated in step (b); (d) contacting the antigen-binding domain or antigen-binding molecule isolated in step (c) with the antigen in the presence of a second small molecule compound; (e) placing the antigen-binding domain or antigen-binding molecule that bound to the antigen in step (d) in the absence or presence of a low concentration of the second small molecule compound; (f) isolating the antigen-binding domain or antigen-binding molecule dissociated in step (e); This includes: wherein the step (c) and the step (d) do not include amplifying a gene encoding the antigen-binding domain or antigen-binding molecule isolated in the step (c). Screening methods. 25.1 The method of

[25] , wherein the library of antigen-binding domains or antigen-binding molecules is a phage library.

[26] A method for screening for an antigen-binding domain or antigen-binding molecule having antigen-binding activity dependent on a small molecule compound, comprising: (a) contacting an antigen with a naive library of antigen-binding domains or antigen-binding molecules in the presence of a small molecule compound; (b) placing the antigen-binding domain or antigen-binding molecule bound to the antigen in step (a) in the absence or presence of a low concentration of the small molecule compound; and (c) isolating the antigen-binding domain or antigen-binding molecule dissociated in step (b); This includes Here, the screening method is such that the naive library is a phage library containing phages that display two or more antigen-binding domains or antigen-binding molecules on their surface.

[27] A method for screening for an antigen-binding domain or antigen-binding molecule having antigen-binding activity dependent on a small molecule compound, comprising: (a) contacting an antigen with a library of antigen-binding domains or antigen-binding molecules in the presence of a small molecule compound; (b) placing the antigen-binding domain or antigen-binding molecule bound to the antigen in step (a) in the absence or presence of a low concentration of the small molecule compound; and (c) isolating the antigen-binding domain or antigen-binding molecule dissociated in step (b); This includes: Here, the screening method is such that the library contains phages having a deletion in the pIII gene derived from a helper phage.

[28] A method for screening for an antigen-binding domain or antigen-binding molecule having antigen-binding activity dependent on a small molecule compound, comprising: (a) contacting an antigen with a library of antigen-binding domains or antigen-binding molecules in the presence of a small molecule compound; (b) placing the antigen-binding domain or antigen-binding molecule bound to the antigen in step (a) in the absence or presence of a low concentration of the small molecule compound; and (c) isolating the antigen-binding domain or antigen-binding molecule dissociated in step (b); This includes: wherein the library comprises phages prepared by increasing the expression of antigen-binding domains or antigen-binding molecules using a small molecule additive that increases the expression level from a promoter that controls the expression of the antigen-binding domains or antigen-binding molecules. 28.1 The screening method according to

[28] , wherein the low molecular weight additive is isopropyl-β-thiogalactopyranoside (IPTG) or arabinose. 28.2 The method according to any one of

[24] to [28.1], wherein the low molecular weight compound is an adenosine-containing compound. 28.3 The method according to any one of

[24] to [28.2], wherein the low molecular weight compound is ATP.

[29] An antigen-binding molecule having antigen-binding activity that depends on the concentration of a tumor tissue-specific compound, wherein the antigen-binding activity in the presence of 100 μM of the compound is at least two-fold higher than the antigen-binding activity in the absence of the compound. 29.1 KD value of 5×10 in the presence of 100 μM of the compound -7 The antigen-binding molecule of

[29] , having a molecular weight of M or less. 29.2 KD value in the absence of the compound is 1 x 10 -6 The antigen-binding molecule of

[29] or [29.1], wherein M or more. 29.3 The antigen-binding molecule of any of

[29] to [29.2], which has neutralizing activity against an antigen. 29.4 The antigen-binding molecule of any of

[29] to [29.3], which has cytotoxic activity against cells expressing the antigen. [29.5] The antigen-binding molecule of any of

[29] to [29.4], wherein the antigen is expressed or secreted by tumor cells, immune cells, or stromal cells in tumor tissue. 29.6 The antigen-binding molecule of any of

[29] to [29.5], wherein the compound is an adenosine-containing compound. 29.7 The antigen-binding molecule of any of

[29] to [29.6], which contains an Fc region. [29.8] An antigen-binding molecule described in [29.7], wherein the Fc region is a mutant Fc region containing amino acid modifications, and the mutant Fc region has enhanced binding activity to at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa compared to a native Fc region. 29.9 The antigen-binding molecule of any of

[29] to [29.8], wherein the antigen-binding molecule is an antibody or an antibody fragment.

[30] A pharmaceutical formulation comprising the antigen-binding molecule of any of

[29] to [29.9] and a pharmaceutically acceptable carrier. 30.1 The pharmaceutical preparation according to

[30] for use in treating tumors. 30.2 The pharmaceutical preparation according to [30.1], which has lower cytotoxic activity in non-tumor tissues compared to a pharmaceutical preparation containing a control antigen-binding molecule. 30.3 The pharmaceutical formulation according to [30.1] or [30.2], which has a lower level of side effects compared to a pharmaceutical formulation containing a control antigen-binding molecule. 30.4 The pharmaceutical formulation according to [30.2] or [30.3], wherein the control antigen-binding molecule is an antigen-binding molecule that does not have antigen-binding activity that depends on the concentration of the tumor tissue-specific compound.

[31] A method for producing an antigen-binding molecule for use in tumor treatment, comprising a step of selecting an antigen-binding molecule whose antigen-binding activity in the presence of 100 μM of a tumor tissue-specific compound is at least two-fold higher than the antigen-binding activity in the absence of the compound.

[32] A method for producing a pharmaceutical formulation for use in tumor treatment, comprising the step of mixing an antigen-binding molecule of any of

[29] to [29.9] with a pharmaceutically acceptable carrier.

[33] An antigen-binding molecule having antigen-binding activity that depends on the concentration of a target tissue-specific compound, wherein the antigen-binding activity in the presence of 1 μM of the compound is at least two-fold lower than the antigen-binding activity in the presence of a sufficient amount of the compound. 33.1 KD value of 2×10 in the presence of 1 μM of the compound -7 The antigen-binding molecule of

[33] , wherein M or more. 33.2 The KD value in the presence of a sufficient amount of the compound is 1 x 10 -7 The antigen-binding molecule of

[33] or [33.1], which has a molecular weight of M or less. 33.3 The antigen-binding molecule of any of

[33] to [33.2], wherein the compound is a tumor tissue-specific compound. 33.4 The antigen-binding molecule of [33.3], wherein the compound is an adenosine-containing compound. [33.5] The antigen-binding molecule of any of

[33] to [33.4], which has a higher plasma retention and / or a lower plasma antigen accumulation capacity compared to a control antigen-binding molecule. 33.6 The antigen-binding molecule of [33.5], wherein the control antigen-binding molecule is an antigen-binding molecule that does not have antigen-binding activity that depends on the concentration of the target tissue-specific compound. 33.7 The antigen-binding molecule of any of

[33] to [33.6], wherein the antigen-binding molecule is an antibody or an antibody fragment.

[34] A pharmaceutical formulation comprising the antigen-binding molecule of any of

[33] to [33.7] and a pharmaceutically acceptable carrier.

[35] A method for producing an antigen-binding molecule that has higher plasma retention and / or lower plasma antigen accumulation capacity compared to a control antigen-binding molecule, the method comprising: (a) producing an antigen-binding molecule whose antigen-binding activity increases as the concentration of a target tissue-specific compound increases; and (b) measuring the plasma retention and / or plasma antigen accumulation capacity of the antigen-binding molecule produced in (a). 35.1 The method of

[35] , comprising a step of selecting an antigen-binding molecule whose antigen-binding activity in the presence of 1 μM of a target tissue-specific compound is at least two-fold lower than its antigen-binding activity in the presence of a sufficient amount of the compound. 35.2 The method of

[35] or [35.1], wherein the control antigen-binding molecule is an antigen-binding molecule that does not have antigen-binding activity that depends on the concentration of the target tissue-specific compound.

[36] A method for producing a pharmaceutical formulation, comprising the step of mixing the antigen-binding molecule of any of

[33] to [33.7] with a pharmaceutically acceptable carrier.

[37] A method for measuring ATP concentration in a solution, comprising the steps of: (i) contacting split Luc / HEK293 cells expressing P2Y11 with the solution; and (ii) measuring luciferase activity in the cells. 37.1 The method of

[37] , further comprising the step of contacting the cells with a solution containing a luciferase substrate. 37.2 The method according to

[37] or [37.1], wherein the solution is intercellular fluid in an in vivo tissue. 37.3 The method described in [37.2], wherein the tissue is tumor tissue. 37.4 The method according to [37.2] or [37.3], wherein step (i) is a step of transplanting split Luc / HEK293 cells expressing P2Y11 into tissue in vivo. [Brief explanation of the drawings]

[0009] [Figure 1] A graph showing the agonistic activity of various anti-CD137 antibodies in the presence or absence of ATP, tested using Jurkat cells. The X-axis represents antibody concentration (μg / mL), and the Y-axis represents relative light emission. [Figure 2]A graph showing the agonist activity of various anti-CD137 antibodies in the presence or absence of ADP, tested using Jurkat cells. The X-axis represents antibody concentration (μg / mL), and the Y-axis represents relative light emission. [Figure 3] FIG. 1 shows the agonistic activity of various anti-CD137 antibodies tested using human T cells in the presence or absence of ADPbetaS. [Figure 4] A graph showing the agonistic activity of dBBAT119-P253 / dBBAT119L-LamLib (small molecule switched anti-CD137 antibody) or NS1-P253 (non-switched anti-CD137 antibody) in the presence or absence of ADPbetaS, tested using human T cells. The X axis represents antibody concentration (μg / mL), and the Y axis represents IFNγ production (ng / mL). [Figure 5] Figure 1 shows the ATP-dependent antigen-binding activity of various anti-CD137 antibodies (switched anti-CD137 antibodies with improved binding activity) tested by phage ELISA. 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 in the presence / absence of antigen. [Figure 6] 1 shows 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. The upper panel shows the binding activity to human CD137 in the absence of ATP, and the lower panel shows the binding activity to human CD137 in the presence of ATP. [Figure 7] Figure 1 shows the agonist activity of dBBAT119H-P253 / dBBAT119L-LamLib, dBBATk119H024-P253 / dBBATk119L020-LamLib, IC17HdK-hIgG1 / IC17L-k0 (control), or NS1-P253 (non-switched anti-CD137 antibody) in the presence or absence of ADPbetaS, as tested using human T cells. Panel (A) shows the results in the absence of ADPbetaS, and panel (B) shows the results in the presence of ADPbetaS. The X axis represents antibody concentration (μg / mL), and the Y axis represents IFNγ production (ng / mL). [Figure 8]Figure 4 shows the agonist activity of various switch anti-CD137 antibodies in the presence or absence of ATP, as tested using the 4-1BB Jurkat reporter gene assay. Panel (A) shows the test results in the absence of ATP, and panel (B) shows the test results in the presence of ATP. [Figure 9] Figure 1 shows the enhanced agonist activity of various switch anti-CD137 antibodies in the presence of ATP due to increased Fcγ receptor-binding activity of the heavy chain constant region, as tested using human peripheral blood mononuclear cells. Panel (A) shows the agonist activity measured using the amount of IL-2 production as an index, and panel (B) shows the agonist activity measured using the amount of IFN-γ production as an index. [Figure 10] Figure 1 shows the enhanced agonistic activity of various switch anti-CD137 antibodies in the presence of ATP, as determined using human peripheral blood mononuclear cells, due to increased Fcγ receptor-binding activity or increased pI of the heavy chain constant region. Panel (A) shows the agonistic activity measured using the amount of IL-2 production as an index, and panel (B) shows the agonistic activity measured using the amount of IFN-γ production as an index. [Figure 11] Figure 1 shows the enhanced agonist activity of various switch anti-CD137 antibodies in the presence or absence of ATP due to increased Fcγ receptor-binding activity of the heavy chain constant region, as tested using human peripheral blood mononuclear cells. Panel (A) shows the agonist activity measured using the amount of IL-2 production as an index, and panel (B) shows the agonist activity measured using the amount of IFN-γ production as an index. [Figure 12] Figure 1 shows the enhanced agonist activity of various switch anti-CD137 antibodies in the presence or absence of ATP due to increased Fcγ receptor-binding activity of the heavy chain constant region, as tested using human peripheral blood mononuclear cells. Panel (A) shows the agonist activity measured using the amount of IL-2 production as an index, and panel (B) shows the agonist activity measured using the amount of IFN-γ production as an index. [Figure 13]Figure 1 shows the enhanced agonist activity of various switch anti-CD137 antibodies in the presence or absence of ATP due to increased Fcγ receptor-binding activity of the heavy chain constant region, as tested using human peripheral blood mononuclear cells. Panel (A) shows the agonist activity measured using the amount of IL-2 production as an index, and panel (B) shows the agonist activity measured using the amount of IFN-γ production as an index. [Figure 14] Figure 1 shows the enhanced agonist activity of various switch anti-CD137 antibodies in the presence or absence of ATP due to increased Fcγ receptor-binding activity of the heavy chain constant region, as tested using human peripheral blood mononuclear cells. Panel (A) shows the agonist activity measured using the amount of IL-2 production as an index, and panel (B) shows the agonist activity measured using the amount of IFN-γ production as an index. [Figure 15] Figure 1 shows the enhanced agonist activity of various switch anti-CD137 antibodies in the presence or absence of ATP due to increased Fcγ receptor-binding activity of the heavy chain constant region, as tested using human peripheral blood mononuclear cells. Panel (A) shows the agonist activity measured using the amount of IL-2 production as an index, and panel (B) shows the agonist activity measured using the amount of IFN-γ production as an index. [Figure 16] Figure 1 shows the enhanced agonist activity of various switch anti-CD137 antibodies in the presence or absence of ATP, as determined using human peripheral blood mononuclear cells, as a result of increasing the pI of the heavy chain constant region. Panel (A) shows the agonist activity measured using the amount of IL-2 production as an index, and panel (B) shows the agonist activity measured using the amount of IFN-γ production as an index. [Figure 17] Figure 1 shows the enhanced agonist activity of various switch anti-CD137 antibodies in the presence or absence of ATP, as determined using human peripheral blood mononuclear cells, as a result of increasing the pI of the heavy chain constant region. Panel (A) shows the agonist activity measured using the amount of IL-2 production as an index, and panel (B) shows the agonist activity measured using the amount of IFN-γ production as an index. [Figure 18]Figure 1 shows the enhanced agonist activity of various switch anti-CD137 antibodies in the presence or absence of ATP, as determined using human peripheral blood mononuclear cells, as a result of increasing the pI of the heavy chain constant region. Panel (A) shows the agonist activity measured using the amount of IL-2 production as an index, and panel (B) shows the agonist activity measured using the amount of IFN-γ production as an index. [Figure 19] Figure 1 shows the enhanced agonist activity of various switch anti-CD137 antibodies in the presence or absence of ATP, as determined using human peripheral blood mononuclear cells, as a result of increasing the pI of the heavy chain constant region. Panel (A) shows the agonist activity measured using the amount of IL-2 production as an index, and panel (B) shows the agonist activity measured using the amount of IFN-γ production as an index. [Figure 20] Figure 1 shows the enhanced agonist activity of various switch anti-CD137 antibodies in the presence or absence of ATP, as determined using human peripheral blood mononuclear cells, as a result of increasing the pI of the heavy chain constant region. Panel (A) shows the agonist activity measured using the amount of IL-2 production as an index, and panel (B) shows the agonist activity measured using the amount of IFN-γ production as an index. [Figure 21] Figure 1 shows the enhanced agonist activity of various switch anti-CD137 antibodies in the presence or absence of ATP, as determined using human peripheral blood mononuclear cells, as a result of increasing the pI of the heavy chain constant region. Panel (A) shows the agonist activity measured using the amount of IL-2 production as an index, and panel (B) shows the agonist activity measured using the amount of IFN-γ production as an index. [Figure 22] Figure 1 shows the enhanced agonist activity of various switch anti-CD137 antibodies in the presence or absence of ATP, as determined using human peripheral blood mononuclear cells, as a result of increasing the pI of the heavy chain constant region. Panel (A) shows the agonist activity measured using the amount of IL-2 production as an index, and panel (B) shows the agonist activity measured using the amount of IFN-γ production as an index. [Figure 23]Figure 1 shows the enhanced agonist activity of various switch anti-CD137 antibodies in the presence or absence of ATP due to increased Fcγ receptor-binding activity of the heavy chain constant region, as tested using human peripheral blood mononuclear cells. Panel (A) shows the agonist activity measured using the amount of IL-2 production as an index, and panel (B) shows the agonist activity measured using the amount of IFN-γ production as an index. [Figure 24] This figure shows the plasma antibody concentrations of various switched and non-switched anti-CD137 antibodies tested using human CD137 knock-in mice. The Fc of each antibody is mIgG1. [Figure 25] This figure shows the plasma antibody concentrations of various switch and non-switch anti-CD137 antibodies tested using human CD137 knock-in mice. The Fc of each antibody is MB110. [Figure 26] This figure shows the plasma antibody concentrations of various switch and non-switch anti-CD137 antibodies tested using human CD137 knock-in mice. The Fc of each antibody is MB492. [Figure 27] A graph showing the antitumor effect of A375-mIgG1 / B167-ml0r in a mouse model transplanted with MC38 cells. Each point represents the mean tumor volume for one group (n=5). [Figure 28] Figure 1 shows organ weights following administration of antibodies (NO1-mIgG1 or A375-mIgG1 / B167-ml0r) in a mouse model transplanted with MC38 cells. Diagram (A) shows the weight of the lymph node, and diagram (B) shows the weight of the spleen. [Figure 29] Figure 1 shows the degree of T cell activation in lymph nodes following administration of NO1-mIgG1 or A375-mIgG1 / B167-ml0r in a mouse model transplanted with MC38 cells. Diagram (A) shows the percentage of PD-1-positive T cells among CD8-positive T cells, diagram (B) shows the percentage of ICOS-positive T cells among CD8-positive T cells, and diagram (C) shows the percentage of Granzyme B-positive T cells among CD8-positive T cells. [Figure 30]Figure 1 shows the degree of T cell activation in the spleen following administration of NO1-mIgG1 or A375-mIgG1 / B167-ml0r in a mouse model transplanted with the MC38 cell line. Diagram (A) shows the proportion of PD-1-positive T cells among CD8-positive T cells, diagram (B) shows the proportion of ICOS-positive T cells among CD8-positive T cells, and diagram (C) shows the proportion of Granzyme B-positive T cells among CD8-positive T cells. [Figure 31] Figure 1 shows the degree of T cell activation in the liver following administration of NO1-mIgG1 or A375-mIgG1 / B167-ml0r in a mouse model transplanted with the MC38 cell line. Diagram (A) shows the percentage of PD-1-positive T cells among CD8-positive T cells, and diagram (B) shows the percentage of Granzyme B-positive T cells among CD8-positive T cells. [Figure 32] This figure shows the antitumor effect of A356-MB110 / B040-ml0r in a mouse model implanted with the MC38 cell line. Each point represents the mean tumor volume for one group (n=5). [Figure 33] Graph showing organ weights following administration of NS2-MB110 or A356-MB110 / B040-ml0r in a mouse model transplanted with the MC38 cell line. (A) shows the weight of the lymph node, and (B) shows the weight of the spleen. [Figure 34] Figure 1 shows the degree of T cell activation in the liver following administration of NS2-MB110 or A356-MB110 / B040-ml0r in a mouse model transplanted with the MC38 cell line. (A) shows the percentage of PD-1-positive T cells among CD8-positive T cells, and (B) shows the percentage of ICOS-positive T cells among CD8-positive T cells. [Figure 35] A graph showing the antitumor effect of A372-mIgG1 / B040-ml0r in a mouse model implanted with the MC38 cell line. Each point represents the mean tumor volume (n=5 per group). [Figure 36] This shows the lymph node cell count (Fig. (A)) and spleen weight (Fig. (B)) following administration of A372-mIgG1 / B040-ml0r in a mouse model transplanted with the MC38 cell line. [Figure 37]FIG. 10 is a graph showing the degree of T cell activation in the liver (proportion of Granzyme B-positive T cells among CD8-positive T cells) following administration of A372-mIgG1 / B040-ml0r in a mouse model transplanted with the MC38 cell line. [Figure 38] A graph showing the antitumor effect of A372-MB110 / B040-ml0r in a mouse model implanted with the MC38 cell line. Each point represents the mean tumor volume for n=5 per group. [Figure 39] Graph showing organ weights following administration of NS2-MB110 or A372-MB110 / B040-ml0r in a mouse model transplanted with the MC38 cell line. (A) shows the weight of the lymph node, and (B) shows the weight of the spleen. [Figure 40] A graph showing the degree of T cell activation in the liver (proportion of PD-1 positive T cells among CD8 positive T cells) following administration of NS2-MB110 or A372-MB110 / B040-ml0r in a mouse model transplanted with the MC38 cell line. [Figure 41] This figure shows the antitumor effect of A372-MB492 / B040-ml0r in a mouse model implanted with the MC38 cell line. Each point represents the mean tumor volume per group (n=5). [Figure 42] Figure 1 shows the lymph node cell count and spleen organ weight after administration of NS1-MB492 or A372-MB492 / B040-ml0r in a mouse model transplanted with the MC38 cell line. (A) shows the lymph node cell count, and (B) shows the spleen organ weight. [Figure 43] A graph showing the degree of T cell activation in the liver (proportion of Granzyme B-positive T cells among CD8-positive T cells) following administration of NS1-MB492 or A372-MB492 / B040-ml0r in a mouse model transplanted with the MC38 cell line. [Figure 44] This figure shows the antitumor effect of A486-MB492 / B167-ml0r or A488-MB492 / B226-ml0r in a mouse model implanted with the MC38 cell line. Each point represents the mean tumor volume per group (n=5). [Figure 45]Figure 1 shows the number of cells per lymph node and spleen weight in a mouse model transplanted with the MC38 cell line after administration of NS1-MB492, A486-MB492 / B167-ml0r, or A488-MB492 / B226-ml0r. (A) shows the number of cells per lymph node, and (B) shows the spleen weight. [Figure 46] This figure shows the level of effector cell infiltration in the liver (proportion of CD3+CD8+ T cells among CD45+ T cells, etc.) following administration of NS1-MB492, A486-MB492 / B167-ml0r, or A488-MB492 / B226-ml0r in a mouse model transplanted with the MC38 cell line. [Figure 47] This figure shows the antitumor effect of A489-MB492 / B223-ml0r in a mouse model implanted with the MC38 cell line. Each point represents the mean tumor volume for one group (n=5). [Figure 48] Figures showing the number of lymph node cells and the number of lymphocyte fraction cells in the spleen following administration of NS1-MB492 or A489-MB492 / B223-ml0r in a mouse model transplanted with the MC38 cell line. Diagram (A) shows the number of lymph node cells, and diagram (B) shows the number of lymphocyte fraction cells in the spleen. [Figure 49] FIG. 1 shows the degree of T cell activation in the liver (proportion of CD8-positive T cells among CD45-positive T cells) following administration of NS1-MB492 or A489-MB492 / B223-ml0r in a mouse model transplanted with the MC38 cell line. [Figure 50] Figures showing the antitumor effects of A548-mIgG1 / B256-ml0r and A551-mIgG1 / B256-ml0r in a mouse model implanted with the MC38 cell line. Figure (A) shows the antitumor effect of A548-mIgG1 / B256-ml0r, and Figure (B) shows the antitumor effect of A551-mIgG1 / B256-ml0r. [Figure 51]Figure 1 shows organ weights following administration of NS1-mIgG1, A548-mIgG1 / B256-ml0r, or A551-mIgG1 / B256-ml0r in a mouse model transplanted with the MC38 cell line. Diagram (A) shows the weight of the lymph node, and diagram (B) shows the weight of the spleen. [Figure 52] Figure 1 shows the degree of T cell activation in the liver following administration of NS1-mIgG1, A548-mIgG1 / B256-ml0r, or A551-mIgG1 / B256-ml0r in a mouse model transplanted with the MC38 cell line. Diagram (A) shows the proportion of PD-1-positive T cells among CD8-positive T cells, and diagram (B) shows the proportion of Granzyme B-positive T cells among CD8-positive T cells. [Figure 53] FIG. 1 shows the antitumor effect of A551-MB110 / B379-ml0r in a mouse model transplanted with the MC38 cell line. [Figure 54] Figure 1 shows organ weights following administration of NS1-mIgG1 or A551-MB110 / B379-ml0r in a mouse model transplanted with the MC38 cell line. Diagram (A) shows the weight of the lymph node, and diagram (B) shows the weight of the spleen. [Figure 55] Figure 1 shows the degree of T cell activation in the spleen following administration of NS1-mIgG1 or A551-MB110 / B379-ml0r in a mouse model transplanted with the MC38 cell line. Diagram (A) shows the percentage of PD-1-positive T cells among CD8-positive T cells, diagram (B) shows the percentage of ICOS-positive T cells among CD8-positive T cells, and diagram (C) shows the percentage of Granzyme B-positive T cells among CD8-positive T cells. [Figure 56] Figure 1 shows the degree of T cell activation in the liver following administration of NS1-mIgG1 or A551-MB110 / B379-ml0r in a mouse model transplanted with the MC38 cell line. Diagram (A) shows the percentage of PD-1-positive T cells among CD8-positive T cells, diagram (B) shows the percentage of ICOS-positive T cells among CD8-positive T cells, and diagram (C) shows the percentage of Granzyme B-positive T cells among CD8-positive T cells. [Figure 57]A graph showing the agonistic activity of various anti-CD137 antibodies in the presence or absence of L-kynurenine, tested using Jurkat cells. The X-axis represents antibody concentration (μg / mL), and the Y-axis represents relative light emission. [Figure 58] A graph showing the agonistic activity of various anti-CD137 antibodies in the presence or absence of small molecule compounds (ATP or ADP) tested using 4-1BB Jurkat cells. The X-axis shows antibody concentration (μg / mL), and the Y-axis shows relative light intensity. [Figure 59] FIG. 1 shows the ATP responsiveness (luciferin luminescence dependent on ATP concentration) of P2Y11 split Luc / HEK293 cells prepared for measuring extracellular ATP levels. [Figure 60] FIG. 1 shows in vivo ATP responsiveness (luciferin luminescence dependent on ATP concentration) when P2Y11 split Luc / HEK293 cells were subcutaneously transplanted into mice. [Figure 61] 1 shows the results of luminescence imaging measurements of mice subcutaneously implanted with a fixed concentration of ATP and P2Y11 split Luc / HEK293 cells, and FM3A tumor-bearing mice subcutaneously implanted with P2Y11 split Luc / HEK293 cells, showing luminescence detected by marks on the ventral side of the mice. [Figure 62] FIG. 10 shows the ATP concentration-dependent binding activity (KD value) of anti-hIL6R antibodies MRAH-G4T1 / MRAL-k0 (control antibody), H0002-G4T1 / L1058-lam1, H0041-G4T1 / L1088-lam1, and H0052-G4T1 / L1083-lam1 (all switch antibodies) to hIL6R. [Figure 63] FIG. 10 shows the ADP concentration-dependent binding activity (KD value) of anti-hIL6R antibodies MRAH-G4T1 / MRAL-k0 (control antibody), H0002-G4T1 / L1058-lam1, H0041-G4T1 / L1088-lam1, and H0052-G4T1 / L1083-lam1 (all switch antibodies) to hIL6R. [Figure 64]FIG. 10 shows the AMP concentration-dependent binding activity (KD value) of anti-hIL6R antibodies MRAH-G4T1 / MRAL-k0 (control antibody), H0002-G4T1 / L1058-lam1, H0041-G4T1 / L1088-lam1, and H0052-G4T1 / L1083-lam1 (all switch antibodies) to hIL6R. [Figure 65] This figure shows the ATP concentration-dependent ADCC activity of the anti-hIL6R antibodies MRAH-mFa55 / MRAL-mk0 (control antibody), H0002-mFa55 / L1058-ml0, H0041-mFa55 / L1088-ml0, and H0052-mFa55 / L1083-ml0 (all switch antibodies). [Figure 66] This figure shows the in vivo antitumor activity of the anti-hIL6R antibodies MRAH-mFa55 / MRAL-mk0 (control antibody), H0002-mFa55 / L1058-ml0, H0041-mFa55 / L1088-ml0, and H0052-mFa55 / L1083-ml0 (all switch antibodies). IC17Hdk-mFa55 / IC17L-mk1 is a negative control antibody. [Figure 67] 1 shows a comparison of the plasma kinetics of the anti-hIL6R antibody MRAH-mFa55 / MRAL-mk0 (control antibody) in normal mice and hIL6R transgenic mice, with the vertical axis of the graph representing the plasma concentration of the antibody. [Figure 68] 1 shows a comparison of the plasma kinetics of the anti-hIL6R antibody H0002-mFa55 / L1058-ml0 (switch antibody) in normal mice and hIL6R transgenic mice, with the vertical axis of the graph representing the plasma concentration of the antibody. [Figure 69] 1 shows a comparison of the plasma kinetics of the anti-hIL6R antibody H0041-mFa55 / L1088-ml0 (switch antibody) in normal mice and hIL6R transgenic mice, with the vertical axis of the graph representing the plasma concentration of the antibody. [Figure 70]1 shows a comparison of the plasma kinetics of the anti-hIL6R antibody H0052-mFa55 / L1083-ml0 (switch antibody) in normal mice and hIL6R transgenic mice, with the vertical axis of the graph representing the plasma concentration of the antibody. [Figure 71] This figure shows antigen accumulation in hIL6R transgenic mice after administration of the anti-hIL6R non-switch antibody MRAH-mFa55 / MRAL-mk0 (control antibody) and the anti-hIL6R switch antibodies H0002-mFa55 / L1058-ml0, H0041-mFa55 / L1088-ml0, and H0052-mFa55 / L1083-ml0 (all switch antibodies). The vertical axis of the graph shows the plasma concentration of soluble hIL6R. IC17Hdk-mFa55 / IC17L-mk1 (referred to as KLH-mFa55 in the figure) was used as a negative control antibody. [Figure 72] This figure shows the in vivo anti-tumor activity of the anti-hIL6R non-switch antibody MRAH-mFa55 / MRAL-mk0 (control antibody), and the anti-hIL6R switch antibodies H0002-mFa55 / L1058-ml0 and H0041-mFa55 / L1088-ml0 (all switch antibodies). IC17Hdk-mFa55 / IC17L-mk1 is a negative control antibody. [Figure 73] Figure 1 shows a comparison of the plasma kinetics of the anti-hIL6R non-switch antibody MRAH-mFa55 / MRAL-mk0 (control antibody) and the anti-hIL6R switch antibodies H0002-mFa55 / L1058-ml0 and H0041-mFa55 / L1088-ml0 (all switch antibodies). The vertical axis of the graph shows the plasma concentration of the antibody. [Figure 74]This figure shows antigen accumulation after administration of the anti-hIL6R non-switch antibody MRAH-mFa55 / MRAL-mk0 (control antibody), and the anti-hIL6R switch antibodies H0002-mFa55 / L1058-ml0 and H0041-mFa55 / L1088-ml0 (all switch antibodies). The vertical axis of the graph shows the plasma concentration of soluble hIL6R. IC17Hdk-mFa55 / IC17L-mk1 (referred to as KLH-mFa55 in the figure) was used as a negative control antibody. [Figure 75] This figure shows the in vivo anti-tumor activity of the anti-hIL6R non-switch antibody MRAH-mFa55 / MRAL-mk0 (control antibody), and the anti-hIL6R switch antibodies H0041-mFa55 / L1088-ml0 and H0052-mFa55 / L1083-ml0 (all switch antibodies). IC17Hdk-mFa55 / IC17L-mk1 is a negative control antibody. [Figure 76] Figure 1 shows a comparison of the plasma kinetics of the anti-hIL6R non-switch antibody MRAH-mFa55 / MRAL-mk0 (control antibody) and the anti-hIL6R switch antibody H0052-mFa55 / L1083-ml0 (switch antibody). The vertical axis of the graph shows the plasma concentration of the antibody. [Figure 77] This figure shows antigen accumulation after administration of the anti-hIL6R non-switch antibody MRAH-mFa55 / MRAL-mk0 (control antibody) and the anti-hIL6R switch antibody H0052-mFa55 / L1083-ml0 (switch antibody). The vertical axis of the graph shows the plasma concentration of soluble hIL6R. IC17Hdk-mFa55 / IC17L-mk1 (referred to as KLH-mFa55 in the figure) was used as a negative control antibody. [Figure 78] FIG. 10 shows the ATP concentration-dependent inhibitory activity of anti-PD1 antibodies mPD1F2VH-mF18 / mPD1F2VL-mk1 (control antibody) and H5029-mFa31 / L3021-ml0 (switch antibody) on PD-1 / PDL-1 binding. [Figure 79] FIG. 10 shows the ATP concentration-dependent inhibitory activity of anti-PD1 antibodies mPD1F2VH-mF18 / mPD1F2VL-mk1 (control antibody) and H5041-mFa31 / L3021-ml0 (switch antibody) on PD-1 / PDL-1 binding. [Figure 80] This figure shows the in vitro neutralizing activity of anti-PD1 antibodies mPD1F2VH-mF18 / mPD1F2VL-mk1 (control antibody), H5029-mFa31 / L3021-ml0, and H5041-mFa31 / L3021-ml0 (all switch antibodies), depending on the AMP concentration. [Figure 81] Fig. 10 shows the ATP concentration-dependent in vitro neutralizing activity of the anti-PD1 antibodies mPD1F2VH-mF18 / mPD1F2VL-mk1 (control antibody), H5029-mFa31 / L3021-ml0, and H5041-mFa31 / L3021-ml0 (all switch antibodies). [Figure 82] This figure shows the in vivo anti-tumor activity of the anti-PD1 antibodies mPD1F2VH-mFa55 / mPD1F2VL-mk1 (control antibody) and H5041-mFa55 / L3023-ml0 (switch antibody). IC17Hdk-mFa55 / IC17L-mk1 is a negative control antibody. [Figure 83] (A) and (B) show the depletion activity of PD-1-expressing cells in the spleen by the anti-PD1 antibodies mPD1F2VH-mFa55 / mPD1F2VL-mk1 (control antibody) and H5041-mFa55 / L3023-ml0 (switch antibody). In the figure, the isotype indicates the negative control antibody (IC17Hdk-mFa55 / IC17L-mk1). [Figure 84] This figure shows the binding mode between the anti-hIL6R switch antibody H0041L1088 Fab fragment and ATP. In the figure, ATP is shown as a ball-and-stick model, and amino acid residues that interact with ATP are shown as stick models. Dashed lines indicate hydrogen bonds between the antibody and ATP. [Figure 85]This figure shows the epitope of the anti-hIL6R switch antibody H0041L1088 mapped onto the amino acid sequence of the hIL6R extracellular domain (shIL6R). In the figure, the amino acid residues shaded in gray indicate amino acid residues of shIL6R (epitope residues) that contain one or more non-hydrogen atoms located within 4.2 Å of either H0041L1088 Fab or ATP in the crystal structure. [Figure 86] This figure shows details of the binding between the ATP-bound H0041L1088 Fab fragment and shIL6R. In the figure, the heavy chain of the antibody is depicted in black, the light chain in gray, and shIL6R in white. ATP is shown as a spherical model, and epitope residues of the antibody or shIL6R within 4.2 Å of ATP, and paratope residues of the antibody within 4.2 Å of the epitope residue are shown as stick models. Dashed lines indicate hydrogen bonds between the antibody and shIL6R. Only F298 of shIL6R is shown as a spherical model to make the interaction with ATP easier to understand. [Figure 87] This is a diagram showing the structure of FIG. 86 rotated 180 degrees (viewed from the back). [Figure 88] FIG. 4 shows the agonist activity of various switch anti-CD137 antibodies in the presence of ATP, as tested using the 4-1BB Jurkat reporter gene assay. [Figure 89] 1 shows a comparison of the plasma kinetics of the anti-CD137 switch antibodies A375-SCF041aPh / B167-Lamlib and A375-MY201aPh / B167-Lamlib, with the vertical axis of the graph representing the plasma concentration of the antibody. [Figure 90] This figure shows the antitumor effects of A375 / B167-SCF041aPh and A375 / B167-MY201aPh in a mouse model generated by transplanting LLC1 / OVA / GPC3 cell lines into hCD137KI / mFcγR2bKO / hFcγR2bTg#90 mice. Each point represents the mean tumor volume for n=5 mice per group. [Figure 91]FIG. 1 shows the agonist activity of various switch anti-CD3 antibodies in the presence of ATP, as tested by a reporter gene assay using T cell activation bioassay (NFAT). DETAILED DESCRIPTION OF THE INVENTION

[0010] I. Definition

[0011] The term "binding activity" refers to the strength of the total noncovalent interactions between one or more binding sites of a molecule (e.g., an antibody) and the molecule's binding partner (e.g., an antigen). Here, "binding activity" is not strictly limited to a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). For example, when the members of a binding pair reflect a monovalent 1:1 interaction, this binding activity is specifically referred to as intrinsic binding affinity ("affinity"). When members of a binding pair are capable of both monovalent and multivalent binding, the binding activity is the sum of these binding forces. The binding activity of a molecule X to its partner Y can generally be expressed by the dissociation constant (KD) or the "amount of analyte bound per unit amount of ligand" (hereinafter sometimes referred to as "binding amount"). Those skilled in the art will understand that, in general, the lower the dissociation constant (KD), the higher the binding activity, and the higher the "amount of analyte bound per unit amount of ligand" or "amount of binding," the higher the 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.

[0012] An "avidity matured" antigen-binding molecule or antibody, or an "avidity enhanced" antigen-binding molecule or antibody, refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs) that result in improved binding activity of the antigen-binding molecule or antibody to its antigen, compared to a parent antigen-binding molecule or antibody that does not have the alterations.

[0013] The terms "anti-CD137 antigen-binding molecule," "anti-CD137 antibody," or "antigen-binding molecule that binds to CD137," "antibody that binds to CD137" refer to an antigen-binding molecule or antibody that can bind to CD137 with sufficient avidity such that the antigen-binding molecule or antibody is useful as a diagnostic and / or therapeutic agent when it targets CD137. In certain embodiments, the anti-CD137 antibody binds to an epitope of CD137 that is conserved among CD137 from different species.

[0014] The term "anti-CD137 antigen-binding molecule or anti-CD137 antibody having small molecule compound-dependent CD137-binding activity" refers to an antigen-binding molecule or antibody whose CD137-binding activity in the presence of the small molecule compound is higher than that in the absence of the small molecule compound. In one embodiment, "in the presence of a small molecule compound" refers to a condition in which the small molecule compound is present at 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. In one embodiment, the degree of binding activity of the anti-CD137 antigen-binding molecule or antibody to an unrelated non-CD137 protein in the presence of a small molecule compound is less than about 10% of the binding of the antigen-binding molecule or antibody to CD137, as measured (for example, by radioimmunoassay (RIA) or surface plasmon resonance (SPR)). In certain embodiments, the anti-CD137 antigen binding molecule or antibody has a cytotoxicity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, e.g. 10 -6 M ~10 -10 M, 10 -7 M ~10 -9 M, for example, 10 -7 M~10 -8 It has a dissociation constant (KD) of 1 M.

[0015] The term "antigen-binding molecule" is used herein 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.

[0016] As used herein, an "agonist antigen-binding molecule" or "agonist antibody" is an antigen-binding molecule or antibody that significantly induces or enhances the biological activity of the antigen to which it binds (e.g., CD137, CD3). Therefore, for example, when the antigen is CD137, an antigen-binding molecule or antibody having such agonistic activity is referred to as a "CD137 agonist antigen-binding molecule" or a "CD137 agonist antibody," respectively. Similarly, when the antigen is CD3, an antigen-binding molecule or antibody having such agonistic activity is referred to as a "CD3 agonist antigen-binding molecule" or a "CD3 agonist antibody," respectively.

[0017] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0018] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0019] An "antigen-binding molecule that binds to the same epitope" or "antibody that binds to the same epitope" as a reference antigen-binding molecule or reference antibody refers to an antibody or antigen-binding molecule that blocks the binding of the reference antibody or reference antigen-binding molecule to its own antigen by 50% or more in a competitive assay. Conversely, a reference antibody blocks the binding of the antibody to its own antigen by 50% or more in a competitive assay. Exemplary competitive assays are provided herein. In one embodiment, when the reference antigen-binding molecule or reference antibody has antigen-binding activity that depends on a small molecule compound, the competitive assay is performed in the presence of the small molecule compound.

[0020] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0021] The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0022] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0023] "Cytotoxic activity" refers to the activity of inhibiting or preventing cellular function and / or causing cell death or destruction. Cytotoxic activity may be, for example, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, or T-cell cytotoxicity, or may be induced by a cytotoxic agent (e.g., a radioisotope or a chemotherapeutic agent) such as an immunoconjugate.

[0024] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain, except that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) residues of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0025] As used herein, the term "variant Fc region" refers to an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution, e.g., about one to about ten amino acid substitutions, preferably about one to about five amino acid substitutions, in the native-sequence Fc region or in the Fc region of the parent polypeptide, compared to the native-sequence Fc region or the Fc region of the parent polypeptide. The variant Fc region herein preferably has at least about 80% homology with the native-sequence Fc region and / or the Fc region of the parent polypeptide, most preferably at least about 90% homology thereto, and more preferably at least about 95% homology thereto.

[0026] Herein, amino acid modifications or substitutions in 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 of serine (Ser) at EU numbering position 424 with asparagine (Asn). EU424N also represents a substitution of any amino acid at position 424 with asparagine (Asn).

[0027] As used herein, the term "Fc region-containing antibody" refers to an antibody that contains an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) or the C-terminal glycine-lysine (residues 446-447) of the Fc region can be removed, for example, during antibody purification or by recombinant engineering of a nucleic acid encoding the antibody. Thus, a composition containing an antibody with an Fc region according to the present disclosure can contain an antibody with G446-K447, an antibody with G446 but without K447, an antibody with G446-K447 completely removed, or a mixture of the above three types of antibodies.

[0028] The terms "full length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a heavy chain with a structure substantially similar to a native antibody structure or containing an Fc region or variant Fc region as defined herein.

[0029] A "human antibody" is an antibody with an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a human antibody repertoire or other non-human source that uses human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0030] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0031] An "acceptor human framework," for purposes of this specification, is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise those same amino acid sequences or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0032] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI according to Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III according to Kabat et al., supra.

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

[0034] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by screening a complementary library of VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to that antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0035] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence (the "complementarity determining region" or "CDR") and / or forms structurally defined loops (the "hypervariable loops") and / or contains antigen-contacting residues (the "antigen contacts"). Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include the following: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) A combination of (a), (b), and / or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra. Also herein, HVR residues and other residues in the variable domain (e.g., FR residues), and amino acid modifications or substitutions at such residues, may be represented by a combination of the Kabat numbering system and amino acids. For example, N99 represents asparagine (Asn) at position 99 of the Kabat numbering system, and N99A represents a substitution of asparagine (Asn) at position 99 of the Kabat numbering system with alanine (Ala).

[0036] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to, cytotoxic agents.

[0037] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes the death or destruction of cells. Cytotoxic agents include, but are not limited to, radioisotopes (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, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes and fragments thereof, such as nucleases; antibiotics; toxins, such as, for example, small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including fragments and / or variants thereof); and various anti-tumor or anti-cancer agents, as disclosed below.

[0038] An "isolated" antibody is one that has been separated from a component of its original environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as measured by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0039] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its original environment. Isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0040] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of effecting the expression of nucleic acids to which they are operatively linked. Such vectors are also referred to herein as "expression vectors."

[0041] "Encoded nucleic acid encoding an anti-CD137 antigen-binding molecule" refers to one or more nucleic acid molecules that encode the polypeptides that constitute the antigen-binding molecule. "Isolated nucleic acid encoding an anti-CD137 antibody" refers to one or more nucleic acid molecules that encode the heavy and light chains (or fragments thereof) of the antibody, and includes nucleic acid molecules carried on a single vector or separate vectors, and nucleic acid molecules present at one or more locations in a host cell.

[0042] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as that for which the original transformed cell was screened or selected are also included herein.

[0043] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variants (e.g., variants containing naturally occurring mutations or variants that arise during the production of a monoclonal antibody preparation; such variants are typically present in small amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present disclosure may be made 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 making monoclonal antibodies are described herein.

[0044] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies may be present in a pharmaceutical formulation.

[0045] "Native antibodies" refer to immunoglobulin molecules with various naturally occurring structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (κ) or lambda (λ), based on the amino acid sequence of its constant domain.

[0046] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, and excluding any conservative substitutions from the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetyx Corporation). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0047] The ALIGN-2 sequence comparison computer program is the copyright of Genentech, Inc., and its source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, one can say that a given amino acid sequence A has or contains a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0048] The term "pharmaceutical formulation" refers to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0049] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0050] An "effective amount" of an agent (eg, a pharmaceutical formulation) refers to an amount, at dosages and for periods of time necessary, effective to achieve a desired therapeutic or prophylactic result.

[0051] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is human.

[0052] As used herein, the term "CD137," unless otherwise indicated, refers to any native form of CD137 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed CD137 as well as any form of CD137 resulting from processing within the cell. The term also encompasses naturally occurring variants of CD137, such as splice variants and allelic variants. The full-length amino acid sequence of an exemplary human CD137 is set forth in SEQ ID NO: 1 (NCBI Reference Sequence: NP_001552.2), and the amino acid sequence of an exemplary extracellular region of human CD137 is set forth in SEQ ID NO: 2. The full-length amino acid sequence of an exemplary mouse CD137 is set forth in SEQ ID NO: 3 (NCBI Reference Sequence: NP_035742.1), and the amino acid sequence of an exemplary extracellular region of mouse CD137 is set forth in SEQ ID NO: 4. The full-length amino acid sequence of an exemplary monkey 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. 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. In addition to its expression on activated CD4+ and CD8+ T cells, CD137 is also expressed on B cells, dendritic cells, natural killer (NK) and NK-T cells, macrophages, monocytes, neutrophils, CD4+CD25+ regulatory T cells, and vascular endothelial cells. It has also been shown to be expressed on cancer cells (Labiano et al., Oncoimmunology, 24:e1062967 (2015)). Its natural ligand, CD137L, is expressed on antigen-presenting cells such as B cells, monocytes / macrophages, and dendritic cells (Watts et al., Annu. Rev. Immunol., 23:23-68 (2005)). Upon interaction with its ligand, CD137 leads to increased TCR-induced T cell proliferation, cytokine production, functional maturation, suppression of apoptosis, and long-term CD8+ T cell survival (Nam et al., Curr. Cancer Drug Targets, 5:357-363 (2005); Watts et al., Annu. Rev. Immunol., 23:23-68 (2005)).

[0053] The terms "cancer," "cancer," and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation.

[0054] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "carcinoma," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive as used herein.

[0055] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0056] As used herein, "treatment" (and its grammatical derivatives, such as "treat," "treating," etc.) refers to a clinical intervention intended to alter the natural course of the individual being treated and can be performed prophylactically or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the present disclosure are used to delay the onset of disease or slow the progression of disease.

[0057] II. Compositions and Methods (Anti-CD137 Agonist Antigen Binding Molecules) In one aspect, the present disclosure is based in part on anti-CD137 agonist antigen binding molecules and their uses. In certain embodiments, antibodies that bind to CD137 are provided. The antibodies of the present disclosure can exert immune cell activation, cytotoxic activity, or anti-tumor activity, and are therefore useful, for example, for the diagnosis or treatment of cancer.

[0058] A. Exemplary Anti-CD137 Antigen Binding Molecules or Antibodies In one aspect, the present disclosure provides an isolated antigen-binding molecule or antibody that binds to CD137. In certain embodiments, the anti-CD137 antigen-binding molecule or antibody is - Small molecule-dependent CD137 binding activity; · Binds to the extracellular domain of CD137; Forms a ternary complex with small molecules and CD137; ·Binds to CD137 of human and monkey origin; ·It is an agonist of CD137 activity; · Exhibits agonist activity against CD137 in the presence of small molecule compounds; Poor agonist activity against CD137 in the absence of small molecule compounds; and / or -In the absence of small molecule compounds, it exhibits virtually no agonist activity against CD137.

[0059] [Binding activity of antigen-binding molecules or antibodies] In certain embodiments, the binding activity of an antigen-binding molecule or antibody provided herein is ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, e.g. 10 -6 M ~10 -10 M, 10 -7 M~10 -9 M, for example, 10 -7 M ~10 -8 It has a dissociation constant (KD) of 1 M.

[0060] In one embodiment, the binding activity of an antigen-binding molecule or antibody is measured by a radiolabeled antigen binding assay (RIA) and expressed as KD. In one embodiment, the RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is measured by measuring the binding affinity of the Fab to the antigen at the lowest concentration ( 125I) Measurement is performed by equilibrating Fab with labeled antigen and then capturing the bound antigen using a plate coated with anti-Fab antibody. (See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish measurement conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 [I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., as in the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although this incubation can be continued for longer periods (e.g., approximately 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl / well of scintillant (MICROSCINT-20™, Packard) is added, and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that gives 20% or less of maximum binding is selected for use in the competitive binding assay.

[0061] In one embodiment, antibody binding activity is measured using a ligand capture method based on surface plasmon resonance analysis, e.g., a BIACORE™ T200 or BIACORE™ 4000 (GE Healthcare, Uppsala, Sweden). BIACORE™ Control Software is used to operate the instrument. In one embodiment, an amine coupling kit (GE Healthcare, Uppsala, Sweden) is used according to the supplier's instructions to immobilize a ligand capture molecule, such as an anti-tag antibody, anti-IgG antibody, or protein A, on a carboxymethyldextran-coated sensor chip (GE Healthcare, Uppsala, Sweden). The ligand capture molecule is diluted with 10 mM sodium acetate solution at an appropriate pH and injected at an appropriate flow rate and injection time. Binding activity measurements are performed using a buffer containing 0.05% polysorbate 20 (also known as Tween®-20) as the measurement buffer, at a flow rate of 10-30 μL / min, and at a temperature of preferably 25°C or 37°C. When measurements are performed by using a ligand capture molecule to capture an antibody as a ligand, the antibody is injected and captured in a desired amount, and then a serial dilution (analyte) of an antigen and / or Fc receptor prepared using the measurement buffer is injected. When measurements are performed by using a ligand capture molecule to capture an antigen and / or Fc receptor as a ligand, the antigen and / or Fc receptor is injected and captured in a desired amount, and then a serial dilution (analyte) of an antibody prepared using the measurement buffer is injected.

[0062] In one embodiment, the measurement results are analyzed using BIACORE® Evaluation Software. Kinetic parameter calculations are performed by simultaneously fitting the binding and dissociation sensorgrams using a 1:1 binding model, and the binding rate (k or ka), dissociation rate (k or k), and equilibrium dissociation constant (KD) can be calculated. When binding activity is weak, particularly when dissociation is rapid and calculation of kinetic parameters is difficult, the equilibrium dissociation constant (KD) may be calculated using a steady state model. Another parameter of binding activity is the "amount of analyte bound per unit amount of ligand," which can be calculated by dividing the amount of analyte bound at a specific concentration (resonance unit: RU) by the amount of ligand captured (RU).

[0063] [Low molecular weight compound-dependent binding activity] In one aspect, the anti-CD137 antigen-binding molecule or antibody has CD137-binding activity that depends on the small molecule compound. In a non-limiting embodiment, the anti-CD137 antigen-binding molecule or antibody has higher CD137-binding activity in the presence of a small molecule compound compared to its CD137-binding activity in the absence of the small molecule compound. In a different embodiment, the anti-CD137 antigen-binding molecule or antibody has higher CD137-binding activity in the presence of a high concentration of a small molecule compound compared to its CD137-binding activity in the presence of a low concentration of the small molecule compound. In a preferred embodiment, the anti-CD137 antigen-binding molecule or antibody has a binding activity to CD137 in the presence of a low molecular weight compound that is 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 15-fold or more, 20-fold or more, 25-fold or more, 30-fold or more, 50-fold or more, 100-fold or more, 200-fold or more, 300-fold or more, 500-fold or more, 1×10 or more, or ... 3 More than twice, 2×10 3 more than twice, above, 3×10 3 More than twice, 5×10 3 More than twice, 1×10 4 More than twice, 2×10 4 More than double, 3×10 4 More than twice, 5×104 More than double, or 1×10 5 In another preferred embodiment, the binding activity of the anti-CD137 antigen-binding molecule or antibody to CD137 in the presence of a small molecule compound is more than 2-fold, more than 3-fold, more than 5-fold, more than 10-fold, more than 15-fold, more than 20-fold, more than 25-fold, more than 30-fold, more than 50-fold, more than 100-fold, more than 200-fold, more than 300-fold, more than 500-fold, or more than 1×10 3 Twice as high as 2 x 10 3 Twice as high as 3x10 3 Twice as high as 5x10 3 Twice as high as 1×10 4 Twice as high as 2 x 10 4 Twice as high as 3x10 4 Twice as high as 5x10 4 times higher, or 1×10 5 More than double.

[0064] The concentration of the small molecule compound can be selected at any 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 concentration of the small molecule compound in the "presence of a small molecule compound" and / or in the "presence of a high concentration of a small molecule compound" can be, 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 herein can be a sufficient amount such that each anti-CD137 antigen-binding molecule or antibody exhibits maximum binding activity. In one embodiment, the concentration of the small molecular weight compound in the presence of a low concentration of the small molecular weight compound can be, for example, 500 μM or less, 250 μM or less, 200 μM or less, 150 μM or less, 100 μM or less, 50 μM or less, 10 μM or less, 1 μM or less, 500 nM or less, 100 nM or less, 50 nM or less, 10 nM, or 1 nM or less. A case where the concentration of the small molecular weight compound is zero or substantially zero can also be selected as an embodiment of a low concentration. Here, "substantially zero concentration" refers to, for example, the presence of a low molecular weight compound, but at an extremely minute concentration that cannot be detected using current technology.

[0065] In one embodiment, the binding activity to CD137 in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM of a small molecule compound is 2-fold or more, 5-fold or more, 10-fold or more, 15-fold or more, 16-fold or more, 17-fold or more, 18-fold or more, 19-fold or more, or 20-fold or more than the binding activity to CD137 in the absence of the small molecule compound. In one embodiment, the binding activity of an anti-CD137 antigen-binding molecule or antibody in the presence of 10 μM or more of a small molecule compound is 2-fold or more, 5-fold or more, 10-fold or more, 15-fold or more, 16-fold or more, 17-fold or more, 18-fold or more, 19-fold or more, or 20-fold or more than the binding activity to CD137 in the absence of the small molecule compound. In one embodiment, the binding activity of the anti-CD137 antigen-binding molecule or antibody to CD137 in the presence of 100 μM or more of a small molecule compound is 2-fold or more, 5-fold or more, 10-fold or more, 15-fold or more, 16-fold or more, 17-fold or more, 18-fold or more, 19-fold or more, or 20-fold or more than the binding activity to CD137 in the absence of the small molecule compound.

[0066] In one embodiment, the anti-CD137 antigen binding molecule or antibody has a binding activity (KD) to CD137 in the presence of 10 μM or more of a small molecule compound of 9x10 -7 M or less, 8x10 -7 M or less, 7x10 -7 M or less, 6x10 -7 M or less, 5x10 -7 M or smaller, or 4x10 -7 A dissociation constant (KD) of 5x10 or less, preferably 5x10 -7 In a further embodiment, the anti-CD137 antigen-binding molecule or antibody has a dissociation constant (KD) of 1×10 M or less, but the binding activity (KD) to CD137 in the absence of the small molecule compound is too great to be calculated by Biacore (weak binding activity) or is less than 1×10 M. -7 M or above, 5x10 -7 M or above, 7x10 -7 M or above, 8x10 -7 M or above, 9x10 -7 M or above, 1x10 -6 M or above, 2x10 -6 M or above, 3x10-6 M or larger, or 4x10 -6 A dissociation constant (KD) of 1x10 or greater, preferably 1x10 -6 In another aspect, the anti-CD137 antigen-binding molecule or antibody has a dissociation constant (KD) of 9x10 in the presence of a small molecule compound of 100 μM or more. -7 M or less, 8x10 -7 M or less, 7x10 -7 M or less, 6x10 -7 M or less, 5x10 -7 M or less, 4x10 -7 M or less, 3x10 -7 M or less, 2x10 -7 M or smaller, or 1x10 -7 A dissociation constant (KD) of 10 or less, preferably 2x10 -7 In a further embodiment, the anti-CD137 antigen-binding molecule or antibody further has a dissociation constant (KD) of 1x10 M or less, where the binding activity (KD) to CD137 in the absence of the small molecule compound is too great to be calculated by Biacore (weak binding activity) or is less than 1x10 -7 M or above, 5x10 -7 M and above, 7x10 -7 M or above, 8x10 -7 M or above, 9x10 -7 M or above, 1x10 -6 M or above, 2x10 -6 M or above, 3x10 -6 M or larger, or 4x10 -6 A dissociation constant (KD) of 1x10 or greater, preferably 1x10 -6 The dissociation constant (KD) is greater than or equal to M.

[0067] In one embodiment, the anti-CD137 antigen binding molecule or antibody has a binding activity (KD) to CD137 in the presence of 10 μM or more of a small molecule compound of 8x10 -8In another aspect, the anti-CD137 antigen-binding molecule or antibody has a dissociation constant (KD) of 2 x 10 or less in the presence of 100 μM or more of a small molecule compound, and the binding activity (KD) to CD137 in the absence of the small molecule compound is so large that it cannot be calculated by Biacore (weak binding activity). -8 The dissociation constant (KD) is less than M, and the binding activity (KD) to CD137 in the absence of the low molecular weight compound is so large that it cannot be calculated by Biacore (weak binding activity).

[0068] In one aspect, the present disclosure provides an anti-CD137 antigen-binding molecule or antibody whose value of [CD137-binding activity (amount of binding) in the presence of 10 μM or more of a small molecule compound] / [CD137-binding activity (amount of binding) in the absence of the small molecule compound] is the same as or greater than that of a reference anti-CD137 antigen-binding molecule. In a different aspect, the present disclosure provides an anti-CD137 antigen-binding molecule or antibody whose value of [CD137-binding activity (amount of binding) in the presence of 100 μM or more of a small molecule compound] / [CD137-binding activity (amount of binding) in the absence of the small molecule compound] is the same as or greater than that of a reference anti-CD137 antigen-binding molecule. In any of the above 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 having the same amino acid sequences as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contained in 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 listed in Table 17.

[0069] 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 set forth in Table 17 as a heavy chain variable region / light chain variable region combination. In a preferred embodiment, the reference antigen-binding molecule is an anti-CD137 antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 with the same amino acid sequences as those of A375 / B167. In a further embodiment, the reference anti-CD137 antigen-binding molecule is an anti-CD137 antibody comprising A375 / B167 as the heavy chain variable region / light chain variable region combination. In another 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 with the same amino acid sequences as those of A551 / B379. In a further embodiment, the reference anti-CD137 antigen-binding molecule is an anti-CD137 antibody comprising A551 / B379 as the heavy chain variable region / light chain variable region combination. In a preferred embodiment, the reference antigen-binding molecule comprises human-derived 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).

[0070] In one aspect, the present disclosure provides an anti-CD137 antigen-binding molecule or antibody whose binding activity (binding amount) to CD137 in the absence of a small molecule compound is the same as or lower than that of a reference anti-CD137 antigen-binding molecule, and whose binding activity (binding amount) to CD137 in the presence of 10 μM or more of the small molecule compound is the same as or higher than that of the reference anti-CD137 antigen-binding molecule under the same conditions. In a different aspect, the present disclosure provides an anti-CD137 antigen-binding molecule or antibody whose binding activity to CD137 in the absence of a small molecule compound is the same as or lower than that of a reference anti-CD137 antigen-binding molecule, and whose binding activity (binding amount) in the presence of 10 μM or more of the small molecule compound is the same as or higher than that of the reference anti-CD137 antigen-binding molecule under the same conditions. In any of the above 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 having the same amino acid sequences as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contained in 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 listed in Table 17.

[0071] In one embodiment, the reference anti-CD137 antigen binding molecule is an anti-CD137 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 set forth in Table 17 as a heavy chain variable region / light chain variable region combination. 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 with the same amino acid sequences as those of A375 / B167. In a further embodiment, the reference anti-CD137 antigen-binding molecule is an anti-CD137 antibody comprising A375 / B167 as the heavy chain variable region / light chain variable region combination. In another 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 with the same amino acid sequences as those of A551 / B379. In a further embodiment, the reference anti-CD137 antigen-binding molecule is an anti-CD137 antibody comprising A551 / B379 as the heavy chain variable region / light chain variable region combination. In a preferred embodiment, the reference antigen-binding molecule comprises human-derived 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).

[0072] In one aspect, the present disclosure provides an anti-CD137 antigen-binding molecule or antibody in which the value of [binding activity (KD) to CD137 in the presence of 1 μM of a small molecule compound] / [binding activity (KD) to CD137 in the presence of 10 μM or more of the small molecule compound] is the same as or greater than that of a reference antigen-binding molecule. In a different aspect, the present disclosure provides an anti-CD137 antigen-binding molecule or antibody in which the value of [binding activity (KD) to CD137 in the presence of 1 μM of a small molecule compound] / [binding activity (KD) to CD137 in the presence of 100 μM or more of the small molecule compound] is the same as or greater than that of a reference antigen-binding molecule. In any of the above 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 having the same amino acid sequences as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contained in 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 listed in Table 17.

[0073] 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 set forth in Table 17, as a heavy chain variable region / light chain variable region combination. 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 with the same amino acid sequences as those contained in A375 / B167. In a further embodiment, the reference antigen-binding molecule is an antibody comprising A375 / B167 as the heavy chain variable region / light chain variable region combination. In a different preferred embodiment, the reference antigen-binding molecule is an antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 with the same amino acid sequences as those contained in A551 / B379. In a further 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 a heavy chain constant region and a light chain constant region of human origin (e.g., G1T3 (SEQ ID NO: 138) as a heavy chain constant region and human λ chain Lamlib (SEQ ID NO: 63) as a light chain constant region).

[0074] In one aspect, the binding activity of an anti-CD137 antibody to CD137 in the presence, absence, high concentration and / or low concentration of a small molecule compound is measured by a ligand capture method using, for example, a BIACORE (registered trademark) T200, which employs surface plasmon resonance analysis as the measurement principle.

[0075] An exemplary method for measuring the binding activity of an anti-CD137 antibody to CD137 is described in detail below. In one embodiment, the binding activity of an anti-CD137 antibody to CD137 is evaluated using a BIACORE™ T200. In a preferred embodiment, the measurement is performed at 37°C using a running buffer containing 20 mM ACES (pH 7.4), 150 mM NaCl, 2 mM MgCl2, and 0.05% Tween 20. In one embodiment, the measurement is performed by capturing the antibody as a ligand on a ligand capture molecule. Specifically, an antibody solution prepared in a running buffer is first allowed to interact with a Series S Sensor Chip CM3 (GE Healthcare) on which Sure Protein A (GE Healthcare) is immobilized, thereby capturing an appropriate amount of antibody (e.g., approximately 100 RU, 200 RU, 300 RU, 400 RU, or 500 RU).

[0076] In a preferred embodiment, approximately 100-500 RU, preferably approximately 250-400 RU, of antibody is captured. Next, a CD137 solution prepared in a running buffer containing a small molecule compound added to a target concentration (e.g., 1 μM, 10 μM, 50 μM, or 100 μM) or a CD137 solution prepared in a running buffer without the small molecule compound is allowed to interact with the antibody, thereby assessing binding activity to CD137 in both the presence and absence of the small molecule compound. The concentration of CD137 in the CD137 solution can be determined as appropriate. For example, when using hCD137-HisBAP (see Example 1-1) as the antigen, measurements are performed at antigen concentrations of 0 nM, 15.625 nM, 62.5 nM, 250 nM, and 1000 nM. In one embodiment, the dissociation constant (KD) of an anti-CD137 antibody to human CD137 is calculated using Biacore T200 Evaluation Software 2.0. Specifically, the sensorgram obtained by measurement is globally fitted using a 1:1 Langmuir binding model to calculate the binding rate constant ka (L / mol / s) and the dissociation rate constant kd (1 / s), and the dissociation constant KD (mol / L) is calculated from these values.

[0077] Further exemplary methods 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 evaluated using a Biacore T200. Binding measurements to human CD137 were performed at 37°C using a running buffer containing 20 mM ACES (pH 7.4), 150 mM NaCl, 2 mM MgCl2, and 0.05% Tween 20. First, an antibody solution prepared in the running buffer was allowed to interact with a Series S Sensor Chip CM3 (GE Healthcare) on which Sure Protein A (GE Healthcare) was immobilized, capturing approximately 250-400 RU of antibody. Next, human CD137 solutions prepared in running buffer containing ATP at the desired concentration (e.g., 1 μM, 10 μM, 50 μM, or 100 μM) or in running buffer lacking ATP were used to evaluate binding activity with human CD137 in both the presence and absence of ATP. The human CD137 antigen, hCD137-HisBAP prepared as described in Example (1-1), was used, 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 with 25 mM NaOH and 10 mM glycine-HCl (pH 1.5), and measurements were performed by repeatedly capturing the antibody. The dissociation constants of each antibody for human CD137 were calculated using Biacore T200 Evaluation Software 2.0. Specifically, the sensorgram obtained by measurement is globally fitted using a 1:1 Langmuir binding model to calculate the binding rate constant ka (L / mol / s) and the dissociation rate constant kd (1 / s), and the dissociation constant KD (mol / L) is then calculated from these values.

[0078] In one embodiment, the binding activity of an anti-CD137 antibody to CD137 (preferably human CD137) can also be expressed as the "amount of CD137 bound per unit amount of antibody." Specifically, using a sensorgram obtained by the above-mentioned measurement method using the BIACORE (registered trademark) T200, the "amount of CD137 bound per unit amount of antibody" is calculated by dividing the amount of CD137 bound to the antibody (RU) by the amount captured by the antibody (RU). In one embodiment, the binding activity of an anti-CD137 antibody to CD137 (preferably human CD137) can also be measured by the method described in Example 5-3 or 6-2.

[0079] The terms "small molecule" and "small molecular compound" as used herein refer to naturally occurring or non-naturally occurring chemical substances other than "biopolymers" present in living organisms. Preferably, they are target tissue-specific compounds or non-naturally occurring compounds, but are not limited thereto. In one aspect, a "small molecule compound" in the present disclosure is a "cancer tissue-specific compound" or a "cancer tissue-specific metabolite." The term "cancer tissue-specific compound" in the present disclosure refers to a compound that is differentially present in cancer tissue compared to non-cancerous tissue. As used herein, the term "cancer" generally refers to a malignant neoplasm, which may be metastatic or non-metastatic. The term "metabolism" refers to chemical changes that occur within the tissues of an organism and includes "anabolism" and "catabolism." Anabolism refers to the biosynthesis or accumulation of molecules, and catabolism refers to the breakdown of molecules. A "metabolite" is an intermediate or product resulting from metabolism.

[0080] The term "target tissue" refers to any tissue in a living body to which the antigen-binding molecule of the present invention is intended to be delivered. The target tissue may be a histologically distinct tissue such as various organs, or a pathologically distinct tissue such as healthy tissue and diseased tissue. In a specific embodiment, the target tissue is tumor tissue. On the other hand, "non-target tissue" refers to a tissue other than the target tissue in a living body.

[0081] The term "tumor tissue" refers to tissue containing at least one tumor cell. Tumor tissue usually consists of a group of tumor cells (parenchyma), which form the main body of the tumor, and the connective tissue and blood vessels (stroma) that exist between them and support the tumor. In some cases, the distinction between the two is clear, while in other cases, the two are mixed. Immune cells and other substances may infiltrate tumor tissue. On the other hand, "non-tumor tissue" refers to tissue other than tumor tissue in the body. Healthy tissue / normal tissue that is not in a diseased state is a typical example of non-tumor tissue.

[0082] A non-limiting embodiment of the cancer tissue-specific compound or cancer tissue-specific metabolite used in the present disclosure is at least one compound selected from the compounds described in detail below. "At least one compound" means that the antigen-binding activity of the same antigen-binding domain described below is dependent on one type of cancer tissue-specific compound or cancer tissue-specific metabolite, as well as on multiple types of cancer tissue-specific compounds or cancer tissue-specific metabolites.

[0083] As used herein, the term "target tissue-specific compound" refers to a compound that is differentially present in target tissue compared to non-target tissue. In some embodiments, a target tissue-specific compound can be a compound defined by qualitative target tissue specificity, such as being present in target tissue but not in non-target tissue, or being present in non-target tissue but not in target tissue. In another embodiment, a target tissue-specific compound can be a compound defined by quantitative target tissue specificity, such as being present in target tissue at a different concentration (e.g., higher or lower concentration) compared to non-target tissue. In certain embodiments, the target tissue-specific compound exhibits a specific activity relative to a non-target tissue, e.g., 1.05-fold or more, 1.1-fold or more, 1.15-fold or more, 1.2-fold or more, 1.25-fold or more, 1.3-fold or more, 1.35-fold or more, 1.4-fold or more, 1.45-fold or more, 1.5-fold or more, 1.55-fold or more, 1.6-fold or more, 1.65-fold or more, 1.7-fold or more, 1.75-fold or more, 1.8-fold or more, 1.85-fold or more, 1.9-fold or more, 1.95-fold or more, 2-fold or more, 2.1-fold or more, 2.2-fold or more, 2.3-fold or more, 2.4-fold or more, 2.5-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 50-fold or more, 10 ... 3 more than twice, 10 4 more than twice, 10 5 more than twice, 10 6 In another embodiment, the target tissue-specific compound is present in the target tissue at a concentration that is, for example, 1.05-fold or more, 1.1-fold or more, 1.15-fold or more, 1.2-fold or more, 1.25-fold or more, 1.3-fold or more, 1.35-fold or more, 1.4-fold or more, 1.45-fold or more, 1.5-fold or more, 1.55-fold or more, 1.6-fold or more, 1.65-fold or more, 1.7-fold or more, 1.75-fold or more, 1.8-fold or more, 1.85-fold or more, 1.9-fold or more, 1.95-fold or more, 2-fold or more, 2.1-fold or more, 2.2-fold or more, 2.3-fold or more, 2.4-fold or more, 2.5-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 50-fold or more, 10 ... 3 more than twice, 10 4 more than twice, 10 5 more than twice, 10 6In certain embodiments, the target tissue-specific compound is present in the target tissue at a concentration that is statistically significantly higher or lower than that of the non-target tissue (i.e., a p-value of less than 0.05 and / or a q-value of less than 0.10, as determined using either a Welch t-test or a Wilcoxon rank sum test). In certain embodiments, the target tissue-specific compound is a tumor tissue-specific compound.

[0084] In certain embodiments, the tumor tissue-specific compound is a metabolite produced by metabolism specific to tumor cells. The metabolite may be a product produced by metabolism essential for vital activities (primary metabolite) or a product produced by metabolism not necessarily required for vital activities (secondary metabolite). Examples of primary metabolites include sugars, proteins, lipids, and nucleic acids. Examples of secondary metabolites include antibiotics and pigments. The metabolite may be a biopolymer or a small molecule. In certain embodiments, the biopolymer is a molecule with a molecular weight of approximately 5,000 or more composed of one or more types of repeating units, such as polysaccharides, polypeptides, and polynucleotides. In certain embodiments, the small molecule is a molecule with a molecular weight of approximately 500 or less and is a chemical substance present in living organisms. In a further embodiment, the tumor tissue-specific compound is a small molecule metabolite produced specifically in tumor cells (Eva Gottfried, Katrin Peter, and Marina P. Kreutz, From Molecular to Modular Tumor Therapy (2010) 3 (2), 111-132). In a further embodiment, the tumor tissue-specific compound is a metabolite specifically produced by cells infiltrating tumor tissue (e.g., immune cells, etc.) or stromal cells present in tumor tissue (e.g., cancer stromal fibroblasts (CAFs)). Examples of immune cells infiltrating tumor tissue include dendritic cells, suppressor dendritic cells, regulatory T cells, exhausted T cells, and myeloma-derived suppressor cells (MDSCs). In a further embodiment, metabolites produced by cells present in tumor tissue (e.g., tumor cells, immune cells, stromal cells, etc.) and released extracellularly upon cell death by apoptosis, necrosis, or the like may also be included in the tumor tissue-specific compounds of the present disclosure.

[0085] To identify tumor tissue-specific compounds, analyses at the transcriptome level (e.g., Dhanasekaran et al. (Nature (2001) 412, 822-826), Lapointe et al. (Proc. Natl. Acad. Sci. USA (2004) 101, 811-816), or Perou et al. (Nature (2000) 406, 747-752)) or proteome level (e.g., Ahram et al. (Mol. Carcinog. (2002) 33, 9-15), Hood et al. (Mol. Cell. Proteomics (2005) 4, 1741-1753)) as well as metabolic (metabolomic) analyses focusing on metabolic profiling are appropriately used. That is, to identify metabolites in a test sample, metabolic profiling using 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)), ELISA, and the like, alone or in combination, can be used as appropriate.

[0086] 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, primary metabolites of sugar metabolism, and nicotinamide and its metabolites. In a further embodiment, the tumor tissue-specific compound is at least one compound selected from the following (1) to (6): (1) Nucleosides having a purine ring structure, such as adenosine (ADO), adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and inosine; (2) Amino acids such as alanine, glutamic acid, and aspartic acid, (3) Metabolites of amino acids such as kynurenine, anthranilic acid, 3-hydroxykynurenine, and kynurenic acid, (4) Metabolites of arachidonic acid such as prostaglandin E2, (5) Primary metabolites of glycolysis or the Krebs cycle, such as lactate, succinate, and citrate, and (6) Metabolites of nicotinamide such as 1-methylnicotinamide.

[0087] (1) Nucleosides with a purine ring structure, such as adenosine (ADO), adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and inosine It is known that upon tumor cell death, a large amount of intracellular ATP leaks out of the cell. Therefore, ATP concentrations in tumor tissues are significantly higher than in normal tissues (PLoS One. (2008) 3, e2599). AMP is metabolized by cell surface enzymes such as eco-5'-nucleotidase (CD73) (Resta and Thompson, Immunol. Rev. (1998) 161, 95-109; Sadej et al., Melanoma Res. (2006) 16, 213-222). Adenosine is a purine nucleoside that is constitutively present in low concentrations in the extracellular environment. However, a significant increase in extracellular adenosine concentrations has been reported in hypoxic tissues found in solid tumors (Blay and Hoskin, Cancer Res. (1997) 57, 2602-2605). CD73 is expressed on the surface of tumor and immune cells (Kobie et al., J. Immunol. (2006) 177, 6780-6786), and its activity has been found to be elevated in breast cancer (Canbolat et al., Breast Cancer Res. Treat. (1996) 37, 189-193), gastric cancer (Durak et al., Cancer Lett. (1994) 84, 199-202), pancreatic cancer (Flocke and Mannherz, Biochim. Biophys. Acta (1991) 1076, 273-281), and glioblastoma (Bardot et al., Br. J. Cancer (1994) 70, 212-218). It has been proposed that the accumulation of adenosine in tumor tissues may be due to increased dephosphorylation of AMP by cytoplasmic 5'-nucleotidase (Headrick and Willis (Biochem. J. (1989) 261, 541-550)). Furthermore, regulatory T cells infiltrating tumor tissues also express ATPase and produce adenosine (Proc. Natl. Acad. Sci. USA (2006) 103(35), 13132-13137, Curr. Med. Chem. (2011) 18, 5217-5223).The produced adenosine is thought to create an immunosuppressive environment in tumor tissue via adenosine receptors such as A2A receptors (Curr. Med. Chem. (2011) 18, 5217-5223). Based on the above, ATP, ADP, AMP, adenosine, and the like, which are thought to accumulate at high concentrations in tumor tissue through the metabolism of purine nucleotides, are examples of tumor tissue-specific compounds used in the present disclosure. Furthermore, adenosine is decomposed into inosine by adenosine deaminase, resulting in the accumulation of inosine at high concentrations.

[0088] In certain embodiments, the nucleoside having a purine ring structure comprises an adenosine-containing compound.In certain embodiments, adenosine-containing compounds can 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), adenosine gamma thiotriphosphate (ATPγS) and the like.In another embodiment, the nucleoside having a purine ring structure comprises inosine, which is a metabolite of adenosine. Furthermore, in certain embodiments, nucleosides having a purine ring structure also include commercially available nucleosides having a purine ring structure, such as ADPbetaS (Sigma).

[0089] (2) Amino acids such as alanine, glutamic acid, and aspartic acid In tumor cells, the rate of intracellular uptake of glutamine, which acts as a nitrogen carrier in vivo, is increased, and this uptake of glutamine and its subsequent conversion to glutamate and lactate (glutaminolysis) is considered to be a characteristic of tumor cells (Mazurek and Eigenbrodt (Anticancer Res. (2003) 23, 1149-1154, and Mazurek et al. (J. Cell. Physiol. (1999) 181, 136-146)). In cancer patients, plasma glutamine levels are decreased, while glutamate concentrations are increased (Droge et al. (Immunobiology (1987) 174, 473-479)). In addition, in lung cancer tissue, 13 Metabolic studies of C-radiolabelled glucose revealed 13 C-labeled succinic acid, 13 C-labeled alanine, 13 C-labeled glutamate, and 13 A correlation was observed between the concentrations of C-labeled citric acid. Based on the above, alanine, glutamic acid, aspartic acid, and the like, which are thought to accumulate at high concentrations in tumor tissues due to glutaminolysis or the like, are listed as examples of tumor tissue-specific compounds used in the present disclosure.

[0090] (3) Metabolites of amino acids such as kynurenine, anthranilic acid, 3-hydroxykynurenine, and kynurenic acid Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-metabolizing enzyme highly expressed in many cancers, including melanoma, colon cancer, and renal 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 in dendritic cells infiltrating tumor tissue, and dendritic cells also produce kynurenine (J. Immunol. (2008) 181, 5396-5404). IDO is also expressed in myeloid-derived suppressor cells (MDSCs) in tumor tissues, and MDSCs also produce kynurenine (Yu et al. (J. Immunol. (2013) 190, 3783-3797)). Kynurenine is converted to anthranilic acid by kynurenidase and to 3-hydroxykynurenine by kynurenine 3-hydroxylase. Both anthranilic acid and 3-hydroxykynurenine are converted to 3-hydroxyanthranilic acid, a precursor of NAD. Kynurenine is converted to kynurenic acid by kynurenine aminotransferase. For these reasons, kynurenine and its metabolites, such as anthranilic acid, 3-hydroxykynurenine, and kynurenic acid, are examples of tumor tissue-specific compounds, particularly tumor cell-specific metabolites, used in the present disclosure.

[0091] (4) Arachidonic acid metabolites such as prostaglandin E2 Prostaglandin E2 (PGE2) promotes the proliferation of colon cancer cells and inhibits their apoptosis (Sheng et al., Cancer Res. (1998) 58, 362-366). Among PGE2 synthases, COX-1 is constitutively expressed in almost all tissues, whereas COX-2 is primarily found to be induced by certain inflammatory cytokines and oncogenes in tumors (Warner and Mitchell, FASEB J. (2004) 18, 790-804). Overexpression of COX-2 has also 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). Regulatory T cells infiltrating tumor tissue also produce PGE2 (Curr. Med. Chem. (2011) 18, 5217-5223). Based on the above, arachidonic acid metabolites such as PGE2 are examples of tumor tissue-specific compounds used in the present disclosure, particularly tumor cell-specific metabolites and immune cell-specific metabolites infiltrating tumor tissue. In addition to PGE2, the production of thromboxane A2 (TXA2) is also increased in tumor tissues such as colon cancer (J. Lab. Clin. Med. (1993) 122, 518-523).

[0092] (5) Primary metabolic products of glycolysis or the Krebs cycle, such as lactate, succinate, and citrate The glycolytic phenotype, characterized by the upregulation of glycolytic (Embden-Myerhof pathway) enzymes such as pyruvate kinase, hexokinase, and lactate dehydrogenase (LDH), has long been known to be a characteristic of solid tumors, known as the Warburg effect. Lactate, the end product of glycolysis, and succinate and citrate produced by the Krebs cycle are known to accumulate in tumor tissue (Teresa et al., Mol. Cancer (2009) 8, 41-59). Based on the above, primary metabolites produced by glycolysis, such as lactate, succinate, and citrate, are examples of tumor tissue-specific compounds, particularly tumor cell-specific metabolites, used in the present disclosure. It is also known that succinate, present at high concentrations within cells, leaks out of cells upon cell death (Nature Immunology, (2008) 9, 1261-1269). Therefore, it is thought that the concentration of succinic acid increases in tumor tissues where cell death occurs frequently.

[0093] (6) Metabolites of nicotinamide such as 1-methylnicotinamide Nicotinamide N-methyltransferase is known to be highly expressed in multiple human tumor tissues. 1-Methylnicotinamide, a stable metabolic product of nicotinamide produced by this enzyme, is known to be secreted extracellularly from tumor cells (Yamada et al. (J. Nutr. Sci. Vitaminol. (2010) 56, 83-86)). Based on the above, 1-methylnicotinamide and the like, which are thought to accumulate at high concentrations in tumor tissues through the metabolism of nicotinamide, are exemplified as tumor tissue-specific compounds for use in the present disclosure.

[0094] The "antigen-binding molecule" of the present disclosure includes an "antigen-binding domain." The "antigen-binding domain" may have any structure, as long as it binds to an antigen of interest. In one embodiment, the antigen-binding domain of the present disclosure includes, for example, the variable regions of the heavy and / or light chains of an antibody, Avimers containing modules (A domains) of about 35 amino acids contained in various cell membrane proteins in vivo (International Publication WO2004 / 044011, WO2005 / 040229), Adnectins containing the 10Fn3 domain of fibronectin, a glycoprotein expressed on the cell membrane (International Publication WO2002 / 032925), Affibodies using an IgG-binding domain consisting of 58 amino acids of Protein A as a scaffold (International Publication WO1995 / 001937), DARPins (Designed Ankyrin Repeat proteins) containing ankyrin repeats (AR), a 33-amino acid repeat sequence, as a scaffold (International Publication WO2002 / 020565), and neutrophil gelatinase-associated lipocalin (neutrophil gelatinase-associated lipocalin). Examples of antigen-binding domains of the present disclosure include anticalin (International Publication No. WO 2003 / 029462), which contains a lipocalin such as NGAL as a scaffold, and variable lymphocyte receptor (VLR), which is a protein that functions in the adaptive immune system of jawless fish such as lampreys and hagfish and contains a leucine-rich repeat (LRR) module (International Publication No. WO 2008 / 016854). In certain embodiments, the antigen-binding domain of the present disclosure comprises the variable regions of the heavy and light chains of an antibody. In further embodiments, examples of antigen-binding domains of the present disclosure include single-chain Fv (scFv), single-chain antibody, Fv, single-chain Fv2 (scFv2), Fab, or F(ab')2.

[0095] [HVR and variable regions] In one aspect, the present 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 NOs: 8, 9, 10, 11, 12, 13, 14, 15, and 16; and (c) HVR-H3 comprising any one amino acid sequence selected from SEQ ID NOs: 17, 18, 19, or 20. In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) an HVR-H2 comprising any one amino acid sequence selected from SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, and 16; and (c) an HVR-H3 comprising any one amino acid sequence selected from SEQ ID NOs: 17, 18, 19, or 20.

[0096] 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 set forth in Table 17 as a heavy chain variable region / light chain variable region combination. In a preferred embodiment, 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 with the same amino acid sequences as those of A375 / B167. In a further embodiment, the anti-CD137 antigen-binding molecule is an anti-CD137 antibody comprising A375 / B167 as the heavy chain variable region / light chain variable region combination. In another preferred embodiment, 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 with the same amino acid sequences as those of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contained in A551 / B379.

[0097] In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises: (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; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17.

[0098] In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises: (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; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17.

[0099] In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises: (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; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17.

[0100] In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises: (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; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18.

[0101] In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises: (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; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18.

[0102] In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises: (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; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18.

[0103] In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises: (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; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18.

[0104] In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises: (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; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19.

[0105] In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises: (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; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20.

[0106] In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises: (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; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20.

[0107] In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises: (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; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17.

[0108] In another aspect, the present 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 any one amino acid sequence selected from SEQ ID NOs: 21, 22, 23, 24, and 25; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3 comprising any one amino acid sequence selected from SEQ ID NOs: 27, 28, and 29. In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises (a) HVR-L1 comprising any one amino acid sequence selected from SEQ ID NOs: 21, 22, 23, 24, and 25; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3 comprising any one amino acid sequence selected from SEQ ID NOs: 27, 28, and 29.

[0109] In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

[0110] In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

[0111] In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28.

[0112] In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 29.

[0113] In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

[0114] In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

[0115] In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

[0116] In another aspect, the anti-CD137 antigen binding molecule or antibody of the present disclosure comprises: (a) a VH domain, wherein at least one, at least two, or all three VH domains are selected from: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (ii) HVR-H2 comprising any one amino acid sequence selected from SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, and 16; and (iii) HVR-H3 comprising any one amino acid sequence selected from SEQ ID NOs: 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) HVR-L1 comprising any one amino acid sequence selected from SEQ ID NOs: 21, 22, 23, 24, and 25; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (iii) HVR-L3 comprising any one amino acid sequence selected from SEQ ID NOs: 27, 28, and 29.

[0117] In another aspect, the present 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.

[0118] In another aspect, the present 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.

[0119] In another aspect, the present 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.

[0120] In another aspect, the present 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.

[0121] In another aspect, the present 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.

[0122] In another aspect, the present 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.

[0123] In another aspect, the present 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.

[0124] In another aspect, the present 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.

[0125] In another aspect, the present 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.

[0126] In another aspect, the present 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.

[0127] In another aspect, the present 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.

[0128] In another aspect, the present 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.

[0129] In another aspect, the present 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.

[0130] In certain embodiments, any one or more amino acids of the above-mentioned anti-CD137 antibodies are substituted at the following HVR positions: - in HVR-H2 (SEQ ID NO: 30): positions 5, 6, 7, 10, 13, 14, and / or 17 - in HVR-H3 (SEQ ID NO: 31): position 3 and / or 6 - in HVR-L1 (SEQ ID NO: 32): positions 4, 5, 9, and / or 11 - in HVR-L3 (SEQ ID NO: 33): positions 6, 7, and / or 8.

[0131] In certain embodiments, the substitutions provided herein are conservative substitutions. In certain embodiments, any one or more of the following substitutions may be made in any combination: - in HVR-H2 (SEQ ID NO: 8): K5H or S; S6G; T7S; E10Y; D13E; S14Q; V17G or L - in HVR-H3 (SEQ ID NO: 17): A3P, K or I; F6E - in HVR-L1 (SEQ ID NO: 21): R4S; Y5T; Y9F; E11N - in HVR-L3 (SEQ ID NO: 27): E6P; H7A; Q8I

[0132] All possible combinations of the above substitutions are encompassed in the consensus sequences of SEQ ID NOs: 30, 31, 32 and 33 for HVR-H2, HVR-H3, HVR-L1 and HVR-L3, respectively.

[0133] In any of the above-described embodiments, the anti-CD137 antigen-binding molecule or antibody is humanized. In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises the HVR of any of the above-described embodiments and further comprises an acceptor human framework (e.g., a human immunoglobulin framework or a human consensus framework). In another embodiment, the anti-CD137 antigen-binding molecule or antibody comprises the HVR of any of the above-described embodiments and further comprises a heavy chain variable region (VH) or light chain variable region (VL) comprising framework (FR) sequences. In one embodiment, FR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 35, FR2 comprises the amino acid sequence of SEQ ID NO: 36, FR3 comprises the amino acid sequence of SEQ ID NO: 37, and FR4 comprises the amino acid sequence of SEQ ID NO: 38. In one embodiment, FR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 39, FR2 comprises the amino acid sequence of SEQ ID NO: 40, FR3 comprises the amino acid sequence of SEQ ID NO: 41, and FR4 comprises the amino acid sequence of SEQ ID NO: 42.

[0134] In another aspect, an 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 to the amino acid sequence of SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-CD137 antigen binding molecule or antibody comprising the sequence retains the ability to bind to CD137. In certain 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 certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., within the FRs). Optionally, the anti-CD137 antibody comprises a 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 certain aspects, the VH comprises one, two, or three HVRs 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 NOs: 8, 9, 10, 11, 12, 13, 14, 15, and 16; and (c) HVR-H3 comprising any one amino acid sequence selected from SEQ ID NOs: 17, 18, 19, or 20. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0135] In another aspect, an anti-CD137 antigen binding molecule or antibody is provided, comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 54, 55, 56, 57, 58, 59, or 60. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-CD137 antigen binding molecule or antibody comprising the sequence retains the ability to bind to CD137. In certain 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 certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., within the FRs). Optionally, the anti-CD137 antigen binding molecule or antibody comprises a VL sequence in SEQ ID NO: 54, 55, 56, 57, 58, 59, or 60, including post-translational modifications of said sequences. In certain embodiments, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1 comprising any one amino acid sequence selected from SEQ ID NOs: 21, 22, 23, 24, and 25; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3 comprising any one amino acid sequence selected from SEQ ID NOs: 27, 28, and 29. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0136] In another aspect, there is provided an anti-CD137 antigen binding molecule or antibody comprising the VH of any of the above embodiments and the VL of any of the above embodiments. In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 43 and SEQ ID NO: 54, respectively, including post-translational modifications of said sequences. In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 44 and SEQ ID NO: 55, respectively, including post-translational modifications of said sequences. In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 45 and SEQ ID NO: 55, respectively, including post-translational modifications of said sequences. In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 46 and SEQ ID NO: 54, respectively, including post-translational modifications of said sequences. In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 47 and SEQ ID NO: 54, respectively, including post-translational modifications of said sequences. In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 48 and SEQ ID NO: 56, respectively, including post-translational modifications of said sequences. In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 49 and SEQ ID NO: 57, respectively, including post-translational modifications of said sequences. In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 50 and SEQ ID NO: 58, respectively, including post-translational modifications of said sequences. In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 51 and SEQ ID NO: 59, respectively, including post-translational modifications of said sequences. In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 51 and SEQ ID NO: 60, respectively, including post-translational modifications of said sequences. In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 52 and SEQ ID NO: 60, respectively, including post-translational modifications of said sequences. In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 50 and SEQ ID NO: 59, respectively, including post-translational modifications of said sequences. In one embodiment, the anti-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 53 and SEQ ID NO: 54, respectively, including post-translational modifications of said sequences. The post-translational modifications mentioned above include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0137] The heavy chain variable region and light chain variable region of preferred anti-CD137 antigen-binding molecules or antibodies of the present disclosure, as well as the amino acid sequences of their HVR1, 2, and 3, and their corresponding SEQ ID NOs are shown in the table below.

[0138] [Table 1]

[0139] When the N-terminal amino acid of the heavy or light chain of an anti-CD137 antigen-binding molecule or antibody provided herein is glutamine, the amino acid may be substituted with glutamic acid.When the N-terminal amino acid of the heavy or light chain of an anti-CD137 antibody provided herein is glutamic acid, the amino acid may be substituted with glutamine.

[0140] In a preferred embodiment, any of the anti-CD137 antigen-binding molecules or antibodies comprising the above-described HVRs, heavy chain variable regions, and / or light chain variable regions has binding activity to CD137 that is dependent on the above-described small molecule compounds.

[0141] [Stationary region] In another aspect, the anti-CD137 antigen-binding molecule or antibody comprises a constant region. The constant region may be a heavy chain constant region (including an Fc region), a light chain constant region, or both. In a further aspect, the anti-CD137 antigen-binding molecule or antibody comprises an Fc region. In some embodiments, the constant region is a native sequence constant region. Exemplary heavy chain constant regions derived from native antibodies include heavy chain constant regions of human IgG1 (SEQ ID NOs: 61 and 62), human IgG2, human IgG3, and human IgG4. Exemplary light chain constant regions derived from native antibodies include human κ chain and human λ chain (e.g., SEQ ID NO: 63).

[0142] As used herein, a "parent constant region" or "parent Fc region" refers to a constant region or Fc region prior to the introduction of the amino acid modifications described herein. A "parent antigen-binding molecule" refers to an antigen-binding molecule comprising a parent constant region or parent Fc region. In some embodiments, the parent Fc region is a native-sequence Fc region (or an Fc region of a native antibody). Examples of antibodies include IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), and IgM. Antibodies may be derived from humans or monkeys (e.g., cynomolgus monkeys, rhesus monkeys, marmosets, chimpanzees, or baboons). Native antibodies may contain naturally occurring mutations. Multiple allotype sequences of IgG due to genetic polymorphisms are described in "Sequences of proteins of immunological interest," NIH Publication No. 91-3242, any of which may be used in the present disclosure. In one embodiment, the parent Fc region is an Fc region derived from the human IgG1 heavy chain constant region of SEQ ID NO: 61, 62 or 182.

[0143] In one aspect, the anti-CD137 antigen-binding molecule or antibody has an increased isoelectric point (pI) compared to an anti-CD137 antigen-binding molecule or antibody comprising a native sequence Fc region or a parent Fc region. In some embodiments, the mutant Fc region comprises at least one amino acid modification. In further embodiments, the amino acid modification increases the isoelectric point (pI) of the mutant Fc region compared to the parent Fc region. Without being bound by theory, it is believed that the pH of biological fluids (e.g., plasma) is within the neutral pH range. In biological fluids, the net positive charge of an antigen-binding molecule or antibody with an increased pI is increased due to the increased pI, and as a result, the antigen-binding molecule or antibody is more strongly attracted to the endothelial cell surface, which has a net negative charge, through physicochemical Coulombic interactions than an antigen-binding molecule or antibody without an increased pI. This brings the agonist antigen-binding molecule (or antibody), or the antigen-bound agonist antigen-binding molecule (or antibody), closer to the surface of Fcγ receptor-expressing cells, thereby increasing the binding of the antigen-binding molecule or antibody to Fcγ receptor-expressing cells. For anti-CD137 agonist antigen-binding molecules or antibodies whose Fcγ receptor-binding activity contributes to CD137 agonist activity, anti-CD137 agonist antigen-binding molecules or antibodies with increased binding to Fcγ receptor-expressing cells due to amino acid modifications that increase pI can exhibit higher CD137 agonist activity than anti-CD137 agonist antigen-binding molecules or antibodies that do not contain the amino acid modifications that increase pI.

[0144] In the present disclosure, pI may be a theoretical pI or an actually measured pI. The pI value can be measured, for example, by isoelectric focusing, a method known to those skilled in the art. The theoretical pI value can be calculated, for example, using gene and amino acid sequence analysis software (such as Genetyx). In this case, antibody characteristics may be reflected in the calculation formula. For example, (i) conserved Cys in antibodies typically form disulfide bonds and do not have a side chain charge. Such Cys may be excluded from the calculation, and only free Cys that do not form disulfide bonds may be included in the calculation. Furthermore, (ii) post-translational modifications may change the charge state, i.e., the isoelectric point, of antibodies. Taking such post-translational modifications into account, the calculation formula may be modified as follows: (a) if the N-terminus of the heavy chain is Q (glutamine), pyroglutamylation is assumed to occur and the N-terminal amino group is excluded from the calculation, (b) if the C-terminus of the heavy chain is K (lysine), cleavage is assumed to occur and K (one residue) is excluded from the calculation, and (c) all C (cysteine) at generally conserved positions are assumed to form disulfide bonds within the molecule and the side chains of these C are excluded from the calculation. In a preferred embodiment, both of the above (i) and (ii) may be reflected in the calculation formula.

[0145] In one embodiment, the pI value may be increased by, for example, at least 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, or more, at least 0.6, 0.7, 0.8, 0.9, or more, at least 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, or more, or at least 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0, or more, compared to before modification.

[0146] In one embodiment, amino acid modifications related to increasing pI and methods for increasing the pI of antigen-binding molecules or antibodies are described in detail in III. Compositions and Methods (agonist antigen-binding molecules comprising a mutant Fc region with an increased isoelectric point (pI)) herein. Those skilled in the art will understand that any amino acid modification and method for increasing pI described in III. Compositions and Methods (agonist antigen-binding molecules comprising a mutant Fc region with an increased isoelectric point (pI)) can be applied to anti-CD137 antigen-binding molecules or antibodies.

[0147] In one embodiment, the anti-CD137 antigen binding molecule or antibody has a variant Fc region with increased pI, wherein the variant Fc region comprises at least one amino acid modification 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 (EU numbering). In a further embodiment, the variant Fc region with increased pI comprises Arg or Lys at each of the selected positions.

[0148] In a further embodiment, the anti-CD137 antigen binding molecule or antibody has a variant Fc region with increased pI, wherein the variant Fc region comprises at least one amino acid modification at at least one position selected from the group consisting of positions 311, 343, and 413 as defined by EU numbering. In a further embodiment, the variant Fc region with increased pI comprises an amino acid modification at position 311, 343, or 413 as defined by EU numbering. In a further embodiment, the variant Fc region with increased pI comprises Arg or Lys at each of the selected positions.

[0149] In another aspect, the present disclosure provides an anti-CD137 antigen-binding molecule or antibody comprising a mutant Fc region with increased pI, which comprises any one of the following amino acid modifications (1) to (3): (1) positions 311 and 343; (2) positions 311 and 413; and (3) positions 343 and 413, as expressed in EU numbering. In a further embodiment, the mutant Fc region with increased pI contains Arg or Lys at each of the selected positions.

[0150] In one embodiment, an anti-CD137 antigen binding molecule or antibody of the disclosure comprises a variant Fc region comprising an amino acid modification as set forth in Table 2 below.

[0151] Amino acid modifications that increase the pI of the Fc region [Table 2]

[0152] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises a mutant Fc region created by adding amino acid modifications to a native-sequence Fc region. In one embodiment, the mutant Fc region has increased binding activity to at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb, compared to a native-sequence Fc region or the parent Fc region. Preferably, the mutant Fc region has increased binding activity to FcγRIIb, compared to a native-sequence Fc region or the parent Fc region. It has been reported that anti-CD137 antibodies comprising a mutant Fc region with increased binding activity to FcγRIIb have increased agonistic activity, compared to anti-CD137 antibodies comprising a native-sequence Fc region. In one embodiment, the amino acid modification that increases FcγRIIb-binding activity may be, for example, the amino acid modification described in WO2012 / 115241, WO2014 / 030728, WO2014 / 163101, and / or WO2017 / 104783. In a preferred embodiment, the modification that increases FcγRIIb-binding activity is an amino acid modification at at least one position selected from the group consisting of positions 234, 235, 236, 237, 238, 264, 268, 295, 326, and 330 (EU numbering).

[0153] The term "Fcγ receptor" (herein referred to as Fcγ receptor, FcγR, or FcgR) refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, and effectively refers to any member of a family of proteins encoded by Fcγ receptor genes. In humans, this family includes FcγRI (CD64), which includes the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 (H type) and R131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any unidentified human FcγR, FcγR isoform, or allotype. FcγRIIb1 and FcγRIIb2 have been reported as splice variants of human FcγRIIb. Furthermore, a splice variant designated FcγRIIb3 has been reported (J Exp Med, 1989, 170: 1369-1385). In addition to these splice variants, human FcγRIIb includes AAI46679.1, registered with NCBI, and all splice variants registered with NCBI, including NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1, and NP_003992.3. Furthermore, human FcγRIIb includes, in addition to FcγRIIb, all genetic polymorphisms that have been reported in the past (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)), as well as all genetic polymorphisms that will be reported in the future.

[0154] There are two allotypes of FcγRIIa: one in which the amino acid at position 131 of FcγRIIa is histidine (H type), and the other in which the amino acid at position 131 is substituted with arginine (R type) (Warrmerdam, J. Exp. Med. 172: 19-25(1990)).

[0155] FcγRs may be derived from any organism, including, but not limited to, FcγRs from humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any mouse FcγR or FcγR isoform.

[0156] In another aspect, the present disclosure provides anti-CD137 antigen-binding molecules or antibodies comprising a mutant Fc region with increased binding activity to FcγRIIb, comprising any one of the following amino acid modifications (1) to (8): (1) positions 234, 238, 264, and 330; (2) positions 234, 238, and 330; (3) positions 234, 237, 238, and 330; (4) positions 236, 268, and 330; (5) positions 235, 236, 268, 295, 326, and 330, as expressed in EU numbering.

[0157] In one embodiment, an anti-CD137 antigen-binding molecule or antibody of the present disclosure comprises a mutated Fc region comprising an amino acid modification described in Table 3 below. In a further embodiment, an anti-CD137 antigen-binding molecule or antibody of the present disclosure comprises a mutated Fc region comprising, in addition to the amino acid modifications described in Table 2 (amino acid modifications that increase the pI of Fc), any combination of amino acid modifications described in Table 3 below.

[0158] Amino acid modifications that increase the FcγRIIb-binding activity of the Fc region [Table 3]

[0159] In one embodiment, the present disclosure provides an altered Fc region comprising an altered Fc region in which at least one amino acid has been altered, wherein the binding activity of the altered Fc region to FcgammaRIIb is the same as or higher than that of a reference Fc region. In one embodiment, the reference Fc region is an Fc region comprising any combination of amino acid alterations listed in Table 3 above. In a preferred embodiment, the reference Fc region is an Fc region contained in the heavy chain constant region of 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 of MY201 (SEQ ID NO: 153) or MY518 (SEQ ID NO: 154).

[0160] In another aspect, the present disclosure provides isolated agonist antigen-binding molecules or antibodies comprising a mutant Fc region with increased Fcγ receptor (preferably FcγRIIb) binding activity and increased pI. In certain embodiments, the mutant Fc region described herein comprises at least two amino acid modifications in the parent Fc region. As described above, antigen-binding molecules or antibodies with increased pI are more strongly attracted to the net negatively charged endothelial cell surface through physicochemical Coulombic interactions than antigen-binding molecules or antibodies without increased pI. Therefore, in agonist antigen-binding molecules or antibodies whose agonist activity is contributed by binding activity to an Fcγ receptor (preferably FcγRIIb), the agonist activity of the antigen-binding molecule or antibody can be increased by combining amino acid modifications that increase Fcγ receptor (preferably FcγRIIb) binding activity with amino acid modifications that increase pI.

[0161] In one embodiment, the anti-CD137 antigen-binding molecule or antibody comprises a mutant Fc region comprising both the above-mentioned amino acid modifications that increase binding activity to an Fcγ receptor (e.g., FcγRIIb) and amino acid modifications that increase the isoelectric point (pI). As described above, antigen-binding molecules or antibodies with an increased pI are more strongly attracted to the net negatively charged endothelial cell surface through physicochemical Coulombic interactions than antigen-binding molecules or antibodies without an increased pI. Therefore, in anti-CD137 agonist antigen-binding molecules or antibodies whose binding activity to an Fcγ receptor (preferably FcγRIIb) contributes to their CD137 agonistic activity, the agonistic activity of the anti-CD137 antigen-binding molecule or antibody can be increased by combining amino acid modifications that increase binding activity to an Fcγ receptor (preferably FcγRIIb) with amino acid modifications that increase pI.

[0162] In one aspect, the present disclosure provides a polypeptide comprising a mutant Fc region with increased FcγRIIb binding activity and increased pI, comprising at least three amino acid modifications including: (a) at least one amino acid modification at at least one position selected from the group consisting of positions 234, 235, 236, 237, 238, 264, 268, 295, 326, and 330, as expressed in EU numbering; and (b) at least two amino acid modifications at at least two positions selected from the group consisting of positions 311, 343, and 413, as expressed in EU numbering.

[0163] In another aspect, the present disclosure provides a polypeptide comprising a mutant Fc region with increased FcγRIIb binding activity and increased pI, comprising any one of the following amino acid alterations (1) to (26): (1) positions 235, 236, 268, 295, 326, 330, 343, and 413; (2) positions 214, 235, 236, 268, 295, 326, 330, 343, and 413; (3) positions 234, 238, 250, 264, 307, 330, 343, and 413; (4) positions 234, 238, 264, 330, 343, and 413; (5) positions 234, 237, 238, 250, 307, 330, 343, and 413; (6) positions 234, 237, 238, 330, 343, and 413; (7) positions 235, 236, 268, 295, 326, 330, 311, and 343; (8) positions 234, 238, 250, 264, 307, 330, 311, and 343; (9) positions 234, 238, 264, 330, 311, and 343; (10) positions 234, 237, 238, 250, 307, 330, 311, and 343; (11) positions 234, 237, 238, 330, 311, and 343; (12) positions 235, 236, 268, 295, 326, 330, and 343; (13) positions 214, 235, 236, 268, 295, 326, 330, and 343; (14) positions 235, 236, 268, 295, 326, 330, and 413; (15) positions 214, 236, 268, 330, and 343; (16) positions 214, 235, 236, 268, 330, and 343; (17) positions 214, 236, 268, 330, and 413; (18) positions 214, 236, 268, 330, 343, and 413; (19) positions 214, 235, 236, 268, 330, 343, and 413; (20) positions 214, 236, 268, 330, and 311; (21) positions 214, 235, 236, 268, 330, and 311; (22) positions 214, 236, 268, 330, 311, and 343; (23) positions 214, 235, 236, 268, 330, 311, and 343; (24) positions 214, 236, 268, 330, 311, and 413; (25) positions 214, 235, 236, 268, 330, 311, and 413; (26) Positions 214, 235, 236, 268, 295, 326, 330, and 311.

[0164] In one embodiment, the variant Fc region of the present disclosure comprises any combination of amino acid modifications set forth in Table 4 below.

[0165] [Table 4]

[0166] In one aspect, a mutant Fc region comprising any combination of amino acid modifications listed in Table 4 above has a deletion of the amino acid at position 447 (EU numbering). In a preferred aspect, a mutant Fc region comprising any combination of amino acid modifications listed in Table 4 above has a deletion of the amino acids at positions 446 and 447 (EU numbering).

[0167] In addition to the modifications exemplified above, it will be understood by those skilled in the art that at least one amino acid modification that increases the binding activity to FcγR, including FcγRIIb, relative to the parent Fc region, as described or suggested in, for example, WO2013 / 047752, WO2013 / 125667, WO2014 / 030728, WO2014 / 163101, or WO2017104783, and at least one amino acid modification that increases the pI relative to the parent Fc region, as described or suggested in, for example, WO2017 / 104783, WO2017 / 046994, and combinations of these amino acid modifications may also be used.

[0168] Additionally, amino acid modifications made for other purposes can be combined in the variant Fc regions described herein. For example, 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); WO2006 / 019447; WO2006 / 053301; and WO2009 / 086320) and amino acid substitutions that improve antibody heterogeneity or stability (WO2009 / 041613) may be made. Alternatively, the mutant Fc regions described herein can be combined with polypeptides having the property of promoting antigen clearance as described in WO2011 / 122011, WO2012 / 132067, WO2013 / 046704, or WO2013 / 180201, polypeptides having the property of specifically binding to target tissues as described in WO2013 / 180200, or polypeptides having the property of repeatedly binding to multiple antigen molecules as described in WO2009 / 125825, WO2012 / 073992, or WO2013 / 047752. Alternatively, the amino acid modifications disclosed in EP1752471 and EP1772465 can be combined in the CH3 of the mutant Fc regions described herein to confer binding activity to other antigens.

[0169] In one embodiment, an anti-CD137 antigen-binding molecule or antibody of the present disclosure comprises a heavy chain constant region comprising any one amino acid sequence selected from SEQ ID NOs: 64 to 85. Preferably, an anti-CD137 antigen-binding molecule or antibody of the present disclosure comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 75 or 82.

[0170] In a preferred embodiment, the anti-CD137 antigen-binding molecule or antibody comprising the above-described mutant Fc region has binding activity to CD137 that is dependent on the above-described small molecule compound.

[0171] In one embodiment, the anti-CD137 antigen-binding molecule or antibody of the present 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 a variant Fc region described above. In a preferred embodiment, the anti-CD137 antigen-binding molecule or antibody of the present disclosure may be any one anti-CD137 antibody selected from the antibodies listed in Table 52.

[0172] In a further aspect, the present disclosure provides antigen-binding molecules or antibodies that bind to the same epitope of CD137 as the anti-CD137 antigen-binding molecules or antibodies provided herein in the presence of a small molecule compound (e.g., in the presence 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 of a small molecule compound). For example, in certain embodiments, antigen-binding molecules or antibodies are provided that bind to the same epitope as anti-CD137 antigen-binding molecules or antibodies comprising the heavy chain variable region / light chain variable region combinations 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 listed in Table 17. In one embodiment, anti-CD137 antigen-binding molecules or antibodies having CD137-binding activity dependent on the antigen-binding activity dependent on a small molecule compound of the present disclosure recognize an epitope formed by a complex of an antigen (e.g., CD137) and a small molecule compound (e.g., ATP).

[0173] In a further aspect, the present disclosure provides antigen-binding molecules or antibodies that compete with the anti-CD137 antigen-binding molecules or antibodies provided herein for binding to CD137 in the presence of a small molecule compound (e.g., in the presence of a small molecule compound at 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 certain embodiments, the antigen-binding molecules or antibodies that compete with the anti-CD137 antigen-binding molecules or antibodies provided herein for binding to CD137 include, for example, the heavy chain variable region / light chain variable region combinations listed in Table 17, A375 / B167, A372 / B040, A356 / B040, A486 / B167, A487 / B167, A488 / B226, A489 / B223, A548 / B376, A551 / B256, A551 / B379, A555 / B379, Competes with anti-CD137 antigen-binding molecules or antibodies, including A548 / B256 and / or A549 / B167.

[0174] In a further aspect of the present disclosure, the anti-CD137 antigen-binding molecule or antibody according to any of the above-described embodiments is a monoclonal antibody, including a chimeric, humanized, or human antibody. In one embodiment, the anti-CD137 antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as a complete IgG1 antibody or other antibody class or isotype as defined herein.

[0175] In further aspects, the anti-CD137 antigen-binding molecule or antibody according to any of the above embodiments may incorporate, alone or in combination, any of the features described in items 1 to 7 below.

[0176] 1. Agonistic activity of anti-CD137 antigen-binding molecules or antibodies In certain embodiments, the anti-CD137 antigen binding molecule or antibody of the present disclosure has CD137 agonist activity. CD137 signaling is known to stimulate not only IFN-γ secretion and proliferation of NK cells (Buechele et al., 2012; Lin et al., 2008; Melero et al., 1998), but also promote DC activation, as indicated by increased survival and cytokine secretion and upregulation of costimulatory molecules (Choi et al., 2009; Futagawa et al., 2002; Wilcox et al., 2002). However, CD137 is best characterized as a costimulatory molecule that regulates TCR-induced activation in both the CD4+ and CD8+ subsets of T cells. In combination with TCR triggering, anti-CD137 agonist antibodies enhance T cell proliferation, stimulate lymphokine secretion, and reduce the susceptibility of T lymphocytes to activation-induced cell death (reviewed in Snell et al., 2011). Among these physiological events observed after CD137 signaling on T cells, the CD137-activated downstream signaling pathways TRAF2, TRAF1, particularly NF-kappaB, JNK, Erk, Akt, survivin, Bcl-XL, and / or Bcl-2 are mediated (Ward-Kavanagh et al., Immunity, 44:1005 (2016)).

[0177] In one embodiment, an "anti-CD137 agonist antigen-binding molecule" or "anti-CD137 agonist antibody" is an antigen-binding molecule or antibody that, upon binding to CD137, transduces CD137 signals and significantly induces or enhances NK cell IFN-γ secretion, proliferation, and increased survival; DC activation as indicated by cytokine secretion and upregulation of costimulatory molecules; TCR induction; T cell proliferation; and / or lymphokine secretion. In a different embodiment, an "anti-CD137 agonist antigen-binding molecule" or "anti-CD137 agonist antibody" is an antigen-binding molecule or antibody that, upon binding to CD137 on T cells, transduces CD137 signals and significantly induces NF-kappaB activation in the T cells. Furthermore, an antigen-binding molecule or antibody "exhibiting CD137 agonist activity" means that any of the physiological phenomena described above is observed upon binding of the antigen-binding molecule or antibody to CD137. The method for measuring CD137 agonist activity is described in detail in the section "C. Measurement Method (Assay)" below.

[0178] In certain embodiments, the anti-CD137 antigen binding molecule or antibody of the present disclosure has CD137 agonist activity that depends on the small molecule compound. In a non-limiting embodiment, the anti-CD137 antigen binding molecule or antibody has higher CD137 agonist activity for CD137 in the presence of a small molecule compound compared to the CD137 agonist activity of CD137 in the absence of the small molecule compound. In a different embodiment, the anti-CD137 antigen binding molecule or antibody has higher CD137 agonist activity in the presence of a high concentration of a small molecule compound compared to the CD137 agonist activity in the presence of a low concentration of the small molecule compound. In a further aspect, the anti-CD137 antigen binding molecule or antibody has a CD137 agonist activity in the presence of a small molecule compound that is 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, 100-fold or more, 200-fold or more, 300-fold or more, 500-fold or more, 1×10 or more, or 1×10 or more, of the CD137 agonist activity in the absence of the small molecule compound. 3 More than twice, 2×10 3 more than twice, above, 3×10 3 More than twice, 5×10 3More than twice, 1×10 4 More than twice, 2×10 4 More than double, 3×10 4 More than twice, 5×10 4 More than double, or 1×10 5 It is more than double.

[0179] The concentration of the small molecule compound can be selected at any 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 signals by binding to CD137 on the cell surface. Therefore, it will be understood by those skilled in the art that an anti-CD137 antigen-binding molecule or antibody that has CD137-binding activity dependent on the small molecule compound also has CD137 agonist activity dependent on the small molecule compound. However, on the other hand, because the measurement methods for binding activity and agonist activity are different, those skilled in the art will understand that the concentration of a small molecule compound at which a difference in binding activity is detected may differ from the concentration of a small molecule compound at which a difference in agonist activity is detected (for example, in an anti-CD137 antigen-binding molecule or antibody whose CD137-binding activity in the presence of 10 μM of a small molecule compound is at least twice as high as that in the absence of the small molecule compound, the CD137 agonist activity (measured) in the presence of 10 μM of the small molecule compound may be less than twice as high as the CD137 agonist activity (measured) in the absence of the small molecule compound). Furthermore, those skilled in the art will understand that the determination of agonist activity may differ depending on the method for measuring CD137 agonist activity (see C. Measurement Method (Assay)).

[0180] In one embodiment, the anti-CD137 antigen binding molecule or antibody (i) exhibits agonist activity against CD137 in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM of a small molecule compound, and (ii) exhibits substantially no agonist activity against CD137 in the absence of the small molecule compound, or exhibits reduced agonist activity against CD137 in the absence of the small molecule compound (compared to in the presence of the small molecule compound).

[0181] In one embodiment, when the agonist activity of an anti-CD137 antigen-binding molecule or antibody is assessed in a) agonist activity assay (PBMC) detailed in C. Assay, the anti-CD137 antigen-binding molecule or antibody (i) exhibits agonist activity against CD137 in the presence of 250 μM of a small molecule compound, and (ii) has reduced agonist 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 agonist activity against CD137 in the presence of 250 μM of a small molecule compound, and (ii) exhibits substantially no agonist activity against CD137 in the absence of the small molecule compound.

[0182] In one embodiment, when the agonist activity of an anti-CD137 antigen-binding molecule or antibody is evaluated in b) agonist activity assay (reporter gene assay) detailed in C. Assay, the anti-CD137 antigen-binding molecule or antibody (i) exhibits agonist activity against CD137 in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM of a small molecule compound, and (ii) exhibits substantially no agonist activity against CD137 in the absence of the small molecule compound, or exhibits low agonist activity (compared to the presence of the small molecule compound). The antibody concentration in the reporter gene assay can be selected arbitrarily; for example, the final antibody concentration is 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.

[0183] In one embodiment, in b) agonist activity measurement method (reporter gene assay) detailed in C. Measurement Method (Assay), when the final antibody concentration is 0.1 μg / mL, (i) the CD137 agonist activity (relative light unit) of an anti-CD137 antigen-binding molecule or antibody in the presence of 10 μM of a small molecule compound is 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, 60-fold or more, 70-fold or more, 80-fold or more, or 90-fold or more higher than the CD137 agonist activity (relative light unit) in the absence of the small molecule compound. In one embodiment, in b) agonist activity measurement method (reporter gene assay) detailed in C. Measurement Method (Assay), when the final antibody concentration is 0.1 μg / mL, (i) the CD137 agonist activity (relative light unit) of an anti-CD137 antigen-binding molecule or antibody in the presence of 100 μM of a small molecule compound is 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, 60-fold or more, 70-fold or more, 80-fold or more, or 90-fold or more higher than the CD137 agonist activity (relative light unit) in the absence of the small molecule compound. In one embodiment, in b) agonist activity measurement method (reporter gene assay) detailed in C. Measurement Method (Assay), when the final antibody concentration is 0.1 μg / mL, (i) the CD137 agonist activity (relative light unit) of an anti-CD137 antigen-binding molecule or antibody in the presence of 250 μM of a small molecule compound is 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, 60-fold or more, 70-fold or more, 80-fold or more, or 90-fold or more higher than the CD137 agonist activity (relative light unit) in the absence of the small molecule compound. In any of the above embodiments, furthermore, 0.1 μg / mL of an anti-CD137 antigen-binding molecule or antibody exhibits substantially no CD137 agonist activity in the absence of the small molecule compound.

[0184] In one embodiment, in b) agonist activity measurement method (reporter gene assay) detailed in C. Measurement Method (Assay), when the final antibody concentration is 1 μg / mL, (i) the CD137 agonist activity (relative light unit) of an anti-CD137 antigen-binding molecule or antibody in the presence of 10 μM of a small molecule compound is 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, 60-fold or more, 70-fold or more, 80-fold or more, or 90-fold or more higher than the CD137 agonist activity (relative light unit) in the absence of the small molecule compound. In one embodiment, in b) agonist activity measurement method (reporter gene assay) detailed in C. Measurement Method (Assay), when the final antibody concentration is 0.1 μg / mL, (i) the CD137 agonist activity (relative light unit) of an anti-CD137 antigen-binding molecule or antibody in the presence of 100 μM of a small molecule compound is 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, 60-fold or more, 70-fold or more, 80-fold or more, or 90-fold or more higher than the CD137 agonist activity (relative light unit) in the absence of the small molecule compound. In one embodiment, in b) agonist activity measurement method (reporter gene assay) detailed in C. Measurement Method (Assay), when the final antibody concentration is 0.1 μg / mL, (i) the CD137 agonist activity (relative light unit) of an anti-CD137 antigen-binding molecule or antibody in the presence of 250 μM of a small molecule compound is 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, 60-fold or more, 70-fold or more, 80-fold or more, or 90-fold or more higher than the CD137 agonist activity (relative light unit) in the absence of the small molecule compound. In any of the above embodiments, furthermore, 1 μg / mL of an anti-CD137 antigen-binding molecule or antibody exhibits substantially no CD137 agonist activity in the absence of the small molecule compound.

[0185] 2. Antibody fragment In certain embodiments, the antibodies provided 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 specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab')2 fragments containing salvage receptor binding epitope residues and having increased half-lives in vivo.

[0186] Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0187] A single-domain antibody is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0188] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of whole antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0189] 3. Chimeric and humanized antibodies In certain embodiments, the antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody whose class or subclass is changed from that of the parent antibody. Chimeric antibodies also include antigen-binding fragments thereof.

[0190] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while maintaining the specificity and affinity of the parent non-human antibody. A humanized antibody usually comprises one or more variable domains, in which the HVRs (e.g., CDRs (or portions thereof)) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues were derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0191] Humanized antibodies and methods for their production are reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and also see, e.g., Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 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) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing resurfacing); Further described in Dall'Acqua et al., Methods 36:43-60 (2005) (describing FR shuffling); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guide selection" approach for FR shuffling).

[0192] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light or heavy chain variable regions (see Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see Baca et al., J. Biol. Chem. 272:10678-10684 (1997)). and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0193] 4. Human antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced by various techniques known in the art. Human antibodies are reviewed in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0194] Human antibodies may be prepared by administering an immunogen to transgenic animals that have been engineered to produce fully human antibodies or complete antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or a portion of human immunoglobulin loci, which either replace endogenous immunoglobulin loci or are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are usually 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, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions from whole antibodies produced by such animals may be further modified, for example, by combining with different human constant regions.

[0195] Human antibodies can also be produced using hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991).) Human antibodies generated via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which describes human-human hybridomas). 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).

[0196] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. These variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0197] 5. Library-derived Antibodies Antibodies of the present disclosure may be isolated by screening combinatorial libraries for antibodies with the desired activity or 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 characteristics. Such methods are reviewed in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described, for example, in 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, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, 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).

[0198] In a specific phage display method, VH and VL gene repertoires are separately cloned by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need for hybridoma construction. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a wide range of non-self and self antigens without immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can be generated synthetically by cloning unrearranged V-gene segments from stem cells and using PCR primers encoding the hypervariable CDR3 regions and containing random sequences to achieve rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent literature describing human antibody phage libraries includes, for example: U.S. Pat. No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0199] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0200] The antigen-binding molecules or antibodies of the present disclosure having antigen-binding activity dependent on a small molecule compound may be selected by screening from a library of antigen-binding molecules. The above-mentioned combinatorial library may be used as the library. The library of antigen-binding molecules may be a library with an unbiased repertoire of antigen-binding molecules (naive library), or a biased library. An example of the latter library is a library of antigen-binding molecules pre-conferred with binding activity to a predetermined compound. In a specific embodiment, the library of antigen-binding molecules is a library of antigen-binding molecules pre-contained with amino acid modifications to confer binding activity to a predetermined compound. Examples of such libraries include the library described in International Publication WO2015 / 083764.

[0201] 6. Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies (e.g., bispecific antibodies). Multispecific antibodies are monoclonal antibodies that have binding specificities at at least two different sites. In certain embodiments, one of the binding specificities is for CD137 and the other is for any other antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of CD137. Bispecific antibodies may be used to localize cytotoxic agents to cells expressing CD137. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0202] In one embodiment, the anti-CD137 antigen-binding molecule or antibody of the present disclosure is a bispecific antibody in which one arm has small molecule-dependent CD137-binding activity and the other arm binds to an antigen other than CD137. The structure of the "antigen" other than CD137 is not limited to a specific structure. In another sense, the antigen may be inorganic or organic. Exemplary antigens are disclosed herein (e.g., "IV. Compositions and Methods (Antigen-binding Molecules with Antigen-binding Activity That Changes Depending on the Compound Concentration), B. Antigens"). In one embodiment, the antigen is preferably an antigen expressed in cancer cells, immune cells, stromal cells, etc. in cancer tissues or inflammatory tissues.

[0203] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and knob-in-hole technology (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can be constructed by manipulating electrostatic steering effects to create Fc heterodimeric molecules (WO2009 / 089004A1); cross-linking two or more antibodies or fragments (see U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to generate antibodies with two specificities (see Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to create bispecific antibody fragments (see Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (Gruber et al., J. Immunol., 152:5368 (1993)). (1994)); and by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991).

[0204] Engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies," are also included herein (see, eg, US Patent Application Publication No. 2006 / 0025576 A1).

[0205] As used herein, antibody or fragment also includes a "dual-acting Fab" or "DAF" that contains one antigen-binding site that binds to CD137 and another distinct antigen (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820).

[0206] 7. Antibody Variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are also contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., antigen binding).

[0207] a) Substitution, insertion, and deletion mutants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of "Preferred Substitutions." More substantial changes are provided in Table 1 under the heading of "Exemplary Substitutions" and are detailed below with reference to classes of amino acid side chains. Amino acid substitutions may be introduced into the antibody of interest, and the products may be screened for a desired activity, such as, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0208] [Table 5]

[0209] Amino acids can be divided into groups according to common side chain properties: (1) Hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile); (2) neutral hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln); (3) Acidic: aspartic acid (Asp), glutamic acid (Glu); (4) Basic: histidine (His), lysine (Lys), arginine (Arg); (5) residues that affect chain orientation: glycine (Gly), proline (Pro); (6) Aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe). Non-conservative substitutions refer to the exchange of a member of one of these classes for one from another class.

[0210] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variant selected for further study will have a modified (e.g., improved) specific biological property compared to the parent antibody (e.g., increased affinity, decreased immunogenicity) and / or will substantially retain the specific biological property of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage-display-based affinity maturation techniques (e.g., those described herein). Briefly, one or more HVR residues are mutated, and the mutated antibodies are displayed on phage and screened for a specific biological activity (e.g., binding affinity).

[0211] Modifications (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such modifications can be made in HVR "hot spots," i.e., residues encoded by codons that frequently mutate during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or residues that contact antigen, and the resulting mutant VH or VL can be tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which 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 scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0212] In certain embodiments, substitutions, insertions, or deletions may be made within one or more HVRs, as long as such modifications do not substantially reduce the antibody's ability to bind to antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such modifications may, for example, be outside the antigen-contacting residues of the HVRs. In certain embodiments of the above-described mutant VH and VL sequences, each HVR is unaltered or contains only one, two, or three amino acid substitutions.

[0213] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues, such as arginine, aspartic acid, histidine, lysine, and glutamic acid) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and it is determined whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to this initial substitution. Alternatively or additionally, a crystal structure of the antigen-antibody complex can be analyzed to identify contact points between the antibody and antigen. Such contact residues and neighboring residues can be targeted as substitution candidates or can be excluded from the list of substitution candidates. Mutants can be screened to determine whether they contain desired properties.

[0214] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as internal insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody of an enzyme (e.g., for ADEPT) or a polypeptide which increases the plasma half-life of the antibody.

[0215] b) Glycosylation variants In certain embodiments, the antibodies provided herein have been modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0216] If the antibody contains an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, usually attached via an N-linkage to Asn297 in the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the present disclosure may be performed to create antibody variants with specific improved properties.

[0217] In one embodiment, antibody variants are provided that have carbohydrate structures lacking fucose added (directly or indirectly) to the Fc region. For example, the amount of fucose 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 in the glycan at Asn297 relative to the sum of all glycostructures (e.g., complex, hybrid, and high-mannose structures) added to Asn297, as measured by MALDI-TOF mass spectrometry, for example, as described in WO2008 / 077546. Asn297 represents an asparagine residue located approximately at position 297 in the Fc region (EU numbering of Fc region residues). However, due to slight sequence variability between multiple antibodies, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Application Publication Nos. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which lack protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. US2003 / 0157108 A1, Presta, L; and WO2004 / 056312A1, Adams et al., especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0218] Further provided are antibody variants having bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody, bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US2005 / 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, in WO1997 / 30087 (Patel et al.); WO1998 / 58964 (Raju, S.); and WO1999 / 22764 (Raju, S.).

[0219] c) Fc region mutants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant (also sometimes referred to as an "altered Fc region"). The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0220] In certain embodiments, antibody variants that retain some, but not all, effector functions are also contemplated by the present disclosure, making them desirable candidates for applications where in vivo half-life is important but certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / lack of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that an antibody lacks FcγR binding (and thus likely lacks ADCC activity) while retaining FcRn binding activity. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., 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); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, e.g., ACT1™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assays (Promega, Madison, WI)).Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of a molecule of interest may be assessed in vivo in an animal model, e.g., as described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be performed to confirm that the antibody is unable to bind C1q and thus lacks CDC activity. See, e.g., the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. CDC measurements may also be performed to assess complement activation (see, e.g., Gazzano-Santoro et al., 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)). Furthermore, determination of FcRn binding and in vivo clearance / half-life may also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0221] Antibodies with reduced effector function include those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with two or more substitutions at amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0222] Certain antibody variants with increased or decreased binding to FcRs have been described (see U.S. Pat. No. 6,737,056; WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0223] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and / or 334 (EU numbering) of the Fc region).

[0224] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either increased or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0225] Antibodies with increased half-lives and increased binding to the neonatal Fc receptor (FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) are described in U.S. Patent Application Publication No. 2005 / 0014934 A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that increase binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (e.g., substitution of Fc region residue 434 (U.S. Patent No. 7,371,826)).

[0226] For other examples of Fc region variants, 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.

[0227] In one aspect, the binding activity of an antibody Fc region (including a mutant Fc region; the same applies below) to each human Fcγ receptor (FcγR) is measured by a ligand capture method using, for example, a BIACORE (registered trademark) T200, which employs surface plasmon resonance analysis as the measurement principle.

[0228] Exemplary methods for measuring the binding activity of antibody Fc regions to each human Fcγ receptor (FcγR) are described in detail below. In one embodiment, the binding activity of antibody Fc regions to FcγRs is assessed using a BIACORE™ T200. In a preferred embodiment, this measurement is performed at 25°C using an assay buffer containing 50 mM phosphate, 150 mM NaCl, and 0.05 w / v% P20, pH 7.4. Specifically, approximately 1000 RU of an antibody comprising a modified Fc region is first captured using a sensor chip immobilized with CaptureSelect™ Human Fab-lambda Kinetics Biotin Conjugate (ThermoFisher Scientific) as a ligand capture molecule. Human FcγRs are diluted in assay buffer to 8 nM for FcγRIa and 1000 nM for other FcγRs, and then allowed to bind to the captured antibody. The binding activity of each antibody to each FcγR is evaluated by calculating the FcγR binding amount per unit antibody amount (RU) using Biacore T200 Evaluation Software 2.0. In one embodiment, the binding activity of an antibody Fc region to each human Fcγ receptor (FcγR) can be measured by the method described in Example 7-4.

[0229] In a preferred embodiment, the FcγR used in the above-mentioned measurement method may be an extracellular domain of FcγR prepared by the following method. First, the gene encoding the extracellular domain of FcγR is synthesized by a method known to those skilled in the art. The sequence of each FcγR is prepared based on information registered with NCBI. Specifically, the FcγRI is prepared based on the sequence in NCBI accession number NM_000566.3, the FcγRIIa is prepared based on the sequence in NCBI accession number NM_001136219.1, the FcγRIIb is prepared based on the sequence in NCBI accession number NM_004001.3, and the FcγRIIIa is prepared based on the sequence in NCBI accession number NM_001127593.1, and a His tag is added to the C-terminus. The polymorphic site of FcγRIIa was prepared with reference to J. Exp. Med., 1990, 172, 19-25, and the polymorphic site of FcγRIIIa was prepared with reference to J. Clin. Invest., 1997, 100, 1059-1070. The resulting gene fragment was inserted into an animal cell expression vector to prepare an expression vector. The resulting expression vector was transiently transfected into FreeStyle293 cells (Invitrogen) derived from human embryonic kidney carcinoma, and the target protein was expressed. The culture supernatant was collected and passed through a 0.22 μm filter. Purification was generally carried out in the following four steps: the first step was cation exchange column chromatography (SP Sepharose FF), the second step was His-tag affinity column chromatography (HisTrap HP), the third step was gel filtration column chromatography (Superdex 200), and the fourth step was sterile filtration. For FcγRI, anion exchange column chromatography using Q Sepharose FF is performed as the first step. The concentration of the purified protein is calculated by measuring the absorbance at 280 nm using a spectrophotometer and using the extinction coefficient calculated from the obtained value using a method such as PACE (Protein Science, 1995, 4, 2411-2423).

[0230] In one embodiment, the binding activity of an antibody Fc region to human FcRn is measured by a ligand capture method using, for example, a BIACORE (registered trademark) T200, which employs surface plasmon resonance analysis as its measurement principle.

[0231] An exemplary method for measuring the binding activity of an antibody Fc region to human FcRn is described in detail below. In one embodiment, the binding activity of an antibody Fc region to human FcRn is evaluated using a BIACORE™ T200. In a preferred embodiment, the measurement is performed at 25°C using an assay buffer containing 50 mM phosphate, 150 mM NaCl, and 0.05 w / v% P20, pH 6.0. Specifically, approximately 400 RU of an antibody containing an Fc region is captured using a sensor chip immobilized with CaptureSelect™ Human Fab-lambda Kinetics Biotin Conjugate (ThermoFisher Scientific) as a ligand capture molecule, and human FcRn diluted with assay buffer is allowed to bind to the antibody. The FcRn-binding activity of each antibody is evaluated by calculating KD(M) using the steady state model using Biacore T200 Evaluation Software 2.0. In a preferred embodiment, the human FcRn protein used in this measurement is prepared by the method described in Reference Example 2 of WO2010107110. In one embodiment, the binding activity of an antibody Fc region to human FcRn can be measured by the method described in Example 7-5.

[0232] d) Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine-engineered antibodies (e.g., "thioMAbs") in which one or more residues of an antibody have been substituted with a cysteine ​​residue. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By substituting these residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody, which may be used to conjugate the antibody to other moieties (such as drug moieties or linker-drug moieties) to generate immunoconjugates, as further detailed herein. In certain embodiments, any one or more of the following residues may be substituted with a cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies may be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0233] e) Antibody derivatives In certain embodiments, the antibodies provided herein may be further modified to contain additional nonproteinaceous moieties known in the art and readily available. Suitable moieties for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3 dioxolane, poly-1,3,6 trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. Polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and if more than one polymer is attached, they can be the same molecule or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, etc.

[0234] In another embodiment, a conjugate of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to, wavelengths that heat the non-protein moiety to temperatures that are not harmful to normal cells but that kill cells in close proximity to the antibody-non-protein moiety.

[0235] B. Recombinant Methods and Constructs Antibodies can be produced using recombinant methods or constructs, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-CD137 antigen-binding molecule or antibody described herein is provided. Such a nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic (e.g., a Chinese hamster ovary (CHO) cell) or a lymphoid cell (e.g., a Y0, NS0, or Sp2 / 0 cell)). In one aspect, a method for producing an anti-CD137 antigen-binding molecule or antibody is provided, comprising culturing a host cell comprising nucleic acid encoding the antibody as described above under conditions suitable for expression of the anti-CD137 antigen-binding molecule or antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0236] For recombinant production of anti-CD137 antigen-binding molecules or antibodies, nucleic acids encoding the antibodies (e.g., as described above) are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids may be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of specifically binding to genes encoding the antibody heavy and light chains).

[0237] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody may be isolated in a soluble fraction from the bacterial cell paste or further purified.

[0238] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0239] Host cells derived from multicellular organisms (invertebrates and vertebrates) are also suitable for expressing glycosylated antibodies. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use in conjugation with insect cells, particularly for transformation of Spodoptera frugiperda cells.

[0240] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0241] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that have been adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7); human embryonic kidney (293 or 293 cells, e.g., as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney (BHK) cells; mouse Sertoli cells (TM4 cells, e.g., as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney (CV1); African green monkey kidney (VERO-76); human cervical carcinoma (HELA); canine kidney (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., as described in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)). (described in

[1999] ); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0242] C. Assay The anti-CD137 antigen-binding molecules or antibodies provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0243] 1. Binding and other assays In one aspect, an antigen-binding molecule or antibody of the present disclosure is tested for its antigen-binding activity by known methods, such as ELISA, Western blot, etc.

[0244] In another aspect, in the presence of a small molecule compound (e.g., 10 A competition assay in the presence of a small molecule compound may be used to identify antigen-binding molecules or antibodies that compete with anti-CD137 antigen-binding molecules or antibodies comprising heavy chain variable region / light chain variable region combinations 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 listed in Table 17 for binding to CD137 (in the presence of a small molecule compound at concentrations of at least 50 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM). In certain embodiments, such a competing antigen-binding molecule or antibody binds to the same epitope (e.g., a linear or conformational epitope) as that bound by an anti-CD137 antigen-binding molecule or antibody comprising the heavy chain variable region / light chain variable region combinations 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 set forth in Table 17. Detailed exemplary methods for mapping epitopes bound by antigen-binding molecules or antibodies are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology, vol. 66 (Humana Press, Totowa, NJ). In one embodiment, an anti-CD137 antigen-binding molecule or antibody having CD137-binding activity dependent on a small molecule compound of the present disclosure recognizes an epitope formed by a complex of an antigen (e.g., CD137) and a small molecule compound (e.g., ATP).

[0245] In an exemplary antibody-based competitive assay, immobilized CD137 is subjected to a competitive assay using a first labeled antibody (e.g., a heavy chain variable region / light chain variable region combination of A375 / B167, A372 / B040, A356 / B040, A486 / B167, or A487 / B167 listed in Table 17) that binds to CD137 in the presence of a small molecule compound (e.g., 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 of a small molecule compound). The immobilized CD137 is incubated in a solution containing a first antibody (anti-CD137 antibody, including A488 / B226, A489 / B223, A548 / B376, A551 / B256, A551 / B379, A555 / B379, A548 / B256, and / or A549 / B167) and a second, unlabeled antibody to be tested for its ability to compete with the first antibody for binding to CD137. The second antibody may be present in hybridoma supernatant. As a control, immobilized CD137 is incubated in a solution containing the first, labeled antibody but not the second, unlabeled antibody. After incubation under conditions that allow binding of the first antibody to CD137, excess unbound antibody is removed, and the amount of label bound to the immobilized CD137 is measured. If the amount of label bound to immobilized CD137 is substantially reduced in the test sample compared to the control sample, this indicates that the second antibody competes with the first antibody for binding to CD137. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Those skilled in the art will understand that this assay can be similarly performed with antigen-binding molecules other than antibodies.

[0246] 2.Activity measurement method In one aspect, a method for identifying anti-CD137 antigen binding molecules or antibodies with biological activity is provided. Biological activity may include, for example, CD137 agonist activity; plasma half-life; anti-tumor activity; and low or suppressed systemic response in tissues other than tumors. Also provided are antigen binding molecules or antibodies that have such biological activity in vivo and / or in vitro.

[0247] In certain embodiments, antigen binding molecules (e.g., anti-CD137 antigen binding molecules) or antibodies of the disclosure are tested for such biological activities.

[0248] a) Agonist activity assay (PBMC) In one embodiment, agonistic activity against CD137 is measured by contacting CD137-expressing cells with an anti-CD137 antigen-binding molecule or antibody in a solution with or without the addition of a small molecule compound. In one embodiment, agonistic activity against CD137 in a solution with or without the addition of a small molecule compound is assessed by measuring cytokine production (e.g., IL-2, IFN-γ, and / or IL-6 production) within 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours after contacting CD137-expressing cells with the anti-CD137 antigen-binding molecule or antibody in the solution. In one embodiment, the solution to which the small molecule compound is added is adjusted so that the concentration of the small molecule compound after adjustment is 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM. In a further embodiment, the CD137-expressing cells are isolated human peripheral blood mononuclear cells (PBMCs) or T cells expanded from isolated human PBMCs.

[0249] In one embodiment, human PBMCs are isolated from blood collected from healthy individuals by centrifugation at 400 x g for 30 minutes at room temperature. Preferably, human PBMCs are isolated in the following two steps. In the first step, Leucosep (greiner bio-one) supplemented with Ficoll-Paque PLUS (GE Healthcare) is centrifuged at 1000 x g for 1 minute at room temperature, followed by the addition of blood diluted with PBS, followed by centrifugation at 400 x g for 30 minutes at room temperature. In the second step, the buffy coat is collected from the tube after centrifugation and washed with 60 mL of PBS (Wako). An exemplary method for measuring CD137 agonist activity using human PBMCs is described in detail below. In the following example, ATP is used as an example of a low molecular weight compound, but other low molecular weight compounds are not excluded. In one embodiment, isolated human PBMCs were cultured in a medium (5% human serum (SIGMA), 95% AIM-V (Thermo Fischer Scientific)) at a cell density of 5x10 6 The isolated human PBMCs are then contacted with an anti-human CD3ε antibody and / or an anti-human CD28 antibody, which induces CD137 expression in the human PBMCs. Preferably, the isolated human PBMCs (cell density 5x10) are diluted to 100%. 6 To a 100 μL plate (100 μL at 100 μL each), 50 μL of 0.04 μg / mL anti-human CD3ε antibody (BD, clone SP34) and 20 μg / mL anti-human CD28 antibody (BD, clone: ​​CD28.2) diluted with medium are added.

[0250] To the human PBMCs to which the anti-human CD3ε antibody and / or anti-human CD28 antibody has been added, (i) a medium containing or not containing ATP; and (ii) an anti-CD137 antigen-binding molecule or antibody are then added. The medium containing or not containing ATP is preferably added in an amount of 25 μL. The anti-CD137 antigen-binding molecule or antibody is preferably added in an amount of 25 μL at 40 μg / mL. More preferably, (i) and (ii) are added approximately 6 hours after contacting the human PBMCs with the anti-human CD3ε antibody and / or anti-human CD28 antibody. In one embodiment, the level of IL-2 production is preferably measured prior to the level of IFN-γ production. In one embodiment, the level of IL-2 production is measured within approximately 24 hours after contacting the human PBMCs with the anti-human CD3ε antibody and / or anti-human CD28 antibody. Preferably, the amount of IL-2 produced is measured approximately 24 hours after contacting human PBMCs with an anti-human CD3ε antibody and / or an anti-human CD28 antibody and approximately 18 hours after addition of an anti-CD137 antigen-binding molecule or antibody.

[0251] In another embodiment, the level of IFN-γ production is measured within about 48 hours after contacting human PBMCs with an anti-human CD3ε antibody and / or an anti-human CD28 antibody. Preferably, the level of IFN-γ production is measured about 48 hours after contacting human PBMCs with an anti-human CD3ε antibody and / or an anti-human CD28 antibody and about 42 hours after adding an anti-CD137 antigen-binding molecule or antibody. In one embodiment, the level of IL-2 production and / or the level of IFN-γ production are measured in the collected culture supernatant. In one embodiment, the human PBMCs to which the anti-human CD3ε antibody and / or the anti-human CD28 antibody has been added are left to stand at 37°C in a 5% CO2 incubator until all measurements are completed.

[0252] Further exemplary details of a method for measuring CD137 agonist activity using human PBMCs are described below. Isolated human PBMCs were cultured at a cell density of 5x10 in a medium (5% human serum (SIGMA), 95% AIM-V (Thermo Fischer Scientific)). 6 Human PBMCs are then diluted to a cell density of 5x10 6 The solution is adjusted to 1 / mL and 100 μL of the solution is seeded into a 96-well flat-bottom multiple-well plate with a lid (Corning). Then, CD137 expression is induced in human PBMCs. For example, 50 μL of 0.04 μg / mL anti-human CD3ε antibody (BD, clone SP34) and 20 μg / mL anti-human CD28 antibody (BD, clone CD28.2) diluted in medium are added to induce CD137 expression in human PBMCs.

[0253] After CD137 expression was induced in human PBMCs, the plate was shaken and placed in a 5% CO2 incubator at 37°C for 6 hours. Then, 25 μL of 2 mM ATP (SIGMA) diluted in culture medium or medium without ATP and 25 μL of 40 μg / mL of each antibody were added to each well. The plate was shaken and placed in a 5% CO2 incubator at 37°C for 18 hours. The culture supernatant was then aliquoted and the amount of IL-2 in the culture supernatant was quantified using the Human IL-2 DuoSet ELISA Kit (R&D systems) or the Human IL-2 ELISA Set (BD Biosciences). After the culture supernatant was collected, the plate was placed in a 5% CO2 incubator at 37°C for 24 hours. A portion of the culture supernatant was then collected, and the amount of IFN-γ contained in the supernatant was quantified using the Human IFN-γ DuoSet ELISA Kit (R&D systems) or the Human IFN-γ ELISA Development Kit (PeproTech). ELISA was performed according to the protocol provided with the kit. For the Human IL-2 DuoSet ELISA Kit (R&D systems) and the Human IFN-γ DuoSet ELISA Kit (R&D systems), color development and quenching were performed according to the protocol using a substrate solution containing H2O2 and tetramethylbenzidine (R&D systems) and 1N H2SO4 (Wako). For the Human IL-2 ELISA Set (BD Biosciences), color development was stopped using 1N H2SO4 (Wako).

[0254] For the IFN-γ ELISA Development Kit (PeproTech), color development and quenching were performed using TMB Chromogen Solution (Thermo Fischer Scientific) and 1N H2SO4 (Wako). Absorbance was then measured using EnVision (PerkinElmer), and the amounts of IL-2 and IFN-γ (pg / mL) in the culture supernatant were calculated using the calibration curves prepared according to the protocol. In this PBMC assay, CD137 agonist activity can be expressed as the fold change in the amounts of IL-2 and IFN-γ in the culture supernatant relative to a negative control antibody (an antibody that does not bind to CD137). In one embodiment, CD137 agonist activity can be measured by the methods described in Examples 5-5-1 and 5-5-2.

[0255] In one embodiment, when the agonist activity against CD137 is evaluated by the amount of cytokine production (e.g., the amount of IL-2, IFN-γ, and / or IL-6 production) in a human PBMC assay, the amount of cytokine production in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM of a small molecule compound when an anti-CD137 antigen-binding molecule or antibody is added is compared with the amount of cytokine production in the presence of a negative control antibody. When the amount of the small molecule compound is 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.05-fold or more, 1.06-fold or more, 1.07-fold or more, 1.08-fold or more, 1.09-fold or more, 1.1-fold or more, 1.11-fold or more, 1.12-fold or more, 1.13-fold or more, 1.14-fold or more, 1.15-fold or more, 1.5-fold or more, 2-fold or more, or 3-fold or more, the anti-CD137 antigen-binding molecule or antibody can be assessed as exhibiting agonistic activity against CD137 in the presence of the small molecule compound.

[0256] In one embodiment, when agonist activity against CD137 is assessed by the amount of IL-2 produced in a human PBMC assay, an anti-CD137 antigen-binding molecule or antibody can be assessed as exhibiting agonist activity against CD137 in the presence of a small molecule compound if the amount of IL-2 produced when an anti-CD137 antigen-binding molecule or antibody is added in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM of a small molecule compound is 1.0...

Claims

1. An anti-CD137 antigen-binding molecule that has CD137-binding activity dependent on the small molecule compound.

2. The anti-CD137 antigen-binding molecule of claim 1, whose binding activity to CD137 in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM of a small molecule compound is at least two-fold higher than its binding activity to CD137 in the absence of the small molecule compound.

3. The anti-CD137 antigen binding molecule of claim 1 or 2, comprising any combination of HVR-H1, HVR-H2, and HVR-H3 selected from the following (a) to (k): (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17; (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, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17; (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, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17; (d) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (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, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (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, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (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, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (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, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19; (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20; (j) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 16, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20; and (k) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:

17.

4. An anti-CD137 antigen binding molecule comprising any combination of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 selected from the following (a) to (m): (a) 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: 17, 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; (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 comprising the amino acid sequence of SEQ ID NO: 7, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 11, 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; (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 comprising the amino acid sequence of SEQ ID NO: 7, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20, 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; (j) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, 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; (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 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: 17, 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.

5. (a) a VH having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 43 to 53; or (b) An anti-CD137 antigen-binding molecule comprising a VL having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 54 to 60.

6. The anti-CD137 antigen binding molecule of any one of claims 1 to 5, comprising a modified Fc region, wherein the modified Fc region comprises any one combination of amino acid modifications selected from the following: 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.

7. An anti-CD137 antigen-binding molecule according to any one of claims 1 to 6, comprising a heavy chain constant region comprising the amino acid sequence of any one of SEQ ID NOs: 64 to 85.

8. An isolated nucleic acid encoding an anti-CD137 antigen-binding molecule according to any one of claims 1 to 7.

9. A vector into which the nucleic acid according to claim 8 has been introduced.

10. A host cell comprising the nucleic acid of claim 8 or the vector of claim 9.

11. A method for producing an anti-CD137 antigen binding molecule, comprising culturing the host cell of claim 10 so that the anti-CD137 antigen binding molecule is produced.

12. An immunoconjugate comprising the anti-CD137 antigen-binding molecule of any one of claims 1 to 7 and a cytotoxic agent.

13. A pharmaceutical formulation comprising an anti-CD137 antigen-binding molecule according to any one of claims 1 to 7 or an immunoconjugate according to claim 12; and a pharmaceutically acceptable carrier.

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